Lipid Nanoparticle-Mediated Delivery of 27-Hydroxycholesterol for Targeting Rhinovirus-Induced Respiratory Diseases

Human rhinovirus (HRV) is a highly widespread pathogen, the most frequent cause of the common cold, and often associated with asthma exacerbation. To date, attempts to develop direct-acting antivirals (DAAs) have proved unsuccessful, also due to their tendency to select resistant variants when challenged with HRV quasispecies. 27-hydroxycholesterol (27OHC), a cholesterol-derived host-targeting antiviral (HTA), inhibits HRV replication and is less prone to selecting resistant variants than the DAAs pleconaril and rupintrivir. In the present study, we developed and evaluated a lipid nanoparticle (LNP)-based formulation for the nasal delivery of 27OHC. The antiviral efficacy of 27OHC-loaded LNPs was assessed on HeLa cells by focus reduction assays and yield reduction assays. The effect on cell viability and the cytotoxicity were determined via MTS and LDH assays to calculate the 50% cytotoxic concentration (CC50). Efficacy and biocompatibility of 27OHC were further validated in a physiologically relevant 3D model of reconstituted human nasal epithelia derived from healthy donors. Cellular uptake and internalization kinetics of LNPs were assessed on HeLa cells with the use of fluorochrome-tagged LNPs and indirect immunofluorescence. Our results demonstrate that 27OHC-loaded LNPs strongly inhibit HRV infectivity at 50% effective concentration (EC50) in the low micromolar range and are characterized by a selectivity index (SIs = CC50/EC50) above 150. Importantly, the adopted formulation suppressed viral replication in the nasal epithelium without cytotoxic effects. The uptake experiments show that LNPs enter cells and are clearly detectable intracellularly at 24 h post-treatment. These findings highlight the therapeutic potential of 27OHC delivered via LNPs as a promising host-targeting strategy against HRV and provide a rationale for further studies aiming to explore its potential in a preclinical setting.