Blocking Viral Entry The last decades have been characterized by increasing investigation on viruses. In particular, their surface charge, protein composition, and host cell entry mechanism have been elucidated, allowing to formulate the first-generation wide spectrum antivirals. Blocking the viral entry is one of the most common known antimicrobial procedure to stop infections at the early stage. In this context, antiviral materials have been used for surface disinfection or for epidemic limitation in humans and animals. Due to their high surface to volume ratio, composition, and tunable surface chemistry, HNMs are now more and more studied as powerful agents in blocking viral entry. As for other biological interactions, the attachment and entry of viruses into host cells are mediated by multivalent interactions between the surface of the virus and cell surface receptors.10 Nanomaterials can display multivalency that makes them able to compete with the host cells on virus attachment, limiting their infectivity. The mechanism of antiviral actions relies on the inactivation of the capsid proteins. As a matter of fact, HNMs have been used for: (1) blocking target proteins for viral entry, (2) capsid protein oxidation, (3) mimicking cell surface, and (4) mechanical rupture of viruses (Figure 1). These strategies target proteins and mechanisms of entry common in most of the viruses, thus allowing the preparation of wide spectrum antiviral agents. In this section, the application of different HNMs as powerful inhibitors of viral entry will be discussed. Figure 1 Illustration of the main mechanisms of blocking virus entry into host cells: capsid denaturation, mimicking cell surface, and mechanical breaking of the virus. Noble Nanoparticles Noble nanoparticles (NPs), made of gold and silver, are attractive as antiviral agents for their surface functionalization versatility and their capacity to cleave disulfide bonds. Their use in disinfection has been extensively studied for different types of viruses. The morphology and the size of the NPs play a crucial role in their ability to efficiently interact with the capsids and in their toxicity for the organism. These nanomaterials are characterized by a very large specific surface area (inversely proportional to the particle diameter). As the particle size becomes smaller and smaller, the percentage of surface atoms increases, creating many unsaturated bonds due to lack of neighboring atoms. As a consequence, AgNPs and AuNPs have unstable atoms with high surface energy. This kind of structure provides a lot of contact adsorption sites and reaction points for further modifications. These chemical features allow to easily combine surface NP atoms with other atoms through chemical bonds. Besides the composition of the metal core, several studies have pointed out the importance of the control of the surface chemistry. The surface groups can: (1) stabilize NPs in the biological media, (2) insert targeting agents, and (3) enhance the circulation time inside the body. Antiviral efficiency can be also enhanced by the multivalency effect, where highly branched ligands are used to locally augment the local concentration of the targeting molecules. In this section the main strategies and results for silver (AgNPs) and gold (AuNPs) nanoparticles in blocking viral entry will be critically discussed. Silver Nanoparticles Many studies have shown that naked AgNPs have a good effect on the control and prevention of a variety of viral diseases (Table 1). However, the antiviral mechanism of nanosilver is still unclear. The antiviral action is associated with the following mechanisms: Nanosilver can prevent the virus from entering the host cells and inhibit the virus from binding to the cell receptor, thereby stopping the virus from infecting the targeted cells. AgNPs may be able to bind the viral surface protein and inhibit the interaction between the virus and the cell membrane receptors (Figure 2, left). However, it has been also reported that AgNPs can inactivate the virus through denaturation of surface proteins containing cysteine and methionine residues present on the viral capsid, in a similar way reported for bacteria. For example, AgNPs smaller than 10 nm were shown to interact with the sulfur-bearing residues of gp120 glycoprotein knobs distributed on the lipid membrane of HIV-1 virus, preventing the virus from binding to CD4 receptor site on the host cells, thus inhibiting the viral infection.11 By means of a viral adsorption assay, it was shown that the AgNP mechanism of anti-HIV action is based on the inhibition of the initial stages of the HIV-1 cycle. To demonstrate that the antiviral effect of AgNPs is due to the particle structure rather than to silver ions present in solution, the antiviral activity of silver sulfadiazine (AgSD) and silver nitrate (known antibacterial silver salts) was evaluated. Both salts showed a much lower therapeutic index than AgNPs in vitro, indicating that silver ions themselves are less efficient.12 These results point out that the antiviral efficacy is not only related to the dose of Ag+ ions present in solution but is also regulated by different other parameters (e.g., size, charge, and surface functionalization) associated with the nanosize dimension. For instance, in the case of Herpesviridae and Paramyxoviridae viruses (both enveloped viruses with embedded viral-encoded glycoproteins), AgNPs can effectively reduce their infectivity, by blocking the interaction between the viral particles and the host cells with an antiviral activity strictly dependent on the size and ζ potential of the AgNPs. As a general observation, it was reported that smaller nanoparticles have better antiviral effect. This effect was associated with the increase of the surface area, where smaller-sized AgNPs could bind more efficiently to the viral particles exerting a higher antiviral activity.13 Another study reported the impairment of Peste des petits ruminants virus (PPRV) replication after incubating infectious viral particles with AgNPs, which did not exhibit any virucidal effect even up to 900 μg/mL. This result suggested that the anti-PPRV activity of the AgNPs is due to the inhibitory effect on viral replication in the target cells. AgNPs do not prevent the binding of PPRV to host cells, but inhibit the entry of