Carbon-Based Nanomaterials: Promising Antiviral Agents to Combat COVID-19 in the Microbial-Resistant Era

Therapeutic options for the highly pathogenic human severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing the current pandemic coronavirus disease (COVID-19) are urgently needed. COVID-19 is associated with viral pneumonia and acute respiratory distress syndrome causing significant morbidity and mortality. The proposed treatments for COVID-19 have shown little or no effect in the clinic so far. Additionally, bacterial and fungal pathogens contribute to the SARS-CoV-2-mediated pneumonia disease complex. The antibiotic resistance in pneumonia treatment is increasing at an alarming rate. Therefore, carbon-based nanomaterials (CBNs), such as fullerene, carbon dots, graphene, and their derivatives constitute a promising alternative due to their wide-spectrum antimicrobial activity, biocompatibility, biodegradability, and capacity to induce tissue regeneration. Furthermore, the antimicrobial mode of action is mainly physical (e.g., membrane distortion), characterized by a low risk of antimicrobial resistance. In this Review, we evaluated the literature on the antiviral activity and broad-spectrum antimicrobial properties of CBNs. CBNs had antiviral activity against 13 enveloped positive-sense single-stranded RNA viruses, including SARS-CoV-2. CBNs with low or no toxicity to humans are promising therapeutics against the COVID-19 pneumonia complex with other viruses, bacteria, and fungi, including those that are multidrug-resistant.

H istory has repeatedly manifested that pathogens cause disastrous effects on human beings. Thus, the recent outbreak of the recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes Coronavirus disease 2019 , that has spread to more than 200 countries, is a clear example. The current confirmed global COVID-19 cases and deaths have reached more than 90 million and more than 2 million, respectively. 1 Nevertheless, experts have suggested that many more undetected or asymptomatic cases exist, 2 especially in underdeveloped countries. COVID-19 continues to spread globally, threatening to collapse the health system of many developed countries such as the United Kingdom and France that have been forced to go to a third lockdown. SARS-CoV-2 is an enveloped positive-sense, single-stranded RNA virus. 3−5 Its origin to this date remains enigmatic; however, multiple hypotheses have been postulated so far. 6 The host tropism/ adaptation pattern raised questions concerning the origin of SARS-CoV-2. 7 SARS-CoV-2 is the seventh coronavirus known to infect humans easily 8,9 and only the third one causing severe pneumonia. 10 Viral pneumonias may be complicated by secondary microbial infections. 11 Thus, coinfection can be caused by viruses in the setting of community-acquired bacterial pneumonia. 12−14 Coinfection of COVID-19 patients is seen with the most common type of bacterial pneumonia caused by Streptococcus pneumoniae. 15 There is a great concern about the rapid spread of pathogens, such as SARS-CoV-2, that can coexist with a broad range of other types of clinically relevant microorganisms, including those which are multidrugresistant. Therefore, the coinfection of SARS-CoV-2 with other viruses, bacteria, or fungi constitutes a real life-threatening situation to humans during the approaching cold season. In this regard, several medications have been proposed which include remdesivir, hydroxychloroquine, lopinavir/ritonavir, interferon β-1a, tocilizumab, favipiravir, plitidepsin, convalescent plasma infusions, and monoclonal antibodies, among many others. 16 However, presently, there is no effective treatment for COVID-19. 17,18 Furthermore, antibiotic resistance in bacterial pneumonia treatment is a widespread problem. 19−21 Therefore, in the quest to finding therapeutics for COVID-19, carbon-based nanomaterials (CBNs) are emerging as promising options that have shown potent antiviral activity against a broad range of enveloped positivesense single-stranded RNA viruses, 22 including SARS-CoV-2, and showed low to no toxicity in humans. 23−28 Also, they exert an effective biocidal action against a broad spectrum of bacteria, viruses, and fungi, including multidrug-resistant strains. 29−31 These CBNs are mainly composed of carbon, an essential element in the human body, 32 and are thus biodegradable and biocompatible and can induce tissue regeneration. 