2.1 Types of Vaccines

To date, massive vaccination has been one of the most effective means of preventing and spreading infectious diseases by stimulating antibody and T cell responses to induce protective long-term immune memory.^[19–21] The use of vaccines leads to a reduction of mortality and disease complications. In the process of vaccine design, the first priority is to define the antigen that affects the body’s immune response, the adjuvant—the co-administered auxiliary substance responsible for prolonging, enhancing, and modulating the immune response against the antigen, and the delivery strategy.^[22] The combination of these data together with the bioinformatic predictions and epitope (antigenic determinant) mapping is extremely important for the modern vaccine design. It should be emphasized that many types of vaccines are either currently in use or under development, which include inactivated and live-attenuated vaccines (first generation), subunit, conjugated, and recombinant vaccines (second generation), as well as DNA and RNA vaccines (third generation) (Figure 3).^[23]

The live-attenuated vaccines comprise weakened forms of the original pathogen. Thus, they induce strong cellular and antibody responses, giving long-term immunity after administration of usually one or two doses. It is easier to develop live-attenuated vaccines with viruses than with bacteria due to the fact that viruses have fewer genes. The greatest limitation of these vaccines is the necessity of refrigeration to maintain potency and the ability of pathogen reversion to the original virulent forms. It is also worth mentioning that the live-attenuated vaccines cannot be taken by immunocompromised individuals because they produce actual disease manifestations.^{[21, 23, 24]} By contrast, inactivated vaccines do not pose a risk to individuals with weakened immune systems, however, they may not provide sufficient protection for people with B cell or combined immunodeficiency.^[21] They are created by the inactivation or killing of a pathogenic agent via its exposure to radiation, chemical, or thermal treatment, resulting in a safety and stability increase (Figure 3). It is proved that these vaccines elicit weaker immune responses, therefore, additional doses are indispensable to retain immunity. Moreover, the possibility of the pathogen’s virulence features recovery is of high concern.^{[21, 23]}

An alternative to live-attenuated and inactivated vaccines is the second-generation vaccines that rely on subunit elements, recombinant or synthetic proteins, nonprotein antigens, and expressed bacterial immunogen or viruses (Figure 3). Subunit vaccines contain the only epitopes (protein–peptides or polysaccharides naturally occurring in pathogenic structure), stimulating the immune system, thus the likelihood of adverse reactions is lower. In the case of conjugate vaccines, proteins are used to enhance the immunogenicity of polysaccharide antigens. These vaccines have helped to reduce the disease incidence, especially in infants.^{[23, 24]} Recombinant vaccines are fabricated using recombinant DNA technology, which enables gene sequencing of the main pathogenic antigen, stimulating an immune response. In order to induce immunogenicity, a small DNA piece from the virus or bacterium is retrieved and incorporated into the manufacturing cells.^[25] It was found that in comparison to other vaccines, recombinant vaccines are safer, particularly for immunocompromised patients. The main limitation of the second-generation vaccines is the weak immunogenicity of the antigens, posing the need for an adjuvant introduction to enhance immune response.

At last, the third-generation vaccines known as genetic ones are based on a plasmid administration, comprising a gene encoding intentional antigen that is expressed in the cells by means of a specific promoter, causing the immune response (Figure 3). DNA vaccines have a lot of advantages but they are still under development for cancer therapies, allergies, autoimmune and infectious diseases. Besides the stability at various temperatures, they are also cost-efficient, safe, facile in manufacturing, and capable of stimulating humoral, mucosal, and cellular immunity. These types of vaccines have some downsides such as the potentiality to be used only for protein antigens, inability to get to target sites, and untimely degradation of the antigens leading to a weakened immune response.^{[26, 27]} Hence, the efforts of the scientific community are currently focused on the development of new vaccines using the benefits of nanotechnology.^[28] Along with the contemporary trend of research aimed at understanding the immune cell interactions at the sub-micrometer level, nanotechnology will play an increasing role in future immunology discoveries.

2.2 The Impact of Nanomaterials on the Improvement of Vaccine Responses

The nanotechnology platforms offer great opportunities in the process of designing new vaccines, being the milestones for medicine and biotechnology. Recently, nanovaccines have drawn huge attention of scientists, who are constantly working on optimizing nanoformulations to efficiently load and safely deliver genetic material (Figure 4). The great achievement reached so far is that both nanoparticles and viruses (including SARS-CoV-2) can act on the same length scale. Moreover, natural and synthesized nanomaterials can simulate the structural features of various viruses.^[20] In contrast to drug release systems that can be designed to target a specific cell type, an immunogenic nanomaterial-based vaccine must interact with the different cell types, e.g., professional antigen-presenting cells, B cells, T cells, macrophages, and neutrophils.^[19] This interplay can take place in many tissues over extended periods of time, making the rational design of nanomaterial vaccines a complex endeavor. One thing is certain, in the case of this type of vaccine, the morphology, and size of nanoparticles, their durability in vivo, the antigen physical orientation, adjuvant specific codelivery, as well as complement activation can be precisely controlled. The listed parameters affect the vaccine pathway to the different lymphoid tissues which determine the quality and potency of the immune response.

When an antigen is encapsulated within nanocarriers or conjugated (bound) to the surface of nanoparticles (Figure 5), its persistence increases at the injection site, in lymphoid tissues, and in circulation.^{[19, 20]} This, in turn, may enhance the antigen immunogenicity which is highly desirable. To achieve this goal, various nanotechnological solutions have been proposed. For instance, it was established that the nanoparticles consisting of the multilamellar lipid vesicles transporting a recombinant malaria antigen, both entrapped in the aqueous core and attached to the lipid bilayer surfaces, increased the stability and the strength of humoral immune B cell responses in a synergistic manner.^[29] Demento et al.^[30] investigated the impact of nanoparticle-mediated antigen delivery on the long-term stability and extent of the vaccine response. It was demonstrated that extended-release of ovalbumin—an antigen model encapsulated within poly(lactic-co-glycolic acid) nanoparticles can improve immunogenicity. Additionally, by controlling the nanoparticle degradation rate, the ovalbumin release can be further prolonged. Thus, it can be suggested that the selection of the appropriate vehicle ensures gradual, lasting availability of the antigen, which is a key factor facilitating a long-term memory T cell response and body recovery after infections with various pathogens.

Reaching a controlled and programmable delivery of nanoparticle-based vaccines to the B cells, T cells, follicular dendritic cells and subcapsular sinus macrophages is an extremely important aspect to consider in the vaccine design process. In recent years, many nanosystems have been developed to act as carriers for antigens. These include liposomes, polymeric nanoparticles, virosomes, inorganic nanoparticles, emulsions, and immune-stimulating complexes which will be comprehensively discussed in the next section.^[31–33]

The nanoparticle systems that are contained in the new generation vaccines have the ability to precisely control, enhance, and optimize the density and orientation of the antigen. The introduction of the antigens onto the nanomaterial surface via physical adsorption is based on the weak electrostatic or noncovalent hydrophobic interactions.^{[35, 36]} This can result in rapid detachment of the antigen from the nanoparticle in vivo. The nanocarriers such as chitosan, dextran sulfate polymeric nanoparticles, gold nanoparticles, and carbon nanotubes have a higher affinity to the amphoteric antigens. Therefore, the antigen release process can be designed by controlling the properties of the biological environment like pH, temperature, or ionic strength.^{[34, 37–39]} In the case of encapsulation and chemical conjugation (Figure 5), the interplay between the antigen and the nanoparticle is significantly stronger. In the first process, the antigens and the nanoparticle precursors are mixed during the synthesis. Hence, only when the nanoparticle is degraded in vivo or inside the cell, antigens can be released.^[39] Contrarily, in chemical conjugation, the antigen is chemically crosslinked to the nanoparticle surface. In this form, it is uptaken by the cell and then released inside the cell.^[40]

The antigens are generally delivered to lymph nodes (LNs) by the antigen-presenting cells (APCs), which gather antigens at the injection site and transport them to the LNs to activate T and B cells and produce adaptive and memory immune response for long-term immunity. However, drainage to the LNs may also occur, which is mostly dependent on the size of the employed nanoparticles. It has been demonstrated that the nanoparticles with sizes in the range of 10–50 nm tend to drain to the LNs,^[41] while those with a diameter below 6 nm drain to the vasculature.^[42] At the same time, other parameters such as flexibility, shape, and surface charge also influence the drainage of the nanoparticles to the LNs.^[43] In order to circumvent the need to take into account all these factors, the best solution could be an intranodal vaccine injection.^[44–46] In this case, the accumulation and degradation of the vaccine at the site of injection are not observed and it is possible to deliver more cargo to LNs. Clinical trials involving this approach have proven to be very promising.^[47–49] Jewell and co-workers^{[46, 50]} studied direct LN injection of biodegradable polymer nanoparticles loaded with antigen and adjuvant. The results confirmed potently enhanced CD8+ T cell response within 7 days after a single injection of vaccine with controlled-release components in small animal models. Strong, long-lasting humoral immunity was also generated. Compared to traditional peripheral injection routes, it was possible to significantly minimize the dose of the vaccine and improve its efficacy. However, it should be mentioned that the anatomical positions and size of the LNs can limit the translation of this vaccination method.

The mucosal immune response is also essential for respiratory, sexual, and oral transmission of viruses.^[51] In this regard, special attention has been paid to the delivery of nanoparticles through the mucosa.^[19] This process depends on the pore size and surface features of the mucosal barriers. Cervicovaginal mucus has the mean pore diameters of 340 nm,^[52] whereas respiratory mucus of up to 200 nm, therefore, only the nanoparticles with sizes smaller than these values can penetrate through the barriers.^[53] Especially, the cationic nanoparticles exhibit high mucoadhesion, so they can be retained in mucus and interact strongly with mucosal immune cells.

