Viral particles and functional nanomaterials crossing point

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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