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.

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Test LO 1.1

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Summary

In the global fight against severe acute respiratory syndrome-CoV- 2 each individual is affected. The people are challenged to protect themselves and others. This combat is far more serious for the frontline and especially for health workers. In this regard, the role of personal protective equipment in this combat is essential. Nanoengineered solutions represent an innovative approach to medicine. Nanomaterials are reported to possess some unique characteristic as small size; improved solubility; surface adaptability; multifunctionality, antiviral/antibacterial activity, hydrophobicity, etc. All these open new opportunities for the development of innovative nano-enabled fabrics and textiles, providing increased personal protection. Furthermore, the application of nanotechnology-based surface coatings, drugs, and vaccines might be one of the possible solutions for limiting the global spread of severe viral diseases.

Key words/phrases: nano-enabled PPE, SARS-CoV-2, facemasks, nanomaterials, virus transmission pathways.

1. Introduction: SARS-Cov-2 – the infection agent of the novel disease COVID-19

In the winter of 2019, a new viral disease was discovered in Wuhan, China. The outbreak of this new and unknown disease forced the scientists to immediately clarify its epidemiology and etiology and to take action to fight it. The symptoms the new viral agent caused to infected humans have been described as fever, dry cough, malaise, rare cases of diarrhea, lymphopenia, and bilateral ground-glass opacity [9, 22]. These symptoms have been reported to be very similar to those caused by previously known pathogenic coronaviruses: SARS-CoV and MERS-CoV [58]. These two beta coronaviruses have been reported during the last 20 years as causative agents of two viral outbreaks: Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) – in 2002, in Guangdong Province, China [67] and the Middle East Respiratory Syndrome coronavirus (MERS-CoV) – in 2012, in the Middle East [64].

The studies of the new viral agent have started with the emergence of the first cases in China. The most important question at that time was how this new virus appeared? However, there have been suggested two opposite theories. Maybe the most common version for the origin of the new disease is that it has originated from the seafood and wet animal wholesale market in Wuhan, Hubei Province, China [45]. Other authors’ statements call into question the assumptions that the wholesale seafood and animal market is the only source of the newly found disease and bring light to the main pathway of disease transmission: human-to-human [9, 22].

The phylogenetic analyses of the novel coronavirus genomes and those of other beta coronavirus showed less than 90% identity, and according to that the new causative agent of viral pneumonia has been stated as a novel beta coronavirus and named 2019-nCoV. The disease caused by this viral agent was firstly named “novel coronavirus – infected pneumonia” (NCIP) [68]. The studies of the newly discovered coronavirus progressed along with its spread across the whole world, infecting millions of people. Based on the obtained information, the Coronavirus Study Group (CGS) of the International Committee on Virus Taxonomy (ICTV) announced, in early February 2020, the newly isolated infection agent as SARS-CoV-2 [19]. At the same time, the World Health Organization (WHO) named the disease caused by this CoV as COVID-19 (CO comes from “corona”, VI comes from “virus” and D comes from “disease”), and a month later, on 11th of March, 2020 – the outbreak of SARS-CoV-2 as a world pandemic [59].

2. The spread of SARS-CoV-2 and the need of personal protective equipment

2.1. Persistence in different environmental niches

The global and fast spread of the SARS-CoV-2 during the last 2 years led to an increase in the studies that aimed to characterize and determine the variety of environmental niches where the virus could present and persist. This knowledge is fundamental for realizing the ways of virus transmission on one hand and for inventing effective and reliable disease control strategies on the other hand.

The presence of COVID-19 infection agent or its genetic material in the water environment has been reported in a great number of studies, which has been stated as expected due to the unimpeded access of infected people to environmental water resources. Summaries of the data from a few studies have reported the presence of viral RNA/viral intact particles in different clinical samples such as urine, sputum, hospital stools, and others, although the nasopharyngeal samples of such patients have been negative, a few days after the infection. According to the research team, this finally might let to an increase of viral titters in hospital wastewater [1]. However, the presence of SARS-CoV-2 in wastewater samples has been demonstrated also in aerosols from wastewater in plant pumping stations during treatment activities [17, 65]. The persistence period of the infective agent in water and sewage has been reported to be 4.5 to 6 days depending on the environmental factors [18].

Other sources of viable virus particles have also been reported: air samples of different origins [28, 29], food samples [12], and tap water samples [7, 30]. The soil is perhaps the least studied environmental niche as a depot of SARS-CoV-2. However, the presence of viral RNA in soil samples, collected near hospitals, where patients infected with SARS-CoV-2 have been admitted for treatment, has been reported [66].

2.2. Presence and persistence of infectious viruses, including SARS-CoV-2, on different inanimate surfaces

The spread of SARS-CoV-2 in various environmental niches is a key prerequisite for the easy and rapid infection of many people simultaneously. The person-to-person transmission pathway is pointed as one of the most invasive [9].

However, the presumption of self-infecting after contact with virus-contaminated dry surfaces needs to be paid attention to as well. This alternative way of transmission has been proposed previously for other types of viruses, long before the COVID-19 pandemic [32]. In a survey published in 2000, Sizun et al. have compared the survival of two strains of human coronavirus (HCoV) on various surfaces and have found that they can persist on aluminium, latex gloves, and cotton gauze sponges for a few hours [48]. The potential sources of transmission of various nosocomial viruses (SARS, coxsackie, influenza, rhinoviruses, etc.) through contamination of different surfaces have also been summarized and it was concluded that they are capable to persist on them for a few days [27]. A similar investigation, that reveals the potential of avian metapneumovirus and avian influenza virus to survive on different solids, has been published, too. The results have shown that viable viral particles could be isolated up to the 9th day from nonporous surfaces, after contamination of the solid [53]. The potential of influenza virus (H1N1) to keep its viability on stainless steel solids has been studied and the results showed that it could be detected after 7 days [40]. Data about the stability of the coronaviruses, responsible for two viral outbreaks during the past two decades (SARS-Co-V and MERS-CoV), on different surfaces, has also been published. The results pointed out that the viruses could persist on solids like metals, glass, or plastic for 9 days [13, 25, 37].

The outbreak of SARS-CoV-2 forced the scientists to immediately study the potential of the virus to remain and persist on different surfaces that could serve as a source of infection. Such potential has been suggested due to the similarity of SARS-CoV-2 to the previously described pathogenic coronaviruses. Data about the virus stability on four different inanimate surfaces has been reported [55]. Thus, the ability of SARS-CoV-2 to survive on stainless steel, copper, plastic, and cardboard has been revealed. Another possible way of transmission of SARS-Co-V has been suggested by Ren and Tang – through coins and banknotes [44]. At that time, no experimental data concerning the potential of the virus to remain stable on banknotes paper had been published. Such hypotheses have been based on the results from studies of SARS-CoV-2 stability on printer and tissue paper [55]. In a recent study, data about banknotes as a reservoir of viable viral particles has been published. They showed that after 24 h at room temperature no viable virus particles have been detected on banknotes. However, greater stability of the virus on bank cards has been reported since viable viral particles have been detected after 48 h [35].

The long-term viability of the pathogenic viruses, including SARS-CoV-2, on different solids, bank cards, and other inert surfaces and their high levels of transmission by aerosols, person-to-person, and other known ways lead to two important issues: i) how an individual could protect oneself from these viruses and ii) how the mass spread of the viruses can be limited.

The application of different disinfecting agents and the use of personal protection equipment (PPE) are among the possible solutions.

2.3. Personal protective equipment’s – the WHO recommendations

Personal protective equipment is considered the last line of personal defense [15]. The basic concept of using PPE has two main aspects:

• to protect the individual from various risk factors during work;
• to protect other people from infections transmitted through air droplets.

In the light of the COVID-19 pandemic, the need for more knowledge on the ways to limit the spread of the viral agent and reduce the number of infected people, increased. As mentioned above, the causative agent of COVID-19 and other nosocomial viruses, including SARS-CoV and MERS-CoV, could be transmitted in different ways: person-to-person, through droplets of saliva (Fig. 1), through touching viral contaminated surfaces, etc.

Figure 1. Transmission of SARS-CoV 2 via saliva droplets

Due to this variety of transmission ways, WHO was forced to publish an Interim guidance – Rational use of personal protective equipment for coronavirus disease 2019 (COVID-19) on 27th February 2020, immediately after the emergence of the first cases of SARS-CoV-2 infected people. The role of this guidance was to give the community actual and specific recommendations for personal and social protection, namely the rational use of PPE. Among the specific recommendations in the guidance, were the following [60]:

• performing hand hygiene frequently with an alcohol-based hand rub if hands are not visibly dirty or with soap and water if hands are dirty;
• avoiding touching eyes, nose, and mouth;
• practicing respiratory hygiene by coughing or sneezing into a bent elbow or tissue and then immediately disposing of the tissue;
• wearing a medical mask if you have respiratory symptoms and performing hand hygiene after disposing of the mask;
• maintaining social distance (a minimum of 1 m) and staying away from those with respiratory symptoms.

According to the setting, personnel, and type of activity, the WHO has listed additional types of PPE, recommended for usage and application (Table 1) [60]:

Table 1. List of PPE recommended by WHO.

Setting	Target personnel or patients	Activity	Type of PPE
Healthcare facilities
Inpatient facilities
Patient room	Healthcare workers	Providing direct care to COVID-19 patients.	Medical mask Gown Gloves Eye protection (goggles or face shield).
		Aerosol-generating procedures performed on COVID-19 patients.	Respirator N95 or FFP2 standard, or equivalent. Gown Gloves Eye protection Apron
	Cleaners	Entering the room of COVID-19 patients.	Medical mask Gown Heavy duty gloves Eye protection (if risk of splash from organic material or chemicals). Boots or closed work shoes
	Visitors	Entering the room of a COVID-19 patient	Medical mask Gown Gloves
Other areas of patient transit (e.g., wards, corridors).	All staff, including healthcare workers.	Any activity that does not involve contact with COVID-19 patients.	No PPE required
Triage	Healthcare workers	Preliminary screening not involving direct contact	No PPE required
	Patients with respiratory symptoms.	Any	Maintain spatial distance of at least 1 m. Provide medical mask if tolerated by patient.
	Patients without respiratory symptoms.	Any	No PPE required
Laboratory	Lab technician	Manipulation of respiratory samples.	Medical mask Gown Gloves Eye protection (if risk of splash)
Administrative areas	All staff, including healthcare workers.	Administrative tasks that do not involve contact with COVID-19 patients.	No PPE required
Outpatient facilities
Consultation room	Healthcare workers	Physical examination of patient with respiratory symptoms.	Medical mask Gown Gloves Eye protection
	Healthcare workers	Physical examination of patients without respiratory symptoms.	PPE according to standard precautions and risk assessment.
	Patients with respiratory symptoms.	Any	Provide medical mask if tolerated.
	Patients without respiratory symptoms.	Any	No PPE required
	Cleaners	After and between consultations with patients with respiratory symptoms.	Medical mask Gown Heavy duty gloves Eye protection (if risk of splash from organic material or chemicals). Boots or closed work shoes
Waiting room	Patients with respiratory symptoms	Any	Provide medical mask if tolerated.
	Patients without respiratory symptoms	Any	No PPE required
Administrative areas	All staff, including healthcare workers.	Administrative tasks	No PPE required
Triage	Healthcare workers	Preliminary screening not involving direct contact	No PPE required
	Patients with respiratory symptoms.	Any	Provide medical mask if tolerated.
	Patients without respiratory symptoms	Any	No PPE required
Community
Home	Patients with respiratory symptoms	Any	Provide medical mask if tolerated, except when sleeping.
	Caregiver	Entering the patient’s room, but not providing direct care or assistance.	Medical mask
	Caregiver	Providing direct care or when handling stool, urine or waste from COVID-19 patient being cared for at home.	Gloves Medical mask Apron (if risk of splash)
	Healthcare workers	Providing direct care or assistance to a COVID-19 patient at home	Medical mask Gown Gloves Eye protection
Public areas (e.g., schools, shopping malls, train stations).	Individuals without respiratory symptoms	Any	No PPE required
Points of entry
Administrative areas	All staff	Any	No PPE required
Screening area	Staff	First screening (temperature measurement) not involving direct contact	No PPE required
	Staff	Second screening (i.e., interviewing passengers with fever for clinical symptoms suggestive of COVID-19 disease and travel history).	Medical mask Gloves
	Cleaners	Cleaning the area where passengers with fever are being screened.	Medical mask Gown Heavy duty gloves Eye protection (if risk of splash from organic material or chemicals). Boots or closed work shoes
Temporary isolation area	Staff	Entering the isolation area, but not providing direct assistance.	Maintain spatial distance of at least 1 m. Medical mask Gloves
	Staff, healthcare workers	Assisting passenger being transported to a healthcare facility	Medical mask Gown Gloves Eye protection
	Cleaners	Cleaning isolation area	Medical mask Gown Heavy duty gloves Eye protection (if risk of splash from organic material or chemicals). Boots or closed work shoes
Ambulance or transfer vehicle	Healthcare workers	Transporting suspected COVID-19 patients to the referral healthcare facility.	Medical mask Gowns Gloves Eye protection
	Driver	Involved only in driving the patient with suspected COVID-19 disease and the driver’s compartment is separated from the COVID-19 patient.	No PPE required
		Assisting with loading or unloading patient with suspected COVID-19 disease.	Eye protection
		No direct contact with patient with suspected COVID-19, but no separation between driver’s and patient’s compartments.	Medical mask
	Patient with suspected COVID-19 disease.	Transport to the referral healthcare facility.	Medical mask if tolerated
	Cleaners	Cleaning after and between transport of patients with suspected COVID-19 disease to the referral healthcare facility.	Medical mask Gown Heavy duty gloves Eye protection (if risk of splash from organic material or chemicals). Boots or closed work shoes
Special considerations for rapid response teams assisting with public health investigations
Community
Anywhere	Rapid response team investigators.	Interview suspected or confirmed COVID-19 patients or their contacts.	No PPE if done remotely (e.g., by telephone or video conference). Remote interview is the preferred method.
		In-person interview of suspected or confirmed COVID-19 patients without direct contact.	Medical mask
		In-person interview with asymptomatic contacts of COVID-19 patients.	No PPE required