It is worth emphasizing that nano-based vaccines can also ameliorate the action of adjuvants and reduce negative side effects, e.g., toxicity and inflammation. Application of the nanoparticles to deliver adjuvants may contribute to their dose reduction through controlled release near or even inside APCs.^[54] In particular, for subunit vaccines, adjuvants are crucial elements to shape immunological memory. It was shown that mice immunization with lipid vesicles incorporating Plasmodium vivax circumsporozoite antigen and delivery in combination with the monophosphoryl lipid A adjuvant stimulated the high-titer antibody responses against VMP001 that lasted for more than 1 year at tenfold lower doses than in the case of traditional adjuvants.^[29] Friede et al.^[55] indicated that liposomes containing nontoxic monophosphoryl lipid A adjuvant can be used as carriers, causing a long-lasting IgG response against peptides. This approach can be helpful for the development of synthetic vaccines as it eliminates a need to employ protein carriers and conventional adjuvants. Finally, some nanoparticles exhibit intrinsic adjuvant activity, resulting from their composition and/or structure, which may facilitate the formulation of novel vaccines.^[37]

The use of stimuli-responsive nanocarriers in the design of new vaccines has also become groundbreaking. These types of smart nanomaterials undergo specific physicochemical changes under the influence of interaction with internal and external stimuli.^[56] The cellular organelles including endosomes and lysosomes reveal sharp pH differences in the intracellular microenvironment.^[57] A pH-sensitive nanocarrier can facilitate the liberation of vaccine components at specific target sites and enable controlled antigen presentation by APCs to elicit desired immune responses. It was found that biomimetic micelles composed of the dendritic cell (DC) membrane, histidine-modified stearic acid-grafted chitosan, and ovalbumin exhibit pH-dependent antigen release resulting in their effective escape from DC lysosomes.^[58] Liu et al.^[59] fabricated pH-responsive poly(d,l-lactic-co-glycolic acid) (PLGA) nanoparticles characterized by thin shells and large inner space containing ovalbumin antigen and NH₄HCO₃ acting as a promoter of antigen release in DCs. NH₄HCO₃ could react with hydrogen ions in DC endosomes and lysosomes (pH ≈5.0 and 6.5) to generate NH₃ and CO₂ causing destruction of nanoparticles and effective release of antigen to the cytoplasm (Figure 6). The immunization of mice with pH-responsive PLGA NPs elicited greater lymphocyte activation, more antigen-specific CD8+ T cells, and enhanced antigen-specific IgG antibodies, demonstrating cellular immunity. The pH-responsive PLGA nanoparticles have great potential to be used as effective vaccine delivery and adjuvant systems for the therapy of virus infections.

A combination of antigen and adjuvant co-delivery with pH-responsive endosomal membrane disruption may provide an even stronger immunostimulatory effect. Wilson et al.^[60] obtained micellar endosomolytic nanoparticles for delivery of immunostimulatory CpG oligodeoxynucleotide adjuvants and ovalbumin antigen to enhance cellular and humoral immunity. Mice immunization with subcutaneous injections of the nanoparticle formulations significantly augmented CD8+ T cell response compared to administration of the free agents.

The temperature change is another intrinsic stimulus that can affect the release of the antigen from the nanocarrier. thermo-responsive materials, which mainly include hydrogel-based polymers are characterized by the unique property of altering solvation state depending on temperature. They can transform from free-flowing liquids at room temperature to gels at physiological conditions without needing chemical or other environmental stimulations.^{[56, 61]} The modified thermo-responsive polymers such as poloxamer 407 and chitosan–methyl cellulose formulations have the ability to create an immobilized biomaterial depot for controlled antigen release, loaded by simple mixing at room temperature, and inducing an immunological response. It was found that the chitosan–methyl cellulose sol–gels sustained liberation of ovalbumin antigen up to at least 14 days after administration and stimulated both cellular and humoral responses in mice.^[62] Future smart vaccination strategies could also exploit thermo-responsive and immunomodulatory properties of hydrogel-based polymers to prolong the release of vaccine adjuvants. It should be emphasized that the sustained vaccine efflux could initiate the innate and adaptive immune response for long-term immunological memory.^{[56, 63]}

2.3 Nanoparticles Used in Vaccine Development

Nanoparticle-based vaccines provide an excellent alternative to conventional vaccines due to numerous advantages, such as tunable sizes and shapes, tailorable surface properties, controllable antigen release kinetics, and improved stability. As previously mentioned, many nanoparticles have been developed for vaccine platforms. Depending on the components creating their structure, they can be divided into organic (e.g., natural and synthetic polymer-based nanoparticles, liposomes, virus-like particles) and inorganic (e.g., gold, mesoporous silica, carbon, and calcium phosphate nanoparticles) (Figure 7). Biocompatibility, nontoxicity, and biodegradability are characteristic features of the majority of organic nanomaterials, whereas inorganic nanoparticles exhibit small particle size, good stability, enhanced permeability, ability to functionalize the external/internal surface, and high sorption capacity (Table 1).^{[64, 65]} In this section, we will describe examples of nanoparticles used in the vaccine design process with particular emphasis on their physicochemical properties, functions, and interaction with antigens.

2.3.6 Inorganic Nanoparticles

Gold nanoparticles of 2–150 nm in size are widely used as carriers in vaccine delivery.^{[39, 72, 81, 82]} They can be easily synthesized by various methods allowing precise control of their diameter and shapes by changing such parameters as temperature, time, type of the reducing agent, and solvent.^{[81, 170]} Both the shape and size of Au nanoparticles have an impact on the immune response of the host cells. The studies of Niikura et al.^[171] on Au nanoparticles coated with West Nile virus envelope protein demonstrated that gold nanorods (40 × 10 nm) are more efficient in macrophage and dendritic cell uptake than spherical (20 and 40 nm in diameter) or cubic (40 × 40 × 40 nm) Au nanoparticles. However, the 40 nm spherical Au nanoparticles elicited the highest level of West Nile virus envelope protein-specific antibodies. Therefore, it can be concluded that antibody production is not determined by uptake efficacy. Au nanorods-treated cells produced significant levels of interleukin-1β (IL-1β) and interleukin-18 (IL-18), indicating that they activated inflammasome-dependent cytokine secretion. Meanwhile, both spherical and cubic Au nanoparticles elicited inflammatory cytokine production (Figure 8).^[171]

Interestingly, it was proved that spherical Au nanoparticles of a diameter ranging from 8 to 17 nm conjugated with synthetic foot-and-mouth disease virus peptide showed the highest antibody response in comparison to the synthetic peptide conjugate.^[172] Au nanoparticles can be utilized as vehicles for antigens originating from the influenza virus^[173] and as an adjuvant for the human immunodeficiency virus DNA vaccine.^[174] It should be mentioned that functionalization of Au nanoparticles with carbohydrate conjugates is another approach used in order to improve the immunogenicity of vaccine. Safari et al.^[175] prepared Au nanoparticles conjugated to synthetic tetrasaccharide epitopes from Streptococcus pneumoniae, which induced specific anti-Pn14PS IgG antibodies, causing cytokine production of TNF-α, IL-2, and IL-5 in mice spleen and activation of memory T-cell.

Carbon-based nanomaterials, such as carbon nanotubes (CNTs), carbon spheres, and graphene have gained great attention in the scientific community corresponding to their unique physicochemical features advantageous for many biomedical-related fields.^[176–180] Particularly, CNTs are the exciting claimants to be used in nanovaccine formulations as scaffolds for carrying the specific antigen and facilitating its presentation to the immune system.^[83–86] CNT carriers possessing diameters in the range of 0.8–2 nm and lengths of 100–1000 nm are relatively inert, stable, nonimmunogenic, and nontoxic.^{[85, 87, 88]} Their distinctive structures enable introducing the combination of a number of antigens onto their surfaces simultaneously. Another advantage is the ability to penetrate into cells, including dendritic cells, which are essential for stimulating efficient immune responses.^{[85, 181]} Bacterial, protozal, and viral antigens beside CpG adjuvants can be conjugated to CNTs. The first reports of the use of CNTs as a scaffold for vaccine development concerned the covalent attachment of the peptide from the foot-and-mouth disease virus, which induces neutralizing antibody responses. It was found that the structure of the peptide’s epitope in its antigenic form is preserved after attachment to CNTs. The peptide linked to CNTs elicited virus-specific immune responses in mice. Moreover, no antibodies against the CNT structure were generated indicating the great potential of CNTs as carriers for antigens.^{[182, 183]} In another work, Plasmodium vivax apical membrane antigen-1 peptide was conjugated to multiwalled CNTs to treat malaria. The specific immune response was directed toward the peptide rather than CNTs, revealing that carbon vehicles are not intrinsically immunogenic.^[184] Meng et al.^[185] developed a therapeutic cancer vaccine based on a combination of tumor lysate protein with single-walled CNTs in mouse model hepatoma. The tumor lysate protein–CNTs conjugate improved noticeably the rate of cure and enhanced the cellular antitumor immune reaction in comparison to the lysate itself. Mocan and Iancu^[186] studied the therapeutic potential of multiwalled CNTs combined with embryonic stem cells as novel vaccine nanobiosystems. They demonstrated that concurrent immunization with embryonic stem cells and multiwalled CNTs elicit significant antitumor responses and enhance tumor rejection in mice with subcutaneous inoculation of malignant colon cells.

Silica-based nanoparticles are one of the most interesting inorganic candidates for biomedical applications such as nanovaccinology,^{[36, 91, 92]} drug delivery,^[187] biosensing,^[188] bone tissue engineering,^[189] cancer therapeutics, and diagnostics.^[190] They possess many beneficial properties, among which the most important are high specific surface area and pore volume,^{[191, 192]} biocompatibility,^{[93, 94]} colloidal and thermal stability,^[193] as well as biodegradability in the cell membranes.^[95] Moreover, silica nanoparticles with sizes in the range of 50–200 nm have tunable structural parameters and morphology determining the cellular uptake and internalization rates.^{[96, 97, 194]} A large amount of silanol groups on the silica surface allows the introduction of various organic/inorganic functionality in order to improve chemical stability and interaction with cells.^[195–198] All these features are of great importance for effective encapsulation, high loading capacity, and controlled release of various biomolecules.^[199] So far, three types of silica nanoparticles (S1, S2, and SBA-15) differing in sizes (430 nm, 130 nm, 1–2 µm) and pore characteristics, were loaded with bovine serum albumin (BSA) model protein to design a vaccine delivery platform. It was established that the immune response was remarkably determined by the uptake and antigen release profile. The structure of the silica was also of great importance. The rate of protein release varied with the size of the silica vehicle pores. The large pores enhanced the interaction between BSA and nanoparticles, stimulating antigen gradual release.^[200] Slowing et al.^[188] suggested that the reason for the slow antigen release from silica nanoparticles may be connected to the high stability and rigidity of a framework, creating a barrier against antigen degradation in the stomach and digestive tract. The hollow mesoporous silica nanoparticles (HMSNs) have been utilized as antigen delivery carriers for Porcine circovirus type-2 (PCV2) ORF2 protein. It was demonstrated that HMSNs improve both humoral and cell-mediated immune responses. The percentage of CD4+ and CD8+ T cells in the mice immunized with HMSN/GST-ORF2-E was higher than that observed for mice immunized using the only HMSNs at 4th and 6th-week of research.^[201] Mahony et al.^[92] studied immunization by the ovalbumin model protein antigen employing MCM-41 mesoporous silica nanoparticles functionalized with amine groups. Functionalization of material led to a 2.5-fold increase in ovalbumin binding capacity compared to the nonmodified sample. Moreover, amine-modified nanoparticles acted as self-adjuvants and induced immune responses at reduced antigen doses in vivo. No local or systemic negative symptoms in animals after injection with silica nanoparticles were observed. The presented examples show that silica nanoparticles have a great potential to be used as vehicles stimulating antigen-controlled release in future vaccine formulations.

