Anti-Coronavirus Activity of Chitosan-Stabilized Liposomal Nanocarriers Loaded with Natural Extracts from Bulgarian Flora

Disease's severity, mortality rates, and common failures to achieve clinical improvement during the unprecedented COVID-19 pandemic exposed the emergency need for new antiviral therapeutics with higher efficacy and fewer adverse effects. This study explores the potential to encapsulate multi-component plant extracts in liposomes as optimized delivery systems and to verify if they exert inhibitory effects against human seasonal betacoronavirus OC43 (HCoV-OC43) in vitro. The selection of <i>Sambucus nigra</i>, <i>Potentilla reptans</i>, <i>Allium sativum</i>, <i>Aesculus hippocastanum</i>, and <i>Glycyrrhiza glabra</i> L. plant extracts was based on their established pharmacological and antiviral properties. The physicochemical characterization of extract-loaded liposomes was conducted by DLS and electrokinetics. Encapsulated amounts of the extract were evaluated based on the total flavonoid content (TFC) and total polyphenol content (TPC) by colorimetric methods. The BALB 3T3 neutral red uptake (NRU) phototoxicity/cytotoxicity assay was used to estimate compounds' safety. Photo irritation factors (PIFs) of the liposomes containing extracts were <2 which assigned them as non-phototoxic substances. The antiviral capacities of liposomes containing medicinal plant extracts against HCoV-OC43 were measured by the cytopathic effect inhibition test in susceptible HCT-8 cells. The antiviral activity increased by several times compared to "naked" extracts' activity reported previously. <i>A. hippocastanum</i> extract showed 16 times higher inhibitory properties reaching a selectivity index (SI) of 58.96. Virucidal and virus-adsorption effects were investigated using the endpoint dilution method and ∆lgs comparison with infected and untreated controls. The results confirmed that nanoparticles do not directly affect the viral surface or cell membrane, but only serve as carriers of the active substances and the observed protection is due solely to the intracellular action of the extracts.