The field of nanotechnology emerged in the mid-20th century to produce new materials, structures and devices on the supramolecular scale, where a nanoparticle (NP) is defined as having a dimension of <100 nm and may encompass a wide variety of shapes (from 0D to 3D) (1). Nanotechnology is likewise proving to be a promising field in the 21st century, with numerous potential novel applications in medical research, including the comprehensive surveillance, control, creation, repair and defense of human biological systems (2). For instance, due to their unique chemical and physical properties, nanomaterials have emerged as novel antimicrobials and have been proposed as a promising antibiotic alternative in the context of the increasing number of multi-resistant bacterial strains that have arisen due to the widespread use of antibiotics (3). Similarly, these materials have become new therapeutic alternatives for viral infections, largely due to the low efficacy of vaccines against this type of pathogen and due to the side effects that certain pharmaceutical products can have, particularly in children (4). Furthermore, nanomaterials have the unique ability to prevent the encapsulated anti-viral drug or agent from deteriorating (5). In the present review article, the available NP-based antiviral strategies were described, specifically for the treatment of the influenza A virus (IAV) (Fig. 1).

IAV is an orthomyxovirus that can adopt either a filamentous or spherical shape; its filamentous form has a dimension of 100 nm in length and 30 nm in diameter (as seen in clinical isolates), while its spherical form has a diameter of ~100 nm (based on laboratory cultures) (6). IAV has a negative-strand RNA genome (3'-5'), which is made up of eight genes that are translated into the following 11 proteins: Hemagglutinin (HA), neuraminidase (NA), matrix 1 (M1), M2, nucleoprotein (NP), nonstructural protein 1 (NS1), NS2 (also known as nuclear protein), RNA-dependent RNA polymerase 1 (PB1) and 2 (PB2) (7) (Fig. 2).

To date, four types of IAV have been described: A, B, C and D. Of these, influenza A and B viruses cause seasonal influenza (8). Type A viruses have numerous combinations of various HA and NA proteins (9). The high mutation rates of this virus along with its ability to rearrange its genome allow for the appearance of new strains that possess the ability to evade the host's immune system (10).

Acute viral respiratory infection caused by IVA affects individuals of all ages and is associated with high mortality (11). Vaccination is the most effective method of preventing influenza infection and its complications. However, the effectiveness of the influenza vaccine varies each year based on the specific influenza strains in circulation and also due to varying rates of individuals who choose to accept the vaccine in a given season.

Currently, four antiviral drugs targeting the NA surface glycoprotein of the influenza virus are available for the treatment and prevention of the disease: Oseltamivir phosphate (trade name, Tamiflu^®), zanamivir (trade name, Relenza^®), peramivir (trade name, Rapivab^®) and baloxavir marboxil (trade name, Xofluza^®) (12). These existing options are up to 60% effective at preventing influenza illness caused by Influenza types A and B.

Regarding vaccines, the following three choices are currently available: Inactivated influenza vaccine, live attenuated influenza vaccine and recombinant influenza vaccine (13).

The existing drugs for IAV often face limitations associated with the emergence of resistant strains, as a result of the constant evolution of the viral genome (14). Therefore, there is a pressing need to develop safe and effective antiviral agents that exert a novel mechanism of action compared to conventional drugs. Due to advances in the field of nanotechnology and its growing applications in drug development, it is now possible to exploit the various biological and antiviral properties of NPs via surface modification (15).

Specifically for the control of viral infections, the usefulness of NPs includes the following mechanisms: As immunity-inducing vaccines, through use in gene silencing, as a drug delivery system and simple NPs for their anti-IAV properties (16). In the following sections, it is first reviewed what is currently known regarding the main types of antiviral NPs (categorized by material) and their applications for the treatment and prevention of IAV, and several conserved IAV protein domains and their roles in pathogenicity are then discussed.

AuNPs can form stable chemical bonds with S-and N-containing groups, allowing them to attach to a wide variety of organic ligands or polymers with a specific function (17). AuNPs are biocompatible and can be functionalized with antigens as carriers for the delivery of vaccines (18). However, AuNPs cannot inhibit viruses alone, as they only possess antiviral outcomes when covalently bound to other molecules that bind to the target virus. In this manner, AuNPs can exponentially increase their antiviral effects through multivalent interactions (19). For instance, it has previously been reported that sialic acid-functionalized AuNPs can inhibit influenza virus infection by multivalent interactions (20).