Calcium phosphate (CaP) nanoparticles prepared by mixing calcium chloride, dibasic sodium phosphate, and sodium citrate under specific conditions have sizes ranging from 50 to 100 nm.^[202–204] Sodium citrate is used as a stabilizing agent and a regulator of crystal growth. Due to the chemical similarity to biological hard tissues, CaP nanoparticles show excellent biocompatibility and low cytotoxicity. They can act as central binding sites for nucleic acids, antigenic proteins, and cells, protecting these biomolecules from external stressors and permitting delivery to endosomes/lysosomes.^[98] After releasing encapsulated biomolecules, CaP nanoparticles are decomposed due to the change in pH of the environment from neutral into acidic during cellular uptake, which makes them ideal vehicles. It was found that these nanoparticles are valuable and easily biodegradable adjuvants for DNA vaccines and mucosal immunity.^{[65, 202, 203]} Moreover, CaP nanoparticles constitute an improved alternative to alum adjuvants in mice immunized with viral proteins.^[203]

The studies utilizing different nanoparticles as delivery systems and/or adjuvants in vaccine design are summarized in Table 2.

Table 2.
Nanoparticle-based vehicles and adjuvants used in vaccine development

Nanomaterial	Immunogen	Disease	Function	Ref.
Alginate-coated chitosan nanoparticles	Hepatitis A surface antigen	Hepatitis A	Adjuvant/delivery system	[99]
Calcium-alginate nanoparticles	Androctonus australis hector venom	Scorpion envenomation	Delivery system	[100]
Chitosan nanoparticles	Newcastle disease virus	Newcastle disease	Delivery system	[113]
Chitosan nanoparticles	Hepatitis B surface antigen	Hepatitis B	Delivery system	[114]
Chitosan nanoparticles	Hepatitis B surface antigen	Hepatitis B	Delivery system	[115]
Inulin	Inactivated virus from H1N1, H3N2, and B strains	Influenza	Adjuvant	[118]
Inulin	Hepatitis B surface antigen	Hepatitis B	Adjuvant	[119]
PLGA	Hepatitis B surface antigen	Hepatitis B	Delivery system	[128]
PCL nanoparticles	Hepatitis B surface antigen	Hepatitis B	Delivery system	[139]
Liposomes	Self-replicating RNA genome	Chikungunya fever	Delivery system	[142]
Cationic lipid DC-Chol	Hepatitis B surface antigen	Hepatitis B	Adjuvant	[144]
Cationic transfersomes	Plasmid DNA encoding hepatitis B surface antigen	Hepatitis B	Delivery system	[145]
ISCOMs	Envelope proteins of Newcastle disease virus	Newcastle disease	Delivery system	[148]
ISCOMs	Influenza virus	Influenza	Adjuvant/delivery system	[149]
ISCOMs	Herpes simplex virus type 2 (HSV-2) antigen	Herpes	Delivery system	[150]
ISCOMs	Chimeric peptide containing V3 loop and transmembrane sequence of gp41 with two glycine motifs	HIV-1	Delivery system	[151]
VLPs	RSV fusion protein	Respiratory syncytial virus infection	Delivery system	[153]
VLPs	HIV-1 envelope protein	HIV-1	Delivery system	[154]
VLPs	Viral capsid formed by the ORF2 protein	Hepatitis E	Delivery system	[158]
Oil-in-water nanoemulsion, MF59	Antigen influenza viral strains	Influenza	Adjuvant	[163–165]
Water-in-oil nanoemulsion, Montanide ISA51	C-terminal 19-kDa fragment of the P. yoelii merozoite surface protein 1	Malaria	Adjuvant	[167]
Water-in-oil-in-water nanoemulsion, Montanide ISA201	Antigen from virus strain of FMDV O/IND/R2/75	Foot-and-mouth disease	Adjuvant	[169]
Au nanoparticles	WNV (West Nile Virus) envelope protein	West Nile fever	Delivery system	[171]
Au nanoparticles	Virus peptide	Foot-and-mouth disease	Delivery system	[172]
Au nanoparticles	Matrix 2 protein (M2e) of influenza A virus	Influenza A	Delivery system	[173]
Au nanorods	HIV-1 Env plasmid DNA	HIV-1	Adjuvant/delivery system	[174]
Au nanoparticles	Spike protein of SARS-CoV	Severe acute respiratory syndrome-related coronavirus infection	Adjuvant/delivery system	[205]
CNTs	Virus peptide	Foot-and-mouth disease	Delivery system	[182, 183]
CNTs	Plasmodium vivax apical membrane antigen-1 peptide	Malaria	Delivery system	[184]
CaP nanoparticles	Viral glycoproteins	Herpes simplex virus type 2 and Epstein-Barr virus infections	Adjuvant	[202, 203]
CaP nanoparticles	Plasmid DNA	Foot-and-mouth disease	Adjuvant/delivery system	[204]

2.4 Nanotechnological Strategies for COVID-19 Vaccine Design

At the outset, it should be noted that vaccines for infectious diseases had never been produced at such a rapid rate as in the case of the COVID-19 vaccines. Moreover, no vaccine existed for preventing infections caused by coronaviruses in humans. The vaccine design process is extremely long and expensive. It usually includes many years of research on different candidates from which the most effective and the safest one is selected. Due to the high costs and failure rates, the vaccine development process follows a linear sequence of steps, such as preclinical vaccine design, efficacy and safety studies, clinical trials—phases I–III, licensure, with breaks for in-depth data analysis and manufacturing process control. Rapid vaccine design requires a new pandemic paradigm of multiple steps in parallel, hence there is an increased financial risk (Figure 9).^[206–209] As soon as China announced in December 2019 that a novel coronavirus was the cause of the Wuhan outbreak, international efforts were focused on the accelerated development of modern vaccine formulations. With the potential for further financial support, vaccine production began when the SARS-CoV-2 genetic sequences were released by the Chinese Center for Disease Control and Prevention.^[210–212]

SARS-CoV-2 is an enveloped single-stranded ribonucleic acid ((+)ssRNA) virus with glycoprotein spikes that protrude from its external membrane surface creating a characteristic “corona.” The coronavirus particle integrity is maintained by four major structural proteins: i) spike (S) protein enabling the attachment of the virus to the host cells; ii) envelope (E) protein playing a structural role and assisting in assembly as well as budding; iii) the abundant membrane (M) protein preserving the integrity of viral particle membrane; and iv) nucleocapsid (N) protein mostly binding to the RNA of SARS-CoV-2 and supporting the formation of the nucleocapsid (Figure 10). In particular, S protein is a splendid target for the design of vaccines, but it is worth highlighting that antigen optimization is necessary to ensure an adequate immune response.^[213] The S protein of SARS-CoV-2 is cleaved into two subunits: S1 and S2. S1 includes the receptor-binding domain (RBD) and is responsible for attachment to the cell. S2 is composed of N and C heptad repeats, a potential fusion peptide, and a transmembrane domain. It facilitates the membrane fusion and entry into the cell.^{[20, 212–215]} By combining information on the structure of SARS-CoV-2 and the knowledge gained during the development of SARS/MERS vaccine candidates, the full length S protein, S1, RBD, and S2 subunit derivatives were designed to contain the main target epitopes to elicit neutralizing antibodies.^[20] From the vaccine technology development perspective, this is an inspiring period in which the novel nano-based approaches can make a great clinical impact for the first time.

Although currently many different COVID-19 vaccines are approved or under development, in this section, we will focus only on nanotechnology-based ones (Table 3).

The mRNA vaccines have many advantages as pandemic-protection platforms, given their flexibility and efficiency in immunogen design and production. As mentioned earlier, for these types of vaccines, lipid nanoparticles are often selected as delivery systems. mRNA complexed with positively charged lipids is characterized by higher stability and resistance to RNase-mediated degradation. They form self-assembled virus-sized particles ensuring the possibility to be administered via various routes. After endocytosis, the lipid nanoparticles release their genetic cargo in the cytosol, where the mRNA is translated into antigenic proteins, stimulating the immune system to produce neutralizing antibodies. The current limitation of this type of vaccine formulation is their long-term storage requiring low temperatures and in turn leading to logistical difficulties in distribution.^[217]

In 2020, BioNTech (Biopharmaceutical New Technologies) German company in conjunction with Pfizer American multinational pharmaceutical corporation started a coordinated program aimed at comparing four RNA-based COVID-19 vaccine candidates in clinical studies performed both in Germany (BNT162-01) and in the United States (C4591001). It was important to choose one vaccine candidate and the dose level for further international safety and efficacy trials. Bearing in mind preliminary clinical-trial results in Germany,^[218] two lipids nanoparticle-formulated^[219] nucleoside-modified RNA vaccines against COVID-19 were assessed in the U.S.^[220] The first one, BNT162b1, encodes the SARS-CoV-2 receptor-binding domain, trimerized by adding a T4 fibritin foldon domain to enhance its immunogenicity. In turn, the second candidate, BNT162b2, encodes the SARS-CoV-2 full-length spike, modified by two proline mutations to lock it in the prefusion conformation and simulate the virus which interacts with the induced virus-neutralizing antibodies. In these vaccine formulations, lipid nanoparticles were used as mRNA carriers, protecting the nucleic acid from degradation and allowing delivery of the mRNA into cells.^[221] BNT162b2 was proved to have a better safety profile than the other vaccine candidates,^[222] and a two-dose regimen provided 95% protection against COVID-19 in individuals 16 years of age or older.^[16] Moreover, Pfizer and BioNTech declared that the vaccine is safe and 100% effective for 12–15 years aged adolescents, which was established based on the phase III trial data. Among all participants of the trial, only 6% reported adverse events after first and second doses.^{[223, 224]}