33−37 Moreover, the development of CBNs as antiviral agents is possible, because they possess a high surface area that allows their functionalization or interaction with biocompatible polymers, which further enhances their biocompatibility and therapeutic efficacy. The epithelial response to viral challenge is well-documented to involve autophagic and apoptotic cell death. 38 Indeed, the primary receptor for SARS-CoV-2, the carboxypeptidase ACE-2, is a protective "survival factor" for human lung alveolar epithelial cells (AECs), 39 but is significantly reduced by SARS-CoV2 infection. 40 The cells which express the most ACE-2 in the human lung alveoli, the type II AECs, normally serve as stem cells for regeneration of lost alveolar epithelia, 41 but are killed by viral challenge. For these reasons, any potential therapeutic that exerts both antiviral activity and the capacity to stimulate tissue regeneration would be expected to promote lung tissue repair in the face of ongoing viral-induced cell death. Research in this area is still in its early stage, though it is predicted to grow exponentially due to the grave consequences caused by the current pandemic. The increasing number of multidrugresistant pathogens announced by the World Health Organization constitutes another real threat to humanity in this microbial resistance era. 42 Therefore, there is an urgent need to find alternative antimicrobial strategies to curb the drug-resistance menace, thus providing a long-lasting treatment for the COVID-19 disease. This Review addresses the application of these broad-spectrum CBNs as antimicrobial agents by analyzing a large number of antiviral studies performed so far with CBNs against enveloped positive-sense single-stranded RNA viruses, looking at their toxicity and biodegradability and deciphering how they could defeat microbial resistance. This vision could surpass substantially any technological paradigms that currently exist or are under development as a part of advanced therapeutics to treat COVID-19.
NEXT GENERATION OF ANTIMICROBIALS: CARBON-BASED NANOMATERIALS Antiviral activity of viral infection compounds can be classified into two different subgroups: (1) virus-inhibiting compounds (usually cellular); or (2) compounds that augment host defense by improving or altering virus infections (immunomodulating agents). 43 Antiviral mechanisms usually require an inhibitory effect of a particular viral cell cycle essential in viral replication. As viral replication mainly depends on the host cell's metabolism as viruses are obligate intercellular parasites, valuable antivirals can disrupt viral-specific functions or at least obstruct virus-directed functions as opposed to the host cell. Therefore, the spectrum of antivirals generally is restricted. Many existing antiviral agents block host cell surface attachment followed by conformational changes of the viral capsid, leading to the uncoating of the viral genome, preventing the virus from penetrating the target cell. Others, such as nucleoside or nucleotide analogues, are antiviral drugs that provide an impairment of nucleic virus synthesis as their mode of action. An essential factor for antiviral activity is the inhibition of virus attachment to a cellular receipt or viral entry, inhibiting the viral from entering the cells. Many viruses involve proteolytic cleavage of polypeptide precursors, utilizing protease inhibitors as antivirals to active, vital viral proteins. Some of the antivirals inhibit the integration of the viral genome with the host cells' genome. Other antivirals inhibit the uncoating (viral disassembly) and therefore disturb the viral life cycle. Finally, the virus release from the host cells is inhibited through the prevention of viral clumping, thus inhibiting cell-to-cell movement of viruses through enzymatic inhibition. 44−46 To control the ever-increasing range of infections caused by multidrug-resistant microorganisms and viruses, 42 the use of antibiotics or alternative antimicrobial agents such as the ionic and/or oxidized form of metals, 47 quaternary ammonium compounds, 48 peptoids, 49 α-peptides, 50 and β-peptides 51 present diverse problems including drug resistance after longterm utilization. In this regard, alternative materials such as CBNs with intrinsic broad-spectrum antimicrobial activity 52−63 represent a promising option that would probably overcome the microbial resistance problem due to their differential antimicrobial mechanisms. Thus, the antimicrobial action of CBNs, such as graphene (G), is often attributed to a combination of several physical and chemical mechanisms: directly on the microbial particle such as (peptidoglycan membrane structure disruption, entrapment of microorganisms, transfer of electrons) and/or indirectly through induction of oxidative stress by reactive oxygen species (ROS). 