viruses into these cells. AgNPs can also interact with the surface and core of PPRV, but this interaction cannot kill the virus directly.12 The same results were then confirmed on other viruses. AgNPs with a diameter of 25 nm inhibited Vaccinia virus replication by preventing viral entry into host cells. However, AgNPs cannot prevent the virus from adsorbing onto the cells, and this virus is still infectious, indicating that AgNPs lack a direct virus-killing effect.13 Figure 2 Potential antiviral mechanism of AgNPs. (1) AgNPs interact with viral envelope and/or viral surface proteins; (2) AgNPs interact with cell membranes and block viral penetration; (3) AgNPs block cellular pathways of viral entry; (4) AgNPs interact with viral genome; (5) AgNPs interact with viral factors necessary for viral replication; and (6) AgNPs interact with cellular factors necessary for productive viral replication. Reproduced with permission from ref (14). Copyright 2016 Taylor and Francis. Table 1 Antiviral AgNPs and Their Possible Mechanisms of Action virus shape size (nm) active concentration mechanism of action ref HIV-1 spherical 1–10 25 μg/mL interaction with gp120 (11) HIV-1 IIIB – 30–50 440 μg/mL interaction with gp120 (19) HSV-1, HSV-2, and HPIV-3 – 20–50 not available possible interaction directly with the viral envelope or its protein (20) Adenovirus type 3 spherical 5–18 25 μg/mL direct destruction of virus particles and DNA structure (15) H1N1 influenza A virus spherical 5–20 12.5 μg/mL inhibition of respiratory enzymes and electron transport components and interference with DNA function (21) HBV spherical 10–50 5 μM interaction with double-stranded DNA and/or binding with viral particles (16) PPRV spherical 5–30 11.1 μg/mL interaction with virus surface and core (12) Vaccinia virus spherical 25 not available preventing viral entry into host cells (13) Monkey pox virus (MPV) – 10–80 12.5 μg/mL blocking virus-host cell binding and penetration (22) Tacaribe virus (TCRV) – 5–10 25 μg/mL inactivation of virus particles before entry (23) Poliovirus spherical 4–9 3.1 ppm preventing viral particles from binding to the receptors of RD cells (24) TGEV spherical <20 12.5 μg/mL direct interaction with TGEV surface protein, such as TGEV S glycoprotein (18) linear 60000–80000 linear 20000–30000 Alternatively, nanosilver can be combined with viral nucleic acids to change the capsid structure, affect the replication of viral genetic material, and make the virus inactive. For example, TEM analyses have shown that NPs can cause a change of the structure of the Ad3 virus from a hexahedral shape to an irregular shape, destroying its fibers and capsid proteins, leading to inhibition of the virus from binding to the host cells and destroying the DNA structure, preventing adenoviral infection.15 Nanosilver can also bind directly to the double-stranded DNA of hepatitis B virus to inhibit its replication.16 In other studies, it has been demonstrated that silver ions released from nanosilver can directly damage the viruses. Based in this property, an interesting application has been proposed. AgNPs were used as a coating on polyurethane condoms, effectively inhibiting the activity of HIV and herpes simplex virus (HSV). The hypothesized mechanism is that silver ions are transferred directly from oxidized NPs to biological targets, such as viral membrane proteins gp120 and gp41. In addition, a small amount of silver ion is also released from the coated contraceptives to improve the antiviral level.17 Although the studies on naked AgNPs to reduce viral infectivity have shown their potential as broad-spectrum antiviral agents, the understanding of the specific antiviral action mechanism still needs to be elucidated in depth. Many studies have shown that the antiviral performance of naked AgNPs is related to their size, and smaller nanoparticles have better antiviral activities.16 In addition to particle size, the antiviral action of AgNP morphology has also attracted interest to fight against coronavirus. AgNPs and two types of silver nanowires were able to significantly cause an inhibitory effect on coronavirus transmissible gastroenteritis (TGEV)-induced host cell infection and TGEV replication. The mechanism is likely based on a direct interaction of AgNPs with TGEV surface proteins (e.g., TGEV glycoproteins) to inhibit the beginning of viral infection. It is possible that AgNPs and Ag nanowires alter the structure of some surface proteins of TGEV and then inhibit their recognition and adhesion to the cellular receptor pAPN.18 Although the potential of AgNPs as antiviral agents has been commonly recognized, unfortunately, their wide biological applications are limited by the risks of self-aggregation and environmental pollution. Silver ions can be released from the surface of AgNPs and potentially pollute the environment, and their agglomeration into bulkier particles or fibers may change their biological characteristics, diminishing the antiviral effect. In several cases, it has been reported that naked AgNPs may affect human health.25 Therefore, research and development of AgNPs whose surface is modified or stabilized by protecting molecular layers is an urgent need to overcome these problems (Table 2). Poly(N-vinyl-2-pyrrolidone) (PVP) is the most commonly used stabilizer of AgNPs. The PVP-coated AgNPs are able to inhibit the activities of HIV-1, herpes simplex 2 virus (HSV-2), and respiratory syncytial virus (RSV).11,26,27 But compared to foamy carbon, small-sized PVP and BSA-coated AgNPs showed poor antiviral activity to the HIV-1 virus.11 For RSV, PVP-coated AgNPs have a specific binding capacity to the viral surface, evidencing a regular spatial arrangement and a clear interaction with G-protein.26 In addition, to improve the stability of AgNPs, their surface modification with antiviral drugs was proved to reduce the drug resistance caused by the drugs administered alone. Tannic acid-modified AgNPs showed good antiviral effects on HSV-2 infection in vitro and in vivo. The viral infection was inhibited only when these NPs directly interacted with HSV-2 virions. Indeed, the pretreatment of host cells with such AgNPs did inhibit the entry of HSV-2. Due to the high affinity of tannins to proteins and sugars, tannic acid can bind glycoproteins on the surface of viruses to make them inert, impairing