Another lipid nanoparticle-encapsulated mRNA vaccine named mRNA-1273 was designed by Moderna American pharmaceutical and biotechnology company and the Vaccine Research Center at the National Institute of Allergy and Infectious Diseases, within the National Institutes of Health.^{[210, 225]} This mRNA-1273 vaccine encodes the SARS-CoV-2 spike (S) glycoprotein stabilized in its prefusion conformation. It should be noted that the S glycoprotein mediates host cell attachment and is necessary for viral entry.^[226] In the case of mRNA-1273, PEGylated lipid nanoparticles act as delivery vehicles. The mRNA-1273 vaccine demonstrated 94.1% efficacy at preventing COVID-19, very similar to that observed for the BNT162b2 vaccine (95%) developed by Pfizer and BioNTech.^[17] Moreover, after its administration, only transient local and systemic reactions were identified.^{[17, 227]}

Preliminary results did not demonstrate any obvious negative signals among pregnant women after administration of mRNA COVID-19 vaccines: BNT162b2 and mRNA-1273. Further long-term monitoring of the large number of women vaccinated during the first trimester of pregnancy should be conducted to determine possible effects on pregnancy and infants.^[228]

Japanese pharmaceutical company Daiichi Sankyo in collaboration with the University of Tokyo’s Institute of Medical Science developed also a gene (mRNA) vaccine for COVID-19 called DS-5670a with the use of nucleic acid delivery technology based on lipid nanoparticle structures. Clinical trials of this vaccine are expected to start in 2021 (NCT04821674).^[229]

Lu et al.^[230] developed three mRNA vaccine candidates for COVID-19 named RQ3011-RBD, RQ3012-Spike, and RQ3013-VLP encoding different antigen forms in vaccinated hosts. The first one encodes the RBD of the spike (S) glycoprotein of coronavirus with N-terminal signal peptide and C-terminal membrane-anchoring helix. The second vaccine RQ3012-Spike encodes the full-length wild-type S. In turn, RQ3013-VLP is prepared with the use of mRNAs cocktail encoding three structural proteins: S, M, and E to create SARS-CoV-2 VLPs with an average diameter of 100 nm. The lipid nanoparticles were utilized to incorporate mRNAs. The efficiency of encapsulation was higher than 98% in the case of all three vaccine candidates. It was demonstrated that although the RQ3012-Spike and RQ3013-VLP contained the same amount of S mRNA, only the latter induced humoral and T cell immune responses. Moreover, it also produced the highest titers of neutralizing antibodies. The least promising vaccine candidate turned out to be RQ3011-RBD, which did not induce sufficient immunity in mice. However, this does not exclude the use of RBD as an antigen in future mRNA vaccines, but some improvements must be made to increase immunogenicity.^[230]

To date, no DNA vaccine for COVID-19 has been licensed. However, it should be mentioned that this kind of vaccine besides antibody and CD4+ T cell responses induces also CD8+ cytotoxic T cell responses which play a crucial role in neutralization of virus infection. Smith et al.^[231] presented the preliminary results on immunogenicity of the synthetic DNA-based vaccine candidate, INO-4800, targeting SARS-CoV-2 S protein. Scientists used their previous experience with the development of vaccines against MERS-CoV (INO-4700) and Zika virus (GLS-5700) that are currently under clinical trials. It was found that INO-4800 is a promising vaccine candidate because it elicits cellular and humoral host immune responses observed within several days after a single immunization in mice and guinea pigs. At present, DNA vaccine leader—Inovio Pharmaceuticals biotechnology company is assessing INO-4800 vaccine against SARS-CoV-2 in the three mid-stage trials in China, collaborating with Advaccine Biopharmaceuticals Suzhou Co., Ltd., in a mid-to-late stage trial in the U.S. and one in South Korea.^[232]

On 11 March 2021, Novavax biotechnology company from Maryland has published interim phase III trial data for a subunit coronavirus vaccine named NVX-CoV2373. Interestingly, this vaccine acts differently compared to other already approved vaccines and it has demonstrated to exhibit final efficacy of 96.4% against mild, moderate, and severe disease caused by the original SARS-CoV-2 strain in the United Kingdom. Moreover, it is also efficient in 55.4% of the HIV-negative trial participants in South Africa where the majority of strains are B1.351.^[233] The data from the 2019nCoV-302 study, conducted with participants aged 18 to 84 in UK, showed that a two-dose regimen of the NVX-CoV2373 vaccine is safe and provides 89.7% protection against the B.1.1.7 coronavirus strain.^[234] This variant is more transmissible and causes higher mortality than previous strains. The Novavax protein-based vaccine was engineered from a modified SARS-CoV-2 spike gene inserted at the beginning into baculovirus, which then infected the culture of Sf9 moth cells. The infected cells produced spike proteins that are harvested and assembled into nanoparticles. Although the nanoparticles simulated the structure of the coronavirus, they can neither replicate nor induce COVID-19 disease.^[235] The NVX-CoV2373 vaccine formulation contains Matrix-M patented adjuvant composed of the nanoparticles of 40 nm in diameter based on saponin with cholesterol and phospholipid, enhancing the immune response and stimulating high levels of neutralizing antibodies.^[236–238] Tian et al.^[235] proved that in mice and baboons, low dose of NVX-CoV2373 vaccine with adjuvant induce high titer anti-S IgG and functional antibodies blocking hACE2 receptor binding and eradicate virus infection.

Subunit vaccines can take the form of VLPs described in detail in Section 2.3.4. They are considered a powerful candidate to prevent spreading the SARS-CoV-2 pandemic. Highly ordered and stable VLP vaccines are manufactured using recombinant expression. An example is the plant-derived COVID-19 vaccine developed by Medicago, a biopharmaceutical company from Quebec City, and GlaxoSmithKline (GSK). The proposed approach utilizes living plants as bioreactors to produce recombinant spike (S) glycoprotein expressed as VLPs which are then administered together with GSK’s adjuvant.^[239] VLPs simulate the native structure of the virus, so they can be readily recognized by the immune system. On 16 March 2021, Medicago and GSK companies have announced that the phase III clinical trials with healthy individuals aged 18–65 years were started. They will continue thereafter with elderly adults (65y +) and adults with comorbidities. All participants of the study will take two doses of vaccine 21 days apart.^[240] A similar approach for the development of several COVID-19 vaccine candidates was applied by the researchers from the University of Bristol and spin-out firm Imophoron. The production of the vaccines relies on the insertion of harmless bits of the viral surface proteins into the ADDomer platform creating synthetic VLPs that mimic coronavirus. The high specificity of the spike (S) parts essential for cell entry decreases the risk of negative side effects. The new highly adaptable platforms for vaccines are extremely stable without refrigeration and enable easy production on a large scale. They will be tested in vivo in a preclinical program.^{[241, 242]}

3.1 Nanocarrier-Based Antiviral Drug Delivery Systems

Clercq and Li^[250] reported that 90 antiviral drugs are approved to treat viral infections, but most of them only work against certain types of virus. Hence, when a completely new virus emerges, its pharmacological control and spread are limited. SARS-CoV-2 mainly attacks the respiratory tract (upper airways, lungs),^[251] however other organs (e.g., gut, kidney) and systems (e.g., vasculature)^[252] can be also impacted. It is believed that the expression of ACE2 influences which organs are subject to infection.^[253] In the case of SARS-CoV-2, attempts have been made to treat patients with antivirals such as lopinavir, ritonavir, ribavirin, remdesivir, chloroquine, hydroxychloroquine, and their various combination.^[254]

Lopinavir and ritonavir belonging to protease inhibitors have been successfully used to treat HIV infection and they have improved the outcomes of MERS-CoV and SARS-CoV patients.^[255–257] The recent studies reported that administration of lopinavir/ritonavir (Kaletra, AbbVie, U.S.) significantly reduced β-CoV titers of the COVID-19 patient in Korea.^[258] However, it was also revealed that lopinavir/ritonavir treatment neither reduced mortality nor diminished throat viral RNA in patients with serious symptoms of COVID-19.^[259] Future trials may confirm or deny the medicinal benefit resulting from the use of lopinavir and ritonavir.

Ribavirin is a purine nucleoside analog exhibiting broad-spectrum antiviral activity which is primarily applied for the treatment of respiratory syncytial virus infections and also in combination with interferon α for the therapy of chronic hepatitis C.^{[260, 261]} Ribavirin has also shown antiviral activity against SARS-CoV and SARS-CoV-2 in vitro. So far, clinical studies performed with ribavirin have resulted in ambiguous outcomes.^{[262, 263]} The reason for this may be that the concentration of ribavirin required to attain inhibition was difficult to achieve in clinical settings. Despite this, when used as a part of combination therapy with the HIV-1 protease inhibitor—lopinavir, the concentration of ribavirin required to inhibit SARS-CoV was reduced to 6.25 µg mL⁻¹.^[255] Hence, ribavirin may still be successfully used as a part of combination COVID-19 therapies with other antiviral drugs, interferons, or HIV-protease inhibitors. However, its use can cause hemolytic anemia.^[264]

Another example of a drug used in COVID-19 therapy is remdesivir (GS-5734), an inhibitor of the viral RNA-dependent RNA polymerase.^[265–267] It exhibits antiviral activity in vitro against the Ebola virus, Marburg virus,^[268] Paramyxoviridae (e.g., Nipah virus, parainfluenza type 3 virus, Hendra virus, measles, mumps viruses), Pneumoviridae (e.g., respiratory syncytial virus),^[269] SARS-CoV-1 and the Middle East respiratory syndrome (MERS-CoV).^[270–272] The clinical trial of intravenous remdesivir in adults has demonstrated potent antiviral activity for SARS-CoV-2, significantly shortening the recovery time of hospitalized patients with lower respiratory tract infection.^[266] The most common side effects connected with the administration of this antiviral drug in people with COVID-19 are nausea, liver inflammation, low blood pressure, and sweating.^[273]

