The last few years have increasingly emphasized the need to develop, through nanoscale technologies, new active antiviral products useful for infection prevention and control. At the same time, advanced computational approaches have found themselves fundamental in the repurposing of active therapeutics or for reducing the very long developing phases of new drugs discovery, which represents a real limitation, especially in the case of pandemics.
The novel coronavirus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), emerged from Wuhan, China in November 2019, is primarily transmitted from person to person via respiratory droplets and aerosols produced when talking, coughing, and sneezing[1], from both symptomatic and asymptomatic people[2][3]. Most patients affected by COVID-19 (Coronavirus disease 2019) develop only mild to moderate symptoms (fever and dry cough). Unfortunately, patients can develop severe illness (dyspnea, respiratory rate ≥ 30 breaths per minute, blood oxygen saturation ≤ 93%, lung infiltrates > 50%), requiring oxygen support, and also become critically ill with respiratory failure, acute respiratory distress syndrome, sepsis, thromboembolism, and multiorgan failure[4][5]. It became evident early on that severe COVID-19 is characterized by uncontrolled systemic hyperinflammation (the so-called cytokine storm), a condition triggered by the release of a big amount of pro-inflammatory cytokines[6].
SARS-CoV-2 shows a continuous evolution due to changes in its genetic code, and multiple variants of this virus have been discovered in the world during this pandemic. A group of variants with similar genetic changes may be designated by public health organizations as Variant of Concern (VOC), Variant of Interest (VOI) and Variant Being Monitored (VBM), if they share characteristics requiring public health action. Many of these variants are mutations concerning the viral Spike protein and therefore, due to the role of this protein in host cell penetration, are the main target of surveillance. Up to today, SARS-CoV-2 still remains a respiratory pathogen with unpredictable viral evolution, aggravated by widespread circulation, intense transmission in humans and appearance of multiple SARS-CoV-2 VOCs, characterized by increased transmissibility and/or virulence, or reduced effectiveness of countermeasures.
Nanoscale technologies are attracting ever-increasing interest from basic and industrial research for their potential employment in diseases due to several viruses[7], and for the development of new effective therapeutic treatments as well as diagnostic tools, innovative materials, and drugs useful for infection prevention and control[8][9][10].
Strategies involving nanodrugs for the treatment and prevention of viral diseases, including COVID-19, are based on the mechanisms that are described below:
Trapping effects: the first step in a viral invasion is its attachment to the host cell membrane. Therefore, it is highly considered when designing anti-infection nanomaterials, including those based on cell membrane properties; in fact, cell-mimicking nanoparticles (NPs), displaying a pathogen’s cognate receptor on their surface, represent an emerging class of therapeutics that are potentially effective against SARS-CoV-2 and other viruses[11].
Inhibition of viral entry: the inhibitory effect of nanodrugs on cell entry by viruses and related mechanisms has been studied extensively. Viral attachment and entry usually require interaction between viral surface protein(s) and receptor(s) on host cell membranes; thus, nanomaterials that interfere with such interactions are promising antiviral agents which can act at a relatively accessible extracellular level, and thus prevent the infection state at an early stage[12]. Some nanodrugs interrupt virus–cell interactions by blocking viral surface proteins and cell membrane receptors[13][14][15]. In particular, SARS-CoV-2 uses the viral Spike protein and the host cell angiotensin-converting enzyme II (ACE2) protein, as the main method of cell penetration, through a “Lock and Key” mechanism that allows the virus access to cells having the corresponding lock. These sites can be found throughout the body, but especially in the lungs, heart, and arteries. This process emphasizes the need to find a treatment that could potentially inhibit this (and similar) “Lock and Key” process(es) altogether.Inhibition of viral replication: nanodrugs can inhibit viral replication by interacting with the viral protein/genome or inducing a suppressive environment for intracellular viral replication.
Viral inactivation effects: nanodrugs, such as metal- and carbon-based nanomaterials, take contact directly with viruses and induce viral inactivation by different mechanisms depending on the nanomaterial and virus. The destruction of the viral envelope or capsid (for example by photocatalytic oxidation) is a common mechanism by which nanomaterials induce the inactivation of viruses.
Transmission methods of SARS-CoV-2 and possible approaches to prevent and treat COVID-19 are shown in Figure 1.