Source: Interim guidance WHO, 2020 [60]

The way a conventional PPE kit looks is shown in Fig. 2. Each component of the kit has its specific function in the overall protection. The connections between the target protection area and a specific PPE component are as follows:

• Full body – coverall suit, aprons, and shoe leggings;
• Eyes – face shield, goggles;
• Nose and mouth – face mask;
• Hands – gloves;
• Head – hood cap.

The most important body areas, which must be completely protected are the eyes, nose, and mouth because these are the main entrance points for nosocomial and air-transmitted viruses entering the human body, including SARS-CoV-2.

Figure 2. The components of a standard PPE Kit

3. The contribution of nanotechnology in the global fight against new coronavirus disease

3.1. The broad concept of the nanotechnology

Viral infections and diseases are difficult to combat for various reasons, among which are the rapid viral mutations and the subsequent emergence of new variants and strains. The lack of reliable treatment challenged the scientists to develop new drugs that could be effective against a large number of viruses [24]. The limitation of this approach is that it takes a long period for the newly developed drugs to be declared effective and safe for use [10]. Thus, the attention has been focused on the prevention and invention of alternative approaches, designed to act at a different stage of the viral life cycle and consequently, efficiently impact the spread and development of viral infections [33]. In this regard, this is the exact place to emphasize the role of nanotechnology as a promising approach to combat viral diseases.

The basic concept of the science branch of Nanotechnology has been given by the Nobel laureate for physics, Richard P. Feynman in his lecture: ‘There’s plenty of room at the bottom’ in 1959 during a meeting of the American Physical Society [16]. In general, nanotechnology can be defined as the “design and application of several materials and devices where at least one dimension is less than 100 nanometres” [8]. Nanotechnology development is known to be in three basic directions [46]: nanomaterials, nanodevices, and nanosystems.

The direction related to the development and invention of nanomaterials is perhaps the most currently evolving. To completely understand the concept of nanotechnology and the related nanoparticles it is essential to clarify the meaning of the term “nano”. It comprises one billionth part (10-9) of a meter i.e., one meter has one billion nanometres. The size of nanoparticles (NPs) can be easily explained compared to different biomolecules and other bigger things like a tennis ball for example (Fig. 3). It is evident that the size of a cancer cell is 100 to 1000 – fold bigger than the size of a nanoparticle. The size of viral particles (including SARS-CoV-2) is approximately equal to those of nanoparticles.

Figure 3. Nanoparticles and their size

There are several reasons nanoparticles to be extensively studied. In this regard, some of their most important properties could be defined as the following [8]:

• small size;
• improved solubility;
• surface adaptability;

All aforementioned properties of the nanoparticles make them applicable in tissue-targeted treatment, personalized medicine, diagnosis, and prevention of viral and bacterial diseases [49]. In brief, the fields in which nanotechnology can be beneficial are far too diverse: medicine (drug development and application), ecology (water decontamination), technical applications (information and communication technologies), and so on [4].

3.2. Nanotechnology in the light of COVID-19

According to Campus et al. [8], the great potential of nanotechnology in the global fight against the SARS-CoV-2 could be realized through five main approaches (Fig. 4):

1. development of nano-enabled PPE;
2. development of nano-based anti-viral disinfectants and surface coatings, capable to catch and destroy the viral particles and thus stopping the spread;
3. the invention of nano-sensors with high specificity designed for quick viral identification and recognition of immunological response in the human body;
4. development of nano-based drugs for target therapy, direct in the affected lungs, for example. Nano-based drugs are considered to have increased effectiveness, decreased toxicity, and sustained release;
5. the invention of nano-based vaccines;

The urgent need for effective treatment of viral infections forced scientists to study different natural sources of active compounds such as plant extracts [5]. However, the efficiency of most of them has been considered weak due to their poor water solubility, low yield, difficult plant cultivation, etc. It is considered that to increase the therapeutic effect of the plant compounds they should be combined with nano-based materials [41].

The development of new generation vaccines, based on nanoparticles is another promising approach. These innovative vaccines have been considered to have some advantages over conventional ones, such as increased antigen delivery and stimulated immune response [2].

Figure 4. Nanotechnology application fields

Several studies have reported the application of nanoparticles in surface covering for the protection of viral and bacterial infections through a self-contaminating pathway [26, 36]. Such an innovative product has been developed by Sisson and Hackemeyer and had the potential to be applied on public surfaces with a high risk of viral contamination: elevator buttons, door handles, etc. The product named NanoTouch is a mineral nanocrystal-based coating activated by light (Fig. 5). The inventors have proven the effectiveness of the product against various viruses, including SARS-CoV-2 [51].

Figure 5. Self-cleaning nano-based surface coating, invented by NanoTouch/nanoSeptic

However, the concept of nano-based surface coating strategy in the fight against pathogenic microorganisms is closely related to the strategy of nano-based technologies for disinfection and sanitation. The information on such nano-based disinfectants, containing engineered water nanostructures, has been reported [56]. Its main advantage is that the applied disinfectant has significantly reduced the quantity of the pathogenic microorganisms after application. Another advantage was that the amount of the active compound required for effective disinfection was extremely low.

A commercially available nano-based disinfectant has been used for building disinfection in Milan during the outbreak of COVID-19. The formulation of the product has been based on Ag and TiO2 nanoparticles and it has been developed by the Italian nanotechnology company Nanotech Surface. The company’s manager claimed that the innovative formulation provides a “self-cleaning” ability for the treated surface for up to two years [52].

Like all new approaches in healthcare, treatment, and prevention, the nano-based products have to be declared safe and effective before their application at a community level. In this respect, the industrial nanobiotechnological companies must provide solid answers to some serious issues, concerning the safety and reliability of nanoproducts, their price, the legal regulation, etc. [8].

4. Nanotechnology-enabled PPE

The easy, fast, and global transmission of pathogenic coronavirus SARS-CoV-2 challenges each individual in his/her fight with the viral agent. In the situation of the global pandemic, two of the most important issues are: how the spread of the virus could be stopped or at least limited and how the people can protect themselves from the infection. The daily use of personal facemasks and gloves all over the world has become normal even in open spaces where the risk of infection is extremely high. However, as it is mentioned above (Table 1) the type of PPE, recommended by WHO could be different due to the current situation and the rate of the existing risk.

In this regard, the quality and effectiveness of the conventional PPE have become a key issue. For instance, many studies and reports demonstrate the negative effects of the long-term use of facemasks. Some of the most common complaints concern the increase in skin damage around the facemask area [14]. Additional restrictions on the prolonged wearing of a conventional facemask have been reported, such as unreliable protective ability and wearing discomfort [43]. It is reported that traditional face masks are produced from material that is ineffective in stopping the viral particles to reach the mouth and nose. The reason is locked in the size of the pores in the mask (10-30 µm) and the distance between the textile fibers that are too large to avoid the penetration of the 100 nm long viral particle. For example, conventional masks used by medical personnel (surgeons, nurses, healthcare staff, etc.) do not provide sufficient protection and the particles measuring up to 80 nm can pass through them. The global market spread face masks N95 and FFP2 protect against particles larger than 100 nm [21]. Nevertheless, some authors reported that if pore size and the distance between the structure fibers are reduced, the breathing will be critically impaired. The consequences of such a situation might be very serious, e.g. increase in body temperature and blood pressure [43].

The development and construction of innovative nano-based materials may be the key to overcoming all the aforementioned limitations in PPE effectiveness and safety. Several reports have shown that PPE, which contains nanomaterials (face masks, aprons, etc.) provides [50, 63]:

• better comfort;
• resistance to microorganisms (provide protection against particles smaller than 50 nm [11];
• increased safety to different chemical agents;
• hydrophobicity;
• no negative effects on materials;
• no negative effects on the breathing.

According to Campos et al., the application of nanomaterial in facemasks production offers two important advantages: i) the facemask acts simultaneously as a barrier and antimicrobial agent, resulting in blockage and destruction of the viral and bacterial pathogens; ii) diminishing the risk for self-infection of the wearer during the process of undressing [8]. Moreover, the viral particles are inactivated after contact with the nano-surface of the masks, i.e. the use of such PPE is environmentally safe.

Some of the patents for the manufacturing of PPE-containing nanomaterials are shown in Table 2.

Table 2. Patents for production of nano-enabled PPE.

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.

Source: Campos et al., 2020 [8]

Nanomaterials encompass each material that contains a “nano” structure: nanowhiskers, nanofibers, and nanoparticles.

4.1. Nanowhiskers

It is known that hydrophobicity is an important feature of the newly designed nano-materials. In the nano-constructed PPE, hydrophobicity has been achieved with the use of extremely tiny hydrocarbon fibers, known as nanowhiskers (Fig. 6). The nanowhiskers are responsible for increasing the surface tension of the textile material and thus, for decreasing its ability to adsorb droplets and other small molecules [8].

4.2. Nanofibers

A promising strategy in face mask manufacturing is the application of nanofibers. Such innovation has been patented by [11]. According to the author “nanofibers” can be different types: electrospun, protein, cellulose, bacterial, inorganic, hybrid, or any suitable combination thereof. The average diameter of the nanofibers can vary between 10 – 20 nm to 400 – 1500 nm. It is thought that the ability of the nanofibers to act as a barrier for small particles is due to two combined processes: i) catching the small particles by the nanofibers and ii) Brownian motion of the particles. Thus, the greater the nanofiber surface area is, the greater the capturing and protective ability of the nanofiber layer will be. The patent for a nanofiber-coated facemask of Conlon [11] is presented in Fig.7. The mask comprises three basic layers marked as 33, 36, and 39 between which the three basic layers are incorporated. Each basic layer has its own inner and outer surfaces.

Figure 6. Nanowhiskers attached to a textile fibre.

In the situation of a global pandemic, people are using PPE every day and sometimes during the whole day. Hence, ensuring physical comfort when wearing PPE is an essential issue. The long-term wearing of conventional facemasks led to discomfort and skin irritations due to the increase in temperature and moisture on the inner side of the mask. Also, these two conditions are prerequisites for increased microbial growth and hence for increased health risk. Frequently, the thermal discomfort is linked to the thickness of the masks. Yang et al. [62] have developed a face mask that ensures the wearer’s thermal comfort. The mask consists of a combination of nanofibers, nanoporous polyethylene, and a layer of silver. The authors have reported that such an innovative combination has an excellent cooling effect and protective abilities.

Figure 7. A facemask with nanofiber layers.

4.3. Nanoparticles

Another promising strategy in the manufacturing of facemasks with improved protective ability is the application of nanoparticles incorporated in the textile material. Various materials can serve as a matrix for the incorporation of nanoparticles: cotton, cellulose, polyamide, polyester, polyaramid, polyurethane, etc. [8].