The drugs representing the inhibitors of viral entry are chloroquine and hydroxychloroquine.^[274] Chloroquine is a derivative of quinine that has been widely used for decades to treat malaria.^[275] In addition to its antimalarial properties, this drug has shown broad-spectrum antiviral effects against a diverse range of viruses, such as the dengue virus,^[276] Zika virus,^[277] chikungunya virus,^[278] and influenza viruses.^[279] It also proved to be an effective antiviral drug against SARS-CoV in vitro when introduced prior to or after the establishment of infection.^{[280, 281]} Recently, it displayed similar, potent antiviral effects against SARS-CoV-2.^[267] In addition to its direct antiviral activity, chloroquine also modulates the host immune system. Preliminary results from two clinical trials have demonstrated the efficacy of chloroquine in reducing SARS-CoV-2 viral load in the majority of patients.^[282] However, it often causes serious side effects, including vomiting, heart problems, headache, vision changes, and muscle weakness. Liu et al.^[283] performed the comparative analysis between hydroxychloroquine and chloroquine against SARS-CoV-2 infected patients. The results show that hydroxychloroquine is less toxic and more effective in inhibiting SARS-CoV-2 infection. Moreover, in a multi-hospital assessment, when controlling COVID-19 risk factors, treatment with hydroxychloroquine alone and in combination with azithromycin was associated with a reduction in COVID-19 associated mortality.^[284]

This short presentation of antiviral examples showed that their administration is very often connected with negative side effects caused by their accumulation in nontarget organs. Furthermore, these drugs are very often characterized by poor aqueous solubility and low bioavailability.^[285] The presented limitations of the drugs used in COVID-19 and other viral therapies can be solved by biocompatible, safe nanocarrier-based drug delivery platforms that will improve their efficiency and bioavailability, reduce toxicity, and maintain the viral spread suppression in plasma (Figure 11). They should also help to keep subtherapeutic concentrations of antivirals in reserve sites.^{[215, 286]} Additionally, by preventing premature release and degradation of drugs, as well as avoiding renal and hepatic clearance, nanocarriers can provide an improvement in their half-life. Thus, they are designed for controlled biodistribution and release of antiviral drug molecules at the target site within a specified time. In general, pharmacokinetic properties (i.e., absorption, distribution, metabolism, excretion) are inherent characteristics of drug molecules that cannot be changed without derivatization. The nanoparticles, which are small enough to circulate in the bloodstream and penetrate into the deep lung, are attractive materials for utilization as drug containers since their pharmacokinetic features can be tuned without affecting the activity of loaded drugs. Key parameters such as size, shape, charge, hydrophobicity, and specific interactions with target cells determine the biodistribution of nanocarriers. However, it should be mentioned that even under normal physiological conditions, effective drug-loaded nanoparticle biodistribution and delivery can be difficult to achieve because they encounter many physical and biological barriers, e.g., shear forces, rapid clearance, and protein adsorption, limiting their fraction reaching target sites.^{[287, 288]} These barriers differ not only across specific disease states, but also on a patient-to-patient basis and may appear at the systemic, microenvironmental, and cellular levels. Their deep understanding is essential for designing optimal drug delivery nanosystems. The appropriate route of administration is also significant because it can improve nanoparticle biodistribution, affect their stability and properties as well as alter antiviral drug efficacy in vivo (Figure 11).^{[289, 290]} For example, in the case of PLGA nanoparticles intravenously injected, their accumulation was observed mainly in the liver and spleen. On the other hand, if they are subcutaneously or intranodal injected, they are more likely to accumulate in local lymph nodes.^[291] Such alternative methods of nanoparticle administration allow them to reach the lymphatic system prior to systemic circulation.^[292] Oral administration is the most common and easiest way to deliver drugs. If encapsulated in nanoparticles, they encounter numerous barriers in the gastrointestinal tract.^[293] Crossing the endothelium by nanoparticles relied on passive diffusion is restricted by concentration gradients and P-glycoproteins excreting drugs from the vasculature into the intestinal lumen. In turn, for nanoplatforms based on endocytosis and subsequent exocytosis, the size is a key factor determining the ability to cross the alimentary canal. Polymeric nanoparticles with a high surface area are beneficial for oral administration due to the enhanced interactions with the gastrointestinal tract.^[294] The optimal size for transcytosis of nanoparticles was found to be around 100 nm.^[294–296] Another, in recent years more and more often considered route of nanoparticle administration, is inhalation. Its advantage is the omission of the systemic circulation and hepatic first-pass metabolism before delivery of nanoparticles directly to the lungs and lymph node (Figure 11).^[289] This administration route could especially be very effective for viral infections. However, in addition to improved transport of nanoparticles to lung tissue, they may face obstacles in the form of mucus and pulmonary surfactants acting as physical barriers to lung delivery which significantly change across patients and pathologies.^{[297, 298]} Due to the different deposition rates and heterogeneous distribution of polymeric nanoparticles in the lungs by intratracheal instillation, intratracheal spraying, and intranasal instillation, there is a need to develop validated methods of nanoparticle pulmonary administration.^[299]

To date, many drug nanocarriers have been developed that enable their rapid or controlled release. Currently, synthesis strategies are based on improving the specificity of the nanostructures to target regions of the organism, and to reduce the immunogenicity by their chemical functionalization or coating with polymers, peptides, natural polysaccharides, antibodies, and tunable surfactants.^{[287, 300–302]} If drugs do not show an affinity for a specific target or do not cross certain barriers, vehicles modified with functional groups/ligands can be used to pass through the cell membrane and allow programmed drug delivery in a particular environment.^[303] Hence, it is crucial to determine the optimal ratio of receptors to ligands and to understand the interactions required to overcome the initial energy barrier for nanostructure uptake.^{[287, 304]} Noteworthy are also stimuli-responsive nanocarriers, which show the ability to liberate drugs in a controlled manner using external factors including ultrasound, heat, light, pH, and ionic strength.^[305–309] They can improve the drug targeting and reduce the frequency of their dosing.

The drug delivery systems based on nanocarriers can be very complex with constituents exhibiting specific functions which can influence one another. Thus, their production should be standardized because even the slightest change in physicochemical properties may entail different targeting, leading to toxic effects. For further clinical use, the mechanism of cytotoxicity associated with a specific type of nanocarriers should be thoroughly investigated. All synthesis steps that may play a role in safety issues should be omitted. Green chemistry approaches of obtaining nanoparticles loaded with antiviral drugs are widely encouraged as they minimize utilizing the hazardous constituents and toxic solvents.^{[303, 310]}

All in all, ideal nanocarriers should consist of biocompatible building blocks to ensure low toxicity, possess a high degree of functionality to tune host–guest interactions, exhibit a high loading capacity (e.g., high pore volume), and be synthesized in a controllable manner to precisely modulate their physicochemical properties (e.g., size and morphology). Their combination with an appropriate therapeutic drug targeting a specific disease state allows for the success of nanomedicine in the fight against viruses. Many types of nanomaterials, including liposomes, dendrimers, polymeric nanoparticles, and solid lipid nanoparticles have been applied as carriers for antiviral drugs. However, despite promising preclinical data on such therapeutic platforms, the vast majority of them fail to reach the stage of clinical trials. They are expected to be commercially successful in the near future due to the improvements in efficacy and safety of antiviral drugs.

Kraft et al.^[311] designed long-acting injectable nanosuspension of lipid nanoparticles containing three antivirals: lopinavir and ritonavir, which are hydrophobic and hydrophilic tenofovir to overcome lymph node insufficiency of oral drug combination therapy. The single injection of the formulation ensured persistent levels of antiviral drugs in macaque lymph node mononuclear cells within 1 week, while in peripheral blood mononuclear cells and plasma for 2 weeks. Furthermore, the lymphocyte-targeting properties of nanosuspension were proved by the higher intracellular drug concentrations in lymph node and peripheral blood mononuclear cells compared to those in plasma. PLGA nanoparticles containing a combination of three antiretroviral drugs (cART): efavirenz, lopinavir, and ritonavir was prepared by a high-pressure homogenization method thanks to which over 79% entrapment efficiency of each drug was achieved. The study has shown significant uptake of the cART nanoparticles in comparison to soluble drugs and the following release of cART in the nuclear, cytoskeleton, and membrane fractions of cells without toxicity for 18 days. The higher intracellular cART delivery by nanocarriers can lead to reducing HIV-1 infectivity via inhibiting HIV replication at lower doses.^[312]

Ribavirin was encapsulated in biodegradable nanoparticles, prepared by blending of PLA homopolymer and arabinogalactan (AG)–poly(l-lysine) conjugate using the solvent diffusion method, in order to modulate the pharmacokinetics of the drug. It was found that ribavirin was accumulated in the mice livers after intravenous administration of the drug-loaded nanoparticles and, subsequently, its content slowly diminished over 7 days. The obtained nanoparticles allowed for delivery of the drug to the target organ (liver) and its sustained release.^[248] PGA polymer nanoparticles are also a promising delivery system for ribavirin to be applied in the treatment of various viral infections. The performed calculations demonstrated that ribavirin-loaded nanoparticles cause three times less accumulation of the drug in the red blood cells and 1180 times more accumulation in the liver cells compared to conventional therapy. However, the efficiency of encapsulation (≥10%) should be improved in future studies.^[313] Stable solid monodispersed PLGA nanoparticles with diameters in the range of 50–200 nm containing ribavirin were synthesized in a microfluidic reactor with flow-focusing geometry. It was shown that the amount of the entrapped ribavirin increases upon the rising of free PLGA content in the precursor solution up to 73.6 µg mg⁻¹ PLGA in the case of microfluidic synthesis, whereas in the bulk nanoformulation, it was possible to introduce only 9.6 µg of drug per mg PLGA. The release of ribavirin from PLGA nanoparticles was relatively rapid.^[249]

