Nanotechnology Tools to Inactivate SARS-CoV-2 in Different Environments Outside the Patient
SARS-CoV is highly stable at room temperature and at 4 °C, but it is inactivated by ultraviolet light at 254 nm, highly alkaline or acidic conditions of pH >12 or pH <3, respectively, or by brief (e.g., 5 min) heat treatment at 65 °C. SARS-CoV-2 is expected to be similarly sensitive.150 Several human coronaviruses can be inactivated by classical disinfectants, including bleach, ethanol, povidone-iodine, chloroxylenol, chlorheximide, and benzalkonium chloride,151 so we expect similar inactivation with SARS-CoV-2. The virus stability on surfaces depends on the composition of the infected material, with inactivation in <3 h on printing and tissue paper, in <2 days on treated wood and cloth, in <4 days on glass and banknotes, and in <7 days on stainless steel and plastic.152 Conversely, active viruses can remain on the outer layer of a surgical mask even after 7 days.152 The surface and aerosol stability of SARS-CoV-2 is comparable to that of SARS-CoV-1,153 with both viruses remaining viable in contaminated aerosols for more than 3 h. Infectious SARS-CoV-1 and SARS-CoV-2 remain viable up to 72 h after inoculation on plastic and stainless steel, whereas both are inactivated on copper in less than 4 or 8 h, respectively, and on cardboards in less than 24 and 8 h, respectively.153 Therefore, the stability of both viruses is similar, and we can hypothesize that surface treatments with NPs that proved to be effective for SARS-CoV-1 could possibly also be effective for SARS-CoV-2.
Nanotechnology can offer alternative methods to classical disinfection protocols used in healthcare settings, which typically rely on chemical-based disinfection using hydrogen peroxide stream or metal-ion-coated surfaces, biological-based strategies including probiotics or biosurfactants, or physical strategies such as irradiation with ultraviolet (UV) light. Nanotechnology may offer pathways to the development of self-disinfecting surfaces that would avoid contamination of the healthcare and housekeeping staff.
The methods proposed here for virus inactivation encompass the use of NPs and nanomaterials known for their intrinsic antipathogenic properties, such as metal-based NPs and graphene, or for their ability to inactivate viruses, bacteria, fungi, or yeasts either photothermally or via photocatalysis-induced ROS generation.

SARS-CoV-2 Inactivation by Nano-Based Tools
Silver, copper, and zinc show intrinsic antimicrobial properties and are already used in medical equipment and in healthcare settings. For instance, Ag is used in wound dressing and in urinary and intravascular catheters. It is advantageous to use NPs composed of these metals rather than bulk materials or the metal ions themselves because NPs release the toxic metal ions slowly and progressively right where the antimicrobial action is needed and because NPs can accumulate within cells without being expelled by specialized efflux pumps. The antimicrobial property of Ag has been used since ancient times for medical applications154 and more recently in commercial products such as silver zeolites in paints155 and in food trays156 as biocide. The antiviral efficiency of Ag NPs has been demonstrated in a variety of viruses, including HIV-1,157 monkeypox virus,158 bacteriophages UZ1 and MS2,159,160 murine norovirus MNV1,159,160 HSV,161 HBV,162 and, recently, in porcine epidemic diarrhea virus (PEDV).163Antiviral properties of Ag NPs arise from three different mechanisms. First, Ag(0) NPs dissolve and release some toxic Ag(I) forms (including Ag+ ions), which could be responsible for their antiviral activity. As a soft metal, Ag shows strong affinity toward sulfur, and therefore, it interacts strongly with thiols from small molecules such as cysteine or glutathione or with sulfhydryl groups in the active sites of many enzymes. Ag(I) may interact with surface proteins of viruses or accumulate in host cells and further interact with thiol-containing enzymes that are involved in virus replication, thus hampering their functions. This hypothesis was proposed by Zodrow et al. to explain the antiviral property of Ag NPs for bacteriophage MS2 and by De Gusseme et al. in response to MNV-1 exposed to Ag NPs.159,164 Moreover, Ag2S nanoclusters (NCs) with diameters of 2.5 and 4 nm showed effective inhibition of PEDV replication in Vero cells via inhibition of the synthesis of viral negative-strand RNA and of virus budding from the cells, but not by preventing their anchorage on cell membranes or their intracellular penetration. Exposing cells to Ag+ ions at the same concentration did not inhibit virus replication, which led the authors to conclude that the antiviral property of Ag NCs was