Singh et al. have explored the potential of a nanocomposite, incorporated in the facemask’s material to determine its antiviral potential. The newly constructed nanomaterial consisted of carbon dots (C-dots) and poly(vinylidene fluoride) (PVDF) – C-dot-PVDF films [47]. The obtained results have shown that this nanocomposite assures hydrophobicity to the mask surface and thus, helps to reduce the moisture. The low moisture levels of the mask diminish the risk of its microbial contamination. Furthermore, the resulted nanoporous material did not affect the respiration through the mask and provided prevention against particles with a size of 100 nm and more (Fig. 8).

Figure 8. Facemask with incorporated C-dot-PVDF films.

It has been reported that carbon-based nanoparticles (for example Graphene and Graphene oxide) can bind to the viral particles and thus, destroy their outer structures [23]. The potential of such Graphene nanoparticles (G) and Graphene oxide nanoparticles (G) as a weapon against antimicrobial causative agents has been studied. Nanoparticles have been added to conventional materials used in PPE production (cotton and polyurethane). A solution containing live viral particles (SARS-CoV-2) has been filtered through the nano-coated material. The authors have found that after filtration the infection ability of the viral particles has been significantly or completely inhibited. Nano-based materials of such kind possess the potential to be implicated successfully in the combat against serious viral infections, including SARS-CoV-2. Furthermore, according to other authors, the incorporation of Graphene into textile materials can make them stronger, more conductive, and resistible to fire, abrasion, and UV light [6]. Various chemical substances, e.g., molybdenum sulfide, copper oxide, manganese dioxide, silicon carbide, etc., have been added to Graphene composites and their antimicrobial effect has been proven, too [42].

Another option is the metal NPs. It has been reported that the mechanism of their antiviral action consists of three basic stages: i) blocking the virus entry into the host cell; ii) stimulation of reactive oxygen species (ROS), radicals, and ions production that inactivates the basic functions of the viral particles, and iii) stimulation of the immune system of the macroorganism [42]. Silver and copper are known to have universal antimicrobial activity. Several studies, summarized by Campos et al., have shown the successful incorporation of these metals into various textile materials [8]. For instance, silver nanoparticles (composited in silica hybrid) have been shown to provide good antiviral activity against the influenza virus when added to filters [39]. Copper nanoparticles added to textile products have also been reported to affect the virus activity of various viruses, including SARS-CoV [69].

The advantages and the perspectives of the nano-engineered materials for the production of PPE are numerous. The summarized information concerning these advantages is shown in Fig. 9.

Figure 9. Main advantages of nano-based PPE.

5. Safety of nano-based products

The potential of nanotechnology is not limited to a single area. Here, the application of nanoparticles, nanofibers, and nanowhiskers in PPE production is discussed. However, nanotechnology possesses a broader potential. In the light of the COVID-19 outbreak and means for combating the disease, the main focuses and hopes are on the new scientific achievements, including in the field of nanotechnology. The development and areas of applications of nanotechnology in medicine (the so-called nanomedicine) can be classified into several main directions: diagnostic, treatment, and prevention of viral/bacterial diseases [8]. Besides the undoubted positives of nanotechnology, there are very important issues, concerning the safety application of nanoparticles, that must be considered. The limitations in using nano-based approaches in medicine could be due to some challenges, concerning their manufacturing, application, and biosafety release in the environment after use. The main aspects, deserving special attention could be classified as the following [8]:

• evaluation of safety application in/on the human body– it has been reported that some nanoparticles, e.g. Ag NPs incorporated in facemasks, can induce damage in the human body (in the lung, blood circulation, and heart) if they were inhaled [20]. Similar data have been reported for TiO2 NPs toxicity in humans, and the cancerogenic effect of the carbon nanotubes [31, 54]. Therefore, more in-depth tests in vivo must be performed to prove the safety of nanoparticles for the human body before their incorporation in PPE materials or drugs, that are in close contact with the outer/inner surface of the human body [8].
• evaluation of environmental safety– the effects of freely released nanoparticles in various environmental niches also must be carefully screened. For instance, silver and copper NPs have been reported to cause serious toxic damage to the marine ecosystem [3]. Comprehensive data on possible negative effects are currently insufficient and need to be improved [54].
• development of universal protocols for broad-spectrum characterization of nanomaterials– biological, physical, chemical, etc. – according to Palmieri et al., the responsibility for this has to be taken by the governments, and all manufacturing companies must follow the stated rules [38].
• adoption of a uniform definitionof the term “nanomaterials” [57];
• careful evaluation of the possibilitiesfor the manufacturing factories to meet the market needs [8].

The need for reliable solutions in human protection and disease treatment is essential. In this regard, nanotechnology might have a very bright future in various fields of medicine, including in PPE production.

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Test LO 1.2

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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.

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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.
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30. URL-4: https://www.microcovid.org/blog/hepafilters
31. URL-5: https://product.statnano.com/product/11653/teqoya-teqair-200-air-ionizer
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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.

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Test LO 2.2

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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].

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Test LO 3.1

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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].

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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.

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Test LO 4.2

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Summary

COVID-19 is disrupting food supply and demand in complex ways and links the health crisis with a food one. The SARS-CoV-2 virus represents a steady danger to food security. Globally, the biggest problem for the food sector is associated with the COVID-19 measures and the necessity of arrangements in the sector to answer those measures. Another challenge is the need to find alternative markets for products related to changed consumption habits in response to COVID-19.

To stop or control the spread of coronavirus infection, different measures have been undertaken at the governmental level. Changes in consumers’ demands as well as changes in their eating habits led to the loss of various food processing sectors like dairy, sugar, meat, and poultry. The ‘Strategic preparedness and response plan’ by WHO predicted the health measures that all countries had to include as an answer to COVID 19 pandemic. At present, there is no proof of SARS-CoV-2, or any other respiratory infection, to be delivered by food or food packaging. However, standard cleaning and sanitizing procedures for SARS-CoV-2, physical distancing, involvement of food business operators and food workers to mind global food safety standards and ensure coherency in the quality and safety of food products are needed.

Key words/phrases: food business, food supply service, food processing, post-pandemic

1. Introduction

Globally, enough food is available but COVID-19 is disrupting supply and demand in complex ways and the health crisis is linked with a food crisis. In addition, the agriculture sector of the economy is assigned by most countries to be excluded from the business restrictive and lockdown measures. The direct impact of the COVID-19 pandemic on primary agriculture has been limited for many countries since the illness does not influence the natural resources on which food production is based. However, the virus represents a steady danger to food security and livelihoods in the countries with lower life standards, where agricultural production systems are with less capacity to cope with such a strong macroeconomic shock.
As food is a basic need, the level of food demand should be influenced less by the crisis than the demand for other goods and services. However, a major change in the structure of demand appeared, linked with strong requirements from restaurants, hotels, and catering services, the closure of open markets, and the surge in demand from supermarkets. Currently, there are indications that businesses along the food chain are already adapting to the changes in demand, using different approaches:
-Changing production lines,
-Enhancing their potential to manage larger catalogues,
-Organizing e-commerce through the use of online platforms,
-Exploiting direct delivery schemes,
-Hiring staff on temporary positions.
Globally, and especially in the poorest countries, the biggest problem for the food sector is associated with the COVID-19 measures and the necessity of arrangements in the sector to answer those measures (which may increase costs). Another challenge is the need to find alternative markets for products related to changed consumption habits in response to COVID-19. How do all these changes become evident within the frame of the food system?

2. Pandemic impact on the food sector

To stop or control the spread of coronavirus infection, different measures have been undertaken at the governmental level. Changes in consumers’ demands as well as changes in their eating habits led to the loss of various food processing sectors like dairy, sugar, meat, and poultry [7].
-During the period of the application of safety measures, different countries in the world involved strong lockdown regulations that influence the dairy industry. The reduction in the demand for dairy products caused a decrease in their price and export level. [37]. This reflects as well the socio-economic status of the dairy workers.
-The operation of the sugar processing industry was also restricted, due to the Covid 19 pandemic. The sugar import and export were strongly influenced, which caused a serious decline in production. This impacted also the production of alcohol. The negative influence on sugar processing industries was linked to the shortage of suitable packaging materials. Such a negative impact was observed also on the dairy industry.
Besides the dairy and sugar industries, the same effect is encountered in the meat, poultry, and seafood industry. Meat and poultry processing industries are particularly vulnerable because of their critical infrastructure in the food sector. The increase of the Corona virus-positive cases (and deaths) in the production facilities of the meat and poultry industry resulted in a full shutdown of plants. According to data from May 2020,, twenty industrial plants in the USA were closed, among which Tyson foods, Plant in Waterloo, Lowa, other plants like JBS, Sanderson farms, Hormel foods, National beef, and Smithfield food after the reports of COVID -19 infections [11]. These alarming data raise panic about the consumption of meat and seafood during the pandemic. For this reason, WHO recommended a diminution of the visits to the live markets and meat consumption of raw or undercooked animal products. To manage the pandemic, hygienic measures for meat production and processing equipment for the workers were implemented. Additionally, training of butchers, and educating them about clean meat production practices, processing, packaging, and quality assurance is promoted. [43].

2.1. Pandemic effects on food and agriculture

The ‘Strategic preparedness and response plan’ by WHO [44] predicted the health measures that all countries had to include as an answer to COVID 19 pandemic. The WHO showed the knowledge adopted about the virus and transformed this information to build a strategic action plan to guide national and international partners to prepare national and regional operational plans. These plans define priority steps and actions in eight major topics:

• Coordinating, planning, and monitoring at the country level;
•  Communication of risk and community participation;
•  Observation, quick-response teams, and case investigation;
•  Entry points;
•  National laboratories;
•  Prevention and control of infection;
•  Situation management;
•  Operational support and logistics.

The fulfillment of these measures is linked with the cut of workplaces and educational institutions, as well as temporary limitation of travel and social meetings. Flexible working from home and online meetings appeared as standard practices today. However, people working in the food industry who have no opportunity for home office performance need to keep the typical actions [29, 18, 45]. As a consequence of the COVID-19 crisis, a solution for food workers was prepared to furnish guidance for a succession of operations in the food processing hardware and COVID-19 virus management in the food industry. The plan discusses control requirements for cleaning, sanitation, disinfection of equipment, screening, and monitoring of people working with COVID-19, managing the ill workers, and use of educational programs for supervisors to cease the spread of coronavirus [8].

Each country in the world needs to find out the way, in which the COVID-19 outbreak can influence the manufacturing industry, and especially the food industry.

The food industry differs from others because it produces products important for daily life and if processors and distributors are infected, all people are at risk [41]. Besides, during pandemics, the food industry is a very important economic branch, as it meets various challenges compared to other sectors, which are not responsible for daily life like tourism and aviation among the others. Currently, due to COVID 19 outbreak, some food companies are obliged to work harder to meet the growing demands of the retailers, while others are facing challenges because of the drop-down in their income. These impose even needs for closing down (temporally or finally) businesses. The current pandemic has shown that companies with a scope of activity within different businesses are closely connected all over the world [11, 37]. Regardless of which sub-branch we are talking about, a major concern of the food companies is:

• To preserve their employees’ health
• To provide a sufficient workforce for enabling normal production cycles and avoiding shortages due to sick or unwilling to work because of coronavirus fear employees.

This means that it is of vital importance to defend and maintain the health of people who are in the food supply during this time of crisis [18, 44].

Holding the distribution chain actively operating with supply management strategies is convenient to answer consumer demands [12]. This process is maintained by the active participation and contribution of all stakeholders by providing the consumers a confidence in food safety and security [18, 44]. During a time of crisis, food security is linked with consumers’ access to food rather than food availability [31]. Generally, consumers are not aware too much regarding the manner of food provision on their tables. However, concerns about food safety during the global pandemic make sense for the huge infrastructure and workforce amenable to forming a safe and reliable food supply in the world. When this global crisis has begun, consumers’ needs for food have raised and some store shelves have been for the time being deserted, and excess purchases of essential products have been realized.

