Biocompatible Films of Calcium Alginate Inactivate Enveloped Viruses Such as SARS-CoV-2

Abstract

The current pandemic is urgently demanding the development of alternative materials capable of inactivating the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the coronavirus 2019 (COVID-19) disease. Calcium alginate is a crosslinked hydrophilic biopolymer with an immense range of biomedical applications due to its excellent chemical, physical, and biological properties. In this study, the cytotoxicity and antiviral activity of calcium alginate in the form of films were studied. The results showed that these films, prepared by solvent casting and subsequent crosslinking with calcium cations, are biocompatible in human keratinocytes and are capable of inactivating enveloped viruses such as bacteriophage phi 6 with a 1.43-log reduction (94.92% viral inactivation) and SARS-CoV-2 Delta variant with a 1.64-log reduction (96.94% viral inactivation) in virus titers. The antiviral activity of these calcium alginate films can be attributed to its compacted negative charges that may bind to viral envelopes inactivating membrane receptors.

Keywords: SARS-CoV-2; bacteriophage; biomaterials; calcium alginate; films; hydrogels; phi 6.

Figure 1

Bacteriophage phi 6 RNA extraction and quantification: Schematic representation of the RNA extraction and quantification process protocol to ensure that the viral particles do not remain attached to the material film instead of being inactivated. Created by Ángel Serrano-Aroca with Biorender.com.

Figure 2

MTT cytotoxicity tests of extracts obtained from the calcium alginate films (AlgCa), negative control (culture medium used to produce the films extracts), and positive control (cytotoxic 1000 μM zinc chloride solution) cultured in human keratinocyte HaCaT cells at 37 °C. ANOVA with subsequent Tukey’s post hoc test: *** p > 0.001; ns, not significant.

Figure 3

Antiviral activity of calcium alginate against bacteriophage phi 6 determined by the double-layer method: (a) Titration images (undiluted samples) for AlgCa film and control after 30 min of viral contact. These images show the reduction in infection capacity (reduction in lighter spots); (b) Viral inactivation in log (PFU/mL) for AlgCa film and control after 30 min of viral contact. Student’s t-test (*** p > 0.001).

Figure 4

Bacteriophage phi 6 RNA extraction and quantification: Bacteriophage RNA concentration before (control) and after being in contact with the calcium alginate (AlgCa) films for 30 min expressed in ng/μL. Student’s t-test (ns, not significant).

Figure 5

Reduction in infection titers of the SARS-CoV-2 Delta Variant in logarithm of TCID50 per mL (log (TCID50/mL)) and TCID50 per mL. Reduction in infection titers in plaque-forming units per mL (PFU/mL) for control and calcium alginate films (AlgCa) at 30 min of viral contact. *** p > 0.001. Student’s t-test (*** p > 0.001).

Figure 6

Inactivation mechanism of SARS-CoV-2 Delta variant and bacteriophage phi 6, in the synthesized negatively charged calcium alginate film: (a) calcium alginate structure in dry state according to the egg-box model Reprinted with permission under a Creative Commons CC BY License from ref. [21]. Copyright 2017 Spinger Nature; (b) calcium alginate in swollen state after being in contact with a viral aqueous solution Reprinted with permission under a Creative Commons CC BY License from ref. [21]. Copyright 2017 Spinger Nature; (c) enveloped RNA viruses: bacteriophage phi 6 and SARS-CoV-2 viral morphologies. Created by Ángel Serrano-Aroca with Biorender.com; and (d) negatively-charged calcium alginate interfering with SARS-CoV-2 Delta variant in viral aqueous solution. Created by Ángel Serrano-Aroca with Biorender.com.