In this work, ionizable and cleavable lipids were designed, synthesized, and characterized. The lipids were derived from the γ-Tisomer of vitamin E and were used to develop novel lipid nanoparticles (LNPs) targeting Bone Marrow on X chromosome (BMX). BMX is a promising and emerging target for the development of advanced cancer therapeutics. Our team recently reported on targeting BMX using small-molecule modalities. Here, we are expanding the modality of targeting employing BMX siRNA LNPs. AI and experimental design (QbD) were used to develop an optimal formulation for the BMX siRNA delivery. AI tools helped in designing the optimal ionizable lipid and further assisted in revealing the desired interaction between the ionizable lipids and the BMX siRNA. The BXM siRNA LNPs developed in this study were initially designed based on the compositions of the FDA-approved Onpattro formulation. The vitamin E-based ionizable and cleavable lipids were synthesized and chemically characterized usingH NMR. QbD played a crucial role in achieving the optimal formulation composition of the LNPs. The optimized BMX siRNA LNP compositions were prepared using methanol dilution and extrusion methods. The developed LNPs were characterized for their physicochemical properties, including particle size, zeta-potential, entrapment efficiency, and pH-dependent release. The in-vitro anticancer activity of the optimized BMX siRNA LPNs was also assessed. The particle size of the optimized BMX siRNA LNPs was in the range of 151 nm, with a zeta potential of 30 mV. The developed BMX siRNA LNPs exhibited a pH-dependent release profile, with the highest release occurring at an acidic pH of 5.5. The in-vitro anticancer activity of the developed BMX siRNA against two prostate cancer cell lines, DU145 and PC3, demonstrated statistically significant anticancer activity, as indicated by ICvalues of 1.1 µM and 1.3 µM, respectively, compared to the control and Onpattro-composition like formulations. Furthermore, the BMX siRNA LNP designed using the novel lipids showed a significant reduction in BMX mRNA expression levels in DU145 and PC3 cell lines compared to the control or the Onpattro-like formulation. Profoundly, the novel LNPs showed significant in-vivo anticancer activity in the Castration-Resistant Prostate Cancer (CRPC) animal model compared to control or the Onpattro-like formulation. The results from this study further support the promise of such novel LNPs in cancer therapeutics development. 3 50 1