Regardless worldwide scale of the pandemic, up to now COVID-9 was not reported to be diffused through food consumption. Hence, as noted by the European Food Safety Authority, there is no data indicating risk to public health related to COVID-19 [16]. Nevertheless, for the infections that have been seen in Xinfandi Market in China, caused by the processing of salmon, it can be decided that the risk for COVID 19 transmission through foods is lower than the comprehended risk. Thinking about the period for survival of SARS-CoV-2 in different materials such as plastic, steel, or cardboard, it is feasible that animal tissues (meat, fish, or poultry) might be considered a steady source for foodborne disease transmission. Food business operators’ hygiene control is intended to cease contamination of food by any pathogen, and in this way – to stop COVID-19 transmission by the foods they are responsible for [3, 10, 15, 32]. It is noted in previous outbreaks that foods are not a source of diffusion of coronaviruses like MERS and SARS-CoV because of the acidic pH of the stomach (pH < 3.5). however, some cooking and eating manners can cause the transmission of the coronavirus from animals to humans [33].

To resume, several problems are determined in the food industry and the food supply chain during the COVID-19 outbreak. The pandemic and the policy responses adopted to address it have affected food systems from the global to the local level. There are many different impacts of the pandemic and pandemic responses on the world’s food systems. Among the major one, we can list the following:

• Lost incomes: the COVID-19 pandemic’s impact on the food security is due mainly to the lost incomes. The World Bank reported a global economy shrank by 5 percent in 2020, with the greatest burden borne by poor people. For instance, the restrictions on global travel and cargo put into place to stop the spread of the virus represented a larger economic cost for poor countries than their own pandemic restrictions. The limitation of the people’s mobility across borders and lockdowns is the reason for the shortages of the labor market for agriculture in many countries, especially those that need peak seasonal labor claims or labor-intensive production. For instance, newly performed travel bans within the European Union, as well as the closure of the Schengen Area, have significantly reduced the available workforce for the fruit and vegetable sector in several European countries.
• Disruption of food supply chains: the food supply chains were disrupted by labor restrictions and falling demand. However, impacts varied along the value chains and between countries and commodities.
• Food insecurity and nutrition losses: Food safety measures must be put into focus to prevent the transmission of coronavirus among producers, retailers, and consumers. The concerns for food security have risen, linked to lockdown restrictions. The impacts of rising poverty and abridged livelihoods are reflected in decreasing diet quality and rising the levels of food insecurity.In addition, in time of pandemics, people need to follow a healthy diet to self-prevent and boost the immune system. This is directly related to the increase in the need for functional foods, containing bioactive ingredients [32].

Food sustainability; magnification of shortcomings and difference: Food sustainability problems have emerged in the COVID-19 pandemic reality [20]. The pandemic proved once again the vulnerability of the poor and other disadvantaged groups of people.

2.2. Guidance for preventing COVID-19 influence on food businesses

Most of the well-known organizations such as Centers for Disease Control and Prevention (CDC), European Commission (EC), Food and Agriculture Organization (FAO), International Food Information Council (IFIC), International Food Policy Research Institute (IFPRI), International Labour Organization (ILO), International Trade Centre (ITC), The Organization for Economic Co-operation and Development (OECD), and World Health Organization (WHO) are engaged with the provision of guidance for coping with the recent challenges for the food supply chain imposed by COVID-19.

The COVID-19 pandemic caused by SARS-CoV-2 is an unprecedented threat to the human population and represents generally a risk to public health. It represents also such a hazard for the occupational safety of workers in any type of business or industry where persons are working in a close relationship. Current data are pointing out that SARS-CoV-2 itself is not accepted as a direct food safety hazard. A lot of countries are pursuing the WHO advice concerning the involvement of physical distancing steps like one to decrease the transmission of the disease [45]. This demand for physical distancing resulted in stopping the activity of many businesses and schools and pose limitations on travel and social events. As a result, some home-working people, teleworking, and such providing online or Internet discussions and meetings currently become a reality in comparison to this practice before the pandemic. Meanwhile, a lot of food industry employees lack the chance for home-working and there is a necessity to proceed with their job in their usual working places.

Large outbreaks of COVID-19 have taken place among farmworkers and in food processing factories. This probably is linked to their working environments having insufficient physical space to ensure personal safety. In the light of this guidance, workers in the food industry comprise all people working in the food business that has contact with food or food surfaces. This comprises all people taking place physically in the food processing, packaging, or handling area. This personnel includes managers, cleaners, maintenance contractors, delivery workers, food inspectors, and others. Thus, a healthy and sufficient workforce is of special importance to keep effective supply chains.

It is also important that food businesses are obliged to consider current food regulations as well as any additional advice from competent authorities and measures that must be implemented linked to the pandemic. All these endeavors are basic to maintain trust and consumer confidence in the safety and availability of food.

2.3. Remote diffusion of COVID-19 through Food

Strangely, people can be infected with COVID-19 from food or food packaging [23, 30]. Up to date findings do not state that food or food packaging is a significant way for the diffusion of human respiratory illnesses, including SARS-CoV-2.

Primarily diffusion of COVID-19 is related to close contact among people through the respiratory way. It is released through droplets, and aerosols produced by actions like coughing, sneezing, shouting, singing, and speaking [43]. The produced respiratory droplets by an infected person can land on different surfaces. Coronaviruses are not able to propagate in food or on lifeless surfaces; they can only multiply in humans and certain animals. During long persistence in the environment, viruses can degrade and reduce their virulence. However, different research reports have visualized the stability of the SARS-CoV-2 kept on different surfaces. It is concluded that the virus can stay viable for up to 72 hours on plastic and stainless-steel surface, up to 4 hours on copper, and up to 24 hours on cardboard [42]. Other investigations evaluated the virus stability on different surfaces at different temperatures or via changing other important parameters [13]. All these findings enriched our perception of viral persistence and survival, which have been studied under laboratory conditions. These investigations have been performed in maintained humidity, temperature, and other factors. Therefore, the obtained data have to be accepted with precaution when evaluating virus stability under given conditions (cool or freezing), high humidity during food processing, as well as behavior in the transportation environment.

Thus, nevertheless the detection of the virus or the viral ribonucleic acid (RNA) in foods and food packaging, giving an indication about former contamination, there is no proof of SARS-CoV-2, or any other respiratory infection, to be delivered by food or food packaging. So, the viral contaminated food products or packaging is not considered real trouble for human health. The SARS-CoV-2 virus is sensitive to most commonly applied disinfectants and sanitizing agents used in the food processing environment.

2.4. Standard cleaning and sanitizing procedures for SARS-CoV-2 virus

In the light of the above-mentioned, the cleaning and sanitizing procedures for the SARS-CoV-2 virus are subjected to the ones, based on sanitizers/surface disinfectants, which are outlined in the manufacturers’ instructions for cleaning purposes.

Commonly, the alcohol-based disinfectants (ethanol, propan-2-ol, propan-1-ol) show major virulence reduction of enveloped viruses like SARS-CoV-2. Therefore, they should be effective at disinfecting the food processing environment.

WHO advocates the application of sanitizers with greater than 70 percent alcohol for adequate contact time for disinfection [44].

Regular disinfectants that contain active compounds, based on quaternary ammonium salts and chlorine, also exhibit viricidal properties and can be applied for sanitizing purposes.

The testing for SARS-CoV-2 in food processing facilities or on food packaging is an expensive procedure and compared to microbiological sampling, which verifies sanitation protocols, it is not of primary significance. Besides, it is time-consuming and does not aid in risk-based decision-making processes for consumer protection. This is the reason it not to be recommended.

The measures for disinfection/sanitizing should be risk-based and relevant to the level of expected workers’ exposure to SARS-CoV-2. In this context, in the places where SARS-CoV-2 is not circulating actively, it is enough to practice the procedures of the food safety management systems that are consistent with the situation. In case of an increase in the threat of COVID-19 infection in the community, the accepted prevention measures have to be expanded. In this latter case, there is a need to highlight measures necessary to control COVID-19 in food operations, to ensure the safety of the workers and the safety of the food supply. These measures should not compromise standard food safety controls and food safety management; rather they should supplement the ongoing food safety measures. As COVID-19 is a global pandemic, the virus occurrence can differ seriously within and between countries, and the measures taken by national and local public health authorities must be relevant to those local or national peculiarities and consequently, well-coordinated.

The following key considerations in the guidance materials of FAO/WHO must be taken into account [18, 43]:

• COVID-19 is not a foodborne disease. See the International Commission on Microbiological Specifications for Foods’ opinion on SARS-CoV-2 and its relationship to food safety [23];
• Businesses must undertake key measures recommended for COVID-19 and complete a COVID-19 Safety Plan and register as a COVID Safe business;
• Businesses need to review their operations and take into account some additional measures to be implemented to reduce the impact of COVID-19;

Businesses with good preparations and procedures in place can minimize the impact of an employee diagnosed with COVID-19.

2.5. Physical distancing

Physical distancing is very important to support the restriction of COVID-19 distribution. The probability of COVID-19 transmission decreases as the distance between individuals increases. Where an intracommunity spread is registered, all food businesses should respect physical distancing and follow the physical contact restrictions’ guidance as far as it is possible in practice. WHO maintains the distance between workers should be at least one meter (ca. three feet). The distance requirements may vary within countries and regions. Where the food manufacture procedures do not allow easily minding the distance requirements, employers need to consider some additional measures to be put in place for employees’ protection. In this respect, the control at the operational and structural level executed to reduce the transmission of the virus encompasses:

• Maintenance of a physical distance of at least one meter (three feet), or as given otherwise by authorities, between individuals;
• Organize workplaces along the processing line in a way that the food workers are not facing one another;
• Ensure enough place between workplaces; this may reflect negatively on the speed of the production lines
• Build-in physical barriers between individual workplaces;
• Assure better air exchange;
• Rearrange break times, where possible, to facilitate temporal distancing.
• Organize staff into working groups or teams to facilitate the reduction of the interactions between the groups;
• Establish various shifts to reduce the presence of a large number of workers in food business premises;
• Subject to cleaning and disinfection of all contact surfaces of general character and specialized equipment devices.
• Distribute written notices and provide explanations and training to staff to promote good hand hygiene, mask-wearing, and physical distancing.
• Encourage vaccination. Vaccines have proven to be effective to lower the severity of COVID-19 and reducing the risk of transmitting the disease. Enable all food workers to become vaccinated, if desired, by providing the necessary flexibility in shift schedule.

Support and mind good personal hygiene. Provide adequate sanitary facilities (soap and warm running water) to ensure that food workers thoroughly and frequently wash their hands. Use hand sanitizers as an additional, not replacing hand washing, measure.

2.6. Involvement of food business operators and food workers

Food Business Operators (FBOs) have to perform necessary corporate measures that are foreseen to keep the safety of their products and to protect their workers. For this reason, FBOs have to develop respective Food Safety Management Systems (FSMS) linked with the Hazard Analysis and Critical Control Points (HACCP) principles to manage food safety risks and safeguard food contamination. If they are not projected particularly for protection from COVID-19 transmission, FSMS undertaken are those from prerequisite programs, including good hygiene practices. Such programs comprise:

• Cleaning and sanitation;
• Zoning of processing areas;
• Supplier control;
• Storage, distribution, and transport;
• Personal hygiene
• Fit for work assessments.

All these fundamental conditions and activities are obligatory for keeping a hygienic food processing environment. In case a food business possesses an FSMS and/or a HACCP body is organized, its personnel has to be involved in appropriate discussions and training to guarantee the minimization of any person-to-person spreading of COVID-19. The aim is to keep the integrity of the FSMS and the efficiency of HACCP measures. The departments in the food business that are liable for the selection and application of adequate measures, and consulting with public health authorities, if needed, are the ‘Occupational health’ and the ‘Human resources’ ones. Besides, the Codex General Principles of Food Hygiene outlines a fundamental basis for the execution of key hygiene controls at each stage of the food processing, production, and marketing network to safeguard against food contamination [43]. Each FBO has to possess suitable plans to stop disease transmission along with the operations and to assure the consent with these measures to prevent workers from COVID-19 and to protect exposition to or transmission of the virus.

The plans for the prevention of disease transmission will involve the following measures [25, 42]:

• To foster workers to stay home in case they are ill;
• To introduce operational and structural controls to enlarge physical distancing between workers;
• To provide separation or barriers between workstations;
• To accept strong hygiene and sanitation measures to prompt effective washing of hands;
• To stimulate mask-wearing, and sanitation at each stage of food production, processing, and marketing.

These measures are important for food businesses where the operating environment is linked with viral transmission between workers, especially among those persons that are exposed to limited ventilation. Application of these measures should be provided through the entire food production chain, including places besides processing lines as well as other areas where workers communicate (meeting rooms, locker or break rooms, dining areas, etc.). The measures concern as well employees’ housing and group transportation provided by the FBO.