64,65 Other nanomaterials such as those based on silver, copper, titanium, or zinc nanoparticles, for example, have shown strong broad-spectrum antimicrobial properties. 47,66−69 However, the existence of microbial resistance to these nanomaterials 70−76 and their high toxicity to mammalian cells 77−81 render these materials less promising for long-term therapeutics. Therefore, CBNs are increasingly proposed as the next generation of antimicrobials against multidrug-resistant infections. They possess unique properties that include very high surface area, excellent electrical and thermal conductivity, biocompatibility, and the possibility to be combined with engineered polymers to develop advanced antimicrobial biomaterial composites. 2829,82,−85 Furthermore, a recent study about the paramount concern of this proposal regarding the interaction of CBNs with the respiratory system showed that a single exposure of several CBNs (at ∼0.3 and 1 μg/cm 2 ) did not manifest any adverse effects under acute exposure scenarios after 24 h. 86 Regarding the biodegradability of CBNs, it has been demonstrated that the human myeloperoxidase, a peroxide enzyme released by neutrophils, degraded graphene and its derivatives, 87,88 leading to biodegradation of graphenebased nanomaterials (GBNs) in the blood after 14 days. 89 Some signals of in vivo degradation of graphene were reported in the lungs, liver, kidneys, and spleen upon 90 days. 90 Oxidized forms of GBNs have shown higher susceptibility to degradation than the reduced forms. 91 The degradation products of graphene oxide (GO) with different dimensions exhibited no genotoxicity to human lung cells. 92 In vivo studies have shown that a concentration of 1.0 mg/kg of small GO particles (148−160 nm) tends to accumulate mainly in the liver with a lower amount in the spleen and lungs and remains longer in circulation than large particles (556−780 nm), 93 which were mostly present in lungs. However, when the concentration of injected GO increased 10-fold, the smaller GO particles accumulated in the lungs instead of the liver, thereby potentially increasing their efficacy for treating pulmonary infections. GO and reduced GO (rGO) of different lateral dimensions (10−800 nm) in a concentration of 1 mg/ kg exhibited long blood circulation times of 14 days after intravenous administration in mice and low uptake by the endothelial reticulum. No pathological variations were detected in the analyzed organs. 85 Biodegradation and cytotoxicity toward different cell lines depended on concentration, exposure time, oxidation degree, lateral size, and cell type 94−97 and could be modified by functionalization. 94,98−101 Smaller and more oxidized GBNs seem to be more cytocompatible than nonoxidized and larger particles. 99 Proteins, such as bovine serum albumin, adsorbed on the GBNs' surfaces, seem to have a protective effect on the hemolytic potential. 102−105 The biocompatibility of CBNs depends on their concentration, oxidation degree, lateral size, and dispersibility. 106−114 The inflammation and other effects on cells and blood components are minimal when a lower concentration of CBNs is applied. Oxidized and smaller GBNs are more biocompatible and accessible to biodegradation in the body. CBNs have shown the potential to support the growth, proliferation, and differentiation of stem cells into different tissue lineages. 35,115 These features potentiate the use of CBNs in combination with stem cell therapies for tissue regeneration as well as for COVID-19 patients. SARS-CoV-2 infection is accompanied by many physiopathological changes and could reach all human organs harnessing several pathways; 116 during this, it reaches and damages the vascular endothelial cells. CBNs can induce the production of angiogenic factors, which initiate a series of signaling pathways to induce angiogenesis through promoting the proliferation and differentiation of endothelial cells or mesenchymal stem cells (MSCs). 117−119 In addition to the MSCs not infected SARS-CoV-2 infection causes many pathophysiological changes such as tissue inflammation, immune system damages (leukopenia, lymphopenia), respiratory microstructure and distal organ injury and secondary infections, and microvascular system damage. CBNs in combination with MSCs have the potential to target these pathophysiological events, acting as an alternative strategy for treating COVID-19 patients.
