Viral filtration using carbon‐based materials

Abstract Viral infections alone are a significant cause of morbidity and mortality worldwide and have a detrimental impact on global healthcare and socio‐economic development. The discovery of novel antiviral treatments has gained tremendous attention and support with the rising number of viral outbreaks. In this work, carbonaceous materials, including graphene nanoplatelets and graphene oxide nanosheets, were investigated for antiviral properties. The materials were characterized using scanning electron microscopy and transmission electron microscopy. Analysis showed the materials to be two‐dimensional with lateral dimensions ranging between 1 and 4 µm for graphene oxide and 110 ± 0.11 nm for graphene nanoplatelets. Antiviral properties were assessed against a DNA virus model microorganism at concentrations of 0.5, 1.0 and 2.0 wt/v%. Both carbonaceous nanomaterials exhibited potent antiviral properties and gave rise to a viral reduction of 100% across all concentrations tested. Graphene oxide nanosheets were then incorporated into polymeric fibres, and their antiviral behaviour was examined after 3 and 24 hr. A viral reduction of 39% was observed after 24 hr of exposure. The research presented here showcases, for the first time, the antiviral potential of several carbonaceous nanomaterials, also included in a carrier polymer. These outcomes can be translated and implemented in many fields and devices to prevent viral spread and infection.

inadequate. A comprehensive review in 2016 identified only 90 antiviral drugs were approved for the treatment of 22 viral infections since 1963, with no treatment for over 200 infectious diseases (De Clercq & Li, 2016). Along with antiviral drugs, vaccines are efficacious in preventing and treating viral infections.
However, the success of vaccines in a pandemic relies on several factors, including early detection and the availability of specific vaccines (Monto, 2006). This phenomenon constitutes a public threat, caused by the increased morbidity and mortality, additional costs due to the excessive use of antiviral medications and the subsequent burden on healthcare systems.
Efforts on the next generation of antivirals have been focused on nanomaterials, particularly metallic nanoparticles (Ganesh, Venkatakrishnan, & Tan, 2019;Kanematsu et al., 2019;Ray et al., 2018;Reddy et al., 2018). Carbon-based nanomaterials, such as graphene and graphene oxide, hold distinctive characteristics that have attracted significant attention in a variety of applications. Graphene oxide is one of the most extensively explored materials for a wide range of applications. Traditionally, graphene oxide is formed from the chemical exfoliation of layered crystalline graphite into mono-sheets (Boehm, Setton, & Stumpp, 1994;Park & Ruoff, 2009); however, alternative methods, such as self-assembly (Tang et al., 2012) and chemical vapour deposition (Liu & Chen, 2016), have been proposed. Graphene oxide is a non-stoichiometric chemical compound comprised of a single atomic plane of carbon molecules arranged in a regular hexagonal lattice with carboxylic groups at its edges and hydroxyl groups in its basal plane (Compton & Nguyen, 2010;Dideikin & Vul, 2019;Park & Ruoff, 2009). Graphene nanoplatelets are composed of layers of carbon atoms arranged in a honeycomb structure (Georgakilas, Perman, Tucek, & Zboril, 2015;Li et al., 2014).
Each atom is attached to three neighbouring carbon atoms in the x-y plane by sigma bonds (Scida, Stege, Haby, Messina, & García, 2011). The atoms also have a weakly delocalized π-electron cloud that is orientated in the z-axis (Greshnov, 2014;Luo, Qiu, Lu, & Ni, 2013;Novoselov et al., 2007). The antibacterial properties of carbonaceous nanomaterials have been widely investigated and reported in the literature; however, their antiviral properties are unknown Matharu et al., , 2020. Studying the interaction between graphene nanomaterials and viral pathogens is of immense interest, to allow for the development of new antiviral treatments. In particular, the use of polymer carriers and their manufacturing techniques for the incorporation of carbonaceous materials are particularly relevant because of the emergence of coronaviruses (Perlman, 2020;Wang, Horby, Hayden, & Gao, 2020).
In this research, the inhibitory effects of graphene nanoplatelets and graphene oxide nanosheets on Escherichia coli T4 bacteriophage (DNA virus) were investigated. The carbonaceous nanomaterials were then incorporated into polymeric fibres, and the antiviral activity of the resulting fibres (carriers) was studied to determine whether or not the materials remain effective.
Poly(methyl methacrylate) (PMMA) (Mw 120,000 g/mol) and chloroform were acquired from Sigma-Aldrich and used during fibre manufacture. All solvents and chemicals were of analytical grade and used as received. ATCC Medium 129 was prepared using sodium chloride, nutrient agar and nutrient broth sourced from Sigma-Aldrich. Phosphate buffer saline was used in the antiviral studies and purchased from Sigma-Aldrich.