independent of the release of Ag(I).163 However, the mechanisms by which Ag+ ions and Ag NCs enter into cells are different and, consequently, their local distribution and handling within cells would also be different. This difference could lead to different modes of toxic action for Ag ions and Ag NCs toward the viruses that have infected cells. For instance, Ag NCs may aggregate in intracellular areas where vital steps of the viral cycle are performed, such as protein or genome production or assembly of nucleocapsids before their release into the extracellular space, whereas Ag(I) could aggregate in other areas of the cells or be rapidly eliminated. Second, the antiviral efficiency of Ag NPs would derive from physical interaction of Ag NPs with the surface of viruses, which would impede their docking on host cells and limit their infectivity. This mechanism was demonstrated by Elechiguerra et al. for HIV-1 exposed to 1–10 nm Ag NPs157 and by Orlowski et al. for HSV-2 exposed to 13, 33, and 46 nm Ag NPs coated with tannic acid.161 Elechiguerra et al. found that the optimal size of Ag NPs was around 10 nm, with larger or smaller NP sizes showing weaker physical interaction with the virus. In contrast, Orlowski et al. found that the larger the NP, the more effective its blocking was of virus attachment to host cell. The same mechanism, combined with the release of Ag(I), was also proposed by De Gusseme et al. to explain the reduced infectivity of MNV-1 virus when exposed to 11.2 nm biogenic Ag NPs.159 Finally, this docking of Ag NPs on the surface of viruses could be associated with the local release of ROS from the Ag NP surface, which would damage the envelope and/or membrane of the virus. Ag NPs are already used in wound dressings, catheters and other medical equipment; their use could also be envisaged to confer biocidal properties to paints used in healthcare settings, or to air filters or face masks. Ag NPs loaded on filters show effective antiviral activity against bacteriophage MS2, which drops with dust loading.165
The antimicrobial activity of Cu has also been known since ancient times,166 and surfaces containing a significant amount of Cu have demonstrated their efficacy to inactivate viruses. Murray et al. showed the efficacy of Cu against poliovirus in 1979.167 More recently, the efficacy of Cu was demonstrated on the HuCoV-229E coronavirus; the effectiveness of Cu to inactivate other forms of coronaviruses suggests potential similar efficacy against SARS-CoV-2.168 Whereas HuCoV-229E persists for more than 6 days in an infectious state on smooth surfaces (Teflon, polyvinyl chloride, ceramic tiles, glass, stainless steel), it is inactivated in less than 60 min on brasses containing at least 70% Cu or Cu–Ni alloys containing at least 90% Cu.168 When incubated on Cu-containing surfaces, the viral genome becomes fragmented, ensuring the irreversibility of inactivation.168 The proposed inactivation mechanisms include both toxicity toward virions of Cu ions released from the Cu-containing surface and attack of viral proteins and lipids by ROS generated from Cu reacting with exogenous hydrogen or molecular oxygen through Fenton-like or Haber Weiss reactions.166 Likewise, both SARS-CoV-1 and SARS-CoV-2 are inactivated on Cu surfaces in less than 4 h, whereas they persist for 48–72 h on plastic and stainless steel and less than 24 h on cardboard.153 In this case, the main inactivation mechanism is also proposed to be damage to viral proteins and lipids by Cu ions and ROS, in particular, envelope proteins.153 Using Cu brasses or Cu-containing alloys rather than stainless steel would provide effective antimicrobial surfaces (doorknobs, bed rails, etc.) in healthcare settings. Supported catalysts composed of Al2O3 impregnated with Ag and Cu to form Ag/Al2O3 (5% Ag) and Cu/Al2O3 (10% Cu) also inactivate SARS-CoV virus in less than 5 and 20 min, respectively, which would be useful for air disinfection.169
Cu and CuO NPs have also been shown to release Cu ions when in contact with live cells.170,171 The large surface that NPs develop due to their small size endows them with a reactivity higher than that of their bulk counterpart and would fasten the kinetics of Cu ion release. The use of nanostructured Cu surfaces would further enhance their antimicrobial activity. Moreover, these NPs could inactivate viruses if sprayed on contaminated surfaces or loaded onto textile fabrics to confer antimicrobial properties (masks, blouses, etc.). Indeed, CuO-impregnated masks have shown remarkable anti-influenza virus (H1N1 and H9N2) activity under simulated breathing conditions,172 and the activity of these materials toward SARS-CoV-2 should be investigated. The viral disinfectant properties of Ag NPs and CuO NPs is further enhanced when they are combined with Fe as bimetallic particles, due to coupled redox reactions between the two metals.173