glycoprotein function and preventing viruses from attaching and entering host cells.28 Table 2 Surface-Modified Antiviral AgNPs and Possible Mechanisms of Action virus shape size (nm) coating mechanism of action ref HIV-1 spherical 1–10 foamy carbon, PVP and BSA interaction with gp120 (11) RSV spherical – PVP, BSA, and recombinant F protein (RF 412) interaction with the G-protein on the virus surface (26) H1N1 influenza virus spherical 2–5 Oseltamivir inhibition of the activity of neuraminidase and hemagglutinin (34) Amantadine (35) Zanamivir (36) inhibition of accumulation of reactive oxygen species (ROS)     HAV, NoV and CoxB4 spherical – polyphosphonium-oligochitosans preventing viral attachment and penetration (29) MPV spherical 10–80 polysaccharide blocking virus-host cell binding and penetration (22) TCRV spherical 10 polysaccharide inactivation of virus particles prior to entry (23) HSV-1 spherical 4 mercaptoethanesulfonate competition for the binding of the virus to the cell (30) Enterovirus 71 (EV71) spherical 2–5 PEI and antiviral siRNA inhibition of the accumulation of ROS and activation of AKT and p53 (37) HSV-2 spherical 13, 33, 46 tannic acid direct interaction and blocking of virus attachment, penetration and spread (28) The surface modification can also exert a synergistic antiviral effect. AgNPs decorated with polyphosphonium-oligochitosan (PQPOC) exhibited moderate to excellent antiviral activity against HAV, NoV, and CoxB4. In addition, AgNPs could interact with the virion glycoproteins and prevent viral attachment and penetration. PQPOC can also serve as an effective virus inhibitor by blocking the interaction of the targeted virus with the host through the electrostatic interaction between the cationic polymers and the negatively charged binding sites of the virus.29 Surface-modified AgNPs can also prevent viral infection by competitive adsorption on host cells. The process of infection of cells by herpes simplex virus type 1 (HSV-1) involves the interaction between viral envelope glycoproteins and heparan sulfate (HS) on cell surface. Therefore, researchers designed AgNPs capped with mercaptoethanesulfonate (Ag-MES) to compete with the cellular HS through the sulfonate end groups, thereby blocking the virus from entering the cells.30 A few years ago, it was shown that curcumin could prevent the replication and the budding of RSV,31 but the disadvantage of poor solubility and low bioavailability limited its clinical application.32 Curcumin was used as a reducing and capping agent to prepare stable curcumin AgNPs (cAgNPs) under physiological conditions. cAgNPs could reduce cytopathic effects induced by RSV and showed efficient antiviral activity against infection by directly inactivating the virus prior to entry into the host cells. Its antiviral effect was higher than curcumin alone or unmodified AgNPs (Figure 3).33 Figure 3 Schematic representation of the synthesis of cAgNPs (A) and a proposed inhibition mode of cAgNPs against RSV infection (B). The inhibition mode of (B) shows that cAgNPs can reduce the binding ability of virus with the binding centers on the surface of cells (b) as compared to those without cAgNPs (a). Reproduced with permission from ref (33). Copyright 2016 Royal Chemistry Society. Alternatively, Zhu et al. prepared AgNPs surface-modified with oseltamivir, amantadine, and zanamivir (Ag@OTV,34 Ag@AM,35 and Ag@ZNV36), by chemical methods. The results showed that these nanoparticles can directly interact with the virions, resulting in viral function damages. Overall different studies have reported the capacity of AgNPs to block viral entry. However, there is not a concerted antiviral mechanism, but their activity differs from case to case, based on viral particle adsorption, capsid structure alteration, or surface protein denaturation. For AgNPs, the antiviral activity can be associated with different parameters including size, shape, surface charge, and functionalization but also to the topical release of silver ions able to disturb the viral cycle replication. As described before, bare AgNPs can be used as disinfectant agents, however their use in biological media is limited by their low colloidal stability and potential cytotoxicity. Surface functionalization can alleviate cytotoxicity, but it can also mask the nanoparticle surface, reducing their affinity for viral particles, thus reducing AgNP antiviral activity. For these reasons, AgNPs at the moment could find application mainly for surface disinfection and for topical administration. Further studies are needed to prepare safer AgNP formulations for systemic administration. In particular, the clarification of the antiviral mechanisms and the use of surface functional groups able to stabilize AgNPs in biological fluids without affecting their prominent antiviral activity are probably the most important challenges to tackle. Gold Nanoparticles Compared to AgNPs, AuNPs exhibit reduced toxicity on healthy cells, making them more attractive for in vivo and clinical applications.38 Indeed, AuNPs have been successfully tested as inhibitors of viral entry into the host cells. AuNPs interact with hemagglutinin (HA), where Au is able to oxidize the disulfide bond of this glycoprotein causing its inactivation, thus impeding the membrane fusion of the virus with host cells. Targeting HA has emerged as an alternative strategy to the actual therapies (e.g., matrix protein 2 and neuramidase), especially to pandemic viruses that show an accelerated mutation speed of their surface proteins, hence a resistance to conventional treatments increasing their infectivity and mortality.38 This strategy has been applied to influenza (e.g., H1N1, HCV) and herpes viruses.39−44 The activity of AuNPs is proportional to the surface area exposed. As a consequence, the size and the morphology of these metal NPs play a substantial role in their antiviral activity. Recently, Kim et al. have reported that porous AuNPs are able to inhibit influenza A infection more efficiently than nonporous AuNPs.39 This effect has been associated with the higher surface area of the porous material that favors their interaction with capsids and thus increases their antiviral activity (Figure 4). Figure 4 Schematic illustration of inactivation of influenza A virus (IAV) treated with porous AuNP (PoGNP). PoGNP