with SARS-CoV-2, it is of note that the stem cells (specially the mesenchymal stem) represent one promising strategy for COVID-19 therapy, specifically those in moderate and/or severe infection. 120−123 Also, MSCs downregulate Th1 and Th17 inflammation immunity and upregulate the antiinflammation immunity of Th2 and Treg cells, and they enhance the recruitment and proliferation of many productive cells and supportive materials (collage and extracellular matrix). Because of its potential, up to 21.02.2021, 9 (out of 91) clinical trials have been completed. 124 MSCs are capable of self-replication and differentiation into specialized functions to replace the disrupted and/or dysfunctioning cells/tissues. 125 SARS-CoV-2 induces its pathological changes via exacerbating cytokine production, which leads to many complications that may become severe in some cases. Stem cells have specific cytokines 126 that tightly derive immunomodulation ( Figure 1), which may not only be helpful in controlling the SARS-CoV-2 infection severity 125,127−129 but also extend beyond patient recovery specifically from severe infection. 130

ANTIVIRAL PROPERTIES OF CARBON-BASED MATERIALS
This section analyzes the antiviral properties of CBNs with different carbon-based structures (Figure 2), such as fullerene, carbon dots, graphene, and derivatives against 13 enveloped positive-sense single-stranded RNA viruses, such as SARS-CoV-2.
Fullerene and Derivatives. Fullerene is a zero-dimensional allotrope of CBNs with antiradical and antioxidant properties. 133,134 Due to the high hydrophobicity character of pristine fullerene, antiviral fullerene derivatives can be synthesized to produce hydrophilic drugs that can be easily dispersed in aqueous media. 30 Studies on fullerenes as antiviral agents started in 1993 on human immunodeficiency virus type 1 (HIV-1) infections. 135 In that study, compound 1 showed effective in vitro antiviral activity. Another study showed that a bis(monosuccinimide) derivative of p,p′-bis(2-aminoethyl)-diphenyl-C 60 was actively inhibiting HIV-1 and also the type 2 (HIV-2) in acutely or chronically infected human lymphocytes and against 3′-azido-3′-deoxythymidine-resistant HIV-1. Another subsequent study showed a water-soluble fullerene−peptide conjugate capable of interacting, albeit weakly, with the HIV-1 protease. 136 In 1996, nine functional derivatives of C 60 -fullerene compounds displayed antiviral capacity at low micromolar concentrations. 137 Furthermore, three of these compounds exhibited antiviral activity at lower concentrations than any fullerene derivative reported to that date. In 1997, nonderivatized fullerene (buckminsterfullerene) showed in vitro antiviral activity against another two enveloped positive-sense single-stranded RNA viruses similar to SARS-CoV-2: the Moloney murine leukemia virus (M-MuLV) and the simian immunodeficiency virus (SIV). 138 However, a study performed in 2003 tested a series of fullerene derivatives (13 in total, compounds 1−13) against HIV-1 and HIV-2. The results of that study showed that some of these CBNs (6 (trans-2), 7 (trans-3), 8 (trans-4), 9 (equatorial), 12 (trans-2)) exhibited potent antiviral activity against HIV-1 but not HIV-2 in the low micromolar concentration range. 139 Nonetheless, cationic fullerene derivatives showed broader anti-HIV properties. 140 In 2007, chlorofullerene was developed as a precursor for the straightforward synthesis of antiviral fullerene derivatives against HIV with high solubility in water. 141 A later study in 2011 showed that derivatives of C 70 -fullerene (i.e., the fullerene molecule consisting of 70 carbon atoms) exhibited high water solubility and virucidal activity against HIV and influenza virus. 26 A series of fullerene derivatives ( Figure 3) showed potent viral inhibition of hepatitis C virus (HCV). 142 Recently, tridecafullerenes appended with up to 360 1,2mannobiose molecules showed outstanding antiviral activity against zika virus (ZIKV) and dengue virus (DENV). 24 Examples of antiviral studies performed with fullerenes and their derivatives are summarized in Table 1.
