Abstract
Nanobiotechnology is increasingly used to control viral diseases such as COVID-19, with selenium nanoparticles (SeNPs) and their composite with chitosan (CS) gaining attention for their broad bioactivity and potential as antiviral agents, but challenges related to synthesis methods, cytotoxicity, and mechanistic understanding remain. In this study, we report a novel, green biosynthesis of SeNPs using Limosilactobacillus fermentum, followed by functionalization with chitosan to produce Se/CS nanocomposites with enhanced antiviral performance against SARS-CoV-2. L. fermentum was used to biosynthesize SeNPs, providing a rapid, safe, and environmentally friendly approach. The production process was optimized by testing different parameters such as concentrations of Na(2)SeO(3), temperature, ratios between cell-free bacterial metabolites and Na(2)SeO(3), and pH. UV-Vis spectroscopy, FT-IR, XRD, Zeta potential, and TEM studies confirmed the successful synthesis of Se/CS NC, with a distinctive peak at 266 nm. FT-IR also showed that proteins were present as capping and stabilizing agents in Se/CS NC. Se/CS NC has a high zeta potential with a negative net surface charge of - 21.84 ± 4.7 mV, giving Se/CS NC great stability. Se/CS NC had an average particle size of 38.19 nm and exhibited a crystalline morphology. Biological assays in SARS-CoV-2-infected Vero E6 cells revealed that SeNPs alone displayed dose-dependent cytotoxicity, reducing cell viability above 125 µg/ml. In contrast, Se/CS NC maintained over 96% cell viability at all tested concentrations and demonstrated potent antiviral activity, achieving over 95% inhibition of viral replication at concentrations ≥ 250 µg/ml. Studies identified virucidal action as the primary antiviral mechanism, with 47.4% inhibition at 500 µg/ml. To the best of our knowledge, this study provides the first experimental evidence that green-synthesized Se/CS NC produced by L. fermentum can effectively inhibit SARS-CoV-2, highlighting their potential as a safe, eco-friendly antiviral candidate for future COVID-19 therapies and pharmaceutical applications. This demonstrates a direct application of nanotechnology in combating COVID-19 by suppressing viral replication and maintaining host cell viability.