The provision of the measures should be risk-based and respective to the level of anticipated food business worker exposition: food safety practice in one efficient FSMS may be reliable to protect food business workers in case there are no community cases of COVID-19. However, when an increasing prevalence of COVID-19 cases in the local community takes place then an increased necessity for additional prevention measures in the local community is shaped.

3. Safety measures in the food sector

After the storming rise of the COVID 19 infections among the operational staff in the food sector, a need emerged for urgent assurance of the safety of the people handling and delivering the food commodity. This imposed the necessity among food operators and workers to keep good manufacturing practices (GMP) and good hygiene practices (GHP).

The manufacture of food permanently demands food manufacturing practices (FMP) to mind global food safety standards.

To ensure coherency in the quality and safety of food products, the attention is focused on five key elements: people, premises, processes, products, and procedures. The latter concerns the basic rules of personal hygiene, e.g. hand washing; sanitizers use; social distancing guidelines keeping [19]. Figure 1 shows the five basic rules of personal hygiene.

Figure 1. Five rules of personal hygiene.

3.1. Measurements performed by operational bodies

• Social distancing: The social distancing provision is reached through suitable marking for the workers. The shift systems have been applied where they are permitted to work in shifts to diminish the chances of mass meetings;
• Health supervision: The early COVID-19 symptoms are fever, cough, headache, and shortness of breath. It is advised a proper thermal screening of all the persons entering the working area be made. The working premises are allowed for workers with good health status kept in the record. In this way, people with severe cases of diseases like asthma, diabetes, or heart ailments are secure and those who are feeling unwell to are excluded from the working process. In this way, the reduction of the exposition of the health workers is ensured, and the risk for other people having COVID 19 infection is identified.
• Safety equipment: Following the official guidelines, the workers in the food industry are furnished with safety kits including face masks, hand gloves, and sanitizers. There are also recommendations for the food operators to keep immunity boosters and ban the use of alcohol, tobacco, and other things that can compromise the staff’s health.
• Work premises sanitization: The food industry strongly depends upon the sanitization process and the proper cleaning. The whole areas: working: production; packaging; stores, and washrooms must be sanitized and cleaned with soap, water, and disinfectants (1% to 2% hypochlorite solution).

Communicative events: campaigns for information and awareness have to be provided to show COVID-19 symptoms, actions in case of infection, and basic rules of personal hygiene used as a norm of different food safety standards.

3.2. Obligations for the food handlers in food processing and packaging industries

• To evade the contact of doors of lifts and use of biometrics;
• To wear the mode of keeping a social distance (at least a 2-meter gap) and proper availability and use of safety equipment like masks, hand gloves, and PPE kits;
• To secure the details of him/her health status for the authority and to keep proper isolation and quarantine rules in case of flu-like symptoms;
• To maintain good practice in the food packaging, proper washing of all the packaging material to be done with soap and water;
• To throw the packaging after removing packaged goods for storage into clean containers;
• To keep up three days of food items before touching them;
• To follow the norms of WHO for proper sanitization.

Although there is no evidence to date contracting the virus after touching food containers and packaged food, safety measures for the handling of packaged food, fresh food, grocery store food, and food containers have to be undertaken (see Fig. 2).

Figure 2. Safety measures for food handling

In case the packaging material is leaking, the products have to be transferred into a secondary container that is sanitized and placed close to the carrying vehicle. If leaks occur, the surface should be thoroughly washed with hot, soapy water or a bleach solution in case it came in contact with raw meat, poultry or seafood, or its juices.

3.3. Food supply and catering services

There are approved guidelines for the workers who are managing food pickup and delivery as handling and delivery of food items was found to be one of the root causes of the spread of infection [18]. The basic measures for proper cleaning, sanitizing, food items maintenance and social distance keeping are listed in Fig. 3, below.

Figure 3. Safety measures for food production auxiliary activities.

 

4. COVID 19 impact on the food processing sector

Along the corona virus infection spreading, governments have implemented different approaches and procedures to handle or to stop the pandemic. These were followed by changes in the demands of the consumers as well as in their eating patterns. Finally, all this resulted in big losses for the most food processing sectors like dairy, sugar, meat, and poultry [7].

Dairy and sugar processing industries

In the process of taking safety measures, various countries across the world implemented stringent lockdown regulations that severely hit the dairy industry and thereby the socio-economic condition of the dairy workers. The price level of dairy products decreased due to the reduction in the demand for dairy products and due to a decrease in the export of dairy products [39]. Pandemic restrictions and measures, imposed difficulties in the normal operation of sugar processing industries. For instance, in India, the import and export of sugar have been severely affected and as the demand got reduced to 50%, there was a sharp decline in the production. Alcohol and jaggery production was adversely affected due to the decline in the sugar processing sector. Sugar processing industries were also hit due to a shortage of consumables like packaging materials [40].

Meat and seafood industries

Apart from the dairy and sugar industries, the meat and seafood industry are the other ones that were largely hit due to the coronavirus pandemic. The increasing positive cases and deaths due to corona virus in the meat and poultry industries resulted in the shutdown of plants. According to the article in Food Processing, 13 May 2020, about 20 industrial plants have been shut down in the US including Tyson foods, a Plant in Waterloo, Iowa, other plants like JBS, Sanderson farms, Hormel foods, National beef, and Smithfield food after the reports of COVID -19 infections [11].

The above facts created panic and rumors around the consumption of meat and seafood during the pandemic. WHO advised on reducing the visits to the live markets and the meat consumption, and asked for caution while eating raw or undercooked animal products. To tackle the crisis during the pandemic, organized retailers invested heavily in the back-end processes, which included introducing hygienic techniques of meat production and processing techniques for the workers of the meat industry, training and educating them about clean meat production practices, processing, packaging, and quality assurance [44].

5. Changes in food consumption during the COVID-19 pandemic

Food production and consumption possesses immersive environmental impact, key to the personal health as well as to the health of the world. On the other hand, afflictions like the COVID-19 pandemic can crash the food system [20] and alter the link with the food. For example, the authorities apply logistic restrictions limiting the flow of goods and people to diminish the spreading of the infection. These measures are increasing the risk of food shortage due to damaged supply chains, involving those related to labour deficit [as can be seen in the US and Europe, [22, 28]. When partial or full lockdown measures are applied at regional and national levels, educational institutions and workplaces are closed, some shops and restaurants as well, events are banned, and traveling is limited. The lockdown measures changed as well the way people received their food, the place they consumed it, and the way the food was prepared. The aforementioned measures are acting as a further obstacle to the food supply to vulnerable populations. For instance, some food programmes providing main meals for school children did not operate during the restrictions. In addition, the quarantine due to disease or contact with infected people further restricted people’s access to food. Also, a variety of COVID-19 linked psychological changes might influence food-related behaviours. Indeed, even in case of relatively low disease risks, people were subjected to serious risk about the threatening dimensions of COVID-19 infection, which caused them stress. This stress is coped with the so called “stress-related eating’”, a condition in which people try to make themselves feel better by eating or drinking when under stress [9, 24]. Thus, during lockdown in Italy, people enlarged the use of processed “comfort foods,” like chocolate, chips, and snacks [5, 36]. This fact was linked to depression caused by COVID-19 and change of their eating habits [14]. An investigation made in Denmark also indicated an increased level of emotional eating during the lockdown, e.g., higher consumption of pastries and alcohol [21]. The same tendency was observed in Norway, where people with increased COVID-19 depression and general psychological distress, higher than the overall population consumed more high-sugar food and beverages [4].

Risk perception linked with COVID-19 effected people’s food purchase and consumption behaviours. Thus, people tried to reduce the risk of the infection through the use of mainly convenient delivery services and buying preferably packaged food. This behaviour is rational since it realises better hygienic impact through purchasing food with a longer shelf-life, limiting the shopping trips, as well as boosting the immune system [34]. The pandemic situation increased people’s concern towards expected food shortages and effected the purchasing behaviour, making them stocking up regarding certain foods [5]. Meanwhile, it was shown that COVID-19 might present additional health risks linked with the metabolic impact of overeating under conditions of home arrest [26].

Ammar et al. [2] reported an evolution of harmful to the health eating patterns, found through an international survey on physical activity and eating behaviour performed with more than 1000 participants in April 2020. Similar results were also found in a Polish national cross-sectional study (N = 1,097) by Sidor and Rzymski [38]. Nearly half of the participating people (mainly overweight individuals) declared more eating and snacking [38]. In Italy, affected by COVID-19 earlier and more seriously than other European countries, after the lockdown in March 2020 about 20% of the patients that passed the corona disease gained weight. These observations were partially confirmed by a food consumption study that investigated changes in the sale of food in over 10,000 Italian stores [5]. The findings of this survey indicated an expansion in the use of pasta, flour, eggs, long-life milk and frozen foods, together with a decrease in the buying of fresh food. The above-mentioned surveys focused on the general population. As regards the specifically targeted younger people, a study of 820 adolescents (10 – 19 years old) from Europe and South America showed that COVID-19 confinement has imposed changes to the dietary habits and consumption of both processed foods and fruits and vegetables [35].

Investigations performed by consulting companies aiming to study the shift in the shopping behaviour during COVID-19 in relation to the variety of product categories (food and others) indicated a marked variation in all categories studied. A defined tendency was observed for a change to “mindful” shopping, “trading-down” to less expensive items [27], with a strong focus on “essentials” [27, 1, 17]. Groceries was the product category in which purchasing across all countries consistently anticipated spending more [27, 36].

The above-mentioned data concerning shifting in food purchase/consumption trends during COVID-19 illustrate general conditions, but does not illustrate the specific changes in people’s state of affairs caused by the pandemic and leading to lockdown. The data are substantial for identification of the mechanisms supporting the described above shifts and for making a precise forecast of the effects of COVID-19. These can define the measures regarding minimization of the adverse effects on food consumption.

6. Beyond the pandemic: transformation of the food systems after COVID-19

The Coronavirus pandemic and the associated political debates and responses led to unprecedented health disaster, economic poverty, services disturbances, unbelievable restrictions on movement. However, this extraordinary situation offers opportunities for the global food system to be transformed in more resilient, inclusive, efficient, sustainable and healthy. With the advancement of the COVID-19 pandemic and the parallel restrictive economic and social measures, a lot of countries introduced measures foreseen to secure the food products flow and introduced programmes developed to ensure food security in new ways. Innovations have been introduced along the food supply chain, developed mainly by the large companies and SMEs from the private sector, to help overcoming the constraints imposed by the recurrent lockdowns. In addition, they stimulated investments in technologies and partnerships to support viable food supply chains.

In the face of the COVID-19 pandemic food systems responded both with a fast pace and flexibility. The post-pandemic reality offered unique option the food systems to be transformed fundamentally in order to protect the fate of billions of people exposed to poverty, food insecurity and malnutrition. In this respect, five major attributes of the food system have been concerned.

• The food system efficiency: delivering proficiency all along the food supply chain, including crop production, infrastructure, food storage and transportation, and food consumption;
• The food system contribution to global health: producing affordable to all food of good nutritious quality and keeping food safety;
• The food system must be inclusive to all: it contributes to building descent livelihood and allows all to benefit – both as participants and consumers of decision making;
• The environmental sustainability of the food system: it is exploiting technological innovations, local and global regulations and governance approaches to conserve and protect natural habitats, resources, and biodiversity;
• The food system must be resilient: it must be able to overcome economic, health, and climate shocks, and provide people stabile livelihood and protection against such shocks. With the tendency the natural disasters (incl. pandemics) to become more frequent, the focus of the food systems must be their resilience from farm to global level.

Consolidation of these five attributes of the food system demands coordinated efforts at the level of food system policies, governance, and accountability.

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Test LO 5.1

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References

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Summary

The corona pandemic, along with human health and wealth, is also affecting the food sector. There is a continuous and enormous rise in the COVID-19 infections on a global scale reported for food workers. Although, there is no report about spreading the virus through food consumption, different gastrointestinal symptoms have been reported, thus the finding of SARS-CoV-2 in food or food packaging may raise concerns about food safety. There is a variety of physical factors and antiviral food components that effect SARS-CoV-2 transmission. The transmission of COVID-19 is also impacted by the food packaging. At present, significant research has been performed concerning antimicrobial food packaging but there is still a shortage in antiviral food packaging development. In this sense, the input of the nanotechnologies and nanomaterials (nanoparticles, and nanocomposites) in exploring antiviral food packaging is undoubtful and promising, since it contributes to the improvement of foodstuff quality, safety, and sustainability.