So far, it has been found that some liposomal formulations can be beneficial for the delivery of chloroquine and ensured better suppression of parasitemia in malaria parasite P. berghei-infected animals than the unformulated antiviral drug.^[314–316] The in vitro and in vivo stability of liposomes are changed by tailoring their size, surface charge, and lipid composition. Since they are very often absorbed rapidly by the reticuloendothelial system, they are modified with the use of different ligands and polymers extending their circulation and improving drug delivery and, consequently, also enabling clinical use. Fotoran et al.^[317] designed highly stable multilayer vesicles for encapsulation of chloroquine inducing a stronger antiparasitic effect than pure chloroquine in a lethal murine model of infection. Moles et al.^[318] used liposomes containing neutral saturated phospholipids for loading chloroquine and antibody targeting to the erythrocyte surface protein glycophorin A. This approach allowed the targeting of the nanocarrier to early intraerythrocytic stages of the malaria parasite and effective robust delivery of chloroquine to uninfected and Plasmodium-infected red blood cells in P. falciparum-infected mice. Baruah et al.^[319] optimized chloroquine-loaded nanostructured lipid nanocarriers employing modified double emulsion method. The nanocarriers obtained are characterized by high stability, minimal toxicity, and prevent the expulsion of the antiviral drug during storage. The in vitro and in vivo studies demonstrated enhanced antimalarial efficiency of the nanoformulations with a better suppression of parasitemia at a lower dose compared to a pure drug. Liu et al.^[320] prepared cholesterol-modified hydroxychloroquine-loaded nanocarriers to treat bleomycin-induced pulmonary fibrosis. This systemic delivery platform containing hydroxychloroquine remarkably inhibited the proliferation of fibroblasts in the rat lungs and decreased inflammation. Novel formulation of liposomal hydroxychloroquine was tested for pulmonary delivery in a preclinical study. Pharmacokinetic analysis after intratracheal administration in Sprague-Dawley rats showed that liposomal formulation enhanced 30 times the drug’s pulmonary exposure, prolonged the half-life in lungs by 2.5-fold over the intravenously and intratracheally administration of free hydroxychloroquine. At the same time, the safety profile of liposomal formulation was improved via reduction of blood and cardiac exposure of the compounds. The main limitation of this study is that the pharmacokinetics were investigated in a rat model using intratracheal instillation to simulate the intended inhalation administration, while the lung deposition efficiency with inhaled aerosols is probably lower. In addition, aerosol-generating procedures such as nebulization should be performed very carefully in patients diagnosed with COVID-19 due to the high risk of contagiousness through the respiratory system.^[321] Sadr et al.^[246] synthesized a novel porous nanocarrier based on the magnetic Fe₃O₄ nanoparticles, utilizing 3-glycidoxypropyltrimethoxysilane as intermediate, and pectin biocompatible polymer for adsorption and release of hydroxychloroquine. It was shown that 32% of hydroxychloroquine was released from the vehicle surface within 90 min reaching 75% after 5 h in a simulated human blood environment (pH 7.4) at 37 °C. The use of the Fe₃O₄@pectin composite as a nanocarrier allowed the drug release in a controlled manner. In 2021, Olejnik and Goscianska^[322] analyzed the relationship between physicochemical properties of functionalized mesoporous silica nanocarriers with different structures and hydroxychloroquine molecules. It was found that the adsorption of the antiviral drug on the surface of nonmodified silica materials was based on the formation of hydrogen bonds, whereas, in the case of 3-aminopropyltriethoxysilane and copper ions modified samples, the complexes with drug molecules were generated. The release behavior of the hydroxychloroquine was highly affected by the pH of the receptor fluid and the presence of functional groups on the surface of silica materials. The profile of drug liberation was the most consistent with Korsmeyer–Peppas kinetic model indicating the Fickian diffusion mechanism. The modification of ordered mesoporous silica nanocarriers with copper ions can enhance their antiviral activity.

Ritonavir was incorporated into solid lipid nanoparticles prepared with the use of solvent emulsification evaporation and double emulsion methods. The encapsulation of the antiviral drug in nanoparticles leads to its sustained release, reaching 40% in 9 days without the initial burst stage. Most likely, the extended-release is associated with drug lipophilicity and homogenous dispersion at lipid core. Moreover, the in vitro experiment indicated that ritonavir-loaded lipid nanoparticles can inhibit HIV-1 virus production like a free drug.^[323] In another study, PLGA nanoparticles were utilized as a delivery system for a combination of drugs: lopinavir and ritonavir. The drugs were either administered alone or in combination with nanoparticles to Swiss strain albino mice infected with RH virulent toxoplasma strain. Parasitological improvement in both mortality rate and parasite count was observed in the case of two forms of drugs. The higher efficiency was reached with the use of lopinavir and ritonavir encapsulated into PLGA nanoparticles which was accompanied by a minimization of the dose. Additionally, the viability and infectivity of parasites were also notably decreased.^[247] Patil and Dhawale^[324] obtained ritonavir nanoparticle formulation by nanoprecipitation technique with Eudragit RL100 polymer to improve drug dissolution and bioavailability. The complete drug release from nanosuspension within 10 min was observed and the rate of the process significantly increased in comparison to the pure drug.

3.2 Nanoparticles for Virus Inhibition

So far, metal nanoparticles have been used as therapeutic or contrast agents, but none have been shown to be beneficial for the delivery and release of antiviral drug molecules due to stable drug–metal interactions. They can directly affect the receptor binding and cell entry of viruses along with inhibition of viral proliferation.

Silver nanoparticles can inactivate different viruses, including HIV-1,^[325] monkeypox virus,^[326] murine norovirus MNV1,^{[327, 328]} HSV,^[329] hepatitis B,^[330] Tacaribe virus,^[331] Rift Valley fever virus,^[332] influenza viruses like H3N2,^[333] and H1N1.^[334] Lara et al.^[335] demonstrated that Ag nanoparticles coated with polyvinylpyrrolidone exhibit anti-HIV activity at an early stadium of viral replication, playing the role of a virucidal agent or viral entry inhibitor. Moreover, it was found that Ag nanoparticles produced from different fungi were capable of controlling viral infectivity by blocking the interaction of the virus with the cell. It was dependent on the Ag nanoparticle size and zeta potential. Ag nanoparticles synthesized via F. oxysporum and Curvularia species, having sizes in the range of 4–13 and 5–23 nm, respectively, exhibited better antiviral activity (80–90% inhibition) against HSV-1 and HPIV-3 and were less cytotoxic than Ag nanoparticles produced by Alternaria and Phoma species, with a size ranging from 7 to 20 nm.^[336] Green synthesized Ag nanoparticles from Lampranthus coccineus and Malephora lutea were compared in terms of potential antiviral effect. The hexane extract of L. coccineus was able to inhibit three viruses including HAV-10, HSV-1, and CoxB4, whereas M. lutea extract showed antiviral activity against only two viruses HAV-10 and CoxB4.^[337] Ag nanoparticles demonstrated antiviral and preventive effects in the infection of the H3N2 influenza virus. Kidney cells of Madin-Darby canine infected with Ag nanoparticles-treated H3N2 influenza virus displayed better viability in comparison to an influenza virus control group. Ag nanoparticles interacted with H3N2 influenza virus which resulted in destruction of morphologic viral structures in a time-dependent manner (30 min to 2 h).^[333] Spherical quasi-silver nanoparticles (size 5–15 nm) obtained with the use of an aqueous extract from Panax ginseng roots appeared to be virucidal against the influenza A virus (strain A/PR/8).^[338] Ag nanoparticles modified with zanamivir showed abilities to inhibit the neuraminidase activity of the H1N1 influenza virus.^[339] Zachar^[340] evaluated the possibility to obtain real effective minimal inhibitory concentration (MIC) of Ag nanoparticles in various respiratory system target locations during viral and bacterial infections by inhalation of standard 5 µm diameter droplets aerosol. The studies were focused on control local outbreaks of COVID-19 at-home treatment and the risk of ventilator-associated pneumonia in hospital ICU. It was found that effective MIC is attainable, both in the bronchial tree and in the alveoli. Due to the fact that respiratory infections begin in the upper airways, it is the best to use the proposed method as the first-line treatment in order to inhibit the progressive infection.

Au nanoparticles are also widely studied in the context of nanomedicine applications due to their excellent electrical, optical, and biological features. They often act as antibacterial and antiviral agents.^{[341, 342]} It was reported that Au nanoparticles conjugated with peptide triazoles (Au NP-PT) indicated a remarkably stronger antiviral activity against HIV-1 in comparison to nonmodified peptide triazoles. Changes in the physicochemical properties of nanoparticles, in particular, an increase in their diameter or the density of PT conjugated to the surface of Au nanoparticles, enhanced the inhibition of infection. The high virolytic activity and adequate irreversible HIV-1 inactivation by nanoconjugates are the result of multivalent contact between the Au NP-PT and metastable envelope spikes on the HIV-1 virus.^[343] Porous gold nanoparticles exhibited stronger antiviral activity against influenza A virus than nonporous sGNP and Ag nanoparticles due to their large surface area corresponding to the unique nanobundled structure. The viral inhibition occurs by blocking of viral attachment associated with membrane fusion, resulting from disulfide bonds cleavage in hemagglutinin.^[344] Au nanoparticles with diameters of 2 and 14 nm functionalized with sialic-acid-terminated glycerol dendron, characterized by high stability and nontoxicity to the cell, demonstrated different antiviral activity toward the influenza virus. The studies have shown that 14 nm sialylated Au nanoparticles were very effective in the inhibition of the influenza virus while analogs with the size of 2 nm did not exhibit a significant effect.^[345] Glutathione-stabilized fluorescent Au nanoclusters were found to selectively inhibit proliferation and protein expression of porcine reproductive and respiratory syndrome virus (PRRSV) as opposed to pseudorabies virus. They directly inactivated PRRSV and blocked its adsorption without impact on viral genome replication.^[346] Zacheo et al.^[347] studied Au nanoparticles coated with ligands ω-terminated with sugars containing multiple sulfonate groups in terms of antiviral activity against the Dengue virus. It was shown that glucose- and lactose-based ligand exhibit a low half-maximal effective concentration (EC₅₀) for Dengue virus inhibition, moderate toxicity, and a virucidal effect in hepatocytes.

Apart from metal nanoparticles, graphene oxide (GO) is also perceived as a potential antimicrobial material due to its unique properties such as large surface area, biocompatibility, high carrier mobility, thermal, and mechanical stability.^{[342, 348, 349]} So far, the antibacterial activity of GO has been widely explored,^[350–352] but only a few papers have presented its potential in inhibiting the entry and replication of enveloped DNA virus (herpesvirus) or RNA virus (coronavirus) in their target cells.^{[353, 354]} GO and partially reduced sulfonated GO were effective against herpes simplex virus type 1 (HSV-1) infections. It was assumed that viral attachment blocking was the primary inhibition mechanism. Moreover, the presence of nanomaterials did not influence the cell-to-cell spread.^[353]

Carbon quantum dots (CQDs) characterized by average diameters below 10 nm, excellent water dispersion, and nontoxicity in animals are also very promising for nanomedical application.^{[86, 355]} Their interesting optical properties enable them to be traced in vivo. It was found that CQDs can be proper scaffolds to interfere with virus entry into cells. CQDs modified using boronic acid inhibited HIV-1 entry via suppressing syncytium formation,^[356] whereas those functionalized simultaneously with boronic acid and amine groups showed antiviral activity against herpes simplex virus type 1.^[357] Ting et al.^[358] reported that uniform and stable CQDs with a size of around 1.5 nm prepared from curcumin can inhibit viral entry and the synthesis of negative-strand RNA of porcine epidemic diarrhea virus. Moreover, they were able to suppress the accumulation of reactive oxygen species by coronavirus reducing cell apoptosis. The CQDs exhibited over 50% of inhibition efficiency on virus entry at an early stage. It has been shown that the cationic CQDs can cause aggregation of the negatively charged virus through electrostatic interaction, resulting in a reduction in its infectivity.