In addition to metal NPs, graphene derivatives have also shown promising viral inactivation properties.174 For example, graphene oxide (GO) sheets and sulfated GO derivatives have been found to be effective against herpes simplex virus type-1 (HSV-1) infections, with viral binding and shielding as the two putative main inhibitory mechanisms.175 Thermally reduced graphene oxide (rGO) sheets functionalized with biocompatible hyperbranched polyglycerol (hPG) and then sulfated have also been generated as graphene-based heparin biomimetics.176−178 Sulfate-rich polymers like heparan sulfate and its equivalent soluble counterpart heparin are widely known as broad antiviral agents,179,180 but their use is limited due to their anticoagulant effects. Sulfated rGO-hPG sheets were found to be effective at inhibiting orthopoxvirus and herpesvirus strains, particularly in the early stages of the infection, although they could not prevent cell-to-cell spread. Additional antiviral activity of graphene derivatives has been attributed to the negative surface charges and sharp edges of the individualized sheets, as the electrostatic interactions promote binding with the positively charged virus particles. Negative charges on sharp-edged single-sheet GO and rGO were shown to bind and to suppress the infection of pseudorabies, PEDV, EV71, and H9N2 viruses.181,182 This mechanism suggests that potentially similar antiviral effects could be offered by other negatively charged, sharp-edged 2D nanomaterials such as Ti3C2Tx MXene, which has shown promising bacterial inactivation effects against both Gram-positive and Gram-negative species due to similar hypothesized mechanisms.118,183
Graphene derivatives linked to virus-specific antibodies have also been adopted in antiviral platforms based on antibody-mediated binding and sensing mechanisms, which have been shown to capture a number of viral species successfully including rotavirus, avian influenza virus subtypes H5 (AIV H5) and H7 (AIV H7), and influenza virus H1N1.184−187

Photothermal Inactivation of SARS-CoV-2
Another example of the use of noble metals for disinfection is the ability of Ag and Au NPs and nanorods to induce heating when illuminated at optimal wavelength, corresponding to the plasmon resonance condition, in a process called plasmonic photothermal treatment.188 This property is currently being evaluated by nanomedical researchers as a method for killing cancer cells with Au NPs because Au NPs are considered much less toxic than Ag NPs. By tuning NP size and shape, it is possible to emit intense heat under solar irradiation of Au NPs and to inactivate viruses, as demonstrated by Loeb et al. with bacteriophages MS2 and PR772 exposed to Au nanorods and nanocubes or to surfaces coated with such NPs.189 Close contact between NPs and the pathogen is necessary for the process to be effective, which supposes that NPs adsorb onto their surface. Elsewhere, Nazari et al. used femtosecond pulsed laser irradiation on Au nanorods to inactivate murine leukemia virus (MLV).190 Here, inactivation did not require contact between Au NRs and viruses, and the addition of ROS scavengers did not reduce the inactivation effect, suggesting that neither heat nor ROS generation accounted for the observed effect. The authors propose that the underlying mechanism is plasmon-enhanced shockwave generation, which alters virus membrane and/or surface groups and, therefore, reduces virus binding and fusion with the host cell.190

Inactivation of SARS-CoV-2 by Photocatalytic Nanoparticles
Photocatalytic NPs offer another possible approach to the inactivation of SARS-CoV-2. The most described NP in this category is titanium dioxide (TiO2), which shows photocatalytic properties when illuminated under UV light, is considered inert, has low toxicity, and is not susceptible to photocorrosion.191 TiO2 is currently used in paints and lacquers,155 in self-cleaning windows, and for water purification.192 TiO2-containing paints have also been envisaged for purifying ambient air because photocatalytic TiO2 successfully removes volatile organic compounds (VOCs) when exposed to UV light. However, recent findings show that VOC disruption is associated with the release of toxins in the air, which questions the prudence of TiO2-doped paints for air purification purposes.155 If effective, the use of TiO2 photocatalysis for SARS-CoV-2 inactivation would be particularly useful for surface decontamination using TiO2-doped paints, aerosol decontamination using air filtration filters and ventilation systems impregnated with TiO2 that can be exposed to UV light, and for wastewater treatment.
