Summary

Viruses are highly ordered supramolecular complexes that have evolved to spread by hijacking the machinery of the host cell. Viruses are extremely diverse, spreading through cells from all kingdoms of life, but they all share common functions and properties. However, to make the best use of viruses and virus-like particles, such as a vehicle for targeted drug delivery or as building blocks in electronics, it is critical to first understand their basic properties and characteristics. The mechanisms affecting viral properties and approaches aimed at utilizing viral particle characteristics are presented in this training unit.

Key words/phrases: Coronaviruses, functional nanoparticles, Virus-Based Nanoparticles (VNPs)

1. Viruses and their importance

1.1. Viruses are found worldwide

Viruses, or molecular nanomachines, infect all cellular life forms, including eukaryotes (vertebrates, invertebrates, plants, and fungi) and prokaryotes (bacteria and Achaea). The presence of viruses is visible in hosts that are displaying illness symptoms. Many healthy species, on the other hand, are hosts to non-pathogenic virus infections, some of which are active while others are dormant. Furthermore, many organisms’ genomes contain fragments of ancient viral genomes that have long since integrated into their host genomes. Viruses can be found in soil, air, and water and can infect species that reside in those habitats, in addition to their hosts [10].

There is still a controversy in the literature about whether viruses are alive or non-living. The point of view decided is determined by how life is defined. Viruses have genes, which are duplicated when they infect cells, making viruses alive in this sense. They are, however, not the same as cellular life forms. When viruses are outside of their host cells, they exist as viral particles (virions), which are non-living and lifeless. [10].

Viruses differ from cells in that they multiply in a different way. A new cell is always generated from a previously formed cell, but a new virion is never formed from a previously formed virion. The replication process, which takes place inside a host cell and involves the synthesis of components followed by their assembly into virions, produces new virions. As a result, viruses are parasites that rely on their hosts for the majority of their needs, such as building components like amino acids and nucleosides, protein-synthesizing machinery (ribosomes), and energy as adenosine triphosphate.

To improve the effectiveness of the reproduction process, a virus alters its host’s intracellular environment. Production of new membrane structures, reduced expression of cell genes, or augmentation of a cell process are examples of modifications. Some enormous phages encode proteins that increase photosynthesis in the cells of their photosynthetic bacterial hosts, hence increasing viral production

1.2 Reasons for studying viruses

1.2.1. Viruses can cause illness

Viruses play a role in a wide range of human diseases, from the minor (e.g., common colds) to the deadly (e.g. rabies). Five pandemic respiratory infections caused by distinct subtypes of influenza virus have attacked the world in the last century, with pigs serving as significant reservoirs for these influenza viruses. The 1918 H1N1 (Spanish flu) killed around 50 million people worldwide, the 1957 H2N2 (Asian flu) killed around 4 million people worldwide, the 1968 H3N2 (Hong Kong flu) killed 1 million people worldwide, the 2005 H5N1 (Bird flu) killed more birds and humans, and the 2009 H1N1 (Swine flu) killed 18,000 people worldwide and encircled over 100 countries infecting humans, pigs, and birds [39].

Another pandemic has emerged from the Coronavirus family. Severe acute respiratory syndrome (SARS) and the Middle East respiratory syndrome (MERS) are two regional epidemics (MERS). SARS claimed the lives of 774 individuals in 2003, whereas MERS claimed the lives of 858 people between 2012 and 2019. (Centres for Disease Control & Prevention, 2005; World Health Organization, 2019). In 2019, a new virus was discovered in China, causing the novel coronavirus disease 2019 (SARS-CoV-2 or COVID-19), which has quickly spread over 216 nations in Europe, North America, Asia, the Middle East, Africa, and Latin America. The World Health Organization declared COVID-19 a pandemic disease on March 11, 2020 [26].

1.2.2. Some viruses can be useful

Some viruses are examined because they have current or future applications that could be beneficial [10].

• Phage typing of bacteria. During outbreaks of diseases caused by bacteria, identifying the phage types of bacterial isolates can provide significant epidemiological information.
• Sources of enzymes. Virus enzymes are used in a variety of molecular biology applications (e.g. reverse transcriptase from retroviruses and RNA polymerases from phages).
• Pesticides.Baculoviruses are employed to control some insect pests, and myxoma virus has already been utilized to control rabbits.
• Anti-bacterial agents. Human phages were utilized to treat various bacterial infections in the mid-twentieth century.
• Anti-cancer agents. The use of genetically engineered viral strains to treat cancer is being researched. These strains have been manipulated with to allow them to infect and destroy specific tumor cells while excluding normal cells.
• Gene vectors for protein production. Viruses are utilised as vectors to introduce genes into cultured animal cells.
• Gene vectors for treatment of genetic diseases. Retroviruses were used as vectors to transfer a non-mutant copy of the mutated gene responsible for the disease into the stem cells of children with severe combined immunodeficiency.
• Virus-based nanomaterials and nanostructuresin energy and biomedical applications.The virus-based biomimetic materials developed are characterized for biosensor and nanocarrier applications [39].

1.3. Classification of viruses

Viruses are currently classified into eight groups by the International Committee on Virus Taxonomy (ICTV) [60]. The first category includes chimeric viruses with double-stranded DNA and single-stranded DNA, such as haloarcula hispanica pleomorphic virus 1. The double-stranded DNA viruses, such as poxviruses, herpesviruses, and adenoviruses, are found in the second compartment. The single-stranded DNA virus, such as parvoviruses, is the third; the double-stranded RNA virus, such as reoviruses, is the fourth. Viruses having positive-sense single-stranded RNA genomes, such as the current SARS-CoV-2 outbreak, enteroviruses, hepatitis A virus, poliovirus, rhinoviruses, hand-foot-and-mouth (HFM) virus, SARS virus, yellow fever virus, hepatitis C virus (HCV), and rubella virus. The sixth group includes viruses with negative-sense single-stranded RNA genomes, such as the deadly Marburg and Ebola viruses, as well as measles, influenza virus, and mumps; the seventh group includes viruses with single-stranded RNA genomes that replicate through a DNA intermediate, such as HIV; and the eighth group includes viruses with double stranded DNA genomes and reverse transcriptase replication, such as the hepatitis B virus (HBV).

1.3.1. Taxonomy of Coronaviruses

Coronaviruses (CoVs) are a significant group of viruses belonging to the Nidovirales order, Cornidovirineae suborder, and Coronaviridae family. Letovirinae and Orthocoronavirinae are two subfamilies of the Coronaviridae family. The Alphaletovirus genus belongs to the Letovirinae family, while the Orthocoronaviridae family is divided into four genera based on phylogenetic analysis and genome structure: Alphacoronavirus (CoV), Betacoronavirus (CoV), Gammacoronavirus (CoV), and Deltacoronavirus (CoV), which contain 17, 12, 2, and 7 distinct species, respectively. Corona is a Latin word that means “crown,” and the virus got its name from the presence of spike extensions on the virus envelope that give it a crown-like form under the electron microscope. The ability of viruses in this order to create a nested set of subgenomic mRNA is referred to as nido [3].

As a result, Coronavirus (CoVs) are categorized into four generations: α-, β-, γ-, and δ-CoV [15]. α- and β-CoVs only infect mammals, while γ- and δ-CoVs can infect birds and some mammals. The most recent classification of the Coronaviridae is shown in Fig 1.

Figure 1. Taxonomy of the SARS-CoV-2 and its close relatives [3]

To date, seven CoVs have been known to cause infections in humans, including CoV-OC43, CoV-229E, HCoV-OC43, CoV-HKU1, CoVNL63, Middle East respiratory disease (MERS)-CoV, and severe acute respiratory syndrome (SARS)-CoV and SARS-CoV-2 or COVID-19 [50, 62].

SARS-CoV-2, a member of the Coronaviridae family, belongs to the -CoV genus and is said to be taxonomically and genetically identical to SARS-CoV, MERS-CoV, and other human coronaviruses [3].

SARS-CoV-2 indicates a separate lineage in the subgenus Sarbecovirus (previously, lineage 2b of CoV), according to Chan et al. [14]. Since there is very limited data about this newly emerged threat, and prevention strategies adopted during previous research and virus epidemics play a significant role in developing new strategies against SARS-CoV-2, it must be accurate and useful for scientists to take those ‘relatives’ of SARS-CoV-2 into account.

Other coronaviruses, however, have produced pandemic infections in domestic and wild mammals and birds, resulting in high fatality rates and significant economic losses. Chicken IBV, Beluga whale coronavirus SW1 (BWCoV-SW1), bat coronaviruses CDPHE15 and HKU10 (ICTV 2018), porcine epidemic diarrhoea virus (PEDV), TGEV, and sudden acute diarrhoea syndrome are among the viruses that have been identified (SADS-CoV) [3].

1.4. The nature of viruses

1.4.1. Viruses are small particles

Most virus virions are too small to see with a light microscope and can only be seen with an electron microscope. They are available in a wide range of sizes, shapes, and forms. Some are huge, while others are small; some are spherical, while others resemble rods. Many of these viruses have a highly symmetrical structure. The standard unit of measurement for virions is nanometres. (1 nm = 10−9 m). Parvoviruses, with dimensions of roughly 20 nm, are among the smallest, whereas the microbe-mimicking virus (mimivirus), identified from an amoeba, is among the largest. Coronavirus virions (CoVs) have a diameter of 60-140 nm and are generally spherical to pleomorphic enclosed particles [1].
Viruses are macromolecular assemblages that are metastable. Except for arenavirus virions, which have cell ribosomes packaged when the virions were produced, they are not cells and do not contain organelles [10].

1.4.2. Viruses have genetic material.

The virus’ genome is contained within the virion. Virus genomes can be double-stranded DNA, single-stranded DNA, double-stranded RNA, or single-stranded RNA, whereas cell genomes can be only double-stranded DNA.
Coronaviruses (SARS-CoV-2) have one of the largest genomes among RNA viruses, with a monopartite single-stranded positive-sense RNA [(+) ssRNA] genome [11]. The coronavirus genome is 29903 nucleotides long and includes two untranslated regions (UTRs) at the 5′ and 3′ ends, as well as 11 open reading frames (ORFs) [14].
A capsid is a protein coat that surrounds the genome. The virion is composed of the genome, capsid, and additional components in many circumstances. The virion’s primary functions are to protect the genome and transport it to a cell where it can multiply. Other proteins known as non-structural proteins are encoded by the viral genome in addition to the proteins that make up the capsid. They are not a part of the organization of the final capsid. These non-structural proteins are required for viral replication to take place within the host cell [59].
The virus’s size is frequently proportionate to the genome’s size. The viral genome, on the other hand, contributes significantly less to the virion’s overall mass than the capsid proteins. As a result, numerous copies of the capsid protein must be linked together to make the capsid (s). The amount of genetic information necessary in such an assembly with repeated subunits is considerably reduced. In certain viruses, a single gene product is involved in capsid development, but in more complicated viruses, numerous gene products are involved [59].
Four structural proteins, Nucleocapsid (N) protein, Membrane (M) protein, Spike (S) protein, and Envelop (E) protein, are encoded by the coronavirus genome, as well as several non-structural proteins (25 nsp) (Fig. 2). The capsid is a protein shell that contains nuclear capsid, or N-protein, which is attached to the virus’s single positive strand RNA and allows it to infect human cells and turn them into virus factories. The N protein covers the viral RNA genome and is necessary for replication and transcription. The viral replication and transcription are processed by the N-terminal of the N protein, which binds to genomic and sub-genomic RNAs [5].

Figure 2. The viral surface proteins (spike, envelope and membrane) embedded in a lipid bilayer.

The M-protein is most prevalent on the viral surface and is thought to be the coronavirus’s key organizer. The S-protein is incorporated into the virus’s surface and facilitates viral entrance into the host cell by mediating attachment of the virus to host cell surface receptors and membrane fusion between the viral and host cell membranes [28]. The E-protein is a tiny membrane protein with 76-109 amino acids that is a minor component of the virus particle. It is involved in virus assembly, host cell membrane permeability, and virus-host cell contact. [24]. The lipid envelop is externally located in some viruses, such as coronaviruses. A lipid bilayer surrounds the viral surface proteins spike, envelope, and membrane. The hemagglutinin-esterase dimer (HE) has been discovered on the viral surface. The HE protein may have a role in virus entry; it is not essential for virus replication, but it appears to be important for natural host-cell infection [34]. The primary antigenic epitopes, notably those identified by neutralizing antibodies, are carried by the envelope glycoproteins, which are responsible for attachment to the host cell. The complete structure of the Spike (S) protein in the closed and open (prefusion) states has been determined by cryo-EM investigations. [61] [67]. This glycoprotein is made up of three identical chains, each with 1273 amino acids, and two well-defined protein domain regions: S1 and S2 subunits, which are involved in cell recognition and membrane fusion, respectively. The latter arises as a result of several protein structural changes that are currently unknown.

1.4.3. Mechanism of viral action

Viruses must reproduce in their host cells, and the process consists of six steps: attachment, penetration, uncoating, replication, assembly, and release [40]. Viruses attach to a specific receptor location on the host cell membrane utilizing attachment proteins in the capsid or glycoproteins embedded in the viral envelope, and the host cells that can be infected by a particular virus are determined by this interaction specificity. In general, only the nucleic acid of bacteriophages penetrates the host cell, leaving the capsid outside. Animal and plant viruses can enter cells by endocytosis, in which the virus is completely enveloped and absorbed by cell membranes. The enveloped viruses will enter their host cells when the viral envelope merges directly with the cell membranes. The viral capsid is destroyed once within the host cells, releasing the viral nucleic acid, which is then available for reproduction and transcription. The viral genome determines the replication mechanism. DNA viruses normally utilise the host cell’s enzymes and proteins to make more DNA, which is then transcribed into messenger RNA (mRNA) and used for direct protein synthesis. The RNA core is commonly used by RNA viruses as a template for the synthesis of viral genomic RNA and mRNA. The release of new virions created in the host cells is the final stage of viral replication, allowing infection of neighbouring cells and self-replication cycles to continue. The viral replication cycle can lead host cells to undergo substantial structural and metabolic changes, as well as harm [69].

Figure 3 depicts the mechanism of SARS-CoV-2 entrance, replication, and RNA packing in the human cell. The spike (S) protein of the coronavirus binds to angiotensin converting enzyme 2 (ACE2) receptors on the surface of numerous human cells, including those in the lungs, facilitating virus entry. Host proteases (trypsin and furin) cleave the coronavirus S protein in two spots at the S1/S2 subunit border (S1/S2 site). The fusion peptide is released once the S2 domain (S2′ site) is cleaved. The membrane fusion mechanism will be activated as a result of this event. Endocytosis is the process through which a human cell ingests the virus. SARS-CoV-2 is assumed to use a unique three-step method for membrane fusion once it enters the cytoplasm, involving receptor-binding and induced conformational changes in Spike (S) glycoprotein, followed by cathepsin L proteolysis by intracellular proteases and further activation of the membrane fusion mechanism within endosomes [52]. The endosome then opens, releasing the virus into the cytoplasm, and the viral nucleocapsid (N) is uncoated by proteasomes, which may hydrolyse endogenous proteins but can also degrade external proteins like the SARS nucleocapsid protein [63]. A novel two-step mechanism has been proposed, in which the virion attaches to a receptor on the target host cell surface via its S1 subunit, the Spike is cleaved by host proteases, and then the viral and host target membranes are anticipated to fuse at low pH via the S2 subunit [25, 33]. Finally, the viral genetic material, which is a single stranded RNA, is released into the cytoplasm in its entirety. The replication and transcription processes take place, which are mediated by the replication/transcription complex (RTC). This complex is made up of non-structural proteins and is encoded in the viral genome (nsp). The RTC is assumed to have produced double-membrane structures in the infected cell’s cytoplasm [58]. Following the positive RNA genome, the open reading frame 1a/b (ORF 1a/b) is translated to generate replicase proteins. These proteins use the genome as a template to generate full-length negative sense RNAs, which are then used to generate additional full-length genomes. M, S, and E structural viral proteins are synthesized in the cytoplasm, inserted into the endoplasmic reticulum (ER), and transferred to the endoplasmic reticulum-Golgi intermediate compartment (Fig. 3). (ERGIC) [37]. In addition, nucleocapsids are formed in the cytoplasm by the encapsidation of replicated genomes by N protein, and as a result, they coalesce within the ERGIC membrane to self-assemble into new virions. At last, novel virions are exported from infected cells by transporting them to the cell membrane in smooth-walled vesicles and then secreting them via a process known as exocytosis in order to infect other cells. Meanwhile, the stress of viral production on the endoplasmic reticulum results in cell death.

Figure 3. The schematic diagram of the mechanism of SARS-CoV-2 entry, replication, and viral RNA packing in the human cell.

2. Functional nanoparticles

2.1. What are nanoparticles?

A nanoparticle, according to the International Organization for Standardization (ISO), is a particle with a size between 1 and 100 nanometres [6]. Nanoparticles, which are invisible to the human eye, can have radically different physical and chemical properties than their bigger material counterparts. As the size of a substance approaches the atomic scale, its properties change. This is due to an increase in the surface area to volume ratio, which causes the surface atoms to dominate the material’s performance. When compared to bulk materials such as powders, plates, and sheets, nanoparticles have a relatively significant surface area to volume ratio due to their extremely small size. When nanoparticles are small enough to confine their electrons and induce quantum effects, they can have unexpected optical, physical, and chemical capabilities.

Metallic nanoparticles differ from bulk metals in terms of physical and chemical properties (e.g., lower melting temperatures, large specific surface areas, specific optical properties, mechanical strengths, and magnetizations), which could be useful in a variety of industrial applications. Copper, for example, is considered a soft material because its atoms cluster at the 50 nm scale, causing bulk copper to bend. As a result, copper nanoparticles smaller than 50 nm are classified as a very hard material, with significantly different malleability and ductility than bulk copper. Gold nanoparticles melt at substantially lower temperatures than bulk gold (1064 °C) (300 °C for 2.5 nm size).

Over the last three decades, activity in the subject of nanotechnology has risen at an exponential rate around the world, transforming it into a major interdisciplinary research topic. The integration of nanotechnology into the area of medical science has driven this rise to a large extent, as nanostructured materials have distinct biological effects.

Nanomaterials are used in a variety of ways in the healthcare industry, one of which is drug delivery.

Figure 4. Biomedical applications of nanoparticles.

One example of this technique is the development of nanoparticles to aid in the delivery of chemotherapy treatments directly to cancerous growths, as well as to deliver drugs to damaged regions of arteries to combat cardiovascular disease. Carbon nanotubes are also being developed to be applied in processes for creation of bacteria sensors through addition of antibodies to the nanotubes.

Nanoparticles have emerged as interesting candidates for optimised therapy through personalized medicine due to their unique qualities, such as huge surface area, structural properties, and extended circulation duration in blood compared to small molecules. The ability to convert unfavourable physicochemical properties of bioactive molecules into desirable biopharmacological profiles, improve therapeutic agent delivery across biological barriers and compartments, control bioactive agent release, improve therapeutic efficacy by selective drug delivery to biological targets, and perform targeted therapy functions by combining multimodal ion channels are all potential advantages of engineered therapeutic nanoparticles [56].

A few nanomaterials and nanoparticles are currently being studied in clinical trials or have already been approved for use in humans by the US Food and Drug Administration (FDA), and many proof-of-concept studies of nanoparticles in cell culture and small animal models for medical applications are underway.

Antiviral drug development is critical for limiting the spread of illnesses and minimizing losses. Many functional nanoparticles, such as quantum dots, gold and silver nanoparticles, graphene oxide, nanoclusters, silicon materials, carbon dots, polymers, and dendrimers, have recently been found to have impressive antiviral properties. These functional nanoparticles-based materials offer unique qualities as possible antiviral candidates, considering their differences in antiviral mechanism and inhibitory efficacy. SARS-CoV-2 is a spike protein encased virus with a diameter of 60–140 nm and particle-like properties. Because of their structural similarities, synthetic nanoparticles can closely resemble the virus and interact aggressively with its pathogenic proteins. Antiviral nanomaterials, such as zinc oxide nanoparticles, have a tetrapod shape that mimics the cell surface when they engage with the viral capsid. Due to a photocatalytic reaction, it inhibited viral proteins when exposed to UV radiation [56].

2.2. Functional nanoparticles as antiviral agents

All parts of viral research have been impacted by nanotechnology. Nanotechnology has demonstrated a powerful potential to solve this issue among other antiviral techniques, and developing nanoparticles have been reported to have outstanding potency against virus infection and reproduction. Firstly, nanotechnology-based probes have been widely utilized in the detection of viruses, leading to the development of a variety of biosensors and bioelectronics based on unique functional nanoparticles [12, 16]. Second, several nanomaterials have been created employing virions and virus-like particles as templates, highlighting the importance of biocompatibility and biosynthetic methods in contemporary biochemical research [31, 35]. Third, significant effort has gone into the production of fluorescent nanoprobes and their use in studying the molecular mechanisms of virus-infected cells [41, 73]. Finally, a growing number of functionalized nanoparticles have been identified as highly effective viral growth inhibitors [66].

2.3. The antiviral activity of functional nanoparticles

Attachment, penetration, replication, and budding are the essential steps in the virus’s infectious process, and antiviral functional nanoparticles are designed to inhibit viruses by inhibiting or reducing some of these steps. We shall classify the various mechanisms of nanoparticles based on their antiviral efficacy in this section. Inactivating viruses is the most direct way to inhibit them, and some nanostructures can interact with viruses, change their capsid protein structure, and subsequently drastically reduce virulence, which can be linked to both physical and chemical mechanisms for reducing the active virus population. Most viral infections begin with the attachment of host cells, which is usually accomplished by binding to the target acceptor protein. The host cells will be free of infection if nanoparticles can effectively prevent the adhesion. Stellacci’s team has developed a series of antiviral nanoparticles with long and flexible linkers that mimic heparan sulphate proteoglycans, the highly conserved target of viral attachment ligands (VALs), that can achieve efficient viral prevention through effective viral association with a binding that is simulated to be strong and multivalent to the VAL repeating units [8]. These particles are non-cytotoxic and have nanomolar irreversible activity against herpes simplex virus, human papillomavirus, respiratory syncytial virus, dengue virus, and lenti virus in vitro. As a result, the functional nanoparticles can be utilized as a broad-spectrum antiviral drug to prevent viral attachment, the first step in the infection process. The second method of virus suppression is to prevent viruses from penetrating and entering host cells by altering the cell surface membrane and protein architecture. Haag and his colleagues created a number of water-soluble fullerene-polyglycerol sulphates (FPS) with various fullerene and polymer weight ratios and polyglycerol sulphate branch numbers [19].

Table 1. Typical antiviral mechanisms of action of nanomaterials.

Nanomaterial	Virus	Mechanism
Graphene oxide	Respiratory syncytial virus	Directly inactivate virus and inhibit attachment
Nanogel	PRRSV	Shield attachment and penetration
Silver nanoparticle	Herpesvirus	Affect viral attachment
Graphene oxide	Herpesvirus	Attachment inhibition
gold nanoparticles	Herpesvirus	Prevent viral attachment and penetration
Nano-carbon	Herpesvirus	Inhibit virus entry at the early stage
Silicon nanoparticles	Influenza A	Reduce the amount of progeny virus
Ag2S nanoclusters	Coronavirus	Block viral RNA synthesis and budding
Gd2O3:Tb3+/Er3+ nanoparticles	Zika virus	As antigen microcarriers for Zk2 peptide of ZIKV
Copper oxide nanoparticles	Herpes simplex virus type 1	Oxidation of viral proteins and degradation of viral genome
NiO nanostructures	Cucumber mosaic virus	Increase the expression of pod, pr1 and pal1 genes
Zirconia nanoparticles	H5N1 influenza virus	Promote the expression of cytokines
Zinc oxide nanoparticles	H1N1 influenza virus	H1N1 influenza virus

FPS, which combines polyanionic branches with a solvent-exposed changeable hydrophobic core, outperforms analogues that only have one of these properties in blocking vesicular stomatitis virus coat glycoprotein contact with baby hamster kidney cells. As a result, developing blockings between viruses and host cells is a good approach to keep virus infections at bay. In the case of virus entry into a cell, the third successful technique to block the virus is to destroy its replication, which is usually accomplished by decreasing the expression of particular enzymes that previously assisted in the completion of virus DNA or RNA replication. The final strategy is to inhibit virus budding and excrete it from host cells. A virus’s offspring may be more virulent than its mother, and if functional nanoparticles prevent the virus from budding and drastically limit the number of offspring viruses, the virus’s virulence will be greatly reduced. Table 1 shows some of the most common antiviral mechanisms for functional nanoparticles.

3. Virus-Based Nanoparticles (VNPs)

The crossing point between viral particles and functional nanomaterials are virus-based nanoparticles. The templated assembly of millions of identical nanoparticles and their creation in live cells are possible with bionanomaterials based on viruses. Viruses infect bacteria, humans, and plants, and they’ve all been utilized to create virus-based nanoparticles (VNPs). Viruses are an excellent place to start because they have evolved to distribute nucleic acids naturally and can thus be manipulated to deliver other compounds like as therapeutics and imaging reagents. Finally, viruses have a high rate of replication, enabling for the mass production of VNPs at a low cost.

VNPs are made up of regular arrays of virus coat proteins with a well-defined three-dimensional structure, making them a better engineering scaffold than manufactured particles. VNPs can also have their structure changed by modifying the nucleic acid template that codes for viral proteins before it is synthesized, as well as chemically decorating them by adding conjugates to certain amino acid side chains. VNPs are known for their biocompatibility, biodegradability, ability to overcome biological barriers, and efficient distribution of cargo to target cells because they are mostly made up of protein. Viruses have evolved to bind with specific cellular proteins, transport nucleic acid cargo, and hijack intracellular machinery to make progeny virus components. These characteristics have led to the development of VNPs based on mammalian viruses for use in gene therapy, but harmful effects stemming from normal virus-host interactions are difficult to rule out [23]. VNPs based on bacteriophages and plant viruses, on the other hand, are considered harmless because even fully functional viruses cannot infect people. As a result, the majority of this lecture will be devoted to the medical applications of VNPs derived from bacteriophages and plant viruses.

Bacteriophages and plant viruses are nucleoprotein assemblies with nucleic acids firmly encased in a capsid made up of many copies of the same coat proteins. Capsids are often icosahedral (approximately spherical), stiff tubes, or flexible filaments, with the latter two categories having a high aspect ratio. Plant viruses and bacteriophages, unlike many mammalian viruses, are not normally wrapped by a frail lipid membrane since they must tolerate harsher environmental conditions in order to successfully infect their hosts.

The virus capsid’s natural function is to protect the viral DNA against nucleases and other physical threats. Virus coat proteins are thus chemically and physically stable, which is advantageous for the development of VNPs because it means they have a long shelf life and can withstand the chemical treatments required for conjugation with targeting ligands or loading with payloads such as drugs, fluorophores, or contrast agents [54].

3.1. Strategies for VNPs modification

Genetic engineering, encapsulation, biomineralization, injection, and bioconjugation are some of the approaches that can be utilized to design and change virus-based products. The fundamental structure of the coat protein can be altered by genetic engineering by inserting, deleting, or swapping specific amino acid residues [42]. Such modifications facilitate functionalization or change of the VNP’s overall physicochemical features [20, 57]. Purification/immunodetection tags, epitope sequences to make the VNP a vaccine, and targeting sequences to make the VNP target specific receptors are all examples of such alterations [70]. Using comparable recombinant expression technologies, it is also possible to incorporate unnatural amino acids as unique handles for subsequent chemical reactions [55].

Under physiological conditions, virus coat proteins self-assemble around nucleic acids, and this property (which VNPs share) can be used to disassemble VNPs and reassemble them into more desired configurations around other cargo molecules. Two basic principles can be used to trigger cargo encapsulation: (a) surface charge and electrostatic interactions or (b) unique binding interactions that occur during self-assembly [18]. A translational repression (TR) operator protein, for example, is found in bacteriophage MS2 and binds to a TR RNA stem loop. Chemically modified TR operator proteins can transport small therapeutic molecules. When undamaged MS2 particles are combined with modified TR operators, the latter diffuse into the VNPs and bind to the capsid in a stable manner. These design techniques have been used to successfully insert therapeutic compounds such as the ricin A chain and 5-fluorouridine into MS2 particles. The transport of payload and the successful killing of target cells have been demonstrated in vitro cell research employing this technique [7, 68].

Biomineralization is the deposition of minerals in and around live organisms’ cells and tissues, but it relates to the ability of viral coat proteins to form around a mineral core or nucleate mineralization in the setting of VNPs. VNP biomineralization has various uses in energy research, but there are also examples in medicine, notably when mineral cargos are utilized as contrast agents [43].

Some materials should be encapsulated by stimulating the formation of capsids around a cargo, while others can diffuse through the viral particle and into the interior cavity, where they can be convinced to stay inside by noncovalent interactions with nucleic acids or internally projecting amino acid side chains, or bioconjugation can permanently link them to handles. [64]. This method has been used to load fluorescent dyes for optical imaging, Gd³⁺ ions for MRI, and small medicinal compounds [45, 71].

The use of classical chemistry to functionalize specific amino acid side chains, such as carboxylate groups on glutamic and aspartic acid residues, reactive amines on lysine residues, sulfhydryl groups on cysteine residues, and phenol groups on tyrosine residues, is one of the most powerful approaches for the modification of VNPs. These groups can be directly attached to specific molecules or changed to include functional groups for more complex conjugation procedures.

3.2. Virus-based nanoparticles in therapeutic interventions

Bacteriophages and plant viruses have the ability to penetrate mammalian cells and replicate without additional reproduction, making them useful therapeutic tools. Virus-based nanomaterials can be designed to target specific cells, such as cancer cells and immune system cells. They can also be employed as vaccinations since they can expose antigens to the immune system. Immunotherapy and immuno/chemo combined therapies benefit from VNP interactions with the immune system, while imaging and drug delivery are typically not. As a result, numerous ways for shielding VNPs from the immune system while guiding them to specific target cells have been devised. VNP clearance via the mononuclear phagocyte system can be circumvented by modifying the surface chemistry or shape of the particles [53]. Surface PEGylation, for example, can reduce nonspecific interactions between VNPs and macrophages, allowing them to circulate longer [30]. The genetic or chemical addition of compounds that bind to receptors highly expressed on specific cell types, such as cancer cells, can be used to target them. The form, size, and aspect ratio of the VNP can also influence tissue specificity, so these are additional properties to consider during the design stage. Tubular or filamentous VNPs, in particular, can exhibit in vivo features superior to spherical VNPs, such as increased flow and margination toward the arterial wall and decreased clearance by the mononuclear phagocytic system, resulting in improved tumour homing and thrombus targeting [51, 65]. VNP structures can be utilized to explore the impact of VNP size and shape on the efficiency of drug administration and imaging since they are monodispersing and can be modified with fine and repeatable spatial control.

3.3. Drug delivery with VNPs

The development of VNPs that target specific cell types has enabled the addition of toxic payloads via conjugation, infusion, and/or encapsulation, resulting in the death of the target cells, allowing for the selective elimination of cancer cells or other diseased cells without off-target effects. Conjugation, as briefly discussed above, entails the selective covalent addition of payload molecules to specific amino acid residues of the coat protein. Infusion is accomplished by incubating the intact VNP in a solution containing the cargo, whereas encapsulation necessitates the assembly of the carrier around the payload [9]. Genes and short interfering RNAs, photoactive molecules that support photodynamic therapy, conventional small-molecule drugs, and even heterologous viral genomes for gene therapy, such as an alphavirus genome encapsulated in a VNP based on CCMV, have all been delivered [4, 17].

Toxic cargos can be loaded preferentially into the VNP cavity rather than coating the external surface, protecting them from enzymatic and chemical degradation in vivo and avoiding interactions with nontarget cells. The capacity and efficiency of loading of VNPs are generally improved by discarding the native viral genome, which can be accomplished by expressing the coat proteins from a plasmid (for bacteriophage VNPs) or a transgene (for plant VNPs) so that the viral nucleic acid is never present; the resulting empty particle is referred to as a virus-like particle (VLP). The viral genome can also be removed through selective chemical or enzymatic degradation.

The covalent attachment of harmful cargo molecules to internally exposed side chains prevents early release, but noncovalent methods usually allow for higher loading efficiency as there is more space within the VNP for more cargo if the whole cavity is used rather than just the internal surface. Polymerization can provide the best of both worlds by forming a branching network of functionalized groups for payload attachment that extends from the VNP’s external surface or pervades its interior [27, 44]. Although most research has focused on VNP design and in vitro toxicity, preclinical testing of a VNP-based drug delivery vehicle has indicated in vivo efficacy and reduced cardiotoxicity of a doxorubicin-loaded VNP, particularly cucumber mosaic virus (CMV) modified with folic acid to target ovarian cancer [72].

VNPs have been loaded with photosensitizers for photodynamic therapy applications, in addition to standard chemotherapy. A VLP based on bacteriophage Q, for example, was loaded with a metalloporphyrin derivative for photodynamic therapy and glycan binding sites targeting cells with the CD22 receptor [47]. Furthermore, as a first demonstration of theranostic VNPs, a multifunctional MRI contrast and photodynamic therapy agent (chelated Gd³⁺ and Zn²⁺ phthalocyanine) was successfully encapsulated in CCMV [38]. In addition, hybrid VNP-based materials containing metal nanoparticles for photothermal therapy have been investigated [21].

3.4. Immunization and immunotherapy based on virus-derived structures

Because virus-based materials have repeated, protein-based structures, they elicit immune responses, making them useful for the development of vaccines and immunomodulators. Particle-based vaccines are classified into four types: (a) chemically inactivated virus vaccines, (b) attenuated virus vaccines with low virulence, (c) genome-free and non-infectious VLPs, and (d) chimeric and nanoparticle vaccines, wherein the pathogen-derived epitopes are presented on a non-infectious carrier such as a plant virus, bacteriophage, or chemically synthesized platform [22]. Particulate vaccines, such as VLPs and other nanoparticle vaccines, have several advantages over DNA vaccines and subunit vaccines [2, 29]. The virus-based carrier provides antigen stability, transports multiple copies of the antigen (multivalent presentation), and has the ability to present two or more different antigens. The formulation encourages passive or active uptake by antigen-presenting cells, which is followed by activation and priming of the appropriate T and B cell responses [32].

3.4.1. Vaccines for Infectious Diseases

VLP vaccines have had great success against viral diseases, particularly when the structure of the noninfectious vaccine formulation closely resembles that of the natural virus (these have been referred to as native VLPs) [46)]. The first successful example was the vaccine against hepatitis B virus (HBV). It has greatly reduced HBV infections in immunized populations. Vaccines against human papillomavirus (HPV) elicit immunity against the virus that in turn protects against HPV-induced cervical carcinoma, and potentially other HPV-induced cancers [48].

Chimeric VLPs express heterologous antigens and can generate antipathogen and neutralizing antibodies, suggesting that immunization may provide protection against pathogen challenge. Many studies on chimeric VLPs based on plant viruses, bacteriophages, insect viruses, and animal polyomaviruses and papillomaviruses have been conducted [48]. Chimeras have also been created from native vaccine platforms (e.g., HBV and HPV), and these platforms have been expanded by displaying additional heterologous epitopes. These native-chimeric VLPs benefit from a vaccine backbone that is approved by FDA.

Flock House virus (FHV), which infects insects, has been used to create chimeric VLPs with complex antigen structures. This multivalent display system has been modified to include fragments of the anthrax toxin receptor (ANTXR2), which serves as a scaffold for displaying the Bacillus anthracis protective antigen. In the absence of adjuvant, the virus-antigen complex activated protective immune responses after a single dose [36]. Additional mechanisms for chemical bonding of multivalent antigens induce immune responses in a similar efficient manner. The FHV system has the capability of accepting protein and peptide insertions in a variety of locations on the capsid surface, as well as the availability of detailed structural and genetic information that allows for precise antigenic domain placement and arrangement. For example, the influenza hemagglutinin (HA) protein is a major antigen for all strains of influenza, but due to antigenic variation, it is difficult to develop broadly neutralizing immune responses. There are some highly conserved regions of the protein, but they are difficult to see in a structural context, which would allow the initiation of specific and neutralizing antibody responses. The induction of these antibodies is enabled by displaying the conserved regions of HA in a trimeric arrangement on FHV. The utility and breadth of native and chimeric VLPs for vaccine applications are expanding. The combination of bioengineering VLP vaccines and administering them into the respiratory tract, for example, has recently been demonstrated as a fundamental strategy for future vaccine development and immunotherapy [49].

3.4.2. Vaccines for cancer

Anti-tumour vaccination has several advantages over chemotherapy, including fewer side effects, avoiding drug resistance, preparation the immune system to eliminate residual drug-resistant cells, and inducing long-term immunological memory to protect against metastases and relapse.

Several VNP-based cancer vaccine strategies have been evaluated, including the patterned display of tumour-associated carbohydrate or peptide antigens. Tn-specific antibodies were produced in high titters after conjugation to the virus-based scaffold and multivalent display. Similarly, antigen-specific IgG and IgM responses can be elicited by Tn antigen conjugated to TMV. The presentation of cancer epitopes on virus-based scaffolds allows these self-epitopes to be presented in a non-native molecular environment, which is a promising strategy for overcoming self-tolerance.

3.4.3. Vaccines for neurological diseases and addiction

VLPs have been used as nanostructures to display the amyloid beta (A) protein, which has been linked to Alzheimer’s disease progression. In the absence of adjuvant, Papillomavirus and Q VLPs containing A antigens elicited anti-A antibodies with limited T cell responses. The antibody subclasses differed depending on whether the whole antigen or peptide antigens were used [13].

A potential nicotine addiction vaccine has recently been developed using a 30-nm icosahedral capsid of bacteriophage Q that has been chemically modified to display nicotine in a multivalent fashion. The Q-based vaccine’s multivalent and particulate nature stimulates the production of antinicotine neutralizing antibodies, lowering blood nicotine levels and limiting transport across the blood-brain barrier.

---

Test LO 1.1

---

References

1. Alimardani V., Abolmaali S. and Tamaddon A. (2021). Recent Advances on Nanotechnology-Based Strategies for Prevention, Diagnosis, and Treatment of Coronavirus Infections. Hindawi J of Nanomaterials, Article ID 9495126, 1-20.
2. Awate S., Babiuk L and Mutwiri G. (2013). Mechanisms of action of adjuvants. Front Immunol, 4, 114.
3. Aydogdu M., Altun E., Chung E., Ren G., Homer-Vanniasinkam S., Chen B and Edirisinghe M. (2021). Surface interactions and viability of coronaviruses. J. R. Soc. Interface, 18, 20200798.
4. Azizgolshani O., Garmann R., Cadena-Nava R., Knobler C and Gelbart W. (2013). Reconstituted plant viral capsids can release genes to mammalian cells.Virology, 441, 12–17.
5. Boopathi, PomaA and Kolandaivel P. (2020). Novel 2019 coronavirus structure, mechanism of action, antiviral drug promises and rule out against its treatment. J Biomol Struct Dyn,39, 9, 1-10.
6. British Standards Institution. (2007). Terminology for Nanomaterials, Publicly Available Specification BS PAS 136, British Standards Institution, London.
7. Brown W., Mastico R., Wu M, Heal K and Adams C. (2002). RNA bacteriophage capsid-mediated drug delivery and epitope presentation. Intervirology, 45, 371–380.
8. Cagno V., Andreozzi P., Alicarnasso M., Silva P., Mueller M., Galloux M., Goffic R., et al. (2018). Broad-spectrum non-toxic antiviral nanoparticles with a virucidal inhibition mechanism. Nature Mater, 17, 195–205.
9. Cao J., Guenther R., Sit T., Opperman C., Lommel S and Willoughby J. (2014). Loading and release mechanism of Red clover necrotic mosaic virus derived plant viral nanoparticles for drug delivery of doxorubicin. Small, 10, 5126–5136.
10. Carter J. and Saunders.V. (2007). Virology. Principales and applications, John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester,West Sussex PO19 8SQ, England.
11. Cascella M., Rajnik M., Cuomo A., Dulebohn S abd Di Napoli R. (2021). Features, evaluation and treatment coronavirus (COVID-19),” in Statpearls, StatPearls Publishing.
12. Caygill R., Blair G and Millner P. (2010). A review on viral biosensors to detect human pathogens. Anal. Chim. Acta, 681, 8–15.
13. Chackerian B. (2010). Virus-like particle based vaccines for Alzheimer disease. Hum Vaccines, 6, 926–930.
14. Chan J., Kok K., Zhu Z., Chu H., To K., Yuan S and Yuen K. (2020) Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan. Emerg Microbes Infect, 9(1), 221-236.
15. Chan J., To K., Tse H., Jin D and Yuen K. (2013). Interspecies transmission and emergence of novel viruses: lessons from bats and birds. Trends Microbiol, 21(10), 544-55.
16. Chen L., Zhang X., Zhou G., Xiang X., Ji X., Zheng Z., He Z. and Wang H. (2012). Simultaneous determination of human enterovirus 71 and coxsackievirus B3 by dual-color quantum dots and homogeneous immunoassay. Anal. Chem, 84, 3200–3207.
17. Choi K., Kim K., Kwon I., Kim I. and Ahn H. (2012). Systemic delivery of siRNA by chimeric capsid protein: tumor targeting and RNAi activity in vivo. Mol Pharm, 10, 18–25.
18. Daniel M., Tsvetkova I., Quinkert Z., Murali A. and De M, (2010). Role of surface charge density in nanoparticle-templated assembly of bromovirus protein cages. ACS Nano,3853–3860.
19. Donskyi L., Druke M., Silberreis K., Lauster D., Ludwig K., Kuhne C., Unger W., et al. (2018). Interactions of fullerene-polyglycerol sulfates at viral and cellular interfaces. Small, 14, 1800189.
20. Douglas T., Strable E and Willits D. (2002). Protein engineering of a viral cage for constrained material synthesis. Adv Mater, 14, 415–418.
21. Everts M., Saini V., Leddon J., Kok R and Stoff-Khalili M. (2006). Covalently linked Au nanoparticles to a viral vector: potential for combined photothermal and gene cancer therapy. Nano Lett, 6, 587–591.
22. Garcea R. and Gissmann L. (2004). Virus-like particles as vaccines and vessels for the delivery of small molecules. Curr Opin Biotechnol,15, 513–517.
23. Guenther C., Kuypers B., Lam M., Robinson T., Zhao J, and Suh J. (2014). Synthetic virology: engineering viruses for gene delivery. WIRES Nanomed Nanobiotechnol,6, 548–58.
24. Gupta M., Vemula S., Donde R., Gouda G., Behera L., and Vadde R. (2021). In silico approaches to detect inhibitors of the human severe acute respiratory syndrome coronavirus envelope protein ion channel. J Biomol Struct Dyn, 39 (7):2617-2627.
25. Hasan A., Paray B., Hussain A., Qadir F., Attar F., Aziz F., and Falahati M. (2020). A review on the cleavage priming of the spike protein on coronavirus by angiotensin-converting enzyme-2 and furin. J Biomol Struct Dyn, 1-13.
26. Helmy Y., Fawzy M., Elaswad A., Sobieh A., Scott P., Kenney S. and Awad A. (2020). The COVID-19 Pandemic: A Comprehensive Review of Taxonomy, Genetics, Epidemiology, Diagnosis, Treatment, and Control. J. Clin. Med, 9.
27. Hovlid M., Lau J., Breitenkamp K. Higginson C. and Laufer B. (2014). Encapsidated atom-transfer radical polymerization in Qβ virus-like nanoparticles. ACS Nano, 8, 8003–8014.
28. Kirchdoerfer R., Cottrell, C., Wang, N., Pallesen, J., Yassine, H., Turner, H., Corbett, et al. (2016). Pre-fusion structure of a human coronavirus spike protein. Nature, 531(7592), 118–121.
29. Klinman D., Takeno M., Ichino M., Gu M., Yamshchikov G. (1997). DNA vaccines: safety and efficacy issues. Springer Semin Immunopathol, 19, 245–256.
30. Kwon O., Kang E., Choi J., Kim S. and Yun C. (2013). Therapeutic targeting of chitosan-PEG-folate-complexed oncolytic adenovirus for active and systemic cancer gene therapy. J Control Release, 169, 257–265.
31. Lee S., Krishnamurthy S., Cho C. and Yun Y. (2016). Biosynthesis of gold nanoparticles using ocimum sanctum extracts by solvents with different polarity, ACS Sustain. Chem. Eng. 4, 2651–2659.
32. Leleux J. and Roy K. (2013). Micro and nanoparticle-based delivery systems for vaccine immunotherapy: an immunological and materials perspective. Adv Healthc Mater, 2, 72–94.
33. Li F. (2016). Structure, function, and evolution of coronavirus spike proteins. Ann. Rev. of Virol., 3 (1), 237–261.
34. Lissenberg A., Vrolijk M., van Vliet A., Langereis M., de Groot-Mijnes J., Rottier, P. and de Groot R. J. (2005). Luxury at a Cost? Recombinant mouse hepatitis viruses expressing the accessory hemagglutinin esterase protein display reduced fitness in vitro. J Virol, 79(24), 15054–15063.
35. Luo K., Jung S., Park K. and Kim Y. (2018). Microbial biosynthesis of silver nanoparticles in different culture media, J. Agric. Food Chem. 66, 957–962.
36. Manayani D., Thomas D., Dryden K., Reddy V. and Siladi M. (2007). A viral nanoparticle with dual function as an anthrax antitoxin and vaccine. PLOS Pathog, 3, 1422–1431.
37. Masters P. (2006). The molecular biology of coronaviruses. Adv. Virus Res., 65(06), 193–292.
38. Millán J., Brasch M., Anaya-Plaza E., de la Escosura A. and Velders A. (2014). Self-assembly triggered by self-assembly: optically active, paramagnetic micelles encapsulated in protein cage nanoparticles. J Inorg Biochem, 136, 140–146.
39. Oh J. and Han D. (2020). Virus-Based Nanomaterials and Nanostructures. Nanomaterials, 10, 567.
40. Oswald M., Geissler S. and Goepferich A. (2017). Targeting the central nervous system (CNS): a review of rabies virus-targeting strategies, Mol. Pharm. 14, 2177–2196.
41. Pan H., Zhang P., Gao D., Zhang Y., Li P., Liu L., Wang C., et al. (2014) Noninvasive visualization of respiratory viral infection using bioorthogonal conjugated near infrared-emitting quantum dots, ACS Nano 8, 5468–5477.
42. Peabody D. (2003). A viral platform for chemical modification and multivalent display. J Nanobiotechnol, 1,
43. Pokorski J., Breitenkamp K., Liepold L., Qazi S. and Finn M. (2011). Functional virus-based polymer-protein nanoparticles by atom transfer radical polymerization. J Am Chem Soc, 133, 9242–9245.
44. Pokorski J. and Steinmetz N. (2011). The art of engineering viral nanoparticles. Mol Pharm, 8, 29–43.
45. Prasuhn D., Jr, Yeh R., Obenaus A., Manchester M. and Finn M. (2007). Viral MRI contrast agents: coordination of Gd by native virions and attachment of Gd complexes by azide-alkyne cycloaddition. Chem Commun, 2007,1269–1271.
46. Pushko P. and Pumpens P. (2013). Grens E. Development of virus-like particle technology from small highly symmetric to large complex virus-like particle structures. Intervirology, 56, 141–165.
47. Rhee J., Baksh M., Nycholat C., Paulson J., Kitagishi H. and Finn M. (2012). Glycan-targeted virus-like nanoparticles for photodynamic therapy. Biomacromolecules, 13, 2333–2338.
48. Roldao A., Mellado M., Castilho L., Carrondo M. and Alves P. (2010). Virus-like particles in vaccine development. Expert Rev Vaccines, 9, 1149–1176.
49. Rynda-Apple A., Patterson D. and Douglas T. (2014). Virus-like particles as antigenic nanomaterials for inducing protective immune responses in the lung. Nanomedicine, 9, 1857–1868.
50. Shen K., Yang Y. and Wang T. (2020). Diagnosis, treatment, and prevention of 2019 novel coronavirus infection in children: experts’ consensus statement World J Pediatr, 16(3), 223-231.
51. Shukla S., Ablack A., Wen A., Lee K., Lewis J. and Steinmetz N. (2013). Increased tumor homing and tissue penetration of the filamentous plant viral nanoparticle Potato virus X. Mol Pharm, 10, 33–42.
52. Simmons G., Gosalia D., Rennekamp A., Reeves J., Diamond S. and Bates P. (2005). Inhibitors of cathepsin L prevent severe acute respiratory syndrome coronavirus entry. PNAS, 102(33), 11876–11881.
53. Singh P., Prasuhn D., Yeh R., Destito G. and Rae C. (2007). Bio-distribution, toxicity and pathology of cowpea mosaic virus nanoparticles in vivo. J Control Release, 120, 41–50.
54. Steinmetz N. (2010). Viral nanoparticles as platforms for next-generation therapeutics and imaging devices. Nanomedicine, 6:634–641.
55. Strable E, Prasuhn D. Jr, Udit A., Brown S. and Link A. (2008). Unnatural amino acid incorporation into virus-like particles. Bioconjug Chem,19, 866–875.
56. Tharayil A., Rajakumari R., Kumar A., Choudhary M., Palit P. and Thomas S. (2021). New insights into application of nanoparticles in the diagnosis and screening of novel coronavirus (SARS-CoV-2). Emergent Materials, 4,101–117.
57. Udit A., Brown S., Baksh M. and Finn M. (2008). Immobilization of bacteriophage Qβ on metal-derivatized surfaces via polyvalent display of hexahistidine tags. J Inorg Biochem,102, 2142–2146.
58. Van Hemert M., Van Den Worm, S., Knoops K., Mommaas A., Gorbalenya A. and Snijder E. (2008). SARS-coronavirus replication/transcription complexes are membrane-protected and need a host factor for activity in vitro. PLoS Pathogens, 4(5).
59. Venkataram P. and Schmid M. (2012). Principles of Virus Structural Organization. Viral Molecular Machines,726, 17–47.
60. Virus taxonomy: the classification and comenclature of viruses, ICTV reports are freely available online: https://talk.ictvonline.org/ictv-reports/ictv_online_report/.
61. Walls A., Park Y., Tortorici M., Wall A., McGuire A. and Veesler D. (2020). Structure, function, and antigenicity of the SARS-CoV-2 Spike glycoprotein. Cell, 181(2), 281–212.
62. Wang,WangY., Ye D, and Liu Q. (2020). A review of the 2019 Novel Coronavirus (COVID-19) based on current evidence. Int J Antimicrob Agents, 55(6), 105948.
63. Wang Q., Li C., Zhang Q., Wang T., Li J., Guan W., Yu J., Liang M.and Li D. (2020). Interactions of SARS Coronavirus Nucleocapsid Protein with the host cell proteasome subunit p42. Virology J, 7(1), 99–98.
64. Wen A., Shukla S, Saxena P, Aljabali A. and Yildiz I. (2012). Interior engineering of a viral nanoparticle and its tumor homing properties. Biomacromol,13, 3990–4001.
65. Wen A., Wang Y., Jiang K., Hsu G. and Gao H. (2015). Shaping bio-inspired nanotechnologies to target thrombosis for dual optical-magnetic resonance imaging. J Mater Chem B, 3, 6037–6045.
66. White K., Jr P., Wang H., Jesus P., Manicassamy B., García-Sastre A., Chanda S., et al. (2018). Broad spectrum inhibitor of influenza A and B viruses targeting the viral nucleoprotein, ACS Infect. Dis, 4,146–
67. Wrapp D., Wang N., Corbett K., Goldsmith J., Hsieh C., Abiona O., Graham B. and McLellan J. (2020). Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science, 367(6483), 1260–1263.
68. Wu M., Brown W. and Stockley P. (1995). Cell-specific delivery of bacteriophage-encapsidated ricin A chain. Bioconjug Chem, 6, 587–595.
69. Yang M., Sunderland K. and Mao C. (2017). Virus-derived peptides for clinical applications. Chem. Rev, 117, 10377–10402.
70. Yildiz I., Shukla S. and Steinmetz N. (2011). Applications of viral nanoparticles in medicine. Curr Opin Biotechnol, 22, 901– 908.
71. Yildiz I., Lee K., Chen K., Shukla S. and Steinmetz N. (2013). Infusion of imaging and therapeutic molecules into the plant virus-based carrier cowpea mosaic virus: cargo-loading and delivery. J Control Release, 172, 568–578.
72. Zeng Q., Wen H., Wen Q., Chen X. and Wang Y. (2013). Cucumber mosaic virus as drug delivery vehicle for doxorubicin. Biomaterials, 34, 4632–4642.
73. Zhang Y., Ke X., Zheng Z., Zhang C., Zhang Z., Zhang F., Hu Q., et al. (2013). Encapsulating quantum dots into enveloped virus in living cells for tracking virus infection, ACS Nano, 7, 3896–3904.

Summary

The virus that causes COVID-19 can be spread from one person to another through tiny particles of water called aerosols and the virus. We make these aerosols when we breathe and more when we talk, shout or sing. Aerosols are different from larger droplets that spread COVID-19. Larger droplets fall to the ground quickly, going three to six meters away from the person making them. Aerosols can float in the air for hours and travel long distances. Aerosols contain fewer viruses than larger droplets, so you have to inhale more aerosols to get sick. Aerosols can build up if the air inside is not circulated properly. Airborne transmission of viruses increases during the winter months because people spend more time indoors and it is often too cold to keep windows open. In winter, the air is drier, especially in heated interiors. Dry air damages the lining of the respiratory tract and can facilitate the entry of the virus into the airways. It also means that smaller aerosols float longer in the air. Therefore, airborne transmission of COVID-19 is expected to be more common during the winter months. If you are not fully vaccinated, wearing face masks and staying at least one meter away from other people, as well as good air circulation (ventilation) in buildings, schools and homes, and air purifiers made using nanotechnology will reduce the spread of COVID-19 in aerosols.