AuNPs conjugated with HA-IAV can bind to a virion, blocking its connection with cellular or viral receptors, thereby inhibiting the initiation of the viral cycle (infection); these NPs can also penetrate the cell membrane and inhibit viral genome replication (21). For viral replication and assembly, viruses remodel the membranes of intracellular organelles to create viral replication complexes, and treatment with AuNPs can trigger disruption of subcellular structures by altering the cytoskeleton (22). In addition, AuNPs with an appropriate size of 100 nm can enter lymph nodes and induce antigen-specific T cell-mediated immunity (23).

In recent years, the use of porous gold NPs (PoGNPs) has been explored to combat IAV, since PoGNPs have greater thermal stability compared to AuNPs. This type of NP has the ability to interact with IAV surface proteins by cleaving disulfide bonds, thereby inhibiting the fusion of the viral membrane to its target cell by blocking the viral entry process through conformational deformation of HA (inactivated viruses exhibit lower cellular infectivity) (24).

The absorption, cytotoxicity and in vivo biodistribution of AuNPs depend on their size as well as the type of target cell (healthy vs. tumor). For instance, an AuNP that is 50 nm in size results in increased AuNP uptake in HepG2 cancer cells, but decreased AuNP uptake in healthy cells (L02, a normal human liver cell line), whereas in cancer cells, an AuNP that is 5 nm in size promotes apoptosis and the production of reactive oxygen species (ROS) (25). Therefore, when designing an AuNP application, it is critical to identify the ideal particle size in the model being used (26).

In mouse models, it has been reported that intranasal administration of the consensus peptide extracellular domain of M2 (M2e; acetylated-SLLTEVETPIRNEWGSRSNDSSDC-amidated; molecular weight, 2,736 Da) of IAV conjugated with AuNPs and the CpG oligonucleotide as soluble adjuvant (immunostimulator) (AuNP-M2e+CpG) induces the activation of lung B cells and elevated anti-M2e immunoglobulin G levels in serum (27). Double-stranded RNA analogues have also been proposed as adjuvants using 40-50 bp of polyinosinic-polycytidylic acid (synthetic origin of ~400 bp) electrostatically conjugated with AuNPs and the HA of the influenza virus for the generation of vaccines. This resulted in the stimulation of interleukin 1β (IL-1β), interferon-β (IFN-β) and tumor necrosis factor (TNF-α) production, which activate the innate immune response, induce apoptosis and stimulate macrophages and natural killer cells (28-31).

CuNPs show much promise due to their low toxicity and good long-term colloidal stability (32). However, CuNPs can become more toxic when they are larger in size (25 vs. 100 nm), and their toxicity is enhanced by a positive charge, which facilitates interactions between cells and NPs. Furthermore, the dissolution of CuNPs depends on the temperature and pH of the solution, which also has a major influence on their toxicity (the lowest toxicity is observed when the pH is between 9 and 11) (33,34). Additional drawbacks are that CuNPs are easily oxidized when exposed to air and that they are unstable in solution (35).

Cu has been proposed to exhibit an antiviral mechanism via inhibition of the attachment and entry of viruses into the host cell through their interaction with cell membrane glycoproteins (36). However, Cu has likewise been postulated to inactivate and degrade viral particles primarily through the generation of ROS, leading to the degradation of the viral genome and its proteins (37). An inactivation assay for H1N1 influenza virus (subtype of IAV) revealed that nucleoprotein (protein visualized through western blot) and HA (RNA quantified via reverse transcription-PCR) are imperceptible after 30 min of treatment with CuNPs (38,39). Furthermore, hybrid silver (Ag)-Cu NPs are capable of binding to glycoprotein gp120 at the HIV cell envelope and inhibiting infection in T cells in vitro (40).

The benefits of AgNPs are that they have high yields, good solubility and high stability (41). AgNPs are likewise able to inhibit the production of viral RNA and extracellular virions likely through a specific interaction between the NPs and the viral genome or via direct binding with viral particles (42). However, AgNPs with particle diameters >800 nm are too toxic to be used in cell culture assays (43).