.The first issue to be underlined to explain nanotechnologies’ great potential for improving prevention and treatment of viral diseases is that nanomaterials[11] possess unique properties—particularly their small size (1–100 nm), but also their high surface-to-volume ratios and modifiable surfaces— which are beneficial for contact with viruses and contribute to multiple antiviral effects. Nanomaterials have been reported to suppress cell entry and viral replication; moreover, their numerous surface binding sites facilitate interactions with target molecules, consequently trapping and inactivating viruses. So, it is evident that modern nanotechnologies do not solely aim to discover new antiviral therapeutics (or to repurpose known products). Nanocomposites can be grouped with different antiviral agents in order to obtain a synergistic effect. Furthermore, nanomaterials may be used to produce innovative drug delivery systems (DDSs) to improve the pharmacokinetic properties of loaded drugs. If drugs are sensible to external stimuli, they may be projected as endowed with auxiliary functions useful for inactivating viruses or mimicking host cells, or they could be functionalized with specific ligands that are able to bind molecular components of the target, and so on (Figure 2). Finally, there is a strong effort toward the development of nanoproducts to be used for nasal/inhalation therapy and taking advantage of antiviral or anti-inflammatory activity of their ingredients[16][17].
The discovery and development of antiviral nanodrugs take enormous advantages from the employment of high-performance computing-based tools[18][19][20][21]. Advanced computational techniques are fundamental in discovering or repurposing, in a rapid and therefore constantly updated way, active therapeutics obtained via chemical synthesis but also derived from natural (particularly vegetable) matrices. These techniques, indispensable for medicine screening to accelerate the development of specific drugs against SARS-CoV-2, are based on the knowledge of the structure of the possible targets (at the level of the virus and of the host cell), to which the potential drug can competitively bind. In particular, molecular docking calculations are based on receptor active site regions to search for whether ligands interact with the target structure and the optimal binding mode between them; molecular dynamics simulation is a method that simulates experimental conditions and can display the microscopic evolution of the system at an atomic level[19][20].
.A particular issue to be underlined is that to defeat COVID-19 (although this can be generalized to other transmissible viral and bacterial infectious diseases), in addition to therapeutic treatments, control measures are essential. The recent outbreak of COVID-19 has demonstrated that the adoption of passive measures helps minimize the impact of current and future infection outbreaks, so that innovative nanomaterials may have a main role in the development of virus spread control measures which would be efficient against SARS-CoV-2. For example, research on antiviral textiles has received considerable attention, since these can effectively inhibit the spread of viruses or the formation of biofilms on their surface, reducing the risk of infection/re-infection[22][23][24][25]. When COVID-19 patients cough or sneeze, tiny virus-containing droplets are emitted and can contaminate surrounding surfaces, contributing to virus spread. This has increased researchers’ interest in developing not only new hand and surface disinfectants, but also self-decontaminating surface materials[26]. Herein wresearchers have reviewed the most recent and innovative nanomaterials proven to be effective against SARS-CoV-2, as well as possibly against other viruses, and useful to develop passive control virus spread measures [from masks and personal protective equipments (PPE) to disinfectants and surface coatings].
Several viruses, including SARS-CoV-2, may be successfully treated with metals (in particular, noble metals) and their complexes. Biomaterials based on metals such as Ag, Cu, gold (Au) and Zn have unique antiviral activities, durability, and efficacy at low concentrations. They are characterized by large-spectrum usage, and can successfully overcome the limitations faced by other conventional medicines since their action is independent of age and comorbidity, no drug resistance is developed, and low cytotoxicity is found[27][28]. However, their antiviral mechanisms are still not completely clear because selective interactions between metals and viral macromolecules, particularly proteins, cannot be easily identified. Experimental data have shown two main antiviral performances of metals: (i) the ability to prevent the viral infection by inhibiting the entrance of virus within the host system; (ii) the ability to affect processes involved in virus replication[29].
Thanks to the ability to slowly release the metal ions from metal NPs, metal-based materials are recently employed as virus spread control tools, providing long-term protection against viruses. For example, it was demonstrated that the use of these materials as coating agents can strongly reduce virus infectivity for several weeks[30]. In a liquid environment, the transport of the virus particles is much slower compared to that of metal ions going from metal NPs, and thus the antiviral effect from metal ions occurs faster[31].
Ag NPs, thanks to their tunable physicochemical properties, easy production routes and remarkable biological effects, including excellent antimicrobial action, are one of the most investigated nanomaterials for biomedicine applications[32]. The recent need for an effective agent against SARS-CoV-2 leads to a growing attention on the Ag NPs’ antiviral activity. As well-known, it is correlated to several mechanisms including interactions with viral envelope and with viral surface proteins (preferentially towards the ones rich in sulfhydryl groups). Other mechanisms regard the interactions with host cell pathways to prevent virus penetration and the interactions with viral factors necessary for virus replication[33][34].