Key words/phrases:  air filtration systems, nanotechnology, COVID-19

1. Introduction

COVID-19 has forced the human population to rethink the way of life. The threat posed by the potential spread of the virus through the airborne mode of transmission through ventilation systems in buildings and confined spaces has been recognized as a major concern. To mitigate this threat, researchers have discovered different technologies and methods that can eliminate or reduce the concentration of the virus in ventilation systems and indoor spaces. Although many technologies and methods have already been researched, some are currently commercially available, but their effectiveness and safety concerns have not been fully investigated. This article contains a brief review of various applicable technologies and methods for combating airborne viruses in ventilation systems and indoor spaces, in order to gain a broader view and overview of the current research and development situation. It includes efficient air filtration, air ionization, environmental control, ultraviolet germicidal irradiation, non-thermal plasma and reactive oxygen species, filter coatings, chemical disinfectants and heat inactivation. In this article, information will be given about air filtration systems that prevent viruses.

COVID-19 has forced the human population to adapt rapidly in the wake of the new and highly contagious virus. The modes of transmission are not fully understood; however, it is accepted that the virus can be transmitted into the air by direct contact with another person or by evaporating respiratory droplets as droplet nuclei that can remain suspended for a long time as aerosols [23, 20, 7]. These aerosols may pass through ventilation systems in buildings and confined spaces, eventually invading other areas away from infected persons [6, 14]. While there is some debate about the seriousness of the threat posed by these airborne droplets, it is recognized that this form of transmission for typically confined spaces cannot be ignored. Moreover, a recent study even suggests that airborne transmission may be the dominant mode of transport (Fig. 1) [6].

Although COVID-19 is not fully understood, many lessons have been learned from previous airborne viruses such as tuberculosis and various strains of influenza [14, 21]. From a very basic understanding of how viruses spread, it follows that a certain amount of virus must enter an uninfected individual in order to increase the viral load and establish a new infection. Traditionally, this is defined in the epidemiological literature as a quantum, the number of infectious airborne particles required to infect 63% of individuals in a confined space [22], and serves as a baseline criterion for many models attempting to quantify the probability of infection without exposure to a pathogen. This model is based on a well-mixed chamber assumption supported in the literature [19, 2, 26]. And it simply assumes that the particles are uniformly dispersed throughout an enclosed space rather than creating a small cloud of aerosols that diffuse around an infected individual. The spread and effect of the infection are determined by factors such as viral load, inhalation rate, droplet volume concentration expelled from the infected individual, the number of viral particles required to initiate an infection, and the volume of the enclosed space.

Figure 1. Risk of infection by airborne droplets.

Mathematically, the quantum emission rate is determined by viral load, inhalation rate, droplet volume concentration expelled from the infected individual, and the number of viral particles required to initiate an infection. The effects to reduce the possibility of infection are factors such as air exchange, air filtration rate, droplet settling, droplet settling rate, inactivation rate, and particle radius.

Therefore, several key factors can be considered as possible methods of removing viral particles from a confined space to reduce the likelihood of an infection. These are (Fig. 2):

• increase the supply of fresh air and consequently decrease the quantum concentration;
• increase the filtration rate for an HVAC system;
• increase the deposition rate of viral particles to surfaces;
• increase viral inactivation.

Figure 2. Improving indoor air quality to prevent COVID-19.

 

Although different in definition, increasing the deposition rate of viral particles can be considered similar to increasing the sedimentation rate. Sedimentation refers to the settling of particles on the ground or other surfaces due to gravitational forces. However, airborne particles can also accumulate on walls and other surfaces due to mechanisms such as unnatural diffusion for particle sedimentation. The Centres for Disease Control and Prevention (CDC) and the World Health Organization (WHO) confirm the removal of viral particles through air exchange [8, 4]. And recommend increasing the supply of fresh air as a simple way to reduce the concentration of viral particles in a confined space. Air ionization can also be used to increase the rate of removal of viral particles from a confined space by increasing filtration efficiency and particle deposition. Various methods are available to sterilize the air and render the virus harmless, thereby increasing the rate of viral inactivation and reducing the need to remove particles from the air. In this regard, the following can be listed.

• • Ultraviolet Germicidal Irradiation (UVGI).This is a traditionally popular technology for fighting airborne viruses (Fig. 3).
  • Control of temperature and relative humidity. It has also been suggested that directly controlling the environmental conditions of an area creates an unfavourable environment for viruses, thereby increasing the natural rate of viral inactivation. This includes controlling the temperature and relative humidity of an area to maintain an especially generally hostile environment.
  • Non-thermal plasma and reactive oxygen species. These offer other alternatives for viral inactivation that have proven effective against bacteria and other microbes.
  • Filter coatings use. Another possible method uses filter coatings that facilitate viral inactivation by mechanisms such as the materials’ inherent antiviral properties or by directly damaging the virus.
  • Chemical disinfectants. Chemical disinfectants have also been proven to effectively remove viruses from surfaces and may provide other solutions to increase viral inactivation.
  • Superheated sterilization. Superheated sterilization may offer another viable solution for inactivating viral particles, although it has traditionally been used to sterilize surgical equipment on a smaller scale.

Figure 3. Improving living and work space ventilation and air filtering to help prevent transmission of COVID-19

2. Air filtration and SARS-CoV-2

In various applications, air filtration has become a critical intervention in managing the spread of the 2019 coronavirus disease (COVID-19). However, the proper placement of air filtration has been hampered by a poor understanding of its principles. These misunderstandings have led to uncertainty about the effectiveness of air filtration in stopping potentially infectious aerosol particles. A proper understanding of how air filtration works is critical for making further decisions regarding its use in managing the spread of COVID-19. The problem is significant because recent evidence has shown that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can stay in the air longer and travel farther than previously expected in the COVID-19 pandemic, with reduced concentrations and viability. SARS-CoV-2 virions are around 60-140 nm in diameter, while larger respiratory droplets and air pollution particles (>1 µm) have been found to harbour virions. Removal of particles that can carry SARS-CoV-2 from the air is possible with air filtration based on natural or mechanical movement of air. Among the various types of air filters, high efficiency particulate trap (HEPA) filters have been recommended. Other types of filters are less or more effective and, accordingly, easier or more difficult to move the air. The use of masks, respirators, air filtration modules and other special equipment is an important intervention in the management of the spread of COVID-19. It is critical to consider air filtration mechanisms and understand how aerosol particles containing SARS-CoV-2 virions interact with filter materials in order to identify best practices for using air filtration to reduce the spread of COVID-19.

There is growing evidence that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can remain suspended in the air for long periods of time. Some of the airborne SARS-CoV-2 virions remain viable for at least 3 hours after aerosolization [31]. Polymerase chain reaction positive SARS-CoV-2 was detected in aerosol particles larger than 1 μm in diameter in rooms where patients with coronavirus disease 2019 (COVID-19) were staying [5]. In another study, SARS-CoV-2 RNA was detected in the aerosol phase at a distance of at least 3 m from infected people indoors [15]. SARS-CoV-2 RNA has also been found in air pollution particles circulating in the air [24].

The diameter of SARS-CoV-2 virions is around 60-140 nm [39]. However, many exhaled respiratory droplets that may contain virions are significantly larger than the virions themselves. However, airborne droplet evaporation reduces their size [39], allowing potentially infectious particles to remain in the air for significantly longer. It was observed that dry droplets with a diameter of about 4 μm formed speech-derived wet droplets of 12 μm to 21 μm due to drying. It took about 8 minutes for these dry droplets to fall only 30 cm in still air [35]. At low ambient temperature, exhaled breath with high humidity can become supersaturated. The moisture then condenses on the particles emitted by a person, causing them to turn into droplets or larger diameter ice crystals. In such droplets or ice crystals, SARS-CoV-2 virions may survive longer, and this is an important hypothesis that future research needs to test. Therefore, environmental conditions and aerosol dynamics can profoundly alter the wide range of inhaled particle sizes and the viability of SARS-CoV-2 virions in aerosol particles that mediate indoor and outdoor airborne transmission. COVID-19 outbreaks in slaughterhouses and ski resorts may be due, at least in part, to cold air aerosol dynamics.

Removing particles that may harbour SARS-CoV-2 from the air using specialized air filtration equipment and masks or respirators is an important intervention in managing the spread of COVID-19. However, a poor understanding of how air filtration works and misunderstandings about the concept of filtration efficiency for aerosol particles of different sizes hinder effective deployment of air filtration. To identify best practices for the use of air filtration in the management of the spread of COVID-19, it is critical to consider air filtration mechanisms and understand how aerosol particles containing SARS-CoV-2 virions interact with filter materials.

For air filtration, efficient air filters (EPA), high efficiency air filters (HEPA) (Fig. 4) filters and ultra-low penetration air filters (ULPA) have been widely used in various industries and applications for many years [25]. HEPA filters are recommended for infection control in healthcare settings [13, 10] based on a balance of higher filtration efficiencies and lower pressure drops compared to ULPA. HEPA filters are also commonly used in non-health environments where airborne infectious agents may be present. Examples include filtration of recirculated air on passenger aircraft and biosafety cabinets in laboratories, including where SARS-CoV-2 research is being conducted [37].

Generally, the abbreviation HEPA is interpreted as “high efficiency particulate air”. Both versions of the underlying term are widely used and there is no difference between them. The United States Department of Energy and the United States Environmental Protection Agency (EPA) define HEPA based on a minimum 99.97% efficiency when tested with an aerosol with a diameter of 0.3 μm [36]. The United States EPA defines a diameter of 0.3 µm as “the most penetrating particle size” (MPPS). However, the MPPS can vary around 0.3 μm with an absolute value depending on the nature of the aerosol particles, the type of filter material and the flow rate [25]. Particles larger or smaller than MPPS are helded with an efficiency greater than 99.97% [32]. The concept of MPPS goes against the common misconception that filtration efficiency drops for particles smaller than MPPS (for example, smaller than 0.3 µm). This misunderstanding contributed to early policies that were misled by the assumption that SARS-CoV-2 virions were too small to be effectively filtered from the air.

Figure 4. HEPA filter.

It is recommended to install HEPA filters at the outlets of ventilators used in the intensive care of people infected with SARS-CoV-2. The use of fixed (building ventilation) and portable HEPA filtration systems with and without air recirculation (indoor air purifiers) is recommended for use in healthcare settings by the United States Centres for Disease Control and Prevention and the World Health Organization, including where SARS-CoV-2 patients are present [10]. National and international standards govern the minimum filtration efficiency specifications of HEPA filters. The two most widely used standards are the international ISO 29463 standard and the European EN1822 standard. The differences between the two standards can be reconciled. For example, a HEPA filter certified to EN 1822, filter class H14, must retain at least 99.995% of aerosol particles in the MPPS. Comparable to EN 1822, filter class H14 standard, ISO 45 H. Multi-step test protocols are available to verify the compliance of filters with the requirements of the standards [12, 18]. When mechanical air movement occurs between filters, it can be important to ensure that strong directional flows or drafts of filtered air do not occur. Recently, concerns have been raised that such directional flows could entrain unfiltered air, which may contain infectious particles, and push them faster and farther than they could diffuse in still air [11].

Antiviral properties can be added to filter materials. However, once the aerosol particles are collected on the filter fibres, almost none of them leaves and passes through the filter during or after proper use [25]. Thus, the antiviral properties of the fibres have almost no effect on airborne removal of live SARS-CoV-2 virions. Particles accumulated on previously collected particles do not come into contact with the filter material, eliminating any antiviral properties. Therefore, imparting antiviral properties to HEPA filter materials may not add value except when people come into direct contact with these filters during or shortly after use.

The mechanisms of aerosol particle filtration in the gas phase—inertial impingement, diffusion, arresting, electrostatic deposition, and sieving [25, 12, 18]—have been explored in depth over decades of research. These mechanisms have varying contributions to the overall particle arresting efficiency of filters, depending on the particle aerodynamic diameter, other particle properties, and the filtration medium. The combined effect of all these filtration mechanisms in HEPA filters explains the high filtration efficiency and MPPS phenomenon across the entire aerosol size spectrum [25]. Various types of aerosol particles are filtered with high efficiency in accordance with relevant standards, regardless of their biogenic or non-biogenic origin [12, 18].

It is known, based on numerous published studies, that some respiratory infections occur more frequently when people breathe more polluted air, and that the healing process and outcomes of some respiratory infections are adversely affected by air pollution. An association between long-term level of particulate air pollution and higher COVID-19 mortality has already been demonstrated [39]. Breathing polluted air is also strongly associated with adverse effects on respiratory and cardiovascular functions [17]. Air filtration-based interventions using adequate equipment should be widely implemented both to reduce the spread of SARS-CoV-2 through the aerosol phase and to improve the health status and outcomes of people exposed and infected with COVID-19.

3. Air purifiers and filters

It is estimated that the use of air filters and purifiers will reduce the viral load in the environment. Air purifiers can be used in patient rooms, which can reduce the likelihood of infection by healthcare workers due to deficiencies in PPE. It can reduce the likelihood of re-infection in a patient due to airborne transmission of viruses. This type of filtration system can also be used in public transport, in the hospital setting, anywhere in the aerosol generating procedure, in closed vehicles and at home. Liquid droplets when coughing or sneezing from an infected person are typically 5 microns or more in size. The smallest particle of concern is the single virion (not attached to any liquid droplet) with a diameter of about 0.12 microns. The smallest particle to worry about is a single virion (not attached to any liquid droplet) with a diameter of about 0.12 microns. These can be reasonably filtered by a HEPA (high efficiency particulate air) filter [3]. ULPA (ultra-low penetration air) filters are more advanced at trapping almost 99.99% of particles 0.12 micron and above. The use of nanotechnology further increases the virus capture capacity and purification of such air purifiers and filters. It has produced an efficient filter based on nickel (Ni) foam to capture and kill airborne viruses and microbes, including SARS-CoV-2 and Bacillus anthracis. Since the SARS-CoV-2 virus cannot survive at temperatures above 70 °C, the air filter is designed to operate at 200 °C by heating Ni-foam. The efficiency of the designed filter is claimed to be 99.8% for SARS-CoV-2 virus and 99.9% for Bacillus anthracis [3].

Recent studies show that, in addition to its use in cleaning products and PPE, nanotechnology has also been used in the development of air cleaners to prevent airborne transmission of the SARS-CoV-2 virus. In this context, the TeqAir 200 air ionizer developed by the France-based company TEQOYA is already on the market (Fig. 5). Since the size of SARS-CoV-2 is close to the median of particle sizes for which TEQOYA air cleaners are efficient, they are expected to reduce the concentration of SARS-CoV-2 in the air.

Figure 5. TeqAir 200 air ionizer.

3.1. Nanofiber technology

Mack Antonoff HVAC has designed air purification and filtration systems using nanofiber technology and UV radiation to combat COVID-19 [16]. Turnkey Environmental Consultants have developed an air filtration system based on a dense nanofiber network (IQAirHyperHEPA® filtration technology) that captures polluting particles of all sizes. It is claimed to capture 99.5% of contaminants, including bacteria and viruses with a size of approximately 0.003 microns [16].

3.2. Photo electrochemical oxidation technology

Researchers from the University of South Florida have developed an air-purifying device “Molekule” that is claimed to effectively destroy air pollutants, including bacteria, mould spores and viruses [9]. The air cleaner uses photo electrochemical oxidation (PECO), in which UV-A light is used to activate a catalyst in the nanoparticle coated filter to generate free radicals that oxidize air pollutants [9]. These PECO-based air purifiers have enormous potential to slow the spread of the virus, predominantly in healthcare facilities.

---

Test LO 2.1

---

References

1. Bazant, M. Z., & Bush, J. W. (2021). A guideline to limit indoor airborne transmission of COVID-19. Proceedings of the National Academy of Sciences, 118(17).
2. Buonanno, G., Stabile, L., & Morawska, L. (2020). Estimation of airborne viral emission: Quanta emission rate of SARS-CoV-2 for infection risk assessment. Environment international, 141, 105794.
3. Chaudhary, V., Royal, A., Chavali, M., & Yadav, S. K. (2021). Advancements in research and development to combat COVID-19 using nanotechnology. Nanotechnology for Environmental Engineering, 6(1), 1-15.
4. Centres for Disease Control and Prevention, Feb. 9, 2021. COVID-19 ventilation FAQs, pp. 1–8 [Online]. Available. https://www.cdc.gov/coronavirus/2019-ncov /community/ventilation.html. (Accessed 4 May 2021).
5. Chia, P. Y., Coleman, K. K., Tan, Y. K., Ong, S. W. X., Gum, M., Lau, S. K., … & Marimuthu, K. (2020). Detection of air and surface contamination by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in hospital rooms of infected patients. MedRxiv.
6. Correia, G., Rodrigues, L., Da Silva, M. G., & Gonçalves, T. (2020). Airborne route and bad use of ventilation systems as non-negligible factors in SARS-CoV-2 transmission. Medical hypotheses, 141, 109781.
7. Dancer, S. J., Tang, J. W., Marr, L. C., Miller, S., Morawska, L., & Jimenez, J. L. (2020). Putting a balance on the aerosolization debate around SARS-CoV-2. The Journal of Hospital Infection, 105(3), 569.
8. Ding, J., Yu, C. W., & Cao, S. J. (2020). HVAC systems for environmental control to minimize the COVID-19 infection. Indoor and Built Environment, 29(9), 1195-1201.
9. Elias, B., & Bar-Yam, Y. (2020). Could air filtration reduce COVID-19 severity and spread. New England Complex Systems Institute, 9.
10. EMW filtertechnik. ISO 29463: new test standard for HEPA filters [cited 2020 Jun 3]. Available from: https://www.emw.de/en/filter-campus/iso29463.html.
11. Ham, S. (2020). Prevention of exposure to and spread of COVID-19 using air purifiers: challenges and concerns. Epidemiology and Health, 42.
12. Hick, J. L., Hanfling, D., Wynia, M. K., & Pavia, A. T. (2020). National Academy of Medicine. Duty to Plan: Health Care, Crisis Standards of Care, and Novel Coronavirus SARS-CoV.
13. Hinds, W. C. (1999). Aerosol technology: properties, behavior, and measurement of airborne particles. John Wiley & Sons.
14. Li, Y., Leung, G. M., Tang, J. W., Yang, X., Chao, C. Y. H., Lin, J. Z., … & Qian, H. (2007). AC 587 Sleigh, H.-JJ Su, J. Sundell, TW Wong, PL Yuen, Role of ventilation in airborne transmission of 588 infectious agents in the built environment-a multidisciplinary systematic review. Indoor Air, 17(589), 2-18.
15. Liu, Y., Ning, Z., Chen, Y., Guo, M., Liu, Y., Gali, N. K., … & Lan, K. (2020). Aerodynamic analysis of SARS-CoV-2 in two Wuhan hospitals. Nature, 582(7813), 557-560.
16. Look, M., Bandyopadhyay, A., Blum, J. S., & Fahmy, T. M. (2010). Application of nanotechnologies for improved immune response against infectious diseases in the developing world. Advanced drug delivery reviews, 62(4-5), 378-393.
17. Nadadur, S. S., & Hollingsworth, J. W. (Eds.). (2015). Air pollution and health effects. Springer.
18. Nazarenko, Y. (2020). Air filtration and SARS-CoV-2. Epidemiology and health, 42.
19. Nicas, M., & Miller, S. L. (1999). A multi-zone model evaluation of the efficacy of upper-room air ultraviolet germicidal irradiation. Applied Occupational and Environmental Hygiene, 14(5), 317-328.
20. Peters, A., Parneix, P., Otter, J., & Pittet, D. (2020). Putting some context to the aerosolization debate around SARS-CoV-2. The Journal of Hospital Infection, 105(2), 381.
21. Riley, R. L. (1982). Indoor airborne infection. Environment international, 8(1-6), 317-320.
22. Riley, E. C., Murphy, G., & Riley, R. L. (1978). Airborne spread of measles in a suburban elementary school. American journal of epidemiology, 107(5), 421-432.
23. Santos, A. F., Gaspar, P. D., Hamandosh, A., Aguiar, E. B. D., Guerra Filho, A. C., & Souza, H. J. L. D. (2020). Best practices on HVAC design to minimize the risk of COVID-19 infection within indoor environments. Brazilian Archives of Biology and Technology, 63.
24. Setti, L., Passarini, F., De Gennaro, G., Barbieri, P., Perrone, M. G., Borelli, M., … & Miani, A. (2020). SARS-Cov-2RNA found on particulate matter of Bergamo in Northern Italy: first evidence. Environmental research, 188, 109754.
25. Stadnytskyi, V., Bax, C. E., Bax, A., & Anfinrud, P. (2020). The airborne lifetime of small speech droplets and their potential importance in SARS-CoV-2 transmission. Proceedings of the National Academy of Sciences, 117(22), 11875-11877.
26. Stilianakis, N. I., & Drossinos, Y. (2010). Dynamics of infectious disease transmission by inhalable respiratory droplets. Journal of the Royal Society Interface, 7(50), 1355-1366.
27. URL-1: https://aeramaxpro.com/covid19-1/
28. URL-2: https://www.usatoday.com/in-depth/graphics/2020/10/18/improving-indoor-air-quality-prevent-covid-19/3566978001/
29. URL-3:https://www.sentryair.com/blog/indoor-air-quality/improving-ventilation-and-air-filtration-to-help-prevent-covid-transmission-in-the-workplace/
30. URL-4: https://www.microcovid.org/blog/hepafilters
31. URL-5: https://product.statnano.com/product/11653/teqoya-teqair-200-air-ionizer
32. S. Department of Energy. DOE-STD-3020-2005, specification for HEPA filters used by DOE contractors; 2015 [cited 2020 Jul 15]. Available from: https://www.standards.doe.gov/standards-documents/3000/3020-astd-2005
33. Van Doremalen, N., Bushmaker, T., Morris, D. H., Holbrook, M. G., Gamble, A., Williamson, B. N., … & Munster, V. J. (2020). Aerosol and surface stability of SARS-CoV-2 as compared with SARS-CoV-1. New England journal of medicine, 382(16), 1564-1567.
34. Vejerano, E. P., & Marr, L. C. (2018). Physico-chemical characteristics of evaporating respiratory fluid droplets. Journal of The Royal Society Interface, 15(139), 20170939.
35. Wells, W. F. (1934). ON AIR-borne infection: study II. Droplets and droplet nuclei. American journal of Epidemiology, 20(3), 611-618.
36. World Health Organization. Laboratory biosafety guidance related to coronavirus disease 2019 (COVID-19): interim guidance, 12 February 2020 [cited 2020 Jun 3]. Available from: https://apps.who.int/iris/handle/10665/331138.
37. World Health Organization. (2020). Severe acute respiratory infections treatment centre: practical manual to set up and manage a SARI treatment centre and a SARI screening facility in health care facilities(No. WHO/2019-nCoV/SARI_treatment_center/2020.1). World Health Organization.
38. Wu, X., Nethery, R. C., Sabath, B. M., Braun, D., & Dominici, F. (2020). Exposure to air pollution and COVID-19 mortality in the United States: A nationwide cross-sectional study. medRxiv 2020.04. 05.20054502.
39. Zhu, N., Zhang, D., Wang, W., Li, X., Yang, B., Song, J., … & Tan, W. (2020). A novel coronavirus from patients with pneumonia in China, 2019. New England journal of medicine.

Summary

Viral epidemics develop due to the emergence of new variants of infectious viruses. The lack of effective antiviral treatments for new viral infections, coupled with the rapid spread of the infection in the community, often results in huge human and financial losses. Viral transmission can occur through close person-to-person contact or contact with a contaminated surface. Therefore, careful disinfection or sanitization is essential to reduce viral spread. There are numerous disinfectant/sanitizing agents/biocidal agents that can inactivate viruses, but their effectiveness depends on many factors such as agent concentration, reaction time, temperature and organic load. Advances in nanotechnology are of great importance in the inactivation of viruses and in the control of the COVID-19 epidemic.

Key words/phrases: disinfection, nanotechnology, COVID-19

1. Introduction

Viral transmissions and infections have historically posed serious threats to human health and well-being and led to widespread socioeconomic disruptions. A century ago, in 1918, the “Spanish flu” pandemic caused a worldwide catastrophe with more than 50 million deaths and 500 million infections [33]. One of the country worst affected by the Ebola epidemic in West Africa in 2014, Liberia’s gross domestic product (GDP) growth decreased from 8.7% in 2013 to 0.7% in 2014 [66]. The threat of a global virus pandemic could lead to greater catastrophes than at any time in human history, as viruses can spread around the world at unprecedented rates due to easier global connections and transportation, and the ease and intensity of human mobility today. Today, however, we see that a similar pandemic has undoubtedly led to more disastrous results [7]. The novel coronavirus, which causes the COVID-19 disease (SARS-CoV-2 or formerly HCoV-19), first reported by China in late 2019, has resulted in an estimated more than six million deaths at the time of this writing. The pandemic is still not over, and new cases and new deaths are reported every day. While governments and states can influence the rates and range of outbreaks [2], individuals can have more important roles in limiting the spread of viruses in public and healthcare settings [47]. Human-to-human transmission of common influenza viruses and coronaviruses can occur through self-inoculation of mucous membranes in the nose, mouth, or eyes by touching contaminated dry surfaces as well as virus-laden bodily fluids [37]. Depending on the surface type and environmental conditions, viruses can remain on inanimate surfaces for 5 minutes or less to more than 28 days [22]. The use of sanitizing agents for personal care and surface disinfection is of great importance in limiting viral transmissions by inactivating viruses before they have a chance to enter the human body.

In this training unit, we provide information on the various types of sanitizing agents used in commercially available formulations with scientifically proven virucidal properties to inactivate viruses in suspension and on surfaces. We also provide information on the antiviral disinfection use of nanotechnological materials, one of the promising new developments that have been shown to inactivate viruses but have not yet reached widespread commercial use.

2. Disinfectants against viruses and general working principles

2.1. Viruses and infectivity

Viruses typically consist of a viral capsid containing nucleic acids (Fig. 1). The nucleic acid serves as template information for replication, while the capsid and its associated proteins function both to protect the nucleic acid and to bind to host cell receptors [29].

Figure 1. Types of viruses.

Viruses cannot reproduce and increase in number outside of a host cell. However, they can often survive for a long time in this condition [67]. When they encounter a suitable host cell, they will become infected and enter the host cell and hijack the cellular machinery for its own replication (Fig. 2). Viruses can infect cells, including bacterial cells, and cause a number of common diseases. This situation is exacerbated by the lack of effective treatment against many of the viruses.

Corona viruses, on the other hand, are enveloped and single-stranded RNA viruses, that is, their genetic material consists of an RNA strand and each viral particle is wrapped in a protein envelope (Fig. 3). All viruses basically follow the same path in infecting their hosts. A virus that invades a cell replicates itself using some components of that cell, and then its copies infect other cells. However, RNA viruses have a different feature. These viruses cannot correct errors that occur during RNA replication because they do not have the error correction mechanisms that cells typically use when copying DNA.

Figure 2. The four stages of the viral infection of a cell in the model.

Coronaviruses are the group of viruses with the longest genome, with 30,000 bases, among the RNA viruses. These pathogens, which lack the ability to correct errors during replication, increase the probability of making mistakes as the amount of base they copy increases. Therefore, every mistake brings with it a new mutation. Some of these mutations may also confer new properties on the virus, such as the ability to infect new cell types, or even new strains. A coronavirus consists of four structural proteins: nucleocapsid, envelope, membrane, and rod-like protrusions (thorns). Since these protrusions are called “corona”, which means crown in Latin, these viruses are called coronavirus (crown virus). The nucleocapsid contains the genetic material in a spherical structure formed by envelope and membrane proteins. The spiny protrusions identify the cells that the virus can infect and attach to the receptors in the cells.

Surfaces, including our hands, play an important role in the spread of viruses. Viruses such as poliovirus and bacteriophage show a much higher survivability when transferred by direct contact of surfaces, as opposed to viruses containing droplet aerosolization or dust. Just 5 seconds of hand and face contact is sufficient to transfer a significant portion of the virus, and viruses can then spread by touching the nasal mucosa or the conjunctiva of the eye. The chance of spread is directly related to the viral survival time on the surface, which differs significantly between different viruses. A very recent study reported that the Covid-19 coronavirus (SARS-CoV-2) can persist the longest on propylene plastic surfaces and stainless steel, and live viruses are present up to 72 hours after initial application, although at a greatly reduced viral titer [60].

Figure 3: Illustration of SARS-CoV-2 virus

2.2. Factors affecting disinfectant efficacy

The main measure of the disinfectant’s effectiveness is the reduction in the infectivity of the virus. Virus infectivity measurement is typically performed by carrier tests and suspension tests. The main parameters affecting the effectiveness of disinfectants against viruses include contact time, concentration of disinfectant and related virus.

In addition, disinfection efficiency may also be affected by environmental factors. If disinfection requires chemical reactions, such as formaldehyde, to occur, the disinfection rate will be higher at higher temperatures. In cold weather, some disinfectants may be ineffective as the disinfection rate will be extremely low. Humidity is another factor that can affect the penetration of the disinfectant into the virus. For reactions such as aldehyde disinfectants, a change in pH will also affect disinfectant effectiveness.

2.3. Factors influencing virus susceptibility

Viruses that affect inactivation by disinfection have certain unique properties. Here there are three main types of viruses with different structures, classified according to the increasing difficulty of chemical disinfectant inactivation: enveloped viruses, large non-enveloped viruses, and small non-enveloped viruses. Although there are exceptions, larger viruses are generally more sensitive to disinfectants [32]. All of the few disinfectant solutions tested are effective against the enveloped viruses Herpes Simplex Virus and Human Immunodeficiency Virus (HIV) type 1, but less effective against the small non-enveloped human coxsackie virus. Enveloped viruses contain a lipid envelope that is essential for infection, and thus interfering with the envelope can potentially reduce virus transmission. Lipophilic disinfectants can often be used to inactivate enveloped viruses. In contrast, non-enveloped viruses use a protein coat for infection, and thus inactivation often requires denaturation of unnecessary viral capsid proteins or essential replicative proteins [36]. Disinfectants that degrade proteins, such as glutaraldehyde or sodium hypochlorite, can be effective in inactivating non-enveloped viruses [32]. Electron microscopy studies show extensive structural damage to the phage, including sodium hypochlorite inactivating bacteriophage PAO1 and damage to capsid proteins. However, since viruses such as polio only maintain infectivity with RNA, the disinfectant may need to penetrate to destroy nucleic acids [32]. While the enveloped virus influenza H1N1 can be inactivated by all disinfectants tested [16], small non-enveloped noroviruses are much more difficult to inactivate and few commonly available disinfectants cannot adequately reduce infection [56].

Viruses also resist disinfection because of the cellular materials with which the viruses are associated. Viruses are normally dependent on host cells for replication, so they are often found in association with materials such as cell debris, soil, and aerosolized droplets. These are called viral aggregation protective factors and they can both reduce the penetration of the disinfectant to the virus and reduce the interaction and activity of the disinfectant agents. This has a great effect on disinfectants and requires a much higher concentration for effective disinfection. Disinfection is often associated with and dependent on cleaning processes, as primarily removal of organic material impurities may allow for a better disinfection process [14]. In addition, viruses can accumulate in the environment when exposed to disinfectants, making it difficult for disinfectants to penetrate and reach viruses [15].

COVID-19 is known to be very contagious and there are many ways of transmission. Recent studies have shown that SARS-CoV-2 spreads mainly through micro-droplets spread from person to person or by touching contaminated surfaces (Fig. 4) [58]. SARS-CoV-2 is known to have the ability to remain in aerosol form for more than 3 hours. It is also stated that depending on the surface, the human corona virus can persist for up to 9 days and at temperatures above 30 °C. In this context, the use of personal protective equipment (PPE), disinfectant and disinfectant is extremely important. The World Health Organization (WHO) recommends the use of physical and chemical factors to reduce contamination through disinfection of surfaces, especially on frequently touched surfaces such as doorknobs, tables, chairs, railings and keys, as well as mask use and hygiene personal care procedures. Different disinfecting agents have been described in the literature, including sodium hypochlorite, hydrogen peroxide, alcohols, soaps/surfactants, etc. [31].

Figure 4. Examples of virus spreading points.

3. Commercially available virucidal sanitizing agents

3.1. Alcohols

Alcohols, particularly isopropyl alcohol (also known as isopropanol and propan-2-ol) and ethyl alcohol (ethanol), can inactivate a broad spectrum of bacterial, fungal and viral activity (Figs. 5-6). These active ingredients play an important role in the healthcare industry for skin antisepsis and disinfection of small medical instruments. Although it has been shown to be effective at destroying infectious microorganisms, alcohols are not sporicidal [44] and are often combined with other major biocidal actives to increase disinfection efficacy.

Potent biocidal agents destroy viruses and bacteria by various mechanisms, such as disrupting cell structure and coagulating and/or denaturing proteins in microorganisms. Although few studies have been conducted to fully understand the biocidal activity of alcohol, it is generally believed that alcohols disrupt cell membranes and denature proteins in general [4]. Viruses and many other microorganisms are generally susceptible to this mode of action. Previous studies have reported that the incorporation of water into the biocidal system increases the effectiveness of alcohol, as water facilitates faster denaturing of proteins [44]. Additionally, the addition of water significantly increases the effectiveness of alcohols as it delays the evaporation of the alcohol and increases its exposure to viruses and bacteria.

However, the virucidal activity of alcohol is highly dependent on the concentration of active substances and the type of test viruses. The effectiveness of alcohols in inactivating viruses largely depends on the surface properties of the microorganism. Non-enveloped viruses are generally known to be more resistant to disinfectants than enveloped viruses, including alcohols.

While alcohols were effective at destroying some types of viruses, other disinfectants such as quaternary ammonium compounds (QAC), glutaraldehyde, and hydrogen peroxide quickly overshadowed its performance [44]. Therefore, disinfectants whose main active ingredients are alcohol are not generally used to disinfect critical equipment or environment in healthcare settings [44]. The use of isopropyl alcohol is also limited, as it only inactivates lipid viruses. This greatly reduces alcohol’s abilities as a broader use disinfectant. Because alcohols are flammable liquids, large amounts of alcohol will increase their risks and hazards as a disinfectant. The flash point of the higher concentration alcohol solution is lower than that of the lower concentration [4]. In addition, prolonged and repeated use of alcohol compromises the integrity of materials such as plastics and paints. Materials that are constantly exposed to alcohol may experience color change, cracking and swelling due to the effects of alcohol. Another challenge with alcohol use is that it evaporates quickly when exposed to air, thus reducing the time of contact with the virus. Maximum disinfection is difficult to achieve unless instruments have been immersed in the bath for some time.

Figure 5. Factors affecting the effectiveness of alcohol-based hand sanitizers against SARS-CoV-2.

Although alcohol’s capabilities are limited, it is still widely used in a variety of active disinfectant procedures. It is imperative to note that the role of alcohol as a disinfectant along with its other properties is still irreplaceable. Alcohols are often used in hospital as an effective disinfectant for thermometers, non-critical instruments, and non-invasive probes [44]. Non-critical surfaces of reusable medical instruments are also disinfected with alcohol. Another advantage of using alcohol as a disinfectant is that it is user-friendly. Alcohol solutions are non-staining, evaporate quickly, have low toxicity compared to other forms of disinfectant, and have a mild, acceptable odor. These features are critical in healthcare environments as they contribute to the efficiency and necessary sanitization of the system.

Figure 6. The antiviral mechanism of action of alcohol against enveloped viruses.

3.2. Surfactants

Surfactants are amphiphilic moieties that have both hydrophilic and lipophilic segments, further classified as cationic, anionic, nonionic, and zwitterionic surfactants. They are active ingredients often found in household disinfectants and detergents and have been shown to be able to inactivate viruses. Enveloped viruses such as the coronavirus family, which includes SARS-CoV-1, MERS, and the novel SARS-CoV-2 viruses, are therefore susceptible to these surfactants. However, some surfactants do not rely on dissolution of their lipid envelope to inactivate viruses.

3.2.1. Cationic surfactants (Quaternary ammonium compounds)

Quaternary ammonium compounds (QACs) form the main mass of cationic surfactants and inactivate viruses mostly by dissolving and breaking down their lipid envelope or membrane. It is reported that they retain virucidal activity better in hard water and also in the presence of anionic residues [38]. QACs are attractive because they are relatively non-toxic, colorless and odorless [19]. They are well known for inactivating enveloped viruses, but their virucidal activity is dependent on concentration, exposure time, and temperature. Effective disinfection with surfactants using QACs is best achieved using warm water and longer reaction times [30]. An advantage of using QAC-based disinfectants is their relatively high tolerance to the presence of contaminating organic matter. This is because their ability to inactivate viruses is generally not reduced by the presence of organic matter, as is seen with other common disinfectants such as alcohol and chlorine-based disinfectants.

3.2.2. Vaccines for cancer

They are some common anionic surfactants found in personal care products such as soap, shampoo, toothpaste and detergents [49].

3.2.3. Vaccines for cancer

Non-ionic surfactants are commonly used as emulsifiers. These non-ionic surfactants inactivate viruses by dissolving the viral envelope and cleaving the nucleocapsid. Zwitterionic surfactants are molecules that carry both cationic and anionic charges, but generally neutrally charged [63]. Researchers have suggested that the mechanism of disinfection by zwitterionic detergent is via viral degradation rather than solubilizing surface proteins [8]. This special ability, which inactivates viruses but preserves the biological activity of surface antigens, allows the use of zwitterionic detergent during the development of vaccines.

3.3. Oxidizing agents

Disinfectants such as sodium hypochlorite, hydrogen peroxide, and peracetic acid use their oxidizing abilities to inactivate viruses. Strong oxidizing agents are among the most effective disinfectants for small non-enveloped viruses that are difficult to disinfect, such as noroviruses [17].

3.3.1. Sodium hypochlorite

Sodium hypochlorite, the active ingredient in household bleach, is a powerful oxidizing agent. It dissolves in water to form hypochlorous acid, which can be reduced to form water and chloride anion [13]. The effectiveness of disinfection decreases with increase in pH, probably due to the decreasing proportion of the hypochlorous acid moiety present [3]. Sodium hypochlorite is fast acting and effective at low concentrations. Its effect was found to be proportional to its concentration and contact time. Sodium hypochlorite can be used for non-enveloped viruses that are difficult to disinfect, such as noroviruses.

3.3.2. Sodium dichloroisocyanurate

Compared to sodium hypochlorite, sodium dichloroisocyanurate has longer lasting disinfectant activity, is more tolerant of the presence of organic material, and has a higher overall disinfectant effectiveness.

3.3.3. Hydrogen peroxide

Hydrogen peroxide is a potent broad spectrum inactivation agent. It decomposes to form water, oxygen, and highly reactive hydroxyl free radicals that can degrade or cross-link a wide variety of biomolecules, including proteins, nucleic acids, and lipids. Hydrogen peroxide is also effective against noroviruses, although it usually requires a higher concentration than sodium hypochlorite.

3.4. Peracetic acid

Peracetic acid similarly decomposes to form highly reactive hydroxyl free radicals as well as acetic acid and oxygen [42]. Peracetic acid forms have been developed to provide greater stability and can be dissolved in situ to form the disinfectant solution.

3.5. Halogenated compounds

3.5.1. Povidone iodine

Povidone-iodine is a broad-spectrum virucidal agent. It is used in clinical applications such as sterilizing agents, surgical swabs, scrubs, and ointments for pre- and post-operative skin cleansing, as well as in everyday products such as antiseptic hand washes, mouthwashes, and mouthwashes containing lower iodophor concentrations [12]. Not suitable for use with silicone products such as povidone-iodine silicone catheters, as iodine may cause the material to degrade faster. Although generally safer and more effective at inactivating viruses than many other antiseptic agents, povidone-iodine can, with prolonged use, cause thyroid dysfunction [27] and allergic contact dermatitis requiring careful medical monitoring [61]. The origins of the broad virucidal activities of povidone iodide have not yet been fully elucidated and are likely to occur by more than one mechanism, reducing the likelihood of chance viral mutations conferring resistance. There is evidence that iodine can block the receptors of the virus responsible for binding to the host cell surface [50]. In addition, iodine can prevent the virus from spreading to other uninfected cells by inhibiting the activity of viral enzymes (eg, neuraminidase) necessary for virus release from host cells [12]. For enveloped viruses, it has also been suggested that the virus membrane can be destabilized by the reaction of iodine, membrane fatty acids with unsaturated C=C bonds.

3.5.2. Chlorhexidine digluconate

Chlorhexidine is a broad spectrum cationic bisguanide biocide found in many antiseptic products. An active ingredient in hand washes, mouthwashes and oral gels, disinfectants and preservatives, chlorhexidine generally has low irritability, good persistence on the skin, and rapid bactericidal activity. However, its activity is highly dependent on its formulation, being reduced by the presence of anionic surfactants and phospholipids as well as organic matter, including serum, and is also pH dependent. Compared to bacteria, its virucidal activity is more variable than povidone-iodine, and it is significantly less potent and slower acting. In general, chlorhexidine is ineffective against non-enveloped viruses (polio and adenoviruses), but shows variable potency to inactivate enveloped viruses

3.5.3. Chloroxylenol

Chloroxylenol is a halogenated phenolic type antiseptic. Widely used for household disinfectants, wound cleaning and disinfecting surgical equipment, it is most effective against bacteria, but its virucidal activity is variable. Despite its extensive commercial use for a long time, surprisingly little is known about its mechanism of action against both bacteria and viruses. Chloroxylenol is generally safe for external use in humans, but has been reported to cause irritant contact dermatitis and contact depigmentation [62].

3.6. Aldehydes

3.6.1. Formaldehyde

Formaldehyde is the simplest aldehyde and is a powerful high-level disinfectant with potent viral inactivation properties. Usually sold as an aqueous solution called formalin, it has been used to inactivate viruses for vaccine production [35] and scientific study [35]. As a top-level disinfectant, it can effectively and rapidly inactivate many different types of viruses, both in suspension and on surfaces, by chemically alkylating the amino and sulfhydryl groups of proteins, as well as the amino groups of nucleic acid bases of DNA and RNA [23]. However, due to its high reactivity, its use makes it harmful to health by causing irritation on exposed body surfaces (e.g. skin and eyes) [43], apart from being a mutagenic and suspected carcinogen [52]. As a result, it is subject to strict regulations regarding human exposure as a disinfectant and sterilizing agent in hospitals and healthcare facilities, except for use in a well-ventilated area, and is therefore not used as a household disinfectant.

3.6.2. Glutaraldehyde

Like formaldehyde, glutaraldehyde (or sometimes also known as glutardialdehyde) is a potent broad-spectrum disinfecting and sterilizing agent that is highly effective against many viruses after short exposure times. Although not suspected to be carcinogenic [54], it is known to cause dermatitis in the eyes, nose, and mouth, and irritation of mucous membranes. For these reasons, it is not used as a household disinfectant. Generally, metals, rubber, plastics, and lens instruments are tolerant to glutaraldehyde, but it is not recommended for use to disinfect non-critical surfaces due to its cost.

3.6.3. Ortho-phthalaldehyde (OPA)

Ortho-phthalaldehyde or 1,2-dicarboxybenzaldehyde is another high-level disinfectant. Like both formaldehyde and glutaraldehyde, its virucidal properties result from reactions that cross-link reactive protein and nucleic acid moieties. OPA has no strong detectable odor and does not irritate the skin, eyes or nasal mucosa [9]. In addition, its excellent material compatibility [1] allows it to be used as a disinfectant in many clinical settings such as endoscopes [45] and urological instruments. However, OPA can turn exposed skin gray and therefore needs to be rinsed with copious amounts of water or used with personal protective equipment (eg gloves and eye protection). For this reason, it is not widely used as a household disinfectant.

4. Nanotechnology

4.1. Nanomaterials for surface decontamination

Nanotechnology offers many opportunities for the development of more efficient and promising disinfectant systems (Fig. 7). The use of nanoparticle-based markers could enable the study of the mechanism by which viruses infect host cells. Today, studies based on nanotechnology for the development of new materials are generally on surfaces with self-cleaning properties [39]. These systems may have antimicrobial activity or slowly release chemical disinfectants, prolonging their duration of action. It may also contribute to the introduction of additional features such as responsive systems that deliver active substances in response to different stimuli, such as photothermal, electrothermal, photocatalytic, and others [10]. It is also known that some metallic nanoparticles have a broad spectrum of action against viruses and other microorganisms [11]. Rai et al. [41] conducted a literature review on the antibacterial, antifungal, and antiviral potential of metallic nanoparticles. According to the results of this study, metallic nanoparticles, especially silver nanoparticles, can be used as a potent and broad-spectrum antiviral agent with or without surface modification. However, the antiviral activity of these nanoparticles is still largely unexplored.

Today, nanotechnology has been a solution to many problems in disinfection applications. Over the past few decades, nanotechnology has emerged as a promising new technology for the synthesis of nanomaterials, which are nanometer-sized particles that exhibit antimicrobial effects due to their high surface area-to-volume ratio and unique chemical and physical properties. Many nanomaterials, such as metal nanoparticles and graphene-based nanosheets, have natural antiviral effects due to their unique physicochemical properties [53]. They generally operate by a common mechanism of action that involves direct interaction with the envelope or capsid proteins of viruses, thereby disrupting structural integrity and inhibiting infectivity. In addition, some nanomaterials may interfere with viral gene replication inside infected cells [20, 28, 18]. Further work is needed on the use of nanotechnology for more efficient disinfectant and sanitizing systems, as well as on achieving self-disinfecting surfaces to increase effectiveness for infection control and health and environmental safety.

Table 1 shows published research and patents on different systems based on nanotechnology for application as disinfectant and disinfectant for viruses.

Figure 7: Schematic representation of SARS-CoV-2 infection and the nanotechnologies tools to prevent and control COVID-19.

The virus entering into cell by the angiotensin-converting enzyme 2 (ACE2) receptor and use the host cell’s machinery to reproduce and contaminate new host cells. Nano-based materials could help in: (i) enhanced the speed and sensitivity of virus detection; (ii) help in the development of more efcient and safer treatment and vaccines and (iii) improve the safety of healthcare workers through the development of nano-based Personal Protective equipment (PPE).

Table 1. Articles and patents in the literature on nanotechnology-based disinfectants and disinfectants.

PPE	Name of the product	Application of nanomaterial	Manufacturing company
Masks
1.	Surgical Masks-ESpin Technologies	Use of nanofibres for particles removal	ESpin Technologies, Inc.-USA
2.	Defenser Series-Respirator masks	The facemask has nanoparticles of silver and copper acting as a blend with antimicrobial activity	Nexera Medical-Canada
3.	The Guardian (valve)- reusable	The valve mask has nanoparticles of silver and copper acting as a blend with antimicrobial activity	Nexera Medical-Canada
4.	The Guardian masks- reusable	The valve mask has nanoparticles of silver and copper acting as a blend with antimicrobial activity	Nexera Medical-Canada
5.	MVX Nano Mask	A self-cleaning surgical mask containing titanium and silver zeolite nanoparticles	MVX Prime Ltd
Gloves
1.	Everyday Protect Gloves L	A product containing silver nanoparticles and the active compounds thiabendazole and zinc pyrithione	Mapa Spontex- United Kingdom
2.	PADYCARE®	Product coated with silver nanoparticles with antibacterial effect	TEXAMED® GmbH-Germany
3.	Chlorhexidine wash gloves	A product containing silver nanoparticles and 2% chlorhexidine; the antibacterial effects last many hours after use	GAMA HEALTHCARE LTD.

4.2. Metal nanoparticles

Silver and its salts have a long history of use as an antiseptic and disinfectant, and their broad-spectrum biocidal properties are well known [21]. Silver nanoparticles are the most studied antiviral nanomaterial and it has been shown that bare or coated AgNPs can inhibit a wide variety of viruses [40]. It is difficult for viruses to develop resistance to this type of treatment, making it particularly attractive to those with a high rate of mutation. AgNPs have been found to be effective in both blocking virion entry from the outside of the cell and inhibiting replication inside infected cells. Overall, AgNPs are effective biocides in small doses [55], but their potential toxicity to humans is still under intense debate [26]. Modern methods have enabled the synthesis of AgNPs with well-defined shapes, particle sizes and polydispersity, which are important parameters determining their ultimate biocidal activities, biological fate and toxicity [25].

The virucidal properties of AgNPs are still largely unexplored, but initial reports are encouraging. AgNPs can inhibit viruses by a number of mechanisms, including binding to and interacting with viral surface proteins and denaturing enzymes by reacting with amino, carboxyl, imidazole, and sulfhydryl groups [6].

AgNP-containing products are increasingly appearing on the market, including clothing, dressings, ointments, and food packaging materials, whose biocidal activities are the result of the slow sustained release of silver nanomaterials [6]. However, it should be noted that, like all the disinfecting agents mentioned above, the virucidal activities of AgNPs differ from virus to virus. Furthermore, the amounts, shapes, sizes, and types of silver nanomaterials released depend on their real-world settings and applications, all of which affect their virucidal properties. Therefore, the efficacy of these AgNP-containing products against viruses in real-life environments as well as their toxicity to humans need to be carefully evaluated and studied.

Apart from AgNPs, gold nanoparticles (AuNPs) are also promising virucidal agents. AuNPs synthesized using garlic extract with an average size of 6 nm showed virucidal activity against measles virus by also binding to surface viral receptors and subsequently preventing host cell attachment and infection [34]. However, due to the cost of gold chemical precursors, AuNPs are unlikely to become inexpensive and commercially widely available disinfectant agents.

The use of metal nanomaterials to create self-disinfecting surfaces has gained attention in recent years due to the long-term persistence of viruses on contaminated surfaces. Self-disinfecting surfaces inactivate viruses that come into contact with them in situ, reducing the possibility of virus transmission through human contact with contaminated surfaces. In one design, the self-disinfecting surface was formed with photoactive metal nanocrystals that required visible light stimulation for viral inactivation. These surfaces, fabricated from CuInZn4S6 (CIZS) nanocrystals with band gaps in the visible light range, can absorb visible light and produce active oxidative species that inactivate influenza A virus by oxidizing amino acid residues presented in viral envelope proteins (Fig. 8). While highly virucidal, visible light must be present to guarantee the self-cleaning effect, thus limiting the practicality of the system.

Figure 8. Illustration of virus disinfection using the self-disinfecting surface powered by visible light.