AgNPs adhere to the viral nucleocapsid where they interfere with viral attachment to the cell membrane by binding to sulfur-containing residues on host cell surface glycoproteins (44,45). This blocks virus entry and also effectively inhibits viral nucleocapsids, which are necessary for the proper assembly of viral progeny (46). In addition, within the host cell, AgNPs can interact with viral genomic material and inhibit genome replication of the virus and can also interrupt certain cellular factors, such as kinases required for phosphorylation of several IAV proteins (47-49). At the same time, the NS1-IAV viral protein has been associated with p53 inhibition and the prevention of host cell apoptosis. However, the virus was found to block p53 activity only in the early stages of epithelial cell infection, while apoptosis increased again in the later stages (8-24 h post-infection) (50). In a study using MDCK cells infected with the H3N2 influenza virus, treatment with AgNPs was found to significantly protect cells against viral infection by increasing cell viability, reducing cytotoxicity, inhibiting viral growth and decreasing cell apoptosis. In addition, within the host cell, AgNPs can interact with viral genomic material and inhibit its genome replication (51). In a different study conducted by Li et al (52), using the same cell type (MDCK), AgNPs combined with oseltamivir (an antiviral drug that blocks the release of new virions from the cell membrane to provide resistance to influenza A) were found to have the ability to inhibit H1N1 infection. This process can be mediated via signaling pathways involving the accumulation of ROS, along with AKT activation and p53 phosphorylation; therefore, the use of AgNPs during the early stages of infection may be used as an antiviral strategy (52).

When Selenium (Se)NPs are modified with functional groups such as polyethylene glycol, they can activate targeting ligands, resulting in enhanced disease site accumulation through controllable surface charge and hydrophobicity of SeNPs in order to modulate cellular uptake, biodistribution and immunostimulation (53). In addition, mesoporous silica NPs can carry antiviral peptides, which help to control the release rate, protection against proteolytic degradation and suppression of binding to serum proteins (54). However, the following negative effects have been observed in vivo after acute exposure: Endothelial dysfunction, hemolysis and neurotoxicity (55).

Clinical investigations have demonstrated the antiviral potential of SiO₂-NPs as both a delivery system for small molecules with antiviral activity and for use in vaccines against viruses such as HIV (56). The antiviral success of SiO₂-NPs is largely attributed to their excellent biosafety profile, which includes good tolerability and minimal side effects (57).

Recombinant H1N1/A/California/4/09 influenza virus HA antigen, conjugated to nano-SiO₂ (as an adjuvant), has been used as a vaccine that stimulated the production of IFN-γ and reactivates T cells in an ‘ex vivo precision-cut lung slices’ model (58). In this system, silica activates the NLR family pyrin domain containing 3 inflammasome that releases IL-1β and IL-18, which provides an important link between the innate and adaptive immune responses (59-61).

ZnO-NPs have the benefit of low toxicity and high UV absorption, making them a good candidate to be used in the biomedical field (62). ZnO-NPs are safe substances and they are easily absorbed by the body. Furthermore, they have demonstrated the ability to inhibit viral protease and polymerase (63). However, certain ZnO-NPs have low penetration and are unsuitable for heterogenous mixtures (64).

ZnO-NPs stimulate the innate and adaptive immune response through the Toll-like receptor 4 signaling pathway, which increases the early activation of NF-κB and triggers the increase of proinflammatory cytokines that keep viral pathogens in check, such as TNF-α, IL-1β and IL-6(65). In addition, ZnO-NPs can absorb UV-vis light, dissociate water molecules and release Zn²⁺ ions, generating ROS (such as hydrogen peroxide, hydroxyl and superoxide radicals), which alters lipids, proteins, carbohydrates and viral DNA and ultimately leads to viral destruction (66). The release of Zn²⁺ ions specifically prevents viral infection by inactivating virus adsorption/entry, blocking uncoating, preventing replication, assembly and release during the viral life cycle and producing ROS (67). Furthermore, Zn²⁺ ion release causes numerous viruses to adopt zinc finger structures during their replication in the cells they infect, increasing the antiviral activity. For instance, the nucleocapsid protein of HIV-1 takes on a zinc finger conformation after ZnO-NP treatment, reducing the efficiency of the chaperone proteins responsible for rearranging nucleic acids into conformations that are thermodynamically stable for retro-transcription, thus highlighting the potential of zinc as an antiviral agent (68).

SeNPs have a high absorption rate, high biological activity and low toxicity, and can be directly absorbed by the human body and converted to organic selenium (69). However, certain SeNPs have poor stability and may be easily converted into gray-black elemental selenium, which may increase their toxicity and result in diminished activity (70). To improve formulation stability and targeted therapeutic effects, the surface of SeNPs could be modified with amino acids, proteins, polysaccharides, folic acid or hyaluronic acid, among others (71).