Key words/phrases: food-mediated corona virus transmission, food nanopackaging, nanoparticles’ antiviral activity

1. Introduction

The corona pandemic, along with human health and wealth, is also affecting the food sector [11]. There is a continuous and enormous rise in the COVID-19 infections on a global scale reported for food workers. Due to the rapid spread of the infection, various myths about the transmission of the virus during consumption of meat and poultry products have been spread. As has been shown by a survey performed in Arabian countries, the majority of the people are not aware of the SARS-CoV-2; they are neither concerned about the food-related transmission. This indicated that the local authorities and government bodies need to control the false rumors about food and its safety [28].

Hence, up to now, there is no report about spreading the virus through food consumption. However, different gastrointestinal symptoms like nausea, vomiting, and diarrhea have been reported [75]. Thus, the finding of SARS-CoV-2 in food or food packaging may raise concerns about food safety, but it doesn’t indicate a risk for public health. It is not a reason for restricting food trade or initiating a food recall but integrating food security and sustainability in a food chain must be considered as an important part of the approaches for controlling putative future pandemics.

2. Predicted modes of food-mediated corona virus transmission

Viruses are intercellular parasite that needs an alive host cell to ensure replication of its genome by the means of a host cell. This specific feature of the viruses does not permit their cultivation in an environment without a living cell.

Generally, viral diagnosis is difficult due to the availability of limited diagnosing and analytical tools for virus detection [74]. It is known that norovirus (gastroenteritis), hepatovirus A (hepatitis A), and orthoreovirus A (hepatitis E) are the typical food-borne viruses that can be transmitted to food commodities via different pathways like contaminated water, oral-faecal route, infected food handlers (Fig. 1).

Figure 1. The typical food borne viruses transmitted by food chain.

Previous outbreaks like SARS and MERS were not established to be transmitted by food. In this respect, there are no clear findings for SARS-CoV-2 to be transmitted also through such a mechanism. While the transmission of SARS-CoV-2 through food is not scientifically proven, this opportunity must not be excluded since SARS-CoV-2 is linked with animal trade and consumption [11]. Following this, the frozen fresh food as a source of SARS-CoV-2, by the analogy of MERS and SARS COV-1, can also be a medium to transmit the virus as it was found that it remains infectious in a frozen state for up to 2 years [33].
The transmission pathway of the viruses via food can take place during the handling in production, processing, packaging, and transportation. Also, cross-contamination from already infected food handlers is one of the major routes of transmission [72]. There is also a risk of spreading COVID-19 infection via food packaging through infected operational staff, for which cases the consumers were highlighted by the EU Commission [25]. Fig. 2 describes the various possible routes for transmission of COVID-19 via food.

Figure 2. Possible modes of transmission of SARS COV-2 in the food production chain.

2.1. Effect of temperature, pressure, humidity, and acidity on SARS-CoV-2 transmission

Food-borne viruses can be disabled using extrinsic and intrinsic factors of the food, and different chemical approaches as well as during technologies for food processing. Factors influencing SARS-CoV-2 virus are multidisciplinary. Hence, it is necessary to take under consideration the various environmental states, comprising temperature, pressure, humidity, and acidity, that forecast the vitality, survival, and infection rate of the virus.
The important extrinsic features of food, like water activity, рН, and frapped and frozen storage temperatures, are considered to control and keep the food microbiologically safe. The effect of these factors is related to their capacity to inhibit microbial contamination of food. When viruses are the contaminating agents, some of these controlling practices can be omitted since the viral infectivity strongly differs in comparison to that of the food-spoilage or pathogenic bacteria. The viruses, like various bacterial pathogens, are comparatively stable under refrigeration and freezing storage. Data are present in the literature for the persistence of various viruses in different food commodities after different storage regimens. For instance, no reduction of murine Norovirus (MNV) in spinach and spring onions was registered after 6 months of frozen storage; after 28 days of storage, а decrease of <1.2 log10N was measured for strawberries. SARS-CoV-2 was found to be stable up to 72 hours during refrigeration (4oC) and 2 years in deep freezing (-20oC). SARS-CoV is deactivated for 15 min at a temperature of 75oC while MERS can be inactivated at 65oC for 1 min.
A thermostability assay performed for SARS-CoV-2 indicated that it is inactivated at a minimum temperature of 70oC for at least 5 minutes. These data pointed out that standard temperatures for cooking are adequate to deactivate the virus. However, fresh and frozen foods may be used as vehicles for virus transmission. For them, rigorous keeping to the safety protocols and strong hand-washing after handling is very important. There is a lower possibility for viral transmission by food when its shipment takes place over а couple of days.
Like the food, drinking water is not treated as a way for transmission of the SARS-CoV-2 according to the reports of the Environmental Protection Agency (ЕРА) and the Centre for Disease Control (CDC). This is because the common water treatment procedures are adequate for the deactivation of the viruses. Thus, the thermal processing of food is considered one of the best useful techniques for foodborne viruses’ deactivation. The HAV, Hepatitis Е (HEV), and NoV viruses are as vulnerable to this treatment as the pathogenic bacteria. During pasteurization temperatures, MNV and HAV were shown to record more than 3.5 log10N at 72oC after 1 minute in the water. Also, MNV and TuV were deactivated after heating at 70°С for 2 min.
Steam blanching of produce like spinach for 1 minute at 80oC reduced the infectivity of MNV by а minimum of 2.4 log10N. Similarly, FCV and HAV were also immensely inactivated after blanching at 95oC for 2.5 minutes.
The strategic combination of acidification (рН decrease) of foods in addition to thermal treatment was also reported to be very effective against the inactivation of HAV.
Regardless of the minimum temperature and time requirements for inactivation of different viruses, in most cases, а minimum of 90 sec of thermal processing at а minimum temperature of 90oC is generally sufficient in inactivating enteric viruses irrespective of the complexity of the food matrix. It is generally believed that а boiling liquid medium such as water is capable of effectively inactivating the virus after about just 1 minute for more than 4 log10N for most enteroviruses including, human NoV, human rhinovirus (HRV), HEV, and HAV. Тhe dried virus was reported to retain its infectivity at 22-25oC and relative humidity of 40-50 % over 5 days. At higher temperatures (38oC) and relative humidity (> 95 %), they quickly lose their viability (> 3 log10N).
Extrinsic changes in рН levels that occur during fermentation or acidification of the carbohydrate substrates and the water activities levels of foods, for example by means of adding solutes like sugar or salt or their combinations, or even coupling of these treatments with dynamics of the storage conditions, have different effects on the viruses’ infectivity. MNV and TuV for example were found to tolerate low рН levels (рН 2) due to lactic acid fermentation. Even though fermentation was reported to be capable of producing compounds with potential usage as food additives, thus stimulating the antiviral properties, the underlying antiviral mechanisms are still yet to be fully understood.

2.2. Effect of irradiation on SARS-CoV-2 transmission

Ultraviolet (UV) radiation has been reported to be effective in activating some viruses. Its efficacy against the SARS-CoV-2 has not yet been tested. SARS coronavirus was extremely susceptive to ultraviolet irradiation. It has been recommended as an additional level of safety during the physical process of disinfection. Comparing the efficiency of disinfection: manual chemical methods reduced the contamination by 36% and а 96 to 99.99% reduction by ultraviolet irradiation from PurpleSun® ЕЗОО system UV system was achieved. At lower viral concentrations, low doses of UVC radiation (200 – 280 nm) completely inactivated the SARS-CoV-2, and higher doses altogether disabled higher concentrations of the virus.
Gamma irradiation between 3 000 to 15 000 rad did not affect the inactivation of the SARS-CoV, indicating that the irradiation dose range was too low to affect the virus. Whereas the virus was inactivated by UVC (unlike UVA D, 320 – 400 nm) after about 6 minutes of exposure. This implies that low wavelength UVC radiation could be effective in deactivating the virus from suspected food products.

2.3. Antiviral food components and food packaging

Food-borne viruses are proven to cause the majority of food-borne outbreaks. In 2010, 15% of food-borne outbreaks reported viruses as the causative agents [21] and according to those reports, Human noroviruses (HuNoVs) and Hepatitis A virus (HAV) was found to be of the greatest concern in the context of food safety. HEV was recently identified as a food-borne virus causing zoonotic transmission from pork meat products to humans upon consumption.
Natural bioactive compounds (flavonoids, polyphenols, tannins, catechins, saponins, polysaccharides, proanthocyanins, proteins, and peptides) are present in plant extract of cranberry, pomegranate, blueberry, black raspberry, grape seeds showed a great extent of antiviral activities by the destruction of viral structure or by preventing the entry of the virus into the host body [18]. Besides plant extracts, there are various essential oils, algal extracts, and proteins that are reported as antiviral. Various algal products such as carrageenan, navicular, and laminarin are also found to have antiviral activity [3]. An extended list of such natural compounds is presented in Table 1.
Currently, there is a lot of ongoing research on antimicrobial food packaging that aims to increase the shelf life of food products and retain food safety and quality. Antimicrobial packaging plays an important role in microbial growth inhibition and food spoilage. In the development of antimicrobial food packaging, there should be direct contact of the packaging material with the surface of the food for the migration of the antimicrobial compounds.
Antiviral food packaging materials are designed with the purpose to control the human enteric virus. Therefore, in the case of antiviral food packaging, it is necessary to inactivate the presence of the human enteric virus in food contaminated by raw and processed food products. Intrinsic material characteristics (which deal with polarity, and chemical composition) are responsible for the release properties of the biocidal compounds [29] and processing conditions for material development, which directly affect its mechanical, thermal, and physical properties (stability and release properties of the antiviral compound) are the relevant factors to be considered while designing antimicrobial packaging material [22]. However, there is less study on antiviral compounds into biopolymers due to their non-compatibility with polymeric structure and the quick release or degradation of antiviral agents.
Martinez-Abad et al. [51] developed 1% silver-PLA film and it was found to eliminate Feline calicivirus (FCV) from lettuce after 6 days of storage. Antiviral packaging materials were also developed by incorporating plant extracts in the biopolymers. A multilayer packaging material developed with cinnamaldehyde and zein ultrathin nanostructure layered covered with an outer layer of polyhydroxy-butyrate (PHB) worked against norovirus surrogates [27]. One of the promising candidates is the chitosan since it possesses excellent antifungal, antimicrobial, and antioxidative properties and is versatile to make coatings or films. It was observed that chitosan coating or films reduced the food-borne pathogens with the addition of essential oils or propolis into it [70].
Recently, the chitosan matrix was utilized in a study for protecting (-) – epigallocatechin gallate [35] having antiviral activity against HAV and MNV. Though only a few packaging materials have been developed which have antiviral activity, many of them were found to change the physicochemical properties of the food products. This change can be controlled through the application of edible films or coating. It is an emerging technology with controlled release of antimicrobial compounds by using various techniques like nanotechnology, encapsulation, and immobilization of antimicrobial agents from the matrix [4, 34]. The development of editable coating or film can be arranged by the addition of antimicrobial compounds in them that can reduce the food-born contamination. Though many pieces of literature are available for antibacterial and antifungal compounds incorporation in edible coating no data is present regarding antiviral edible coating or films. Therefore, research on antiviral edible coating is expected to have a great future. For making antiviral edible packaging materials, carvacrol, green tea extract, and grape seed extract can be used as natural antiviral agents. Similarly, many natural compounds such as clove, and oregano which were found to have antiviral activity against MNV, FCV can be incorporated into the development of antiviral food packaging [23]. Many researchers are fascinated by the micro- and nano-encapsulating sensitive antiviral compounds. Indeed, it not only helps to stabilize the compounds but also enhances their activity. Encapsulation is found to increase the stability of antimicrobial compounds even in irradiation [34]. However, there is limited data about the encapsulation of antiviral compounds and their applications in the food industry.

Table 1. Typical antiviral mechanisms of action of nanomaterials.