The underlying mechanism of this photocatalytic process relies on the excitation of an electron from the valence band (VB) of the photocatalytic material to the conduction band (CB) when exposed to UV light, which leaves a positive hole (h+) in the VB. The e–/h+ charge carriers migrate to the surface of the photocatalyst and initiate reactions leading to the production of ROS, including the superoxide anion, hydrogen peroxide, the hydroxyl anion, and hydroxyl radical.193 The production of hydroxyl radicals by the oxidation of water molecules on the photocatalysts’ surface accounts for their disinfection activity, owing to their capacity to oxidize many organic constituents of microorganisms, such as lipid peroxidation, leading to damage to cell wall and cell membrane, protein alteration, and/or DNA damage.193 Bare TiO2 exposed to UV light is effective against a broad spectrum of Gram-positive and Gram-negative bacteria, including multi-drug-resistant strains but also against some fungi, viruses, and yeasts. As discussed by Bogdan et al., according to some authors, viruses would be more susceptible to inactivation than bacteria.194 Among viruses, some researchers have found that enveloped viruses would be more protected from photocatalytic inactivation than non-enveloped viruses, whereas other authors reported the opposite.194 Only one article reported the usefulness of this inactivation strategy for treatment of SARS-CoV, using a photocatalytic titanium apatite filter (PTAF). This filter showed effective inactivation of SARS-CoV when exposed for 6 h to UV light.195 One could also imagine that photocatalysts coupled to UV light could damage spike proteins and lead to decreased infectious capacity of the virus.
Because TiO2 shows low solar light activity and a high recombination rate of electron–hole pairs, researchers have developed second-generation photocatalysts in which TiO2 is used in combination with other components, such as metals. This new generation of photocatalysts shows high efficiency of inactivation of a wide range of bacteria and some viruses. Among them, S-doped and N-doped TiO2 show photocatalytic properties when exposed to visible light and, therefore, would possibly be effective under interior lightning. The antimicrobial properties of these photocatalysts have been tested with a variety of bacteria, sometimes indicating good disinfection efficiency (for reviews, see refs (192) and (193)), but to our knowledge, they have not been tested on viruses. Moreover, depositing some Ag NPs on the surface of TiO2 NPs increases their antiviral efficiency against MS2 by means of increased production of hydroxyl radicals.196 A Ag- and Cu-doped TiO2 nanowire membrane is more active in eliminating bacteriophage MS2 from drinking water than are TiO2, Ag-TiO2, or Cu-TiO2 membranes, both in the dark and when exposed to UV light. The underlying mechanism is thought to combine both enhanced photoactivity due to the lower band gap of (Ag, Cu)-TiO2 than that of TiO2197 and antimicrobial activity of free Ag and Cu ions released into the treated water.198 Another strategy to improve the antiviral capability of TiO2 is by increasing its potential to absorb viruses, which has successfully been achieved by mixing TiO2 NPs with SiO2 NPs. Due to the large specific surface area of SiO2, the mixture of NPs inactivated bacteriophage MS2 more effectively than did TiO2 alone, in spite of reduced hydroxyl production.199 Glass slides coated with TiO2 doped with Pt show slightly better efficiency in inactivating aerosols containing influenza A (H3N2) virus than do surfaces coated with only TiO2 when irradiated with UV-A,200 owing to their increased oxidizing photocatalytic properties. Finally, as described by Byrnes et al.,193 new photocatalytic materials that show efficient antibacterial activity have been developed and could be tested for the inactivation of SARS-CoV-2. These new materials include (among others) BiVO4, CuFeO2, CuYxFe2–xO4, LaFeO8, CuMn2O4, ZnMn2O4, BaCr2O4, SrCr2O4, NiCo2O4, CuCo2O4, LaCoO3, and La0.9Sr0.1CoO3. Importantly, before being used for SARS-CoV-2 inactivation, their nontoxicity should be ensured.

Nanotechnology-Based Solutions to Increase the Efficiency and Safety of Protective Devices
Cryoelectron microscopy (cryo-EM) studies show that SARS-CoV-2 virions are particles near the larger end of the NP size range (70–90 nm).21,201 However, when dispersed into the air, the infectious particles exist as functionally larger particles. Initially, liquid droplets containing coronavirus virions originating from the respiratory tract of infected patients are emitted during normal breathing, forced expiration (e.g., coughing and sneezing), or aerosol-generating medical procedures (e.g., intubation and suctioning). Liquid droplets emitted into the air through these mechanisms originate from points throughout the respiratory tract and carry within them virions as well as other materials associated with the airways, including bacterial cells and epithelial cells (Figure 5). Droplets are emitted over a wide size range, and their potential viral burden is a cubic function of particle diameter (Figure 6). Thus, larger droplets have the potential to carry a significantly larger burden of virions according to their size and are substantially more hazardous than small droplets. Once emitted into the air, water from droplets immediately begins to evaporate. Water loss is associated with a rapid decrease in both particle diameter and terminal settling velocity (the equilibrium rate of fall of a particle in still air). The rate of evaporation of the largest droplets (>150 μm) is often insufficient to slow their swift descent in air, and they impact on nearby surfaces. However, the rapid water loss and sharp slowing of settling velocity of smaller droplets enables them to avoid a similar fate. Their constituent solid residues are instead drawn together during evaporation and cemented with dried respiratory secretions, and they remain aloft as droplet nuclei. Several epidemiological studies have supported the potential for droplet nuclei to be an important means of transmission for SARS-CoV-2.202,203
Figure 5  Droplets and droplet nuclei as important mechanism for transmission of infection. Liquid droplets containing SARS-CoV-2 virions originating from the respiratory tract of infected patients are emitted into the air and carry other materials including bacterial cells and epithelial cells. They are reduced in size by evaporation to small, dry particles resulting in droplet nuclei.