interacts with IAV surface proteins and cleaves their disulfide bonds. Inactivated viruses exhibit lower infectivity to cells. Reproduced with permission under a Creative Commons CC-BY license from ref (39). Copyright 2020 BioMed Central Ltd., Springer Nature. Besides the per se antiviral activity, AuNP surface modifications have been developed in order to enhance their overall therapeutic benefits. The engineering of tailored AuNPs with selected ligands has allowed the preparation of efficient antiviral nanoagents. The target ligands can be introduced directly during the particle synthesis via ligand exchange reactions or ligand modifications. For instance, direct reduction of gold ions in the presence of gallic acid produced homogeneous AuNPs able to sensibly reduce herpes simplex virus infection in vitro.40 Compared to free ligand NPs, functionalized AuNPs benefit from the multivalency effect and higher circulation times, decreasing the needed therapeutic concentrations.39 Functionalized AuNPs can present organic groups that mimic host cell surfaces or other specific molecular patterns that selectively target the virus. Normally, negative charges are used to mimic cell surfaces and favor the interaction between the particles and the capsid. In particular, sulfonates and organic sulfates have been used for their capacity to attract the virus via capsid protein interaction and block the HA activity.41 AuNPs functionalized with sulfonates showed an increasing inhibition of influenza A compared to the nanoparticles capped with succinic acid.42 This study also demonstrated that there is not a correlation between the negative charge and the antiviral activity, but instead the inhibition depends mainly on the organic groups used. Thiol-capped AuNPs also displayed powerful inactivation of bovine viral diarrhea virus in vitro.43 Multivalency has been exploited in more complex systems using dendrons as capping agents. This strategy allows to generate higher concentrations of the target ligand in close proximity to the AuNPs and to increase the binding efficiency of the nanoparticles to the capsid. The driving force of the antiviral efficiency relies on the concentration of the targeting agent onto the particles. Sulfonated dendrons were grafted to AuNPs via a sulfide bond and tested for HIV inhibition.44 The results showed that the decorated AuNPs exerted a higher affinity to the virus. Additionally, comparing AuNPs functionalized with different generation dendrons, those with a third generation displayed the highest inhibition performance with an IC50 below 0.1 μmol/mL, thus making them attractive for in vivo translation. It is worth noting that the inhibition efficiency is strictly dependent on the available sulfonate groups present on the surface of the NPs, making crucial a thorough characterization of the material.44 The size of the AuNPs clearly plays an important role in the concentration of targeting ligands exposed per particle.45 Indeed, too big NPs have a limited surface area, while too small would not allow an efficient grafting of the dendrons due to steric hindrance. For instance, it has been shown that dendron-functionalized AuNPs showed a size-dependent antiviral activity for influenza virus, where 14 nm particles exhibited a higher efficiency than 2 nm AuNPs. This has been associated with the low functionalization grade of the small nanoparticles and to the inappropriate spatial distribution of the interacting ligand/receptor pairs. The development of viral proteomics has profoundly transformed the antiviral and disinfection strategies. In particular, small molecules and peptides able to target and block the viral biochemical machinery have been developed. However, despite these efforts into the drug design, many of these molecules suffer from poor biological effect, low concentration in the diseased areas, and undesired side effects. In this context, AuNPs have been coupled to biologically inactive small molecules to create biologically active multivalent AuNP therapeutics. A bright example has been reported by Bowman et al., where the authors functionalized AuNPs with SDC-1721, a small membrane fusion inhibitor of HIV.46 The results demonstrated that, while pure SDC-1721 has low activity, functionalized AuNPs are able to inhibit HIV replication at μM concentrations. Similar results have been reported using targeting peptides. In particular, it was evidenced that the functionalized AuNPs can sensibly reduce the IC50 up to 2 orders of magnitude compared to pure peptides.47 Preliminary results in vivo confirmed the biosafety of the AuNPs.48 Nanoparticles Generating Reactive Oxygen Species One of the main advantages of using NPs compared to oxidized metals relies on the slow release of ions and clusters from these particles, leading to an enhancement of the antiviral activity. Additionally, the use of metal NPs containing Cu or Fe in ionic form catalyzes the generation of radicals via Fenton and Fenton-like reactions oxidizing the capsid proteins and consequently blocking the viral infection at early stage. For instance, copper ions (derived from sulfates or iodide salts) have been widely used as antiviral agents because of their activity on several kinds of enveloped and non-enveloped viruses including influenza virus,49−51 herpes simplex virus52−54 and hepatitis A virus.55 Their mechanism of action relies on the formation of Cu+ ions (from soluble salts or nanoparticles) that generate hydroxyl radicals.56 The use of metallic copper nanostructures in the form of particles or sheets has shown only a moderate efficiency due to the low concentration and low release of Cu+.56 For these reasons, Cu+ salts, where the copper ions are readily present in their active monocationic form, have been favored. In particular, CuI nanoparticles (stable at room temperature) have been extensively studied for deactivation of feline calicivirus56 and H1N1 pandemic influenza virus.57 However, the use of copper salts at high concentrations can irreversibly alter reactive oxygen species (ROS) homeostasis of healthy cells, provoking a general toxicity for the organism, limiting their applications to disinfection.56 Nanostructured cuprous and cupric oxides have been also extensively employed as antiviral agents for in vitro applications. For instance, cuprous oxide nanoparticles (CuONPs) were successfully employed against hepatitis C.58 In particular, it was found that these NPs exerted a favorable antiviral activity with no cytotoxic effects. CuONPs target the binding and entry step of viral infection to hepatic cells (Figure 5). Similar results were reported on the use of CuONPs against HSV-1, however without any profound investigation on the antiviral mechanism.59 Figure 5 Huh7.5.1 cells at 72 h post-infection were stained with HCV-positive serum from patients (green signal) and with DAPI (blue signal). Cuprous oxide NPs (CuONPs) are able to reduce viral infection in vitro. Reproduced with permission from ref (58). Copyright 2015 Elsevier B.V. Alternatively, zinc salts have been successfully used as antimicrobial agents from research up to clinical trials for viral warts.60,61 More recently, ZnO nanoparticles (ZnONPs) were developed for the treatment of HSV-2. ZnONPs were prepared with a tetrapod morphology.62 The results showed that they can mimic cell surface interacting with the HS present on the viral capsid. Additionally, these particles have been used for photocatalysis showing to efficiently destroy the viral proteins upon UV irradiation.62 Besides all these interesting examples, in vivo applications are still needed to validate this therapeutic modality. Due to the generation of high levels of ROS, the toxicity of copper nanoparticles has been widely debated. The antiviral activity of copper nanoparticles is generally associated with the release of Cu+ ions in solution, thus the leakage of cytotoxic cationic species can be modulated by surface functionalization before in vitro and in vivo applications. On the other side, the use of nanomaterials generating ROS can find applications in textile and surface coating. The general broad virucidal efficiency of copper oxide nanoparticles shown for H1N1 pandemic influenza57 should be tested on SARS-Cov-2 and might be used for improving mask protection efficiency. Carbon Nanomaterials Due to their diversity, versatility, and tunable surface chemistry, carbon nanomaterials have been attractive for several types of applications. In particular, the past decade has seen a tremendous raise in the preparation of performant carbon-based nanomaterials in the antiviral field. Fullerene and its derivatives are the most studied carbon nanomaterials for their virucidal activity. Due to the lack of solubility of pristine fullerene, functionalization strategies have been developed to prepare water-soluble drugs. Investigations in the biomedical field evidenced the membranotropic capacity of fullerene derivatives.63 By modulating shape and functions, fullerene derivatives have been shown to possess antiviral properties through inhibition of viral entry and blockage of viral replication. From these results, the attention has been directed also to other carbon nanomaterials. In particular, functional carbon dots (CDs) and graphene oxide (GO) have been investigated for their ability to block viral entry into host cells. Glycofullerenes The emerging of mortal viruses, like Ebola or Zika, and the lack of suitable treatments led the academic and the industrial communities to look for alternative therapeutic routes. Most of these pathogens are RNA enveloped viruses, and they share common infection mechanisms that can be targeted for the preparation of wide spectrum antivirals. The external surface of the envelope of these viruses is covered by glycans that tightly interact with lectin receptors on host cells.64 This strong interaction allows the attachment of the virions to the cells, followed by internalization and infection. Blocking lectin receptors is a general strategy used to stop viral infection at an early stage. Fullerenes have been widely investigated as antiviral molecules, drug carriers, or tissue scaffolds.65 Fullerene applications have been recently extended to the design of mannosylated derivatives to block the entry of viral particles into host cells. Mannose, due to the high affinity with lectin receptors, competes with the virus in the interaction with the host cells. For example, one of the targets is the inhibition of viral particles through the interaction of mannose with the dendritic cell-specific ICAM-grabbing non-integrin (DC-sign). DC-sign receptors mediate the interactions between DCs and T cells.66,67 To exploit these characteristics, mannose was combined with fullerene in the design of the so-called glycofullerenes to study their capacity to inhibit Ebola, Dengue, and other pathogens. For this purpose, different glycofullerenes were synthesized by changing the number of mannose units (from 12 to 36) and the spacers between the fullerene moieties and by varying steric hindrance in order to obtain a library of molecules.66 The synthetic route is composed of three steps based on “click chemistry”: (1) assembly of glycodendrons by Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC), (2) synthesis of alkyne-substituted Bingel-Hirsch hexakis-adducts, and (3) the coupling between the last two products again by CuAAC. To increase the number of mannose moieties up to 36, the glycodendron core was changed from malonate to trialkynyl pentaerythritol. In order to compare the different derivatives, in vitro studies were performed. Jurkat cells (lymphocyte T CD4 immortalized cells) expressing DC-sign were used to prove the inhibition capacity of the glycofullerenes on viral infection of Ebola (Figure 6, route A). The study revealed an IC50 in the μM range for the 12 mannose fullerene, a lower efficiency with the 36 mannose fullerene with a short spacer (PEG, with 2 ethylene oxide units), while a nanomolar IC50 was achieved with 36 mannose fullerenes with a longer spacer (PEG, with 3 ethylene oxide units) (Table 3). This first proof-of-concept study was then expanded, aiming to obtain a better antiviral activity by increasing the valence and inserting longer and flexible spacers.68 Figure 6 Chemical design and general scheme to block the viral entry by glycofullerenes. Three different shapes of glycofullerenes can act as inhibitor of viral infection: (A) shape composed of monodisperse fullerene bearing mannose, (B) assembly as “sugar balls” of tridecafullerenes exhibiting mannose on the edges, and (C) supramolecular micellar aggregates of fullerenes bearing mannose. Table 3 Antiviral Activity