Rodrigues et al.[35] investigated, through a computational approach, the spontaneous interaction among silver ions (Ag+) and five amino acids (glutamate, isoleucine, leucine, threonine, and lysine) present in the structure of the SARS-CoV-2 spike protein. The theoretical studies demonstrated that the interactions between Ag+ and -NH2 groups are more favorable rather than those with -C=O. The negative values of Gibbs free energies and the negative enthalpy energy variation (DH < 0) indicate that the interactions Ag+-amino acids are spontaneous and exothermic.
Jeremiah et al. evaluated the efficiency of Ag NPs with different diameters (2 to 15 nm) necked or capped with polyvinylpyrrolidone. This material was proposed as a virus spread control measure (to be used on inanimate and nonbiological surfaces) against SARS-CoV-2 infection, since it is able to disrupt viral integrity[36]. However, these Ag NPs could also interact with ACE2 receptors and/or intracellular mechanisms.
Merkl et al. have tested the antiviral activity against SARS-CoV-2 of glass and porous filter media (glass fiber filters and FFP3 filters) coated with Ag as well as CuO[31]. The antiviral activity of Ag was significantly higher than that of CuO; in fact, Ag reduced the viral load up to 75% after 5 min and 98% after 120 min instead the viral load using CuO was reduced to 54% and 76% after 30 and 120 min, respectively. One can hypothesize that Ag nanomaterial has a direct antiviral activity releasing Ag+ ions which bind to viral proteins or directly damage membranes, while CuO nanomaterials act through an indirect antiviral action by inducing the generation of reactive oxygen species (ROS).
Thanks to their good biocompatibility, poor immunogenicity, and ability to bind biological ligands, Au NPs are interesting candidates as antiviral agents[37]. The antiviral mechanism of Au NPs is based on the ability to prevent virus binding to cell membranes, and on the capability to inhibit virus proliferation. Cirri et al. found that also the organo-gold (III) compound Aubipyc possesses antiviral properties[29]. As evidenced by a computational study, the antiviral action of Aubipyc appeared due to the metalation of suitable metal-coordinating sites on viral proteins, that is the deprotonated forms of cysteine and selenocysteine, evidencing the main role played by the pH of the milieu in determining the occurrence of metalation. Although Aubipyc showed a low selectivity index and so it is not very suitable for in vivo tests, it could find useful applications in the field of viral spread control,
Zn has shown virucidal and antiviral properties[38][39][40]. Recenty, several authors have studied the effectiveness of Zn2+ ions against SARS-CoV-2 and the involved mechanisms[41][42]. Pormohammad et al.[43] used molecular modeling to demonstrate that Zn can bind COVID-19 RdRp (RNA-dependent RNA polymerase) and 3CLpro (3C-like proteinase). This bond influences the folded conformation and/or activity of these viral proteins, modulating viral replication. It was demonstrated that Zn2+ ions are able to bind the catalytic dyad of SARS-CoV-2 Mpro (main protease) via metal coordination bonds with His41 and Cys145 residues[44]. Tao et al. demonstrated the ability of Zn gluconate (a common zinc supplement) to inhibit the proteolytic activity of PLpro (papain like protease) and Mpro[45]. Crystallographic studies revealed two potential Zn2+-binding sites, one in the dyad catalytic center, and the other located on the surface of the protein structure. A lower affinity towards Mpro compared to other SARS-CoV-2 targets, including the ACE2 receptor and SARS-CoV-2 RdRp, was also confirmed studying the interaction of these proteins with ZnO by in silico docking studies, showing this descending order: ACE2 > RdRp > Mpro[46] (Figure 3).
Figure 3. (a) Docking results of ZnO NPs interacting with amino acids of COVID-19 RdRp and (b) corresponding zoom. (c) The related ligand-protein interaction diagrams. The dashed green lines indicate hydrogen bonds. Figure reprinted from Ref.[46] under the terms of the Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) license.
Zn2+ ions embedded in polyamide fibers was projected by Gopal et al. as a hybrid material useful in the fabrication of PPE; this material has the ability to decrease the SARS- CoV-2 titer by approximately 100-fold[41]. Two porous coating materials based on submicrometer ZnO particles bound with silica menisci and on ZnO tetrapods bound with polyurethane were projected by Hosseini et al.; the findings showed that infectivity is strongly related to material porosity and capability to absorb aqueous droplets[42].