| Nanomaterial antiviral activity
T4 bacteriophage suspensions containing 0.5, 1.0 and 2.0 w/v% of graphene nanoplatelets or graphene oxide nanosheets in PBS were incubated for up to 24 hr. An actively growing broth culture of E. coli was prepared by incubating a single colony in sterile medium, and a plaque assay was performed to determine the virus concentration at 0, 3 and 24 hr.

| Fibre manufacture and antiviral activity
PMMA fibres containing 2 wt% of either graphene nanoplatelets or graphene oxide nanosheets were manufactured using pressurized gyration as previously reported Matharu, Porwal, et al., 2018;Matharu et al., 2020). The antiviral activity of the prepared fibres was assessed by incubating 0.1 g of fibres in 10 ml of T4 bacteriophage suspension for 24 hr. The number of virions present at 0 and 24 hr was counted using a plaque assay, and viral reduction was calculated. Virgin PMMA fibres were used as a control. Antiviral studies were completed in triplicate.

| Nanomaterial antiviral activity
This research reports the effect graphene-based nanomaterials, graphene nanoplatelets and graphene oxide nanosheets (Figure 1), have on the inhibition of viruses. Analysis showed the graphene oxide nanosheets had a thickness of 0.85 ± 0.12 nm and lateral dimensions in the range of 1-4 µm (Matharu et al., 2020), while the graphene nanoplatelets had an average width of 110 ± 0.11 nm and average length of 170 ± 0.08 nm Matharu, Porwal, et al., 2018). This study involves testing both nanomaterials, incorporating them into polymeric fibres and testing the resulting antiviral efficacy. Both materials were exposed to the virus for up to 24 hr, and viral reduction was calculated using a plaque assay.
Graphene nanoplatelets and graphene oxide nanosheets significantly reduced virion numbers and were able to inhibit infection by 100% after just 3 hr of exposure ( Figure 2). The potent antiviral activity of both graphene-based nanomaterials is attributed to (a) firstly, its unique morphological structure and/or (b) secondly, oxidative stress caused by the production of reactive oxygen species (Krishnamoorthy, Umasuthan, Mohan, Lee, & Kim, 2012;Ye et al., 2015). A key feature of viral inactivation by carbonaceous materials is that it occurs prior to entry to the host cell (Ye et al., 2015).
This subsequently prevents viral attachment and entry into the host cell, thus preventing the release of the capsid and its content. It is therefore plausible that graphene-based nanomaterials directly interact with the virions and demolish their structure, resulting in the disruption of viral function.
Another mechanism in which graphene-based nanomaterials cause viral inactivation is through reactive oxygen species-related damage (Paiva & Bozza, 2014). Research has shown the inactivation of DNA viruses when exposed to hydrogen peroxide (a form of reactive oxygen species) (Hayashi, Hooper, Okuno, Takada, & Hooks, 2012). Typically, viruses produce catalase (a protective enzyme that catalyses the decomposition of oxidative stress) to prevent damage caused by reactive oxygen species. However, it is thought that most DNA viruses do not have catalase, therefore offering no protection against reactive oxygen species-induced stress (Newcomb & Brown, 2012).

| Fibre antiviral activity
Graphene nanoplatelets and graphene oxide nanosheets were incorporated into PMMA fibres as previously described in our antibacterial studies Matharu, Porwal, et al., 2018;Matharu et al., 2020), to determine whether or not they remain F I G U R E 1 (a) Scanning electron micrograph of graphene oxide nanosheets and (b) transmission electron micrograph of graphene nanoplatelets effective. Several methodologies have been proposed to prepare nonleaching antimicrobial materials, but in most cases, they require a multistep procedure and are only specific for certain chemistries. Pressurized gyration approaches have demonstrated the successful manufacture of nanocomposite materials in a single step (Matharu, Charani, Ciric, Illangakoon, & Edirisinghe, 2018;Matharu, Porwal, et al., 2018;Matharu et al., 2020).
The resulting fibre morphologies are described in Table 1. Figure 3 shows SEM images of the 8 wt % loaded fibres, where a smooth topography can be observed Matharu, Porwal, et al., 2018;Matharu et al., 2020). As none of the nanosheets are protruding from the fibre surface, the antiviral activity of the fibres is thought to be chemical rather than physical. These physical and chemical scenarios are further discussed in our antibacterial work Matharu, Porwal, et al., 2018;Matharu et al., 2020). Figure 4, pure PMMA fibres (loaded with 0 wt %) showed a moderate decrease in virions. This minor reduction is likely owed to two factors: (a) the lack of host cells in the PBS to allow for viral survival and reproduction and (b) viral destruction triggered by the hydrophobic interaction between PMMA and the proteins present on the virus surface (Karlin & Brendel, 1988;Shi & Tarabara, 2018).