Carbon Dots and Derivatives. Carbon dots (CDs), also known as carbon quantum dots (CQDs) or Cdots, are other ACS Nano www.acsnano.org Review members of the CBN family with small dimensions up to 10 nm in diameter that are cost-effective and environmentally inert. 23,156 They possess an organic molecule's chemical functionality, show a very high surface-to-volume ratio, and can be homogeneously dispersed in water. Many CDs are explored in several fields, such as chemical sensing, bioimaging, electrocatalysis, and other applications. 62,157 The antiviral activity of functionalized CDs produced from 4-aminophenylboronic acid against human coronavirus (HCoV) infections has been recently demonstrated in human Huh-7 liver cells 22 (see Table 1). Benzoxazine-monomer-derived carbon dots (BZM-CDs) were effective against the Japanese encephalitis virus (JEV), ZIKV, and DENV. 146 The antiviral activity of highly biocompatible glycyrrhizic-acid-based carbon dots (Gly−CDs) synthesized from glycyrrhizic acid was demonstrated against large-enveloped RNA viruses by using the porcine epidemic diarrhea virus (PEDV) as a viral model of the coronavirus (CoVs). 54 This virus belongs to the alphacoronavirus genus, and it cannot be transmitted to humans. 158 Graphene and Related Carbon-Based Nanomaterials. Graphene and its oxidated form, GO, are 2D CBNs with excellent physical and biological properties 159−163 that can be used successfully to detect and capture viruses 147,164 (see Table 1). The antiviral activity of carbon nanofibers (CNFs) incorporated into alginate was reported using a nonenveloped double-stranded viral model. 165 However, the antiviral capacity of CNFs has never been tested against an enveloped virus belonging to the same Baltimore group 166 such as SARS-CoV-2. Functionalized multiwall carbon nanotubes (MWCNTs), another type of carbon-based filamentous nanomaterial, exhibited potent viral inhibition against HIV. 148 In this study, the effect of hydrophilicity and dispersibility of the nanomaterials showed to be the key to control the antiviral activity of MWCNT-based nanomaterials. Carboxylated MWCNT (ox-MWCNT sample) and drug-conjugated MWCNT (MWCNT−C-CHI36) showed high antiviral activity contrary to pristine MWCNT. GO has also shown potent antiviral activity with nonionic polyvinylpyrrolidone (PVP) in contrast to its combination with the cationic poly(diallyl dimethylammonium chloride) (PDDA). 28 The combination of silver nanoparticles (AgNPs) with GO inhibited the infectivity of enveloped feline coronavirus (FCoV) by 25% compared to 16% for GO. 151 AgNPs are well-known alternative antiviral agents that interact with cell surface receptors and block virus entry into the host cells. 66 In the same research line, GO−AgNP nanocomposites showed better viral inhibition capacity than AgNPs or GO using a PRRSV pattern on the replication of virus. 152 Water-soluble graphene quantum dots (GQD) synthesized from MWCNT through oxidation and exfoliation with and without conjugated antiretroviral agents exhibited efficient viral inhibition of the HIV. 154 Very recently, graphene derivatives with long aliphatic chains have shown inhibition capacity against FCoV and SARS-CoV-2 replication. 155 Therefore, these results confirm the potential utilization of CBNs in the fight against viruses such as SARS-CoV-2.

CARBON-BASED NANOMATERIALS AGAINST VIRUSES
This current report analyzes the antiviral potentials of CBNs against 13 viruses. The antiviral activities were detected in 40 out of 44 studies as depicted in Figure 4.
The number of studies is greater than the 22 published papers shown in Table 1, because several CBNs and/or types of viruses were studied in some publications. To perform this count of studies, only CBNs with different chemical structural forms were considered. Thus, fullerene and its derivatives showed antiviral activity in nine studies against HIV-1, 26, 135, 137, 139− 141, 143, 144 five studies against HIV-2 26,139−141,143,144 out of six, and against other viruses such as SIV, 138 M-MuLV, 138 HCV, 142 ZIKV, 24 and DENV. 24 Carbon dots and their derivatives exhibited antiviral activity against HCoV, 22 HIV-1, 61 JEV, 146 ZIKV, 146 DENV, 146 PEDV 54 and two studies against PRRSV. 54,63 Graphene and related CBNs have shown antiviral activity against HIV-1 in four studies out of five, 147,148,154 five studies against PEDV out of seven, 28,152 two studies against FCoV, 151,155 and one study against PRRSV 152 and SARS-CoV-2. 155 All these viruses are enveloped positive-sense single-stranded RNA viruses (IV Baltimore group 166 ). Therefore, the antiviral properties of CBNs have been tested against a wide range of viruses similar to SARS-CoV-2, which suggests that CBNs are promising nanomaterials as alternative antiviral agents against this pathogen. The viruses tested against the CBNs are shown in Table 2 with all their characteristics such as name, abbreviation, genus, family, who the virus infects, disease/action, and references.