Virus	Natural source	Type
-PIV 3 - Parainfluenza virus type 3 -FIPV – Feline infectious peritonitis virus -VSV – Versicolor stomatitis virus -HSV – Herpes simplex virus -FHV – flock house virus -PR8, H1N1 and H6N1respiratory syncytial virus	Curcuma longa (L.)	Rhizome extract and Curcumin
-CHIKV - Chikungunya virus	Kalanchoe pinnata (L.) Pers.	Whole plant extract
-CHIKV - Chikungunya virus	Aristolochia tomentosa Sims	Whole plant extract
-CHIKV - Chikungunya virus	Paris polyphylla Sm.	Whole plant extract
-Yellow fiver virus	Clerodendrum serratum (L.) Moon	Whole plant extract
-Enterovirus 71	Terminalia chebula Retz.	Whole plant extract
-HRV 3 – Human rhinovirus 3	Chamaecyparis obtuse (Siebold & Zucc.) Endl.	Whole plant extract
-HRV 3 – Human rhinovirus 3	Chrysanthemum boreale (Makino)	Whole plant extract
-HRV 3 – Human rhinovirus 3	Cryptomeria japonica (L.f.) D.Don	Whole plant extract
-HSV – Herpes simplex virus	Swertia chirayita (Roxb. ex Fleming) H. Karst.	Whole plant extract
-VHSV - Viral Haemorrhagic Septicaemia Virus	Olea europaea L.	Leaf extract
-HIV – Human immunodeficiency virus	Salvia Rosmarinus Spenn.	Whole plant extract
-HSV-1 – Herpes simplex virus type 1	Camellia sinensis (green tea)	Whole plant extract
-HSV-1 – Herpes simplex virus type 1	Melaleuca alternifolia	Essential oils
-HSV-1 – Herpes simplex virus type 1	Thymus sp.	Essential oils
-Influenza viruses	Origanum acutidens. (Hand.-Mazz.) Ietsw.	Essential oils
	Artemisia obtusiloba var. glabra	Essential oils
	Houttuynia cordata Thunb.	Essential oils
	Salvia sclarea L.	Essential oils
	Cynanchum stauntonii (Decne.) Schltr. ex H.Lév.	cinnamaldehyde
-Corona virus	Nigella sativa L.	Essential oils
	Anthemis hyalina DC.	Essential oils
	Citrus × sinensis (L.)	Essential oils

Source: Jyoti and Bhaswati (2021) [45]

The lessons learned on the COVID-19 pandemic indicated helped issue some valuable recommendations for future outbreaks. There is а need for а cross-platform strategy to mitigate the spread of the virus along the food chain. This may involve international and governmental agencies, the food industry, retailers, food handlers, and consumers. The use of artificial intelligence (AI) to monitor and trace any exposure to SARS-CoV-2 is recommended, especially in the industry dealing with fresh products such as meat and vegetables.

3. COVID-19 impact on food packaging

3.1. Food packaging

As per definition, food packaging is the way, in which the food products are prepared for transportation, distribution, and retailing in a manner that assures their safe delivery to the consumer [64]. The systems for packaging are organized into three definite groups concerning their functions and levels of packaging. These are as follows [60].

• Primary packaging: this is the first packaging level. It comprises the layer of packaging that is in direct contact with the packed food products;
• Secondary packaging: this packaging involves a series of primary packages and serves to protect them from damage during transportation and storage. This group of packaging is designed in a way to assure a customer-friendly view of the package since the second packaging is displayed on the retail shelves.
• Tertiary packaging: this packaging executes the role of a distribution carrier that contains several primary and secondary packages.

The purposes of food packaging are multifunctional. Its major roles are related to [71]:

• Protect and maintained food safety from physical, chemical, and biological deterioration;
• To keep food products’ quality by extending their shelf-life;
• To ensure food products safety by diminishing the risk of interference and contamination;
• To serve as convenient containment, protection, and/or preservation device that is easily communicated among producers, retailers, and consumers;
• To help reduce the disposal of municipal solid waste, i.e. to reduce the cost of the food products by promoting the efficiency of bulk distribution, hence facilitating large-scale production.

Besides these important advantages, several drawbacks have to be considered as well. They are related mainly to non-biodegradable food packaging. It raises environmental concerns since non-biodegradable food packaging contributes to changes in the carbon dioxide cycle, raises composting problems, and elevates the levels of toxic emissions [26].

All these negative environmental impacts are directly related to the health safety concerns of consumers, and a lot of research studies are currently focused on the development of biodegradable packaging. Being renewable and environmental-friendly, biodegradable polymers will be progressively becoming the preferred choice of the packaging industry. For instance, the natural biopolymer sources, such as starch and chitin (polysaccharides), waxes and paraffins (lipids), collagen and gelatin (proteins), or their mixtures are subjected to comprehensive research nowadays [38, 73]. Among them, the potential of the proteins to be applied for packaging purposes is the greatest one due to their specific characteristics to form films with good barrier and mechanical properties [77].

3.2. Food packaging and transmission of COVID-19

The understanding of the transmission of COVID 19 through food packaging is based on the Good Manufacturing Practices (GMPs) regarding SARS-CoV-2 to avoid cross-contamination and transmission. Although there is no report of transmission of COVID-19 through food and food packaging, the importance of following Good Manufacturing Practices (GMPs) to avoid the cross-contamination and transmission of SARS-COV-2 still holds good. An individual may get infected if touches a surface or object, including food packaging. According to a report, the persistence of the coronavirus on plastic (72 hours) and steel (48 hours) is for a longer period than on the cardboard surface (24 hours). The persistence on the surface of copper (4 hours) is shorter possibly due to its antimicrobial actions [6].
During this pandemic, according to the report of the Food Packaging Forum, the reusable system is claimed to be much safer than the single-use packaging system; however, it affects the target of zero–a waste lifestyle. In fact, it is not possible to trace the handling of the single-use packaging products; at the same time reusable packaging can be washed with soap and hot water, and thus, can be used again.
Implementation of lockdown and execution of stringent government rules resulted in the shutdown of dine-in restaurants. Although food delivery chains operated, consumers avoided visiting them as well; that in turn negatively effectuated the food packaging industry [10]. As per a survey in the U.S. on COVID-19 risk perceptions about food packaging and food delivered in restaurants, more than 50% of consumers were moderately concerned about the food packaging in dine-in restaurants and about 23% of the consumer population were found to be seriously concerned about the restaurant packaging of food [10]. According to a study conducted in China’s five-star hotels regarding the transformation from offline to online food delivery systems as a pandemic response, people were concerned about the packaging and service delivery quality as compared for instance to the food taste, freshness, and brand credibility. About $900 billion per year of the food packaging industry is on the front line worldwide, where the Corona pandemic showed the sharpest decline in the eco-friendly food packaging market. According to a Jewish market report from 17 July 2020, the global eco-friendly food packaging market was valued at $163.5 billion in 2018 which was expected to gain revenue of $248.7 by 2026 but due to the current scenario, it reached only $159.8 billion. Market analysts have expected the market to restore from the losses and grow significantly by the third or last quarter of the year 2022.

4. Nanotechnology for safe food packaging

Nanotechnology progress has contributed to various aspects of food science and industry. One of its major applications is the production of food packages with improved physical properties and enhanced safety. These applications offer new promises for improvement of the efficiency of food packaging. Thus, nanostructures embedded in food packaging systems are used to transform them in intelligent packaging ones because they can detect and neutralize chemical, biochemical, and microbiological alterations and make the consumers aware for these problems [69].

World economy is strongly effectuated by packaging industry. In the USA, about 55–65% of $130 billion are invested in food and beverage packaging [9]. Recently, the use of operative and smart packaging systems for the food, based on meat products, which can be easily contaminated, showed remarkable market increase. The packaging of meat products aimed to suppress the blundering, shunt the contamination, increase the delicacy by enabling enzymatic activity, diminish the weight loss, and keep the specific ‘cherry red’ color in the red meats [36].

4.1. Use of nanomaterials in food packaging

The use of nanomaterials improves packaging flexibility and gas barrier properties – two of the most important characteristics of the process. The innovative nano-packages possess additional exclusive properties, e.g. capabilities to destroy microorganisms that are present in the food substances [41, 42]. Nanomaterials applied in wrapping matters allow the existence of food products for longer periods without causing any harmful modifications of the products’ inherent features [58].

An overview of the applications of nanomaterials in the food industry indicates that concerning food packaging, the electrochemical nano-sensors, nano-films, fluorescent particles, and antimicrobials are the most exploited ones.

Various types of nanomaterials find application in food packaging. Among the pleura, the nanomaterials, nanoparticles, and nanocomposites are those that have a major contribution to the improvement of foodstuff quality and safety. Both possess low molecular weight, mechanical strength, and high barrier capacity against O2, CO2, moisture, UV radiation, and volatiles.

4.1.1. Nanoparticles

Nanoparticles (Fe, Ag, MgO, ZnO, TiO, SiO2) are generated and applied in the industry because of their ability to encapsulate in active compounds; the latter possessing enhanced functionality, stability, and bioavailability [2]. The nanoparticles, incorporated into packaging materials designed for food products, provide for a longer shelf life of these products and their enhanced quality [16]. Those nanoparticles have an antimicrobial effect, that is why they have captured the attention of both the R&D and business for practical applications. Thus, metal nanoparticles, in particular the Ag ones, have been incorporated in polymer coverings (films) due to their antimicrobial properties via an active system (see below) [30]. Ag nanoparticles have been used for material packaging for a long time. It is well known that substances covered by Ag nanoparticles are preserved from contamination. In food packaging, many researchers focused their attention on Ag nanoparticles. However, just a few methods for the application of Ag nanoparticles are certified by European Food Safety Agency (EFSA) to be subjectable to recycling [20]. Various chemical modifications and deposition techniques have been introduced to enhance the attachment of Ag nanoparticles to the surface of plastic materials that help slow the release of the metal ions and their accumulation in the packed food [7].

Using of silicate nanoparticles in food packaging is acting as a hurdle for gases or moisture, and in this way – is reducing considerably the food spoilage and drying.

The application of a big number of nanoparticles in the food industry is characterized by significant antimicrobial power. They can also work as carriers of antimicrobial polypeptides and ensure protection against microbial food damage. For instance, a packaging material is prepared through a coating of starch colloids with an antimicrobial agent, which is acting as a hurdle to microbes by controlling the release of antimicrobials from the packaged material [46].

Nanoparticles are employed as vehicles of different substances: enzymes, antioxidants, anti-browning agents, flavors, and other bioactive materials aiming to improve their shelf life even after the package is opened [23, 24]. The formed reactive oxygen species (ROS) by TiO2 nanoparticles is destroying the pathogenic microbes, thus making them an effective antimicrobial agent.

The above-mentioned inorganic nanoparticles (iron, silver, zinc oxides, carbon, magnesium oxides, titanium oxides, and silicon dioxide) are broadly applied not only as antimicrobial agents but in some cases as food ingredients as well [61].

Nanoparticles are used as well for packaging food substances for modification of the penetration abilities of different packaging foils, increasing their mechanical properties, resistance to heating, and biochemical and microbial obstacle effects [59].

4.1.2. Nanocomposites

The nanocomposites are overtime reactive natural components compared to their macroscale counterparts, a property that is linked to the high surface/volume ratio [49]. Various nanocomposites (Fe-Cr/Al2O3, Ni/Al2O3, ZnO, SiO2) are used for packaging and coating purposes [17, 58]. Among them, SiO2 clay and nanoplates, carbon nanotubes, starch nanocrystals, grapheme, chitin or chitosan nanoparticles, cellulose-based nanofibers, and other inorganic nanocomposites are utilized. Generally, they are filled in a polymeric matrix, and in this way, the matrix becomes lighter and fire-resistant with better thermal properties and low permeability to gases [32]. The charging of active nanoparticles into the polymer matrices rises the completion of the food packaging material and gives functional opportunities like anti-oxidant, antimicrobial, and scavenging, resulting in the longer shelf life of the packed food products [43].

Nanoparticles (< 100 nm) may be involved in different plastics to result in polymer nanocomposites with improved characteristics. For instance, the so-called thermoplastic polymers contain 2–8% nano-scale chargers, such as carbon nanoparticles, nanoclays, polymeric resins, and nanoscale metals and oxides.

The silver in the silver zeolite is amenable to the antimicrobial activity, due to the ROS production. Silver zeolite coated ceramics are applied in food preservation, material decontamination as well as and disinfection of medical products. The extended antimicrobial activity of silver-based nanocomposite is unique to the silver zeolite [8].

The use of carbon nanotubes causes the removal of CO₂ or nasty flavors. The nanoclay in the nanocomposites (bentonite), put-upon in the manufacture of bottles and other packaging materials for food, considerably increases the gas barrier features. In this way, it inhibits oxygen and moisture from diffusion, drink destabilization, and food spoilage. The use of nanocrystals entrapped in nanocomposite plastic beer bottles, proposed by Nanocor (Arlington Heights, USA), diminishes the loss of CO2 and inflow of O2 into the beer bottles, like the natural biopolymer-based nanocomposites [53].