Figure 6  Maximum theoretical viral burden versus droplet nucleus size (nm). Assuming a median virion size of about 100 nm, a droplet nucleus of 1 μm diameter could contain up to 370 randomly packed virions. A similar 10 μm diameter droplet nucleus could contain up to 360,000. In practical terms, however, the size-defining element of a droplet nucleus is determined by the component item with the largest volume, which is often a bacterial cell or an epithelial cell. Thus, droplet nuclei in the 1–10 μm size range and above contain far fewer virions than this theoretical maximum.
Filter media, such as those used in N95 masks and in mechanical ventilation systems, consist of myriad interwoven fibers through which air is moved. Their purpose is to arrest particles as they move through the matrix. Filters capture particles chiefly by three mechanisms: impaction, interception, and diffusion. Impaction occurs when the momentum of a particle propelled toward a filter fiber prevents the particle from diverging around the fiber along the flow lines of the air stream, causing the particle to collide with the fiber. Impaction is the primary mechanism responsible for removing particles greater than 500 nm in diameter. Interception occurs when a particle diverges around a fiber along the flow lines of the airstream, the distance between the vector of the airstream and the centroid of the particle is smaller than the radius of the particle, and the particle touches the fiber. Interception operates efficiently on particles greater than 200 nm in diameter. Diffusion is the final important mechanism of particle removal, and it is most effective at removing very fine particles less than 200 nm, especially at low flow rates. Particles around 300 nm in diameter are least subject to these three removal mechanisms, and they are considered the “most penetrating” particles for a majority of filter types.204
When virus-laden droplet nuclei are deposited on filter media, they penetrate the filter matrix to different depths depending on their size characteristics: larger particles tend to become impacted or intercepted nearer the surface of the intake-facing side, whereas smaller particles penetrate more deeply into the fibrous matrix. In the case of filtering facepiece masks, cyclical breathing can cause changes in the physical characteristics of particles after they have been deposited. Humid exhaled air causes hygroscopic droplet nuclei to swell, becoming larger than they were when captured on filtration media. This size change can affect the ability of the filter fibers to retain particles and can lead to redistribution, shedding, or even breakthrough of particles.
From the standpoint of COVID-19, there are many opportunities for nanotechnology-based solutions to increase the efficiency and safety of air filter and mask devices. Some specific opportunities include (i) improving particle capture and retention characteristics, particularly, in the 300 nm diameter size range; (ii) reducing the effects of exhaled humid air on particle redistribution; (iii) rapid inactivation of membrane-bound microbes including enveloped viruses upon capture; and (iv) thin, high-efficiency filtration media for personal masks that are able to be reused repeatedly without loss of efficiency (e.g., novel electrospun nanofibers). In this context, recent findings exploring the performance of several fabrics commonly used in cloth masks, alone or in combination, suggest that the combined mechanical and electrostatic effect observed in hybrids enabled enhanced performance with a filtration efficiency >80 and >90% for particle sizes <300 and >300 nm, respectively.205
Electrospinning is a technique that is widely used to produce nanofibers with diameters smaller than a micrometer (typically, ∼100 nm). Even a micron-thin layer of nanofibers can capture the smallest droplets containing viruses and bacteria and prevent them from traveling through the mask. TiO2-coated nanofibers deposited on a filter surface by the electrospinning process can capture submicrometer droplets and destroy the virus upon UV irradiation or under natural sunlight. After a micrometer-thick film of polyamide 11 nanofibers was deposited on polypropylene filter fabric, TiO2 NPs were directly electrosprayed onto the nanofibers.206 Scanning electron microscopy (Figure 7) demonstrated that nanofibers were uniformly coated by TiO2 NPs without agglomeration. TiO2-coated filters showed excellent photocatalytic and bactericidal activity and photoinduced hydrophilicity.
Figure 7  Scanning electron microscope images of electrospun nanofibers on polypropylene filter fabric (a) and titania-coated electrospun nylon nanofibers (b). Reprinted with permission from ref (206). Copyright 2010 Springer Nature.