Design and in vitro validation of Brome mosaic –virus-like particles for gene delivery and immunomodulation of melanoma

RNA interference (RNAi) is a powerful tool for post-transcriptional gene silencing, yet its clinical translation remains limited by the lack of safe and efficient delivery systems. In this study, we evaluated the potential of plant virus-like particles (VLPs), derived from Brome Mosaic Virus (BMV), as a biodegradable and biocompatible nanocarrier for small interfering RNA (siRNA) delivery aimed at modulating immune checkpoints in melanoma. Recombinant BMV capsid proteins were expressed in <i>E. coli</i> and self-assembled <i>in vitro</i> into uniform VLPs encapsulating siRNA directed against the programmed death-ligand 1 (PD-L1). The VLPs displayed high structural stability, efficient siRNA encapsulation, and excellent biocompatibility. <i>In vitro</i>, A high cellular uptake was confirmed by confocal microscopy and flow cytometry. Besides, no cytotoxicity was observed and functional siRNA delivery was demonstrated <i>in vitro</i> by knockdown of eGFP in macrophages and PD-L1 in B16F10 melanoma and JAWS II dendritic cells, with significant knockdown efficiencies comparable to controls. Beyond molecular knockdown, PD-L1 silencing mediated by BMV-VLPs induced functional immunomodulation, increasing CD8⁺ T-cell-mediated cytotoxicity in melanoma and CMT64 tumor models and enhancing allogeneic T-cell proliferation in dendritic cell-based mixed leukocyte reactions. Overall, these findings indicate that plant-derived BMV-VLPs can safely mediate RNA-based checkpoint modulation and could complement antibody-based PD-1/PD-L1 blockade strategies. Owing to their intrinsic biocompatibility, scalability, and modular design, BMV-VLPs represent a sustainable and versatile alternative to synthetic lipid systems for RNA immunotherapy.