Loczechin et al.^[359] studied the antiviral activity of different functional CQDs against HCoV-229E coronavirus. The first group of antiviral CQDs was obtained by hydrothermal carbonization of ethylenediamine/citric acid acting as carbon precursors and subsequent modification with boronic acid ligands. They exhibited virus inactivation with an EC₅₀ of 52 µg mL⁻¹. In turn, CQDs derived from 4-aminophenylboronic acid constituted the second group of anti-HCoV nanomaterials with an EC₅₀ reduced to 5.2 µg mL⁻¹. It was demonstrated that CQDs inhibit virus entry at the early stage of infection which may be the result of interaction between CQDs functional groups with HCoV-229E entry receptors (Figure 12).

Nowadays, nanotechnology offers great prophylactic and therapeutic possibilities aimed at combating various viral infections, including those caused by SARS-CoV-2. Many different functional nanoparticles provide excellent platforms for the development of completely new, safe, and effective antiviral drugs. They can also act as delivery systems for therapeutics that have already been approved. However, there are still many challenges and barriers to overcome in the near future to successfully exploit the full power of medicinal nanotechnology. It should be emphasized that the amount of nanomedicines available to patients is below all expectations. This is partly due to the translation gap between animal and human studies resulting from a lack of understanding of the differences in their physiology and pathology that affect the behavior and functionality of nanomedicines in the body. The divergence across the individual species and patients is a limiting factor in clinical translation. Amongst drug-loaded nanoparticles that have already been approved, only a few are recommended as first-line treatment options. Moreover, many of them bring about improvement in a small group of patients due to underexplored heterogeneity in the biological underpinnings of diseases which can substantially alter the therapeutic effectiveness of nanoparticles. Currently, for the controlled synthesis of nanosystems, new, advanced techniques are used allowing for the incorporation of complex architectures, bioresponsive moieties, and targeting agents to improve their delivery. Some temperature and pH-sensitive nanosystems are designed to react to the local environment and provide targeted drug delivery. The complex platforms can maximize the therapeutic efficacy, target particular phases of the cell cycle or overcome mechanisms of drug resistance.

5.1 Electrochemical Nanobiosensors

Electrochemical nanobiosensors have many advantages including very high sensitivity, good stability, reproducibility, lower limits of detection, and a wider range of linear response in comparison to other sensors.^[439] The improved physicochemical features resulting from discrete nanoelectrodes or functionalization of the electrode surface with different nanostructures make these devices very attractive for pharmaceutical and medical applications. In electrochemical nanobiosensors, the detection of the signal takes place at the interface of the electrode and pathogen-containing electrolyte solution. Thus, the interaction requires a redox process followed by electron transfer in voltammetric devices (dynamic methods) while the charged species concentration, as a function of the electrochemical potential, is determined by potentiometric nanosensors (static methods).^{[433, 440, 441]} In short, the mechanism of action of electrochemical nanobiosensors relies on the change in the electrical conductance, resistance, or capacitance of the sensing surface via the nanomaterials, which occurs when the target element for biological recognition is binding to the electrode. It should be emphasized that appropriate bioreceptors must be integrated with the transducer to identify the presence of any virus in the analyzed samples.^{[434, 442]} The undoubted advantage of nanobiosensors with electrochemical transduction is the possibility of their easy miniaturization into inexpensive and integrated platforms, which resemble handheld electrochemical readers useful in point-of-care diagnostics. Besides, they can be multiplexed by introducing multiple individually accessible electrodes on the same platform, enabling simultaneous detection of target elements.^[443]

Mahari et al.^[444] fabricated a cost-effective and highly specific biosensor device (eCovSens) by immobilization of nCovid-19 monoclonal antibody on the screen-printed carbon electrode for the detection of nCOVID-19 spike antigen in saliva samples. It was compared with a potentiostat-based immunosensor composed of a fluorine-doped tin oxide (FTO) electrode integrated with Au nanoparticles and nCOVID-19 monoclonal antibody. Au nanoparticles were used as a catalyst and electrochemical signal amplifiers by enhancement of electrical conductivity. Moreover, they provide a platform that enables the attachment of nCOVID-19 monoclonal antibodies as a result of electrostatic interactions or physical adsorption. Both sensors demonstrated high sensitivity for the detection of nCOVID-19 spike antigen ranging from 1 × 10⁻¹⁵ m to 1 × 10⁻⁶ m in the standard buffer. The limit of detection (LOD) in saliva samples was shown to be 90 × 10⁻¹⁵ and 120 × 10⁻¹⁵ m for eCovSens and FTO/Au NPs sensors, respectively. eCovSens ensures fast results within 10–30 s and requires only 20 µL of sample volume, therefore it can be applied as a diagnostic tool for detection of nCovid-19 antigen traces. A competitive electrochemical immunosensor based on Au nanoparticle modified carbon electrodes array was also designed for detection of MERS-CoV and other coronaviruses within 20 min in spiked nasal samples. It was demonstrated that it exhibited a low detection limit of 1.0 and 0.4 pg mL⁻¹ for MERS-CoV and HCoV, respectively, as well as good stability. The proposed immunosensor was found to be highly selective against nonspecific proteins such as influenza A and B. Its sensitivity can be connected with using carbon array electrodes modified with Au nanoparticles causing enhanced efficiency of electron transfer.^[445]

A low-cost and highly sensitive cobalt-functionalized TiO₂ nanotube-based electrochemical sensor was developed for SARS-CoV-2 identification. It specifically detected the spike receptor-binding domain of SARS-CoV-2 even at a very low concentrations (14 × 10⁻⁹–1400 × 10⁻⁹ m) within 30 s with a linear relationship between sensor response and protein concentration with the LOD as low as ≈0.7 × 10⁻⁹ m.^[446]

Pathogenic virus detection in medical samples can be performed employing field-effect transistor (FET)-based biosensing devices that enable highly sensitive and immediate measurements even at small concentrations of bioanalytes. This type of biosensor has great potential to be applied in clinical immunological diagnosis and point-of-care testing. The most frequently used nanomaterial for FET sensing platforms is graphene, characterized by high electronic conductivity, carrier mobility, and the large specific surface area, which provides an optimal environment for ultrasensitive detection.^{[447, 448]} Ono et al.^[449] demonstrated the graphene FET functionalized with sialoglycan for the selective detection of the influenza virus. It exhibited very high sensitivity and specificity toward two lectins derived from Sambucus sieboldiana and Maackia amurensis used as alternatives to the human and avian virus. Another example presented microfluidic integrated reduced GO (rGO) transistor chip for detection of H5N1 influenza virus gene in the flowing environment. It was shown that immobilization of extended long capture DNA probe on the surface of rGO ensured both high stability and sensitivity. In turn, a short capture DNA probe was characterized by low stability. The developed transistor sensor based on rGO reached a LOD of 5 × 10⁻¹² m with a detection time of about 1 h and can be a promising candidate for the identification of nucleic acid in clinical diagnostics and environmental monitoring.^[450]

Seo et al.^[451] fabricated FET biosensor by coating graphene sheets with a specific antibody against SARS-CoV-2 spike protein for detecting coronavirus in clinical samples. The SARS-CoV-2 antibody was immobilized onto the sensing surface with the use of 1-pyrenebutyric acid N-hydroxysuccinimide ester as a probe linker (Figure 18). The performance and efficiency of the FET device were assessed employing antigen protein, cultured virus, and nasopharyngeal swab specimens taken from patients infected with COVID-19. It was found that the developed biosensor can detect SARS-CoV-2 spike protein at concentrations of 1 and 100 fg mL⁻¹ in phosphate-buffered saline and transport medium, respectively. Its great advantage is a high-speed detection of antigens in clinical samples (>1 min) without any preparation or preprocessing while molecular diagnosis with the use of real-time RT-PCR takes at least 3 h and for loop-mediated isothermal amplification method <1.5 h. The promising results were also obtained in studies conducted in culture medium (LOD of 1.6 × 10¹ pfu mL⁻¹) and clinical samples (LOD of 2.42 × 10² copies mL⁻¹). For reference, the detection limit of PCR-based assay in sputum and for viral RNA equals 8.31 × 10¹ and 1.50 × 10¹ copies mL⁻¹, respectively.

Interestingly, the biosensor did not show cross-reactivity with the MERS-CoV antigen. Thus, the proposed highly sensitive technology can be successfully used for the diagnosis of COVID-19 and other viral diseases. At the same time, Grapheal company as a spin-off from the Institut Néel of the CNRS in Grenoble introduced an all-electronic test based on a graphene biosensor sensitized by antibodies that directly detects SARS-CoV-2 particles in saliva samples.^[452]

Another very promising and rapid gold nanoparticles decorated graphene FET sensor functionalized with phosphorodiamidate morpholino oligos was developed by Li et al.^[453] It was used for the detection of SARS-CoV-2 RNA in human throat swab specimens without preamplification. The sensor featured a low limit of detection in nuclease-free phosphate buffer saline (0.37 × 10⁻¹⁵ m), throat swab (2.29 × 10⁻¹⁵ m), and serum (3.99 × 10⁻¹⁵ m), but also a short response time (2 min) to COVID-19 patients’ samples. The fabricated nanosensor was capable to analyze RNA extracts from 30 real clinical samples and differentiate healthy people from infected ones. The test results obtained were consistent with the RT-PCR results (Kappa index = 0.92). The proposed approach provides more rapid analysis and lower LOD compared to colorimetric, fluorescence, and optomagnetic biosensing platforms.

Summary of various electrochemical nanobiosensors applied for the detection of SARS-CoV-2 are collected in Table 4.