MECHANISM OF ACTION OF CBNS AGAINST VIRAL INFECTION
The antiviral mechanisms of fullerenes, carbon dots, graphene, and related carbon nanomaterials are still not completely understood. However, some progress in understanding the mechanism of action of these promising antiviral agents has been achieved. Details of these suggested mechanisms are discussed below.
Fullerene and Derivatives. Antiviral fullerene derivatives can inhibit viral entry, modify its morphology and functions, and block viral replication. 30 Studies about the antiviral mechanism of action of C 60 -fullerene derivatives against viruses such as HIV-1 and HIV-2 suggest that the C 60 carbon sphere fits well to the active site of some HIV enzymes such as the HIV-protease. 135,137,143 In the same research line, a diamido diacid diphenyl fulleroid derivative (2c) was found to be an inhibitor of HIV-1 and HIV-2 protease and reverse transcriptase at low micromolar concentrations. 143 More recently, the series of fullerene derivatives shown in Figure 3 exhibited potential inhibition of hepatitis C virus (HCV) NS5B polymerase and HCV NS3/4 protease. 142 On the other hand, the mechanism of aqueous fullerene preparations of C 60fullerene combined with PVP (C 60 /PVP) against influenza A virus has been reported. 167 Influenza A virus is a negative-sense single-stranded virus that belongs to the Baltimore group V. 166 However, it is an enveloped RNA virus like SARS-CoV-2. The antiviral mechanism of action of the C 60 /PVP complexes is       167 Thus, the round and oval morphology of the vial particles before being in contact with the C 60 /PVP preparation was observed by transmission electron microscopy. The well-defined surface glycoproteins in the form of "brush" lost their integrity of viral envelopes when they were in contact with the C 60 /PVP complexes by breaking the lipoprotein envelope structure, and these complexes fused with the virion-like aggregates. Carbon Dots and Derivatives. In the context of antiviral mechanisms, CDs could inhibit viral replication by activating the interferon response for the porcine reproductive and respiratory syndrome virus (PRRSV). 63 Furthermore, CDs conjugated with carboxyl phenylboronic acid (CBBA) prevented the entry of HIV-1 viruses into cells by suppressing syncytium (Figure 5). 61 The antiviral studies of functionalized CDs produced from 4-aminophenylboronic acid against human coronavirus (HCoV) infections in human Huh-7 liver cells showed inhibition of the viral entry and effects at the replication steps, which were ascribed to the interaction of the CBNs' functional groups with the viral entry receptor DPP4 22 (see Figure 6).
Graphene and Related Carbon-Based Nanomaterials. Graphene and GO can destroy the virus surface proteins and extract their RNA by bioreduction. 164 The high binding affinity of graphene to the essential target proteins HIV Vpr, Nef, and   ACS Nano www.acsnano.org Review Gag during HIV infections was reported in 2014. 147 Evaluation of the antiviral capacity of GO and rGO against PEDV showed significant viral inhibition by inactivating the virus before entering the cell (Figure 7). 28 It was reported that the sharp edges of the GO or rGO nanosheet inactivated the virus by physical disruption of its biological structure through direct interaction, which resulted in the outflow of intracellular metabolites. 28 Since rGO and GO exhibited a similar viral inhibition capacity in that study, the surface functional groups present in these CBNs may play a minor role. The antiviral activity was concentration-and timedependent incubation. Therefore, the potent antiviral activity of both GO and rGO can be attributed to the negative charge, which favors the electrostatic interaction with the positive charge of the virus and the single-nanosheet-layer structure. 30 On the other hand, graphite (Gt) showed no viral inhibition, and graphite oxide (GtO) exhibited weaker viral inhibition than monolayer GO and rGO, suggesting that the nanosheet form plays a significant role in the antiviral activity. 28 In the same research line, GO−AgNP nanocomposites prevented PRRSV from entering the host cells (∼59.2% inhibition) and improved the production of interferon-α (IFN-α) and ISGs, which directly block the proliferation of PRRSV. 152 Furthermore, graphene sheets with defined dual sulfate/alkyl functionalities have shown potent antiviral activity against FCoV and SARS-CoV-2. 155 Here, graphene acted as a 2D platform to allow the interaction of the negatively charged polyglycerol sulfate (PGS) branches with the positively charged patches of the virus particles, and then, the long aliphatic chains (C11) ruptured the membrane of the lipid envelope. Thus, G-PGS-C11 displayed the strongest antiviral activity against SARS-CoV-2 without exhibiting significant toxicity against eukaryotic host cells (Figure 8).