Adhikari et al. proved that the favorable interactions between SARS-CoV-2 spike protein, spherical ZnO NPs, and different facets of the ZnO nanostructure induce a denaturation of spike proteins, which may lose the capability to bind the ACE2 receptors in human cells[47]; this non-toxic Zn nanomaterial was duly added to nanoceutical cotton fabric to produce a membrane filter to be employed in face mask fabrication (Figure 4).
Figure 4. (A) Schematic representation showing how ZnO petals added to nanoceutical cotton fabric may induce a denaturation of the spike protein; (B) The five steps for growing ZnO nanoflowers on cotton cellulose fibers. Reprinted with permission from Adhikari et al. Nanoceutical Fabric Prevents COVID-19 Spread through Expelled Respiratory Droplets: A Combined Computational, Spectroscopic, and Antimicrobial Study. ACS Applied Bio Materials, 2021 4 (7), 5471–5484. Copyright 2021 American Chemical Society[47].
Sportelli et al. verified the anti-SARS-CoV-2 activity of ZnO NPs ecofriendly produced in the presence of both cationic and anionic stabilizers (cetyltrimethylammonium bromide - CTAB, poly-diallyl-(dimethylammonium) chloride - PDDA, poly (sodium 4-styrenesulfonate) - PSS). When tested in vitro, the PDDA-ZnO NPs induced a decrease in viral load between 70% and 90%; however, no contribution was given by PDDA itself to the antiviral activity of the system. Then, PDDA-ZnO NPs were embedded into polyethylene oxide (a biodegradable and nontoxic polymer) film to reach a coating for frequently touched surfaces, with promising results despite the polymeric material limits the active surface of ZnO NPs to exert ionic release and subsequent antiviral activity[48].
A nano-spray disinfectant against SARS-CoV-2 based on ZnO NPs (with a size of about 50 nm) was designed by El-Megharbel et al.[49]. ZnO NPs showed anti-SARS-CoV-2 activity with potent antiviral activity at low concentrations (inhibitory concentrations [IC50] = 526 ng/mL) but with some cytotoxic effect to the cell host (cellular cytotoxicity [CC50] = 292.2 ng/mL), which limits its employment. To overcome the cytotoxicity issue and to reach high performing anti-SARS-CoV-2 systems, the functionalization of ZnO NPs with biocompatible materials (for example, polyethylene glycol) might be an interesting strategy[50].
Cu was the first metal to be declared as an effective metallic anti-microbial agent in 2008 by the U.S. Environmental Protection Agency (EPA). Unlike bacteria, viruses do not develop mechanisms of resistance to copper ions, leading to their high susceptibility towards this metal and its derivatives[51]. Antiviral activity of copper compounds seems to depend on the release of Cu2+ ions in solution, which generates ROS, leading to the loss of genome integrity, lipid peroxidation and deactivation of viral enzymes. A contact mechanism, based on metal ion binding and especially effective on the enveloped virus, has been described[52]. However, other mechanisms leading to an antiviral effect cannot be excluded. For example, Almalki et al. have synthetized a new thiazole derivative to inhibit COVID-19, by using Co(II) and Cu(II) complexes[53]. The results of their spectral analysis have been confirmed by theoretical calculations, explaining the details of the interactions with two COVID-19 proteins (namely the structures 6lu7 and 7bz5 from Protein Data Bank). Furthermore, Refat et al.[54] studied, via molecular docking analysis, the interaction of SARS-CoV-2 protease with Cu(II) complexes of deoxycholic acid (used in various fields of human medicine and food industry), evidencing their ability to interact with residues of this target protein.
A computational study by Aallaei et al.[55] demonstrated the correlation between the shape of Cu NPs (to be used as disinfectant) and their interaction with SARS-CoV-2 Mpro and spike glycoprotein. Cylindrical and conical Cu NPs were more efficient than spherical ones.
SARS-CoV-2 inactivation may be also induced by treatment with copper iodide (CuI) NPs[56], to be used as be applied to produce coatings for high-touch surfaces, masks, protective clothing and hand hygiene products. This virucidal action, maintained also using CuI doped film and fabric, is mainly due to the capability to destruct viral spike and nucleocapsid proteins and may be mediated through direct and indirect mechanisms related to cuprous ions (Cu+) release and ROS production. In fact, Cu+ generates hydroxyl radicals both in the presence of H2O2 by a Fenton-like reaction and in the absence of H2O2[57].
