PRELIMINARY RESULTS ON MICROPLASTIC AND NANOPLASTIC IMPACT IN VIRUS AND BACTERIAL REPLICATION

Nicoletta CAPUANO^1,2, Alessia GIANNATTASIO³, Noemi CAFA’^1,2, Anna BARBATO^1,2, Mariarosa SCOGNAMIGLIO⁴, Vincenzo BELGIORNO⁵, Luca RINALDI^6, Veronica FOLLIERO^1,2, Antonio BUONERBA³, Gianluigi FRANCI^1,2 | ¹Dipartimento di Medicina, Chirurgia e Odontoiatria, Scuola Medica Salernitana, Università degli Studi di Salerno, Baronissi (SA), Italia; ²UOS di Microbiologia e Virologia, AOU San Giovanni di Dio e Ruggi D’Aragona, Salerno, Italia; ³Dipartimento di Chimica e Biologia, Università degli Studi di Salerno, Fisciano (SA), Italia; ⁴Dipartimento di Ingegneria Industriale, Università degli Studi di Salerno, Fisciano (SA), Italia; ⁵Dipartimento di Ingegneria Civile, Università degli Studi di Salerno, Fisciano (SA), Italia; ⁶Dipartimento di Medicina e Scienze della Salute, Università degli Studi del Molise, Italy

Introduction: Microplastics and Nanoplastics (MP-NPs) are a global concern due to their significant environmental impact and potential effects on human health. These particles originate from a variety of materials and can act as "Trojan horses," carrying on their surface heavy metals, additives, and microorganisms, including viral particles. Viral adsorption onto MPs may extend viral shelf-life, increasing their persistence and infectivity over time. Moreover, NPs can also affect bacterial survival by exerting a moderate impact on their growth.

Materials and Methods: Oxidized Polyethylene microplastics (PE-Ox-MPs) were sterilized using UV light and dispersed in aqueous solution containing Tween-20 and Sodium Dodecyl Sulfate (SDS), resulting in improved particle dispersion and solution homogeneity. PeOx-MPs were characterized by Nuclear Magnetic Resonance (NMR, Bruker AVANCE 600 MHz), Diffusion-Ordered Spectroscopy (DOSY), and Field Emission Scanning Electron Microscopy (FE-SEM, Carl Zeiss, model Supra 35). Particle size distribution was verified via Dynamic Light Scattering (DLS, Zetasizer Nano S). Cytotoxicity was assessed in VERO-76 cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in a concentration range of 10 to 0.039 μg/mL. Bisphenol A (BPA), an additive used in the production of plastic material, served as an internal control for microplastics. The effect of MPs on viral shelf-life was evaluated through a plaque assay after viral pre-treatment for 1 h, 2 h, and 6 h in response to PE-Ox-MPs. Instead, Nanoplastics of PolyLactic Acid (PLA-NPs) were dissolved in PBS 1X with a final concentration of 1 mg/mL. Before use, the solution was processed in a sonicator bath according to the following parameters: temperature 4°C, power 100 W and frequency 37 Hz. Antibacterial activity of PLA-NPs was assessed by plate microdilution method against both Gram-positive (S. aureus and E. faecalis) and Gram-negative bacteria (K. pneumoniae and E. coli), in a concentration range from 300 to 0.6 µg/mL.

Results: Size analysis revealed an average diameter of ~300 nm. MTT assay results indicated low cytotoxicity, with cell viability exceeding 80% at the highest concentration tested, suggesting minimal direct cytotoxic effects. Pre-treatment plaque assay demonstrated that MPs extended viral survival, with infectivity increasing by approximately 20% after 2 h and 40% after 6 h, compared to 1 h exposure. PLA-NPs showed a better antibacterial effect against Gram-positive bacteria rather than Gram-negative, with about 20% of inhibition at the highest concentration tested in the first case and a reduction of less than 10% in the second one.

Conclusions: This study confirms that viruses can adsorb onto the surface of MPs, thereby prolonging their survival and infectivity. The different responses of bacteria to NPs depend on factors related to structural differences as well as intrinsic characteristics of the materials, such as charge and size. Moreover, the cellular response is influenced by the ability of NPs to accumulate within the cytosol, leading to oxidative stress. Therefore, MP-NPs pose a dual threat, environmental and biological, due to their contaminant nature and their role in enhancing viral transmission and bacterial abundance.