Carbon-based nanomaterials may exercise multiple mechanisms against positive-sense single-stranded RNA viruses according to their physical configuration, chemical modifications, and metabolism. Hence, CBNs could work directly against the virus particle by distorting the envelope or the capsid organization; additionally, they may exert a steric hindrance effect by physically occupying a catalytic site of an  essential viral enzyme or a receptor cavity. 136 When chemically modified with charged residues or metabolically activated, CBNs could perform both a direct disrupting effect on the virion structure and an indirect antiviral activity due to tuning of the redox signaling and the homeostatic innate/inflammatory response of the target cells. 30 All of the above functions, coupled with a negligible toxicity, may account for the anti-SARS-CoV-2 therapeutic potential of carbon compounds. 155 An even synergistically increased inhibitory effect may be played by CBNs when loaded with specific antiviral drugs. 30 Thus, the direct and intrinsic antiviral activities of the compounds plus the loaded antiviral drug will work synergistically against the viral particles, which may increase the viral destruction potential. 168 Another characteristic feature of the CBNs is based on the immunostimulatory potentials, 169,170 which will derive and propagate the antiviral-cell-mediated immunity, which also works synergistically with the CBN− drugs leading to an increase in the virally infected cells and/or viral particle accessibility for CBNs or CBNs−drug achieving a more efficient viral infection clearance (Figure 9) through many suggested scenarios. 171 The most versatile interesting feature of CBNs is selecting one or more entities of them and functionalizing the selection base on the target function. The planning interactions between empty or loaded CBNs and the immune system, including the complement system, can be reciprocal in the context of which immune system components would involve. 171,172

TOXICOLOGICAL ASPECTS OF CARBON-BASED NANOMATERIALS
There are widespread concerns on the toxicological aspects of CBNs, because some studies have reported that depending on the type of CBN, dimensions, oxidation degree, functionalization, concentration, and exposure time, CBNs may exert cytotoxicity effects on host cells. 94−101 Nevertheless, we focused our attention on the toxicological studies performed with the CBNs tested against the 13 enveloped single-stranded positive-sense RNA viruses analyzed in this Review ( Table 1). Details of these studies are discussed below.
Fullerene and Derivatives. An anti-HIV-1 fullerene derivative (compound 1) showed no cytotoxicity on human PBM cells in 1993. 135 Another study reported noncytotoxicity for concentrations up to 100 μM of an anti-HIV bis-  (monosuccinimide) derivative of p,p′-bis(2-aminoethyl)diphenyl-C 60 in peripheral blood mononuclear (PBM) cells and H9 human embryonic stem cells, Vero, and CEM cells. 144 In 1996, nine anti-HIV functional derivatives of C60-fullerene showed no cytotoxicity for concentrations also up to 100 μM. However, a series of anti-HIV-1 bis-functionalized fullerene derivatives were developed after that in 2003, and only one of them (derivative 1) exhibited moderate toxicity. 139 Nonetheless, low cytotoxic properties were reported for anti-HIV cationic fullerene derivatives and anti-HIV water-soluble fullerene derivatives in 2005 and 2007, respectively. 140,141 A later study in 2011 showed that anti-HIV derivatives of C 70fullerene also exhibited low toxicity in vitro and in vivo. 26 More recently, an anti-HCV fullerene derivative (derivative 1a, Figure 3) showed no cytotoxicity in the low micromolar range. 25 Furthermore, very recently, anti-ZIKV and anti-DENV tridecafullerenes appended with up to 360 1,2-mannobiose molecules showed no cytotoxicity in the picomolar range. 24 Carbon Dots and Derivatives. CDs are highly promising nanomedicine tools for antiviral applications due to their low toxicity and potent antiviral activity. 