of Different Fullerenes Functionalized with Mannose material functional groups core function number of mannose moieties virus number of atoms between fullerene and mannose IC50 ref glycofullerene monosaccharide malonate 12 Ebola 8 2 μM (66) glycofullerene monosaccharide trialkynyl pentaerythritol 36 Ebola 22 68 μM glycofullerene monosaccharide trialkynyl pentaerythritol 36 Ebola 28 0.3 μM trideca-fullerenesa monosaccharide malonate 120 Ebola 8 20.375 nM (69) trideca-fullerenesb monosaccharide malonate 120 Ebola 8 0.667 nM glycofullerene disaccharide trialkynyl pentaerythritol 36 Zika 29 8.35 nM (70) Dengue 7.71 nM trideca-fullerenes disaccharide trialkynyl pentaerythritol 120 Zika 29 0.52 nM Dengue 0.098 nM trideca-fullerenes disaccharide trialkynyl pentaerythritol 360 Zika 48 0.067 nM Dengue 0.035 nM micellar glycofullerenes monosaccharide trialkynyl pentaerythritol 6 Ebola 23 424 nM (72) micellar glycofullerenes monosaccharide trialkynyl pentaerythritol 12 Ebola 25 196 nM a Small spacer between core C60 and surrounding fullerenes. b Large spacer between core C60 and surrounding fullerenes. Based on these studies, another class of multivalent fullerene dendrimers was then designed. A fast and controlled synthetic route was developed to achieve giant globular multivalent fullerenes, containing hundreds of functional groups. The first study was performed with tridecafullerenes containing 120 mannoses.69 The molecular structure is composed of 12 hexakis C60 surrounding a C60 core (Figure 6, route B). Compared to the previous study, an IC50 3 orders of magnitude lower was measured on the inhibition of Ebola virus (Table 3).66,68 In order to present more carbohydrates at the periphery of the dendrimer, a trialkynyl pentaerythritol derivative allowed to afford a tridecafullerene with 360 carbohydrates. In this case, the molecule was synthesized with a C60 tridecafullerene bearing α(1,2)mannobioside.70 The use of this disaccharide was already investigated, showing an increase of affinity with DC-sign receptors by a factor of 3–4.71 The synthetic strategy exploited also the use of strain-promoted copper-free cycloaddition of azides to alkynes (SPAAC) for the coupling of the core fullerene to the surrounding fullerenes. SPAAC allows an easier purification avoiding the removal of cytotoxic copper ions. The inhibition performance of this molecule was studied in vitro with viral pseudoparticles of Dengue and Zika. The comparison was made between 360 and 120 disaccharides tridecafullerenes and 36 disaccharide monofullerene. The results highlighted a picomolar IC50 inhibition on both Zika and Dengue models for the 360 disaccharide glycofullerene (Table 3). The ability to inhibit other types of viruses allows the use of glycofullerenes as broad spectrum antiviral drugs. Moreover, the negligible toxicity to other cells proved the biocompatibility of these molecules. Following an alternative strategy, a supramolecular assembly of monodisperse glycofullerenes, leading to the formation of micelles, was achieved and tested.72 These micelles present a uniform and spherical shape (Figure 6, route C). The aggregation synthetic route is faster compared to a controlled synthesis of giant glycofullerenes, but it might suffer from low reproducibility and batch-to-batch differences between each formulation. This self-assembled C60 functionalized with 6 or 12 mannoses exposes a large amount of carbohydrate at the surface, leading to an inhibition of Ebola virus in the nanomolar range (IC50 of 424 nM for six mannoses and 196 nM for 12 mannoses, respectively, Table 3). Further in vitro studies evidenced again a good biocompatibility of these glycofullerenes. While there are no in vivo studies yet, these promising results enlarge the panel of molecules in the fight against new emerging viruses. Functionalized fullerenes have been used for their ability to compete with viral particles through lectin receptors in host cells. There has been a tremendous advancement in the functionalization of fullerenes leading to the preparation of derivatives with a high amount of mannose, capable to enhance the multivalency effect and thus to increase the therapeutic outcome. First, glycofullerenes do not have an intrinsic virucidal activity. They can reduce the infectivity, but they are not able to completely inactivate the virus. Second, the mechanism of action of glycofullerenes relies on their interaction with host cells and not with viral particles. Thus, for a therapeutic application, they should be injected at different time points, ensuring that the local concentration is therapeutically relevant to prevent the virus from invading the host cells. In addition, glycofullerenes can be internalized into host cells losing their viral “shield” activity. On the other hand, the well-developed surface chemistry of glycofullerenes can be used for other key receptors involved in viral entry. For instance, in the case of the current SARS-Cov-2 pandemic, a similar click chemistry strategy can be used to anchor ligands recognized by human lung ACE2 receptors and so inhibiting viral entry.73 Other Carbon Nanomaterials Alongside fullerenes, other carbon nanomaterials (NMs) have been scrutinized for their ability to block viral entry. CDs and GO are the most known and studied carbon NMs with marked antiviral properties. CDs are zero-dimensional carbon nanoparticles. They are generally produced via hydrothermal decomposition of carbon containing “low-cost” precursors. The use of CDs in the biomedical field has been encouraged by their easy preparation, low toxicity, fluorescence properties, and easy surface functionalization. Pristine CDs have shown moderate viral blocking activity for HIV infection in vitro.74 This has been associated with the surface of the material rich in carboxylic and hydroxyl groups prone to form noncovalent interaction with viral membranes. Moreover, due to the complexity of the biological systems these nonspecific interactions could not be so effective in vivo, likely reducing the antiviral efficacy. Therapeutic targeting molecules can be grafted onto a CD surface to enhance their antiviral activity. In this context, the design of multifunctional CD platforms can be obtained through two different strategies. The first consists in a single-step reaction that foresees the insertion of the