key: cord-0254109-d9kqjbp3 authors: Sanmartín-Santos, Isaías; Gandía-Llop, Sofía; Serrano-Aroca, Ángel title: Low-cost alginate-based nanocomposite films showed high antiviral activity date: 2020-08-18 journal: bioRxiv DOI: 10.1101/2020.08.18.255646 sha: ba13da1483d5de22663238e80eaa3cc30e5b8f1b doc_id: 254109 cord_uid: d9kqjbp3 Due to the current COVID-19 situation caused by the spread of the new SARS-Cov-2 coronavirus, new alternative materials with antiviral activity are encouraged to be fabricated by the World Health Organization for present and future pandemics. Previous studies of low-cost alginate-based nanocomposite films produced with very low amounts (0.1% w/w) of carbon nanomaterials such as graphene oxide (GO) or carbon nanofibers (CNFs) have shown very low transparency reduction, enhanced mechanical performance, improved water diffusion and wettability, and similar biological properties than neat alginate in terms of cell adhesion and non-cytotoxicity. However, only the nanocomposite containing CNFs have shown antibacterial activity. Herein, the antiviral properties of these nanocomposite biomaterials are explored for the first time in literature using a double-stranded DNA viral model. The results of this study showed that neat calcium alginate films possess antiviral activity and the incorporation of that low percentage of CNFs significantly enhanced its antiviral action from ~55.6% to 96.33% inhibition after 48 hours. Nevertheless, the addition of this minuscule amount of GO did not improve the antiviral activity of calcium alginate. Nonetheless, both antiviral composite biomaterials possess excellent physical and biological properties with great potential in biomedical applications. Sodium alginate (SA) has been authorized by the US Food and Drug Administration for human biomedical applications due to its excellent properties such as biodegradability, renewability, cost-effectiveness, non-toxicity and biocompatibility [1] . This biopolymer can be cross-linked with Ca 2+ cations to form hydrogels [2] with physical properties such as mechanical performance, water diffusion and wettability that can be enhanced by the incorporation of low amounts of carbon nanomaterials (CNMs) such as graphene oxide (GO) [3] [4] [5] or carbon nanofibers (CNFs) [6, 7] . These nanocomposites possess similar biological properties than neat calcium alginate in terms of cell adhesion [8] and cytotoxicity [9, 10] . CNFs are hollow carbon filaments of one dimension with antibacterial activity in pure form and when incorporated into calcium alginate [9] as well as GO [10] . However, in these previous studies, the calcium alginate/GO nanocomposite films required a higher percentage of CNMs (from 0.5% w/w) in contrast to the calcium alginate/CNFs nanocomposite films (from 0.1% w/w) to acquire antibacterial activity. The antiviral properties of calcium alginate has been hardly explored [11, 12] . However, as far as we know, the antiviral activity of calcium alginate with carbon nanofibers and graphene oxide has never been studied before. Thus, herein, we explore the antiviral activity of these nanocomposites using a tailed double-stranded DNA viral model [13] . Sodium alginate (Panreac AppliChem, Darmstadt, Germany), calcium chloride (anhydrous, granular, ≤7.0 mm, ≥93.0%, Sigma-Aldrich, Saint Louis, Missouri, USA), graphene oxide (Ref: 796034, powder, 15-20 sheets, 4-10% edge-oxidized, Sigma- Aldrich, Saint Louis, Missouri, USA) and carbon nanofibers (Ref: 13/0248, Graphenano, Yecla, Spain) were used as received. Alginate nanocomposite films of approximately 0.25 g were prepared with a composition of 99.9% w/w of SA and 0.1% w/w of GO or CNFs following a recently reported new engineering route to produce more homogenous alginate-based composites with enhanced physical properties [4] . Thus, mixing of GO/SA or CNFs/SA in 22 ml of distilled water was performed by magnetically stirring for 1 hour at room temperature (26±0.5ºC). After that, another aqueous solution containing 6% (with respect to the SA mass) of CaCl2 in 10 ml of distilled water was mixed with the GO/SA or CNFs/SA aqueous solution for 10 additional minutes. Thus, thin films were produced in Petri dishes after 24 h of drying at 37±0.5ºC in an oven by solvent evaporation. Finally, the films were cross-linked by immersion in an aqueous calcium chloride solution (2% w/v) for 2 hours. After rinsing with distilled water, the films were vacuum dried at 60°C±0.5ºC. The calcium alginate films without CNMs were produced following the same chemical procedure but without adding any CNMs. These films will be referred to as Alginate, GO0.1% and CNFs0.1% films. The sodium alginate used in this study was characterized by nuclear magnetic resonance (NMR), high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and size exclusion chromatography with multi angle light scattering detection (SEC-MALS) by the NOBIPOL research group at the Norwegian University of Science and Technology (NTNU). The GO nanosheets and CNFs were observed by high-resolution transmission electron microscopy (HR-TEM) in a JEM 2100F (JEOL, Japan) 200 kV electron microscope. The sample preparation consisted of dispersing a very low amount of CNMs in dichloromethane placed inside an ultrasound bath for ten minutes and subsequent drying at ambient temperature before HR-TEM observation. A JEM-1010 (JEOL, Japan) 100 kV transmission electron microscope (TEM) was utilized to observe the CNFs and GO nanomaterials incorporated into the calcium alginate films. Ultrathin sample preparation with sections of 60 nm was performed with a Leica Ultracut UC6 ultramicrotome (Leica Mikrosysteme GmbH, Austria) and a Diatome diamond knife (Diatome Ltd., Switzerland). The specimens were placed on TEM grids (300 mesh) coated in carbon film. The Escherichia coli B, living, bacteriophage host (Reference: 