---

Test LO 2.2

---

References

1. 1. Akamatsu, T., Minemoto, M., & Uyeda, M. (2005). Evaluation of the antimicrobial activity and materials compatibility of orthophthalaldehyde as a high-level disinfectant. Journal of international medical research, 33(2), 178-187.
   2. Bell, D. M. (2004). Public health interventions and SARS spread, 2003. Emerging infectious diseases, 10(11), 1900.
   3. Block, S. S. (Ed.). (2001). Disinfection, sterilization, and preservation. Lippincott Williams & Wilkins.
   4. Boyce, J. M. (2018). Alcohols as surface disinfectants in healthcare settings. infection control & hospital epidemiology, 39(3), 323-328.
   5. Campos, E. V., Pereira, A. E., De Oliveira, J. L., Carvalho, L. B., Guilger-Casagrande, M., De Lima, R., & Fraceto, L. F. (2020). How can nanotechnology help to combat COVID-19? Opportunities and urgent need. Journal of Nanobiotechnology, 18(1), 1-23.
   6. Castro-Mayorga, J. L., Martínez-Abad, A., Fabra, M. F., Lagarón, J. M., Ocio, M. J., & Sánchez, G. (2016). Antimicrobial Food Packaging.
   7. Christophersen, O. A., & Haug, A. (2006). Why is the world so poorly prepared for a pandemic of hypervirulent avian influenza? Microbial ecology in health and disease, 18(3-4), 113-132.
   8. Conley, L., Tao, Y., Henry, A., Koepf, E., Cecchini, D., Pieracci, J., & Ghose, S. (2017). Evaluation of eco‐friendly zwitterionic detergents for enveloped virus inactivation. Biotechnology and bioengineering, 114(4), 813-820.
   9. Cooke, R. P. D., Goddard, S. V., Whymant-Morris, A., Sherwood, J., & Chatterly, R. (2003). An evaluation of Cidex OPA (0.55% ortho-phthalaldehyde) as an alternative to 2% glutaraldehyde for high-level disinfection of endoscopes. Journal of Hospital Infection, 54(3), 226-231.
   10. Dalawai, S. P., Aly, M. A. S., Latthe, S. S., Xing, R., Sutar, R. S., Nagappan, S., … & Liu, S. (2020). Recent advances in durability of superhydrophobic self-cleaning technology: a critical review. Progress in Organic Coatings, 138, 105381.
   11. Dyshlyuk, L., Babich, O., Ivanova, S., Vasilchenco, N., Prosekov, A., & Sukhikh, S. (2020). Suspensions of metal nanoparticles as a basis for protection of internal surfaces of building structures from biodegradation. Case Studies in Construction Materials, 12, e00319.
   12. Eggers, M. (2019). Infectious disease management and control with povidone iodine. Infectious diseases and therapy, 8(4), 581-593.
   13. Fukuzaki, S. (2006). Mechanisms of actions of sodium hypochlorite in cleaning and disinfection processes. Biocontrol science, 11(4), 147-157.
   14. Gallandat, K., Wolfe, M. K., & Lantagne, D. (2017). Surface cleaning and disinfection: efficacy assessment of four chlorine types using Escherichia coli and the Ebola surrogate Phi6. Environmental Science & Technology, 51(8), 4624-4631.
   15. Gerba, C. P., & Betancourt, W. Q. (2017). Viral aggregation: impact on virus behavior in the environment. Environmental science & technology, 51(13), 7318-7325.
   16. Jeong, E. K., Bae, J. E., & Kim, I. S. (2010). Inactivation of influenza A virus H1N1 by disinfection process. American journal of infection control, 38(5), 354-360.
   17. Girard, M., Mattison, K., Fliss, I., & Jean, J. (2016). Efficacy of oxidizing disinfectants at inactivating murine norovirus on ready-to-eat foods. International journal of food microbiology, 219, 7-11.
   18. Huang, S., Gu, J., Ye, J., Fang, B., Wan, S., Wang, C., … & Cao, S. (2019). Benzoxazine monomer derived carbon dots as a broad-spectrum agent to block viral infectivity. Journal of colloid and Interface Science, 542, 198-206.
   19. Heuschele, W. P. (1995). Use of disinfectants in zoos and game parks. Revue Scientifique et Technique (International Office of Epizootics), 14(2), 447-454.
   20. Jackman, J. A., Lee, J., & Cho, N. J. (2016). Nanomedicine for infectious disease applications: innovation towards broad‐spectrum treatment of viral infections. Small, 12(9), 1133-1139.
   21. Jung, W. K., Koo, H. C., Kim, K. W., Shin, S., Kim, S. H., & Park, Y. H. (2008). Antibacterial activity and mechanism of action of the silver ion in Staphylococcus aureus and Escherichia coli. Applied and environmental microbiology, 74(7), 2171-2178.
   22. Kampf, G., Todt, D., Pfaender, S., & Steinmann, E. (2020). Persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents. Journal of hospital infection, 104(3), 246-251.
   23. Kamps, J. J., Hopkinson, R. J., Schofield, C. J., & Claridge, T. D. (2019). How formaldehyde reacts with amino acids. Communications Chemistry, 2(1), 1-14.
   24. Lara, H. H., Garza-Treviño, E. N., Ixtepan-Turrent, L., & Singh, D. K. (2011). Silver nanoparticles are broad-spectrum bactericidal and virucidal compounds. Journal of nanobiotechnology, 9(1), 1-8.
   25. Lee, S. H., & Jun, B. H. (2019). Silver nanoparticles: synthesis and application for nanomedicine. International journal of molecular sciences, 20(4), 865.
   26. Liao, C., Li, Y., & Tjong, S. C. (2019). Bactericidal and cytotoxic properties of silver nanoparticles. International journal of molecular sciences, 20(2), 449.
   27. Lithgow, K., & Symonds, C. (2017). Severe thyrotoxicosis secondary to povidone-iodine from peritoneal dialysis. Case Reports in Endocrinology, 2017.
   28. Liu, H., Bai, Y., Zhou, Y., Feng, C., Liu, L., Fang, L., … & Xiao, S. (2017). Blue and cyan fluorescent carbon dots: one-pot synthesis, selective cell imaging and their antiviral activity. RSC advances, 7(45), 28016-28023.
   29. Lodish, H., Berk, A., Zipursky, S. L., Matsudaira, P., Baltimore, D., & Darnell, J. (2000). Viruses: Structure, function, and uses. In Molecular Cell Biology. 4th edition. WH Freeman.
   30. Louie, W., & Reuschlein, D. (2011). 8 Cleaning and Disinfection in the Bottled Water Industry. Technology of Bottled Water, 223.
   31. Lukasik, J., Bradley, M. L., Scott, T. M., Dea, M., Koo, A., Hsu, W. Y., … & Farrah, S. R. (2003). Reduction of poliovirus 1, bacteriophages, Salmonella Montevideo, and Escherichia coli O157: H7 on strawberries by physical and disinfectant washes. Journal of food protection, 66(2), 188-193.
   32. McDonnell, G. E. (2007). Antiseptics, disinfection, and sterilization. Types, action, and resistance.
   33. Martini, M., Gazzaniga, V., Bragazzi, N. L., & Barberis, I. (2019). La pandémie de grippe espagnole: une leçon de 100 ans après 1918. J Prev Med Hyg, 60, E64-E67.
   34. Meléndez-Villanueva, M. A., Morán-Santibañez, K., Martínez-Sanmiguel, J. J., Rangel-López, R., Garza-Navarro, M. A., Rodríguez-Padilla, C., … & Trejo-Ávila, L. M. (2019). Virucidal activity of gold nanoparticles synthesized by green chemistry using garlic extract. Viruses, 11(12), 1111.
   35. Möller, L., Schünadel, L., Nitsche, A., Schwebke, I., Hanisch, M., & Laue, M. (2015). Evaluation of virus inactivation by formaldehyde to enhance biosafety of diagnostic electron microscopy. Viruses, 7(2), 666-679.
   36. Nuanualsuwan, S., & Cliver, D. O. (2003). Infectivity of RNA from inactivated poliovirus. Applied and environmental microbiology, 69(3), 1629-1632.
   37. Otter, J. A., Donskey, C., Yezli, S., Douthwaite, S., Goldenberg, S., & Weber, D. J. (2016). Transmission of SARS and MERS coronaviruses and influenza virus in healthcare settings: the possible role of dry surface contamination. Journal of hospital infection, 92(3), 235-250.
   38. Perry, K., & Caveney, L. (2012). Chemical disinfectants. Veterinary infection prevention and control, 129-143.
   39. Querido, M. M., Aguiar, L., Neves, P., Pereira, C. C., & Teixeira, J. P. (2019). Self-disinfecting surfaces and infection control. Colloids and Surfaces B: Biointerfaces, 178, 8-21.
   40. Rai, M., Kon, K., Ingle, A., Duran, N., Galdiero, S., & Galdiero, M. (2014). Broad-spectrum bioactivities of silver nanoparticles: the emerging trends and future prospects. Applied microbiology and biotechnology, 98(5), 1951-1961.
   41. Rai, M., Deshmukh, S. D., Ingle, A. P., Gupta, I. R., Galdiero, M., & Galdiero, S. (2016). Metal nanoparticles: The protective nanoshield against virus infection. Critical reviews in microbiology, 42(1), 46-56.
   42. Rokhina, E. V., Makarova, K., Golovina, E. A., Van As, H., & Virkutyte, J. (2010). Free radical reaction pathway, thermochemistry of peracetic acid homolysis, and its application for phenol degradation: spectroscopic study and quantum chemistry calculations. Environmental science & technology, 44(17), 6815-6821.
   43. Rovira, J., Roig, N., Nadal, M., Schuhmacher, M., & Domingo, J. L. (2016). Human health risks of formaldehyde indoor levels: an issue of concern. Journal of environmental science and health, part a, 51(4), 357-363.
   44. Rutala, W. A., & Weber, D. J. (2008). Guideline for disinfection and sterilization in healthcare facilities, 2008.
   45. Rutala, W. A., & Weber, D. J. (2015). Disinfection, sterilization, and control of hospital waste. Mandell, Douglas, and Bennett’s principles and practice of infectious diseases, 3294.
   46. Santos, I. D. A., Grosche, V. R., Bergamini, F. R. G., Sabino-Silva, R., & Jardim, A. C. G. (2020). Antivirals against coronaviruses: candidate drugs for SARS-CoV-2 treatment? Frontiers in microbiology, 1818.
   47. Saunders-Hastings, P., Crispo, J. A., Sikora, L., & Krewski, D. (2017). Effectiveness of personal protective measures in reducing pandemic influenza transmission: A systematic review and meta-analysis. Epidemics, 20, 1-20.
   48. Singh, D., Joshi, K., Samuel, A., Patra, J., & Mahindroo, N. (2020). Alcohol-based hand sanitisers as first line of defence against SARS-CoV-2: a review of biology, chemistry and formulations. Epidemiology & Infection, 148.
   49. Sirisattha, S., Momose, Y., Kitagawa, E., & Iwahashi, H. (2004). Toxicity of anionic detergents determined by Saccharomyces cerevisiae microarray analysis. Water Research, 38(1), 61-70.
   50. Sriwilaijaroen, N., Wilairat, P., Hiramatsu, H., Takahashi, T., Suzuki, T., Ito, M., … & Suzuki, Y. (2009). Mechanisms of the action of povidone-iodine against human and avian influenza A viruses: its effects on hemagglutination and sialidase activities. Virology journal, 6(1), 1-10.
   51. Straughn, J. C., & Barker, F. B. (1987). Avoiding glutaraldehyde irritation of the mucous membranes. Gastrointestinal endoscopy, 33(5), 396-397.
   52. Swenberg, J. A., Moeller, B. C., Lu, K., Rager, J. E., Fry, R. C., & Starr, T. B. (2013). Formaldehyde carcinogenicity research: 30 years and counting for mode of action, epidemiology, and cancer risk assessment. Toxicologic pathology, 41(2), 181-189.
   53. Szunerits, S., Barras, A., Khanal, M., Pagneux, Q., & Boukherroub, R. (2015). Nanostructures for the inhibition of viral infections. Molecules, 20(8), 14051-14081.
   54. Takigawa, T., & Endo, Y. (2006). Effects of glutaraldehyde exposure on human health. Journal of occupational health, 48(2), 75-87.
   55. Tian, X., Jiang, X., Welch, C., Croley, T. R., Wong, T. Y., Chen, C., … & Yin, J. J. (2018). Bactericidal effects of silver nanoparticles on lactobacilli and the underlying mechanism. ACS applied materials & interfaces, 10(10), 8443-8450.
   56. Tung, G., Macinga, D., Arbogast, J., & Jaykus, L. A. (2013). Efficacy of commonly used disinfectants for inactivation of human noroviruses and their surrogates. Journal of food protection, 76(7), 1210-1217.
   57. URL-1: https://www.genome.gov/genetics-glossary/Virus
   58. URL-2: https://news.arizona.edu/story/continuously-active-surface-disinfectants-may-provide-barrier-against-spread-viruses
   59. Vafadar, S., Shahdoust, M., Kalirad, A., Zakeri, P., & Sadeghi, M. (2021). Competitiveexclusionduringco-infection as a strategytopreventthespread of a virus: A computationalperspective. PloSone, 16(2), e0247200.
   60. Van Doremalen, N., Bushmaker, T., Morris, D. H., Holbrook, M. G., Gamble, A., Williamson, B. N., … & Munster, V. J. (2020). Aerosol and surface stability of SARS-CoV-2 as compared with SARS-CoV-1. New England journal of medicine, 382(16), 1564-1567.
   61. Velázquez, D., Zamberk, P., Suárez, R., & Lázaro, P. (2009). Allergic contact dermatitis to povidone-iodine. Contact Dermatitis, 60(6), 348-349.
   62. Verma, G., Mahajan, V., Shanker, V., Tegta, G., Jindal, N., & Minhas, S. (2011). Contact depigmentation following irritant contact dermatitis to chloroxylenol (Dettol). Indian Journal of Dermatology, Venereology and Leprology, 77(5), 612.
   63. Viana, R. B., da Silva, A. B., & Pimentel, A. S. (2012). Infrared spectroscopy of anionic, cationic, and zwitterionic surfactants. Advances in physical chemistry, 2012.
   64. Weng, D., Qi, H., Wu, T. T., Yan, M., Sun, R., & Lu, Y. (2012). Visible light powered self-disinfecting coatings for influenza viruses. Nanoscale, 4(9), 2870-2874.
   65. Wilton, T., Dunn, G., Eastwood, D., Minor, P. D., & Martin, J. (2014). Effect of formaldehyde inactivation on poliovirus. Journal of virology, 88(20), 11955-11964.
   66. World Bank. (2016). 2014–2015 West Africa Ebola crisis: impact update.
   67. Yeargin, T., Buckley, D., Fraser, A., & Jiang, X. (2016). The survival and inactivation of enteric viruses on soft surfaces: a systematic review of the literature. American journal of infection control, 44(11), 1365-1373.

Summary

To battle with the current COVID-19 pandemic, nanomaterials can be deemed excellent candidates against viral infections, particularly CoVs, because of their capability to penetrate cells easily, interact with viruses, and avoid viral genome reproduction. In addition, nanoparticles’ use permits the detection of contagious agents in tiny sample volumes instantly in a susceptible, precise, and quick format at lower costs than current in-use technologies. This advancement in early detection allows accurate and fast treatment.

Key words/phrases: nanomaterials, COVID-19, detection

1. Introduction

The continuing explosion of the novel coronavirus disease COVID-19 attracts worldwide considerations due to its prolonged incubation duration and substantial infectivity. The fast worldwide spread of the pandemic, driven by the harsh acute respiratory SARS-CoV-2, has created a pressing need for its diagnosis and treatment. As a result, many researchers have sought to find the most efficient and suitable methods to detect and treat the SARS-CoV-2. Real-time reverse-transcriptase polymerase chain reaction (RT-PCR) testing is presently used as one of the most reliable approaches to detect the new virus; however, this process is time-consuming, labour-intensive, and demands trained laboratory workers. Moreover, despite its high perceptiveness and specificity, false negatives are documented, particularly in non-nasopharyngeal swab samples that yield lower viral loads. Consequently, developing and utilising faster and more reliable methods seems crucial. In recent years, many attempts have been made to manufacture various nanomaterial-based biosensors to detect viruses and bacteria in clinical samples [27, 46].

A discreet way for diagnosing coronavirus disease COVID-19 is highly demanded to fight the existing and forthcoming global health hazards. Nanoparticles offer favourable implementation and significant prospects to function as a platform for quickly diagnosing viral infection with elevated sensitivity. Nanoparticles such as gold nanoparticles, magnetic nanoparticles, and graphene (G) were applied to detect SARS-CoV 2. They have been employed for molecular-based diagnosis processes and serological approaches. Nanoparticles enhanced explicitness and shortened the time demanded the diagnosis. They may be executed into tiny devices that encourage self-diagnosis at home or in places such as airports and shops. Nanoparticles-based methods can be employed for the analysis of virus-contaminated samples from a patient, surface, and air [1].

2. Current methods of detection of SARS-CoV-2

Conventional methods for the detection of SARS-CoV-2 are the reverse transcription polymerase chain reaction (RT-PCR), computed tomography (CT) scan and next-generation sequencing (NGS) [1, 26, 40] (Fig. 1). RT-PCR and chest CT imaging are the most typical diagnostic techniques in detecting COVID-19. In addition, several diagnostic methods such as clustered regularly interspaced short palindromic repeats (CRISPR)–specific high-sensitivity enzymatic reporter unlocking (SHERLOCK), reverse transcription loop-mediated isothermal amplification (RT-LAMP), enzyme-linked immunosorbent assay (ELISA), and sequencing are under development for enhanced detection of the virus in a minimum amount of time [1, 9]. RT-PCR has been acknowledged as the leader and most effective method for coronavirus detection [1, 26].

Figure 1. Conventional methods currently being used for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. (A) Reverse transcription polymerase chain reaction (RT-PCR). cDNA, complementary DNA. (B) Computed tomography scan. (C) Enzyme-linked immunosorbent assay.

2.1. Reverse Transcription Polymerase Chain Reaction (RT-PCR)

RT-PCR is vastly used for COVID-19 detection. It is based on cDNA synthesis from genomic RNA and is followed by amplification [26, 38]. Amplification of minimal amounts of viral genetic material in a mixture of other nucleic acid series is effectively done by RT-PCR. It is presently the standard gold technique of SARS-CoV-2 detection in upper respiratory tract samples. Several studies have used serum, ocular, and stool specimens for the RT-PCR-based detection. A contemporary method has used self-collected salivary samples as a non-invasive and secure technique for healthcare providers before doing RT-PCR. In this method, the reverse transcriptase first alters the RNA viral genome into DNA using a small sequence succession primer and the complementary DNA (cDNA) generation. Then, a fluorescent dye or a fluorescent-labelled sequence-specific DNA probe observes the amplification of DNA in real-time. Finally, a fluorescent or electrical signal displays the viral cDNA after successive amplification cycles [1, 9, 22].

Conventional RT-PCR procedures included one-step or two-step approaches. While one-step methods entangle a single primer-contained tube, the two-step procedure utilizes more than one tube to conduct the reactions. Still, it provides a more prudent and flexible track. Also, it can stock cDNA for the quantification of diverse targets with fewer starting materials. However, the standard method in detecting SARS-CoV-2 is the one-step approach since it is swifter, demands less sample handling, reduces bench time, and lowers pipetting errors [9, 43].

RT-PCR-based detection is also linked with false-negative results, which might be due to the low viral load in patients’ throats, improper handling of RNA samples, or lack of sufficient internal controls [8, 9, 16]. The main issue of RT-PCR is its low sensitivity to chest scans due to the inadequate number of viruses in the blood of RT-PCR. In addition, it is low sensitivity to chest scans due to the insufficient number of viruses in the blood or the laboratory kit’s inaccuracy [26].

2.2. Computed tomography scan (CT)

Another method for detecting and managing COVID-19 is the chest CT scan, which applies X-ray imaging of a patient’s chest at different angles. As per radiological reports, any uncommon features on the CT scan print may be due to COVID-19 infestation. Typically observed characteristics on a chest scan of a patient with COVID-19 are ground-glass opacification (GGO), especially on the peripheral and more inferior lobes, consolidations (rise in the opacity of the parenchyma, which results in coverage of the underlying vessels), crazy-paving pattern (GGO with intralobular and interlobular septal thickening), and linear opacities. The high-resolution CT could help detect GGOs in the early stages of infection [9, 26, 40].

CT sensitivity appears to be increased in patients with positive RT-PCR (86–97% in various case studies) and lower in patients with only constitutional and nonrespiratory symptoms (about 50%). Ultrasound has been used as a diagnostic tool in a minimal number of cases. Ultrasound has very low specificity, and, despite being influenced by factors such as disease stringency, patient weight and operator dexterity, sensitivity is estimated to be around 75%. However, ultrasound may play a role in observing the advancement of the disease via detection of interstitial lung disease features [26].

2.3. SHERLOCK

Further than RT-PCR and CT scans, various other detection techniques have also been developed for SARS-CoV- 2 detections. As it is described in Gupta at all SHERLOCK has been developed by Zhang et al. [45] “to detect RNA fragments of SARS-CoV- 2 with 10–100 copies/μl of the input. The basic principle of SHERLOCK-based diagnosis is CRISPR-based detection. This test can be performed in < 60 min, without requiring specific instruments. They chose two targets, the S gene and Orf1ab gene, from the SARS-CoV-2 genome. To minimize cross-reactivity with other respiratory virus genomes, they also selected specific guide sequences.” [9].

2.4. RT-LAMP

An optimized RT-LAMP-based detection method has more sensitivity than traditional PT-PCR methods and needs less time (Fig. 2). As a result, this process can be utilized to rapidly diagnose coronavirus and increase the testing capacity by 2–2.5-fold [9, 13].

Figure 2. Workflow comparison of our RT-LAMP assay relative to qRT-PCR for emergency cases (outpatients) and inpatients. Our RT-LAMP assay is 2–2.5 times faster than the qRT-PCR assays and can be shipped at room temperature.

Gupta et al. summarized the current techniques used to detect SARS-CoV-2 infection in Fig. 3 [9].

Figure 3. Current techniques used for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection.

3. Nanomaterials for SARS-CoV-2 detection

Nucleic acid testing by reverse transcription-polymerase chain reaction (RT-PCR) is the current method for detecting COVID-19 infection. Although RT-PCR is widely used to detect COVID-19, there are several issues [3, 14, 29, 35].

1. False-negative results;
2. Long response times / Time consuming;
3. RT-PCR is unable to detect asymptomatic patients, as it demands the existence of observable SARS-CoV-2 in collected samples;
4. Poor analytical sensitivity;
5. Labor intensive;
6. Healthcare centers in non-urban settings lack adequate PCR infrastructure to accommodate increased sample throughput;
7. Expensive;
8. The availability of RT-PCR kits and reagents cannot meet the augmented demand.

The present situation requires developing detection techniques that are rapid, cost-effective and easy to operate. To overcome the limitations of traditional methods, an improved multidisciplinary approach is needed. Nanomaterial based technological solutions present diverse possible applications to battle against the virus [10, 32].

3.1. Properties of nanomaterials

The unique characteristics of nanoparticles play a critical role in tackling pandemic and mitigating future outbreak. Nanoparticles show distinctive properties such as:

• tiny size;
• solubility;
• multifunctionality;
• target-ability;
• stimulus-responsive features;
• large surface area;
• surface adaptivity.

Therefore, they have been used widely for several applications in a variety of fields such as analytical chemistry, pharmacy, sensing/biosensing, biotechnology, nanomedicine, drug delivery, biological detection, gene transfer, optics, wound healing, energy-based applications, agriculture and environmental applications. Nanoparticles enhanced these applications by delivering increased performance with a significant prospect for enactment into a miniaturized machine, including wearable electronics. Hence, they show the tremendous potential to enhance the quality of life via regulating the viral spread via premature detection. Nanoparticles have at least one dimension in the nanometer range (1 nm =10^-9) (Fig. 4) [1, 15, 30, 36].

Figure 4. Scale of nanoparticles with some examples.

The nanoparticles’ high surface-to-volume ratio, high adsorption, quantum size effects and high reactivity allows for efficient interaction with sample analytes. Furthermore, they have exceptional multiplexing abilities, rendering them appropriate for incorporation into state-of-the-art technologies for virus detection. Moreover, nanoparticles offer ease of surface functionalization, suggesting that multiple ligands can be attached via covalent or noncovalent bonding, which further improves selectivity and particularity and decreases detection time. In addition, nanomaterials can also be used as labels for improving the signals, which helps detect very low-magnitude signals [32].

3.2. Categories of nanomaterials

A variety of nanomaterials for virus detection and tracking have been created, contributing to the illumination of virus infection mechanisms, such as [11]:

• Metallic nanoparticles, e.g., gold nanoparticles (Au NPs), silver nanoparticles (Ag NPs);
• Metal oxide nanoparticles, e.g., iron oxide magnetic nanoparticle (Fe₃O₄NPs);
• Carbon nanomaterials including 0-dimensional (0D, e.g., fullerenes (C60), carbon dots (C-dots)), 1D (carbon nanotunes (CNTs), 2D (e.g., graphene (G), graphene oxide (GO), and 3D (e.g., graphite);
• Quantum dots (QDs): CdS QDs, CdTe QDs, carbon QDs;
• Porous materials: metal–organic frameworks (MOFs), covalent organic frameworks (COFs);
• Polymers: natural polymers (e.g., chitosan, cellulose), and synthetic (e.g., polythiophene, polypyrrole);
• Lipid nanoparticles (LNPs): triglycerides, fatty acids, steroids, and waxes.

Figure 5. Schematic diagrams showing different examples of nanomaterial-based COV detection methods. (a) Fluorescent Zr QDs and magnetic nanoparticles are conjugated with antibodies that specifically bind to COV. In the presence of COV, a magnetic fluorescent complex is formed, which is isolated magnetically and detected by fluorescence measurements. (b) Nanotraps are used to concentrate COV and improve their stability, hence facilitating their detection. (c) Reverse transcription PCR is carried out in the presence nanoparticles, improving the efficacy of the polymerase chain reaction, and resulting in a better detection sensitivity of this method. (d) COV detection method, which is based on the interactions between complementary DNA originating from COV and acpnPNA probe at the surface of Ag NP, which results in a separation between Ag NPs, and a yellow color associated with the luminescence of well dispersed Ag NPs, further revealing COV presence.

Figure 6. Different nanoparticles.

Nanomaterials can be utilized in a variety of roles for COVID-19. Rasmi et all summarize the functions and primary role of nanomaterials in the below table (Fig. 7) [30].

Figure 7. Summary of the role on nanomaterials for COVID-19.

3.2.1. Gold NPs (Au NPs)

Gold nanoparticles (AuNPs) have been increasingly employed in SARS-CoV-2 detection platforms due to their remarkable optical properties such as increased extinction coefficients and tunable localized surface plasmon resonance (LSPR), allowing a separate color readout with a simple equipment or the naked eye. For SARS-CoV-2 antibody detection (IgG, IgM, or IgA), AuNP- and fluorescent nanoparticle-based assays have been suggested [17].

Gold nanoparticle (AuNPs) is one of the most typically utilised nanomaterials for quick diagnostics. The gold nanoparticle was employed to detect target viruses’ double-stranded DNA (dsDNA). Specifically, single-stranded DNA (ssDNA) or ssRNA can interact with citrate ions on the AuNP surface. Adding salt to the solution can stabilise the particles and change colour. Furthermore, a simple colourimetric hybridisation assay was applied to detect dsDNA of SARS-CoV based, developed from ssRNA. This assay can see the target at 4.3 nM in 10 min without needing any cumbersome device [18, 30].

Colour change approach

Another analysis introduced a method to visually detect the COVID-19 virus without sophisticated tools. Colourimetric detection was designed using thiol-modified antisense oligonucleotides (ASOs)-coated AuNPs explicitly intended for the. Thiol-modified ASO-cap AuNPs were selectively aggregated in the existence of the SARS-CoV-2 target RNA sequence and delivered a modification in its surface plasmon resonance. The result can be observed in 10 min with a detection limit of 0.18 ng/μL [23, 30].

Figure 8: Schematic representation for the selective naked-eye detection of SARS-CoV-2 RNA mediated by the suitably designed ASO-Capped AuNPs.

Effective detection of COVID-19 was developed by immobilizing proteins on the surface of Au using the Au-binding polypeptides. Using the improved green fluorescent protein, SARS-CoV-E protein, and core streptavidin of Streptomyces avidinii as examples, the Au-binding polypeptide fusion protein was immobilized explicitly on AuNP, and the protein nanopatterns on the bare Au surface were demonstrated. These complexes interact with the antibody, resulting in absorbance and colour change [25, 30].

 Non-invasive approach

The detection of COVID-19 using non-invasive approaches has been proposed from exhaled breath using an AuNP-based sensor. The sensor consisted of different AuNP attached to organic ligands and inorganic nanomaterial film. The inorganic film is accountable for electrical conductivity. Therefore, when exposed to the volatile organic compounds (VOCs) from exhaled breath, the organic film reacts with the VOCs, resulting in the inorganic film swelling or shrinkage and the changes in electrical conductivity. Therefore, this non-invasive sensor could potentially be used to rapidly screen COVID-19 [30, 32].

Electrochemical hybridization approach

An AuNP-based electrochemical hybridization method was defined using a gene-sensor consisting of a thiolated-DNA probe-immobilized on the AuNPs carbon electrode to hybridize biotinylated-target DNA. An electrochemical chip was presented via a carbon electrode composed of AuNP array. The coronavirus protein was bound on an AuNP-electrode, and both coronavirus protein and free viruses compete for binding sites in the existence of antibodies. There was an excellent linear reaction between the sensor response and the concentrations of coronavirus ranging from 0.001 to 100 ng mL−1. The assay achieved the detection limit of as low as 1.0 pg mL−1. The method was single-step, sensitive and precise (Fig. 9) [17, 30].

Figure 9. COV immunosensor array chip (a), The immunosensor fabrication steps (b), the detection process of the competitive immunosensor for the virus (c).

Immunochromatogrphy approach

A lateral flow assay for the rapid detection of IgM against COVID-19 was designed through the indirect immunochromatography approach. The SARS-CoV-2 nucleoprotein (SARS-CoV-2 NP) was coated on an analytical membrane for target capturing, and anti-human IgM was conjugated to AuNP, operating as a detection reporter. AuNP-LF analysis exhibited remarkable selectivity in the IgM detection without interference from other viruses. Each assay only needs 10–20 μL serum, and the result can be received within 15 min [12, 30].

3.2.2. Quantum Dots (QDs)

Quantum dots (QDs) are multifaceted mechanisms that can battle against COVID-19 virus. Quantum dots (QDs), likewise known as “semiconductor nanomaterials,” play a vital role in COVID-19 detection. QDs have been recognised as a new fluorescent probe for molecular imaging. The size of the QDs varies from 1 to 10 nm. The exceptional characteristics of QDs, including great optical and semiconductor properties, exemplified photo- stability, high quantum yield, and narrow emission spectrum with adjustable size, have made them a significant candidate to operate as a fluorescent label. Because of these outstanding properties, QDs can be considered a great agent to fight against viral infections. Moreover, incorporating possible biocompatible carriers can aid interdisciplinary study and permit clinical approaches to fighting the virus. Owing to their superior properties, QDs are now dominant imaging probes (chemosensors and biosensors) for sensing [21, 30].

Figure 10. Description of Operation Principle of the AuNP=LF Strip.

QDs are employed due to their traceability under a specific wavelength of light. In addition, QDs can be tunable into the desired size (1–10 nm) and shape that efficiently targets/penetrates SARS-CoV-2 with a size span between 60 and 140 nm. Furthermore, the positive surface charge of carbon-based QDs could be utilised to sequester/disable the S protein of SARS-CoV-2. In addition, cationic surface charges exhibited by QDs interact with the negative RNA strand of the virus, directing to the production of reactive oxygen species within SARS-CoV-2 [21].

A QD-conjugated RNA aptamer-based chip was introduced for sensitive and rapid detection of SARS-CoV N protein with a detection limit of 0.1 pg mL−1 on a developed chip. The QD- conjugated RNA aptamer can bind to the SARS-CoV N protein immobilized on the chip, creating an optical signal. The use of fluorescent-based QDs may help researchers in designing an easy, sensitive and rapid diagnostic tool for COVID-19 [30, 31].

Figure 11. Schematic representation of the actions exerted by QDs on SARS-CoV-2. QD, quantum dot; S protein, spike protein; SARS-CoV-2, severe acute respiratory syndrome coronavirus type 2

Carbon quantum dots

Carbon quantum dots (CQDs) can be utilized to sense microbes, biomolecules and infections. In addition, they can be used as biocompatible inactivation systems for pathogenic human coronavirus infections as dominant imaging probes (chemosensors and biosensors) with antiviral activity. The CQDs are about 10 nm with high solubility in water, were fabricated via hydrothermal carbonization of carbon precursors.  Some innovative approaches for detecting coronaviruses have focused on the application of CQDs. In one method, the antiviral activities of seven types of CQDs were used to cure human coronavirus contagions. Different kinds of CQDs by hydrothermal carbonization and conjugation of boronic acid were used. It was disclosed that the virus inhibition is possibly owing to the interchange between CQDs operating groups with entry receptors of the virus [10].

Zirconium quantum dots (Zr QDs)

Zirconium, due to its properties such as mechanical stability, thermal resilience and UV light capture, has been utilised in many biomedical areas as a nontoxic transition. Besides, the nanosize of Zr has unique physical and chemical aspects due to its high surface area and the captivity of electronic states in comparison with its bulk regime [10].

In general, the employment of QDs against coronavirus is one of the most suitable choices due to its outstanding curative efficiency. Moreover, QDs can be employed as a robust imaging probe and sensor in diagnosis and prognostic. In addition, the drugs can be coated on the surface of QDs to target COVID-19. Nevertheless, caution should be exerted to avoid renal filtration and additional side consequences.

3.2.3. Carbon-Based Nanomaterials

Carbon materials are functional in every aspect of our everyday life because they are plentiful and weightless fabrics that can be used for a combination of applications. Carbon-based nanomaterials can be categorised based on their dimensionalities (D) as zero-D (0D) such as carbon dots, one-D (1D) as in CNTs, and two-D (2D) in graphene nanostructures. These nanomaterials maintain more exhaustive operational temperature, perceptiveness and vaster dynamic transducing signal range even in extreme environmental situations [24].

Carbon-based nanomaterials have been extensively employed in developing a platform for COVID-19 detection. Their outstanding physicochemical and antiviral characteristics suggest that nanomaterials play a vital role against COVID-19. These nanomaterials, including graphene and graphene oxide, carbon quantum dot, carbon nanotube, and fullerene with excellent properties mainly sensing, antiviral and antimicrobial properties, are superior options with potential applications against COVID-19 in biosensor for diagnosis, antiviral coating, airborne virus filtration, facemask, and drug delivery [10, 24, 30].

 Graphene and graphene oxide

The antimicrobial and antiviral properties of the nanomaterial graphene and graphene oxide has two dimensions that captured a lot of awareness and examination. First, graphene-based field-effect transistors (FET) as potable sensors have been developed to analyze COVID-19 viral load in clinical nasopharyngeal samples, utilizing unique antibodies against its spike protein. The fabricated FET sensors can catch the SARS- CoV-2 spike protein in phosphate-buffered saline and 100 fg mL− 1 medical transfer system, at the level of 1fgmL− 1 concentration and limit of detection ~1.6 × 101 pfu mL-1 and ~2.42 × 10² pfu mL-1 for the cultured sample and medical test, respectively. This sensor shows is highly sensitive to screening and diagnosis of novel coronavirus disease 2019 without any sample pretreatment. The existence of graphene leads to an improved signal-to-noise ratio [10].

Figure 12. Schematic illustration of certain allotropes of carbon nanomaterials for nanobiosensor development.

Carbon nanotubes

Carbon nanotubes (CNTs) were widely applied for biology and biomedical sciences due to the following properties and open new horizons for scientific development [10]:

• 10− 100 nm dimensions;
• antiviral and antimicrobial activity;
• good light-heat conversion efficiency;
• large surface volume ratio;
• slight density;
• small pore size;
• flexibility;
• resistance to acids and bases;
• great mechanical strength;
• ability to create reactive oxygen species;
• resistance to respiratory droplet;
• biological compatibility with several drugs.

Carbon dots were found in 2004, and they usually have photoluminescence, bio-compatibility, and high resilience, predisposing them to diverse applications, including biosensing and bio-imaging. Carbon nanotubes (CNTs), graphene, and carbon dots (CDs) can be classified as zero-(0D), one-(1D), and two-(2D) dimensional carbon nanomaterials [10, 30].

Figure 13. Future prospects of CNTs in the prevention, diagnosis and treatment of SARS-CoV-2 infection.

High storage space, high surface area, high biocompatibility, excellent permeability of biological barriers, reasonable bio absorption rate, multi-energy surface/tube chemical functional group capability, and targeted biomolecule modification potency are excellent properties of CNTs that provide novel suggestions encountering COVID-19. Similarly, CNTs are used as diagnosis systems, filtering and virus inactivation agent [10].

A CNT size-tunable enrichment microdevice (CNT-STEM) was designed to enrich and concentrate viruses from raw samples. CNTs can be utilised to diagnose respiratory viruses, including SARS-CoV-1 and SARS-CoV-2. The channel sidewall in the microdevice was manufactured by nitrogen-doped multiwalled CNTs, where the intratubular space between CNTs is optimised to correspond to the size of different viruses. By using this device, the avian influenza virus strain was determined. The CNT-STEM significantly improves virus isolation rates and detection perceptiveness. Because of the ease and trustworthiness of this technique, it can be adjusted to detect SARS-CoV-2 RNA or proteins [30, 34].

Figure 14. The working principle of virus enrichment and concertation from field samples. (A) A filed sample containing viruses (purple spheres) is collected by a cotton or as a tissue sample. (B) The supernatant of the field sample flows through the CNT-STEM, and the viruses are enriched within the device. Inset (right): Illustration of size-based virus enrichment by the aligned N-MWCNTs. Inset (bottom right): SEM image (scale bar, 100 nm) of the H5N2 AIV virions trapped inside the aligned N-MWCNTs. Inset (bottom left): Dark-field TEM image (scale bar, 100 nm) of enriched H5N2 AIV after the aligned N-MWCNTs structures were retrieved from the CNT-STEM.

Nanodiamonds

Due to its high stability and low cytotoxicity, nanodiamonds have received significant attention for COVID-19 diagnostics. Therefore, fluorescent nanodiamonds were utilised for COVID-19 lateral flow immunoassay as an ultrasensitive label. These nanodiamonds were immobilised on the test line, and a microwave field was used to selectively split their fluorescence signal from the background signal, which significantly enhanced the detection sensitivity. This assay was 105 more sensitive than the traditional gold-nanoparticle-based lateral flow assay. Carbon-based nanomaterials can be employed as an antiviral therapeutic agent for COVID-19 [30].

3.2.4. Magnetic NPs (MNPs)

Before detection, magnetic NPs (MNPs) are typically used to detect SARS-CoV-2, host antibody response, and nucleic acid separation. It was shown that silica-coated iron oxide NPs have a significant association with SARS-CoV-2 RNA, as the cracked open the virus. The magnet was utilised to isolate the RNA coated NPs from the sample solution. This method is economical and straightforward, enabling to extract RNA from patient samples efficiently [15, 30].

Precise detection demands efficient extraction and separation of nucleic acids from samples, allowing target purification. Superparamagnetic nanoparticles (80 nm) conjugated with a complementary probe to the target sequence SARS-CoVs was employed in one study. Utilizing a magnet, the functionalized superparamagnetic nanoparticles can extract target cDNA from specimens. The amount of extracted DNA was boosted through PCR which was tested employing silica-coated fluorescence nanoparticles conjugated with a complementary sequence. Silica-coated fluorescence NPs produce fluorescence signals directly correlated to the concentration of the target cDNA [30].

The surface functionalized MNP’s adsorbs the nucleic acid from the lysis solution and are fast separated from most of the contaminations with the assistance of an external magnetic field. Following this short procedure, the nucleic acid can be additionally separated from the functionalized surface of MNP’s by the desorption process in the eluent. However, although this process is much easier and shorter than traditional procedures, MNP’s assisted extraction process still consists of several stages, which is inadequate for practical detection. The zinc ferrite nanoparticles were synthesized by discharge, and the nanoparticle surfaces were functionalized with silica and carboxyl-modified polyvinyl alcohol. This platform shows the capability to automatically remove the viral RNA from diverse sample types. It decreases the functional steps, which presents a significant prospect for COVID-19 molecular-level diagnostics [30, 34].

A more straightforward and contemporary MNP’s assisted RNA-extraction protocol is suggested for possible extraction and RT-PCR-based diagnosis of COVID-19. The MNP’s of zinc ferrite (ZNF) were manufactured by the cost-efficient sol-gel auto-combustion route, and after that, its surface was functionalized with carboxyl containing polymers (CPoly). Among the magnetic materials, zinc ferrite was selected due to its high chemical resilience, smooth magnetic behaviour, uncomplicated preparation and biocompatible character. Due to the robust interface among nucleic acids and carboxyl groups, the surface-functionalized MNP’s promote fast and possible viral RNA’s adsorption. This cost-effective and straightforward technique may provide a qualified alternate for conventional methods [34].

Figure 15. Schematic procedure for surface functionalised MNP’s assisted RNA-extraction protocol.

In addition, there is a one-step nucleic acid extraction procedure that particularly ties viral RNA using polycarboxyl-functionalized amino group-modified MNPs (PC-coated NH2-MNP). Nucleic acids were gathered using a magnetic field, and then they were released from the MNPs by adding wash buffer. By catching COVID-19-pseudoviruses, polycarboxyl-functionalized MNPs exhibited perfect absorption and paramagnetic properties via fast capture (30 s magnetic capture) of targets [30, 46].

Figure 16: A schematic representation of the pcMNP-based viral RNA extraction method.

3.2.5. Nanozymes

Nanozymes are unnatural enzymes composed of nanomaterials with similar efficiency as natural enzymes. In addition, nanozymes have superior catalytic activities, quick response and self-assembly capability, extensively employed for disease diagnostics and treatment. A novel nanozyme-based chemiluminescence paper assay for rapid and acute detection of SARS-CoV-2 spike antigen combines nanozyme and enzymatic chemiluminescence immunoassay with the lateral flow strip created.

Figure 17: (A) Schematic illustration of the nano- zyme chemiluminescence paper test for SARS-CoV-2 S-RBD antigen. Recognition, separation and cata- lytic amplification by nanozyme probes.

Conventional chemiluminescence immunodiagnosis utilises natural proteases such as HRP or alkaline phosphatase that reveal constraints such as scarce storage resilience, complicated preparation methods and high cost. The suggested biosensor employed peroxidase-mimic Co-Fe@hemin nanozyme rather than natural horseradish peroxidase (HRP) that could greatly boost the chemiluminescent signal reaching the detection limit of 0.1 ng/mL. The Co-Fe@hemin nanozyme was demonstrated to have better stability for temperature and acerbity or alkalinity as compared to HRP, which can be stably held at room temperature. This testing can be conducted within 16 min, much quicker compared to the usual 1-2 h needed for currently employed nucleic acid tests. Furthermore, signal detection is possible using the camera of a typical smartphone. Components for nanozyme synthesis are easy and readily obtainable, considerably reducing the overall expense [20, 30].

3.2.6. Metal-Organic Framework

Porous nanomaterials can be used for the detection of different pathogens. The analyte, pathogen, does not require to be absorbed by the porous nanomaterials; however, the pathogen needs to interact with the surface of the MOF that different NPs modify. By this interaction, additional Off–On or On–Off optical mechanisms can be optimized to detect the pathogen, and in this case, various optical active components can be employed as quenchers or activators. In the issue of SARS-CoV-2, there is no necessity to detect the same genetic material and genetic sequence on the surface of the mask or even clothes due to the significant discrepancies between the concentrations of the SARS-CoV-2 with others. Instead, using a fingerprint fluorescence pattern, which has been optimized before, the same range of attention of SARS-CoV-2 on the contact surface of the gas and solid phases can be measured by optical changes. Moreover, if the MOF based biosensors successfully work for HIV-1, H1N1, ZIKA, and other pathogens detections with considerable precision and LOD, then the morphology and optical-based biosensor for detection SARS-CoV-2 should function as well. [26].

4. Challenges and Limitations of nanomaterials

Nanomaterials can be significantly valuable for biomedical applications. However, they have some constraints, such as toxicity. One of the significant challenges is to ensure the safe use of nanomaterials. Another challenge is that the behaviour of nanomaterials in the body can change when they reach blood circulation due to protein corona formation. Thus, faithful in vivo models are required to sufficiently comprehend the toxicokinetic behaviour of the nanoparticles in the body, particularly for long-term exposure.

Another problem is the absence of standardized protocols for nanomaterials’ physicochemical and biological definition and the lack of a universally agreed-upon definition of a nanomaterial. Capacity for large-scale manufacturing is another hurdle that needs to be overcome for the broader commercialization of nano-based formulations. Due to the multi-faceted interchanges between nanomaterials and biological systems, it is very demanding to foresee the behaviour of these materials under physiological conditions. Once within the body, the nanoparticles reach the blood circulation, a complex matrix containing ions, small molecules, proteins and cells. [37].

---

Test LO 3.1

---

References

1. Abdelhamid H., and Badr G., (2021). Nanobiotechnology as a platform for the diagnosis of COVID‐19: a review. Nanotechnology for Environmental Engineering 6:19 https://doi.org/10.1007/s41204-021-00109-0
2. Abraham AM., Kannangai R., and Sridharan G., (2008). Nanotechnology: A new frontier in virus detection in clinical practice. Indian Journal of Medical Microbiology, 26(4): 297-301
3. Alhalaili B., Popescu I.N., Kamoun O., Alzubi F., Alawadhia S., Vidu R. (2020) Nanobiosensors for the detection of novel coronavirus 2019-nCoV and other pandemic/Epidemic Respiratory viruses: A review. Sensors 2020, 20, 6591. [CrossRef]
4. Alimardani V., Abolmaali S., Tamaddon A., (2021). Recent Advances on Nanotechnology-Based Strategies for Prevention, Diagnosis, and Treatment of Coronavirus Infections. Journal of Nanomaterials Volume 2021, Article ID 9495126, 20 pages https://doi.org/10.1155/2021/9495126
5. Alphandery E., (2020). The Potential of Various Nanotechnologies for Coronavirus Diagnosis/Treatment Highlighted through a Literature Analysis. Bioconjugate Chem. 2020, 31, 1873−1882. https://dx.doi.org/10.1021/acs.bioconjchem.0c00287
6. Bendavid E., Mulaney B., Sood N., Shah S., Ling E., Bromley-Dulfano R. (2020). COVID-19 Antibody Seroprevalence in Santa Clara County, California. MedRxiv
7. Carter L. J., Garner L. V., Smoot J. W., Li Y., Zhou Q., Saveson C. J., et al. (2020). Assay techniques and test development for COVID-19 diagnosis. ACS Cent. Sci. 6, 591–605. doi: 10.1021/acscentsci.0c00501
8. Di Paolo M., Iacovelli A., Olmati F., Menichini I., Oliva A., Carnevalini M., et al. (2020). False-negative RT-PCR in SARS-CoV-2 disease: experience from an Italian COVID-19 unit. ERJ Open Res. 6, 324–2020. doi: 10.1183/23120541.00324-2020
9. Eftekhari A., Alipour M., Chodari L., Maleki Dizaj S., Ardalan M., Samiei M., Sharifi S., Zununi Vahed S., Huseynova I., Khalilov R. et al. (2021). A Comprehensive Review of Detection Methods for SARS-CoV-2. Microorganisms, 9, 232. https://doi.org/10.3390/ microorganisms9020232
10. Ghaemi F., Amiri A., Bajuri M., Yuhana N., Ferrara M., (2021), Role of different types of nanomaterials against diagnosis, prevention and therapy of COVID-19. Sustainable Cities and Society 72 (2021) 103046
11. Gupta R., Sagar P., Priyadarshi N., Kaul S., Sandhir R., Rishi V. and Singhal N.K. (2020). Nanotechnology-Based Approaches for the Detection of SARS-CoV-2. Front. Nanotechnol. 2:589832. doi: 10.3389/fnano.2020.589832
12. Huang C., Wen T., Shi F.J., Zeng X.Y., Jiao Y.J., (2020). Rapid Detection of IgM Antibodies against the SARS-CoV-2 Virus via Colloidal Gold Nanoparticle-Based Lateral-Flow Assay. ACS Omega 2020, 5, 12550–12556.
13. Jiang M., Pan W., Arasthfer A., Fang W., Ling L., Fang, H., et al. (2020). Development and validation of a rapid, single-step reverse transcriptase loop- mediated isothermal amplification (RT-LAMP) system potentially to be used for reliable and high-throughput screening of COVID-19. Front. Cell. Infect. Microbiol. 10:331. doi: 10.3389/fcimb.2020.00331
14. Jindal S., and Gopinath P., (2020) Nano Ex. 1 022003
15. Kang J., Tahir A., Wang H., Chang J. (2021). Applications of nanotechnology in virus detection, tracking, and infection mechanisms. WIREs Nanomed Nanobiotechnol. 2021;13:e1700. https://doi.org/10.1002/ wnan.1700
16. Kelly J. C., Dombrowksi M., O’Neil-Callahan M., Kernberg A. S., Frolova A. I., and Stout M. J. (2020). False-negative testing for severe acute respiratory syndrome coronavirus 2: consideration in obstetrical care. Am. J. Obstet. Gynecol. 2(Suppl. 3):100130. doi: 10.1016/j.ajogmf.2020.100130
17. Layqah L.A., Eissa S. (2019). An Electrochemical Immunosensor for the Corona Virus Associated with the Middle East Respiratory Syndrome Using an Array of Gold Nanoparticle-Modified Carbon Electrodes. Microchimica Acta 2019, 186, 224.
18. Lew T., Aung K., Ow S., Amrun S., Sutarlie L., Ng L., and Su X., (2021). Epitope-Functionalized Gold Nanoparticles for Rapid and Selective Detection of SARS-CoV‐2 IgG Antibodies. ACS NANO, https://doi.org/10.1021/acsnano.1c04091
19. Li H., and Rothberg L., (2004). Colorimetric detection of DNA sequences based on electrostatic interactions with unmodified gold nanoparticles. Proc. Natl. Acad. Sci. USA 2004, 101, 14036–14039
20. Liu D., Ju C., Han C., Shi R., Chen X., Duan D., Yan J., Yan, X. (2021). Nanozyme Chemiluminescence Paper Test for Rapid and Sensitive Detection of SARS-CoV-2 Antigen. Biosens. Bioelectron. 2021, 173, 112817
21. Manivannan S., Ponnuchamy K. (2020). Quantum Dots as a Promising Agent to Combat COVID-19. Appl. Organomet. Chem. 2020, 34, e5887
22. Mizumoto K., Kagaya K., Zarebski A., Chowell G.J.E. (2020). Estimating the asymptomatic proportion of coronavirus disease 2019 (COVID-19) cases on board the Diamond Princess cruise ship, Yokohama, Japan, 2020. Eurosurveillance , 25, 2000180
23. Moitra P., Alafeef M., Dighe K., Frieman M.B., Pan D. (2020). Selective Naked-Eye Detection of SARS-CoV-2 Mediated by N Gene Targeted Antisense Oligonucleotide Capped Plasmonic Nanoparticles. ACS Nano, 14, 7617–7627.
24. Ozmen E., Kartal E., Turan M., Yazicioglu A., Hiazi J., and Qureshi A., (2021). Graphene and carbon nanotubes interfaced electrochemical nanobiosensors for the detection of SARS-CoV-2 (COVID-19) and other respiratory viral infections: A review. Materials Science & Engineering C 129 (2021) 112356
25. Park T.J., Lee S.Y., Lee S.J., Park J.P., Yang K.S., Lee K.-B., Ko S., Park J.B., Kim T., Kim S.K., (2006). Protein Nanopatterns and Biosensors Using Gold Binding Polypeptide as a Fusion Partner. Anal. Chem. 2006, 78, 7197–7205
26. Pascarella G., Strumia A., Piliego C., Bruno F., Del Buono R., Costa F., et al. (2020). COVID-19 diagnosis and management: a comprehensive review. J. Intern. Med. 288, 192–206. doi: 10.1111/joim.13091
27. Pishva P., and Yuce M., (2021). Nanomaterials to tackle the COVID-19 pandemic. Emergent Materials https://doi.org/10.1007/s42247-021-00184-8
28. Rabiee N., Bagherzadeh M., Ghasemi A., Zare H., Ahmadi S., Fatahi Y., Dinarvand R., Rabiee M., Ramakrishna S., Shokouhimehr M., Varma R.S. (2020). Point-of-Use Rapid Detection of SARS-CoV-2: Nanotechnology-Enabled Solutions for the COVID-19 Pandemic. Int. J. Mol. Sci. 2020, 21, 5126. https://doi.org/10.3390/ijms21145126
29. Rai M., Bonde S., Yadav A., Bhowmik A., Rathod S., Ingle P., Gade A. (2021). Nanotechnology as a Shield against COVID-19: Current Advancement and Limitations. Viruses 2021,13,1224. https:// doi.org/10.3390/v13071224
30. Rasmi Y., Saloua K.S., Nemati M., Choi J.R. (2021). Recent Progress in Nanotechnology for COVID-19 Prevention, Diagnostics and Treatment. Nanomaterials 2021, 11, 1788. https://doi.org/10.3390/ nano11071788
31. Roh C., Jo S.K., (2011). Quantitative and Sensitive Detection of SARS Coronavirus Nucleocapsid Protein Using Quantum Dots- Conjugated RNA Aptamer on Chip. J. Chem. Technol. Biotechnol. 2011, 86, 1475–1479.
32. Shan B., Broza Y.Y., Li W., Wang, Y., Wu S., Liu Z., Wang J., Gui S., Wang L., Zhang Z., et al. (2020). Multiplexed Nanomaterial-Based Sensor Array for Detection of COVID-19 in Exhaled Breath. ACS Nano 2020, 14, 12125–12132.
33. Singh P., Singh D., Sa P., Mohapatra P., Khuntia A., and Sahoo S. (2021). Insights from nanotechnology in COVID-19: prevention, detection, therapy and immunomodulation. Nanomedicine (Lond.) (2021) 16(14), 1219–1235
34. Somvanshi S., Kharat P., Saraf T., Somwanshi S., Shejul S., and Jadhav K. (2021). Multifunctional nano-magnetic particles assisted viral RNA-extraction protocol for potential detection of COVID-19, Materials Research Innovations, 25:3, 169-174, DOI: 10.1080/14328917.2020.1769350
35. Talebian S., Wallace g., Schroeder A., Stellacci F., and Conde J. (2020). Nature Nanotechnology, Vol 15, August 2020, 618-624, www.nature.com/naturenanotechnology
36. Tavakol S., Zahmatkeshan M., Mohammadinejad R., Mehrzadi S., Joghataei M., Alavijeh M., Seifalian A. (2021). The role of nanotechnology in current COVID-19 outbreak. Heliyon 7 (2021) e06841
37. Tharayil A., Rajakumari R., Chirayil C., Thomas S. and Kalarikkal N., (2021) A short review on nanotechnology interventions against COVID-19. Emergent Materials (2021). 4:131–141  https://doi.org/10.1007/s42247-021-00163-z
38. To K.K.-W., Tsang O.T.-Y., Yip C.C.-Y., Chan K.-H., Wu T.-C., Chan J.M.-C., Leung W.-S., Chik T.S.-H., Choi C.Y.-C., Kandamby D.H. et al. (2020). Consistent Detection of 2019 Novel Coronavirus in Saliva. Clin. Infect. Dis
39. Toledo G., Toledo V., Lanfredi A., Escote M., Champi A., Da silva M., Nantes-Cardoso I., (2020), Promising Nanostructured Materials against Enveloped Virus. An Acad Bras Cienc (2020) 92(4): e20200718 DOI 10.1590/0001-3765202020200718
40. Udugama B., Kadhiresan P., Kozlowski H. N., Malekjahani A., Osborne M., Li V. Y. C., et al. (2020). Diagnosing COVID-19: the disease and tools for detection. ACS Nano 14, 3822–3835. doi: 10.1021/acsnano.0c02624
41. Varghese R., Salvi S., Sood P., Karsiya J., Kumar D. (2021). Carbon nanotubes in COVID-19: A critical review and prospects. Colloid and Interface Science Communications 46 (2022) 100544
42. Waller J. V., Kaur P., Tucker A., Lin, K. K., Diaz M. J., Henry T. S., et al. (2020). Diagnostic tools for coronavirus disease (COVID-19): comparing CT and RT-PCR viral nucleic acid testing. Am. J. Roentgenol. 215, 834–838. doi: 10.2214/AJR.20.23418
43. Wong M.L., Medrano J.F. (2005). Real-time PCR for mRNA quantitation. Biotechniques 39, 75–85.
44. Yeh Y.-T., Tang Y., Sebastian A., Dasgupta A., Perea-Lopez N., Albert I., Lu H., Terrones M., Zheng, S.-Y., (2016). Tunable and Label-Free Virus Enrichment for Ultrasensitive Virus Detection Using Carbon Nanotube Arrays. Sci. Adv. 2016, 2, e1601026.
45. Zhang F., Abudayyeh O. O., Gootenberg J. S., Sciences C., and Mathers L. (2020). A Protocol for Detection of COVID-19 Using CRISPR Diagnostics.
46. Zhao Z., Cui H., Song W., Ru X., Zhou W., Yu X. (2020). A Simple Magnetic Nanoparticles-Based Viral RNA Extraction Method for Efficient Detection of SARS-CoV-2. bioRxiv 2020.

