Other Carbon Nanomaterials
Alongside fullerenes, other carbon nanomaterials (NMs) have been scrutinized for their ability to block viral entry. CDs and GO are the most known and studied carbon NMs with marked antiviral properties. CDs are zero-dimensional carbon nanoparticles. They are generally produced via hydrothermal decomposition of carbon containing “low-cost” precursors. The use of CDs in the biomedical field has been encouraged by their easy preparation, low toxicity, fluorescence properties, and easy surface functionalization. Pristine CDs have shown moderate viral blocking activity for HIV infection in vitro.74 This has been associated with the surface of the material rich in carboxylic and hydroxyl groups prone to form noncovalent interaction with viral membranes. Moreover, due to the complexity of the biological systems these nonspecific interactions could not be so effective in vivo, likely reducing the antiviral efficacy. Therapeutic targeting molecules can be grafted onto a CD surface to enhance their antiviral activity. In this context, the design of multifunctional CD platforms can be obtained through two different strategies. The first consists in a single-step reaction that foresees the insertion of the therapeutic molecule directly into the step of preparation. Target molecules are decomposed with the other precursors, generating the desired functional CDs. This protocol is fast and efficient, however the drug loading as well as its activity are hard to estimate. Indeed, the hydrothermal treatment can alter the chemical structure of the active molecule, thus vanishing its therapeutic effect. For these reasons, the reaction conditions must be carefully controlled.75 The second method is a two-step reaction and implies the postfunctionalization via amide formation on the surface of the CDs rich in carboxylic groups. This strategy offers a better chemical control, but the yield and the drug loading may not be quantitative and high, respectively. Different functionalized CDs were prepared to hamper host cell viral entry. For instance, benzoxazine (a low water-soluble antiviral agent) was incorporated into the CD structure during their preparation (Figure 7). The as-prepared CDs showed a broad spectrum viral blocking capacity in vitro for enveloped (e.g., Japanese encephalitis virus, Dengue virus, and Zika virus) and non-enveloped viruses (e.g., porcine parvovirus and adenovirus-associated virus).76 These positive results were explained by the efficient binding and deactivation induced by the multivalent effect of the CDs to the viral particles
Figure 7  Illustration of benzoxazine-functionalized CDs and their broad antiviral entry activity. Reproduced with permission from ref (76). Copyright 2019 Elsevier B.V. Amino-functionalized CDs were also tested for the treatment of human norovirus. In this study, CDs were functionalized with 2,2′-(ethylenedioxy)bis(ethylamine) (EDA) and 3-ethoxypropylamine (EPA) via amide bond formation.77 These NMs exerted a good viral blockage. In particular, EPA-functionalized CDs were able to inhibit 100% of viral infection at concentration of 2 μg/mL, while in the case of CDs prepared with the other amines, 80% of inhibition was reported. These effects have been associated with the higher positive charge of CD-EDA compared to CD-EPA. Another surface group used for viral targeting is boronic acid (BA), which can bind glycosylated surfaces forming boronic esters. This strategy was successfully adopted to treat HIV where the boronic groups, linked to different nanoparticles (e.g., silica nanoparticles and nanodiamonds) can target gp120 receptors on the viral envelope inhibiting the infection.78 Another recent study proposed the use of CD functionalized with phenylboronic acid for prevention of HIV infection.74 The functional materials showed good inhibition properties compared to nonfunctionalized CDs by preventing the binding to the target cell in vitro. Overall, the use of CDs for stopping host cell viral entrance has shown good results in vitro. However, there is a lack of proofs in vivo limiting their applications to surface disinfection or masks. In addition, most of the in vitro studies foresee first the contact of the CDs with the viral particles and then their incubation with host cells. Deeper investigations should be performed adding the NMs at other time points (for instance in infected cells) to understand if the antiviral activity is maintained.
In addition, CDs have been successfully used for photodynamic therapy (generating radicals upon light irradiation) in cancer treatment. The same approach may be used to combat viral infections, where the antiviral activity induced by the surface modification can be sensibly enhanced by ROS generation under irradiation.
Graphene materials, and in particular GO and reduced GO (rGO), have been used for different biomedical applications including drug delivery, biosensing, and tissue engineering.5 GO platforms have shown also interesting antimicrobial activity.79 Regarding viral infection, GO was used to block the virus entrance in host cells. GO and rGO can be considered as two-dimensional materials that contain hydrophilic and hydrophobic domains allowing to adsorb many biological molecules including nucleic acids and proteins. GO showed low interaction with viruses, however its surface functionalization with target molecules can sensibly enhance its affinity for the viral particles. Additionally, GO can be used as photothermal agent (generation of heat by NIR irradiation) or photodynamic therapy (using visible light irradiation) inactivating the capsids by local thermal shock or by radical formation during irradiation, respectively. The use of phototherapies may significantly augment the antiviral properties of the materials. However, we must keep in mind that these therapeutic modalities can be applied only to disinfection, since the radical/heat production may be harmful for the healthy tissues in vivo. Photodynamic therapy has been successfully exploited using bacteriophage MS2 as a model virus.80 In this study, GO was functionalized with an aptamer recognized by the viral surface. The results showed that this functionalization is able to enhance the binding efficiency of the MS2 capsids onto the GO surface compared to nonfunctionalized GO. Subsequently, irradiation in the visible light was able to disinfect the solution, while nonfunctionalized GO showed much less activity due to the lack of adsorption. Despite these interesting results, this pioneer work remains at an early research stage since the use of high light dose (300 W for 10–140 min) and the lack of material recovery and reuse make its application for surface disinfection difficult.
GO can be also used as a platform to link antiviral agents. Encouraging results were reported using GO with hypericin for the treatment of a recently appeared duck reovirus.81 More recently, Deokar et al. reported an original rGO-based multifunctional platform for HSV-1 treatment.82 In this work, the authors functionalized the material with organic sulfate groups and iron oxide magnetic nanoparticles (FeNPs). The rGO functionalized with the sulfate is able to mimic the host cell surface and to bind HSV-1. Subsequently, the viral particles captured onto the rGO-FeNP surface can be concentrated via magnetic precipitation and destroyed via photothermal therapy. This approach is highly efficient for disinfection with low energy (1.6 W/cm2 for 7 min) and cost effectiveness. HS is a common entry receptor in various types of viruses (e.g., herpes viruses, human papillomavirus, Dengue virus).83 The use of organic sulfate-functionalized graphene sheets mimicking HS, like GO and rGO, has been already explored. However, it is worth noting that these NMs are prone to strongly adsorb proteins in culture environments (coronation), likely inhibiting their antiviral efficacy.84 High loadings of sulfate groups were introduced onto rGO using polyglycerol sulfate.85,86 This approach has been used for inhibition of orthopoxvirus, pseudorabies virus, and African swine fever virus in vitro.85,86
Graphene has also been used as antiviral material. Polysulfates and fatty amines were grafted onto graphene surface via triazine chemistry for the treatment of herpes simplex virus.87 This strategy promotes the synergy between the electrostatic and hydrophobic interactions, showing incredibly high inhibition efficacy. Overall, graphene materials have shown a good capacity to block host cell viral entry. Disinfection with graphene family materials is also promising, offering the possibility to couple high viral binding with phototreatments. Regarding the GO and rGO activity in cellular environments, different parameters must be considered such as protein coronation, blood circulation time, and activity in vivo. So far, the use of sulfonic groups introduced via diazonium salt decomposition has been largely privileged. We take this opportunity to encourage future studies using other targeting groups (e.g., boronic acids) and grafting methods (e.g., epoxide ring opening or hydroxyl esterification reactions).