12-4300) and the coliphage T4r (Reference: 12-4335) from Carolina (Burlington, North Carolina, USA) was used for the antiviral tests. The sample films of alginate, GO0.1% and CNFs0.1% were cut into 1 mm diameter discs and sterilized by washing the films during 30 minutes in a beaker with 50 µl of 70% ethanol under magnetic stirring at 450 r.p.m. with fresh ethanol every 10 minutes. After that, the discs were dried at room temperature and exposed to ultraviolet light 1 hour per each side of the disc. Finally, the antiviral activity of the films was analyzed by a "contact test" of the bacteriophages with the discs in 96- The statistical analyses were performed by ANOVA followed by the Tukey's posthoc test ( ⁎⁎⁎ p > 0.001) with the GraphPad Prism 6 software. The results of the sodium alginate characterization by NMR, SEC-MALS and HPAEC-PAD showed: a guluronic acid content of 43% and a proportion of alginate consisting of guluronic acid in blocks of dimers and trimmers of 27 and 23% respectively, a weight-average (Mw) and a number-average (Mn) molecular weight of 379.5±9.5 and 170.7±3.1KDa respectively. The GO and CNFs in the form of powder were observed by high-resolution transmission electron microscopy (HR-TEM) and showed a morphology of irregular nanometric sheets and micrometer length hollow fibers with a wide range of nanometric diameters respectively (see Figure 1 (a&b)). The EDS results of the CNFs and GO showed C/O ratios of 31.3 and 15.4, respectively in a previous study performed with the same CNMs [8] . The Raman spectra of these CNMs were also analyzed in that study (see whole spectra in the supplementary material of reference [8] ) and exhibited an ID/IG ratio of 0.92 for the hydrophilic 2D material (GO nanosheets) and 1.51 for the hydrophobic 1D filamentous CNFs due to their higher degree of molecular disorder [14] . The TEM micrographs (Figure 1(c&b) ) shows that the GO nanosheets and CNFs (dark phase) are embedded and randomly distributed in the alginate polymer matrix (clear phase). [7] nor with that of graphene oxide [10] . The antiviral properties of calcium alginate with a low percentage of carbon nanofibers and graphene oxide were studied here for the first time in literature. Thus, Figure 2 shows the plaque forming units measured in the antiviral tests performed after 0, 18 and 48 hours of contact with the coliphage T4r. The antiviral activity of these samples at 0 hours is not statistically significant as expected. These results at time zero indicate that the virus is able to go out of the sample films after the sonication and vortex treatment, and some of contact time is required for them in order to be deactivated. However, when the coliphages T4r are in contact with the alginate film for 18 hours, a decrease of ~56-55.6% in the viable phage counts after 18 hours occurs (see Figure 2 and 3). This antiviral action of calcium alginate did not improve either after more time (48 hours), or with the incorporation of graphene oxide (see Figure 2 and 3). However, the calcium alginate films with 0.1% of carbon nanofibers achieved a viral inactivation of ~85.51 after 18 hours, which was enhanced up to 96.33% after 48 hours. These antiviral results are in good agreement with the antibacterial tests performed with the same calcium alginate films produced with 0.1% w/w of GO [10] or CNFs [9] , which also showed only enhancement of antimicrobial activity with the incorporation of CNFs. Furthermore, these antiviral composite materials have a very low reduction of transparency [7, 10] and thus, they can be very promising for biomedical applications with transparency requirements such as ophthalmology and odontology. We have demonstrated that a minuscule amount of carbon nanofibers (0.1% w/w) can significantly enhance the antiviral activity of calcium alginate contrary to graphene oxide. Nonetheless, both alginate-based nanocomposites possess antiviral activity, and enhanced physical and biological properties which render them great potential in biomedical applications with antimicrobial requirements. Biomaterials Science: An Introduction to Materials in Medicine Enhancement of water diffusion and compression performance of crosslinked alginate films with a minuscule amount of graphene oxide Green synthetic routes to alginate-graphene oxide composite hydrogels with enhanced physical properties for bioengineering applications Synthesis of irregular graphene oxide tubes using green chemistry and their potential use as reinforcement materials for biomedical applications Low-Cost Advanced Hydrogels of Calcium Alginate/Carbon Nanofibers with Enhanced Water Diffusion and Compression Properties Physical and biological properties of alginate/carbon nanofibers hydrogel films Study of 1D and 2D carbon nanomaterial in alginate films Carbon Nanofibers in Pure Form and in Calcium Alginate Composites Films: New Cost-Effective Calcium alginate/graphene oxide films: reinforced composites able to prevent Staphylococcus aureus and methicillin-resistant Staphylococcus epidermidis infections with no cytotoxicity for human keratinocyte HaCaT cells Cytotoxicity and antiviral activity of calcium alginate fibers and zinc alginate fibers Alginate hydrogel protects encapsulated hepatic HuH-7 cells against hepatitis C virus and other viral infections Molecular architecture of tailed double-stranded DNA phages Raman spectra of carbon nanotubes and nanofibers prepared by ethanol flames The authors would like to acknowledge the NOBIPOL group at the Norwegian University of Science and Technology (NTNU) for the characterization of the sodium alginate used in this study. The authors would also like to acknowledge the Universidad Católica de Valencia San Vicente Mártir for their financial support through the Grants 2019-231-001UCV and 2020-231-001UCV (awarded to Á.S-A). The authors declare no conflict of interest.