Summary

The advances of nanotechnology are of significant importance in the diagnosis of COVID-19. Protection and diagnosis are essential for controlling the spread of infection. Nanotechnology offers novel techniques for rapid diagnosis, early-stage infection detection, and identification of COVID-19. Due to their smaller size and larger surface area, nanotechnology products can detect the disease with high precision. As the symptoms of COVID-19 are very comparable to those of other respiratory diseases, it is essential to have precise, sensitive and fast diagnostic tools to detect the infection at an early stage.

Key words/phrases: nanotechnology, COVID-19, diagnosis

1. Introduction

The coronavirus disease 2019 (COVID-19) induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global health issue that the WHO announced a pandemic. COVID- 19 has resulted in a worldwide lockdown and endangered the global economy. SARS-CoV-2 has spread fast worldwide, generating a global pandemic outnumbering. This virus can be transferred human-to-human via droplets and tight contact, and people of all ages are exposed to this virus. The COVID-19 outbreak put international pressure on modern societies, especially the infrastructure linked to health care. Therefore, diagnostic tests special to this disease are urgently required to ensure positive cases, screen patients and execute viral surveillance. Diagnostics may play an influential role in preventing COVID-19, permitting quick execution of management actions that limit the spread by detecting and isolating cases and via contact tracing. Consequently, the world is encountering a new challenge: to create ultra-rapid, ultra-sensitive devices, and nanoscale analytical tools, or sensing systems (e.g., nanobiosensors) that are highly efficacious at detecting the 2019 novel coronavirus (COVID-19) or severe acute respiratory syndrome (SARS) [3, 10, 17].

With the advancements in nanotechnology, their exceptional properties, including their capability to strengthen signal, can be employed for the development of nanobiosensors and nanoimaging processes that can be utilised for early-stage detection along with other diagnostic tools. nanotechnology is being thoroughly examined for its prospect in the development of , diagnostic techniques, therapeutics, vaccines and strategies to ease the healthcare burden [10].

2. Current laboratory methods for diagnosis of SARS-CoV-2

The diagnosis of COVID-19 relies on the analysis of the patient’s reaction due to the disease or the study of virus contents, e.g., RNA or their protein. The patient’s temperature (boosted temperature), feeling exhausted, and difficulty breathing suggest infection. Nevertheless, these symptoms are lack particularity and may be observed due to the infection with other pathogens. The patient’s pathological modifications in organs such as the chest can be observed via computerised tomography (CT) scan. A CT scan may be a reliable test for screening SARS-COV 2 cases like other pneumonia types. However, the analysis demanded specialised equipment and failed to meet a considerable scale of requirements. COVID-19 can be diagnosed via laboratory measurements. These methods are usually utilised for the study of patients. They cannot be used to analyse contaminated samples such as surface and air [1].

Figure 1. Diagnosis methods for COVID-19.

Several methods have been developed for the diagnosis of COVID-19. The main tests for diagnosis can be classified in three main categories [1]:

1. Genetic tests (viral nucleic acid tests):analysis of viral genome employing methods such as real-time- quantitative reverse transcription-polymerase chain reaction (RT-qPCR), isothermal amplification (e.g., Loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), rolling circle amplification (RCA), Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)), and nanopore targeted sequencing (NTS).
2. Antigen tests:analysis of the viral proteins (mem- brane-bound spike proteins or the nucleocapsid proteins) using techniques such as colorimetric, field-effect transistor (FET), enzyme-linked immuno- sorbent assay (ELISA), and mass spectrometry (MS)
3. Serological tests: analysis of the antibodies (Immu- noglobulin M (IgM) and Immunoglobulin G (IgG)) against the virus [18, 19]. The study of patient’s antibodies can be achieved using methods such as electrical (EC) biosensors, localized surface plasmon resonance (LSPR), surface-enhanced Raman scat- tering (SERS), quartz crystal microbalance (QCM), fluorescence-based biosensor, colorimetric biosensor, gold immunochromatography, ELISA, chemilumi- nescence immunoassay, and piezoelectric microcan- tilever sensors (PEMS).

Unfortunately, many traditional detection methods of respiratory viruses, such as RT-PCN have many disadvantages. These include time-consuming, costly, are not always determinable or reproducible, and demand qualified staff and other technical facilities [3].

Figure 2. Disadvantages of traditional methods.

3. Nanotechnology

Nanotechnology offers new techniques for rapid diagnosis, early-stage infection detection, and identification of virulent pathogens causing the pandemic, particularly in improving the efficiency and quality of the detection process by employing nanobiosensors. Furthermore, new nanostructures and nanosensors display properties and performances unseen at the macroscopic level, significantly for detecting and sensing occasions at a nanoscale level [3].

Nanotechnology can enhance the diagnosis of COVID- 19 and suggest a state-of-the-art diagnostic method based on a Point Of Care (POC) sensing technology. Moreover, it can be interfaced with artificial intelligence (AI) techniques and the internet of medical things (IoMT)-integrated biosensors for studying practical informatics via data storage, sharing, and analytics. Also, they can bypass traditional processes such as low sensitivity, low selectivity, high cost, and extended diagnostic time. New methods can be utilised for no pain sample analysis, such as analysing the patient’s saliva using graphene oxide (GO)/Au/Fiber Bragg grating (FBG) probe. Nanotechnology can advance technologies such as label-free biosensors, paper lateral flow assays, optical technologies, and digital technologies [1].

3.1. Nanobiosensors

The higher preponderance of viral outbreaks can be attributed to the improper detection tools utilised to detect the contagious agents. Consequently, this requires a detection or diagnostic tool that is vigorous, quick, demanding, and precise in its biosensing properties. The biosensors can be characterised as analytical instruments that can assess low concentrations of an analyte in biological samples (like the human serum, blood, tears, saliva, etc.). Compared to traditional qualitative and quantitative test kits, these biosensors are favourably precise and susceptible to the directed target [15].

Figure 3. Classification and applications of various biosensors.

The need for accuracy and rapidity in diagnosing COVID-19 is not fulfilled by the traditional methods of serology-based tests and reverse transcription-polymerase chain reaction (RT-PCR), which are routinely utilised to detect and diagnose COVID-19. That condition can be fulfilled by employing ultrasensitive nanobiosensors that play a significant role in detecting novel coronavirus. Nanobiosensors deliver a quick, cost-effective, precise, and miniaturised platform for the detection of SARS-CoV-2 [10]. Biosensors typically include a biological recognition molecule immobilised onto the surface of a signal transducer and can be utilised for analysis, diagnosis, protection, security, and testing of larger populations [3].

Nanobiosensors provide several advantages that cause detection effective, such as [3]:

• Cost effective;
• Long self-life;
• Easy to use;
• Autonomous;
• Precision;
• Portability;
• Quick response;
• High sensitivity;
• Multiplexing capabilities;
• Viable process.

Nanobiosensors are devices in which the transducer is altered to catch the target component, convert the biological answer into electrical signals, and fast detect it with high precision. The physical responses can be calculated by determining the appropriate bioreceptors, such as nucleic acids, antigens, DNA probe, peptide, whole-cell, micro-organism, and tissue. These receptors are easily recognizable, highly sensitive, and detect specific bioanalyte. Various types of bioreceptors have been investigated to catch the viruses, such as nucleic acids (NA), immunoaffinity and protein in multiple nanobiosensors based on electrochemical, impedance, quartz crystal microbalance, and optical and surface plasmon resonance. The target molecule binds to the bioreceptor to catch a biological molecule by an unusual reaction. Then, the transducer with integrated molecule by a particular response. Then, the transducer with integrated nanostructures transforms the detection into an electrical signal defined by the detector (Fig. 4) [3].

Figure 4. The schematic diagram of different analytes, bioreceptors for biorecognition elements, transducers with integrated nanostructures as parts of a typical nanobiosensor design for respiratory viruses.

Nanobiosensors utilised for the detection of SARS or MERS coronaviruses can be categorised based on the biological molecule of the viral target (nucleic acids, antigens, or antibodies) into nucleic acid-based into nucleic acid- biosensor, antigen-based biosensor, and antibody-based biosensor (Fig. 5) [3].

Figure 5. Schematic representation of different biosensors classifications for the detection of SARS and MERS coronaviruses

3.1.1. Electrochemical Nanobiosensors

Electrochemical biosensors are the most widely used and favourably favoured type of sensing venues. According to the International Union of Pure and Applied Chemistry (IUPAC) definition, electrochemical biosensor is “a self-contained integrated device which is capable of providing specific quantitative or semi-quantitative analytical information using a biological recognition element (biochemical receptor) which is retained in direct spatial contact woth an electrochemical transduction element.” [8, 13].

An electrochemical nanobiosensor is a molecular sensing appliance that pairs a biological recognition event with an electrode transducer to create a usable electrical signal. Because electrochemical nanosensors include electrodes, the semiconductors properties, dielectric properties, and charge distribution are critical elements [3].

Figure 6. Scheme showing biosensor design components employed for sensing of target analyte samples, with special emphasis to utilizing an electrochemical bio- sensing platform that transforms biochemical information into current or voltages signals on an electrochemical transducer surface.

The main benefits of electrochemical biosensors are [5, 11]:

• Easy development;
• Possibility of miniaturization;
• High sensitivity;
• Relatively low cost.

Electrochemical sensors are an appealing choice for detecting a variety of biomolecules because they can be smoothly combined with multiple modules such as a. low-cost microelectronic circuits b. miniaturized lab-on-a-chip, c. interfacing with electronic read-out, and d. a signal processing unit. Electrochemical biosensors being sensitive, easy to miniaturize, require little analyte volumes, the superior limit of analyte detection and display on-site results are most preferred in medical diagnostics and many other research areas, including food safety and environmental monitoring [8].

The electrochemical biosensor can be categorised based on the transducer modes utilised for signal measurements. These include conductometry and surface charge, amperometry and potentiometry transduction platforms. The general principle of electrochemical biosensing (bio-electrochemistry) is established on electrochemical response taking place on or at the proximity of electrode and or between the electrodes that lead to; (i) a measurable current signal (amperometric), (ii) cumulate charge or potential(potentiometric), or (iii) changes in the conductivity of the medium (conductometric). [8].

Figure 7. Classification of the electrochemical biosensors based on type of transducer and signal modes.

Electrochemical nanobiosensors can also be employed to identify viral nucleic acids. An electrochemical genosensor developed for detecting SARS was developed using a monolayer of thiolated oligonucleotides self-assembled on gold nanoparticles-coated carbon electrodes. The oligonucleotide sequences are precise to the nucleocapsid protein of SARS, and the viral infection is detected via enzymatic amplification of viral DNA. The nanobiosensor helps the susceptible detection of SARS. An electrochemical nanobiosensor manufactured utilising gold nanoparticles changed with a carbon electrode and recombinant spike protein S1 as a biomarker was designed to detect MERS-CoVs; nevertheless, this approach also keeps promise for detection coronaviruses. Because of its electrical conductivity, the biosensor was created using fluorine-doped substrate and gold nanoparticles as a signal amplifier. [10].

Modifying electrochemical sensing interfaces with gold nanoparticles (AuNPs) shows improved applications and can be used to detect MERS-CoV. AuNPs act as working interfaces possessing electrocatalytic properties and permit amplification of the electric reaction (Figure 8). An immunosensor was designed to detect the MERS-CoV virus connecting the prospect of electrochemical sensors and gold nanoparticles. The nanobiosensor is developed with a bunch of carbon electrode-coated gold nanoparticles.

Figure 8. Operation steps for the COVID-19 electrochemical sensing platform: (A) sample collection via the nasal swab or saliva, (B) RNA extraction, (C) immobilization of RNA extract on the top of the graphene- ssDNA-AuNP platform, (D) incubation of 5 min, and (E) record the digital electrochemical output.

It has been observed that the recombinant spike (S1) protein gets immobilized to gold nanoparticles and competes with the virus particles for binding to the antibody. When virus infection is absent, it attaches to the immobilized spike protein. Because this nanobiosensor method has a group of electrodes, it can be used to detect various coronaviruses [5, 10].

 Graphene interfaced electrochemical detection of SARS-CoV-2

Electrochemical transducing platforms can detect viruses or any living microbial pathogens using their specific biorecognition elements. There are several alternative ways for electrochemical detection of disease-causing mechanisms. However, detecting genetic markers employing electrochemical sensing platforms is not susceptible for viral detections due to their undetectable viral titers, particularly at the early onset of viral infections [8, 14].

In current years, several attempts have been made toward applying strategies similar to electrochemical glucometers to detect viruses or viral infections. Torrente-Rodríguez et all developed low-cost graphene integrated portable electrochemical biosensor for rapid diagnosis and biochemical monitoring markers in serum and saliva samples for COVID-19 [8, 14].

The electrochemical sensor electrodes were graphene inscribed on a flexible polyimide (PI) polymeric substrate for multiplexed detection of viral infection biomarkers (antigens and antibodies). Torrente-Rodríguez et all demonstrated quantitative detecting specific biomarkers of COVID-19, such as SARS-CoV-2 spike protein (S1), nucleocapsid protein of SARS, CRP, a protein biomarker for inflammation within physiologically relevant ranges in both blood and saliva and specific immunoglobulins (Igs) such as S1-IgM and S1-IgG. This venue uses seized antigens and antibodies on graphene electrodes with increased sensitivity with a multiplexing capacity for sensing multiple SARS-Co- V2 makers, while the resulting response data is transmitted wirelessly to a portable mobile device. This type of miniaturized electrochemical platform shows a grand promise for the future PoC electrochemical and personalized health care devices [8, 14].

3.1.2. Optical Nanobiosensors

Due to the exceptional features of optical biosensors, such as high sensitivity, being label-free, robustness, immunity to electromagnetic interference, having computable optical outputs, being amenable to miniaturisation, integration capabilities, portability, multiplexing capacity and delivering simultaneous detection of various targets, optical biosensors are employed as diagnostic tools for respiratory virus infection. Thus, optical biosensors are eligible for the point-of-care zone [9, 10].

Figure 9. A Wireless Graphene-Based Telemedicine Platform (SARS-CoV-2 RapidPlex) for Rapid and Multiplex Electrochemical Detection of SARS- CoV-2 in Blood and Saliva (A) Schematic illustration of the SARS-CoV-2 RapidPlex multisensor telemedicine platform for detection of SARS-CoV-2 viral proteins, antibodies (IgG and IgM), and inflammatory biomarker C-reactive protein (CRP). Data can be wirelessly transmitted to a mobile user interface. WE, working electrode; CE, counter electrode; RE, reference electrode. (B) Mass-producible laser-engraved graphene sensor arrays. (C) Photograph of a disposable and flexible graphene array. (D) Image of a SARS-CoV-2 RapidPlex system with a graphene sensor array connected to a printed circuit board for signal processing and wireless communication.

Carbon nanotubes, gold nanoislands, and graphene are majorly employed in optical and electrochemical biosensors. Gold nanoislands made of tiny gold nanostructures can be constructed with artificially synthesised DNA receptors and complementary RNA sequences of SARS-CoV-2 on a glass substrate. Because COVID-19 is a single-stranded RNA virus, the receptor of the nanobiosensor acts as a complementary succession to the RNA sequence of the coronavirus and detects the virus. LSPR (localised surface plasmon resonance) was utilised to detect RNA sequence binding to the sensor. After binding the molecules on the surface of the nanobiosensor, the local infrared index changes, and an optical nanobiosensor calculates the modifications and determines the existence of RNA strands [9, 10].

Notably, highly effective optical biosensor-based detection of SARS-CoV-2 has been presented with surface plasmon resonance and fluorescence. When an optical biosensor is conjoined with the surface plasmon resonance method, the resulting method is valuable for rapid diagnosis of SARS infection, more so than enzyme-linked immunosorbent assays (ELISA). A fiber-optic-enabled biosensor based on localized surface plasmon associated fluorescence (LSPCF) can sense the recombinant N protein (SARS-CoV-N) using AuNPs. It was marked that a viral stock as small as 106 particles/mL can be detected by using a fiber-optics-based nano-enabled biosensor within 15 min. These surveys indicate that viral respiratory infections can be diagnosed rapidly and promptly by utilising nanomaterial-enabled [9].

Figure 10. A schematic of an optical biosensor

3.1.3. Graphene-based biosensors

A grapheme-based FET (field effect transistor) device is employed to determine SARS-CoV-2 viral burden in nasopharyngeal swabs of COVID-19 patients. The graphene-based FET nanobiosensor consists of a graphene sheet as the sensing area, moved to a SiO2/Si substrate and SARS-CoV-2 spike antibody immobilized on the graphene sheet. The biosensors help detection of SARS-CoV-2 antigen spike even at the concentration of 1 fg/mL in phosphate buffer [10].

Graphene-based biosensors are valuable for testing and cutting-edge detection of [9]:

• blood glucose;
• respiration rate;
• real-time body temperature;
• blood pressure;
• virus;
• small molecules.

Due to the cost-effectiveness, high association, and ease of fabrication, graphene-based nanomaterials are the most appealing materials for biosensors. For example, a transistor-based biosensor has been successfully developed to detect SARS-CoV-2 (spike protein). The biosensor was manufactured using field-effect transistor (FET) coated graphene sheets with a specific antibody (Figure 9). Graphene and its derivatives show suitable integrity FET-based biosensing devices for capturing viruses as they have benefits over other diagnostic methods which are at the present available [9].

FET-based biosensing devices can make susceptible and instantaneous measurements by using small amounts of analytes. Furthermore, FET-based biosensors have probable and utility in clinical diagnosis, on-sight detection and point-of-care testing. An unamplified and quick nanosensing platform was created to detect SARS-CoV-2 RNA in human throat swab specimens. A graphene field-effect transistor (G-FET) sensor was designed to illustrate gold nanoparticle (AuNP). On the surfaces of AuNPs, complementary phosphorodiamidate morpholino oligo (PMO) probes were immobilized. This sensor directs to a low background signal, as the PMO is highly sensitive to SARS-CoV-2 RdRp. When a graphene field-effect transistor is connected with a CRISPR-Cas9-based biosensor, it will be capable of detecting unamplified target genes, and thus, it could be evaluated for viral targets, such as the nucleic acids of SARS-CoV-2 [9]:

3.1.4. Chiral Nanobiosensors

Chiral biosensors will soon be at the bionanotechnology domain’s vanguard due to their ultra-sensitivity and rapid response time. They will be instrumental in the SARS-CoV-2 pandemic. The popularity of nano-chiroptics has burst because of novel methods to manufacture engineered metallic nanostructures with a tunable surface morphology and complete their nano-assembly. This offers unparalleled power over their electronic and optical properties. The most important benefit of such nanohybrid structures is that they improve the chiroptical reaction, which could be of significant interest in various applications linked to chiral biosensing, opening up new research areas. In comparison to natural chiral molecules, chiral plasmonic nanostructures not only lead to significant chiroptical effects, but also present completely unique ideas of superchiral light in technological applications [2, 5].

Figure 11. Detection of SARS-CoV-2 using FETs: The schematic shows a collection of biological samples from a patient and their application to the graphene-based sensing area of a FET biosensor. Binding events associated with the SAR-CoV2 virus can be captured by the sensor in real time.

Ahmed et al. developed a self-assembled technique for the development of a chiral immunosensor using gold nanoparticles and quantum dots. Zirconium quantum dots and magnetic nanoparticles were conjugated with coronavirus specific antibodies and mixed. In the presence of a viral target, both the quantum dots and nanoparticles will attach to the viral target and develop magneto plasmonic- fluorescent nanohybrids, which an exterior magnet can divide. The analyte concentration was then decided by calculating the fluorescence assertiveness of the diverged nanohybrids. This sensing process has a limit of detection of 79.15 EID/50 μl [2, 5, 10].

3.1.5. Aptamer-Based Biosensor

Due to the robust screening method, aptamers can detect viral genes, proteins, or any other viral infection markers. By adjusting the developed assays, aptamer-based sensors can distinguish between infected and uninfected host cells or active and inactive viral forms. Because of their properties, aptamer-based detection has significant benefits over antibodies, including high resilience at a vast range of temperatures and situations, straightforward synthesis through a systematic evolution of ligands by exponential enrichment (SELEX) method, and easy transformation according to the needs of the assay [4].

Figure 12. Detection of SARS-CoV and SARS-CoV-2 using aptamer-based biosensors.

Biosensors employ antibody- and aptamer-based detection mechanisms. Aptamers are more durable, more affordable and quicker to synthesize than antibodies. Aptamers, also known as ‘chemical antibodies’ or ‘artificial antibodies’, are often analogised to antibodies regarding their critical particularity towards their targets. Some aptamers have been isolated in SARS-CoV-2 and incorporated in aptasensing platforms [7].

Aptamers are oligonucleotide sequences that can be designed to recognize and bind to different biomolecules specifically:

• diminutive molecules such as amino acids, nucleotides, and antibiotics;
• 67,68 macromolecules such as nucleic acids and proteins;
• 69 and even surface-epitope bearing whole bacteria, viruses70 and other cells.

Aptamers form unique three-dimensional (3D) structures while securing specifically to analytes. These can be modelled quickly and can be immobilized stably on the surface of biosensors. Aptamer-based biosensors (aptasensors) can quantitatively detect target analytes by calculating the signal developed from the coupled chemical and/or biochemical surface interactions. Aptamers have been considered as a promising diagnostic tool for detecting viruses [7].

Aptasensors are aptamer-based biosensors designed to explore and quantify target analyte biomolecules via distinct biochemical reactions related to a quantifiable signal generation mechanism. The interchange of specific aptamers with target biomolecules represents the biorecognition and capturing the event, which is additionally transduced into a proportionate signal. Aptasensors recently reported for detecting SARS-CoV-2 can be broadly divided into two categories based on the nature of signal transduction: optical and electrochemical aptasensors [7].

3.2. Point-of-Care Testing

Point-of-Care testing as it is defined by the Centers for Disease Control and Prevention Center are “diagnostic tests performed at or near the place where a specimen is collected, and they provide results within minutes rather than hours. These may be Nucelic Acid Amplification Test (NAAT), antigen, or antibody tests.” [18].

The point-of-care testing (POCT) infectious disease market represents an auspicious and substantial increase in the industry’s global in-vitro diagnostics (IVD). The increasing spread of human immunodeficiency virus (HIV), tuberculosis (TB), and malaria in developing countries, and the danger of emerging and reemerging contagious diseases such as the Middle East respiratory syndrome (MERS), severe acute respiratory syndrome (SARS), ZIKA, a variety of influenza strains, and the West Nile virus are factors that boost the need for POCT [6].

Contagious diseases pose a substantial threat to human health and lead to more than half of deaths worldwide. Further, widespread contagious diseases have continuously increased fatality rates in developing countries. The most efficient way to contain the epidemic is an early diagnosis, which is challenging to utilize common approaches because of expensive and extensive equipment, specialists, and slow data output. Thus, rapid POCT methods are essential for overcoming these burdens by miniaturizing and decreasing the device expense and delivering accessible, quick, easy-to-use diagnostic tests without specialized training [6].

POC testing enables the diagnosis of infected individuals without sending patient specimens to laboratories. This is extremely important for places or residents that lack appropriate laboratory infrastructure for specimen testing. The essential part of PoC testing is the biosensor, which is utilised to achieve a biochemical assay to detect the pathogen.

The advantages of using PoC testing are [4].

1. minimal space condition for testing and storage;
2. wide-scale analysis;

• testing can be achieved in a variety of locations;

1. adaptable in meeting various medical needs.

Figure 13. Schematic illustration of the quantitative evaluation of SARS-CoV-2 using the SERS-based aptasensor. (a) After SARS-CoV-2 lysates release the target spike proteins, they are recognized by the aptamer DNAs on the Au nanopopcorn surfaces. The S protein–bound aptamers move away from the Au nanopopcorn surfaces, leading to a decreased Raman peak intensity of Cy3 reporters. (b) Cy3-tagged aptamer DNAs are hybridized with capture DNAs on the Au nanopopcorn substrate. The internal standard 4-MBAs are immobilized along with aptamer DNAs on the Au nanopopcorn substrate. (c) Recognition of the SARS-CoV-2 S protein induces a conformational change of aptamer DNAs, enabling the aptamer DNAs to bind with the RBD on the spike protein.

One of the most attractive POCTs is the ones that are based on the colometric biosensors as they permit detection of the analyte through easy color changes that are observable to the unassisted eye [5].

Figure 14: Nanoparticle based colorimetric detection of virus. This figure depicts the mechanism by which virus causes aggregation of nanoparticles, leading to color change from red to purple.

Kim et al. created a colorimetric assay employing gold nanoparticles to detect the MERS-CoV virus. They suggested a colorimetric assay based on an extended structure of double-stranded DNA (dsDNA) self-assembly shielded gold. This assay utilises two thiol modified probes and citrate capped gold nanoparticles (AuNPs) nanoparticles (AuNPs) under positive electrolyte (e.g., 0.1 M MgCl2) [5, 6].

The gold nanoparticle-based colorimetric test makes a gold nanoparticle solution that collects the virus and shows an observable colour change in the liquid. This gives a rapid test for COVID-19 by turning the colour of gold nanoparticles. This low-cost test acts much better than the other diagnostic techniques, similar to the standard PCR tests. The main benefit of this test is that gold nanoparticles show specific colours because they absorb particular wavelengths. To the gold nanoparticles, the sample containing SARS-CoV-2 is counted, resulting in the accumulation of the virus, and it provokes a transformation in the absorption height that results in a change of colour of the solution. This shift in the colour will be observable to the naked eye, and the downside is that it is feasible only when a load of the virus is very high [12].

The probes are conjugated to AuNPs via substantial Au-S interchanges. In the absence of a target, the AuNPs total (in a positive electrolyte) leads to colour change, either envisioning with the naked eye or detecting by localised surface plasmon resonance (LSPR) shift. Nonetheless, the existence of viral target causes comprehensive self-assembly of double-stranded DNA, controlling the accumulation of gold nanoparticles in the existence of positive electrolytes, discouraging shift in optical properties of AuNPs [5, 6].

The potential detection limit of this assay is 1 pmol μl−1, permitting the detection of lower amounts of the viral target. Furthermore, using such kind of colorimetric based assay enables low-cost and rapid disease diagnosis without the requirement for sophisticated tools [5].

3.3. Nanopore target sequencing (NTS)

The nanopore metagenome method (NTS) has been demonstrated to detect respiratory bacterial infection and viruses instantly from clinical samples. In addition, pathogens and antibiotic resistance genes can be recognised in several hours, much faster than conventional culture processes as the real-time data generation of nanopore sequencers. Moreover, nanopore sequencing was utilised to direct sequences in the transcriptome of SARS-CoV-2. The NTS method simultaneously detects SARS-CoV-2 and ten other respiratory viruses within only 6–10 hours. Therefore, it is suitable for the current diagnosis of COVID-19; nonetheless, the framework can be extended to diagnose other viruses and pathogens. NTS is based on amplifying 11 SARS-CoV-2 virulence-related and exceptional gene fragments (e.g., orf1ab) utilising an inner primary panel followed by sequencing the boosted fragment on a nanopore platform. This task uses a nanopore platform for sequencing to sequence long nucleic acid fragments and simultaneously analyze the data output in real-time. This allows verifying SARS-CoV-2 infections within minutes of sequencing by mapping the sequence reads to the SARS- CoV-2 genome and analyzing the output sequence’s originality, validity, and read number sequence [16, 17].

4. Challenges and Limitations of Nanotechnology in COVID-19

Nanotechnology-based systems, despite their advantages, encounter multiple barriers before they can be safely presented to the market. The most common issues are:

1. Scalability and production costs;
2. ,Intellectual and regulatory properties;
3. Potential toxicity and environmental effects.

Some problems in nanotechnology applications must be handled before they are widely adopted in the healthcare system. The primary task will be to secure the safety of nanomaterial via in vitro studies of their biocompatibility. The destiny of nanomaterials can be transformed into the body when they travel through blood due to the protein corona formation. Hence, in vivo studies need to be executed carefully to understand better the toxicity of nanoparticles in the body. Because of constraints, generic protocols have been utilised for categorization at an early stage of research and development that miscalculate the chances of failures in clinical translation of nanotechnology-based therapy. Closer cooperation between regulatory agencies, experts in material science, pharmacology, and toxicology are needed to overcome other limitations [10].

---

Test LO 3.2

---

References

1. Abdelhamid H., and Badr G.
2. (2021). Nanobiotechnology as a platform for the diagnosis of COVID‐19: a review. Nanotechnology for Environmental Engineering 6:19 https://doi.org/10.1007/s41204-021-00109-0.
3. Ahmed SR., Nagy É., and Neethirajan S. (2017). Self-assembled star-shaped chiroplasmonic gold nanoparticles for an ultrasensitive chiro- immunosensor for viruses RSC Adv. 7 40849–57
4. Alhalaili B., Popescu I.N., Kamoun O., Alzubi F., Alawadhia S., Vidu R. (2020). Nanobiosensors for the detection of novel coronavirus 2019-nCoV and other pandemic/Epidemic Respiratory viruses: A review. Sensors, 20, 6591.
5. Gupta R., Sagar P., Priyadarshi N., Kaul S., Sandhir R., Rishi V. and Singhal N.K. (2020). Nanotechnology-Based Approaches for the Detection of SARS-CoV-2. Front. Nanotechnol. 2:589832. doi: 10.3389/fnano.2020.589832
6. Jindal S., and Gopinath P. (2020). Nano Ex. 1 022003
7. Kim H.,Park M.,Hwang J.,Kim J.H., Chung D-R., Lee Kand Kang M. (2019). Development of label-free colorimetric assay for MERS-CoV using gold nanoparticles ACS Sens.
8. Mandal M., Dutta N., and Dutta G. (2021). Aptamer-based biosensors and their implications in COVID-19 diagnosis. Anal. Methods, 2021, 13, 5400
9. Ozmen E., Kartal E., Turan M., Yazicioglu A., Hiazi J., and Qureshi A. (2021). Graphene and carbon nanotubes interfaced electrochemical nanobiosensors for the detection of SARS-CoV-2 (COVID-19) and other respiratory viral infections: A review. Materials Science & Engineering C 129 (2021) 112356
10. Pradhan A., Lahare P., Sinha P., Singh N., Gupta B., Kuca K., Ghosh K.K., Krejcar O. (2021). Biosensors as Nano-Analytical Tools for COVID-19 Detection. Sensors 2021, 21,7823. https://doi.org/10.3390/ s21237823
11. Rai M., Bonde S., Yadav A., Bhowmik A., Rathod S., Ingle P., Gade A. (2021). Nanotechnology as a Shield against COVID-19: Current Advancement and Limitations. Viruses 2021,13,1224. https:// doi.org/10.3390/v13071224
12. Satvekar R. (2021). Electrochemical nanobiosensors perspectives for COVID 19 pandemic.  J. Electrochem. Sci. Eng. 00(0) (2021) 000-000; http://dx.doi.org/10.5599/jese.1116
13. Tavakol S., Zahmatkeshan M., Mohammadinejad R., Mehrzadi S., Joghataei M., Alavijeh M., Seifalian A. (2021). The role of nanotechnology in current COVID-19 outbreak. Heliyon 7, e06841
14. Thevenot D.R., Toth K., Durst R.A., Wilson G.S., Biosens. (2001). Bioelectron. 16:121–131.
15. Torrente-Rodríguez R.M., Lukas H., Tu J., Min J., Yang Y., Xu C., Rossiter H.B., Gao W., (2020). Matter. 3: 1981–1998.
16. Varghese R., Salvi S., Sood P., Karsiya J., Kumar D. (2021). Carbon nanotubes in COVID-19: A critical review and prospects. Colloid and Interface Science Communications 46: 100544
17. Wang M., Fu A., Hu B., Tong Y., Liu R., et al. (2020). Nanopore target sequencing for accurate and comprehensive detection of SARS-CoV-2 and other respiratory viruses. medRxiv.
18. Waris A., Ali M., Khan AU., Ali A. (2020). Baset A. Role of nanotechnology in diagnosing and treating COVID-19 during the Pandemic. Int J Clin Virol. 2020; 4: 065-070.
19. https://www.cdc.gov/coronavirus/2019-ncov/lab/point-of-care-testing.html

Summary

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is the causal agent of COVID-19. The infection with SARS-CoV-2 usually produces mild-to-moderate respiratory symptoms but some patients may need hospitalization and intensive care. The mainstay of conservative therapy for COVID-19 is a combination of corticosteroids, anticoagulants or anti-aggregants and, in cases of bacterial pneumonia, antibiotics. Several specialised treatments for COVID-19 (antivirals, monoclonal antibodies, and others) have been developed that may be used as pre-exposure prophylaxis or as treatment in the early stages of the infection in patients at high risk for complications. Modern nanotechnology offers a variety of high-tech solutions for the purposes of prevention of infection, diagnostics, post-exposure prophylaxis, as well as effective treatments in cases when SARS-CoV-2-related disease has already developed. It may be reasonably expected that nanomaterial-based sensors, drugs and vaccines will play a critical role in the management of the pandemic in the near future.

Key words/phrases: COVID-19, antivirals, monoclonal antibodies, vaccines, nanotechnology

1. Vaccines and treatments for COVID-19 – an overview

1.1. SARS-CoV-2 and COVID-19 – an overview of causality, pathogenesis and potential approaches to decrease the burden of COVID-related complications

On 31 Dec 2019, the World Health Organization (WHO) was informed about a rapidly growing number of cases of pneumonia of yet unknown origin in Wuhan City, China. The causal agent was identified by the local authorities on 7 Jan 2020 as a hitherto unknown member of the beta-coronavirus family.

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is the causal agent of an infectious disease named COVID-19 (abbreviated from Coronavirus Disease of 2019). In most cases, the infection with SARS-CoV-2 will produce only mild-to-moderate symptoms. These may include any or all of the following: asthenoadynamia, respiratory symptoms, anosmia/dysosmia, headaches, joint and muscle pains, diarrhoea and/or abdominal pain, and others. In most cases the recovery is spontaneous, within several days up to two weeks. Some patients, however, may develop acute respiratory distress syndrome (ARDS) and/or progressive respiratory failure. Compared to its relatives SARS and MERS (Middle East respiratory syndrome), COVID-19 has milder clinical symptoms and lower fatality rates. Nevertheless, anyone can contract and may die from SARS-CoV-2, regardless of sex and age (although the risk for death is higher in males and children, and adults with underlying disease).

1.2 Pathogenesis

 

SARS-CoV-2 enters the cell by endocytosis via the ACE2 receptor on the cell surface, similarly to most coronaviruses. The process is mediated by binding of the receptor binding domain (RSD) of the S (spike) protein to the ACE2 receptor of the target cell [67]. When the virus enters the cell, it uncoats its single-stranded, positive-sense genome. The genomic RNA is translated into two replicase polyproteins that are subsequently cleaved into 16 non-structural proteins that form the RNA replicase-transcriptase complex that produces the viral genome and the subgenomic copies carrying open reading frames for protein synthesis. The viral RNA and the structural proteins are then assembled into new viral particles that are released by exocytosis to infect other cells [66, 38]. Since the ACE2 receptor gene is expressed in many tissues besides the lung (namely, the gastrointestinal tract, the kidneys and others [74, 15], the patients may develop lung injury and/or injury to multiple tissues and organs.

1.3. Pathomorphology and laboratory findings

The main pathomorphological finding in patients with COVID-19 is diffuse alveolar damage combined with pathological changes in the pulmonary vascular bed and alveolar hemorrhagic syndrome [Pathology of COVID-19: Atlas, 2020]. Systemic inflammation with inflammatory lymphocytic and monocytic infiltration also accounts for the impact on the target tissues, in particular the lung and the heart [22, 38]. Typical laboratory findings of severe COVID-19 are lymphopenia (although some patients may exhibit lymphocytosis, thrombocytopenia, elevated fibrinogen, D-dimer, LDH, ferritin, IL‑2, IL‑7, IL‑6, monocyte chemoattractant protein 1 (MCP1), macrophage inflammatory protein 1‑alpha (MIP‑1‑alpha), and tumour necrosis factor (TNF‑α) levels.

1.4. Transmission and prevention of infection

SARS-CoV-2 is unstable outside the human body and is transmitted by close contact with the infected person. It is spread mainly via airborne mechanisms. The most effective ways to prevent or, at least, to slow down its transmission are self-isolation, maintaining safe distance from others, washing hands often (and/or using 70 % ethanol-based hand disinfectants), cleaning regularly the surfaces and objects that are frequently touched, and wearing properly fitted facemasks when indoors. The World Health Organization (WHO) release regularly updated guidelines on prevention of the infection with SARS-CoV-2: https://www.covid19treatmentguidelines.nih.gov/about-the-guidelines/whats-new/

1.5. Pathogenetic targets for treatment of the SARS-CoV-2 infection

The SARS-CoV-2 genome encodes 4 major structural proteins, namely: spike (S), envelope (E), membrane (M), and nucleocapsid (N), along with non-structural and accessory proteins. Both the viral RNA and the structural proteins of the virus have been viewed as targets for treatment. The most commonly used antiviral drugs usually target the RNA-dependent RNA polymerase of the virus, while the monoclonal antibodies are more commonly targeted at the structural proteins of the virus.

1.6. Risk factors for complications of SARS-CoV-2 infection

Major risk factors for adverse outcomes are male gender and underlying medical conditions such as cardiovascular disease, renal disease, diabetes, chronic respiratory disease and cancer [51]. Obstructive sleep apnea is a specific risk factor that increases the risk for hospitalization and respiratory failure almost 8-fold [36]. Advanced age was initially believed to be a major risk factor for adverse outcomes but later it was recognised that the presence of underlying disease had significantly more weight than age alone [https://www.cdc.gov/aging/covid19/covid19-older-adults.html; https://gis.cdc.gov/grasp/COVIDNet/COVID19_5.html].

1.7. Treatments of SARS-CoV-2-infection and its complications

1.7.1. Conservative management

Uncomplicated cases of COVID-19 are best managed with home self-isolation from others, rest and symptomatic treatment. About 10-20 % of the infected are at risk of becoming seriously ill and may need specialised medical care [https://www.uptodate.com/contents/covid-19-clinical-features]. Complicated cases are managed according to designated WHO and Centre for Control of Disease (CDC) protocols, and local modifications of these protocols. As COVID-19 is associated with hyperinflammation and prothrombotic states, anti-inflammatory and anti-coagulation and/or anti-aggregation therapy are vital parts of virtually any protocol [https://covidprotocols.org/en/chapters/inpatient-management/]. The mainstay of anti-inflammatory therapy for COVID-19 are corticosteroids, mainly long-acting such as dexamethasone. In patients with severe respiratory failure, methylprednisolone may be added. Low-molecular weight heparin is most commonly used for anticoagulation in the protocols for treatment of COVID-19, but other options are available such as unfractionated heparin, indirect-acting anticoagulants, novel oral anticoagulants, aspirin, clopidogrel, dihydropyridamole, and others. The potent anti-inflammatory properties of corticosteroids come, among others adverse effects, at the price of gastric irritation; therefore, use of gastric protection by H2 antagonists or proton pump inhibitors is usually the norm. Antibiotics come into use in COVID-19 patients when there is evidence of secondary bacterial pneumonia. The latter is a common complication of severe COVID-19, as the virus produces, besides its other effects, a severe suppression of the innate immunity and a profound dysregulation of the immune signalling in the host [76]. Thus, treatment of severe COVID-19 often includes wide-spectrum antibiotics. Bactericidal antibiotics (e.g. penicillins, 4-quinolones and aminoglycosides) are typically being preferred over bacteriostatics (e.g. macrolides and tetracyclines). About 2 % of the infected patients may develop progressive respiratory failure and may need intensive care [https://www.uptodate.com/contents/covid-19-clinical-features].

1.7.2. Specific treatments against SARS-CoV-2 related disease

A variety of antivirals and monoclonal antibodies have been tried and tested for activity against SARS-CoV-2. Mostly, these are pre-existing drugs that have been previously tried for other disease caused by RNA-based viruses, such as favipiravir, remdesivir, lopinavir/ritonavir, tocilizumab, and others. Other, such as nirmatrelvir (a compound of Pfizer’s recently approved drug Paxlovid) and sotrovimab were specifically developed for the purposes of treating COVID-19, albeit on the base of previously known drugs or antibodies.

The advent of vaccines against SARS-CoV-2 brought new possibilities for management of infections and the prognosis for those who have developed COVID-19. The virus was isolated by the end of 2019 [70] and the first genetic sequence was published less than 2 weeks later [71]. Thus, the scientific and medical community along with the pharmaceutical industry had an early warning and could integrate and target their efforts toward developing effective treatments for COVID-19 and vaccines against infections with SARS-CoV-2 before the first wave of the pandemic hit the globe in March and April 2020. The first experimental anti-SARS-CoV-2 vaccine (Convidecia, developed by CanSino Biologics) was approved in China in late June 2020. Within the next few months about a dozen vaccines of the vector, peptide and mRNA type were fast-tracked and approved for use in adults, and, later, in children as well. By December 21, at least 14 vaccine products against SARS-CoV-2 have had their assessment finalized and about 10 more were currently under development (e.g. EpiVac Corona by Vector State Research Center of Virology and Biotechnology, and others) or under final assessment (e.g. vaccinal products by BioCubaPharma, CanSinoBio, Sanofi, Clover Pharmaceuticals, and others). More data may be viewed at Status_COVID_VAX_23Dec2021.pdf (who.int).

Applications for several products have been withdrawn. One of these was, unfortunately, the first entirely nanoparticle-based vaccine against infection with SARS-Cov-2 (zorecimeran, by CureVac). It was issued an expression of interest (EOI) by the WHO, but the application was later withdrawn by the manufacturer, as the demonstrated efficacy of protection of the vaccine against symptomatic disease was below 50 % (namely, 48%) in all age groups. The vaccine showed efficacy of 100% against hospitalization and death in the study group, but as it was comprised of 134 objects only, the final analysis pointed out that at least 80 additional cases were needed for proper assessment [https://www.reuters.com/business/healthcare-pharmaceuticals/curevacs-covid-19-vaccine-misses-efficacy-goal-mass-trial-2021-06-16/]. Thus, CureVac abandoned the zorecimeran project and started cooperation with Sanofi-GlaxoSmithCline in the work on their own brand of vaccine [https://www.reuters.com/business/healthcare-pharmaceuticals/curevac-withdraw-first-generation-covid-19-vaccine-candidate-2021-10-12/]. By January 2021, WHO has listed a total of 63 candidate vaccines in clinical development and further 173 in preclinical development.

2. Nano insights into treatment and prevention of COVID-19

Nanotechnology, as defined by the Centre for Disease Control in Atlanta, USA is… “the manipulation of matter on a near-atomic scale to produce new structures, materials and devices… using materials with a length scale between 1 and 100 nanometres…at which size materials begin to exhibit unique properties that affect [their] physical, chemical, and biological behaviour” https://www.cdc.gov/niosh/topics/nanotech/default.html#:~:text=Nanotechnology%20is%20the%20manipulation%20of,new%20structures%2C%20materials%20and%20devices.&text=Nanotechnology%20refers%20to%20engineered%20structures,between%201%20and%20100%20nanometers. Nanomaterials usually have good solubility in biological liquids and a high surface-to-volume ratio that permits them to interact well with biological membranes and allows for better control of drug release than classic pharmaceuticals.

SARS-CoV-2 is a medium-sized virus (its particle is within the range of 60-140 nm), which fits very well into the nanoscale range. Modern nanotechnology offers a variety of high-tech solutions for the purposes of prevention of infection (development of efficient personal protection equipment (PPE); development of vaccines; pre-exposure prophylaxis, etc.); diagnostics (detection of the virus in biological and environmental samples), and management of the early stages of the infection (post-exposure prophylaxis), as well as effective treatments in cases when SARS-CoV-2-related disease has already developed. It may be reasonably expected that nanomaterial-based sensors, drugs and vaccines will play a critical role in the management of the pandemic in the near future [52, 41].

To date, a wide variety of nanomaterials have been employed for use in the detection of SARS-CoV2, prevention of transmission, drug and vaccine delivery. One of the best and most comprehensive reviews of the use of nanomaterials may be viewed here: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8308319/table/nanomaterials-11-01788-t001/?report=objectonly

2.1. Nanomaterials for prevention of transmission of SARS-CoV-2

The highest load of SARS-CoV-2 viral particles is found in fine respiratory droplets that are released when the patient (or the presymptomatic carrier) talks, coughs and sneezes; but large droplets containing viral particles also have high infective potential, when present on surfaces and objects that are frequently touched [https://www.cdc.gov/coronavirus/2019-ncov/science/science-briefs/sars-cov-2-transmission.html]. The significant infective potential of SARS-CoV-2 was acknowledged at an early stage of the pandemic, although it was significantly lower than the levels of infectivity of the later variants Delta and Omicron [https://www.cdc.gov/coronavirus/2019-ncov/variants/omicron-variant.html].

The size of the SARS-CoV-2 viral particles allows them to penetrate easily through usual protective equipment that has proven effective when used against other common respiratory diseases (influenza, chickenpox, and others). The development of modified types of PPEs called for the use of nanotechnology. Novel nanofibres and nanofibre webs were developed for use in protective facemasks worn by the medical personnel and first responders. High-performance facemasks (FFP2, FFP3, and N95 standard) use a combination of nanofiber webs and electrostatic charge that could protect from large respiratory droplets dispersed by the patient [9]. FFP-standard masks are 3D-printed to fit tightly the face of the wearer and may include a molded nosepiece to create an airtight seal. The filtration efficiency for high-performance masks for the highly virus-laden larger droplets (>0.3 μm) is expected to be at least 78% for FFP1, 92% for FFP2, and 98% for FFP3 masks [https://www.protectivemasksdirect.co.uk/ffp3-masks-hse-guidance].

Additional protection may be obtained by impregnation of the layers of the mask with compounds with antimicrobial and antiviral activities. Nanomaterials such as silver and copper oxide nanoparticles, alone or carrying other bacteriocidal and/or viricidal agents (e.g. iodine), have been used with success [6, 1].

Special nanocoatings (silicon-based, graphene based and others) have been developed to ensure that the masks are reusable after cleaning [14, 75].

Additionally, the size of the pores in the filter of the mask may be decreased by impregnation with polymers such as ß-cyclodextrin. It must be noted, however, that while the safety for the wearer of the mask is crucial, a perfectly fitting facemask may actually result in decreased compliance of the wearer. Typical surgical masks and respirators from the pre-nanotechnology era were known for their low air permeability, causing the wearer to fidget and try to touch and adjust the mask. Modern nanofibers produced by electrospinning create an exquisite web with excellent air permeability, improving the functionality of masks and increasing the antibacterial effects.

Similarly to facemasks, nanotechnology has also contributed to manufacturing medical gloves and other PPEs. Gloves with silver nanoparticles have shown bactericidal and virucidal effects.

A very interesting proposal about nanotechnology-based capture of viral particles by ligand-receptor interaction was made in 2021. It was based on the fact that the SARS-CoV-2 binds and enters the cell via the ACE2 receptor. Thus, it was proposed that nose filters, masks, gloves and other PPEs could be impregnated with nanotechnology-engineered ACE2-receptor that could bind the offensive agent and minimise the risk for the wearer [2]. The results, however, are yet to come.

Since SARS-CoV-2 (as well as influenza virus, HCV, HBV and HIV) is an enveloped virus, most currently used hand and surface disinfectants are ethanol or 2-isopropanol-based. The critical concentration of ethanol that completely inactivates the virus is believed to be over 35 % v/v [44]. Ethanol and isopropanol are, nevertheless, volatile, flammable, may contain compounds that are harmful. They are also relatively expensive and may have adverse effects such as headaches, nausea and dizziness when used as sprays, liquids and rubbing gels. Several nanoparticle-based sanitizers have been developed using titanium oxide and silver nanoparticles as well as engineered water nanostructures [49, 7]. Other types of nanotechnology-based hand sanitizers utilize hydrogen peroxide to ensure bactericidal and virucidal effect [65].

2.2. Nanomaterials in the diagnostics of SARS-CoV-2 viral particles

Almost all aspects of SARS-CoV-2 infection present a challenge. The virus is highly contagious, but not all people exposed to the virus will become infected. The infected person, even when symptomatic, does not produce a distinct clinical and laboratory phenotype that would allow COVID-19 to be reliably diagnosed on clinical appearance or results of laboratory tests. The incubation period may vary between 2 and 14 days (although it is, at present, believed that remaining asymptomatic beyond 7 days means that it is unlikely that the person is infected). The differential diagnosis of mild and moderate cases may include virtually any other respiratory infection and, in severe cases, may mimic other causes for rapid deterioration of the clinical status such as inflammatory disease of any origin, sepsis, vascular accidents, and others. A significant proportion of cases may present with radiological evidence only, repeatedly producing negative tests for viral RNA. Thus, development of a rapid, highly sensitive and highly specific COVID-19 diagnostic test is yet to come, especially in the view of the enormous potential of the virus to mutate further, evading routine diagnostics.

As COVID-19 is a modern pandemic, nanotechnology has been employed in order to improve the reliability of diagnosis of infection with SARS-CoV-2 since the first months after the initial outbreak. Most of the technologies and devices used in detection of SARS-CoV-2 are based on those previously developed for the detection of SARS and MERS.

Various nanomaterials have been tested and approved for sensitive and specific detection of viral agents prior to the outbreak of COVID-19. Among these are silver and gold nanoparticles loaded with gold-binding polypeptides and proteins [32, 47]; DNA probes immobilized onto a carbon electrode overlaid with gold nanoparticles; antisense oligonucleotides-covered gold nanoparticles coupled with colour substrate in order to ensure rapid visual detection [39, 30, 42], and others.

The typical SARS-CoV-2 test uses material from a nasopharyngeal or throat swab. This method of sampling, although scoring low in the invasiveness scale, may be viewed by the patients as an intrusion on their privacy. This may result in poor swab taking technique in order to avoid complaints and, respectively, in increased rate of falsely negative results. Less invasive approaches have been proposed for detection and rapid screening for SARS-CoV-2 in exhaled air using a biosensor-based gold nanoparticles [57, 19].

Magnetic nanoparticles have been used as a tool in molecular diagnostics of coronaviruses for extraction of nucleic acids from biological and environmental samples [20]. This was the basis for development of a specialised extraction protocol for SARS-CoV-2 using zinc ferrite nanoparticles [58; 76].

Quantum dots are nanoscale-sized crystals of artificial origin that contain excitable electrons with excitation and emission wavelengths that are subject to variance by the user. They possess semiconductor and fluorescent properties. The optical properties of quantum dots and, specifically, the correlation between size and optical characteristics allows them to be used for as imaging probes in biosensors. After the first SARS outbreak quantum dots have been used in a biosensor for detection of SARS nucleocapsid protein antigen [56]. In 2020, Liu et al., proposed that the same technology may potentially be adapted for detection for SARS-CoV-2 [33].

Carbon dots, carbon nanotubes and nanodiamonds have been used for enrichment and concentration of samples for the purposes of improved sensitivity of low-copy viral nucleic acid [72].

Nanozymes are nanoscale-sized molecules of artificial origin that possess catalytic activity. Recently, a rapid and sensitive paper strip-based assay for SARS-CoV-2 using Co-Fe hemin peroxidase nanozyme instead of natural peroxidase was developed [33]. The test provided, according to the authors, a lower detection limit as low as 10 pg/mL of the S protein antigen, with the test complete in about 15 minutes.

Serological detection of SARS-CoV-2 has also benefited from the development of nanotechnology. In 2020, Huang et al. developed an assay for rapid detection of IgM against SARS-CoV-2 using viral nucleoprotein conjugated to gold nanoparticles, obtaining results in less than 15 min [23].

3. Nanotechnology to combat COVID-19: therapeutics research

Nanotechnologies have the potential to become an integral component of development of treatments for COVID-19, providing novel platforms for drug delivery as well as regulation and coordination of temporal and spatial release of bioactive substances such as antivirals and monoclonal antibodies. The field is rapidly developing, although results lag behind the development of pharmaceuticals with antiviral and immunomodulatory properties. Still, research, technology and clinical medicine have presented a front as united as never before when humanity became face to face with SARS-CoV. It could be reasonably expected that development of novel nano-based drugs and vaccines against the virus would be significantly accelerated in the near future.

3.1. Antivirals for COVID-19

3.1.1. Favipiravir

Favipiravir is a guanosine analogue inhibitor of RNA-dependent RNA polymerase. It has been marketed in Japan as a treatment for influenza under the brand name Avigan (Fujifilm Toyama Chemical Co, Ltd.) Later, after the outbreak of SARS-CoV-2, favipiravir was entered into a trial drug for COVID-19 and in Feb 2020 was granted the status of a repurposed anti-COVID-19 drug in China (Favilavir, Zhejiang Hisun Pharmaceutical) https://www.pharmaceutical-technology.com/news/china-favilavir-testing-approval/. Later in 2020, favipiravir was approved in Italy for experimental use against COVID-19 [13]. Favipiravir is administered orally and its use is prescription-only. There have been reports that favipiravir may provide advantage in mild cases of COVID-19, especially in patients with fever [27, 62], but the drug is still not considered efficacious enough. The official statement of Fujifilm Toyama Chemical Co. is: ”… at this stage, clinical application of Avigan Tablet to treat Coronavirus disease (Covid-19) is under study and preparation in order to obtain clear evidence of the drug’s efficacy and safety” [https://www.fujifilm.com/fftc/en/avigan].