The inclusion of clay nanoparticles in the ethylene-vinyl alcohol copolymer and polylactic acid (PLA) biopolymer was found to refine the oxygen gate and enhance the shelf life of food materials [5]. The modified nanoclays, included in a polymer matrix, ensure mechanical strength and serve as a barrier to gases, volatiles, and moisture. Besides, PLA bionanocomposite, obtained through the inclusion of nanofillers into the biodegradable polymer PLA, indicated faster bio-degradation than its counterpart PLA without nanofillers [44].

Mechanical, thermal, and barrier features of the packaging material have been raised considerably by charging polymer–clay nanocomposites [56]. Hampering of oxidation, tuning of moisture migration, respiration rate, microbial growth, volatile flavor, and aromas are considerably affected by the use of nanotechnology in the packaging industries [13]. Such killing activities towards pathogenic microorganisms were founded also on the chitosan-based nanocomposite films, especially Ag-containing nanocomposites [48]. Essential oil from garlic stiffed with PEG-coated nanoparticles can be used to limitation of stored-product pests [15].

The enhanced shelf life of the food products was efficiently achieved through the use of phytoglycogen octenyl nanoparticles incorporated within Ɛ-polylysine.

Carbon-based graphene nanoplates are resistant to heat and have potential applications in the packaging of food products in the food industry [76].

An oxygen reduction of packaging material is achieved through the application of water-based nanocomposites formed by 1–2-µm nano-coatings on the surface. Other nano-preparations are the nanoemulsions used in food packaging as well as the disinfection of food packaging equipment. Nanomicelle-based products including glycerine restrict pesticide residues in fruits and vegetables and oil/dirt from cutlery. The supplementation of nanoemulsified bio-actives and flavors to beverages mustn’t alter the products’ look [67, 37]. Various food pathogens like gram-negative bacteria must be strongly enthralled by the nanoemulsions.

For everyday applications, zinc oxide is termed a safe material, certified by FDA, and is considered a food additive [50]. These nanoparticles are incorporated in matrices of polymers to provide nanocomposites with good properties such as antimicrobial activity and enhanced packaging properties [52].

Nanocomposites are also used in the packaging of food products. They possess specific characteristics to resist thermal stress during food processing, and transportation and storage of food products, as well. For instance, nanocomposites are used in beer bottles, enhancing their shelf life by up to 6 months.

4.2. Food nanopackaging approaches

The implementation of nanotechnologies in food production leads to smart packaging approaches and systems constituting the so-called active and intelligent packaging. Both systems foresee the improvement of food quality, from production to consumption, applying nanoparticles/nanocomposites for assurance of food protection and safety (Fig. 3).

Figure 3. Various food nanopackaging approaches.

4.2.1. Active food packaging systems

Active packaging is one of the innovative approaches used for packaging foodstuff. Its main characteristic is that the status of the packaged food items changes to improve the sensory quality and the safety of the food products, thus increasing their shelf life [66]. Bioactive packaging provides a positive impact on the health of consumers with the production of packaged foods that are good for health [1].

The systems for active packaging contain agents for moisture regulation, scavengers of CO₂ and O₂, as well as emitters and antimicrobials.

The active packaging systems are used mainly for storage purposes [13]. For instance, envelop systems for packaging are applied for short-term chilled storage. The modified atmosphere packaging (MAP) systems, vacuum packaging, MAP systems utilizing 100% CO₂, and bulk gas flushing ones are used for long-term chilled storage. The commercially used polymeric films for packaging are inert and hydrophobic and have less surface energy in comparison to low-density poly-ethylene (LDPE) and polypropylene (PP). To include antimicrobial substances for stopping food spoilage, modifications of the packaging material surface with functional properties and polar groups are exploited [13].

The modified atmosphere packaging (MAP) system founds wide applications and is used for the distribution, storage, and maintenance of meat products under low temperatures [14]. The lipid oxidation, dehydration, discoloration, and loss of aroma are factors associated with the potential spoilage of processed meats and should be taken into account to extend and maintain the shelf life of meat products. MAP promotes the shelf life and quality of the meat products by saturation the packaging environment of the meat products with formulated gas mixtures. Commonly, in the MAP technology, the non-inert gases such as O₂ and CO₂ are used. Their profiles are changing depending on the factors like the type of product, respiration, materials used for packaging, size of the pack, storage conditions, and package integrity. The uniform dispersion of clay nanoparticles on the transparent plastic film produced by the chemical giant Bayer (Leverkusen, Germany) prevents O₂, CO₂, and moisture from reaching fresh meats and other foods. There are several patents on the applications of nanomaterials in the food packaging filed in the USA, Europe, and Asia, and most of them report the utilization of nanoclays and nanosilver [40].

Another example of active food packaging systems is the use of allyl isothiocyanate and carbon nanotubes to limit microbial contamination and color changes, regulate oxidation, and support the storage of shredded, cooked chicken meat for 40 days [19].

4.2.2. Smart / Intelligent food packaging systems

The smart packaging includes nanosensors that are intended to sense microbial as well as biochemical alterations and provide signals for them [43]. Nanosensors detect microorganisms, toxic substances, and contaminants present in different foodstuffs because of their high resolution and detection capacity [39]. The application of nano-sensors for detecting toxins, pesticides, and microbial contamination in food products, provides a versatile alarm tool for the consumers to detect food spoilage or contamination linked with flavor production and coloring [31].

Improvements in the sensor technology present in the smart packaging of food materials provide information on the quality and safety as well as the half-life of materials [62].

Nanoparticles are applied in the preparation of nanosensors to disclose food contaminants. The nanosensors made for definite purposes are necessary for food analysis, determination of flavors or colors, drinking water, and clinical diagnosis [24]. The usage of nanosensors in food packaging helps in spotting the physical, chemical, and biological modifications during food processing. Nanosensors and nanodevices with the specialized design used in smart packaging assist in finding toxins, chemicals, and food pathogens [57]. This system with sensors and indicators is also applied to follow and display information concerning the quality of the packaged foods over the storage and transport.

Different functional nanomaterials can be used as nanosensors and active packaging materials. They possess considerable technical and hurdle properties, therefore are resourced and targeted to nutrient delivery systems [66]. It was shown that smart or intelligent packaging saves food quality during distribution.

The nanosensors work by monitoring the changes linked with the internal or external environmental stimuli. In brief, the following indicators are used in food packaging:

– Integrity (package integrity determination);

– Freshness (quality of the packaged products);

– Time-temperature (time and temperature-dependent changes).

They are recorded during the production and distribution chain to keep the quality and increase the shelf life of products.

Another smart nano-packaging system is the so-called nanobarcodes, ID tags that are introduced on the base of barcodes developed by nanoparticles [47].

The application of nanosensors in packaging encompasses as well enzymes raising the breakdown of food compounds that makes food improper for human consumption.

The application of nanosensors in the smart packaging systems encompasses as well the identification of gases, chemical contaminants, aromas, temperature and light intensity, pathogens, or the products of microbial metabolism [55].

Advanced analytical techniques like GC/MS, portable headspace O2, and CO2 gas analyzers are used to study the gas phases in the MAP products. However, these methods have certain blemishes; in real-time processes, optical sensor-based methods are more effective than these methods. The same is the effect in the large-scale usage [13].

The filing of food is the main trouble in the food industry and it is mainly a result of bacterial contamination. It is due to leakage of adverse odor but is not easily detected by the human nose, and sometimes causes poisoning. To indicate such kinds of odors linked with food poisoning, strongly sensitive biosensors are necessary [39]. In advanced food packaging, an integrated electronic ‘tongue’ comprises a set of nanosensors that are predominantly sensitive to the gases released from food waste. The device comprises sets of chemical sensors attached to a data processing system that gives a clear and visible signal indicating whether the food is fresh or not, using a sensor strip that changes color [54]. This device is used for a highly accurate determination of volatiles and monitoring the quality control processes in the food industry. Nanosensors were applied in the European project GOODFOOD (2004–2007) for food safety and quality control applications [65].

The intelligent food packaging nano-based systems aim to minimize food losses due to microbial contamination. A variety of nanoparticles (TiO2, MgO, ZnO, Ag, Fe0, C-nanotubes, and fullerene derivatives) have proved their effectiveness as antimicrobial agents [61]. In addition, the application of specific nanomaterials for both detection and elimination of destructive chemicals and pathogenic bacteria has been extensively used recently.

5. Antiviral mechanism of nanoparticles’ activity

The factors like product nature (formulation), processing conditions (intrinsic factors), type of package, and storage and distribution strongly influence the shelf life of a food product.

• The common intrinsic factors include water activity, pH, microbes, enzymes, and the level of reactive compounds. They can be tuned by the utilization of specific raw materials and components and convenient processing parameters.
• The common extrinsic factors are temperature, total pressure, light, partial pressure of various gases, relative humidity, and mechanical stress (human handling). They impacted the rate of degradation reactions during food material storage [76]. Exploiting antimicrobial packaging resulted in effective decontamination in parallel to the usage of antimicrobials as food additives. This result is because the surface microbial growth on the contaminated products is easily accessible for sterilization by surface-acting antimicrobial substances.

Meanwhile, antimicrobial packaging strongly interplays with the food product as well as the environment [12].

Antimicrobial nanoparticles, namely Cu, CuO, MgO, Ag, ZnO, Pd, Fe, and TiO₂, or nanoemulsions/nanoencapsulations, enclosing natural anti-microbial substances that can be adhered to via electrostatic, hydrogen bonding, and covalent interactions are developed to produce antimicrobial packaging systems.

The nanocomposites of organic (chitosan and essential oils) and inorganic (ZnO, TiO₂, and Ag) nature are successfully applied for food preservation through innovative packaging. The polymer coatings due to their chemical structure control the release of the active compounds and thus, regulate the function of the nanocomposites. Polymer matrixes that have been used for nanocomposite production are polyolefins, nylons, ethylene-vinyl acetate (EVA) copolymer, polyethyl-eneterephthalate (PET), polystyrene (PS), polyamides, and polyimides. Following this principle, extended antimicrobial efficiency against E. coli and Staphylococcus aureus has been observed with Ag nanoparticles immobilized in cellulose and collagen coatings for sausages. Although their bactericidal activity, these nanocomposites were not harmful to the consumers and the environment. [12]. Similar antimicrobial activity that lasted for 28 days was found for Ag nanoparticles entrapped in a polyamide matrix. Films of low-density polyethylene (LDPE) coated with Ag nanoparticles exhibit remarkable antimicrobial potential against Gram-positive and Gram-negative bacteria. The same effect was observed for chitosan–silver nanocomposite [29]. Antimicrobial activity coupled with significant mechanical stability was found in ZnO-encapsulated halloysite–polylactic acid nanocomposites. Extended shelf life, slowed down bacterial growth, and lipid oxidation has been observed for meat and poultry products packed in LDPE/ZnO+Ag nanocomposites coatings.

These findings indicate that the antimicrobial characteristics of the nanopackagings comprising nanocomposites depend to a great extent on the characteristics of both the polymer matrix and the nanoparticles. In addition, the storage conditions and storage duration impact the antimicrobial activity of the nanocomposite coating films. For instance, a study of the stability and antimicrobial activity of pullulan films with incorporated Ag or ZnO nanoparticles and essential oils of oregano and rosemary was performed for 7 weeks at various storage temperatures (4, 25, 37, and 55°C). Its findings indicated that the antimicrobial potential of the pullulan nanocomposite films against the common food pathogens L. monocytogenes and S. aureus was maintained at temperature < 25°C, and reduced significantly at > 25°C. [68].

Food nanopackaging with low Ag concentration, with enhanced and stable bioavailability, is a challenge for the application of Ag in food packaging. At present, the citrate-mediated silver complex is the most frequently used standardized silver formulation with antimicrobial action.

6. Conclusion

The respiratory virus SARS-CoV-2 has completely changed the scenario of food industries whether in production, processing, or packaging. There is a need to understand the transmission route of SARS-CoV-2 through food, where street food and openly sold items are of the main concern. Although various vaccines are currently available, there is a strong need to spread awareness regarding the pandemic to enforce the rules for personal hygiene, and avoiding cross-contamination. At present, significant research has been performed concerning antimicrobial food packaging but there is still a shortage in antiviral food packaging development. This imposes the great need to explore the antiviral food packaging incorporated with natural antiviral bioactive compounds to ensure both food safety and sustainability.

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Test LO 5.2

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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.

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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].

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Test LO 6.1

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References

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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.

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Test LO 6.2

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