therapeutic molecule directly into the step of preparation. Target molecules are decomposed with the other precursors, generating the desired functional CDs. This protocol is fast and efficient, however the drug loading as well as its activity are hard to estimate. Indeed, the hydrothermal treatment can alter the chemical structure of the active molecule, thus vanishing its therapeutic effect. For these reasons, the reaction conditions must be carefully controlled.75 The second method is a two-step reaction and implies the postfunctionalization via amide formation on the surface of the CDs rich in carboxylic groups. This strategy offers a better chemical control, but the yield and the drug loading may not be quantitative and high, respectively. Different functionalized CDs were prepared to hamper host cell viral entry. For instance, benzoxazine (a low water-soluble antiviral agent) was incorporated into the CD structure during their preparation (Figure 7). The as-prepared CDs showed a broad spectrum viral blocking capacity in vitro for enveloped (e.g., Japanese encephalitis virus, Dengue virus, and Zika virus) and non-enveloped viruses (e.g., porcine parvovirus and adenovirus-associated virus).76 These positive results were explained by the efficient binding and deactivation induced by the multivalent effect of the CDs to the viral particles Figure 7 Illustration of benzoxazine-functionalized CDs and their broad antiviral entry activity. Reproduced with permission from ref (76). Copyright 2019 Elsevier B.V. Amino-functionalized CDs were also tested for the treatment of human norovirus. In this study, CDs were functionalized with 2,2′-(ethylenedioxy)bis(ethylamine) (EDA) and 3-ethoxypropylamine (EPA) via amide bond formation.77 These NMs exerted a good viral blockage. In particular, EPA-functionalized CDs were able to inhibit 100% of viral infection at concentration of 2 μg/mL, while in the case of CDs prepared with the other amines, 80% of inhibition was reported. These effects have been associated with the higher positive charge of CD-EDA compared to CD-EPA. Another surface group used for viral targeting is boronic acid (BA), which can bind glycosylated surfaces forming boronic esters. This strategy was successfully adopted to treat HIV where the boronic groups, linked to different nanoparticles (e.g., silica nanoparticles and nanodiamonds) can target gp120 receptors on the viral envelope inhibiting the infection.78 Another recent study proposed the use of CD functionalized with phenylboronic acid for prevention of HIV infection.74 The functional materials showed good inhibition properties compared to nonfunctionalized CDs by preventing the binding to the target cell in vitro. Overall, the use of CDs for stopping host cell viral entrance has shown good results in vitro. However, there is a lack of proofs in vivo limiting their applications to surface disinfection or masks. In addition, most of the in vitro studies foresee first the contact of the CDs with the viral particles and then their incubation with host cells. Deeper investigations should be performed adding the NMs at other time points (for instance in infected cells) to understand if the antiviral activity is maintained. In addition, CDs have been successfully used for photodynamic therapy (generating radicals upon light irradiation) in cancer treatment. The same approach may be used to combat viral infections, where the antiviral activity induced by the surface modification can be sensibly enhanced by ROS generation under irradiation. Graphene materials, and in particular GO and reduced GO (rGO), have been used for different biomedical applications including drug delivery, biosensing, and tissue engineering.5 GO platforms have shown also interesting antimicrobial activity.79 Regarding viral infection, GO was used to block the virus entrance in host cells. GO and rGO can be considered as two-dimensional materials that contain hydrophilic and hydrophobic domains allowing to adsorb many biological molecules including nucleic acids and proteins. GO showed low interaction with viruses, however its surface functionalization with target molecules can sensibly enhance its affinity for the viral particles. Additionally, GO can be used as photothermal agent (generation of heat by NIR irradiation) or photodynamic therapy (using visible light irradiation) inactivating the capsids by local thermal shock or by radical formation during irradiation, respectively. The use of phototherapies may significantly augment the antiviral properties of the materials. However, we must keep in mind that these therapeutic modalities can be applied only to disinfection, since the radical/heat production may be harmful for the healthy tissues in vivo. Photodynamic therapy has been successfully exploited using bacteriophage MS2 as a model virus.80 In this study, GO was functionalized with an aptamer recognized by the viral surface. The results showed that this functionalization is able to enhance the binding efficiency of the MS2 capsids onto the GO surface compared to nonfunctionalized GO. Subsequently, irradiation in the visible light was able to disinfect the solution, while nonfunctionalized GO showed much less activity due to the lack of adsorption. Despite these interesting results, this pioneer work remains at an early research stage since the use of high light dose (300 W for 10–140 min) and the lack of material recovery and reuse make its application for surface disinfection difficult. GO can be also used as a platform to link antiviral agents. Encouraging results were reported using GO with hypericin for the treatment of a recently appeared duck reovirus.81 More recently, Deokar et al. reported an original rGO-based multifunctional platform for HSV-1 treatment.82 In this work, the authors functionalized the material with organic sulfate groups and iron oxide magnetic nanoparticles (FeNPs). The rGO functionalized with the sulfate is able to mimic the host cell surface and to bind HSV-1. Subsequently, the viral particles captured onto the rGO-FeNP surface can be concentrated via magnetic precipitation and destroyed via photothermal therapy. This approach is highly efficient for disinfection with low energy (1.6 W/cm2 for 7 min) and cost effectiveness. HS is a common entry receptor in various types of viruses (e.g., herpes viruses, human