In 2020, a novel way of delivery of favipiravir directly to the pulmonary epithelium by means of a nanoemulsion was proposed [55]. In 2021, solid lipid nanoparticles have been developed for the purposes of local delivery of favipiravir by nebulization [64]. These trials are exclusively in in vitro settings. In vivo studies in the field have yet to come.

3.1.2. Molnupiravir (Lagevrio by Merck)

Molnupiravir is a pyrimidine ribonucleoside analogue that inhibits RNA-dependent RNA polymerase of SARS-CoV-2 [46]. Similarly to favipiravir, molnupiravir was initially developed for treatment of influenza. The Emergency Use authorization (EUA) for molnupiravir was granted by the Federal Drug Agency of US at Dec 23 2021, on the next day after the EUA of Paxlovid (see below) was granted [https://www.fda.gov/news-events/press-announcements/coronavirus-covid-19-update-fda-authorizes-additional-oral-antiviral-treatment-covid-19-certain]. Molnupiravir is administered orally. Its use is prescription-only and is indicated in non-hospitalised patients with “…mild-to-moderate coronavirus disease in adults with positive results of direct SARS-CoV-2 viral testing, and who are at high risk for progression to severe COVID-19, including hospitalization or death, and for whom alternative COVID-19 treatment options authorized by the FDA are not accessible or clinically appropriate”. At present, the EUA for Molnupiravir does not extend its use in children. Treatment with Molnupiravir must be initiated within five days of symptom onset in eligible patients. Current studies report that molnupiravir may reduce the risk of hospitalization or death by 50% in patients at risk [37, 25, 69]. The incidence of drug-related adverse events is comparable to the incidence occurring with other treatments [69]. Molnupiravir is one of the anti-COVID drugs that are considered for development in the direction of use of nanotechnologies to ensure better delivery and higher bioavailability [25], although trials of novel nano-formulations are yet to come.

3.1.3. Remdesivir

Remdesivir (Veklury, Gilead Sciences) was among the first specific treatments that exhibited significant effects in patients with COVID-19. Remdesivir was initially developed for treatment of hepatitis C and was found to be effective in the suppression of replication of virus RNA in animals infected with the Ebola virus [68] but inferior to other therapeutics for Ebola in humans [43]. It is an adenosine analogue that binds to the viral RNA-dependent RNA polymerase and inhibits viral replication by premature termination [21]. Unlike most other antivirals for COVID-19, Remdesivir is only used parenterally, as a slow IV infusion. Also, unlike other antivirals used against COVID, Remdesivir is indicated for use in hospital settings and in patients with severe disease only. It was initially approved for use in hospitalised adult and paediatric patients (provided they were aged ≥12 years and weighed  ≥40 kg) with COVID-19 that had severe disease and respiratory failure (evidenced by need to use supplemental oxygen) and were already on dexamethasone [https://www.covid19treatmentguidelines.nih.gov/management/clinical-management/hospitalized-adults–therapeutic-management/].

Those eligible for treatment with Remdesivir must have documented COVID-19 evidenced by positive results of direct SARS-CoV-2 testing (although, in cases where there is no positive test, evidence from radiographic or CT scan image such as ground-glass lung opacities may be enough for justification of the use of Remdesivir). Later, Remdesivir was approved for use in smaller children and in certain groups of non-hospitalized patients [https://www.fda.gov/news-events/press-announcements/fda-takes-actions-expand-use-treatment-outpatients-mild-moderate-covid-19]. Remdesivir use is subjected to review in each individual case but is generally contraindicated in patients with liver and kidney failure and is only administered after a negative cutaneous skin test for hypersensitivity.

Few research papers on use of nanotechnology to improve the delivery of remdesivir have been published so far. One, based on use of remdesivir-loaded nanovesicles made of poly(lactic-co-glycolic) apparently may serve as a base for creation of platform for improved delivery of remdesivir but results are yet not far away from the computer design [71].

3.1.4. Nirmatrelvir/Ritonavir (Paxlovid, Pfizer)

In late December 2002, the FDA issued an EUA for Pfizer’s combined anti-COVID drug Paxlovid (nirmatrelvir and ritonavir). Nirmatrelvir is a 3C-like protease inhibitor that inhibits the main viral protease 3CLpro that cleaves the polyprotein generated by translation of the viral mRNA in order to generate separate protein products [45]. It was developed onto the template of another covalent protease inhibitor, lufotrelvir. The latter was also previously a candidate drug in preclinical trials for treatment of COVID-19 [5]. Ritonavir, a well-known protease inhibitor commonly used in co-formulations in highly active antiretroviral therapy (HAART) for treatment of HIV infection works by inhibiting CYP3A. This results in decreased breakdown of nirmatrelvir by proteases and thereby increasing the its serum levels [40].According to a recent press release by Pfizer, Paxlovid reduced the risk of hospitalization or death by 89% [https://www.pfizer.com/news/press-release/press-release-detail/pfizers-novel-covid-19-oral-antiviral-treatment-candidate]. Paxlovid is administered orally and is indicated for the treatment of mild-to-moderate coronavirus disease (COVID-19) in adults and paediatric patients (12 years of age and older weighing at least 40 kilograms or about 88 pounds) that are at high risk for progression to severe COVID-19, including hospitalization or death [https://www.fda.gov/news-events/press-announcements/coronavirus-covid-19-update-fda-authorizes-first-oral-antiviral-treatment-covid-19]. For best results, treatment with Paxlovid must be initiated within five days of symptom onset. It is available by prescription only.

The studies of the effect of Paxlovid on the course of COVID-19 in high-risk patients are still scarce, calling for further studies to elucidate whether it really decreases the risk for hospitalization and death. One meta-analysis showed that patients on Paxlovid did experience lower incidence of adverse outcomes and that the rate of adverse events related to therapy with Paxlovid was close to the rate in the placebo group [69], but the amount of data is still deemed insufficient to draw conclusions on the efficacy and safety of the drug. Yet, nanotechnologies have not played a significant role in development of novel formulations of Paxlovid or its components.

3.1.5. Lopinavir/Ritonavir

Lopinavir/ritonavir (Kaletra or Aluvia by AbbVie) is another combo of protease inhibitors that has been tried in the treatment of COVID-19. It is a well-known and safe drug for treatment of HIV-infection. One of its known disadvantages is its poor water solubility, resulting in erratic bioavailability. In 2016, development of lopinavir granules that spontaneously produced drug-loaded self-assembling nanoparticles upon contact with water, greatly improving the delivery of the drug, was reported [48]. This could serve as a basis for further development of nanoparticle-based antivirals with improved safety profile and more effective delivery. Kaletra was, at one time, repurposed as a COVID-19 drug in hospitalized patients. Studies (including the RECOVERY study) show that the use of Kaletra in COVID-19 patients does not significantly decrease the risk for complications and death [53]. Later, Patel et al. demonstrated that the rate of adverse events was higher in the patients treated with lopinavir/ritonavir compared to patients receiving other treatments [60]. Therefore, its use is mostly discouraged at present.

3.2. Monoclonal antibodies (mAbs) for the treatment of SARS-CoV-2 infection and COVID-19

Monoclonal antibodies have been successfully used in the treatment of immune and auto-immune disease such as rheumatoid arthritis, psoriatic arthritis, ulcerative colitis and Krohn’s disease, some types of cancer, cytokine storms occurring in the course of infections and in transplanted patients, etc. The most common adverse effects of monoclonal antibodies are infusion-related reactions, including allergic reactions and anaphylaxis.

All monoclonal antibodies currently used in the therapy for COVID-19 are authorized for patients with mild and moderate disease that are at risk of developing severe complications. Most mAbs used for the treatment of SARS-CoV-2 infection are targeted towards structural proteins of the virus or may be directed against key players of the pro-inflammatory pathways that are inherent to the pathogenesis of systemic inflammation in COVID-19 (as is tocilizumab, see below). At present, only monoclonal antibodies that target the spike protein have demonstrated to have clinical benefit in infected patients [28]. Other monoclonal antibodies may be used as pre-exposure prophylaxis (for details, see below).

Several neutralising monoclonal antibodies have received EUAs from FDA for use in outpatient settings in patients with confirmed SARS-CoV-2. Four of these are used in combination (bamlanivimab and etesevimab; casirivimab and imdevimab), while sotrovimab, bebtelovimab and redganvimab may be used alone. Two antibodies are authorized for use as pre-exposure prophylaxis only.

3.2.1. Bamlanivimab and etesevimab (by Ely Lilly)

Bamlanivimab and etesevimab (generic only) bind to different, although overlapping epitopes in the spike protein receptor-binding domain of SARS-CoV-2. They are administered in an intravenous infusion. The BLAZE-1 trial showed a clinical benefit of the combination in patients with mild-to-moderate disease aged 2 years or older [12]. However, since the two combinations have shown reduced activity against the Omicron variant of concern, their use has been limited by the FDA [https://www.fda.gov/news-events/press-announcements/coronavirus-covid-19-update-fda-limits-use-certain-monoclonal-antibodies-treat-covid-19-due-omicron].

3.2.2. Casirivimab and imdevimab ((REGEN-COV by Regeneron, Ronapreve by Roche)

Casirivimab and imdevimab bind to non-overlapping epitopes of the receptor-binding domain of the spike protein.  The Human Medicines Committee of the European Medicine Agency (CHMP) stated that the combined preparation of Ronapreve significantly reduces hospitalisation and deaths in COVID-19 patients at risk of severe COVID-19 [COVID-19: EMA recommends authorisation of two monoclonal antibody medicines | European Medicines Agency (europa.eu)].

3.2.3. Sotrovimab (Xevudy by GlaxoSmithKline)

Sotrovimab targets an epitope in the receptor-binding domain of the spike protein that is highly conserved between the related SARS-CoV and SARS-CoV-2. So far, Sotrovimab has shown efficacy against the Omicron variant of concern (VOC) [https://www.fda.gov/media/149534/download]. It is administered within 10 days of symptom onset as a single IV infusion or subcutaneously in non-hospitalized patients with mild-to-moderate COVID-19 that are at high risk of clinical progression. Sotrovimab is only administered to adult patients or children aged ≥12 years and weighing ≥40 kg.

3.2.4. Bebtelovimab (generic, by Ely Lilly)

Bebtelovimab is another spike-protein targeting mAb that reportedly retains its activity against the Omicron variant. It was granted an EUA by the FDA in February 2022 for use in adult and paediatric patients wirh mild-to-moderate disease [https://investor.lilly.com/news-releases/news-release-details/lillys-bebtelovimab-receives-emergency-use-authorization]. Bebtelovimab is administered to as a single IV injection.

3.2.5. Tixagevimab and cilgavimab (Evusheld by Astra Zeneca)

Tixagevimab and cilgavimab bind to non-overlapping epitopes of the receptor-binding domain of the spike protein [10]. FDA has granted an EUA for the combination tixagevimab and cilgavimab for pre-exposure prophylaxis. Eligible for treatment are individuals that have not been infected with SARS-CoV-2 and have not been in recent contact with an infected person, but may be at high risk for an inadequate immune response to COVID-19 vaccination, or have a documented history of severe adverse reaction to an available COVID-19 vaccine or any of its components [https://www.fda.gov/news-events/press-announcements/coronavirus-covid-19-update-fda-authorizes-new-long-acting-monoclonal-antibodies-pre-exposure].

3.2.6. Regdanvimab (Regkirona, by Celltrion)

Regdanvimab is a human monoclonal antibody directed against the spike protein of SARS-CoV-2. It is administered parenterally in a single infusion. Regdanvimab is indicated for the treatment of adults with mild-to-moderate COVID-19 who do not require supplemental oxygen and who are at increased risk of progressing to severe COVID-19 [https://www.ema.europa.eu/en/documents/product-information/regkirona-epar-product-information_en.pdf]. The CHMP stated that  the use of Regkirona significantly reduces hospitalisation and deaths in COVID-19 patients at risk of severe COVID-19 [COVID-19: EMA recommends authorisation of two monoclonal antibody medicines | European Medicines Agency (europa.eu)].

3.2.7. Tocilizumab (Actemra, RoActemra by Roche)

Tocilizumab has been successfully used as a biological treatment for a variety of autoimmune diseases, including rheumatoid arthritis and systemic juvenile idiopathic arthritis. It is a humanized monoclonal antibody against the interleukin-6 receptor. Since IL-6 plays a major role in inflammation and is among the early inflammatory markers in COVID-19, it was initially proposed that targeting IL-6 could reduce the rate of deaths among hospitalised patients. At the time, it was believed that the main pathogenetic mechanism of COVID-19-related complications was the cytokine storm resulting from dysregulation of the immunity signalling pathways. Tocilizumab was granted an EUA by the FDA for the treatment of COVID-19 in the in June 2021 [https://www.fda.gov/news-events/press-announcements/coronavirus-covid-19-update-fda-authorizes-drug-treatment-covid-19]. Several large trials (RECOVERY, REMAP-CAP) showed that the reduction of deaths produced by use of tocilizumab was added to those produced by use of dexamethasone [73]. The initial enthusiasm, however, was soon tempered down, as significant transaminase elevation (a known complication of treatment with tocilizumab) was consistently occurring in most hospitalized patients with COVID‐19 treated with tocilizumab. Indeed, many patients experience transaminase elevation as a result of treatment for COVID-19, but the use of tocilizumab added to the risk of serious liver injury. In July 2020, Hoffmann-LaRoche announced that tocilizumab treatment did not improve clinical status for patients with COVID-19-associated pneumonia and did not reduce patient mortality: [Roche provides an update on the phase III COVACTA trial of Actemra/RoActemra in hospitalized patients with severe COVID-19 associated pneumonia”]. Thus, tocilizumab is not recommended for use in patients with COVID-19. In the spring of 2021, a trial of a combination preparation of favipiravir-tocilizumab encapsulated in mucoadhesive vesicles and administered via nebulizer was started [61]. So far, no encouraging results have come through.

3.3. Other drugs with potential for use in patients with COVID-19

3.3.1. SQAd/VitE nanoparticles

A study published in 2020 described the development of anti-inflammatory drug based on nanoparticles [11]. These nanoparticles were made by conjugating the natural compounds squalene (a compound with anti-inflammatory activities) and adenosine (a natural immunomodulator), encapsulating them together with another biological compound, α-tocopherol (an antioxidant). The authors proposed that their nano-based drug serve as a novel therapeutic approach for safe treatment of inflammation. The practical applications are yet to come.

3.3.2. Baricitinib

In November 2020, FDA issued an authorization for the use of the combination remdesivir-baricitinib in hospitalized adults and paediatric patients two years of age or older with COVID-19 and severe respiratory failure [https://www.fda.gov/news-events/press-announcements/coronavirus-covid-19-update-fda-authorizes-drug-combination-treatment-covid-19]. Baricitinib (Olumiant, by Ely Lilly) is a pre-existing drug based on inhibition of the janus kinase (JAK)-mediated signalling pathways. It is usually used as a second-line treatment of rheumatoid arthritis. Baricitinib was not initially authorized as a stand-alone treatment for COVID-19. Baricitinib plus remdesivir was shown in 2021 to be superior to remdesivir alone in patients with severe COVID-19, especially those notably among those receiving non-invasive ventilation and had a satisfactory safety profile [26]. In July 2021 the EUA for the combination was revised. Presently, baricitinib alone may be used for the treatment of COVID-19 [https://www.thepharmaletter.com/article/fda-authorizes-baricitinib-alone-as-treatment-for-covid-19, https://www.fda.gov/media/143822/download]

3.3.3. Fluvoxamine (Fevarin by Mylan and Luvox by Solvay Pharmaceuticals, Inc.)

Fluvoxamine is a selective serotonine selective reuptake inhibitor that has been in use in clinical psychiatry for decades. It is usually prescribed for major depressive disorder and obsessive-compulsive disorder (OCD) but may also have its use in anxiety disorders [17]. It has a good safety profile, with lower incidence of the typical adverse effects associated with SSRI use – headaches, anxiety, irritation, sexual and sleep problems and cardiovascular complications. Psychiatric drugs usually have multiple targets and many potential adverse effects. Thus, the potential for delivery of drugs specifically to the target site has always been a major issue in clinical psychiatry. Design of fluvoxamine maleate-loaded solid lipid nanoparticles have been reported in 2019, demonstrating high entrapment efficiency and effective release of the drug [29].

At least three randomized trials have been conducted in order to study the effects of fluvoxamine in the treatment of outpatients with COVID-19. The first (STOP COVID) – showed reduction of the rate of clinical deterioration in the patients treated with fluvoxamine [69]. The second was stopped for futility by the data safety monitoring board as the treatments effects were significantly lower than expected [69]. The third was the TOGETHER study, which showed in 2022 that fluvoxamine may result in reduction of the severity of COVID-19-associated symptoms [54]. Namely, when used orally by non-hospitalized patients with early diagnosed COVID-19 and high risk for complications, fluvoxamine in standard therapeutic doses apparently reduced the risk for hospitalisation and death [54, 69]. The effects of fluvoxamine in COVID-19 patients remain to be seen in further trials. An application for an EUA for fluvoxamine use in patients with COVID-19 was submitted to the FDA [https://www.medpagetoday.com/special-reports/exclusives/96431] but, by present day, there is no information about an EUA being granted.

4. Nanoparticles’ vaccines

4.1. History and basics of vaccination as a method of prevention of transmissible disease

Vaccines have become a main weapon in the arsenal of medicine against infectious disease since the late XVIII century, when Edward Jenner applied his practical observations on the apparent immunity to smallpox of those that have previously had cowpox. This was not, strictly speaking, Jenner’s own invention, as he must have been familiar with the Eastern practice of ‘variolisation’, which was introduced in Britain earlier in the century by Mary Wortley Montagu and the studies of Dr. John Fewster that prevented contraction of smallpox by previously infecting his test subjects with cowpox [63]. In any case, the glowing reputation of Jenner and high social status undoubtedly contributed to the positive reception of his vaccinal practices. In the XX century, vaccines were rapidly developed for a variety of common infectious diseases and became an obligatory part of the vaccination calendar throughout the world. Vaccines against endemic infectious diseases such as yellow fever, tick-borne encephalitis, Japanese encephalitis, typhoid fever, and others are also available and some are obligatory for travelers in areas with vaccine preventable endemic disease.

Vaccines work by presenting an antigen typical of the infectious agent to the immune system of the host without occurrence of infection. Thus, the host may build a potent mechanism of defense against the offending agent prior to encounter or at very early stages of infection. Vaccination and immunization are different terms that are, nevertheless, very commonly confused. Immunity against a certain agent may be innate or acquired and may be developed either after vaccination or after the host has met with the infectious agent and has run the natural course of the infectious disease. Vaccination involves presentation only of the antigen (or more than one antigen, as is the case of inactivated vaccines) to the host’s immune system without the risk of (or, as with live vaccines, at a very low risk) of contracting the associated disease.

4.2. Types of vaccines

Multiple types of vaccines may exist for a single disease. Historically, there have been four major types of vaccines:

• Inactivated (e.g. Salk vaccine against poliomyelitis, rabies vaccine, Hepatitis A vaccine, and others);
• Live attenuated (Sabin vaccine against poliomyelitis, vaccines against measles, mumps, infectious parotitis, and others);
• Toxoid vaccines (e.g. tetanus and diphtheria toxoids);
• Subunit vaccines (e.g. some of the vaccines against influenza (e.g FluMist Quadrivalent by MedImmune), vaccines against hepatitis B, and others). These include peptide polysaccharide and conjugate vaccines.

Recently, two more types of vaccines have been added to this list: namely, vector vaccines (e.g. Zabdeno (against Ebola) and several vaccines against SARS-CoV-2) and mRNA vaccines (against SARS-CoV-2).

For some of oldest vaccines (such as the polio vaccine), several types may have been tried (in the case of polio – a live attenuated or inactivated vaccine, which are administered via different routes). Different countries may have a different policy regarding the type of vaccine used on the general population and in special cases.  Up to the COVID-19 era, however, there have not been such a large choice of vaccines against the same infectious agent.

mRNA vaccines have shown a comparable or superior safety profile to most other vaccines and, when administered in at least two doses, provided highest levels of protection (94-95 %) against development of COVID-19 and associated complications [50, 3]. The humoral immune response induced by most currently used mRNA vaccines against SARS-CoV-2 tends to decrease beyond 6 months of the second dose which was temporally overcome by the use of booster dose [31]. Also, booster dose was reported to increase vaccine effectiveness in terms of about 75 % less COVID-19 related emergency care department visits and 80 % less hospitalizations [16].

4.3. Vaccines against SARS-CoV-2

Presently, authorized SARS-CoV-2 vaccines belong to three of the four major ‘classic’ types listed above or may be of the newer mRNA or vector types.

4.3.1. Vaccines developed using ‘classic’ techniques

Inactivated vaccines against SARS-CoV-2 are both vaccines developed by Sinopharm (Sinopharm BIBP and Sinopharm WIBP); Turkovac (by Health Institutes of Turkey), CoronaVac (SinoVac Biotech), Covaxin (by Bharat Biotech), QazVac (by Kazakh Biosafety Research Institute).

Subunit vaccines are usually based on presentation of the S (spike) protein to the immune system of the vaccinated host. Such are: EpiVacCorona (by VECTOR center of biotechnology, Corbevax (by Biological E. Ltd.), Novavax, Soberana and the yet unnamed Sanofi-GSK vaccine;

Live attenuated vaccines against SARS-CoV-2 are, notably, among the least popular choices among the vaccines against SARS-CoV-2. At present, the only live attenuated vaccine is COVI-VAC by Codagenix Inc., in a phase 1 trial.

4.3.2. Nanotechnology for development of vaccines against SARS-CoV-2 infection

The newer generation of vector vaccines is strongly represented in the list of anti-SARS-CoV-2 vaccines. Such are Oxford/Astra Zeneca’s Vaxzevria, the vaccine developed by Janssen-Cilag, Sputnik V, Convidecia (CanSino Biologics), and others.

mRNA-based vaccines have been in development for at least 20 years but until the pandemic spread of SARS-CoV-2, the effort was targeted mainly at development of anticancer vaccines and novel vaccines against influenza. Representative members of the mRNA vaccines are Comirnaty (by Pfizer-BioNTech) and Spikevax (by Moderna). Basically, they both create and boost the host’s immunity against SARS-CoV-2 via presentation of artificially created and modified mRNA encoding parts of the protein sequence of the S protein of SARS-CoV-2. Development of the mRNA vaccines is, par excellence, a nanotechnology, as the mRNA encoding the S protein is packed into nanoparticles (e.g. liposomes). The particles are taken up by dendritic cells and the mRNA is readily translated by the host ribosomes. The foreign protein is exported on the surface of the antigen-presenting cells and presented to the T cells in order to mount an immune response.

Liposomes are closed vesicles composed of one (or more) phospholipid bilayers. Their structure resembles the structure of cell membranes. The phospholipids may be natural or artificial origin. Various compounds may be encapsulated within the vesicles and may be delivered to a specific target tissue or in a time-dependent manner, allowing liposomes to be used for controlled drug delivery. The phospholipids in the bilayers may be modified in order to achieve the properties desired by the user – increased lipo- or hydrophilicity (depending on the target), slow or rapid disintegration of the vesicle upon reaching their target (in order to control drug release), etc. Polyethylene glycol (PEG)-conjugation (PEGylation) is commonly used in drug design. It increases their hydrophobic properties and prolongs the circulation time of the liposomes in living tissues [24] (although repeated administration may result in the exact opposite – see below). PEG3350 (polyethylene glycol [PEG])-2000]-N,N-ditetradecylacetamide) is a common ingredient in many routinely used preparations – e.g. tablets, injectable depot preparations, oral laxatives, and others. PEG3350-conjugated liposomes are currently used in both Comirnaty (Pfizer BioNTech) and Spikevax (Moderna) vaccines. When injected into muscle, the active substance forms a depot that is slowly released over several hours up to several days, allowing for migration of dendritic cells to the place of deposition and uptake, processing and presentation of the antigen in order to mount an immune response.

In general, liposomes are biocompatible and non-toxic carriers for drug delivery. They have been successfully used in vivo for drug delivery to specific targets, mainly in clinical oncology [34, 35]. However, modifications (including PEGylation) may result in increased immunogenicity of the resulting preparation. Anaphylactic and other immune reactions have been described for a variety of PEG-containing preparations (e.g. laxatives, depot steroids) [59]. mRNA vaccines usually are described as having excellent safety profile that is readily demonstrated by an overall 0.2 % incidence of adverse events after vaccination [https://www.cdc.gov/mmwr/volumes/70/wr/mm7002e1.htm]. Anaphylactoid reactions, however, have been reported both for Comirnaty and Spikevax [4, 8].  Presently, almost all cases of anaphylaxis after administration of the Pfizer-BioNTech vaccine are believed to be associated with the presence of PEG3350 in the preparation [18]. Since Moderna does not list PEG in the safety sheet for their Spikevax, it is difficult to know whether the reported cases of anaphylaxis after vaccination are linked to the PEG content or some other compound. At present, the official recommendation of FDA is: “If you are allergic to PEG, you should not get an mRNA COVID-19 vaccine…If you are allergic to polysorbate, you should not get the J&J/Janssen COVID-19 vaccine.” https://www.cdc.gov/coronavirus/2019-ncov/vaccines/recommendations/specific-groups/allergies.html

Liposomes and the related encapsulation technology are only early precursors to the much wider variety of technologies based on the use of lipid nanoparticles. Potential next steps are: improving target specificity, better control of drug release, development of stimuli-responsive liposomes (sensitive to triggers such as changes in temperature, pH, binding of specific ligand, etc.) and many others.  Use of nanotechnology undoubtedly improves the defense against the new pandemics at all levels and allows for creation of reliable, more robust and easily accessible diagnostic tests, personal protection equipment, drugs and vaccines.

---

Test LO 4.1

---

References

1. Albaz A., Rafeeq M., Sain Z., Almutairi W., Alamri A., Aloufi A., Almalki W., et al. (2021). Nanotechnology-based approaches in the fight against SARS-CoV-2. AIMS Microbiol, 7(4), 368-398.
2. Aydemir D. and Ulusu N. (2020). Angiotensin-converting enzyme 2 coated nanoparticles containing respiratory masks, chewing gums and nasal filters may be used for protection against COVID-19 infection. Travel Med Infect Dis, 37, 101697.
3. Baden L., El Sahly H., Essink B., Kotloff K., Frey S., Novak R., Diemert D., et al., and Zaks T, for the COVE Study Group. (2021). Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine. N Engl J Med, 384(5), 403-16.
4. Bigini P., Gobbi M., Bonati M., Clavenna A., Zucchetti M., Garattini S. and Pasut G. (2021). The role and impact of polyethylene glycol on anaphylactic reactions to COVID-19 nano-vaccines. Nature Nanotechnol, 16, 1169-1171.
5. Boras B. Jones R., Anson B., Arenson D., Aschenbrenner L., Bakowski M., Beutler N., et al. (2021). Preclinical characterization of an intravenous coronavirus 3CL protease inhibitor for the potential treatment of COVID19. Nature Communications, 12 (1), 6055.
6. Borkow G., Zhou S., Page T. and Gabbay J. (2010). A novel anti-influenza copper oxide containing respiratory face mask. PLoS One, 5(6), e1.
7. Campos E., Pereira A., de Oliveira J., Carvalho L., Guilger-Casagrande M., de Lima R. and Fraceto L. (2020). How Can Nanotechnology Help to Combat COVID-19? Opportunities and Urgent Need. J Nanobiotechnol 18, 1-23.
8. CDC COVID-19 Response Team and Food and Drug Administration. (2021). Allergic Reactions Including Anaphylaxis After Receipt of the First Dose of Moderna COVID-19 Vaccine — United States, December 21, 2020–January 10, 2021. MMWR Morb Mortal Wkly Rep, 70(4), 125-129.
9. Chua M., Cheng W., Goh S., Kong J., Li B., Lim J., Mao L., et al. (2020). Face Masks in the New COVID-19 Normal, Materials, Testing, and Perspectives. Review Research (Wash DC), 7286735. doi, 10.34133/2020/7286735. eCollection 2020.
10. Dong J., Zost S., Greaney A., Starr T., Dingens A., Chen C., Chen R., et al. (2021). Genetic and structural basis for SARS-CoV-2 variant neutralization by a two-antibody cocktail. Nature Microbiol 6 (10), 1233-1244.
11. Dormont F., Brusini R., Cailleau C., Reynaud R., Peramo A., Gendron A., et al. (2020). Squalene based multidrug nanoparticles for improving mitigation of controlled inflammation in rodents. Sci Adv, 6(23), eaaz5466.
12. Dougan M., Azizad M., Mocherla B., Gottlieb R., Chen P., Hebert C., Perry R., et al. (2021). A randomized, placebo-controlled clinical trial of bamlanivimab and etesevimab together in high-risk ambulatory patients with COVID-19 and validation of the prognostic value of persistently high viral load. Clin Infect Dis, ciab912.
13. Dube T., Ghosh A., Mishra J., Kompella U. and Panda J. (2020). Drugs, Molecular Vaccines, Immune-Modulators, and Nanotherapeutics to Treat and Prevent COVID-19 Associated with SARS-CoV-2, a Deadly Nanovector. Review Adv Ther (Weinh), 2000172.
14. El-Atab N., Qaiser N., Badghaish H., Shaikh S. and Hussain M. (2020). Flexible Nanoporous Template for the Design and Development of Reusable Anti-COVID-19 Hydrophobic Face Masks. ACS Nano, 14(6), 7659-7665.
15. Fan C., Li K. and Ding Y. (2021). ACE2 expression in kidney and testis may cause kidney and testis damage after 2019-nCoV infection.  Front Med (Lausanne), 13, 7:56389.
16. Ferdinands J., Rao, Dixon B., Mitchell P., DeSilva M., Irving S., Lewis N., et al. (2022). Waning 2-Dose and 3-Dose Effectiveness of mRNA Vaccines Against COVID-19-Associated Emergency Department and Urgent Care Encounters and Hospitalizations Among Adults During Periods of Delta and Omicron Variant Predominance – VISION Network, 10 States, August 2021-January 2022. MMWR Morb Mortal Wkly Rep, 71(7), 255-263.
17. Figgitt D. and McClellan K. (2000). Fluvoxamine. An updated review of its use in the management of adults with anxiety disorders. Drugs, 60 (4), 925-54.
18. Garvey L. and Nasser S. (2021). Anaphylaxis to the first COVID-19 vaccine – is polyethylene glycol (PEG) the culprit? Br J Anaesth, 126(3), e106-e108.
19. Giovannini G., Haick H. and Garoli D. (2021). Detecting COVID-19 from Breath, A Game Changer for a Big Challenge. ACS Sens, 6, 1408-1417.
20. Gong P., He X., Wang K., Tan W., Xie W., Wu P. and Li H. (2008). Combination of Functionalized Nanoparticles and Polymerase Chain Reaction-Based Method for SARS-CoV Gene Detection. J Nanosci Nanotechnol, 8, 293-300.
21. Gordon C., Tchesnokov E., Woolner E., Perry J., Feng J., Porter D. and Götte M. (2020). Remdesivir is a direct-acting antiviral that inhibits RNA-dependent RNA polymerase from severe acute respiratory syndrome coronavirus 2 with high potency. J Biol Chem, 295(20), 6785-6797.
22. Huang C., Wang Y. and Li X. (2020). Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet (London, England), 395(10223), 497-506.
23. Huang C., Wen T., Shi F., Zeng X. and Jiao Y. (2020). Rapid Detection of IgM Antibodies against the SARS-CoV-2 Virus via Colloidal Gold Nanoparticle-Based Lateral-Flow Assay. ACS Omega, 5, 12550-12556.
24. Harris J. and Chess RB. (2003). Effect of pegylation on pharmaceuticals. Nat Rev Drug Discov, 2(3), 214-221.
25. Imran M., Arora M., Asdaq S., Khan S., Alaqel S., Alshammari M., Alshehri M., et al. (2021). Discovery, Development, and Patent Trends on Molnupiravir, A Prospective Oral Treatment for COVID-19. Review Molecules, 26(19), 5795.
26. Kalil A., Patterson T., Mehta A., Tomashek K., Wolfe C., Ghazaryan V., Marconi V., et al., and ACTT-2 Study Group Members. (2021). Baricitinib plus Remdesivir for Hospitalized Adults with Covid-19. Randomized Controlled Trial N Engl J Med, 384(9), 795-807.
27. Krishnan H., Leema M., Gopika G., Hari Prasad P., Rajan A., Anil A., Dev A. and Pillai Z. (2021). SARS CoV-2, Progression and treatment protocols – An overview. Mater Today Proc, 46, 3144-3147.
28. Kumar R., Lee M., Mickael C., Kassa H., Pasha Q., Tuder R. and Graham B. (2020). Pathophysiology and potential future therapeutic targets using preclinical models of COVID-19. ERJ Open Res, 6(4), 00405-2020.
29. Kumar A., Mancy S., Manjunath K., Kulkarni S. and Jagadeesh R. (2019). Formulation and Evaluation of Fluvoxamine Maleate Loaded Lipid Nanoparticle. Int J Pharm Sci Nanotechnol, 12(4), 4593-4600.
30. Layqah L. and Eissa S. (2019). An Electrochemical Immunosensor for the Corona Virus Associated with the Middle East Respiratory Syndrome Using an Array of Gold Nanoparticle-Modified Carbon Electrodes. Microchimica Acta, 186, 224.
31. Levin E., Lustig Y., Cohen C., Fluss R., Indenbaum V., Amit S., Doolman R., et al. (2021). Waning Immune Humoral Response to BNT162b2 Covid-19 Vaccine over 6 Months. N Engl J Med, 385(24), e84.
32. Li H. and Rothberg L. (2004). Colorimetric Detection of DNA Sequences Based on Electrostatic Interactions with Unmodified Gold Nanoparticles. Proc Natl Acad Sci USA, 101, 14036-14039.
33. Liu S., Wang Z., Xie H., Liu A., Lamb D. and Pang D. (2020). Single-Virus Tracking, From Imaging Methodologies to Virological Applications. Chem Rev, 120, 1936-1979.
34. Liu D., Ju C., Han C., Shi R., Chen X., Duan D., Yan J., et al. (2021). Nanozyme Chemiluminescence Paper Test for Rapid and Sensitive Detection of SARS-CoV-2 Antigen. Biosens Bioelectron, 173, 112817.
35. Lu L., Ding Y., Zhang Y., Rodney J., Ho Y., Zhao Y., Zhang T. and Guo C. (2018). Antibody-modified liposomes for tumor-targeting delivery of timosaponin AIII. Int J Nanomedicine, 13, 1927–1944.
36. Maas M., Kim M., Malkani R., Abbott S. and Zee P. (2021). Obstructive Sleep Apnea and Risk of COVID-19 Infection, Hospitalization and Respiratory Failure. Sleep Breath, 25(2), 1155-1157.
37. Mahase E. (2021). Molnupiravir reduces risk of hospital admission or death by 50% in patients at risk, MSD reports. BMJ, 375, n2422.
38. Malone B., Urakova N., Snijder E. and Campbell E. (2022). Structures and functions of coronavirus replication–transcription complexes and their relevance for SARS-CoV-2 drug design. Nature Rev Mol Cell Biol 23, 21-39.
39. Martínez-Paredes G., González-García M.B. and Costa-García A. (2009). Genosensor for SARS Virus Detection Based on Gold Nanostructured Screen-printed Carbon Electrodes. Electroanal. Int J Devoted Fundam Pract Asp Electroanal, 21, 379-385.
40. McDonald E. and Lee T. (2022). Nirmatrelvir-ritonavir for COVID-19. CMAJ, 194(6), E218.
41. Mohammed S. and Shaaban E. (2019). Efficacious nanomedicine track toward combating COVID-19. Randomized Controlled Trial N Engl J Med, 381(24), 2293-2303.
42. Moitra P., Alafeef M., Dighe K., Frieman M. and Pan D. (2020). Selective Naked-Eye Detection of SARS-CoV-2 Mediated by N Gene Targeted Antisense Oligonucleotide Capped Plasmonic Nanoparticles. ACS Nano, 14, 7617-7627.
43. Mulangu S., Dodd L., Davey R. Jr., Mbaya O., Proschan M., Mukadi D., Manzo M., et al., and PALM Consortium Study Team. (2019). A Randomized, Controlled Trial of Ebola Virus Disease Therapeutics. N Engl J Med, 381, 2293-2303.
44. Nomura T., Nazmul T., Yoshimoto R., Higashiura A., Oda K. and Sakaguchi T. (2021). Ethanol Susceptibility of SARS-CoV-2 and Other Enveloped Viruses. Biocontrol Sci 2021, 26(3), 177-180.
45. Owen D., Allerton C., Anderson A., Aschenbrenner L., Avery M., Berritt S., Boras B., et al. (2021). An oral SARS-CoV-2 M(pro) inhibitor clinical candidate for the treatment of COVID-19. Science 374(6575), 1586-1593.
46. Painter W., Holman W., Bush J., Almazedi F., Malik H., Eraut N., Morin M., et al. (2021). Human Safety, Tolerability, and Pharmacokinetics of Molnupiravir, a Novel Broad-Spectrum Oral Antiviral Agent with Activity Against SARS-CoV-2. Antimicrob Agents Chemother, 65, e02428-20.
47. Park T., Lee S., Lee S., Park J., Yang K., Lee K.-B., Ko S., et al. (2006). Protein Nanopatterns and Biosensors Using Gold Binding Polypeptide as a Fusion Partner.  Anal Chem, 7197–7205.
48. Pham K., Li D., Guo S., Penzak S., and Dong X. (2016). Development and in vivo evaluation of child-friendly lopinavir/ritonavir pediatric granules utilizing novel in situ self-assembly nanoparticles. J Control Release, 226, 88-97.
49. Pini M., Cedillo González E., Neri P., Siligardi C., and Ferrari A. (2017). Assessment of Environmental Performance of TiO2 Nanoparticles Coated Self-Cleaning Float Glass. Coatings, 7, 8.
50. Polack F.P., Thomas S.J., Kitchin N., Absalon J., Gurtman A., Lockhart S., Perez J.L., et al., and C4591001 Clinical Trial Group. (2020). Safety and Efficacy of the BNT162b2 mRNA Covid–19 Vaccine. N Engl J Med, 383(27), 2603-2615.
51. Rashedi J., Poor B., Asgharzadeh V., Pourostadi M., Kafil H., Vegari A., Tayebi-Khosroshahi H., et al. (2020). Risk Factors for COVID-19. Infez Med, 28(4), 469-474.
52. Rasmi Y., Saloua K., Nemati M., and Choi J. (2021). Recent Progress in Nanotechnology for COVID-19 Prevention, Diagnostics and Treatment. Review Nanomaterials (Basel), 11(7), 1788.
53. RECOVERY Collaborative Group. (2020). Lopinavir-ritonavir in patients admitted to hospital with COVID-19 (RECOVERY), a randomised, controlled, open-label, platform trial. Randomized Controlled Trial Lancet, 396(10259), 1345-1352.
54. Reis G., Dos Santos Moreira-Silva E.A., Silva D.C.M., Thabane L., Milagres A.C., Ferreira T.S., Dos Santos C.V.Q., et al., and TOGETHER investigators. (2022). Effect of early treatment with fluvoxamine on risk of emergency care and hospitalisation among patients with COVID-19, the TOGETHER randomised, platform clinical trial. Lancet Glob Health, 10(1), e42–e51.
55. de M Ribeiro N. and Fonseca B. (2020). The role of pharmaceutical nanotechnology in the time of COVID-19 pandemic Review Future Microbiol, 15, 1571-1582.
56. Roh C. and Jo S. (2011). Quantitative and Sensitive Detection of SARS Coronavirus Nucleocapsid Protein Using Quantum Dots-Conjugated RNA Aptamer on Chip. J Chem Technol Biotechnol, 86, 1475-1479.
57. Shan B., Broza Y., Li W., Wang Y., Wu S., Liu Z., Wang J., et al. (2020). Multiplexed Nanomaterial-Based Sensor Array for Detection of COVID-19 in Exhaled Breath. ACS Nano, 14, 12125-12132.
58. Somvanshi S., Kharat B., Saraf S., Somwanshi S., Shejul S. and Jadhav K. (2020). Multifunctional Nano-Magnetic Particles Assisted Viral RNA-Extraction Protocol for Potential Detection of COVID-19. Mater Res Innov, 25, 169-174.
59. Stone C., Liu Y., Relling M.V., Krantz M., Pratt A., Abreo A., Hemler J. and Phillips E. (2019). Immediate hypersensitivity to polyethylene glycols and polysorbates – more common than we have recognized. J Allergy Clin Immunol Pract, 7(5), 1533‐1540.
60. Patel T., Patel P., Barvaliya M., Saurabh M., Bhalla D. and Khoslae P. (2021). Efficacy and safety of lopinavir-ritonavir in COVID-19. A systematic review of randomized controlled trials. J Infect Public Health, 14(6), 740-748.
61. Thakur V., Ratho R., and Panda J. (2021). Respiratory delivery of favipiravir-tocilizumab combination through mucoadhesive protein-lipidic nanovesicles, prospective therapeutics against COVID-19. Virusdisease, 32(1), 131-136.
62. Tiwaskar M., Dhar R., Talwar D., Ansari A., Lakhe M., Panchal S., Bhagat S., et al. (2022). Real-world Experience with Favipiravir for Treatment of COVID-19 among Indian Healthcare Professionals. J Assoc Physicians India, 69(12), 11-12.
63. Thurston L. and Williams G. (2015). An examination of John Fewster’s role in the discovery of smallpox vaccination.  J Royal Coll Phys Edinburgh, 45, 173-79.
64. Tulbah A. and Lee W-H. (2021). Physicochemical Characteristics and In Vitro Toxicity/Anti-SARS-CoV-2 Activity of Favipiravir Solid Lipid Nanoparticles (SLNs). Pharmaceuticals (Basel), 14(10), 1059.
65. Vaze N., Pyrgiotakis G., McDevitt J., Mena L., Melo A., Bedugnis A., Kobzik L., et al. (2019). Inactivation of Common Hospital Acquired Pathogens on Surfaces and in Air Utilizing Engineered Water Nanostructures (EWNS) Based Nano-Sanitizers. Nanomed. Nanotechnol Biol Med, 18, 234-242.
66. V’kovski P., Kratzel A., Steiner S., Stalder H. and Thiel V. (2021). Coronavirus biology and replication, implications for SARS-CoV-2. Nature Rev Microbiol, 19, 155-170.
67. Wang Q., Zhang Y., Wu L., Niu S., Song C., Zhang Z., Lu G., et al. (2020). Structural and Functional Basis of SARS-CoV-2 Entry by Using Human ACE2. Cell. 181 (4), 894-904.
68. Warren T., Jordan R., Lo M., Ray A., Mackman R., Soloveva V., Siegel D., et al. (2016). Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys. Nature, 531(7594), 381-385.
69. Wen W., Chen C., Tang J., Wang C., Zhou M., Cheng Y., Zhou X., et al. (2022). Efficacy and safety of three new oral antiviral treatment (molnupiravir, fluvoxamine and Paxlovid) for COVID-19: a meta-analysis. Ann Med, 54(1), 516-523.
70. Wu F., Zhao S., Yu B., Chen Y-M., Wang W., Song Z-G., Hu Yi., et al. (2020). A new coronavirus associated with human respiratory disease in China. Nature, volume 579, 265-269.
71. Wu J., Wang H. and Li B. (2020). Structure-aided ACEI-capped remdesivir-loaded novel PLGA nanoparticles, toward a computational simulation design for anti-SARS-CoV-2 therapy. Phys Chem Chem Phys, 22, 28434-28439.
72. Yeh Y-T., Tang Y., Sebastian A., Dasgupta A., Perea-Lopez N., Albert I., Lu H., et al. (2016). Tunable and Label-Free Virus Enrichment for Ultrasensitive Virus Detection Using Carbon Nanotube Arrays. Sci Adv, 2, e1601026.
73. Zarębska-Michaluk D., Jaroszewicz J., Rogalska M., Martonik D., Pabjan P., Berkan-Kawińska A., Bolewska B., et al. (2021). Effectiveness of Tocilizumab with and without Dexamethasone in Patients with Severe COVID-19, A Retrospective Study. J Inflamm Res, 14, 3359-3366.
74. Zhang H., Kang Z., Gong H., Xu D., Wang J., Li Z., Cui X., et al. (2020). Digestive system is a potential route of COVID-19, an analysis of single-cell coexpression pattern of key proteins in viral entry process. Gut, 69, 1010-1018.
75. Zhong H., Zhu Z., Lin J., Cheung C.F., Lu V.L., Yan F., Chan C.Y., et al. (2020). Reusable and Recyclable Graphene Masks with Outstanding Superhydrophobic and Photothermal Performances. ACS Nano, 14(5), 6213-6221.
76. Zhou Y., Liao X., Song X., He M., Xiao F., Jin X., Xie X., et al. (2021). Severe Adaptive Immune Suppression May Be Why Patients With Severe COVID-19 Cannot Be Discharged From the ICU Even After Negative Viral Test. Front Immunol, 12, 755579.

Summary

The novel generation of nano-carriers holds a great promise for effective delivery of drugs and vaccines to their targets with significantly lower risk for adverse effects, modulation of immune reactions to self and non-self antigens, and development of sensitive imaging technologies for early detection of human diseases. COVID-19 was (and presently is) a challenge to medicine as well as to science. Novel nanotechnology-based biosensors, diagnostic devices, antiviral drugs, and vaccines have been developed or repurposed in order to combat the modern pandemic. The outbreak of SARS-CoV-2 triggered the research and development sectors to work together to create novel, versatile, and efficient ways to prevent and treat human diseases that are likely to continue their rapid development beyond the present pandemic situation.

Key words/phrases: COVID-19, antivirals, monoclonal antibodies, vaccines, nanotechnology

1. SARS-CoV-2 and COVID-19 – an overview of origins of a modern pandemic and its effect on thinking in research and clinical medicine

Human infection with SARS-CoV-2 occurred a zoonotic spill-over in the weeks and months preceding the first reported case in early December 2019 [69]. On 31 Dec 2019, the World Health Organization (WHO) was informed about a rapidly growing number of cases of pneumonia of yet unknown origin in Wuhan City, China. The causal agent was identified by the local authorities on 7 Jan 2020 as a hitherto unknown member of the beta-coronavirus family. In the end of January 2020, the WHO Director General D-r Tedros Adhanom Ghebreyesus declared that the novel coronavirus outbreak is a public health emergency of international concern [https://www.who.int/news/item/30-01-2020-statement-on-the-second-meeting-of-the-international-health-regulations-(2005)-emergency-committee-regarding-the-outbreak-of-novel-coronavirus-(2019-ncov)]. Remarkably, at this time there were a total of about 100 cases and no deaths yet, but the virus has managed to spread in almost 20 countries outside China. Research on the potential of development of etiologic treatment and prevention started immediately, uniting the efforts of clinicians, scholars and biotechnological and pharmaceutical companies.

COVID-19 was (and presently is) a challenge to medicine as well as to science. Nevertheless, it is also an opportunity to learn (yet again) from Nature about the unlimited variety of mechanisms of evolution, the causal relationship between changes occurring on nano-and micro-scale and the magnitude of the effects they may have on the macroworld. This modern pandemic triggered the research and development sectors to work together to create novel, versatile and efficient ways to prevent and treat human disease that are likely to continue their rapid development beyond the present epidemic. A prime example is the development of vector and mRNA vaccines. By the end of 2019 there was, at best, one vector vaccine approved for use in humans (rVSV-ZEBOV, or Ervebo, by Merck) and no approved mRNA vaccines, although the first clinical trials for a rabies mRNA vaccine started in 2013.

The timeline of development of vaccines against SARS-CoV-2 is, indeed, striking, but not because they were developed ‘too fast’, rather because apparently all the knowledge was already there and ‘a big push’ was all that was needed to invent novel types of vaccines that could be safely and efficiently used to prevent severe disease in millions of people. Same applies to the development of safe and efficient anti-COVID-19 drugs. The antivirals that have shown significant effects in the treatment of patients with COVID-19 in present day (remdesivir, favipiravir, and others) are, in their majority, repurposed drugs that were previously tried in the treatment of other viral diseases. Thus, the novelty of the anti-COVID treatments of present day is not exactly in their principle of action on their targets, but rather in the manner in which these targets are addressed.

2. Development of versatile nano-scale carrier platforms

The well-tried conservative therapies work, very generally speaking, by saturating the environment of living cells with a drug so that the chance for its binding to the target (e.g. a cellular receptor) is greatly increased. The majority of drugs enter the living cells via a receptor-ligand interaction. As receptors of a certain type are expressed on more than one type of cell and the same drug ligand may bind to more than one type of receptor, there is a (sometimes significant) risk for adverse effects related to the effects of the drug on tissues different from the target tissue. This is especially true for cytostatic and psychiatric drugs. Thus, safe and effective drug treatments (and vaccines, for that matter) do not depend solely on the active substance in the drug but also on its carrier and the manner this carrier interacts with the target tissue.

Nanotechnology is… “the manipulation of matter on a near-atomic scale to produce new structures, materials and devices… using materials with a length scale between 1 and 100 nanometres…at which size materials begin to exhibit unique properties that affect [their] physical, chemical, and biological behaviour”, according to the definition provided by the Centre for Disease Control (CDC) in Atlanta, USA: https://www.cdc.gov/niosh/topics/nanotech/default.html#:~:text=Nanotechnology%20is%20the%20manipulation%20of,new%20structures%2C%20materials%20and%20devices.&text=Nanotechnology%20refers%20to%20engineered%20structures,between%201%20and%20100%20nanometers.

The size (comparable to the size of biological macromolecules) and the significant surface-to-volume ratio of the nano-scale particles increases their solubility and may be used to modulate the segregation of nano-carriers loaded with bioactive substances into different cell and tissue compartments (depending on the desired effect). The novel generation of nano-carriers for pharmaceuticals for human use ensure effective delivery of the active substances to the target with significantly lower risk for adverse effects. Nanotechnology may provide ways to overcome the nature-provided barriers in order to improve penetration of drugs in certain target sites (e.g. the blood-brain barrier), to modulate the immune reactions to self and non-self antigens and may assist in the development of sensitive imaging technologies for early detection of human disease. There are also many other applications of nanotechnology in modern research and development, some of which may be viewed in Fig. 1\

Figure 1. Some of the applications of nanotechnology in modern research and development.

The size of most viruses may vary between 20 and 250 nm, which makes nanotechnology-based technology an excellent option for the purposes of antiviral research.

SARS-CoV-2 possesses high infectivity potential (comparable to the infectivity of the influenza and measles virus). Roentgen imaging, computerized tomography and laboratory (molecular biology) tests are routinely used to assist screening for infections and in clinical diagnosis of COVID-19. X-ray and CT findings that are considered ‘typical’ for COVID-19 (disseminated interstitial changes, ground-glass opacities) lag significantly behind the onset of symptoms and may persist after the clinical status has improved. Somewhat paradoxically, the first laboratory test developed for COVID-19 was the highly sensitive but elaborated, expensive and time-consuming RT-PCR test. PCR-based tests for detection of disease are, in essence, nanotechnology-based test devices (although the technology dates back to the 90-ties of the XX century). Antigen tests for SARS-CoV-2 (which are another example of nanotechnology-based tests) were developed later in order to speed up the diagnostics and make it easily available for patients and carers, but besides the common limitations of using nasopharyngeal and throat swabs, they have additional issues such as varying brand-to-brand sensitivity and specificity. Serological tests (for the presence of IgG, IgM and, sometimes, IgA) may reveal present and past SARS-CoV-2 infections but are less applicable for new infections, as they must be performed with significant delay from the onset of symptoms and may be unreliable in the case of previously vaccinated symptomatic patients. Biosensors (another nanotechnology-based type of devices) have been developed for the purposes of diagnosis relatively early (first similar report by Seo et al. was published in June 2020) [76] but are still mainly for research use due to their high cost.

The size of SARS-CoV-2 virion is about 100 nm (medium range). Thus, the potential of COVID-19 as a target for nanoparticles-based treatments has been considered from the very beginning of the pandemic. So far, nanotechnology-based approaches have been developed and tried for the purposes of prevention of infection, diagnostics and treatment of COVID-19.

Several nanotechnology-based devices have been developed for the sensitive and specific detection of COVID-19. At present, they augment the routinely used approaches based on RT-PCR and antigen tests. A novel biosensor based on gold nanoparticles has been proposed for detection of SARS-CoV-2 in exhaled air [75, 78, 27]. Magnetic nanoparticles were used in a specialised extraction protocol for SARS-CoV-2 [81, 97]. Nanoscale-sized crystals with semiconductor properties (commonly known as quantum dots) have been successfully used in a biosensor for detection of SARS-CoV-1 nucleocapsid protein antigen [70] and may well be used for detection for SARS-CoV-2 [40]. It could be expected that nanomaterial-based detection devices, sensors, drugs and vaccines will be the mainstay of the management of the pandemic in the foreseeable future [65, 50].