157 Thus, CDs showed antiviral activity against PRRSV and low cytotoxicity in MARC-145 cells. 63 Furthermore, the modified CDs or their combination with other compounds significantly decreased the cytotoxicity of these carbon-based antiviral nanomaterials. 22,54,61,146 For example, in the cytotoxicity tests of both Cdots and CBBA−Cdots at high concentrations (up to 300 mg·mL −1 ), the proliferation of human cells was as fast as that of the untreated cells (control) after 24 h of incubation, suggesting the absence of cytotoxic effects for both types of nanomaterials. 61 However, this study did not assess ROS or cytokine generation. Immunomodulatory effects are known to be induced by other carbon-based nanoparticles such as graphene (see the next paragraph). Anti-HCoV CDs produced from 4-aminophenylboronic acid, BZM-CDs with anti-JEV, anti-ZIKV, and anti-DENV properties, and anti-PEDV and anti-PRRSV Gly−CDs manifested no cytotoxicity. 22,54,146 Graphene and Related Carbon-Based Nanomaterials. The toxicological analysis of pristine MWCNT, ox-MWCNT, and MWCNT−C-CHI36 with anti-HIV-1 properties showed opposite results. 148 Thus, ox-MWCNT and MWCNT−C-CHI36 showed low cytotoxicity contrary to pristine MWCNT, which exhibited high cytotoxicity. However, GO showed potent anti-PEDV using a noncytotoxic concentration (≤6 μg/ mL). 28 Anti-FCoV GO−AgNP nanocomposites and neat GO could also be prepared at very low or noncytotoxic concentrations. 151 In the same way, anti-PRRSV GO−AgNP nanocomposites could be prepared at a noncytotoxic concentration (≤4 μg/mL). 152 Finally, a water-soluble GQD with a conjugated antiretroviral agent (GQD−CHI499) was selected as an excellent potential candidate due to its high antiviral activity against HIV and low cytotoxicity. 154 Anti-FCoV and anti-SARS-CoV-2 graphene platforms have shown a large concentration window without significant toxicity against human cells. 155 Therefore, the lack of toxicity could provide great potential in the development of safe therapeutics based on carbon-based nanomaterials to combat COVID-19 in the microbial-resistant era.

CONCLUSIONS
Carbon-based nanomaterials have been evaluated for their antiviral activity against 13 enveloped viruses (HCoV, PRRSV, PEDV, HIV-1, HIV-2, FCoV, JEV, SIV, M-MuLV, ZIKV, DENV, HCV, and SARS-CoV-2), all single-stranded positivesense RNA viruses belonging to the Baltimore group IV. Most of the studies have shown a potent antiviral activity and from low to no toxicity, supporting the potential for the use of CBNs to combat SARS-CoV-2. As a revolutionary technology approach to treat COVID-19, these carbon-based therapeutics can provide a significant breakthrough, as these nanomaterials allow the targeting of microbial resistance issues and can potentially induce tissue regeneration at the same time. Furthermore, these antimicrobial nanoweapons could be employed to deal with SARS-CoV-2 alone or with other types of viruses, bacteria, or fungi causing pneumonia, including multidrug-resistant strains. The chance of success in applying these wide-spectrum antimicrobial nanomaterials is very high because of the preliminary antiviral results reported for 13 viruses and the fact that the proposed approach could be extended to other types of pneumonia caused by other important pathogens.

Notes
The authors declare no competing financial interest. All authors are members of the Self-Assembled CoVid Research and Education Directive (SACRED) Consortium.
VOCABULARY coronavirus disease 2019 (COVID- 19), viral infectious disease that has spread throughout the world causing the current COVID-19 pandemic; acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the causative pathogen responsible for the COVID-19 disease; carbon-based nanomaterials (CBNs), are materials mainly constituted by carbon atoms that possess a broad range of antimicrobial properties; enveloped positive-sense single-stranded RNA viruses, are viruses that belong to the IV Baltimore group similar to SARS-CoV-2; pneumonia, is an infection of the lungs caused by bacteria, viruses, or fungi.