papillomavirus, Dengue virus).83 The use of organic sulfate-functionalized graphene sheets mimicking HS, like GO and rGO, has been already explored. However, it is worth noting that these NMs are prone to strongly adsorb proteins in culture environments (coronation), likely inhibiting their antiviral efficacy.84 High loadings of sulfate groups were introduced onto rGO using polyglycerol sulfate.85,86 This approach has been used for inhibition of orthopoxvirus, pseudorabies virus, and African swine fever virus in vitro.85,86 Graphene has also been used as antiviral material. Polysulfates and fatty amines were grafted onto graphene surface via triazine chemistry for the treatment of herpes simplex virus.87 This strategy promotes the synergy between the electrostatic and hydrophobic interactions, showing incredibly high inhibition efficacy. Overall, graphene materials have shown a good capacity to block host cell viral entry. Disinfection with graphene family materials is also promising, offering the possibility to couple high viral binding with phototreatments. Regarding the GO and rGO activity in cellular environments, different parameters must be considered such as protein coronation, blood circulation time, and activity in vivo. So far, the use of sulfonic groups introduced via diazonium salt decomposition has been largely privileged. We take this opportunity to encourage future studies using other targeting groups (e.g., boronic acids) and grafting methods (e.g., epoxide ring opening or hydroxyl esterification reactions). Mechanical Disruption of the Capsid The most direct way to suppress viruses and stop the spreading of viral infection is to inactivate them before the attachment to the host cells, by binding to the acceptor proteins.88 One of the most conserved targets of viral attachment ligands is the heparan sulfate proteoglycan (HSPG), previously mentioned. HSPGs are expressed on the surface of almost all eukaryotic cell types, and many viruses like HIV-1, HSV, human papilloma virus (HPV) exploit HSPGs as the target of their viral attachment ligands. Bearing in mind this behavior, different studies have used HSPG-mimicking materials to target this type of virus–cell interaction and to achieve broad spectrum efficacy. For example, in one key study, AuNPs were functionalized with mercaptoethanesulfonate (MES) based on its mimicry of HS (Au-MES NPs). Au-MES NPs were shown to interfere with viral attachment, viral entry, and cell-to-cell spreading.89 The importance of the polyvalent interactions with the virus makes these NPs a good candidate for antiviral therapy. However, Au-MES NPs presented virustatic activity, meaning that upon dilution of the NPs, the virus recovers its infectivity due to the reversibility of the cell-virion interaction. This problem was solved by Stellacci and colleagues who developed NPs coated with mercapto-1-undecanesulfonate (MUS) ligands (Au-MUS NPs). The long aliphatic and flexible linkers provide stronger associations with the viral particles compared to Au-MES NPs, leading to local distortions and eventually inducing a global deformation and breaking of the capsid that inactivates its contagion irreversibly (Figure 8).90 These Au-MUS NPs were tested against different HSPG-dependent viruses, showing a high viricidal activity over HSV, HPV, and RSV (Figure 9A). Furthermore, the activity of the Au-MUS NPs was studied in vivo using mice infected with RSV, indicating that the material can prevent pulmonary dissemination of the infection and showing potential use as medically relevant virucidal drugs to fight viral infections. More recently, the concept was further extended to cyclodextrins modified with mercapto-1-undecanesulfonate, proposing this system as a broad spectrum virucidal macromolecule.91 Besides Au-NPs, a similar mechanism of disruption was studied with other materials like graphene or GO. In a study, in which the toxicity of graphene was evaluated theoretically, it was also shown that graphene nanosheets can interrupt the hydrophobic protein–protein interaction, which is essential to biological functions.92 This feature was attributed to the hydrophobic nature of graphene. Thus, it seems energetically favorable for graphene to slide between the interface of two proteins in contact, due to hydrophobic interactions. In another study inspired by this behavior, the authors performed molecular dynamics simulations of graphene nanosheets in the proximity of the surface of the Ebola viral matrix protein VP40 showing that the nanosheets can break the hydrophobic interactions in VP40, a key protein for the replication and stability of Ebola virus (Figure 8).93 These findings suggest that graphene nanosheets might have potential antiviral activity against Ebola; however, there is a lack of experimental evidence that corroborates this mechanism of disruption. On the other hand, GO was tested experimentally against Pseudorabies virus and Porcine epidemic diarrhea virus (PEDV), showing significant decrease in the infectivity.94 It was found that the negatively charged surface of GO is important for the adsorption of the virus, whose surface is positively charged, and that GO could directly interact with the viral particles and destroy their structures due to the sharp edges of the material (Figures 8 and 9B). Figure 8 Mechanical disruption mechanisms of different NMs. Left: Au-MUS NPs inducing mechanical forces in the virus capsid leading to inactivation.90 Center: Graphene NSs disrupting VP40 hydrophobic interactions in Ebola.93 Right: Interaction between negatively charged surface of GO and positively charged capsid.94 Figure 9 Interaction of NMs with viral capsid. A) Gold NPs acting on HSV-2 virus. After 90 min, the percentage of destroyed virus was significantly increased. Scale bars: 100 nm. Reproduced with permission from ref (90). Copyright 2018 Springer Nature Limited. (B) GO acting on Pseudorabies virus. After incubation with GO for 1 h, part of the virus envelope and spikes were destroyed. Scale bars: 200 nm. Reproduced with permission from ref (94). Copyright 2015, American Chemical Society. The mechanical disruption of the capsid is a peculiar antiviral mechanism associated with some NMs. In particular, the use of specific sulfonates able to mimic heparan sulfate can be also used to target SARS-Cov-2 infection.95