3. Nano-based delivery systems for antiviral drugs

3.1. Nanotechnology formulations – types and potential applications in the treatment of viral infections

A nanopharmaceutical, is, by definition, a material with particle size in the nanoscale range that has therapeutic potential. Nanoparticles may have different shapes and chemical compositions. The active compound in the nanoparticle may be dissolved, entrapped, encapsulated, adsorbed, conjugated or chemically attached, thereby the vehicle of the active compound is referred to as a nanocarrier. A schematic representation of the main types of nanocarriers is shown on Fig. 2.

Figure 2. Types of nanocarriers.

The most common types of nanocarriers are briefly listed below. Special attention is paid to liposomes, as they were the first and are, at present, the most commonly used type of drug nanocarrier.

3.1.1. Lipid-based nanoformulations

Lipids are, by far, the safest type of carriers. They are easier and cheaper to produce, are biodegradable, biocompatible, non-toxic, and, generally, non-immunogenic [61]. The lipids in the formulation are used together with other agents such as surfactants and solvents. Commonly, used lipid-based nanoformulations include liposomes, solid lipid nanoparticles (SLN), nanoemulsions, and nanosuspensions.

Liposomes

Liposomes are vesicles in which an aqueous core is entirely enclosed by a membranous lipid bilayer composed of natural or synthetic phospholipids. Depending on the method of preparation, lipid vesicles can be multi-, oligo- or unilamellar, containing many, a few, or one bilayer shell(s), respectively. The diameter of the vesicle may vary widely – between 10 and 1000 nm.

The aqueous core of the first-generation (conventional) therapeutic liposomes contained the hydrophilic bioactive compound. Later-generation liposomes may entrap hydrophobic compounds as well and may carry additional modifications to increase the time of circulation in vivo and to ensure targeted and timed delivery of the medicinal agent [37]. A schematic of the different types of liposomes may be seen in Fig. 3.

Figure 3. A representation of the different types of liposomal drug delivery systems.

Liposomes are prepared from an aqueous solution of phospholipids by a variety of methods: ultrasound sonication, dehydration-rehydration, reverse phase evaporation, freeze-thaw cycles, vesicle extrusion, etc. [31]. The resulting entrapment efficiency may be very different. Optimal control in the size and lamellarity of the resulting liposomes is usually achieved by extrusion using filters with pores of defined size. Targeting therapeutic liposomes to the desired site is achieved by adding ligands to the surface of the vesicle [37].

Liposomes have their drawbacks as nanocarriers. Among these are their relatively low drug-loading capacity and instability, as well as potential risk of immunogenicity due to various adjuncts (e.g. PEG3350) [11].

At present, several liposomal formulations are in clinical use. Among the applications are targeted delivery of cytotoxic agents, intraocular applications, post-irradiation treatments for patients with extreme UV sensitivity with preparations of T4 endonuclease V [95, 13, 49].

Polyethylene glycol (PEG)-conjugated liposomes are currently used in both Comirnaty (Pfizer BioNTech) and Spikevax (Moderna) vaccines.

Solid lipid nanoparticles (SLNs)

SLNs are composed of a solid lipid matrix (triglycerides, steroids, fatty acids, waxes, etc.). Unlike liposomes, SLNs are industrially scalable. SLNs have a history of use as carriers for a variety of antiviral drugs (ritonavir, maraviroc, darunavir, efavirenz, zidovudine, lopinavir, dolutegravir and others- for details see below).

A later generation of SLNs are the nanostructured lipid carriers (NSLCs). Unlike SLNs, the lipids in the NSLCs are in liquid state, conferring increased stability and improved controlled release pattern.

Nanoemulsions

Nanoemulsions (NE) are globular single-phase systems consisting of emulsified oils, water and surfactants. NEs have a high loading capacity, increased hydrophilicity and enhanced bioavailability. Nanoemulsions have been used to develop formulations of anti-HIV drigs such as protease inhibitors (saquinavir, indinavir) [90].

Self-nanoemulsifying drug delivery systems (SNEDDS) are another type of formulation of single-phase oil-water-surfactant systems that are used for carriers of hydrophobic drugs, such as the NNRTI drug nevirapine (see below) [Selvam and Kulkarni, 2014]. To date, a nanoemulsion [68] and a suspension of solid lipid nanoparticles [87] containing the antiviral drug favipiravir have been developed for targeted delivery to the pulmonary epithelium. So far, the results obtained in vitro studies are encouraging.

3.1.2. Polymer-based nanoformulations

A variety of natural and synthetic polymers may be used in the preparation of nanoformulations. Hydrophilic polymers may include gelatin, albumin, alginate, dextran, chitosan, agarose and others. Hydrophobic polymers used in nanoformulations may include polylactic acid (PLA), polylactide-co-glycolic acid (PLGA), polystyrene, polycaprolactone (PECL), polymethacrylate (PMMA) and others. Surface-modifications may be introduced in order to improve the pharmacokinetics, reduce potential immunogenicity and introduce stimulus-triggered release of the drug in response to changes in pH, temperature and other stimuli. Special polymers inhibiting P-gp and other multidrug resistance (MDR) proteins have been developed specifically for the purposes of oncology.

Polymer-based nanoformulations includes polymeric micelles, polymeric solid nanoparticles, nanocapsules, and nanospheres.

Polymeric micelles

Polymeric micelles are nano-sized structures made of amphiphilic copolymers, each structure (unimer) consisting of a hydrophobic and a hydrophilic subunit. Unimers associate to form structures with a hydrophobic core and a hydrophilic shell called micelles. The hydrophobic core contains drugs with poor aqueous solubility while the hydrophilic shell drug cargo is responsible for the transport and targeted release of the drug.

The hydrophobic core-forming polymers may be polyesters, polycaprolactone, poly(l-amino acids) and others. Polyethylene glycol is usually the major component of the hydrophilic shell. Various modifications such as adding sialic acid residues to the hydrophilic shell have been tried. In one study, sialylated outer shell was used to bind the haemagglutinin of the influenza virus in order to prevent viral entry [1].

Polymeric nanoparticles

Polymeric nanoparticles may be manufactured with a variety of natural or synthetic polymers. Nanocapsules are polymeric nanoparticles that entrap the drug into their core. When the drug is adsorbed onto the surface or embedded in the matrix of the nanoparticle, the structure is termed as a nanosphere. Polymeric nanoparticles have been used for developing newer formulations of anti-HIV drugs, such as efavirenz, lopinavir, ritonavir, and others.

Polymer drug conjugates

Polymer drug conjugates are comprised of a polymer and a covalently bound drug. The drug is typically a small molecule but may be a larger molecule, e.g. a protein. Most applications of polymer drug conjugates are in clinical oncology (e.g. daunorubicin, doxorubicin, methotrexate, melphalan) and ophtalmology (e.g. daunorubicin) [62, 20] but also in antiviral therapy. PEGylated interferons were, for a long time, the only highly effective treatment of HCV [17]. Zidovudine (AZT) conjugates typically exhibit longer plasma half-lives than conventional AZT [38]. In 2018, Andersen et al. used N-(2-hydroxypropyl) methacrylamide (PHPMA)-albumin copolymer for delivery of a combination of anti-HIV drugs of ART to primary human T cells with very promising results.

Nanocapsules

A nanocapsule is comprised of a core (where the drug is entrapped) and a shell. Nanocapsules may be loaded with significantly larger amounts of drug than liposomes and may be used for controlled and targeted drug delivery. Nanocapsules of poly(iso-butylcyanoacrylate) core, entrapping AZT triphosphate and polyethyleneimine shell have been designed.

Nanospheres

Nanospheres are spherical structures where the drug is not enclosed within but, is, rather, dispersed in the matrix. Nanospheres are smaller in size than nanocapsules and may be subjected to rapid drug clearance. Topical formulations of acyclovir in nanospheres has been tried for treatment of infections caused by herpes simplex virus.

Lipid-polymer hybrid nanoformulations

More often than not, a single drug is not enough for treatment of a viral infection.  On the other hand, patient compliance decreases when the patient is asked to take more than two drugs. Thus, more than one drug with different physicochemical properties may need to be delivered by single delivery vehicle. This may be achieved using lipid-polymer hybrid nanoparticles. Several hybrid nanocarrier systems have been developed, including polymer core-lipid shell nanoparticles, hollow core/shell lipid–polymer-lipid hybrid nanoparticles, lipid bilayer-coated polymeric nanoparticles, and polymer-caged nanoparticles. Basically, hybrid lipid-polymer nanoformulations consist of an inner polymeric core enclosed in one or more outer layers of lipid or lipid-polymer layers. Polymer-caged nanoparticles are somewhat different, as they are based on liposome technology with the liposome surface being modified by cross-linkage with polymers. Lipid-polymer hybrid nanoformulations exhibit very high capacity for drug loading and high encapsulation efficiencies.

A variant of lipid-polymer hybrid nanoformulations are biomimetic lipid-polymer hybrid nanoformulations, in which the surfaces of the nanoparticles are modified so that they mimic cell surface proteins. This category comprises virus-like particles (VLPs) and virosomes (see below).

Stimuli-based lipid-polymer hybrid nanoparticles are yet another variety of lipid-polymer hybrid nanoformulations that are capable of releasing the encapsulated drug in response to various stimuli such as pH, temperature, and magnetic field. Thus, timed delivery to the desired site is feasible. In 2012, methylcellulose stearate was proposed as a thermosensitive nanocarrier for slow intravaginal delivery of tenofovir [38]. Similarly, a hybrid thermosensitive hydrogel was developed for the co-delivery of theaflavin (an antioxidant, which is hydrophilic) and nifeviroc (an antiviral, which is hydrophobic) for intravaginal application as a pre-exposure prophylactic of transmission of HIV. Thermosensitive hydrogels have also been tried for intranasal delivery of antiviral drugs.

Nanoparticles may have different sizes and shapes. Shape-based lipid-polymer hybrid nanoformulations are based on the observation that shape of a nano-scaled particle may have significant impact on cell-cell interaction, drug uptake, and biodistribution [8]. For example, non-spherical nanoparticles have been found to provide higher therapeutic efficacy in comparison with spherical particles in the treatment of cancer [74]. Thus, different shapes and builds have been explored in order to improve the pharmacokinetic properties of drugs.

3.1.3. Dendrimers

Dendrimers are highly branched three-dimensional synthetic nano-architectures, 2–10 nm in diameter consisting of a central core, an inner shell, and an outer shell carrying various modifications [83]. A schematic of a very simple dendrimer may be seen in Fig. 4.

The dendrimer core allows for entrapment of various molecules and the functional groups on the surface allow interaction with the desired targets. Dendrimers exhibit longer circulation times, and enhanced solubility and stability and targeted delivery. Dendrimers are synthesized by sequential addition of building blocks molecules to an initiator molecule. Commercially available, at present, are dendrimers made of polyamidoamine, polypropylene imine, and poly-l-lysine dendrimers. The outer shell may be covalently modified (e.g. esterified, glycosylated, etc.) or conjugated to proteins, peptides, etc. Polyanionic carbosilane dendrimers have been used as topical microbicides for barrier protection against HIV and herpes simplex viruses. García-Gallego et al. in 2015 demonstrated that carbosilane dendrimers may inhibit the internalization of HIV-1 into the epithelial cells and the entry into peripheral blood mononuclear cells. Thiolated dendrimers exhibited sustained release of acyclovir.

Figure 4. Polyethylenimine dendrimer generation.

3.1.4. Carbon-based nanoformulations

Carbon-based nanoformulations comprise carbon nanotubes, graphene oxide nanoparticles, and fullerenes.

Graphene

Graphene is a two-dimensional planar derivative of graphite. For biomedical purposes, graphene-based materials are modified with various functional groups to improve biocompatibility and to reduce toxicity. Both hydrophilic as well as hydrophobic drugs may be encapsulated. The functional groups may be designed to provide sites for attachment of diverse biological molecules.

Carbon nanotubes (CNTs) are hollow cylindrical nanotubes with walls made of one or more graphene sheets. The cylinder may be capped with fullerene on one or both ends. CNTs have excellent properties in terms of are drug loading and potential for controlled release but their medical applications are quite limited due to pulmonary toxicity and high hydrophobicity. Newer types of CNTs exhibit decreased toxicity and increased biodegradability.

Fullerenes

Fullerenes are carbon structures forming a nano-sized hollow cage. Fullerenes have been found to inhibit HIV, HCV and influenza virus replication by steric blocking  and inhibited the expression of viral nucleoprotein.

3.1.5. Inorganic nanoformulations

Quantum dots (QDs) are nanocrystals with semi-conductor properties. They are comprised of an inorganic core (made of silicon, cadmium selenide, cadmium sulphide, or indium arsenide, responsible for the semi-conductor properties), a hydrophilic shell, and a cap. Quantum dots find their applications predominantly in biomedical imaging, as they can absorb and emit light at preset wavelengths. As drug-carrier systems, modified QDs have been used in the treatment of HIV [42].

Metal and metal oxide nanoparticles

Metal nanoparticles may have microbicidal and antiviral activity of their own. Such are silver, gold, copper, titanium, cerium oxides and others. The surface of metal nanoparticles may be functionalized with various groups in order to improve molecular interaction, bioavailability and release of the conjugated drug. Small interfering RNAs have been conjugated to AuNPs and the resulting formulation inhibited dengue virus replication and the release of the virion. Silver nanoparticles were found to inhibit haemagglutination and H1N1 influenza A virus-induced apoptosis in cell cultures. Silver nanoparticles conjugated with tannic acid were shown to reduce HSV-2 infection vitro and in vivo. Zinc oxide nanoparticles have been studied in mouse models of vaginal infections with HSV-2 with promising results with regard to suppression of viral activity.

The use of inorganic nanoparticles is largely limited due to concerns for toxicity [43]. Newer formulations are being developed, but clinical trials in humans are still severely limited.

3.1.6. Nucleic acid-based nanotechnologies

Aptamers

Aptamers are, basically, short nucleic acid (oligonucleotides) or protein sequences (peptides) that can specifically bind to other molecules such as other nucleic acids and proteins [34]. In a specific third category fall X-aptamers that are a combination of natural and chemically-modified DNA or RNA oligonucleotides. Aptamers may have thiophosphate backbone to enhance nuclease stability and binding affinity [45].

Aptamers have been tried for therapeutic potential for at least 20 years. For example, PEGylated aptamers have been used to target vascular endothelial growth and the resulting preparation (pegaptanib, Macugen by Bausch & Lomb) has been approved as an intravitreal injection factor in macular degeneration [85]. Aptamers targeting key coagulation cascade members such as factor IXa, thrombin, and von Willebrand factor have been tested as potential anticoagulants [57].

Oncology is a very wide field for development of therapeutic aptamers.  Aptamers against nucleolin, prostate-specific membrane antigen (PSMA), carcinoembryonic antigen (CEA), MUC1, ErbB-2 and other proteins with significant roles in pro-carcinogenesis have also been investigated as cancer treatment strategies [72, 43, 22, 86]. Nevertheless, no aptamer has been approved for treatment of cancer yet.

In the field of infectious disease, aptamers have been found to inhibit various stages of HIV infection by binding to reverse transcriptase and integrase [19, 71]. The hemagglutinin of the influenza virus has been successfully targeted by aptamers [53]. Aptamer-based biosensors for SARS-CoV and SARS-CoV-2 been designed [16, 70; 63]. In 2021, aptamers were used by Sun et al. to prevent the binding of the spike protein to the ACE receptor [82].

Antisense nucleotides (ASOs)

Antisense nucleotides are short, single-stranded, artificially created nucleic acids that have been designed specifically to bind and inhibit mRNAs. ASOs with potential for applications to combat viral disease target mRNAs coding for proteins critically important for the deployment of the infective potential of the virus. The first approved member of this family was fomivirsen (Vitravene, by Novartis), a 21 bp oligodeoxyribonucleotide complementary to a sequence encoding the major early proteins responsible for cytomegalovirus (CMV). Vitravene was withdrawn ‘for commercial reasons’ from the market in 2002 [Public Statement on Vitravene (fomivirsen) (europa.eu)].

There are three generations of ASOs, differing in their chemical characteristics with regard to potency, pharmacokinetics, toxicity. First generation ASOs feature a phosphorothioate backbone in order to increase resistance to hydrolysis. Fomivirsen was a first-generation ASO. Second-generation ASOs are modified at the position 2 of the sugar moiety allowing them to act as steric blockers. One of the better known second-generation ASOs, Mipomirsen (Kynamro, by Genzyme) was rejected by the European Medicines Agency shortly after it was released due to severe adverse effects [http://www.fiercebiotech.com/regulatory/ema-committee-shoots-down-sanofi-s-cholesterol-drug-mipomersen]. Third-generation ASOs (e.g. eterlipsen, marketed as ExonDys by AVI BioPharma International for treatment of Duchenne’s muscular dystrophy) have superior nuclease resistance, increased target affinity and pharmacokinetics compared to previous generations.

In 2004, shortly before the first human beta-coronavirus outbreak, several second-generation ASOs against the causal agent SARS-CoV have been reported [56]. They were designed to target the open reading frame of the main polyprotein of the virus (ORF1) and its 5′-UTR regulatory sequence. In vitro, they demonstrated significant antiviral activity. Afterwards, ASOs targeting the translation initiation site of ORF1 and the site where frameshift occurs in order to transcribe more than one polyprotein (commonly termed as ‘pseudoknot’) were designed [1]. Later, AVI Biopharma developed ASOs targeting the 3-UTR of the viral RNA [AVI BioPharma’s NeuGene antisense drugs inhibit SARS coronavirus (pharmabiz.com)]. In 2020, a rapid and sensitive detection system for the presence of SARS-CoV-2 RNA based on ASOs bound to gold nanoparticles (AuNPs) was reported [51]. The ASOs belonged to the first generation and were specific for the nucleocapsid phosphoprotein gene of SARS-CoV-2. The authors claimed that their colorimetric naked-eye assay could detect several copies of the SARS-CoV-2 viral genome without need for special equipment.

Small interfering RNAs (siRNAs)

siRNAs are small double-stranded RNA molecules that show a promise in the treatment of COVID-19. Key members of the siRNA family are patisiran and givosiran that are presently used for treatment of genetic disease. Both patisiran and givosiran were first-in-class medications [9]. According to the definition by FDA, a ‘first in class’ is a drug that works by “new and unique mechanism of action” to treat a particular medical condition [FDA approves first-of-its kind targeted RNA-based therapy to treat a rare disease | FDA].

Patisiran (Onpattro, by Alnylam) is approved for threatment of polyneuropathy in patients with hereditary transthyretin (hTTR) amyloidosis [Onpattro | European Medicines Agency (europa.eu)]. Patisiran targets the mRNA of a mutant form of the TTR gene that produces a misfolded protein whose degradation products are deposited in many tissues and organs. A newer siRNA drug manufactured by Alnylam for treatment of hTTR amyloidosis is revusiran. Givosiran (Givlaari by Alnylam) is another siRNA drug used in the treatment of acute hepatic porphyria, a genetic disease caused by mutations in the ALAS1 gene. Givosiran is believed to decrease the incidence of attacks of acute porphyria in the affected patients and to decrease chronic pain, which is the cardinal symptom of AHP [Balwani et al., 2020]. One of the Ebola vaccines developed earlier, namely, TKM-Ebola (by Arbutus Biopharma Corporation) was comprised of siRNA encapsulated in LNPs. TKM-Ebola, however, did not reach later phases of clinical trials due to increased risk for allergic reactions.

Nanotechnology has already demonstrated its power for improvement of drug release with all three Alnylam siRNA formulations. Patisiran is delivered to the liver (where the mutant gene is normally expressed) in a lipid-nanoparticle formulation that increases nuclease resistance degradation and facilitates the delivery to the target cells. The siRNA in givosiran and revusiran is covalently linked to a ligand containing syalic acid residues to ensure better binding to the target and is packed into lipid nanoparticles for efficient hepatic delivery. siRNAs are transfected into cells using polymer carriers or cationic lipids (liposomes or lipid nanoparticles).

siRNAs packed in solid lipid nanoparticles have been highly effective against SARS-CoV-2 when used in animal models [33, 4]. The siRNA targets specifically the conserved regions of the SARS-CoV-2 genome. The formulation allows that the pharmaceutical is delivered preferentially to the lungs as a prime site for SARS-CoV-2 replication but by variation of parameters the authors have shown that the loaded nanoparticles may target the liver and the spleen and, possibly, other tissues. This versatility may be very valuable in COVID-19, as it is a multisystem disease and may extend its damage well beyond the lungs. Presently (as of Jan 2022), a database of siRNAs targeting SARS-CoV-2 is being established, containing more than 100 000 siRNAs ranging from 18 to 21 nucleotides in length [47]. This may be expected to speed up the process of design of novel siRNA-based drugs against COVID-19.

3.2. Nanoformulations developed specifically for the delivery of antiviral drugs

Modern antiviral treatment is very effective but has its limitations. Many patients will need a multidrug regimen, but antiviral drugs may interact with each other as well as with other prescription medicines. Some patients will require long-time treatment (COVID-19 makes no exception, as the incidence of long COVID syndrome reaches 40-50 % in some studies [54]). The antiviral drugs may have poor bioavailability and short half-lives, resulting in a need of multiple dosing and, respectively, poor patient compliance. Administration of higher doses in attempt to compensate for the low bioavailability may result in toxic effects. Development of drug resistance is common in patients who require treatment beyond several days or weeks. Many viruses may spread into sites that are typically inaccessible for most drugs (usually termed as virus sanctuaries, such as the CNS, lymphatic system, and synovial fluid. Lastly (but not in importance) is the fact that as the virus uses the host’s cellular apparatus to synthesize its proteins and nucleic acids, the differential selectivity of antiviral agents toward the virus and the target cells may be crucially important.

Nanotechnology-based novel platforms for drug delivery may assist in finding solutions to many of these issues. The largest amount of data we have about drug treatment of diseases caused by RNA-containing viruses are HCV and HIV-associated states and influenza.  Many drugs used successfully for treatment of HIV and HCV infections have been tried for activity against SARS-CoV-2 and several have been repurposed in COVID-19.

Antivirals approved for the treatment of HCV and HIV (and, some of them, for COVID-19) may, very broadly, be divided in several classes depending on their site of action and their principal mechanism:

1. Interferons (IFNs);
2. Entry blockers;
3. Nucleoside/nucleotide analogues;
4. Non-nucleoside analogues;
5. Protease inhibitors;
6. Integrase inhibitors;
7. Antisense nucleotides;
8. Small interfering RNAs (siRNAs);
9. Others.

The ASOs and siRNAs presently tested for activity against COVID-19 were already mentioned in the previous section.

The latter group, “Others”, is comprised of a variety of drugs that are normally used in the clinic for other purposes, such as antimalarials (e.g. hydroxychloroquine), antihelminthics (levamisole, ivermectin), antidepressants (fluvoxamine), etc. They may exhibit activities typical of the former groups (inhibitors of replication, protease inhibitors, etc.). Some of these, such as ivermectin, have antiviral activity in vitro, but the results in vivo have not come through yet or are not encouraging [39]. Others (such as bromhexine hydrochloride, with potential protease inhibitor activity) have shown some activity in vivo in terms of preventing symptomatic disease but the effects were not significant enough to justify more research in the field or the early-phase clinical trials have not been finished yet [48]. Chloroquine was shown to interfere with virus-endosome fusion of SARS-CoV, to inhibit the glycosylation of ACE2 receptors [93, 94] and to possess anti-inflammatory effects via inhibition of phospholipase A2 activity and blocking cytokine production and release [2], but significant antiviral effects have not been observed for SARS-CoV-2. Fluvoxamine, an antidepressant of the selective serotonine selective reuptake inhibitor (SSRI) group, initially showed more promise as a treatment for patients with COVID-19, but ongoing trials do not show anything except that larger studies may be needed to clarify whether there is an effect of fluvoxamine of disease severity [91]. A report about a fluvoxamine formulation containing solid lipid nanoparticles that reportedly ensured effective targeted release of the drug was published in 2019 [35]. No commercial preparation based on these studies has come out yet.

The COVID-19 pandemic caused significant psychological distress to those affected by the virus as well to those who have witnessed loved ones’ struggle with the disease. As is typical of such times, ‘wonder treatments’ emerged, widely proclaimed by the media. Most of these did not show, in the long term, any significant benefits to those taking them. Instead, many affected patients got worse after they used their ‘wonder cures’ and, respectively, missed the right time to seek proper treatment. Hydroxychloroquine, ivermectine, bromhexine and others all enjoyed their short periods of (yet) undeserved fame. None of them has shown significant clinical benefits and their use by patients with COVID-19 ought to be discouraged until reliable further studies have proven conclusively that they lower the rates of hospitalization and mortality.

3.2.1. Interferons

Interferons are signaling proteins normally produced by the immune system of the infected host and interfere (hence their name) with the replication of the virus within the cell. Previously used in the therapy of viral hepatitis, the capacity of IFNs to interfere with the synthesis of proteins needed for replication is, at present, utilized predominantly in the treatment of multiple sclerosis and various cancers. PEGylation of interferons as a basic nano-scale technology has been used to increase their clearance time in order to ensure that the drug binds effectively with its target [89]. PegIntron (by Merck, discontinued) is a commercial preparations of interferon alpha-2b that was used in the treatment of hepatitis B and C until 2016. Pegasys (Genentech) remains, to this day, the only PEGylated interferon approved for use by the FDA and EMA for treatment of hepatitis B in adults [https://www.natap.org/2005/HBV/051605_02.htm; https://www.ema.europa.eu/en/documents/product-information/pegasys-epar-product-information_en.pdf]. PEG-interferon alpha-2a (Roferone by Roche, used in the treatment of leukemia, malignant melanoma, polycythemia vera, essential thrombocythemia and others) is another interferon drug that was recently discontinued due to severe adverse reactions and the availability of safer drugs [https://pharmac.govt.nz/medicine-funding-and-supply/medicine-notices/interferon-alfa-2a/]. Interferon beta-1a (Rebif by Merck and Pfizer) is presently approved in the treatment of relapsing multiple sclerosis [https://www.pfizer.com/news/press-release/press-release-detail/emd_serono_and_pfizer_announce_fda_approval_of_rebif_rebidose_interferon_beta_1a#:~:text=announced%20today%20that%20the%20U.S.,of%20multiple%20sclerosis%20(MS).].

Gold nanoparticles have been studied as a potential carrier of interferon alpha for the clinical treatment of hepatitis C virus (HCV) infection [36]. A sustained drug delivery IFN-β-1a formulation based on nanoparticles composed of poly(lactic-co-glycolic acid and polymers was developed and tested in vitro in 2020 [24]. The authors report that the formulation increased the efficiency of interferon delivery and had the potential to decrease the associated adverse effects of IFN-β-1a. There have been numerous reports about trials of IFNs in the treatment of patients with COVID-19 [32, 64, 52]. IFN-alpha was shown to reduce viral shedding and suppress the inflammation associated with COVID-19, while IFN-beta was associated with faster viral clearance [46]. Sodeifian et al. point out that data from animal studies show that the administration of IFNs administration at the early stages of COVID-19 or in mild-to-moderate disease may have protective effect, but during the later stages and in severe disease may cause significant harm to the patients [80]. The effect of IFNs on mortality from COVID-19 may be reported as drastically different in different studies. One randomized controlled trial of the use of IFN-beta in patients with severe COVID-19 reported a lower mortality rate in the group receiving IFN, but the patients received other drugs as well, including standard antivirals (lopinavir/ritonavir, or atazanavir/ritonavir [44]. Another trial reported that the use of IFN beta showed no significant difference in the mortality [21]. Studies of the use of PEGylated IFN-alpha 2b as an addition to standard therapy showed that it resulted in rapid viral clearance and improved clinical status [12]. It is possible that the nanotechnology-based systems developed previously may be used for efficient delivery of antivirals in patients with COVID-19, but the controversial results from the use of interferons have prevented deployment of large clinical trials yet.

3.2.2.Nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs)r

NRTIs work by interfering with the replication of viral nucleic acid, usually presenting a nucleoside analogue that, upon being added to the growing polynucleotide chain, would terminate the elongation of the chain inhibit the replication of the nucleic acid of the virus. Reverse transcriptase inhibitors have been extensively studied in relation to their use in the treatment of HIV. Current clinical practice of antiretroviral therapy (ART) proposes first-line anti-HIV therapy being constituted by a combination of two (or more) NRTIs and a NNRTI or a protease or integrase inhibitor. Depending on the characteristics of the patient, an entry inhibitor may be added, although they are expensive, cause compliance issues (e.g. subcutaneous application two times a day, which may be problematic) and may have severe adverse effects. Lamivudine, stavudine, zidovudine, emtricitabine, zalcitabine, tenofovir, and others are some of the commonly used NRTIs.

NRTIs and NNRTIs are very efficient drugs. Nevertheless, they have their shortcomings, such as short half-life, high first-pass effect (resulting in low bioavailability), and variation in serum levels due to individual variation on the activity of the CYP system. A wide variety of technologies have been tried for improving the delivery and the stability of nucleoside analogues.

Nanotechnology has provided a number of highly efficient vehicles for delivery of NRTs used in the treatment of HIV infection. Liposomes, dendrimers, micelles, solid lipid nanoparticles, nanosuspensions, and polymeric nanoparticles have all been used to improve drug delivery in HIV [73, 29].

The data about the use of nanotechnology to improve the delivery of anti-COVID-19 antivirals is still sparse but the field is rapidly expanding.  Most of antivirals used in COVID-19 are NRTIs. Such are: favipiravir (a guanosine analogue); molnupiravir (a pyrimidine ribonucleoside analogue) and remdesivir (an adenosine analogue). Most of them are approved for patients with mild-to moderate disease at high risk of complications and must be started early in the course of the disease.  Only remdesivir is approved for treatment of hospitalized patients with severe disease and respiratory failure [https://www.covid19treatmentguidelines.nih.gov/management/clinical-management/hospitalized-adults–therapeutic-management/] although later it was approved for use in selected groups of outpatients also [https://www.fda.gov/news-events/press-announcements/fda-takes-actions-expand-use-treatment-outpatients-mild-moderate-covid-19]. In a study from 2020, HIV-positive patients receiving tenofovir/emricitabine exhibited a lower risk for COVID-19 and related hospitalization than those receiving other therapies [5]. Remdesivir-loaded polymer nanovesicles, a favipiravir nanoemulsion and favipiravir-loaded SLNPs intended to deliver the drug directly to the pulmonary epithelium have been developed and tried in vitro [68, 92, 87]. In vivo studies in the field have yet to come.

3.2.3. Non-nucleoside reverse transcriptase inhibitors (NNRTIs)

The non-nucleoside reverse transcriptase inhibitors (NNRTIs) directly inhibit the HIV-1 reverse transcriptase by binding in a reversible and non-competitive manner to the enzyme. The currently available NNRTIs are nevirapine (Viramune, by Boehringer Ingelheim), delavirdine (Rescriptor, ViiV Healthcare, and efavirenz (generic, by Mylan and Systiva, by Bristol Myers Squibb). Nano-based formulations have been tried to improve the bioavailability and the controlled release of NNRTIs. Manyarara et al. in 2018 developed a nevirapine nanoemulsion for pediatric uses. A self nanoemulsifying drug delivery system (SNEDDS) has been successfully developed for nevirapine in order to increase its bioavailability [60]. NNRTIs have been evaluated for anti-COVID-19 activity through molecular docking studies, but their clinical efficiency in COVID-19 remains to be proven [15, 25].

3.2.4. Entry inhibitors (fusion blockers)

Entry inhibitors, basically, prevent a virus from entering a cell by blocking the viral entry point, e.g. a preferred receptor. Presently, entry blockers such as maraviroc (Celzentry, by GlaxoSmithKline), enfivurtide (Fuzeon, by Genentech), bulevirtide (Hepcludex, by Gilead Sciences), and others) are used in the treatment of HIV and hepatitis D infections.  In 2015, a iron oxide nanoparticles coated with amphiphilic polymer were used for delivery of enfivurtide to the HIV sanctuaries in mice [23]. In 2020, polymer-lipid hybrid nanoparticles (PLN) loaded with a combination of a non-nucleoside reverse transcriptase infibitor (efavirenz) and enfivurtide were used to study the potential for delivery of the drugs to T-cells and macrophages sequestered in virus sanctuaries.

3.2.5. Protease inhibitors (PIs)

Protease inhibitors inhibit the protease that cleaves the viral polyprotein in order to form separate proteins. PIs are, similar to the above group of nucleoside analogues, are very well studied because of their common use in the treatment of HIV. Again, similarly to nucleoside analogues, PIs are subject to extensive first-pass metabolism and are substrates of CYP, hence the wide variance of the levels of the same drug in the same dosage taken by a different person. This is partly overcome by co-administering them with serum level enhancers (essentially, CYP3A blockers) such as ritonavir or cobicistat. Nanotechnology-based approaches have been developed in order to enhance the bioavailability of the most common PIs, such as lopinavir (solid lipid nanoparticles, polymer-based nanoparticles), atazanavir (nanoemulsion, nanosuspension), darunavir (lipid nanoparticles), nelfinavir (polymer-based nanoparticles) and others [55, 66, 88, 28, 79, 7, 18]. Saquinavir-conjugated quantum dots have been tried to improve bioavailability of the drug in virus sanctuaries [42].

Protease inhibitors are one of the exchangeable components of the standard antiretroviral therapy for HIV, as either protease or integrase inhibitor may be added to the reverse transcriptase blockers in ART. The PIs, however, seem to have risen to the heights of a novel drug for SARS-CoV-2 infection. In 2020, Beck et al. used a deep learning-based drug-target interaction prediction model to predict the potential anti-COVID-19 activity of a group of drugs of different classes that are used for treatment of HIV infections (remdesivir, efavirenz, ritonavir, and dolutegravir) [10]. They found that atazanavir had the highest affinity for the viral protease of SARS-CoV-2. A new oral drug (Paxlovid, by Merck) was only approved for use in patients with COVID-19 in December 2020. It consists of nirmatrelvir, a protease inhibitor and ritonavir, a well-known augmenting agent used in ART [59]. Undoubtedly, after enough data has been collected about the efficiency of the oral preparation, better and more efficient nanotechnology-based formulations will be developed shortly.

3.2.6. Integrase inhibitors

Integrase inhibitors work by preventing the insertion of a DNA copy of the RNA genome into the genome of the host cell. Typically, an integrase inhibitor such as dolutegravir (Tivicay, by ViiV Healthcare), elvitegravir (part of the fixed-dose combination Stribild, by Gilead Sciences) or raltegravir (Isentress, by Merck) is a key component in ART for HIV infections. Nanoformulations of integrase inhibitors have been developed in order to increase their solubility and for controlled delivery. Myristoylated dolutegravir was proposed as a long-acting formulation against HIV infection with slow-release potential [90]. In 2015, it was demonstrated that gold nanoparticles are readily internalized by lymphocytes and macrophages and an AuNP-conjugate of raltegravir was proposed that could inhibit the replication of HIV in peripheral mononuclear cells [26]. In 2020, dolutegravir was identified to have a potential inhibiting activity for the viral protease of SARS-CoV-2 [10]. The studies in the field are ongoing.

4. Virus capsids used for drug delivery

Virus-like particles (VLPs) are self-assembling particles that are formed by incorporating viral proteins of the capsid or the viral envelope into various naturally occurring proteins like ferritin or bacterial encapsulin. The proteins are typically produced in heterologous systems. VPLs may potentially be used in a wide range of applications, including development of drugs, immunotherapies, vaccines, gene therapies, imaging and others. Virosomes are VLPs that feature an additional phospholipid bilayer that incorporates the viral envelope glycoproteins (e.g. haemagglutinin). A schematic may be viewed in Fig. 5.

The proteins may be modified or conjugated with peptides, toxins, chemotherapeutics, siRNA, quantum dots, etc. in order to achieve targeting to a specific site and/or timing of release of the encapsulated agent.

Natural viral particles may, in response to triggers such as pH or temperature change, release units or parts of their capsid or envelope lacking the genome of the virus. Similarly, viral particles may be disassembled and reassembled into VLPs under specific conditions [58]. VLPs resemble the morphology of the wild-type viral particles, exhibit the same tropism and intracellular distribution, but do not include the nucleic acid of the virus that makes them, in fact, genome-free equivalents of viruses. Thus, the cargo of the vesicle (drugs, antibodies, fluorescent dyes, contrast agents, etc.) may be delivered to a specific type of the target cell using cell-cell interactions similar to the virus-cell interaction. VNPs may be used for development of novel materials such as catalysts, biomimetics or selectively targeted imaging agents [14, 58].

Figure 5. Schematic representation of a virus-like particle (the phospholipid envelope is not shown).

VLPs have been used to design vaccines against human disease since 2017, when Kanekiyo et al. created a haemagglutinin-ferritin fusion protein that would spontaneously self-assemble to create nanoparticles presenting trimers of haemagglutinin on its surface. Similarly, an anti-RSV vaccine has been developed later that presented the F-antigen of RSV [30].

Virus-like particles hold a great promise among the modern nano-based approaches to improve the safety and the efficiency of drugs. VLPs are, generally, safer to use than viral vectors in the process of vaccine development. A potential drawback of VLPs is their immunogenicity due to the presence of viral proteins. Since the proteins are produced in bacteria, however, their expression does not typically include post-translational processing that may actually decrease the risk for immune response following translation [58, 3].

Drugs with high toxicity may safely and efficiently be delivered to the target using VLPs. Ashley et al. used VLPs to deliver encapsulated doxorubicine to human hepatocellular carcinoma cells (Hep3B) that were known to use the Pgp efflux mechanism to expel chemotherapeutic drugs [6]. The results showed that the doxorubicine, cisplatine and 5-fluorouracil loaded viral particles could inhibit the growth of cancer cells at significantly lower levels of the drug.

Modification (functionalization, decoration) of the proteins in VLPs may increase their affinity to the target and improve the release of the drug. Decoration of VLPs with biotin moieties in order to ensure targeting via avidin-biotin interaction was reported in 2007 [67].

In 2020, three types of mRNA vaccine candidates based on VLPs were tested in mouse models. The VLPs presented the spike, membrane and the envelope protein of SARS-CoV-2. The expressed proteins were encapsulated in lipid nanoparticles and transfected into cultured cells, then VLPs were secreted by the cells in the culture medium. The results were encouraging, although the antibody levels of the vaccinated mice did drop rapidly shortly after the vaccination [41]. In 2022, Yilmaz et al. reported the first results of development and preclinical evaluation of VLP-based vaccine against SARS-CoV-2 tried in cultured cells ad in animal models. The VLPs expressed all 4 structural proteins of the SARS-CoV-2. The results showed that the vaccine triggered a potent T-cell response and significantly reduced viral load in the vaccinated infected animals [96].

Covifenz (by Medicago and GSK) is a vaccine based on VLPs. Its Phase III trials were finished in early February 2022. According to the manufacturers, Covifenz is already approved for use by Health Canada [Medicago’s Canada-made COVID-19 vaccine approved by Health Canada | Globalnews.ca].

As of February 2022, no VLP-based drug formulations for the treatment of COVID-19 are reported or approved. Future research about the possible applications of VLPs in the treatment of SARS-CoV-2 and the associated conditions may be needed before any products may be approved.

---

Test LO 4.2

---

References

1. Ahn D., Lee W., Choi J.K., Kim S.J., Plant E.P., Almazán F., Taylor D.R., et al. (2011).  Interference of ribosomal frameshifting by antisense peptide nucleic acids suppresses SARS coronavirus replication. Antiviral Res, 91(1), 1-10.
2. Al-Bari M. (2015). Chloroquine analogues in drug discovery: new directions of uses, mechanisms of actions and toxic manifestations from malaria to multifarious diseases. J Antimicrob Chemother, 70, 1608–1621.
3. Aljabali A., Hassan S., Pabari R., Shahcheraghi S., Mishra V., Charbe N., Chellappan D., et al. (2021). The viral capsid as novel nanomaterials for drug delivery. Future Sci OA, 7(9), FSO744.
4. Ambike S., Cheng C., Feuerherd M., Velkov S., Baldassi D., Afridi S.Q., Porras-Gonzalez D., et al., (2022). Targeting genomic SARS-CoV-2 RNA with siRNAs allows efficient inhibition of viral replication and spread. Nucleic Acids Res, 50(1), 333-349.
5. Del Amo J., Polo R., Moreno S., Díaz A., Martínez E., Arribas J., Jarrín I., et al., and The Spanish HIV/COVID-19 Collaboration. (2020). Incidence and Severity of COVID-19 in HIV-Positive Persons Receiving Antiretroviral Therapy: A Cohort Study Ann Intern Med, 173(7), 536-541.
6. Ashley C., Carnes E., Phillips G., Durfee P., Buley M., Lino C., Padilla D., et al. (2011). Cell-specific delivery of diverse cargos by bacteriophage MS2 virus-like particles. ACS Nano, 5(7), 5729-45.
7. Augustine R., Ashkenazi D.L., Arzi R.S., Zlobin V., Shofti R. and Sosnik A. (2018). Nanoparticle-in-microparticle oral drug delivery system of a clinically relevant darunavir/ritonavir antiretroviral combination. Acta Biomater, 74, 344-359.
8. Auría-Soro C., Nesma T., Juanes-Velasco P., Landeira-Viñuela A., Fidalgo-Gomez H., Acebes-Fernandez V., Gongora R., et al. (2019). Interactions of Nanoparticles and Biosystems: Microenvironment of Nanoparticles and Biomolecules in Nanomedicine.Nanomaterials (Basel), 9(10), 1365.
9. Balwani M., Sardh E., Ventura P., Peiró P., Rees D., Stölzel U., Bissell D., et al., and ENVISION Investigators. (2020). Phase 3 Trial of RNAi Therapeutic Givosiran for Acute Intermittent Porphyria. Clinical Trial N Engl J Med, 382(24):2289-2301.
10. Beck B., Shin B., Choi Y., Park S. and Kang K. (2020). Predicting commercially available antiviral drugs that may act on the novel coronavirus (SARS-CoV-2) through a drug-target interaction deep learning mode. Comput Struct Biotechnol J, 18, 784-790.
11. Bigini P., Gobbi M., Bonati M., Clavenna A., Zucchetti M., Garattini S. and Pasut G. (2021). The role and impact of polyethylene glycol on anaphylactic reactions to COVID-19 nano-vaccines. Nature Nanotechnol, 16, 1169-1171.
12. Bhushan B., Wanve S., Koradia P., Bhomia V., Soni P., Chakraborty S., Khobragade A., et al., and Study Investigators Group. (2021). Efficacy and safety of pegylated interferon-α2b in moderate COVID-19: a phase 3, randomized, comparator-controlled, open-label study. Clinical Trial Int J Infect Dis, 111, 281-287.
13. Chakarov S., Petkova R., Russev G. (2008). Rapid and efficient method for production of T4 endonuclease V by heterologous expression in E. coli. Biotechnol Biotechnol Equip, 22(4), 1011-1012.
14. Chakravarty M. and Vora A. (2021). Nanotechnology-based antiviral therapeutics. Drug Deliv Transl Res, 11(3), 748-787.
15. Chang Y., Tung Y., Lee K., Chen T., Hsiao Y., Chang H., Hsieh T., et al. (2020). Potential Therapeutic Agents for COVID-19 Based on the Analysis of Protease and RNA Polymerase Docking. Preprint. February 2020, DOI:10.20944/preprints202002.0242.v1.
16. Cho S., Woo H., Kim K., Oh J. and Jeong Y. (2011). Novel system for detecting SARS coronavirus nucleocapsid protein using an ssDNA aptamer. J Biosci Bioeng, 112(6), 535-40.
17. Davoudi-Monfared E., Rahmani H., Khalili H., Hajiabdolbaghi M., Salehi M., Abbasian L., Kazemzadeh H. and Yekaninejad M. (2020). A Randomized Clinical Trial of the Efficacy and Safety of Interferon β-1a in Treatment of Severe COVID-19. Antimicrob Agents Chemother, 64(9), e01061-20.
18. Desai J. and Thakkar H. (2018). Darunavir-Loaded Lipid Nanoparticles for Targeting to HIV Reservoirs. AAPS PharmSciTech, 19(2), 648–660.
19. Ditzler M., Bose D., Shkriabai N., Marchand B., Sarafianos S., Kvaratskhelia M., Burke D. (2011). Broad-spectrum aptamer inhibitors of HIV reverse transcriptase closely mimic natural substrates. Nucleic Acids Res, 39(18), 8237-8247.
20. Duncan R., Vicent M., Greco F. and Nicholson R. (2005). Polymer–drug conjugates: towards a novel approach for the treatment of endrocine-related cancer. Endocr Relat Cancer, 12(Suppl. 1), S189–S199.
21. Estébanez M., Ramírez-Olivencia G., Mata T., Martí D., Gutierrez C., de Dios B., Herrero M., et al., and COVID 19 Central Defense Hospital “Gomez Ulla” Team. (2020). Clinical evaluation of IFN beta1b in COVID-19 pneumonia: a retrospective study. Preprint from medRxiv, 19 May 2020, DOI: 10.1101/2020.05.15.20084293.
22. Ferreira D., Barbosa J., Sousa D.A., Silva C., Melo L.D.R., Avci-Adali M., Wendel H.P., et al. (2021). Selection of aptamers against triple negative breast cancer cells using high throughput sequencing. Sci Rep, 11(1), 8614.
23. Fiandra L., Colombo M., Mazzucchelli S., Truffi M., Santini B., Allevi R., Nebuloni M., et al. (2015). Nanoformulation of antiretroviral drugs enhances their penetration across the blood brain barrier in mice. Nanomedicine, 11(6), 1387-1397.
24. Fodor-Kardos A., Kiss A., Monostory K. and Feczkó T. (2020). Sustained in vitro interferon-beta release and in vivo toxicity of PLGA and PEG-PLGA nanoparticles. RSC Adv, 10, 15893.
25. Frediansyah A., Tiwari R., Khan S., Dhama K. and Harapan H. (2021). Antivirals for COVID-19: A critical review. Clin Epidemiol Glob Health, 9, 90-98.
26. Garrido C., Simpson C., Dahl N., Bresee J., Whitehead D., Lindsey E., Harris T., et al. (2015). Gold nanoparticles to improve HIV drug delivery. Future Med Chem, 7(9): 1097–1107.
27. Giovannini G., Haick H. and Garoli D. (2021). Detecting COVID-19 from Breath: A Game Changer for a Big Challenge. ACS Sens, 6(4), 1408–1417.
28. Ghosh A., Osswald H., Prato G. (2016). Recent Progress in the Development of HIV-1 Protease Inhibitors for the Treatment of HIV/AIDS. J Med Chem, 59(11), 5172–5208.
29. Gong Y., Chowdhury P., Nagesh P., Cory T., Dezfuli C., Kodidela S., Singh A., et al. (2019). Nanotechnology approaches for the delivery of cytochrome P450 substrates in HIV treatment. Expert Opin Drug Deliv, 16(8), 869–882.
30. Ha B., Yang J., Chen X., Jadhao S., Wright E. and Anderson L. (2020). Two RSV Platforms for G, F, or G+F Proteins VLPs. Viruses, 12(9), 906.
31. Huang L., Luo Y., Sun X., Ju H., Tian J. and Yu B.Y. (2017). An artemisinin-mediated ROS evolving and dual protease light-up nanocapsule for real-time imaging of lysosomal tumor cell death. Biosens Bioelectron, 92, 724-732.
32. Hung I., Lung K., Tso E., Liu R., Chung T., Chu M., Ng Y., et al. (2020). Triple combination of interferon beta-1b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with COVID-19: an open-label, randomised, phase 2 trial. Lancet, 395(10238), 1695-1704.
33. Idris A., Davis A., Supramaniam A., Acharya D., Kelly G., Tayyar Y., West N., et al. (2021). A SARS-CoV-2 targeted siRNA-nanoparticle therapy for COVID-19. Mol Ther, 29(7), 2219-2226.
34. Kinghorn A., Dirkzwager R., Liang S., Cheung Y., Fraser L., Shiu S., Tang M., Tanner J. (2016). Aptamer Affinity Maturation by Resampling and Microarray Selection. Anal Chem., 88(14), 6981-5.
35. Kumar A., Mancy S., Manjunath K., Kulkarni S. and Jagadeesh R. (2019). Formulation and Evaluation of Fluvoxamine Maleate Loaded Lipid Nanoparticle. Int J Pharm Sci Nanotechnol, 12(4), 4593-4600.
36. Lee M., Yang J., Jung H., Beack S., Choi J., Hur W., Koo H. et al. (2012).  Hyaluronic Acid–Gold Nanoparticle/Interferon α Complex for Targeted Treatment of Hepatitis C Virus Infection. ACS Nano, 6(11), 9522–9531.
37. Li H., Tatematsu K., Somiya M., Iijima M. and Kuroda S. (2018). Development of a macrophage-targeting and phagocytosis-inducing bio-nanocapsule-based nanocarrier for drug delivery. Acta Biomater, 73, 412-423.
38. Li W., Wu J., Zhan P., Chang Y., Pannecouque C., De Clercq E. and Liu X. (2012). Synthesis, drug release and anti-HIV activity of a series of PEGylated zidovudine conjugates. Int J Biol Macromol, 50(4), 974-980.
39. Lim S., Hor C., Tay K., Mat Jelani A., Tan W., Ker H., Chow T., et al., and I-TECH Study Group. (2022). Efficacy of Ivermectin Treatment on Disease Progression Among Adults With Mild to Moderate COVID-19 and Comorbidities: The I-TECH Randomized Clinical Trial. JAMA Intern Med, 2022 Feb 18. doi: 10.1001/jamainternmed.2022.0189. (online ahead of print).
40. Liu S., Wang Z., Xie H., Liu A., Lamb D. and Pang D. (2020). Single-Virus Tracking, From Imaging Methodologies to Virological Applications. Chem Rev, 120, 1936-1979.
41. Lu J., Lu G., Tan S., Xia J., Xiong H., Yu X., Qi Q., et al. (2020). A COVID-19 mRNA vaccine encoding SARS-CoV-2 virus-like particles induces a strong antiviral-like immune response in mice. Cell Res, 30(10), 936-939.
42. Mahajan S., Roy I., Xu G., Yong K.T., Ding H., Aalinkeel R., Reynolds J., et al. (2010). Enhancing the delivery of anti retroviral drug Saquinavir across the blood brain barrier using nanoparticles. Curr HIV Res, 8, 396–404.
43. Kawata Mahlknecht G., Sela M. and Yarden Y. (2015). Aptamer Targeting the ERBB2 Receptor Tyrosine Kinase for Applications in Tumor Therapy. Methods Mol Biol, 1317, 3-15.
44. Malhani A., Enani M., Saheb Sharif-Askari F., Alghareeb M., Bin-Brikan R., AlShahrani S., Halwani R., et al. (2021). Combination of (interferon beta-1b, lopinavir/ritonavir and ribavirin) versus favipiravir in hospitalized patients with non-critical COVID-19: A cohort study. PLoS One, 16(6), e0252984.
45. Mann A., Somasunderam A., Nieves-Alicea R., Li X., Hu A., Sood A., Ferrari M., et al. (2010). Identification of thioaptamer ligand against E-selectin: Potential application for inflamed vasculature targeting. PLoS ONE, 5, e13050.
46. Mary A., Hénaut L., Macq P.Y., Badoux L., Cappe A., Porée T., Eckes M., et al. (2020). Rationale for COVID-19 Treatment by Nebulized Interferon-β-1b–Literature Review and Personal Preliminary Experience. Front Pharmacol, 11, 592543.
47. Medeiros I., Khayat A., Stransky B., Santos S., Assumpção P. and de Souza J. (2021). A small interfering RNA (siRNA) database for SARS-CoV-2. Nature Sci Rep, 11, 8849.
48. Mikhaylov E., Lyubimtseva T., Vakhrushev A., Stepanov D., Lebedev D., Vasilieva E., Konradi A., et al. (2022). Bromhexine Hydrochloride Prophylaxis of COVID-19 for Medical Personnel: A Randomized Open-Label Study. Interdiscip Perspect Infect Dis, 2022, 4693121.
49. Mishra S., Malhotra P., Gupta A.K., Singh P.K., Mishra A.K., Javed S. and Kumar R. (2014). Novel method for screening of radioprotective agents providing protection to DNA ligase against gamma radiation induced damage. Int J Radiat Biol, 90(2), 187-192.
50. Mohammed S. and Shaaban E. (2019). Efficacious nanomedicine track toward combating COVID-19. Randomized Controlled Trial. N Engl J Med, 381(24), 2293-2303.
51. Moitra P., Alafeef M., Dighe K., Frieman M. and Pan D. (2020). Selective Naked-Eye Detection of SARS-CoV-2 Mediated by N Gene Targeted Antisense Oligonucleotide Capped Plasmonic Nanoparticles. ACS Nano, 14, 7617-7627.
52. Monk P., Marsden R., Tear V., Brookes J., Batten T., Mankowski M., Gabbay F., et al., and Inhaled Interferon Beta COVID-19 Study Group. (2021). Collaborators, Safety and efficacy of inhaled nebulised interferon beta-1a (SNG001) for treatment of SARS-CoV-2 infection: a randomised, double-blind, placebo-controlled phase 2 trial. Clinical Trial Lancet Respir Med, 9(2), 196-206.
53. Musafia B., Oren-Banaroya R. and Noiman S. (2014). Designing anti-influenza aptamers: novel quantitative structure activity relationship approach gives insights into aptamer-virus interaction. PLoS One, 9(5), e97696.
54. Nasserie T., Hittle M. and Goodman S. (2021). Assessment of the Frequency and Variety of Persistent Symptoms Among Patients With COVID-19: A Systematic Review. JAMA Network Open, 4 (5), e2111417.
55. Negi J., Chattopadhyay P., Sharma A.K., and Ram V. (2013). Development of solid lipid nanoparticles (SLNs) of lopinavir using hot self nano-emulsification (SNE) technique. Eur J Pharm Sci, 48(1–2), 231-239.
56. Neuman B., Stein D., Kroeker A., Bestwick R., Iversen P., Moulton H., and Buchmeier M. (2006). Inhibition and Escape of SARS-CoV Treated with Antisense Morpholino Oligomers. In: The Nidoviruses, 581, 567–571.
57. Nimjee S., Povsic T., Sullenger B. and Becker R. (2016). Translation and Clinical Development of Antithrombotic Aptamers. Nucleic Acid Ther, 26(3), 147-155.
58. Nooraei S., Bahrulolum H., Hoseini Z., Katalani C., Hajizade A., Easton A. and Ahmadian G. (2021). Virus-like particles: preparation, immunogenicity and their roles as nanovaccines and drug nanocarriers. J Nanobiotechno, 19, 59.
59. Owen D., Allerton C., Anderson A., Aschenbrenner L., Avery M., Berritt S., Boras B., et al. (2021). An oral SARS-CoV-2 M(pro) inhibitor clinical candidate for the treatment of COVID-19. Science 374(6575), 1586-1593.
60. Panner Selvam R. and Kulkarni P. (2014). Design and Evaluation of Self Nanoemulsifying Systems for Poorly Water Soluble HIV Drug. J PharmaSciTech, 4(1).
61. Puri A., Loomis K., Smith B., Lee J-H., Yavlovich A., Heldman E. and Blumenthal R. (2009). Lipid-based nanoparticles as pharmaceutical drug carriers: from concepts to clinic. Crit Rev Ther Drug Carrier Syst, 26, 523–580.
62. Rabowsky J., Dukes A., Lee D. and Leong K. (1996). The use of bioerodible polymers and daunorubicin in glaucoma filtration surgery. Clin Trial Ophthalmol, 103(5), 800-807.
63. Samson R., Navale G. and Dharne M.  (2020). Biosensors: frontiers in rapid detection of COVID-19. J Biotech, 10(9), 385.
64. Rahmani H., Davoudi-Monfared E., Nourian A., Khalili H., Hajizadeh N., Jalalabadi N., Fazeli M., et al (2020). Interferon beta-1b in treatment of severe COVID-19: A randomized clinical trial. Int Immunopharmacol, 88, 106903.
65. Rasmi Y., Saloua K., Nemati M., and Choi J. (2021). Recent Progress in Nanotechnology for COVID-19 Prevention, Diagnostics and Treatment.  Review Nanomaterials (Basel), 11(7), 1788.
66. Ravi P., Vats R., Dalal V., Nitin Gadekar N. (2015). Design, optimization and evaluation of poly-ɛ-caprolactone (PCL) based polymeric nanoparticles for oral delivery of lopinavir. Drug Dev Ind Pharm,41(1), 131–140.
67. Reches M. and Gazit E. (2007). Biological and chemical decoration of peptide nanostructures via biotin-avidin interactions.  J Nanosci Nanotechnol, 7(7), 2239-2245.
68. de M Ribeiro N. and Fonseca B. (2020). The role of pharmaceutical nanotechnology in the time of COVID-19 pandemic Review Future Microbiol, 15, 1571-1582.
69. Roberts D., Rossman J. and Jarić I. (2021). Dating first cases of COVID-19. PLOS Pathogens, June 24, 2021, https://doi.org/10.1371/journal.ppat.1009620.
70. Roh C. and Jo S. (2011). Quantitative and Sensitive Detection of SARS Coronavirus Nucleocapsid Protein Using Quantum Dots-Conjugated RNA Aptamer on Chip. J Chem Technol Biotechnol, 86, 1475-1479.
71. Rose K., Alves Ferreira-Bravo I., Li M., Craigie R., Ditzler M., Holliger P. and DeStefano J. (2019). Selection of 2′-Deoxy-2′-Fluoroarabino Nucleic Acid (FANA) Aptamers That Bind HIV-1 Integrase with Picomolar Affinity. ACS Chem Biol, 14(10), 2166-2175.
72. Rosenberg J., Bambury R., Van Allen E., Drabkin H., Lara P. Jr., Harzstark A., Wagle N., et al. (2014). A phase II trial of AS1411 (a novel nucleolin-targeted DNA aptamer) in metastatic renal cell carcinoma. Invest New Drugs, 32(1), 178-187.
73. Roy U., Rodríguez J., Barber P., das Neves J., Sarmento B. and Nair M. (2015). The potential of HIV-1 nanotherapeutics: from in vitro studies to clinical trials. Nanomedicine (London, England), 10(24), 3597–3609.
74. Sabourian P., Yazdani G., Ashraf S., Frounchi M., Mashayekhan S., Kiani S. and Kakkar A. (2020). Effect of Physico-Chemical Properties of Nanoparticles on Their Intracellular Uptake. Int J Mol Sci, 21(21), 8019.
75. Shan B., Broza Y.Y., Li W., Wang Y., Wu S., Liu Z., Wang J., et al. (2020). Multiplexed Nanomaterial-Based Sensor Array for Detection of COVID-19 in Exhaled Breath. ACS Nano, 14(9):12125-12132.
76. Seo G., Lee G., Kim M., Baek S., Choi M., Ku K., Lee C., et al. (2020). Rapid Detection of COVID-19 Causative Virus (SARS-CoV-2) in Human Nasopharyngeal Swab Specimens Using Field-Effect Transistor-Based Biosensor. ACS Nano, 14(4), 5135-5142.
77. Sercombe L., Veerati T., Moheimani F., Wu S.Y., Sood A.K. and Hua S. (2015). Advances and Challenges of Liposome Assisted Drug Delivery. Front Pharmacol, 6, 286.
78. Shan B., Broza Y., Li W., Wang Y., Wu S., Liu Z., Wang J., et al. (2020). Multiplexed Nanomaterial-Based Sensor Array for Detection of COVID-19 in Exhaled Breath. ACS Nano, 14, 12125-12132.
79. Singh G. and Pai RS. (2016). Atazanavir-loaded Eudragit RL 100 nanoparticles to improve oral bioavailability: optimization and in vitro/in vivo appraisal. Drug delivery, 23(2), 532–539.
80. Sodeifian F., Nikfarjam M., Kian N., Mohamed K. and Rezaei N. (2022). The role of type I interferon in the treatment of COVID-19. J Med Virol, 94(1), 63-81.
81. Somvanshi S., Kharat B., Saraf S., Somwanshi S., Shejul S., and Jadhav K. (2020). Multifunctional Nano-Magnetic Particles Assisted Viral RNA-Extraction Protocol for Potential Detection of COVID-19. Mater Res Innov, 25, 169-174.
82. Sun M., Liu S., Wei X., Wan S., Huang M., Song T., Lu Y., et al. (2021). Aptamer Blocking Strategy Inhibits SARS-CoV-2 Virus Infection. Angew Chem Int Ed Engl, 60(18), 10266-10272.
83. Svenson S. and Tomalia DA. (2005). Dendrimers in biomedical applications-reflections on the field. Adv Drug Deliv Rev, 57(15), 2106-2129.
84. Tran S., DeGiovanni P-J., Piel B. and Rai P. (2017). Cancer nanomedicine: a review of recent success in drug delivery. Clin Transl Med, 6, 44.
85. Trujillo C., Nery A., Alves J., Martins A. and Ulrich H. (2007). Development of the anti-VEGF aptamer to a therapeutic agent for clinical ophthalmology. Clin Ophthalmol, 1(4), 393-402.
86. Tsogtbaatar K., Sousa D.A., Ferreira D., Tevlek A., Aydın H.M., Çelik E. and Rodrigues L. (2022). In vitro selection of DNA aptamers against human osteosarcoma. Invest New Drugs, 40(1), 172-181.
87. Tulbah A. and Lee W-H. (2021). Physicochemical Characteristics and In Vitro Toxicity/Anti-SARS-CoV-2 Activity of Favipiravir Solid Lipid Nanoparticles (SLNs). Pharmaceuticals (Basel), 14(10), 1059.
88. Venkatesh D., Baskaran M., Karri V., Mannemala S., Radhakrishna K. and Goti S. (2015). Fabrication and in vivo evaluation of Nelfinavir loaded PLGA nanoparticles for enhancing oral bioavailability and therapeutic effect. Saudi Pharm J, 23(6), 667–674.
89. Veronese F. and Mero A. (2008). The impact of PEGylation on biological therapies. BioDrugs, 22(5), 315-329.
90. Vyas T., Shah L and Amiji M. (2006). Nanoparticulate drug carriers for delivery of HIV/AIDS therapy to viral reservoir sites. Expert Opin Drug Deliv, 3(5), 613–628.
91. Wen W., Chen C., Tang J., Wang C., Zhou M., Cheng Y., Zhou X., et al. (2022). Efficacy and safety of three new oral antiviral treatment (molnupiravir, fluvoxamine and Paxlovid) for COVID-19：a meta-analysis. Ann Med, 54(1), 516-523.
92. Wu J., Wang H. and Li B. (2020). Structure-aided ACEI-capped remdesivir-loaded novel PLGA nanoparticles, toward a computational simulation design for anti-SARS-CoV-2 therapy. Phys Chem Chem Phys, 22, 28434-28439.
93. Vincent M., Bergeron E., Benjannet S., Erickson B., Rollin P., Ksiazek T., Seidah N., et al. (2005). Chloroquine is a potent inhibitor of SARS coronavirus infection and spread. Virol J, 2, 69.
94. Yang Z., Huang Y., Ganesh L., Leung K., Kong W., Schwartz O., Subbarao K., et al. (2004). pH-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through DC-SIGN. J Virol, 78(11), 5642-5550.
95. Yarosh D. (2002). Enhanced DNA repair of cyclobutane pyrimidine dimers changes the biological response to UV-B radiation. Mutat Res, 509(1-2), 221-226.
96. Yilmaz I., Ipekoglu E., Bulbul A., Turay N., Yildirim M., Evcili I., Yilmaz N., et al. (2022). Development and preclinical evaluation of virus-like particle vaccine against COVID-19 infection. Allergy, 77(1), 258-270.
97. Zhao Z., Cui H., Song W., Ru X., Zhou W. and Yu X. (2020), A Simple Magnetic Nanoparticles-Based Viral RNA Extraction Method for Efficient Detection of SARS-CoV-2. bioRxiv, 2020, doi: 10.1101/2020.02.22.961268.