As shown in
Fibres containing graphene nanoplatelets and graphene oxide nanosheets exhibited antiviral properties across all conditions tested. Only a slight increase in viral reduction can be observed between the 3-hr and 24-hr incubation periods. This suggests a 3-hr exposure time is sufficient for viral deactivation. After 24 hr of exposure, fibres containing the lowest nanoparticle concentration (2 wt%) exhibited viral reductions of 20.8 ± 1.6% and 22.4 ± 1.3% for graphene nanoplatelets and graphene oxide, respectively. Further increasing the loading to 4 wt % of graphene nanoplatelets or graphene oxide nanosheets resulted in viral reductions of 26.0 ± 1.7% and 28.9 ± 1.2%, respectively. Fibres containing 8 wt % graphene nanoplatelets or graphene oxide nanosheets displayed the strongest potencies with average viral reductions of 33.6 ± 0.9% and 38.7 ± 1.1%, respectively. These results indicate that the antiviral activity of the nanocomposite fibres is in fact concentration-dependent. Increasing the loading increases the concentration of graphene-based nanomaterials on the surface of the fibres. Therefore, more of the active agent is exposed for the virions to interact with. A study by Mori et al (2013) showed a 45% titre after exposure to silver nanoparticle/chitosan composites (embedded with 5 wt % silver nanoparticles). This result is comparable to the results obtained in this study.
Though the antiviral properties of the graphene loaded fibres were less effective when compared to pure nanoparticles, incorporating them into a polymer broadens their applications and F I G U R E 2 Antiviral activity of graphene nanoplatelets and graphene oxide nanosheets at 0.5, 1.0 and 2.0 w/v% against T4 bacteriophage after (a) 3 hr and (b) 24 hr of exposure. Control samples were exposed to no treatments. Error bars represent standard deviation (n = 3)  demonstrates how they can be translated into different applications. As the graphene nanomaterials are entrapped in the fibres and not free floating, viral deactivation through direct interaction is not plausible. Therefore, only the secondary mechanism of action, production of reactive oxygen species, is functioning (Matharu et al., 2020). The unfavourable ramifications of viral pathogens are widespread, with major implications on societies, economies and political systems, especially in developing countries, not only because of the associated morbidity and mortality, but also the high cost that represents their prevention and treatment. The cytotoxicity of carbonaceous nanomaterials remains underdetermined, with existing literature giving contradictory judgements. Some studies have shown graphene nanomaterials to be toxic to mammalian cells, while other research has shown composite graphene nanomaterials to be non-cytotoxic and can be used in a range of biomedical constructs (Gonçalves, Cruz, Ramalho, Grácio, & Marques, 2012;Liao, Lin, Macosko, & Haynes, 2011;Liu, Cui, & Losic, 2013;Pahlevanzadeh, Bakhsheshi-Rad, & Hamzah, 2018). As the prepared composites are not intended for consumption or direct implantation into humans, toxicity is not a major concern. This study shows these nanocomposites can be used in a variety of applications, such as surface treatments, filters and more, as effective antiviral treatments.

| CON CLUS IONS
In the present study, graphene nanoplatelets and graphene oxide nanosheets exhibited antiviral activity towards DNA viruses after 3 hr of exposure. The antiviral mechanism of both materials is attributed to physical and chemical interactions, with direct nanoparticle interaction being the predominant mode of action. Both graphene nanoplatelets and graphene oxide nanosheets showed comparable antiviral activity, suggesting the oxygen functional group is expendable in the antiviral mode of action.
The graphene nanoplatelet and graphene oxide polymer fibres also showed antiviral activity. The fibres show much weaker antiviral activity than the pure nanomaterials, suggesting that direct physical interaction is important for antiviral activity.

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest. The funders had no role in the design of the study; collection, analyses or interpretation of data; manuscript writing; or decision to publish the results.