It has previously been reported that selenium (Na₂SeO₃) can suppress hepatitis B virus replication and transcription in liver cancer cells (72).

Li et al (73) observed that adding SeNPs to oseltamivir can increase the antiviral properties against the H1N1 influenza virus in MDCK cells by activating phosphorylation of p53 and Akt.

SeNPs regulate superoxide radicals, thereby possessing the ability to inactivate a virus. For instance, an evaluation carried out in Marc-145 cells infected with porcine reproductive and respiratory syndrome virus treated with SeNPs found a substantial reduction in the rate of apoptosis, as attributed to decreased ROS, lowered phosphorylation levels of c-Jun and degradation of both caspase-3 and poly ADP-ribose polymerase (74).

Furthermore, a study carried out with SeNPs plus amantadine showed reduced NA activity of the H1N1 influenza virus and inhibition of apoptosis in H1N1-infected MDCK cells (75,76). The authors attributed these effects to decreased levels of ROS via activation of the PI3K/Akt pathway, which negatively regulates the JNK pathway through apoptosis signal regulating kinase 1, as mediated by Bax and dependent on JNK (77). It should be noted that cells infected with the H1N1 influenza virus displayed an increase in viability after being treated with SeNPs plus amantadine (32.34 vs. 79.6%) (78).

The HA protein of IAV is an excellent target for the development of new treatments, largely because it stimulates the production of neutralizing antibodies during natural infection or vaccination. It should be noted that the HA2 subunit (stem domain) is more conserved compared to the HA1 subunit (globular head domain) (79). The conserved regions are very relevant as possible treatment targets, since effective antivirals can be designed on them (80). The use of NPs as antivirals could exert more targeted effects if the NPs are coupled to molecules capable of recognizing these targets. This has already been achieved for diagnostic purposes, to detect various viruses in samples, including influenza (81). Likewise, AuNPs have been designed to which molecules capable of acting as receptors for the influenza virus have been coupled, inhibiting its hemagglutinating capacity and thus its infectious capacity (45). These capabilities have been achieved with the successful coupling of the viral HA receptor glycans onto the NPs (82). However, it would also be relevant to use NPs coupled with broad-spectrum influenza virus neutralization antibodies as a means of recognition (83). The neutralization capacity of these antibodies has revealed the presence of epitopes that are conserved among various subtypes of influenza, both group 1 and group 2 of IAV (84) and even influenza B virus (IBV) (85). Therefore, the use of these antibodies would facilitate a specific action of the NPs on viruses or cells that express HA, a product of viral infection, on their membrane, with the great advantage of the broad recognition capacity of influenza subtypes.

M2 is a tetrameric membrane protein with pH-controlled selective proton channel activity, which promotes the entry of virus constituents into the cytosol of the infected cell. Specifically, a 54-amino-acid sequence within the M2 protein has previously been found to localize to the cytosol, where it has a key role in the assembly and release of viral particles by interacting with the M1 protein. In addition, M2e is highly conserved among various IAVs, making it an important antigenic target for developing a universal influenza vaccine (86). The coupling of M2e to the surface of AuNPs has shown to be very useful as an immunogen; it has been successfully tested in a murine model to avoid the lethal consequences of infection with influenza virus A/PR/8/34 (H1N1) (87), which has shown the relevance of the use of NPs as carriers of relevant antigens for possible universal vaccines.

In the present review, the antiviral activities of six NPs were described, which may serve as an aid for the development and manufacture of novel and safe biopharmaceuticals targeted against the influenza virus (Table I). NP medicines offer high storage and transport stability. However, it is necessary to optimize the biocompatibility of each medication and to consider their inhibitory effects on resistant viral strains (88). The following parameters should be carefully considered when designing nanomedicines: Particle composition, charge, size, surface functionalization, stability, morphology and cellular target (89). To date, only a small number of clinical trials have been conducted using metal NPs (90). Most of the data available have been acquired via in silico, in vitro and in vivo assays (91). This is largely due to the fact that metal NPs exhibit immunogenicity and inflammation after systemic administration, which causes damage to the cardiovascular system via oxidative stress, systemic inflammation, endothelial dysfunction, thrombosis and arrhythmia (92). Therefore, these limitations need to be considered and future research is warranted to address these limitations before safely incorporating metal NPs into future clinical trials.