Summary

The methodology of nanomedicine constitutes perhaps the most important “key technology” of the future: no other field is accompanied by so many hopes and will entail comparable social consequences as the expected developments in nanomedicine. And in this context, the use of RNA technologies in the therapeutic, diagnostic and preventive (immunological) fields will certainly play an essential role (be it in the form of mRNA, cRNA or even “free RNA”). The use of mRNA vaccines is merely the prelude to this development. While the ethical problem is still relatively simple here (especially since pure safety risks can only be clarified empirically and therefore do not fall within the focus of bioethics), the circle of (nano-) ethical questions will expand enormously as soon as RNA technology has also gained a foothold in other fields of genetic engineering-based nanomedicine.

Key words/phrases: Bioethics, nanoethics, technology assessment, precautionary principle, social acceptance, safety of mRNA vaccines, data protection.

1. Introduction: Nanotechnology and “Nanoethics”

Nanotechnological processes and products have been playing a considerable role – largely unnoticed by the general public¹  – for many years now. And this also applies to the field of medicine, in which more and more primarily genetically engineered products (such as human insulin obtained via genetically modified bacteria) are being used in diagnostics and therapy. This is even indispensable for so-called “somatic gene therapy”, since here therapeutically effective gene sequences are supposed to compensate for the malfunction of “sick” genes with the help of gene shuttles (mostly viruses rendered incapable of reproduction), for example by coding for vital proteins that the diseased organism itself is either unable to produce or is unable to produce in sufficient quantities. In this case, entities on the nanoscale are even used twice: on the one hand in the form of the “healthy” gene sequence and on the other hand through the use of viral transfer systems (vectors).

But apart from such often rather “exotic” areas of application² , the public has only become aware of the importance of nanotechnology (and here in particular with the means of genetic engineering) through the development of a completely new class of vaccines: namely through the mRNA vaccine to combat the Covid-19 or SARS-CoV2 pandemic. Previously, genetic engineering products had come to general attention mainly in the field of agriculture and food production and were then criticized, sometimes very strongly, because of possible environmental and health risks. It is therefore not surprising that the innovative mRNA vaccines were – at least initially – viewed with great suspicion by many people, since genetic engineering products do not enjoy a good reputation. In addition, little was (and still is) known about the potential side effects of these vaccines due to the lack of long-term clinical studies at the beginning of the vaccination campaigns. Only when the efficacy and relative safety of the mRNA vaccine gradually became apparent in the course of the mass vaccination campaigns did public acceptance of this new procedure also improve. And it is possible that the success of the new vaccines will help to improve the overall perception and acceptance of genetic engineering, so that it could also be trusted more than it has been so far in other (non-medical) fields of application. Nevertheless, by no means all “critical” questions in connection with so-called “nanomedicine” based on genetic engineering have yet been clarified: in particular, not all ethical questions. And the debate on genetic engineering will continue to occupy bioethicists and nanoethicists for a long time to come.

The continuing need for clarification also has to do with the fact that nanotechnologies are applied on a microscopic scale, i.e., they elude immediate visibility, and, moreover, they intervene in the highly complex system of cells and organisms, whose structures and mechanisms are also microscopic in size and are still far from being understood in detail. Above all, proteomics, which describes the dynamic behavior of proteins expressed by genes within the cell, is still in its infancy. Therefore, no one knows for sure whether a DNA or RNA molecule, if introduced into an organism, will really only have the desired effect (if at all) or whether it may also have adverse (unintended) consequences. Outside the laboratory with its safe “containment” conditions, namely tested directly on living humans (as in the case of mRNA vaccination), a clinical or even everyday medical use of genetically engineered remedies resembles a “real experiment” with society [25]. Such real experiments are otherwise only known from the construction of innovative nuclear power plants or unique structures (e.g. bridges, landfills or airports), whose stability and inherent dynamics can often hardly or not at all be simulated under laboratory conditions.

In principle, therefore, extreme caution is always called for here: but in view of the terrible, often fatal effects of the pandemic, there was already very rapid (and sometimes by way of “emergency approvals”) widespread use of the innovative vaccine in order thereby to prevent worse. However, although the overall study situation was very uncertain (especially since long-term data were not yet available), the approval of the new vaccine was of course preceded by a detailed evaluation by the responsible institutions and committees (including national ethics committees), in which above all ethical aspects were also taken into account: in particular, it had to be weighed up whether, in view of the serious pandemic situation, it was permissible to shorten the implementation of clinical studies. However, it cannot be said that the approval was given lightly or completely “blindly”. All in all, the scientific community of immunologists, epidemiologists, infectiologists and virologists, as well as the political decision-makers, can be said to have a considerable ethical awareness. And as it looks at present, the use of the mRNA vaccine can be considered a great success – as well as a breakthrough for nanomedicine as a whole.

On what scale do nanotechnologies operate and what purposes do they serve? To answer this question, we have to broaden our view and look at the entire field of nanotechnological developments. In general, we can say that nanotechnologies describe structures that are 80,000 times smaller than the diameter of a human hair (1 nanometer = 10^-9 meters). However, the classifications of materials as nanomaterials often differ, for example, when the British government assumes a size of up to 200 nanometers, while the USA allows a size of up to 1,000 nanometers [18]. Whatever the case, these technologies will in any case enable fundamental relationships to be explored at the molecular and atomic level and new materials with promising properties to be developed. Nanotechnologies are therefore considered key technologies of the 21st century, which are our “tickets” to the future [16].

In the following, we will again focus more on the field of medically significant nanotechnology. Among the numerous promising applications of nanotechnologies, the field of medicine occupies a special position, as it is particularly associated with high expectations and hopes. New cancer therapies are already being tested in clinical trials³ , and innovative nano-transport systems for drugs make more efficient treatment with fewer active substances possible. Undesirable side effects should thus be reduced. Miniaturized mobile diagnostic units for rapid tests in doctors’ offices and imaging methods for diagnosing diseases that are less stressful for patients are being tested. And innovative surface coatings for implants or new materials in dental technology could help to significantly improve tolerability and durability and thus reduce costs.

This small excerpt from the breadth of applications in the field of medicine illustrates the great potential of nanotechnologies⁴ . Quite a few observers even speak here of a “paradigm shift in healthcare”. In the EU, around 100 million euros have been allocated to nanomedicine projects for the period 2007-2013 under the 7th Framework Research Program. The funding volume is likely to increase even more dramatically in the future in view of the success of mRNA vaccines. In the USA, too, the Project on Emerging Nanotechnologies and the National Cancer Institute [24] have also developed comprehensive funding programs for the application of nanotechnologies. National and international policy makers are thus focusing on research and location promotion in the field of nanomedicine.

2. “Nanoethics” as a new bioethical subdiscipline

All this is not just about promoting basic research and product development, because in its new Code of Conduct [12], the EU requires all research projects to take account of possible risks and to be embedded in social and ethical issues. This is because the possibility of exceeding the limits of current forms of therapy simultaneously raises questions about new boundaries. And this brings into play a special field of application of ethics (or practical philosophy), which is generally called “bioethics” and which itself has many subfields.

In connection with the development of nanotechnological processes, a new field of research and reflection has been established within bioethics: so-called “nanoethics”. This field is essentially concerned with monitoring the effects of a new nanotechnology from the perspective of sustainability and evaluating its results with regard to the well-being of society. The focus of nanoethical expertise is thus on the “interest of the common good” in the sense of improving the quality of life of the community.

In order to be able to better classify nanoethics, it is first necessary to understand the goals and tasks of bioethics. For just as nanoethics represents a subfield of bioethics, bioethics for its part can be understood as a subfield of Technology Assessment (TA) in the area of the application of biotechnological processes. This is especially true when TA is related to the application of genetic engineering processes in biomedical, food technology or agricultural fields of application. In this context, the scope of TA includes not only ethical issues in the narrower sense, but also issues of reliability and safety, as well as social and political aspects, by asking, for example, “Are the social effects of a new technology politically and socially acceptable?” For example, if it should one day become possible to extend human life far beyond the normal lifespan with the help of genetic engineering. Would this be desirable at all? Are we not embarking on a fundamentally “slippery slope” that could have devastating consequences for the future of society? And what would it mean for our image of man if we were able to eradicate all hereditary diseases by means of genetic engineering or to shape or optimize the genetic makeup of human beings at will?

In other words, the field of bioethics or bioethically sensitive TA encompasses all ethical, legal and social implications (abbreviated to ELSI) arising from the application of biotechnological processes. And it is only by locating them in the context of the broader ELSI issues of TA that the questions of bioethics can be adequately addressed at all, so that ethical reflection does not take place in a “vacuum”, i.e., detached from other factual issues. There may thus be basic ethical principles that arise in any application of technology, but their meaningful application to particular subject areas (such as nanomedicine) should never be detached from the specifics of the particular field of technology.

However, the aim of bioethics or TA is not to hinder or even prevent new biotechnological developments simply because they are novel and unclear in terms of their hazard potential, but to serve as a kind of “early warning system” that draws attention in good time to undesirable developments or ethically and socially precarious applications of new biotechnological methods. It is therefore important to include bioethical reflections as far as possible from the outset in the research and development of novel biotechnologies (in the sense of accompanying research that is already involved in the research process). This not only prevents ethically questionable developments, but also avoids unnecessary costs and protects the public image of biotechnology. In any case, it would be ideal if ethical reflection would contribute to the design of technology “ex ante” and not only “ex post” [17]. Here, ethical analysis and evaluation would have to focus primarily on (1.) the goals and purposes of the technical innovation, (2.) the instruments and means (e.g., animal or field testing), and (3.) the unintended side effects (i.e., establishing the risk profile, e.g., with regard to possible toxicity, and adherence to the precautionary principle in the face of lack of knowledge).

3. Why do we need ethics in view of the introduction of new technologies?

Every ethics is always based on a certain value system. Without reference to those value determinations and ideals which are decisive for a society, no decisions of action could be made which could be justified before other persons. Every form of responsible action always takes place within a horizon of legitimized value systems that can be invoked as arguments for a particular decision. Many of these values have found their way into legal regulations (laws and regulations), so that they serve the courts as normative criteria for the adjudication of legal conflicts or claims brought before them. In Western culture, it is above all humanistic ethics, often combined with Christian values, that serves as the basis for finding and justifying decisions and that has found expression, for example, in general human rights and in democratic rights to freedom (as rights of defense against the state).

On the basis of the value system, ethics asks what man should do or what he may do in a given situation. This can be about the observance of certain fundamental value principles, which must be adhered to unconditionally (without exception) (thus, according to “deontological ethics”, not even white lies are allowed), or it is about possible undesirable consequences of a certain behavior (thus, “consequentialist ethics” tries to assess the potential effects of actions). In this context, ethics serves primarily to resolve conflicts of values (which, however, is not always successful or possible) by rationally weighing the arguments in favor of or against a particular decision to act. Finally, ethics is that area in which the often only unconsciously valid value concepts are made explicit, so that the establishment of a value standard (or value canon) becomes possible, to which people can orient themselves. Ethics can serve as “ethics of attitude” for the enforcement of ‘ideological’ value attitudes, in which a certain image of man or also a social ideal (a “utopia”) is expressed, or it can attempt as “ethics of responsibility” to do justice to the empirical peculiarities of the respective situation of action by subjecting all circumstances and possible consequences to an evaluation. Either way, ethics is always about answering questions of justice (e.g., about the fair distribution of scarce goods or opportunities and rewards) and about avoiding possible harm (e.g., to life and limb) or a restriction of liberties by weighing different interests, legal claims and expectations against each other.

This was particularly clear in the case of the political justification of socially drastic “lockdown” measures in connection with the Corona pandemic: the right to freedom of economic activity and freedom of movement was in conflict here with the right to physical integrity and protection against infection, which are among the highest legal rights and the most important tasks of the democratic state. Although there is no ranking of fundamental rights in the constitutions – such as in the Basic Law of the Federal Republic of Germany – a decision had to be made in view of the pandemic as to which fundamental right should be given priority. In the end, it was decided that ethical priority should be given to the protection of health and life, since seriously ill or even deceased persons are no longer able to exercise their other fundamental rights. An additional argument in favor of restricting other civil liberties by imposing a “lockdown” or even the mask and quarantine obligation was that this would not only be a matter of self-protection for individuals, but above all of protecting third parties who could be unintentionally infected. On the other hand, it is more difficult to call for a general obligation to vaccinate in order to be able to include those who refuse vaccination, since such an obligation would seriously interfere with the right to self-determination. The ethical evaluation of the admissibility of the new mRNA vaccines must therefore not only concern the safety aspects of these vaccines, but must also take into account the social context in which these vaccines are to be used: be it voluntarily or be it due to a legal obligation: How could a vaccination obligation be justified if neither the possible vaccination risks nor the long-term protective effect of the new vaccines are already sufficiently known?

4. Nanoethical question areas: Acceptance problems and safety risks

As already mentioned above, more and more nanotechnological methods and their products are finding their way into everyday medical practice. And this is mainly due to the growing importance of genetic engineering in the field of medical diagnostics⁵  and therapy or prophylaxis, with mRNA vaccines falling into the field of preventive medicine, insofar as they are used to prevent the outbreak of a disease. However, the development of these vaccines would not have been possible without the prior molecular genetic elucidation of the viral pathogen, so that genetic engineering carried out on the nanoscale is used here both in the descriptive sequencing of the viral RNA and in the constructive development of the vaccines (especially since the immunologically active mRNA sequence must also be packaged in a shell of nanolipid particles in order to be able to enter the human organism safely and stably). It is therefore not sufficient to consider the new vaccines alone from a bioethical point of view: on the one hand, the entire research and production process and, on the other hand, the totality of the effects of vaccination must be included in the reflection, including not only the possible physiological side effects, but also the social and economic consequences of widespread use of the vaccines. And likewise, consideration must be given to what would be involved in not using these new vaccines. Technology-related ethics must always seek to assess and evaluate the risks, on the one hand, and the opportunities, on the other, of an innovative technology (which is why it is best considered as a subfield of technology assessment, as suggested above).

As a rough approximation, the ethically relevant aspects arising (1) from the application of mRNA vaccines within medical practice in the form of a broad-based vaccination campaign can be distinguished from those ethically relevant aspects of these vaccines arising (2) from the application of genetic engineering procedures in the nanotechnological size range. In the following, both sets of questions will be dealt with in detail, whereby, in the context of our “Nanocode” project, the ethical aspects mentioned under (2) are of particular importance.

4.1. Medical and socio-ethical aspects of the vaccination campaign

There is still much discussion and even dispute in the various societies affected by the covid pandemic about for whom vaccination with mRNA vaccines is useful, i.e. beneficial. There is widespread agreement that especially so-called “vulnerable groups” can benefit from vaccination with mRNA vaccines: this applies especially to elderly people, whose immune system is often already considerably weakened, and people with certain pre-existing conditions, so that a particularly severe (possibly even fatal) course of a corona disease can be expected in them. Pregnant women, on the other hand, are advised against vaccination for good reasons. It is also known that in people with certain rheumatic diseases no or at best moderate vaccination success is to be expected. Finally, one must also evaluate whether there may be adverse “cross-effects” between the vaccine and medications that a patient must take regularly because of his or her current or chronic illnesses. However, all these are not ethical questions, but purely medical or pharmacological questions that can only be clarified empirically as well as in relation to the individual case (anamnesis). Therefore, the usual requirements for clinical testing of any new drug (including vaccines) correspond to this: only when the “candidate” has successfully passed all clinical tests for efficacy and safety, only then can it receive a patent-protected marketing authorization for its use in medical practice. And in the process, it may very well be that a new drug only receives limited approval if it is not effective or safe for every possible patient. For this very reason, clinical trials must always be carried out on different groups of subjects: e.g. on women and men, on adolescents and children, on pregnant women and diabetics, etc., in order to be able to ascertain all possible risks. As a rule, such clinical trials (even carried out on laboratory animals in the first preclinical phase) drag on for many years, with most “candidates” failing and having to be abandoned so that they do not even reach market maturity.

In the case of the innovative mRNA vaccine, however, a shortened clinical trial procedure was chosen due to the urgency and extraordinary danger of the pandemic, in particular by foregoing long-term studies in order not to lose any time. After all, the use of the vaccine was initially restricted to vulnerable groups and the very old in order to gather extensive experience (i.e., data), on the basis of which further vaccination recommendations could then be made for other adults. Such prioritization or differentiation of the patient population (of all potential beneficiaries) is necessary from both a medical and ethical perspective to minimize potential adverse effects. But should, for example, adolescents or even children also be vaccinated? The extent of the protective effect of vaccination in children and adolescents, or the mildness of the course of covid disease in the absence of such vaccination, can of course only be determined by empirical research. This is therefore not an ethical question. Accordingly, it can also only be determined empirically whether, in children and adolescents, the potential side effects of vaccination (the vaccine symptoms) outweigh the possibly severe disease symptoms in the event of infection. Perhaps it is better to leave it to the “nature” of the normally robust immune system of children and adolescents to cope with a Corona infection themselves. On the other hand, children and adolescents can also be carriers of Covid 19 viruses to adults, so that one could be of the opinion that the vaccination of children and adolescents is at least able to reduce the viral load in such a way that a transmission of the pathogens to non-vaccinated adults should relevantly reduce their risk of suffering a severe course of the disease.

From an ethical perspective, it should be noted here that vaccination of children and adolescents, which primarily serves to protect unvaccinated adults (and less their own protection), is only permissible if the possible harmful side effects of vaccinating children and adolescents are not more significant than the health benefits that the children and adolescents themselves can derive from vaccination. It should not be the case that children and adolescents are exposed to unnecessary potential vaccination risks simply to better protect unvaccinated adults from infection⁶ . Instead, it could be argued that an adult who refuses to be vaccinated must bear the risk of infection and thus also of a potentially severe course of the disease on his or her own responsibility.

However, the validity of this argument depends on there already being sufficient empirical evidence that vaccination with the novel mRNA vaccines is both sufficiently effective and safe with regard to dangerous long-term effects of the vaccine. The problem in this case is that this is a completely new class of vaccines with which medical science has not yet been able to gain experience. It is therefore ultimately up to clinical research to prove that the protective effect of the mRNA vaccine is high and that (beyond statistically insignificant harmful vaccine reactions⁷ ) no late effects of the vaccination are to be expected (e.g. in that the mRNA molecules could permanently latch onto the human genome, trigger cancer or dementia at some point, reduce fertility or cause lasting damage to the immune system). So far, however, it looks quite encouraging that researchers are able to confirm both the high efficacy and health safety of the mRNA vaccines. And this also includes possible long-term late effects in that no physiological mechanism has yet been discovered that could give serious cause for concern that the mRNA molecule not only serves the immune system as a blueprint for the production of the viral antigen (in order to then generate antibodies against it), but could also stimulate undesirable metabolic processes or cellular tissue changes. This is because the mRNA molecule apparently neither enters the genomic cell nucleus nor remains in the organism for any length of time before it is degraded again, i.e. breaks down into its nucleic bases and thus becomes ineffective.

Furthermore, there is currently a heated debate about how often and at what intervals such a vaccination should be repeated in order to both ensure and increase the protective effect⁸ : again, these are questions that can only be answered on the basis of immunological studies and statistical evaluations of the vaccination success. From an ethical point of view, it can only be said that everything possible must be done to increase the protective effect of an otherwise harmless vaccine as far as possible. This also applies to the further development of the vaccine: for example, its modifying adaptation to new virus variants⁹ .

As already indicated above, questions of efficacy and safety are in principle not ethical but purely scientific questions. The situation is somewhat different with the question of whether the “precautionary principle” should always apply by insisting that the safety of a new drug be tested in advance. But this is already fulfilled by the requirement of multi-phase clinical trials, i.e. regulated in detail in pharmaceutical law. This aspect will therefore not be discussed in detail here, especially as it is dealt with in the training module “Legal and Social Aspects”. There, it is also discussed who (and in what respect) is to be held liable in the event of vaccine damage occurring (the treating physician, the manufacturer or the health authorities).

However, “precaution” also concerns the question of whether larger stocks of vaccines should be stockpiled and whether it should be ensured that the production of vital vaccines is safeguarded within a national framework in order, on the one hand, to be able to monitor the quality assurance of the substances on one’s own and, on the other hand, to be able to contain the risk of a “rupture” of the supply chains. From an ethical point of view, the state’s health care for its population also includes a certain degree of self-sufficiency in the supply of medicines, so it must be considered risky to move production abroad (to India or China, for example) for purely logistical and economic reasons (cost savings). Only within the framework of a national and thus relatively autonomous drug supply can situations of scarcity be prevented, which could force physicians to make ethically highly questionable “triage” decisions (as is familiar from military hospital medicine, where in extreme situations it must often be decided for which wounded patients the drugs that have become scarce can be used most promisingly; and for which patients not, so that they are withheld from them). However, this concerns not only the available quantity of high-quality drugs, but also the other infrastructure of medical care: for example, the number of intensive care beds available in hospitals, or the capacity of the medical and nursing staff needed to operate the apparatus (such as ventilators) and to provide physical care to patients. However, these are general questions of medical ethics that concern the organization of medical care and therefore go beyond the scope of the ethically correct use of mRNA vaccines, so they need not be discussed further here.

Another point concerns issues of distributive justice and access to the new nanomedical possibilities: For example, given the initial scarcity of mRNA vaccines, it could not be overlooked that financially strong countries could obtain them more easily than poorer countries. Although the WHO reserved a certain quota of vaccines for the “Third World”, this proved to be completely insufficient. The majority of the manufacturers of the new vaccines also refused, for reasons of profit, to allow the poorer countries to produce the vaccines themselves without paying patent fees, i.e. to set up their own production facilities. This, too, put the developing countries at a serious disadvantage. In general, there was initially also fierce competition between the richer countries for the purchase of the rare vaccines, which must be viewed negatively from an ethical perspective, since a more concerted approach would also have been possible to ensure fair distribution. Basically, the question arises here how it can be achieved that costly nanotechnologies can also be made accessible to poorer beneficiaries, e.g. to prevent a “two-class medicine”.

In any case, as far as the efficacy and safety of mRNA-based vaccines are concerned, only empirical studies can provide information on this. Ethics has a say in this context only insofar as one can ask according to which criteria the benefit of a vaccine is to be evaluated: the prevention of a serious, perhaps even lethal disease is certainly the decisive criterion here, provided it is actually fulfilled. Against serious diseases such as smallpox and the plague in the past or zika fever or Ebola today, the existing vaccines are certainly the “means of choice”. But there is also a minority view that vaccination is too much and too hasty (e.g., against the seasonal flu), so that our “natural” immune system tends to be overloaded (stressed) and thus hindered in the development of its spontaneous “self-healing power”. Of all things, the great successes of vaccination campaigns – especially in the case of less threatening diseases – could ultimately prove to be “Pyrrhic victories”, since we would rely too much on modern pharmacology and apparatus medicine and accordingly neglect other (“gentler”) ways of maintaining and increasing health. In the case of Covid-19, however, there seems to be no way around vaccination, especially since there are no really effective therapeutics yet, so that a possible infection could be met with some equanimity. In general, there may be many ways to strengthen the innate immune system (such as a healthy diet, sufficient exercise and sleep, and a stress-reducing lifestyle), but against a really serious infectious disease, probably only a suitable vaccination will help in advance.

On the other hand, especially in the case of Corona, one could consider whether epidemics and pandemics originating in animals could not also be prevented by limiting the occasions when a virus (or any other dangerous pathogen: a bacterium or a parasite) can jump from animals to humans. Indeed, the covid-19 pathogen is, after all, a “zoonosis” (at least, there is little to suggest that it escaped unintentionally from a Chinese laboratory¹⁰ ) that was probably facilitated by the fact that pangolins or certain bats were offered for consumption at a market in Wuhan, which are excellent hosts for numerous viruses that can be potentially dangerous to humans if transmitted. This is to say: Changing our dietary habits can also prevent the outbreak of serious infectious diseases. The idea is that in the exchange area of human civilization and nature, we should limit or at least control the risk of transmission as much as possible. Indeed, the (illegal) wildlife trade, for example, as well as new forms of technology-intensive forest management, increase the likelihood of human contact with previously unknown pathogens through intrusion into previously largely untouched wilderness areas. Of particular sociological interest, moreover, are the often domestic cohabitation with farm animals (such as poultry) and the often inadequate local hygiene standards (e.g., in drinking water quality control or waste disposal). In general, human settlements and the associated road construction are apparently expanding further and further into the wilderness; just as, conversely, wild animals (incl. birds and insects) are increasingly being displaced from their ancestral natural habitats and settling in the settlements.

Thus, in order to avoid zoonotic diseases, the “epidemiological management” of the diverse human-nature relationships in the border area to wilderness becomes more and more urgent. In addition to scientific monitoring of the possible spread of wild species with zoonotic potential, legal and practical measures are thus also required: e.g. in the area of settlement and infrastructure development, economic exploitation of rainforests, improvement of hygiene, (nature-oriented) food production and health education. This is where medical bioethics meets environmental ethics. But these are all broader issues that are, to a certain extent, in the forefront of mRNA strategies against the Covid 19 pandemic: because once a pandemic has broken out, all considerations of preventive measures against zoonotic risks come too late, so that we must now try, on the one hand, to contain the further spread of the infectious event as far as possible (e.g., by wearing protective masks, by using a protective clothing, etc.). (e.g., by wearing protective masks, disinfecting hands and surfaces, ventilating indoor areas, temporary quarantine, and even a temporary “lock down”) and, secondly, through broad-based vaccination campaigns. And in the case of the latter, we are fortunate that the mRNA vaccines could be developed to operational readiness so amazingly quickly, which has certainly saved countless lives.

4.2. Nanoethical Aspects of mRNA Vaccines as Genetic Engineering Products

After briefly discussing the general medical and socio-ethical implications of the practical use of the novel mRNA vaccines, we will now consider the possible problems that could arise from the genetic engineering character of these vaccines: i.e. from the fact that these agents are, on the one hand, the result of constructive operations (so to speak, “RNA engineering”) on a molecular genetic scale; and that, on the other hand, they are intended to intervene in the biological functions of cells or in the immune system of a living organism (i.e. the human organism). in the immune system of a living being (namely the human organism). As already mentioned above, the field of nanoethics will be limited to the field of human medicine in the context of this training unit. Whereby – narrowing down the field even further – nanoethics will be related mainly (but not only) to the development of mRNA vaccines. In fact, all “manipulations” of RNA and DNA molecules, i.e. also all constructions of gene sequences (and the mRNA molecule, after all, also codes for a specific spike protein on the envelope of the covid-19 virus, i.e. for a viral gene) can be considered nanotechnical procedures. The development of mRNA-based vaccines is only a special case here. However, since ethical questions also arise in this special case, which arise overall for genetic engineering procedures or for the medical application of the products of these procedures, it makes sense to broaden the focus of ethical reflection accordingly, i.e. to include the entire spectrum of genetic engineering-based developments in the field of human medicine in the ethical assessment. Indeed, it will become apparent that in this special case, too, virtually all the ethical questions that arise in connection with genetic engineering in the health sector will come up¹¹ .

It can certainly be disputed that a special “nanoethics” as a special discipline is necessary, insofar as it would be merely a further application of “bioethics” or “gene ethics” and thus the questions raised by nanobiotechnology are already very well-known from other contexts of ethical reflection. Indeed, one should not misjudge the cross-cutting nature of ethical reflection, since even (nano-) technologies that are completely different in substance often face quite similar ethical and social challenges.

In principle, the use of genetic engineering methods to combat (infectious) diseases is certainly to be welcomed. However, in connection with the genetic engineering production (construction) of mRNA vaccines and with their handling during transfer into the human body, there are not only safety issues, but also, e.g. However, in connection with the genetic engineering production (construction) of mRNA vaccines and their handling during transfer into the human body, not only safety issues arise, but also questions of social acceptance, insofar as genetic engineering (both as a process and in terms of its products) does not enjoy a particularly good reputation: it is often argued that man would interfere with “God’s creation”, even “play God”, by changing the “blueprint of life” (which, however, is hardly the case in the case of mRNA molecules, since they merely provide the human immune system with templates for its own activity). Even the environmental compatibility of genetically engineered drugs is sometimes doubted (although, for example, human insulin obtained via genetically modified bacteria is readily accepted by diabetics). In any case, it is difficult to be dismissive of the construction of mRNA molecules to fight serious infections, since their advantages obviously clearly outweigh any concerns. One would have to be a fundamental opponent of technology, or at least an “ideologically” convinced enemy of genetic engineering, not to be able to see and appreciate the health benefits of precisely this application of genetic engineering. In the case of the agricultural use of GM plants, this may be somewhat different, since the safety situation and the environmental compatibility under “field conditions” are not yet clear in the last consequence; and also, in the case of the reproductive cloning of farm animals as well as the “reconstruction” of organisms with the help of “synthetic biology” or “genome editing”, not all risk and ethical questions have yet been cleared up (we will come back to this later).

Interestingly, in the case of the “tailor-made” mRNA vaccines, we are actually dealing with two nanostructures: on the one hand, the mRNA itself, i.e. the active substance, and on the other hand, the lipid nanoparticles into which the mRNA is “packaged” and subsequently introduced into the human organism. The nanotechnological procedure thus takes place on two different levels of construction, thus forming an exceedingly complex process.

Although the focus of this training module is the search for vaccines against the Corona virus in its different variants (as well as their ethical evaluation), the medical use of tailor-made mRNA hosts is capable of more than just combating infectious diseases: there is justified hope that artificial mRNA products can also be successfully used for innovative approaches in the field of gene therapy; or in the treatment of cancer as well as cardiovascular diseases. However, in order to be able to intervene in the genetic material of diseased cells in a targeted manner using mRNA, suitable insertion procedures (“erase and paste”) are required. And this is where the CRISPR-Cas technology of “genome editing” comes into play. Finally, in an at least indirect way, mRNA sequences could also become important in the development of diagnostic methods in the future (for example, in genome analysis, in the detection of tumor markers, etc.). Therefore, these areas of application will also be addressed subsequently, because a full assessment of mRNA nanotechnology can also only be made from an ethical perspective if this technique is considered in the broader context of other RNA- and DNA-based genetic engineering applications. This broadening of the scope of reflection is also justified by the fact that RNA technology is likely to soon open up further fields of application: such as in cancer or gene therapy, but also in diagnostics. And at the latest when this happens, the focus of ethical consideration will also have to expand, as ethical aspects will then become relevant that do not only concern the use of this technology for vaccination purposes: because only then will the enormous potential of this method become apparent. However, since any ethical reflection on the social implications of a new technology should take place as early as possible, it makes sense to try to evaluate these implications in the various fields of application of RNA technology already today. The advantage of a “prospectively” pursued nanoethics also consists in preparing a “proactively” oriented technology policy, in that nanoethics draws attention to possible risks or disadvantages of the new technology at an early stage.

In any case, the expected different medical applications of (RNA-based) genetic engineering raise particular scientific (empirical) and ethical problems, depending on the scope of construction and the depth of intervention in the organism, or depending on their objectives. The variation and weight of these problems depend, for example, on the level of construction reached by the manipulation of those molecular structures or organisms that are intended either to produce pharmacologically valuable proteins (e.g. in the bacterial production of human insulin) or to serve as “ferries” (vectors) for the introduction of therapeutic agents into the human body. However, even genetic medical procedures already applied at the nanoscale for purely diagnostic purposes produce data that are often very personal (e.g., genetic data that are characteristic of a particular person, making that person partially “genetically transparent”) and that could therefore be misused (e.g., by insurance companies if the data collected indicate future illnesses due to certain genetic dispositions; or also by government authorities to identify certain individuals even though there is no law enforcement connection). In this case, appropriate precautions must be taken under data protection law: e.g. by means of suitable procedures for anonymization or at least pseudonymization of the data (or also by means of high access barriers or by holding the data for a limited period of time). Also, the use of genetic data, for example for epidemiological purposes, must not take place without the express consent of the data donor (an “informed consent”) (this applies, for example, to clinical tissue collections or research biobanks in which genetically meaningful tissue samples are stored and evaluated).

Thus, it can be seen that the development of mRNA techniques should be viewed in the broader context of the development of molecular genetic tools, all of which are, or will be, effective at the nanoscale: be it

(a) for diagnostic purposes (e.g., in genomic analysis for the detection of inherited disease predispositions);

(b) or for therapeutic purposes (e.g., in the performance of somatic or even germline-interfering gene therapy);

(c) or for immunological purposes (e.g., in the construction of mRNA sequences that are “tailored” to combat specific pathogens);

(d) or for bioconstructive purposes, where the aim is to design whole organisms (single-celled organisms) in such a way that they can be used for the production of diagnostically or therapeutically effective drugs (e.g. by means of “genome editing” in the field of “synthetic biology”, for example, in order to incorporate new metabolic pathways “top down” into a given organism; in addition, however, the completely new construction of a living organism would also be conceivable “bottom up”, which could even have nucleic bases in its DNA or RNA that do not occur in nature).

Unfortunately, it is not possible within the limited scope of this paper to present here all the relevant areas in which genetic engineering is used within medicine. Therefore, in conclusion, we will only take a look at the vectors with the help of which the mRNA vaccines are introduced into the human organism.  In addition to the vaccines themselves, these transport systems represent the second application of nanotechnological methods in the context of combating covid-19.

5. “Nano Delivery Systems”: Functions and Risks

Since the safe transport of the mRNA agent into the human immune system is crucial for vaccination success, this aspect will first be considered in some detail. It has already been mentioned above that the mRNA active ingredient must be packaged in a shell of lipid nanoparticles in order to be able to enter the human immune system in a stable manner so that it can serve there as a template (antigen) for the production of antibodies against covid-19. However, this is only one example of a large number of so-called “delivery systems” at the nanoscale that can perform very different transport functions.

Nanomaterials are used in a wide variety of ways in the human body. Two particularly promising areas of application will be discussed in the following section: First, the group of various nano-transport systems (“nano delivery systems”), which serve to distribute active substances in the body. On the other hand, various metallic nanoparticles are used in cancer therapy, where alternating magnetic fields provide heating and destruction of tumor cells (hyperthermia process). Here, only the first case will be considered in more detail.

Nanoscale systems are used to transport active ingredients in the body (drug delivery). The nanomaterials enclose the active substance with tiny protective shells, which are then referred to as encapsulated systems or micelles. They enable the active substances to be protected or disguised by biological mimicry [6] in such a way that they can be transported to specific areas of application. Depending on their structure, they can overcome biological barriers such as cell walls, the gastrointestinal wall or the blood-brain barrier [19]. It is precisely the blood-brain barrier that has so far prevented a readily usable pharmaceutical approach to effectively treat diseases such as Alzheimer’s disease. Accordingly, the hopes associated with the use of nanomaterials are high. Depending on the objective and the desired site of application, the nano-transport systems fulfill different tasks. For example, they envelop poorly water-soluble or fat-soluble vitamins and active ingredients [2], making them more readily available to the body.

Other processes allow the release of active ingredients to be timed or substances that would decompose too quickly in the body to be released only at the point of use or evenly distributed over a very long period of time. There is a whole range of encapsulation systems, e.g. for cosmetics, for new pharmaceutical products or for contrast agents. Many systems use natural materials that are easily broken down by the body, but their nanoform gives them more stability or makes them more easily absorbed by the body. These include tiny fat droplets (nanolipid structures), natural protein compounds such as those that can be obtained from the extracts of shellfish (chitosan), or gelatin. Many systems copy nature, such as degradable polylactogluconates (protein-sugar compounds) or dendrimers (tree-like polymer structures), which are to be used in cancer therapies, herpes and difficult-to-treat fungal diseases.

Other systems work with materials such as carbon. These form non-degradable, nanometer-sized football-like structures (fullerenes) or tiny carbon nanotubes in which the active substances can be transported [7, 22]. Another development step that researchers are working on is targeted delivery systems, which can be equipped with specific receptors for cell types, viruses or other pathogens to “recognize” their target location [30, 13]. This would ensure that active substances act at the intended site of action, e.g. at specific organs such as the liver or at specific tumor cells, but not in other regions of the body. Monoclonal antibodies, which attach themselves to the tumor cells, are usually used. What the different types of delivery systems have in common is that improved or more targeted uptake could significantly reduce the amount of drug and undesirable side effects [1].

The societal benefits of drug delivery systems are seen primarily in improved medical cures and increased quality of life for patients [11]. Other benefit aspects include the potential reduction in health care costs and the expected positive economic development. Various attempts to quantify these benefits are summarized below.

First and foremost are approaches to cancer treatment. Cancer represents one of the leading causes of death worldwide, with approximately 7.6 million deaths in 2005. In industrialized nations, cancer is the second leading cause of death. The WHO predicts that cancer-related deaths will rise to 9 million in 2015 and increase to 11.4 million by 2030 [32]. Any therapeutic advances could mean cures or time delays for millions of sufferers and their families, and the greater efficiency of treatment methods could potentially lead to a reduction in healthcare costs.

In the research report “Nanotechnology pro Health: Opportunities and Risks”, written in 2004 for the German Federal Ministry of Education and Research [3], the authors refer to American studies [14] which, using the example of virial carcinomas, calculated possible cost reductions through the use of nanomaterials, since the lower side effects required fewer follow-up treatments. This was especially true for older female patients with a higher susceptibility to side effects. However, the BMBF study advises that the estimates of potential economic savings should be viewed with caution because of the poor comparability of the various international treatment methods and health care systems, as well as possible price trends for drugs and procedures [3].

Overall, undesirable side effects are a serious problem. In the USA, for example, they were responsible for an estimated 100,000 deaths within one year, making them the tenth most common cause of death [33].

Most quantitative estimates of the benefits of nanomaterials in the pharmaceutical industry relate to projections of market growth. They predict an increase of about 50% per year within the period from 2005 to 2012. At the same time, a steadily increasing share of nanotechnology in the overall pharmaceutical market is forecast. The forecast of a market volume of 4.8 billion US dollars in 2012 shows the optimistic assessment of the market potential of nanotechnology in this area [23]. The question would then remain open as to whether the high growth figures are associated with high drug prices, which would cancel out some of the cost savings in the health care system.

A general risk assessment of nano delivery systems is not possible in view of the wide range of applications and materials used as outlined above. Statements on the hazardousness or non-hazardousness of nanomaterials in this field of application should always be related to the individual case. Not only the forms of nanomaterials used, but also their possible bonding or decomposition processes (agglomeration and deagglomeration) must be taken into account [4].

When used in the medical field, specific safety tests apply before a product is approved. Of course, this also applies to products containing nanomaterials as active ingredients or as excipients, or to medical devices. Active ingredients are understood to be natural or synthetically produced chemical elements, their compounds, and mixtures or solutions that produce a pharmacological effect. They must be tested in preclinical trials to determine whether they have a long-term toxic effect on animals or humans (acute and chronic toxicity), whether they cause cancer (carcinogenicity), affect genetic material (mutagenicity) or have negative effects on unborn children (teratogenicity). As a rule, an additional risk assessment for environmental effects is required. Excipients, on the other hand, refer to substances that are necessary to give the drug a certain form, to make it durable, to flavor it, to color it, or to otherwise improve it with regard to its use. The pharmaceutical association Interpharma lists starch, sugar, gelatine, fats, oils, water and alcohols as examples of excipients [20].

Depending on the context of application, nanomaterials can fall under active substances as well as excipients if they are only used as a transport system. The German Medicines Act (AMG; 14th amendment AMG) and the Ordinance on the Application of Good Clinical Practice in the Conduct of Clinical Trials of Medicinal Products for Human Use (GCP Ordinance) specify precisely the extent to which safety tests must be carried out for active substances and excipients. This concerns the consultation procedures and clinical trials prior to approval, the approval procedures themselves, and the ongoing monitoring and reporting (pharmacovigilance) after approval, which documents the occurrence of side effects. Included in the review procedures for approval are consultations by ethics committees, which must approve clinical trials.

Currently, a discussion is taking place in expert circles on the extent to which nanomaterials as transport systems are sufficiently tested by approval procedures for excipients. Since 2002, however, the Notice on Marketing Authorization under Section 21 of the German Medicines Act has been in effect, requiring information on the bioavailability and bioequivalence of medicines. The improved bioavailability resulting from the use of nanomaterials in excipients must therefore be stated in new approvals, even if existing formulations are modified.

In the various scientific papers on drug delivery systems, there are usually detailed descriptions of functions and benefits, but only a few references to possible risk potentials. A distinction is made between degradable and non-degradable delivery systems. The majority of experts assume that degradable nanotransport systems such as the fat, protein or sugar compounds described above are processed by the body in the same way as larger compounds and do not pose a nano-specific risk [7]. At the heart of the concerns expressed are the possible overdose and entrainment effects of toxic substances from the environment, which could enter the organism with the drug delivery systems virtually by piggyback principle. However, these are all questions that need to be answered empirically and are only indirectly of ethical relevance.

Non-degradable (persistent) nanomaterials have been assessed as far more problematic. Various studies show negative health effects, e.g., for fullerenes [26] and carbon nanotubes, which do not recommend their use for transport systems in medicine [34]. However, again, recent studies on nanotubes indicate that a risk assessment is highly dependent on the form and application chosen and can only be made on a case-by-case basis. For nondegradable, persistent nanomaterials, questions also arise about environmental risks-even if they should be harmless to humans. Here, it is necessary to examine how they behave after excretion in the environment, i.e., what effects they might have on water, soil, and air. However, research in this area is still in its infancy.

Various ethical and social issues arise for nanotransport systems in medicine. In general, applications in medicine are considered a special case in the social risk assessment of nanomaterials. The core question of how much risk a society is willing to take when using new technologies in view of still existing knowledge gaps is considered in a very individualized way in the field of medicine [17]. Here, the health of the individual and the potential benefits from the use of nanomaterials are weighed against the individual risks of side effects. Depending on the severity of the disease and previous failures in therapy, the risk tolerance is very high if therapy with nanomaterials is seen as a promising method or “last resort”. This certainly applies in particular to cancer therapies, but in a broader sense also to the other applications where nanomaterials increase the efficacy of drugs and reduce side effects.

Ethicists are paying particular attention to the crossing of the blood-brain barrier and the resulting potential fields of application [21, 15]. The possibility of positively influencing brain performance in the case of Alzheimer’s disease could be used to increase performance in healthy people. An important topic of the ethical debate is therefore the possibility of misuse of this application for non-therapeutically indicated improvement of humans (human enhancement) by drugs. In its Code of Conduct, the EU excludes research on procedures or materials to improve healthy humans and makes reflection on ethical and social aspects of research projects mandatory for all EU projects [12].

The problem of misuse is also addressed in connection with military applications. Here, the main issue is the medication of soldiers to increase concentration or for continuous use without the need for sleep, as well as, in a broader sense, the use of nano delivery systems in the development of biological warfare agents [15]. What is problematic about the debate on military use or misuse of nanomaterials is that it remains predominantly in the realm of speculation due to the secrecy surrounding the actual projects.

In addition to the individual risk assessment and the possibilities of misuse, the critical question for nano-transport systems is the possible entry into the environment. Environmental organizations and ethicists alike address the open questions of risk assessment for the environment [29]. This concerns research, production and disposal of the products as well as possible entry into the environment through human or animal excretions. As there are currently no long-term studies on the use of nanomaterials in medicine, it is difficult to assess possible hazards. Until reliable findings are available, the principle of avoiding contact between humans and the environment with nanomaterials throughout the entire product life cycle applies in both the pharmaceutical and chemical industries. Particular attention is being paid to the use of non-degradable carbon-based nanomaterials (fullerenes and carbon nanotubes). Ethicists therefore appeal for a responsible approach to nanomaterials and focus on a critical debate about necessary approval criteria [15].

*

In the training unit 6.2 on the “legal aspects” of Covid 19 molecular genetic strategies, we will then see how some of the ethical issues are addressed by the legislation of different states as well as by the EU.

