Multifunctional Lipid Nanoparticles for Protein Kinase N3 shRNA Delivery and Prostate Cancer Therapy.

Abstract

Protein kinase N3 (PKN3), by virtue of its abnormal expression in prostate cells, has been widely used as a target of RNAi (shRNA, siRNA, miRNA) therapy. The major challenges of PKN3 RNAi therapy lie in how to design effective interference sequences and delivery systems. Herein, new PKN3 shRNA sequences (shPKN3-2459 and shPKN3-3357) were designed, and bioreducible, biodegradable, ionizable lipid-based nanoparticles were developed for shPKN3 delivery. First, an ionizable lipid (DDA-SS-DMA) bridged with disulfide bond and ester bonds was synthesized by a three-step reaction and confirmed by MS, 1H NMR, and 13C NMR. The ionizable lipid was mixed with cholesterol, DSPC, PEG-lipid, and shPKN3 by a microfluidic mixer to prepare lipid nanoparticles (LNP-shPKN3) which were characterized by DLS and TEM. Afterward, the pH and glutathione (GSH)-responsiveness of the DDA-SS-DMA based LNP delivery system were investigated by lysosome escape and gel electrophoresis assays. Compared with the commercial transfection reagent Lipo2000, the DDA-SS-DMA based delivery system showed higher transfection efficiency and lower toxicity. Western blot analysis, invasion tests, and migration assays were performed to evaluate the silencing effect of shPKN3 in vitro. In in vivo studies, high tumor suppression (65.8%) and treatment safety were evident in the LNP-shPKN3-2459 treatment group. Taken together, the DDA-SS-DMA based delivery system encapsulating shPKN3-2459 showed significant antitumor efficacy and might be a promising formulation for the treatment of prostate cancer.