Jung et al.[58] showed the excellent antiviral performance of a copper-coated polypropylene (PP) filter face mask prepared by depositing a copper thin film on a spunbond PP filter surrounding a KF94 face mask. Oxygen ion beam pretreatment was employed to improve the film adhesion on the PP fibers and avoid copper film detachment (which should be a significant hazard for the possible inhalation of film particles).
Clay-based materials may be used for different biomedical purposes, due to their interesting properties which include large surface areas and adsorption capacity, thermal and chemical stability, good biocompatibility and low-cost[59]. Materials based on kaolin [Al2Si2O5(OH)4], a 1:1 clay mineral, can retain the virus on their surface, damaging viral proteins and thus blocking cell virus penetration[60]. Rius-Rocabert et al. developed a nanohybrid system consisting of Ag or CuO NPs supported on kaolin plates[61], to be used as a disinfecting antiviral. Both materials showed a strong reduction of viral infectivity against SARS-CoV-2, with a mechanism related to virus adsorption on kaolin plates and virucidal activity of the released metallic NPs and ions Another advantage of this nanocomposite is that kaolinite plates act as a dispenser for the metallic NPs, avoiding a discharge in the environment.
Pan et al. prepared a copper–zinc nanowire (CuZnNW) ink to be sprayed on high-touch surfaces[62]. Cu NW inactivates SARS-CoV-2 faster with respect to bulk Cu, and the addition of a lower amount of Zn (0.16 at % zinc) leads to an improvement of the virucidal activity; Zn stabilizes the copper ions release and thus the coating is effective for a longer time. Zhou et al.[63] have developed novel plastic films finalized to increase the effective contact area between virus particles and the surface coatings and deactivate SARS-CoV-2. The system is based on Ag NPs and Cu NPs (ranging between 10 and 40 nm), combined with nanoscale conical pillars, together with the addition of sodium dodecyl sulfate and polyvinyl acetate to ameliorate the features of the coating deposited on polyethylene terephthalate and polyethylene films (Figure 5).
Figure 5. Simplified scheme about the design of functionalized nanoscale conical pillars with antiviral properties. Figure reused from Ref.[63] under the terms of CC BY 4.0 license.
Bello-Lopez et al. demonstrated the efficacy against SARS-CoV-2 spread of nanometric layers of bimetallic AgCu deposited on polypropylene fibers to prepare reusable cloth masks[64]. Quantum chemistry calculations confirmed that the addition of Ag-Cu NPs makes the polymeric fiber a better electron acceptor, producing damage of viral phospholipids and genetic material. Similarly, Mosselhy et al.[65] showed that two Cu-Ag nanohybrids inhibited SARS-CoV-2 efficiently when used as surface coating; it contained a higher amount of Cu and lower amount of Ag (around 65 and 78 wt% and 7 and 9 wt%, respectively).
2.2 Carbon-Based Nanomaterials
Carbon based nanomaterials thanks to their interesting properties are deeply investigated for several therapeutic applications including antiviral performances[66][67][68][69]. The potentiality as biomedicine tools is mainly ascribed to their capability to cross the cellular membrane by different pathways and to directly interact with several biomolecules such as proteins[70][71].
The high ability of graphene oxide (GO) nanosized sheets to interact with spike protein, ACE2 and the ACE2-bound spike complex was recently studied by molecular docking. The results showed that GO binds strongly ACE2 and spike protein[72]. This is explained taking into account the 12 hydrogen bonds, 2 hydrophobic, and 1 electrostatic interaction computed for ACE2, in comparison with the 7 hydrogen bonds and 2 hydrophobic with ACE2-bound spike complex. GO nanosheets are also capable to hinder virus infectivity, showing in vitro antiviral activity against three different clades of SARS-CoV-2.
The antiviral activity of graphene (G) and GO on Vero cells infected with SARS-CoV-2 was also reported by De Maio et al. Furthermore, in this study, graphene and GO were used to functionalize polyurethane or cotton to obtain new hybrid materials (Figure 6). These latter are able to in vitro eradicate SARS-CoV-2 infectivity and thus potentially useful for the production of PPE[73]. GO could be employed also for water treatment and air purification due to its hydrophilic properties.
Figure 6. Schematic preparation and potentential applications of nanohybrid systems based on (A) hydrophilic GO and (B) hydrophobic graphene materials employed to defeat SARS-CoV-2. Figure reprinted from Ref.[73] under the terms of the Creative Commons CC-BY-NC-ND license.