____________________

¹ The fact that nanotechnology still takes place largely in the shadows can certainly be viewed critically: for although nanomaterials are not infrequently a component of marketable products, the manufacturer has so far almost nowhere been required to provide information (in contrast, for example, to the duty to notify in the case of additives in foodstuffs). A labeling obligation – such as applies within the EU for products with genetically modified ingredients – would therefore be desirable for cosmetics and textiles containing nanoparticles, for example [9].

² The application potential of RNA vaccines is enormous: in the future, for example, it could be possible to use them to effectively combat such recalcitrant diseases as tuberculosis, AIDS and malaria; as well as to significantly improve the annual flu vaccination [10].

³ Some of the hopes here go very far: molecular machines introduced into the body will one day be able to make autonomous diagnoses and then take action, for example, to remove detected deposits in the arteries or to reconstruct damaged tissue in a targeted manner. However, it is precisely such ideas of serviceable “nanorobots” that frighten many people. What would happen if these robots got out of control? Could they then be deactivated again or “recalled” from their own bodies? There is obviously a considerable trust deficit here.

⁴ It should not go unmentioned that the relatively easy-to-produce mRNA active substances could also contribute to opening up healing opportunities for rare (and often hereditary) diseases, the so-called “orphan diseases”. These diseases in particular often receive little attention from the pharmaceutical industry, as it is hardly profitable to combat them.

⁵ For example, a blood test for expectant mothers has recently been developed which, with the help of “free” RNA, makes it possible to determine the risk of a dangerous pregnancy complication (the “pre-eclampsia”) at an early stage.

⁶ But a recommendation to vaccinate children (around the age of 6 to 11 years) can of course already be made: and this applies especially to children who have contact with vulnerable older adults. After all, what child wants to put his or her grandparents at risk? However, the decision to vaccinate always ultimately rests with the parents with parental authority.

⁷ For example, with the Moderna vaccine, occasional attacks of fatigue, fever, and muscle pain occur.

⁸ Here it is not decisive that the vaccination against Corona is not able to protect absolutely (i.e. to ensure long-term immunity): it is sufficient to prove that the vaccination can significantly attenuate the symptoms of the disease in case of infection.

⁹ It is extremely helpful here that RNA vaccines can be developed and modified very quickly. As Ron Renaud, CEO of the company Translate Bio, said: “You can change the sequence almost in the blink of an eye and adapt it to the currently circulating pathogen strains” (quoted from Dolgin, 2021 [10])

¹⁰ It is curious, however, that Wuhan, of all places, is home to a laboratory that has the world’s largest collection of coronaviruses [5].

¹¹ The references of coronavirus research to “synthetic biology” and to so-called “gain-of-function” (GoF) research, in which an organism is endowed with new capabilities, will not be discussed in detail here. It is worth noting, however, that in 2015 a team of researchers led by biologist Ralph Baric produced an artificial coronavirus by combining spike proteins from a bat pathogen with a Sars-CoV derivative: Such constructive research projects are not harmless unless there is absolute assurance that the modified organisms cannot escape into the environment and perhaps cause a pandemic there. On the other hand, GoF experiments can also help prevent widespread epidemics by showing which modifications of a potential pathogen could become dangerous. According to German GoF expert Silke Stertz, “In the current pandemic, we are also benefiting from the fact that researchers have been studying Sars-CoV and other coronaviruses for decades and exploring ways to vaccinate against them” (quoted from Spektrum der Wissenschaft, 2022[27]).

¹² The importance of these lipid nanoparticles should not be underestimated. As the Norwegian expert Nick Jackson put it: “Lipid nanoparticles have finally allowed RNA molecules to be used against a broad spectrum of diseases” (quoted from Dolgin, 2021 [10])

¹³ In the future, for example, mRNA-based cancer therapies should specifically block signals and signaling pathways by making so-called “small molecules” on the surface of tumor cells effective as receptor blockers. It would be advantageous here that the sequence of the mRNA could easily be individually adapted [28].

---

Test LO 6.1

---

References

1. Bilati, U., et al. (2005): Development of a nanoprecipitation method intended for the entrapment of hydrophilic drugs into nanoparticles. In: European Journal of Pharmaceutical Sciences. Volume 24, Issue 1, Jan 2005, pp. 67–75.
2. Bisht, S., et al. (2007): Polymeric nanoparticle-encapsulated curcumin (»nanocurcumin«): a novel strategy for human cancer therapy. In: Journal of Nanobiotechnology 2007, 5:3
3. BMBF – Bundesministerium für Bildung und Forschung (2004): Nanotechnologie als Wachstumsmarkt.
4. Borm, P. J. A., et al. (2006): The potential risks of nanomaterials: a review carried out for ECETOC. In: Part Fibre Toxicol., Aug. 14, pp. 3-11.
5. Casper, J.A. (2021): Made in China? In: Die ZEIT, No. 43, 21. Oct. 2021.
6. Chow, E. K.-H., et al. (2008): Copolymeric Nanofilm Platform for Controlled and Localized Therapeutic Delivery. In: ACS Nano 2 (1), pp. 33–40.
7. Conano (2007): Comparative Challenge of Nanomaterials. A Stakeholder Dialogue Project.
8. Council of Europe(1996): Convention for the Protection of Human Rights and Dignity of the Human Being with Regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine, Strasbourg.
9. Deutsches Ärzteblatt (2007): Nanotechnologie: Viele Chancen, unbekannte Risiken. A-548 / B-480 / C-464.
10. Dolgin, E. (2021): Siegeszug der RNA-Impfstoffe. In: Spektrum der Wissenschaft, 3/2021, pp. 53-57.
11. ETP – European Technology Platform on NanoMedicine (2006): Nanotechnology for Health. Strategic Research Agenda for Nanomedicine.
12. European Commission (2008): Commission’s recommendation on a Code of Conduct for Responsible Nanoscience and Nanotechnologies Research, 07 February 2008.
13. Farokhzad, O. C., et al. (2006): Targeted nanoparticle-aptamer bioconjugates for cancer chemotherapy in vivo.
14. Forbes, C., et al. (2002): A systematic review and economic evaluation of pegylated liposomal doxorubicin.
15. Gammel, S. (2007): Ethische Aspekte der Nanotechnologie. In: Interfakultäres Zentrum für Ethik in den Wissenschaften. Eberhard-Karls-Universität Tübingen.
16. Grobe, A., et al. (2008): Nanomedizin – Chancen und Risiken. Eine Analyse der Potentiale, der Risiken und der ethisch-sozialen Fragestellungen um den Einsatz von Nanotechnologien und Nanomaterialien in der Medizin. Gutachten im Auftrag der Friedrich-Ebert-Stiftung. Bonn (Germany).
17. Grunwald, A. (2004): Ethische Aspekte der Nanotechnologie. Eine Felderkundung. In: Technikfolgenabschätzung – Theorie und Praxis Nr. 2, 13. Jg., Juni 2004, pp. 71–78.
18. Hagengruber, R. / Dasch, Th. (2021): Was ist und wozu Nanoethik?
19. Hoet, P. et al. (2004): Nanoparticle – known and unknown health risks. In: Journal of Nanobiotechnology 2 (1), pp. 12–27.
20. Interpharma (2008): FQA Medikamente, Herstellung; available under: htts.//interpharma.ch/de/1718.asp
21. Kreuter, J. (2004): Influence of the Surface Properties on Nanoparticle-Mediated Transport of Drugs to the Brain. In: Journal of Nanoscience and Nanotechnology, Vol. 4, Nr. 5, Okt. 2004, pp. 484–488.
22. Marcato, P. D. / Durán, N. (2008): New Aspects on Nanopharmaceutical Delivery Systems. In: Journal of Nanoscience and Nanotehnology 8, pp. 1–14.
23. Moradi, M. (2005), zitiert nach: Hullmann, A. (2006): The economic development of nanotechnology – An indicator based analysis.
24. NCI 2008: National Cancer Institute (2008): Definition Soft Tissue.
25. Nordmann, A. (2013): Nanotechnologie. In: Handbuch Technikethik, ed. By Grunwald, A, et al., Stuttgart (Germany), pp. 338-342.
26. Sayes, C. M., et al. (2004): The Differential Cytotoxicity of Water-Soluble Fullerenes. In: NANOLETTERS, Vol. 4, No. 10, pp. 1881-1887.
27. Spektrum der Wissenschaft (2022): Kritiker überschätzen die Risiken extrem. Interview with Silke Stertz. April 2022, pp. 48-52.
28. Thiem, J. (2022): Kleiner Pieks mit großer Wirkung. In: zukunft Medizin, 2022, p. 14.
29. Tiefenauer, L. (2004): Nanotechnologie in der Medizin. In: Technikfolgenabschätzung – Theorie und Praxis Nr. 2, 13. , Juni 2004, pp. 52–57.
30. Torchilin, V. P., et al. (2003): Immunomicelles: Targeted pharmaceutical carriers for poorly soluble drugs. In: PNAS, Vol. 100, Nr.10, pp. 6039–6044.
31. UNESCO(1996): Vorläufiger Entwurf einer Allgemeinen Erklärung zum Menschlichen Genom und zu den Menschenrechten, Paris.
32. WHO – World Health Organization (2008): Cancer; available under:  http://www.who.int/mediacentre/factsheets/fs297/en/index.html
33. Woodrow Wilson Center (2007): Nano Frontiers. On the Horizons of Medicine and Healthcare. In: Issue 1, May 2007.
34. Yan, L., et al. (2007): The Latent Toxic Effects of Carbon Nanotube Serving As Biomedicine. In: Bioinformatics and Biomedical Engineering, 2007. The 1st International Conference on ICBBE 2007, pp. 342–345.

Summary

Despite the lack of specific regulatory guidelines, many nanomedicines are on the market and their number is growing steadily. These are mainly used in cancer therapy because they require persistent toxic compounds and the tumor landscape is very difficult, which hinders effective drug treatment. The lack of formal regulation of nanomedicines and the manufacture of nanomaterials for health-related applications is a worldwide problem. Inconsistency among different government agencies results in some nanomedicines being classified as medical devices and others as drugs.
Therefore, a global consortium for nanomaterials regulation should be formed to advance these agendas and issue formal guidance to the research communities. Currently, in 2 the context of nanomaterials in the European Union, we are dealing with both binding legal acts and non-binding legal acts, such as recommendations on the fair conduct of scientific research or on the application of a uniform definition of nanomaterials.

Key words/phrases: Legal regulation, safety of nanomaterials, European Union: REACH, precautionary principle, international developments.

1. Introduction

The COVID-19 pandemic has become one of the greatest global health threats in modern history. Advances in nanotechnology development have provided relief in the form of rapid diagnostic tests and rapidly developed vaccines against SARS-CoV-2 [43]. But legal considerations must be factored into the development of nanotechnology solutions to global health problems [43, 23]. In the European Union (EU), this has been recognized as a key technology that can provide new and innovative medical solutions to unmet medical needs [44]. There are an increasing number of applications and products that contain nanomaterials or at least nanotechnology-based claims. The use of nanotechnology in the development of new drugs is now a part for pharmaceutical research [2, 35, 37, 47].

The application of nanotechnology for medical purposes is called nanomedicine. It is defined as the use of nanomaterials for the diagnosis, monitoring, control, prevention, and treatment of disease [47]. However, more research with specific relevance to regulatory issues is needed, particularly with respect to implementation of the definition of nanomaterials, enforcement of product labeling, development of methods for safety testing and risk assessment, and improved availability of quality data on nanomaterials for regulatory purposes [39]. While the definition of nanomaterials is controversial among various scientific and international regulatory agencies, some efforts have been made to find a consensus definition.

Due to their small size, nanomaterials possess novel physicochemical properties that differ from those of their conventional chemical equivalents. These physicochemical properties open up a number of opportunities for drug development. Some examples regarding the application of nanomaterials include.

• The physicochemical properties of the nanoformulation, which can lead to changes in pharmacokinetics, i.e., absorption, distribution, elimination and metabolism,
• the potential to more easily overcome biological barriers,
• toxic properties, and
• their persistence in the environment, and
• pathways in the human body [2, 47].

As the research community continues to explore nanomedicine, its efficacy, and related safety issues, it will be critical to address the scientific and regulatory gaps to ensure that nanomedicine can reach the next generation of biomedical innovation.

First, it is appropriate to establish a clear definition for the presence of nanomaterials. The European Commission (EC) has established a definition based on the Joint Research Centre of the European Commission and the Scientific Committee on Emerging and Newly Identified Health Risks. This definition is used only as a reference to determine whether or not a material is considered a nanomaterial. The European Commission maintains that it should be used as a reference for additional regulatory and policy frameworks related to quality, safety, efficacy, and risk assessment [4]. The EMA Working Group introduces nanomedicines as purposefully designed systems for clinical applications that have at least one component at the nanoscale and have reproducible properties and characteristics related to the specific nanotechnology application and characteristics for the intended use (route of administration, dose) associated with the expected clinical benefits of nanotechnology (e.g., preferential organ/tissue distribution) [35]. According to the former definition, there are three basic aspects to determine the presence of a nanomaterial, namely.

• Size,
• Particle size distribution (PSD), and
• Surface Area [10].

Ideally, characterization of a nanomaterial should be performed at different stages of its life cycle, from design to evaluation of its in vitro and in vivo performance. Interaction with the biological system or even sample preparation or extraction procedures may alter some properties and affect some measurements. In addition, the determination of in vivo and in vitro physicochemical properties is important for understanding the potential risk of nanomaterials [7].

2. Overall legal regulations

Nanotechnology is thus a very broad field that encompasses a number of nanoscale technologies, including pharmaceuticals, biotechnology, genomics, neuroscience, robotics, and information technologies [1]. Regulators have therefore started to address the potential risks of nanoparticles since 2000 [41]. Since 2004, the EU has been developing a regulatory policy to tighten control and improve regulatory adequacy and knowledge of nanotechnology risks [30].

Currently, there are specific regulations on nanomaterials for biocides, cosmetics, food additives, food labelling, and food contact materials [1]. The observation is that nanotechnologies inevitably raise questions, such as.

• public confidence,
• potential risks,
• environmental impact issues,
• transparency of information,
• responsible nanoscience and
• nanotechnology research.

Nanotechnology is defined as a dual-use technology, as it brings opportunities for human progress and development, but it can also pose a serious threat to human health and life and to the environment [1, 13, 30]. It is a very young field, and the effects of nanomaterials on the human body and the environment, especially in the long term, are not always known [38]. Nanomaterials may have increased biological activity as they readily penetrate biological membranes and they may therefore have toxic properties and pose a hazard to humans and animals.

Relevant legal regulations in the European Union do not directly address nanotechnology, so new instruments should be created to prevent the possible harmful effects of nanomaterial use [38]. The possibility of creating a collective legal order in the future is usually not considered, as it can be done only under the condition of obtaining complete knowledge about the properties of nanomaterials. Thus, it is a matter of application of a unified definition of nanomaterial, development of measurement tools related to nanomaterials, development of safety tests and methods of risk assessment.

The aim of the EU legislation is to,

• to provide the public with access to innovative applications of nanotechnology, and

at the same time ensure safety and the protection of health and the environment.

3. COVID-19 impact on food packaging

The European Union (EU) regulatory framework, which consists of several horizontal and sector-specific pieces of legislation, covers nanomaterials explicitly or implicitly. Thus, nanomaterials, and in particular the potential risks associated with them, are in principle covered by existing legislation, even if nanomaterials are not explicitly mentioned [39]. In addition, recently updated product-specific EU legislation, as well as newly proposed legislation, explicitly addresses nanomaterials, including specific nanomaterial information requirements, authorization of nanomaterials for specific uses, and safety assessment that takes into account nanospecific characteristics [39]. The European Commission’s review of nanomaterials legislation concluded that nanomaterials are similar to normal chemicals/substances in that some may be toxic while others are not. Potential risks are related to specific nanomaterials and specific uses. Risk assessment of nanomaterials should be conducted on a case-by-case basis using relevant information. Current risk assessment methods are applicable, although further research is needed from certain aspects of the risk assessment [39].

Although the European Commission has adopted a recommendation for a definition of nanomaterial, this term is currently not clearly defined in a legally binding way, but its definition and implementation depend on the specific legal context.

• First, the question arises whether “it will probably be necessary to base future law concerning [nanotechnology] on prior law concerning analogous prior products or processes,” that is, whether they are compatible with biotechnology, for example [6, 33].
• Some authors see the possibility of increasing the safety of nanomaterial use through non-judicial means [29].
• Because of the dynamics of change in this area, others see an urgent need for the use of soft law [29]. The main question raised in the international literature is whether nanotechnology should be included in the legal framework or whether the so-called soft law mechanisms can be used.
• Currently, in the context of nanomaterials in the European Union, we are dealing with both binding legal acts (regulations, directives) and non-binding legal acts, such as recommendations on the fair conduct of scientific research or the application of a uniform definition of nanomaterials [45]¹ .

Soft law includes:

• Resolutions,
• Guidelines,
• Declarations,
• Messages,
• Programs,
• Plans, usually issued by the authorities involved in the legislative procedures, the result of which is a generally applicable legal act.

The construction of soft law instruments is intended to provide a basis for the future hard legal regulations.

• The literature also points to the need to regulate the intellectual property aspects related to nanotechnology. Also, in the context of patent law, the question arises whether nanotechnology inventions should be excluded from patenting because of the unknown potential risks to human health or the environment, just as in the case of biotechnology inventions [6, 33]. Later, if it is a technology, some of the problems facing nanotechnology will be specific and therefore “can only be addressed by creating entirely new rules.” [18, 50].
• Incidentally, the voluntary programs aimed at gathering information useful for the design of legal instruments are also important.
• In line with the position of the EU bodies, some authors suggest strengthening existing forms of cooperation in the field of nanotechnology and encouraging states to create internal legal regulations [16, 17].
• A difficult issue is the ability to regulate nanotechnology at the international level under future framework agreements [28]. Some authors agree that regulating nanotechnology at the international level is a major challenge because nanomaterials are used in different ways.  However, it seems that comprehensive regulations in the field of nanotechnology will be created in the future. The creation of best practices for handling nanomaterials to be applied at the international level could significantly influence this. Nanotechnologies are an interdisciplinary subject, which is reflected in a very wide range of possible applications. Nowadays, nanotechnologies encompass most areas of technology [28].

The concept of nano-ethics, defined as moral reflection on the development and application of nanotechnology or the manipulation of matter at the molecular level, is emerging in the literature. In this context, there are beginning to be dilemmas regarding the potential harmfulness of nanoparticles. In the case of nanotechnology, there is the question of its availability and its impact on the welfare of humanity. There is a well-founded fear that nanotechnology will become another element that reinforces the division of the world into developed and backward countries or controlled and controlled countries [43]² .

4. Legal regulations in the field of nanotechnology

4.1. Precautionary principle as the basis for nanotechnology regulation

According to Principle 15 of the Rio Declaration, the lack of complete scientific certainty should not be a reason for postponing cost-effective measures to prevent environmental damage [42]. In short, the precautionary principle reflects a “better safe than sorry” approach to potential environmental risks [36]. That is, based on current knowledge, nanomaterials are similar to natural substances in that some may be toxic while others are not [21]. The potential risk is related to specific nanomaterials and specific applications. Therefore, in the case of nanomaterials, a risk assessment is required and should be conducted on a case-by-case basis using relevant information. Currently, the main challenge is to develop validated methods and tools for the detection, characterization and analysis of nanomaterials, to obtain complete information on the risks associated with nanomaterials and to develop methods for assessing exposure to nanomaterials.

Despite the studies conducted to date, we are not able to quantitatively assess the associated risks. The toxicity of individual nanoparticles varies widely, making it difficult to establish a common criterion. Nanoparticle toxicity is evaluated in relation to individual organisms: mammals, protozoa, crustaceans, algae, and plants.  Toxicity to mammals is tested in rodents.  There have been few attempts to evaluate the health status of humans who are occupationally exposed. Sparse studies on human cell lines showed significant DNA damage [27].

Since the risks posed by nanomaterials are not yet fully understood, they should be covered by multi-layered and diverse legislation.  The new regulations need to be drafted based on the precautionary principle and the producer responsibility principle to ensure the safe manufacture, use and disposal of nanomaterials before they are placed on the market.  The precautionary principle allows for a rapid response to potential risks to human, animal or plant health or to the protection of the environment. According to the Commission, the precautionary principle can be invoked when the phenomenon, product or activity poses potential safety risks identified through a scientific and objective assessment, if such an assessment makes it possible to identify such a threat with reasonable certainty [24].

In the case of nanomaterials, we certainly have to deal with the situation where there is no complete knowledge of the risks arising from their use. This principle is enshrined in the legal systems of many countries.  The European Union has included it in Article 191(2) of the Treaty on the Functioning of the European Union (TFEU), resulting in the obligation of Member States to apply this principle in their legislation.  The implementation of this provision helps in assessing the potential risk. It is explicitly stated that even if the presence of nanoparticles in the elements of the environment or waste can be detected, it would be technically difficult to eliminate them. Therefore, the measures taken at the end of the contamination chain, so as to prevent the possible negative consequences for the environment and human health, cannot be effective [21].

As early as 2009, the European Parliament recommended that Member States invest in adequate assessment of the risks arising from the use of nanomaterials in order to fill the knowledge gaps and rapidly develop and implement assessment methods and appropriate and harmonized metrology and nomenclature. There are no methods to assess the risks associated with nanomaterials, making it impossible to develop effective regulatory mechanisms in this area [40].

Only with more detailed and comprehensive scientific research can scientists evaluate the potential risks of nanomaterials and conduct an appropriate risk assessment, as required by the precautionary principle [36]. Consequently, the precautionary principle supports the improvement of new technologies and only prevents the use of new technologies that are harmful to the environment. The precautionary principle does not hinder new technologies. Rather, it strikes a more prudent balance between technological advancement and environmental safety, giving the environment the benefit of the doubt.

Legally non-binding regulations, however, have many advantages that binding regulations do not. First, it may be easier to reach consensus on a particular issue, as the inhibition threshold is reached because of the lower inhibition threshold for parties to agree to nonbinding regulations [3]. This lower inhibition threshold can be very helpful in initiating a joint discourse on possible regulations. Second, soft laws are less costly and more flexible in terms of their negotiation and implementation [46]. Third, the coercive nature of a law is no guarantee that affected individuals will abide by it. Rather, there are are many reasons why individuals adhere to legally non-binding rules, how their socialization, their self-interest, the moral codes of their society prevent them from doing so.

The international community should not only keep in mind the importance of protecting the environment from the negative from the negative effects of nanomaterials, but also think about future regulations for other new technologies. Since the risks posed by new technologies will be one of the greatest environmental challenges of the future, the international community must demonstrate its ability to successfully address the challenges posed by these new technologies. Therefore, effective international regulation of nanomaterial risks must take an important step into a new era of environmental law.

4.2. Treatment in the European Union

In the European Commission (EC) Communication “Nanosciences and nanotechnologies: an action plan for Europe for 2005-2009. Second implementation report for 2007-2009”, it was stated that nanotechnology offers significant potential to improve the quality of life and industrial competitiveness in Europe [26]. Its development and use should not be delayed, unbalanced or left to chance [25]. At that time, a first review of the regulatory framework in the field of nanotechnology was carried out to investigate whether new regulatory measures were needed to cover the risks associated with nanomaterials.

Preliminary results showed that existing regulations in principle cover health issues and environmental impacts. Member State regulators were tasked with assessing national legislation and identifying gaps therein.  Even then, a preventive approach to nanotechnology was recommended. In the absence of complete knowledge on nanomaterials, it was recommended to use existing legal mechanisms related to thresholds, authorization of substances and ingredients, classification of hazardous waste, strengthening of conformity assessment procedures, restriction of the marketing of chemical substances and preparations and their use. However, given limited resources and rapidly evolving technology, it is more likely that a self-regulatory approach will be taken, “perhaps supported by strong incentives in the form of tort liability or criminal laws.” [18].

In 2009, the European Parliament (EP) adopted a report on the regulatory aspects of nanomaterials, which also took into account the Commission Communication of 17 June 2008 entitled “Regulatory aspects of nanomaterials” (COM (2008) 366). The report shows that the European Commission sees the benefits of nanotechnological development, but at the same time is aware of the risks that this development poses to humans and the environment. The European Commission confirmed that knowledge about the potential risks of nanomaterials is incomplete. There is no evidence of the risks posed by specific nanomaterials, and there is an overall lack of methods for adequately assessing the risks associated with concerns about nanomaterials. Given the many doubts about the use of nanomaterials, it seems urgent to include this area in the regulatory framework.

For this reason, since 2008, the European Commission has been reviewing the existing regulations on the use of nanomaterials and identifying the actions that should be taken in the future.  In the second review of nanomaterials legislation, the European Commission highlights the need to improve EU legislation to ensure the safe use of nanomaterials.  The Communication highlights the diverse nature and types of nanomaterials, ranging from everyday materials,as they have been used for decades (e.g., in tires or as anticoagulants in food), to advanced materials used in industry and cancer therapies. More and more is becoming known about the hazardous properties of nanomaterials.

They cannot be categorized, which justifies the need to assess the risk associated with specific applications.  The European Commission emphasizes that an individualized approach should be taken to risk assessment, using strategies based on information about the potential risks in terms of exposure or hazard.  In recent years, the majority of member states have been working on legislation to regulate the use of nanomaterials. The current regulations on the use of nanomaterials consist mainly of two regulations,

• the so-called REACH Regulation (Registration, Evaluation and Authorization of Chemicals) ((EC) No. 1907/2006) and
• the CLP Regulation (Classification, Labelling and Packaging) ((EC) No. 1272/2008).

Nanomaterials are already used in numerous products of everyday use, but the risks have not yet been sufficiently researched, because for many nanomaterials there are hardly any reliable data to assess their potential risks. Closing this knowledge gap is actually the task of European chemicals legislation. However, most of the laws regulating chemicals and products have so far contained no, or only limited, specifications on the handling of nanomaterials.

REACH, the abbreviation for Registration, Evaluation and Authorization of Chemicals, is a milestone in the protection of people and the environment from substances that are harmful to health. The EU regulation required industry to submit data on the environmental and health impacts of its chemicals for the first time starting in June 2007 – as a prerequisite for them to be marketed at all [21]. Until then, harmful effects had to be proven by the legislator before a chemical could be banned. REACH has now reversed the burden of proof. The principle applies: no data, no market.

REACH has also strengthened consumers’ rights to information. Consumers have the right to ask the manufacturer or distributor of a product whether it contains a particularly hazardous substance. Companies are obliged to respond.

REACH lays down strict rules for the use of particularly hazardous substances. They may only continue to be used if a special authorization is granted for this purpose or if there are no safe alternatives. Chemicals are considered “substances of very high concern” if they:

• Cause cancer and damage genetic material or reproductive ability,
• do not degrade in the environment, accumulate in humans and animals, and are toxic,
• are practically not degraded in the environment and accumulate very strongly in the body, but for which no toxic effect has yet been proven,
• have similar hazardous effects, e.g. hormonal effects.

In addition, the ECHA Nanomaterials Working Group (NMWG), composed of experts from EU Member States, the European Commission, ECHA and recognized stakeholder organizations, informally advises on scientific and technical issues related to the implementation of REACH and the Classification, Labeling and Packaging (CLP) legislation with respect to nanomaterials. In addition, ECHA organized a Working Group on the Assessment of Already Registered Nanomaterials (GAARN) to address best practices for assessing and managing the safety of nanomaterials under REACH. ECHA takes into account the findings of these expert groups when developing new or updated guidance. The work of the scientific committees also feeds into the work of other EU bodies such as EFSA, the European Medicines Agency (EMA) and ECHA. All scientific opinions of the Scientific Committees are published on the Internet.

Together with the Center for International Environmental Law (CIEL) and the advocacy organization ClientEarth, BUND in Germany submitted its own proposal for the regulation of nanomaterials [5]. This proposal provides for a new, horizontal EU regulation. On the one hand, it contains general principles for the regulation of nanomaterials and, on the other hand, concrete adaptations of individual EU regulations, in particular the European chemicals regulation REACH.

This is what BUND demands with regard to the provisions of REACH:

• Adoption of the definition for nanomaterials already proposed by the EU Commission in 2011 in all relevant laws. However, these have so far been non-binding.
• A general obligation to report all nanomaterials and nanoproducts. These are to be kept in an EU-wide nano register.
• Labeling of nanomaterials on products in the list of ingredients.
• Closing the gaps for nanomaterials in REACH.

A nano-register [5].

4.3. Regulations

Provisions on nanomaterials can also be found in regulations.  Since nanotechnologies are also used in medicine, a directive on the Community code relating to medicinal products for human use appeared in 2001 (Directive 2001/83/EC) [41]. Procedures for the authorization of medicinal products were also established ((EC) No. 726/2004) [8]. To ensure safety, it is advisable to establish a register of nanomaterials and products containing nanomaterials. Such a register facilitates the monitoring of companies placing nanomaterials on the market and ensures transparency of product data for purchasers. It should be recognized that the European Union is working consistently to regulate nanotechnology.

Individual EU member states have begun implementing initiatives aimed at better informing the public about nanotechnology developments. In the United Kingdom, the DEEPEN (Deepening Ethical Engagement and Participation with Emerging Nanotechnologies) project was launched to provide a basis for social acceptance of nanotechnology development [11]. In the Netherlands, it was the Nanopodium program, one of the most important social dialogue programs in the European Union [49]. Belgium launched the Nanosoc program, which aimed to create a common platform for discussion on nanotechnology for researchers, companies and society. Although nanotechnology does not generate the kind of controversy that biotechnology does, and societies tend to be more positive about the diagnostic and therapeutic possibilities of nanotechnology, they are increasingly demanding detailed information about the long-term effects of nanoparticles on the body.

In Germany, the Nanologue project was launched to highlight the benefits and consequences of nanotechnology, explain the ethical, social and legal issues associated with its use, and promote dialogue between the public and other interested parties [48]. Social acceptance and the elimination of concerns about nanomaterials will have a positive impact on the future and development of nanotechnology. Social dialogue should include civil society representatives and scientists, as well as other stakeholders.

The EU4Health program is the EU’s most ambitious health policy response to the COVID-19 pandemic, which is having a significant impact on patients, medical and healthcare professionals, and healthcare systems in Europe [15]. EU4Health will go beyond a mere crisis response to make health systems more resilient to crises, according to Regulation (EU) 2021/522. The established program will provide funding to eligible institutions, health organizations and NGOs from EU countries or non-EU countries associated with the program. EU4Health paves the way to a European Health Union and focuses on urgent health priorities [15].

The ten specific objectives formulated under the four general objectives are: Improving and promoting health in the Union; Disease prevention and health promotion; Health initiatives and cooperation at international level; Combating cross-border health threats; Prevention, preparedness and rapid response with regard to cross-border health threats; Supplementing national stockpiles with essential crisis-related products, Establishing a reserve of medical, health and support staff, Improvements in medicines, medical devices and crisis-related products, Available and affordable medicines, medical devices and crisis-related products, Strengthening health systems, their resilience and resource efficiency, Strengthening health data, digital tools and services; Digitizing health systems, improving access to care, developing and enforcing EU health law, and evidence-based decision-making and alignment between national health systems [15].

EU countries are consulted on the program’s priorities and strategic orientations and, together with the Commission, ensure the coherence and complementarity of national health policies through the “EU4Health Steering Group.” Before the adoption of the annual work programs, they give their opinion in the EU4Health Program Committee.

The European Commission prepares, adopts and implements the annual work programs. It also monitors and reports on progress toward program objectives. It may seek the opinions of the relevant decentralized agencies and independent health professionals on technical or scientific issues relevant to the implementation of the program. The Health and Digital Executive Agency (HaDEA) will implement the program [15].

Finally, in 2018, the European Commission made changes to REACH Annexes I, III, VI, VII, VIII, IX, X, XI, and XII that took effect on January 1, 2020 [9]. Under the revised REACH requirements, all nanoforms must be registered, with a focus on chemical safety assessment. Nanoforms of substances must additionally be identified and characterized as part of the registration process. Most importantly, risks to the environment and human health must be assessed using OECD-mandated guidelines that are either in place or being developed. Further refinement of the guidelines may be required as industry and its knowledge base continue to grow, but the EU has, as always, set high standards when it comes to maintaining the quality of human health and the environment.

5. International developments

Activities to regulate nanotechnology have also been undertaken in the international arena, as the potential risks arising from developments in this area are seen by many countries [23].

According to the OECD definition, “regulatory frameworks consist of the norms and rules that govern a particular group of persons, actions, or objects and that are enacted by governmental bodies pursuant to statutory authorization.” [34]. At the global level, the Organisation for Economic Co-operation and Development (OECD) launched a strategic program in 2006 within its Framework for Chemical Safety to provide a global forum for discussion of manufactured nanomaterials, particularly their safety assessment and risk evaluation, and to promote the responsible development of these technologies. The OECD Working Party on Manufactured Nanomaterials (WPMN) promotes international cooperation on human health and environmental safety aspects of manufactured nanomaterials and focuses on developing appropriate methods and strategies to ensure safe use of nanotechnology [12]. Under the WPMN program for testing manufactured nanomaterials, OECD WPMN members, together with non-OECD countries and industry, have tested a selected list of manufactured nanomaterials for endpoints relevant to physicochemical properties, environmental fate and toxicology, mammalian toxicology, and material safety [12]. Data obtained under these guidelines are covered by the OECD Mutual Acceptance of Data (MAD) agreement in the evaluation of chemicals. MAD is an essential component of the international harmonization of approaches to chemical safety through regulatory acceptance of these testing guidelines. Therefore, data on nanomaterials obtained under the OECD testing guidelines applicable to nanomaterials are equally covered by MAD.

5.1. The USA

In the United States, the U.S. National Research Council published a report in 2008 that also called for greater regulation of nanotechnology.

The wide range of devices and products produced by nanotechnology companies can send many agencies on their way, such as the Food and Drug Administration, the Environmental Protection Agency, the National Institute of Health, or the Department of Health and Human Services Health and Human Services, either taking their own regulatory approach or developing a coordinated regulatory approach [50].

As part of its leadership in this area, the federal government has authorized the 21st Century Nanotechnology Research and Development Act (“Act”), 15 U.S.C.A. §7501- §7509, authorized $4.7 billion between 2004 and 2008 for the National Nanotechnology Initiative, a nanotechnology initiative composed of nine agencies: the National Science Foundation, the Department of Energy, the National Aeronautics and Space Administration, the National Institutes of Heath, the National Institute of Standards and Technology, the Environmental Protection Agency, the Department of Justice, the Department of Homeland Security, and the Department of Agriculture. The programs of these agencies are overseen by external and intergovernmental committees, while the Office of Science and Technology Policy is responsible for coordinating and managing the National Nanotechnology Initiative (see www.nano.gov for more information). One of the main goals of the Act is to create a collaborative effort between government and industry to develop and commercialize nanotechnology in a coordinated and efficient manner.

Another organization currently addressing the impact of nanotechnology is the American Bar Association. The Section’s Standing Committee on Nanotechnology is currently organizing fora to try to identify the potential risks and hazards associated with nanotechnology and where scientists, advocates, and legislators can discuss the ethical and social implications of nanotechnology [12]. In the medium term, the best approach is to discuss regulation or changes in protections in a more temperate manner and based on the experience gained from addressing problems. In the long term, as nanotechnology matures and becomes established as an industry, it is likely that circumstances will be so different from those in the world today that any current “proposals are bound to overshoot the mark by a wide margin.”

The overview of the major legislation that has been passed related to nanotechnology shows that the priority of the U.S. government to date has been to promote and fund nanotechnology research and development.

Arguably, the biggest problem with regulating nanomedicines is the fact that regulatory agencies such as the FDA use safety data based on bulk materials that do not have the same pharmacodynamic and pharmacokinetic activity as nanomedicines [22]. This means that the safety and efficacy data collected are not representative of what might actually happen if the nanomedicine is used in clinical situations once they receive approval. This leads to problems in establishing regulations for the safety and efficacy parameters of nanomedicines, as a non-nanoversion may meet regulatory standards, but a nanomedicine may not. This means that a nanomedicine may be classified as a drug in one country and a medical device in another, so the regulations that must be met change depending on the classification. Thus, the specific safety and efficacy standards that the product must meet in order to be marketed vary, so a nanomedicine may be used in some countries that may not meet regulatory standards in another [29]. Because of their highly complex structures and properties, it is difficult to establish a sound and consistent manufacturing process that defines the quality, efficacy, stability, and safety of nanomedicines.

Nanomedicine products would be evaluated on a product-specific basis. Manufacturers are advised to coordinate with FDA in the development of their nanotechnology products to establish a mutual understanding of regulatory issues.

This inaction in the changing landscape has led to much criticism of the FDA. As a result, nanoformulations consisting of already approved building blocks appear to be fast-tracked through the system without new drug approval or full premarket review. This strategy is extremely risky and only time will tell if it is appropriate [19].

There have been very few legislative efforts to regulate nanotechnology. In fact, no laws have been passed that directly address the regulation of nanotechnology. Perhaps this is the right approach to take for an emerging technology; however, it leaves room to question whether this is an approach that has allowed nanotechnology to develop in a blind spot. The reason for this blind spot is the lack of accumulated research to determine the safety of these nanomaterials.

Despite these concerns, regulation through legislation is not recommended as a solution for a regulatory framework. This means that regulation by legislation is taken in response to the discovery of a major risk or after a disaster. Thus, it is easy to see the failures of oversight and regulation in the asbestos industry, but it was not easy to see this deficiency when companies were using asbestos in almost all new construction. Because regulatory laws are typically enacted only in response to a reaction to a catastrophic event or overwhelming evidence, they are not considered a viable element of the regulatory framework recommended in this paper [14]³ .

5.2. Australia, Canada and other countries

Australia and Canada are also quite active in nanoregulation.  Both countries have major Environmental Health and Safety (EHS) research programs and have published in-depth reviews of their regulations to identify any limits in the use of nanotechnology.  Although no specific legislation has been enacted, both countries provide for the application of the precautionary principle in the use of nanotechnology.

Japan, China, Korea, and Taiwan, which are heavily involved in nanotechnology, also have important research initiatives at various levels that address EHS issues such as risk assessment and risk management of nanomaterials and nanoproducts. While they participate in the global debate on nanoregulation, no specific initiatives on this topic have been undertaken in these countries [14]. Currently, nanotechnology regulation activities are focused at the national level, and initiatives in the nature of joint research programs can be observed at the international level.

Health Canada has established a working definition of nanomaterials, which states that “any manufactured product, material, substance, ingredient, device, system, or structure is considered a nanomaterial if it is at the nanoscale (1-100 nm) in at least one spatial dimension or is smaller or larger than the nanoscale in all spatial dimensions and exhibits one or more nanoscale phenomena.” [20]. Regarding the approval of nanotechnology products, Canada relies on the existing regulatory framework. Health Canada advises manufacturers to consult with the appropriate regulatory authority early in the development phase to identify and assess the risks and characteristics of the product. In Canada, the Health Portfolio Nanotechnology Working Group was established to gather and discuss issues related to nanotechnology. It consists of representatives from regulatory agencies such as Health Canada and the Canadian Institutes of Health Research (CIHR). A general guidance document on the review of nanotechnology-based biomaterials for health products and foods has also been issued by Health Canada [20].

In Japan, pharmaceuticals are regulated by the Ministry of Health, Labour and Welfare (MHLW)/Pharmaceuticals and Medical Devices Agency (PMDA) [31]. Japanese regulators have yet to develop a definition and nanomedicine-specific regulations for nanomedicines. In 2016, a guide for liposome drug development was published. Nanomedicines are regulated under the Pharmaceutical Affairs Law, a general drug legislation, on a case-by-case basis. It should be noted that regulatory agencies and reviewers are collecting and analyzing data on nanomedicines. The MHLW/PMDA has also collaborated with the EMA in issuing reflection papers, particularly on the development of block copolymer micelle-based drugs and nucleic acid (siRNA)-loaded nanotechnology-based drugs.

Although there is little regulation in this area in Asia, countries such as India, Japan, China, and Thailand are currently establishing government and regulatory policies to address the growing issues in nanotechnology. In India, the Ministry of Science and Technology and the Indian government have set up a group to regulate nanotechnology and have drafted a set of guidelines that have created a three-tiered regulatory framework to help policymakers develop a pathway to regulate nanomedicine. This will ensure the continued growth of this technology while addressing the risks associated with nanomedicine.

6. Conclusions and future prospects

Despite the lack of specific regulatory guidelines, many nanomedicines are on the market and their number is growing steadily. These are mainly used in cancer therapy because they require persistent toxic compounds and the tumor landscape is very difficult, which hinders effective drug treatment. Among the best known are the liposomal preparations Doxil®, AmBisome® and the more recent successes with albumin drug nanoparticles such as Abraxane® and polymeric micelles such as Eligard®, to name a few.
The lack of formal regulation of nanomedicines and the manufacture of nanomaterials for health-related applications is a worldwide problem. Inconsistency among different government agencies results in some nanomedicines being classified as medical devices and others as drugs. What is considered appropriate in one jurisdiction does not translate to other countries, and while small molecules are often not approved globally for this reason, the nanomedicine community is in dire need of a unified approach so that development can continue in line with expectations. The formation of clusters and working groups has not yet made a difference because nanomaterials are not new, and the need and urgency for treatments for specific diseases or conditions cannot be met with the current regulatory structure.
While there have been some efforts by academic communities and government agencies to establish national characterization laboratories, more explicit and stringent guidance is needed from key agencies such as the FDA and MHRA.
Therefore, a global consortium for nanomaterials regulation should be formed to advance these agendas and issue formal guidance to the research communities. Billions of dollars have been poured into nanomedicine development over the past two decades. Without clear leadership and guidance from regulators, these efforts will not lead to product launches, and future investments may be made elsewhere.
Currently, in the context of nanomaterials in the European Union, we are dealing with both binding legal acts and non-binding legal acts, such as recommendations on the fair conduct of scientific research or on the application of a uniform definition of nanomaterials.
An important measure is the regulation of activities in the field of nanotechnology, which is unfortunately not easy due to the application of nanotechnology in various economic sectors. In particular, a unified definition of the term “nanomaterial” should be sought, which will facilitate the identification of materials for the application of the relevant regulatory provisions. Important challenges mainly concern the introduction of validated methods and tools for detection, characterization, and analysis, the completion of information on nanomaterial hazards, and the development of methods for assessing exposure to nanomaterials. The importance of public debate on nanotechnology has been highlighted by several bodies in the EU. The European Commission states that Member States should increase public debate on the benefits, risks, and uncertainties associated with nanotechnology. Individual EU Member States have begun to implement initiatives aimed at better informing the public about nanotechnology developments. The societal dialogue should involve representatives of civil society and scientists.

_______________

¹ This also applies to the liability, see [45]: “This has changed with the new EU regulation on medical devices. In the legislative proposal of the European Commission (autumn 2012) 78 includes, in addition to a specific definition for nanomaterials, regulations for labeling, and a classification for Products containing nanomaterials are envisaged. Accordingly, all products that contain or consist of nanomaterials will be assigned to Class III (highest hazard category), unless the nanomaterial is unless the nanomaterial is encapsulated or fixed in such a way that it cannot be released during the is not released when the product is used as intended.”

² Nanojustice and the E3LSC challenges: However, this is not just about identifying technical problems and developing technological solutions to overcome them. One of the most serious moral failures in addressing global health problems and implementing measures to combat the current pandemic has been the lack of equity in the distribution and use of COVID-19 nanovaccines, i.e., a patent issue: not only have wealthy populations and countries been favored in the introduction of the vaccines, but developed countries have hoarded the pre-ordered doses of vaccine to the detriment of the vast majority of people in developing countries. Dr. Tedros Adhanom Ghebreyesus, Director General of the World Health Organization (WHO), stated, “The world is on the brink of a catastrophic moral failure, and the price for that failure will be paid in lives and livelihoods in the world’s poorest countries. ”
Sociopolitical issues may also raise concerns about class differences between wealthier societies and countries that develop or access the benefits of nanotechnology and those that cannot [50].

³ Despite the lack of a specific legal tool for accessing information about new technologies and products in development . . companies facing [drug approval or biologics approval requirements] have a significant incentive to provide the FDA with the information the agency needs to understand and efficiently review new products, because the products are also weighted very differently later.

---

Test LO 6.2

---

References

1. Baran A. (2016). Nanotechnology: Legal and ethical issues. Economics and Management, 8, 1: pp. 47-54, doi: 10.1515/emj-2016-0005.
2. Bleeker E.A., de Jong W.H., Geertsma R.E., Groenewold M., Heugens E.H., Koers-Jacquemijns M., et al., (2013). Considerations on the EU definition of a nanomaterial: science to support policy making. Reg. Toxicol. Pharmacol. 65, 119-12, doi: 10.1016/j.yrtph.2012.11.007.
3. Beyerlin U., Marauhn T. (2011). International and Environmental Law, 220, 47.
4. Boverhof D. R., Bramante C. M., Butala J. H., Clancy S. F., Lafranconi M, West, J., et al., (2015). Comparative assessment of nanomaterial definitions and safety evaluation considerations. Regul. Toxicol. Pharmacol. 73, 137–150. doi: 10.1016/j.yrtph.2015.06.001.
5. BUND, Friends Of The Earth, Mensch & Umwelt, Höchste Zeit, Nanomaterialien zu regulieren!, 15.02.2022
6. Castro F. (2004). Legal and Regulatory Concerns Facing Nanotechnology. Chicago-Kent Journal of Intellectual Property, 4, 1, Article 5.
7. Choi H. and Han H. (2018). Nanomedicines: current status and future perspectives in aspect of drug delivery and pharmacokinetic. J. Pharm. Investig. 48, 43. doi: 10.1007/s40005-017-0370-4.
8. Commission Regulation (EC) No 507/2006 of 29 March 2006 on the conditional marketing authorisation for medicinal products for human use falling within the scope of Regulation (EC) No 726/2004 of the European Parliament and of the Council.
9. Commission Regulation (EU) 2018/1881 of 3 December 2018.
10. Commission Recommendation of 18 October 2011 on the definition of nanomaterial 2011/696/EU. Off. J. Eur. Union L. 275, 38–40.
11. DEEPEN – Deepening Ethical Engagement and Participation in Emerging Nanotechnology, Posted on 27/03/2014
12. DGUV, Nano-Portal: Sicheres Arbeiten mit Nanomaterialien, 07.08.2018, OECD-Dokument zu internationalen Entwicklungen im Bereich Nano-Sicherheit. www. abanet.org/scitech/specomm.html
13. Dorocki S. and Kula A. (2015). Spatial diversity of nanotechnology development in Europe. Development of industry in selected countries, 29, 1. doi: https://doi.org/10.24917/20801653.291.2.
14. Duvall M. N. (Editor), FDA Regulation of Nanotechnology, Taylor, footnote 23, p. 44. http://www.nanowerk.com
15. European Commission, Public Health, EU4Health 2021-2027 – A vision for a healthier European Union Regulation (EU) 2021/522
16. Falkner R., Breggin L., Jaspers N., Pendergrass, J. and Porter, R. D. (2010). International Handbook on Regulating Nanotechnologies, in: G. A. Hodge, D. M. Bowman, & A. D. Maynard (Eds.)
17. Falkner R., Stephan H., Vogler J. (2010). International Climate Policy after Copenhagen: Towards a ‘Building Blocks’ Approach. Global Policy, 1, 3: 252-262. doi: 10.1111/j.1758-5899.2010.00045.
18. Fiedler F. and Reynolds G. (1994). Legal Problems of Nanotechnology: An Overview, 3, S. Cal. L.J., p. 593.
19. Foulkes R., Man E., Thind J., Yeung S., Joya, A. and Hoskins C. (2020). The regulation of nanomaterials and nanomedicines for clinical application: current and future perspectives. Biomater. Sci., 8, 4653, doiI: 10.1039/d0bm00558d rsc.li/biomaterials-science
20. Government of Canada, Departments and agencies, Health Science and Research Reports and Publications – Science and Research Nanomaterial, Policy Statement on Health Canada’s Working Definition for Nanomaterial, 10.1.2022.
21. Hansen S. F. (2018). Registration, Evaluation, Authorisation, Categorisation and Tools to Evaluate. Nanomaterials – Opportunities and Weaknesses (REACT NOW), Technical University of Denmark.
22. Hertog J. (1999) General theories of regulation., in Encyclopedia of Law and Economics, Edward Elgar and the University of Ghent, UK, p. 223.
23. Karim E., Bakar bin Munir A. and Hajar Mohd Yasin, S. (2016) Nanotechnology and International Law Research Guide, Hauser Global Law School, Program. NYU Law School.
24. KOM (2000) 1 endg.
25. KOM (2009), 607.
26. Kommission der Europäischen Gemeinschaften, Mitteilung der Kommission an den Rat, das Europäische Parlament und denEuropäischen Wirtschafts- und Sozialausschuss, Nanowissenschaften und Nanotechnologien: Aktionsplan für Europa 2005-2009. Zweiter Durchführungsbericht 2007.
27. Langauer-Lewowicka H. and Pawlas K. (2014). Nanoparticles, nanotechnology – the potential environmental and occupational hazards, Medycyna Środowiskowa, 17(2): 7-14.
28. Marchant G. E. and Doug J. S. (2006). Transnationale Modelle für die Regulierung der Nanotechnologie. Zeitschrift für Recht, Medizin und Ethik, 714-725.
29. Matsuura J. H. (2012). Nanotechnology Regulation and Policy Worldwide, Boston/London.
30. Maynard A. D. (2007). Nanotechnology: the next big thing, or much ado about nothing? Ann Occup Hyg. 51(1): 1-12, doi: 10.1093/annhyg/mel071.
31. Ministry of Health, Labour and Welfare, MHLW Pharmaceuticals and Medical Devices Safety Information, Policy Information, Outline of the Law for Partial Revision of the Pharmaceutical Affairs Law (Act No.84 of 2013), 26.2.2022.
32. Nanotechnology and International Law Research Guide, in: Globalex.
33. Nogueira de Sousa Branquinho Nordberg, A. R (2009). Nanotechnology patents in Europe: Patentability Exclusions and Exceptions, Stockholm University.
34. OECD ‘Regulatory Frameworks for Nanotechnology in Foods and Medical Products: Summary Results of a Survey Activity’, OECD Science, Technology and Industry Policy Papers, No. 4 (24 April 2013), OECD Publishing, at 11 <http://dx.doi.org/10.1787/5k47w4vsb4s4-en>.
35. Ossa D. (2014). Quality Aspects of Nano-Based Medicines SME, Workshop: Focus on Quality for Medicines Containing Chemical Entities, London, Available online at: http://www.ema.europa.eu/docs/en_GB/document_library/Presentation/2014/04/WC500165444.pdf.
36. Picecchi D. (2018). Tiny Things with a Huge Impact: The International Regulation of Nanomaterials. Michigan Journal of Environmental & Administrative Law, 7, 2.
37. Pita R., Ehmann R., Papaluca M. (2016). Nanomedicines in the EU—regulatory overview, Oud M. (2007). A European perspective, in: G. A. Hodge, D. M. Bowman, & K. Ludlow (Eds.), New global frontiers in regulation, The age of nanotechnology, pp. 97-109. AAPS J. 18, 1576–1582, doi: 10.1208/s12248-016-9967-1.
38. Ponce Del Castillo A. (2009). The EU Approach to Regulating Nanotechnology, SSRN Electronic Journal, doi: 10.2139/ssrn.2264056.
39. Rauscher H., Rasmussen K., Sokull-Klüttgen B. (2015). Regulatory Aspects of Nanomaterials in the EU, https://doi.org/10.1002/cite.201600076.
40. Regulatory aspects of nanomaterials. 2008/2208(INI), (2010/C 184 E)
41. Richtlinie 2001/83/EG des Europäischen Parlaments und des Rates vom 6. November 2001 zur Schaffung eines Gemeinschaftskodexes für Humanarzneimittel (ABl. L 311 vom 28.11.2001).
42. Rio Declaration (1992). https://www.un.org/en/development/desa/population/migration/generalassembly/docs/globalcompact/A_CONF.151_26_Vol.I_Declaration.pdf
43. Salamanca-Buentello F. and Daar A. S. (2021). Nanotechnology, equity and global health. Natur Nanotechnology, 16, 358-361.
44. Soares S., Sousa J., Pais A. and Vitorino C. (2018). Nanomedicine: Principles, Properties, and Regulatory Issues. Front. Chem., https://doi.org/10.3389/fchem.2018.00360.
45. Spindler, G. (2009). Nanotechnologie und Haftungsrecht, in: Hendler/Marburger/Reiff/Schröder, Nanotechnologie als Herausforderung für die Rechtsordnung.
46. Timothy F., Malloy, (2012). Soft Law and Nanotechnology, A Functional Perspective. Jurimetrics 52, 347.
47. Tinkle S., McNeil S. E., Mühlebach S., Bawa R., Borchard G., Barenholz Y. C., et al., (2014). Nanomedicines: addressing the scientific and regulatory gap. Ann. N. Y. Acad. Sci. 1313, 35–56. doi: 10.1111/nyas.12403.
48. URL 1: http//cordis.europa.eu/home_pl.html, 12.01.2022
49. Van Est R., Walhout B., Rerimassie V., Stemerding D. and Hanssen, l. (2012). Governance of Nanotechnology in the Netherlands –Informing and Engaging in Different Social Spheres. International Journal of Emerging Technologies and Society, 10, 6–26. http://www.swin.edu.au/ijets.
50. Wolfson J.R. (2003). Soziale und ethische Fragen der Nanotechnologie: Lehren aus der Biotechnologie und anderen Hochtechnologien, 22 Biotechnology, L. Rep., pp. 376.