5.2 Optical and Magneto-Optical Nanobiosensors

Optical biosensors are devices in which the analyte-bioreceptor interaction causes changes in optical features such as luminescence, fluorescence, absorbance, or reflectance corresponding to the analyte concentration. They provide an alternative method of virus detection thanks to simple use, safe, and cost-effective technology.^{[443, 457, 458]} So far, optical biosensors have been used to detect HIV,^[459] Ebola,^[460] norovirus,^[461] SARS-CoV,^[462] MERS-CoV,^[463] and influenza virus.^[464]

Surface plasmon resonance (SPR) sensing is a promising label-free method with huge potential for viral detection. The viral particles captured by the biorecognition element immobilized on the SPR sensor chip surface induce the change in the plasmon resonance wavelength or intensity measured by an optical system.^[465] The general advantages of this method are high sensitivity, simple sample preparation, reusability, broad linear range, and rapid response time. Huang et al.^[466] reported a one-step method for rapid and direct optical studies of SARS-CoV-2 particles with the use of a spike protein-specific nanoplasmonic resonance sensor obtained by a replica molding process. A tapered nanopillar array on a silicon wafer was employed as the original mold on which 10 nm of titanium and 70 nm of gold were deposited. It was found that the developed sensor allows detection of SARS-CoV-2 from 370 vp mL⁻¹ within 15 min, while in the case of other viruses such as SARS, MERS, and VSV, the response is negligible. The concentration of SARS-CoV-2 can be quantified linearly in the range of 0 to 107 vp mL⁻¹. A similar sensing capability was observed for cheap handheld optical equipment connected with the smartphone App. In addition, a dual-functional plasmonic biosensor that combines the plasmonic photothermal effect and localized SPR sensing transduction was developed for the diagnosis of COVID-19. 2D gold nanoislands self-assembled on the glass surface and modified with complementary DNA receptors provide highly sensitive detection of the selected genome sequences of SARS-CoV-2 in a multigene mixture via hybridization of nucleic acid. The LOD has been decreased to the concentration of 0.22 × 10⁻¹² m.^[467]

Colorimetric biosensors are widely used to detect the target element through a color change easily detectable with a naked eye or an optical detector. Therefore, they are excellent candidates for point-of-care identification of viral diseases.^{[458, 468]} In order to diagnose positive COVID-19 cases from isolated RNA samples within 10 min, a colorimetric assay relied on Au nanoparticles capped with thiol-modified antisense oligonucleotides specific for N-gene of SARS-CoV-2 were applied (Figure 19). The functionalized Au nanoparticles selectively agglomerating in the presence of the target RNA sequence of the virus cause visual change in surface plasmon resonance amplified after RNaseH addition. The selectivity of the COVID-19 biosensor has been assessed against MERS-CoV viral RNA and no meaningful change in absorbance was noticed. Hence, the presented method allows for reproducible, reliable, and selective naked-eye detection of SARS-CoV-2 without the requirement of any advanced instrumental techniques.^[469] However, this assay had some limitations, such as a relatively high detection limit that resulted in the misidentification of samples as a false negative. Therefore, the protocol has been improved by introducing an isothermal nucleic acid amplification step so that it can detect samples characterized by much lower viral copy numbers. Additionally, specific primer sets for the targeted region of the SARS-CoV-2 N-gene were also designed. The versatility of the presented approach was evaluated in nasal and oropharyngeal swabs. It was demonstrated that the accuracy, sensitivity, and specificity of the assay were >98.4%, >96.6%, and 100%, respectively, with LOD of 10 viral copies per µL. Importantly, the results of the test in clinical samples were observed by the naked eye within 40 min and no color change occurred for COVID-19 negative samples.^[470]

Today, rapid and automated nucleic acid extraction methods are highly desirable for diagnosing COVID-19 with the use of RT-PCR. Therefore, Zhao et al.^[471] proposed the synthesis of poly(amino ester) with carboxyl groups (PC)-coated magnetic nanoparticles (pcMNPs) to improve the viral RNA extraction method leading to shortening the turnaround time and lowering operational requirements. This simplified approach connected lysis and binding steps. The created pcMNPs-RNA complexes were directly added into the following RT-PCR reactions without elution stage and were found to be compatible with different isothermal amplification methods (e.g., LAMP – loop-mediated isothermal amplification, RPA – recombinase polymerase amplification). Purification of viral RNA from multiple samples was achieved within 20 min. Moreover, a linear correlation between 10 and 10⁵ copies of SARS-CoV-2 particles was observed.

A comparison of different optical and magneto-optical nanosensors used for the detection of SARS-CoV-2 is presented in Table 5.

5.3 Piezoelectric Immunosensors

Piezoelectric immunosensors are highly sensitive analytical devices exhibiting superior performance compared to other biosensors. The detection mechanism exploits variations in the resonance frequency of a piezoelectric material (PZ) correlating to mass changes. If mass increases as a result of interactions between biomolecules (e.g., antibody–antigen), the frequency monitored through the alternating current voltage decreases.^{[457, 476, 477]} Mass response-type piezoelectric immunosensors have gained considerable attention for the virus detection due to their ability to identify antigens in the picogram range without labeling. A schematic illustration of the piezoelectric immunosensor operation is depicted in Figure 20. Probe antibodies are placed on the upper electrode surface of the piezoelectric material, whose resonation is driven by upper and lower electrodes. Then target antigen is bound by probe antibodies. The mass change on the surface of the electrode causes a time-dependent frequency shift that is represented by the frequency change Δf of the material in the oscillation circuit.^[476]

Piezoelectric immunosensors can be successfully combined with piezoelectric energy-harvesting devices for the IoT, giving the opportunity to recognize viruses by controlling mechanical vibration. Importantly, it is easy to attach or embed them into smart clothing.^[476] Contemporary piezoelectric biosensors are designed by employing new nanostructured materials and advanced fabrication methods to avoid the toxicity of conventional lead-based piezoelectric materials.^[478]

Wang et al.^[479] produced a nanowell-based quartz crystal microbalance (QCM) aptasensor for rapid and selective identification of the H5N1 avian influenza virus. The nanoporous gold film having a pore size of ≈20 nm was immobilized onto a gold electrode surface utilizing a self-assembled monolayer. It was shown that this aptasensor significantly reduced the detection time up to 10 min without the need to apply labels. No interference from other avian influenza virus subtypes of H1N1, H2N2, H7N2, and H5N3 was noted.

The first piezoelectric immunosensor for SARS-CoV detection in sputum was developed by Zuo et al.^[480] They used horse polyclonal antibody elicited by SARS-CoV for coating PZ crystals. The antigen sample was atomized into an aerosol so it could be specifically adsorbed by the PZ surface covered by the antibody. The change in crystal mass led to a frequency shift linearly dependent on antigen concentration ranging from 0.6 to 4 µg mL⁻¹. It should be noted that the sensor was characterized by good reproducibility (use up to 100 times without activity change), stability, and a short time of analysis (less than 2 min). Albano et al.^[481] fabricated an aptamer-PQC nanobiosensor for the selective detection of helicase protein obtained from SARS-CoV replication. The coupling of paramagnetic nanoparticle technology with nanobiosensor allowed for the identification of target proteins in assay within 1 min with LOD of 3.5 ng mL⁻¹.

The interactions between the SARS-CoV-2 spike protein and the sensing surface are crucial for the detection process. Due to the fact that binding sites of the spike S1 protein are composed of hydrophobic and positively charged amino acid residues, in particular, the negatively charged surfaces should adsorb/attach them through hydrophobic and electrostatic interactions. It was found that especially mixed self-assembled monolayers forming nanostructures modified with CH₃ and COOH groups are the most suitable for this purpose. These engineered surfaces implemented on the QCM-based sensor can be evaluated for the diagnosis of COVID-19 reaching sensitivity up to ng level.^[482]

In summary, the design of modern nanobiosensors to satisfy the demands of rapid, convenient, and large-scale diagnostics of viral diseases has captured the imagination of researchers with considerable progress being made in this field. Current conventional diagnostics methods are increasingly supplanted by sensing devices constructed with the use of advanced nanotechnological solutions at a laboratory scale, and they start to penetrate successfully medical praxis. Despite the significant progress, the application of nanobiosensors in virus detection still faces many challenges that need to be considered. First, much effort must be put into decreasing detection limits. Thus, in order to overcome this limitation, different nanomaterials (e.g., Au nanoparticles, Ag nanoparticles, QDs) can be applied as labels to enhance the signal to be easily detectable. Nanobiosensor design should leverage unique optical, electrical, and magnetic features of nanoparticles, which will enable specific and selective detection of target viruses.^[438] Such approaches characterized by simplicity, cost-effectiveness, fast response, and real-time diagnostic procedures can replace PCR-based assays—the gold standard in the diagnosis of viral infections. It should be mentioned that nanobionsensors exhibit high stability and sensitivity at the lab scale but this can change when analyzing real samples. Many parameters will affect the obtained results, ranging from the characteristics of the target elements, the physicochemical properties of nanomaterials, to interfering molecules present in tested samples. Additionally, the nanobiosensors should be designed for the rapid identification of viruses in clinical samples, such as blood, saliva, urine, and nasopharyngeal swabs. In this context, the greatest limitation may be the costly and time-consuming procedure of extraction/preparation of the target biological elements. Hence, the fabrication of nano-based biosensors that do not require additional extraction procedures is highly desirable. Another solution can be the integration of the extraction system into the biosensor to be wearable and at the same time user-friendly. The designed platforms could be translated into microneedles-based biosensors that permit for continuous monitoring of SARS-CoV-2 antigens, antibodies, or nucleic acid in the dermal interstitial fluid of population infected by coronavirus symptomatically and asymptomatically. Wearing such devices would provide key information about the spread of viral infection in a community, the development or loss of immunity, and the immune response to vaccination. It should be noted that antibody levels fluctuate over time, which may be due to natural infection or vaccination. Continuous measurements would help to track these changes, indicating a need for a booster vaccination. Additionally, it would be possible to predict the likelihood of subsequent waves of the pandemic.

It is expected that the collaboration between researchers, engineers, and industrial partners will allow the technological breakthrough necessary to bring nanobiosensors to the medical community. These sensors will overcome false negative/positive results, validation processes, cost-effectiveness, detection speed, and selectivity. Future efforts should focus on developing innovative, fast, affordable, and miniaturized biosensing devices for diagnostic applications to control the spread of viral infectious diseases.