Optimizing Exosome Lipid Hybrid Nanoparticles for Enhanced siRNA Delivery and Improved Therapeutic Anticancer Efficacy In Vivo.

Exosome lipid hybrid nanoparticles (ELNs) have emerged as promising drug delivery vehicles, integrating the innate targeting capabilities of exosomes with efficient cytosolic delivery of lipid nanoparticles. However, despite growing interest, the development of ELNs for nucleic acid delivery remains a formidable challenge, compounded by diverse production methods and a lack of systematic approaches to optimize their formulation and performance. This study employed a Box-Behnken design and two fabrication methods: freeze-thaw and sonication, to optimize the formulation of ELNs derived from exosomes of five distinct cancer cells. Formulation criteria focused on maximizing the fusion efficiency while minimizing particle size. The impact of the fusion method on cellular association and gene silencing of promising therapeutic targets, CD24, CD44, and CD47, was evaluated. The optimized formulations were subsequently assessed for therapeutic efficacy in 4T1 and B16F10 tumor models. Through careful manipulation of formulation variables, we obtained optimal ELNs with fusion efficiencies exceeding 50% and particle sizes under 170 nm while preserving exosomal markers CD9, CD63, and CD81. Cellular association studies revealed that ELNs specifically targeted their parental cell line, achieving ∼2.5-fold higher siRNA association compared to LNPs. Furthermore, the optimized ELNs facilitated the delivery of therapeutic siRNAs, resulting in robust gene silencing and consequently improved the in vitro macrophage-mediated phagocytosis of treated cancer cells. In vivo studies using 4T1 and B16F10 tumor models highlighted the enhanced therapeutic potential of the optimized ELNs, as evidenced by significant tumor targeting and growth inhibition. These findings underscore the importance of systematic formulation and method optimization in advancing ELNs as effective nucleic acid delivery platforms for cancer therapy.