(257f) Efficient RNA Interference by Intracellularly Targeted and Enhanced Release of siRNA by Acid-Degradable Linear Polyethylenimine: In Vitro and In Vivo Evidence

Silencing a target gene using small interfering RNA (siRNA) has been of great interests due to its scientific clarity and broad targets in biological studies and medicine. Like delivery of other types of therapeutic genes (except the requirement for nuclear localization in plasmid DNA delivery), overcoming extracellular and intracellular biological barriers is a key design parameter in achieving efficient, biocompatible, and specific gene silencing. In this study, linear polyethylenimine (L-PEI), which generates low toxicity with simultaneously limited siRNA delivery efficiency, was ketalized to accomplish 1) efficient endosomal escape of siRNA-containing polyplexes, 2) selective cytoplasmic localization of released siRNA, and 3) rapid unpackaging of the complexed siRNA from the carrier polymers, upon the hydrolysis of acid-degradable ketal branches. A confocal laser scanning microscopic study demonstrated that siRNA-complexing ketalized linear PEI (KL-PEI) was efficiently internalized and the delivered siRNA was exclusively released into the cytoplasm where its target mRNA is present. More importantly, complxed siRNA was found efficiently dissociated from KL-PEI (enhanced unpackaging) in the cytoplasm and no siRNA was found in the nucleus (selective intracellular localization in the cytoplasm). On the contrary, siRNA and unmodified linear PEI were colocalized not only in the cytoplasm but also in the nucleus with no obvious dissociation. Consequently, KL-PEI/siRNA polyplxes showed much higher gene silencing efficiency than unmodified linear PEI/siRNA polyplexes in a serum independent manner, implying the possibility of achieving high RNA interference in vivo. Two different models were employed to demonstrate efficient gene silencing in vivo. Anti-eGFP siRNA was polyplexed with KL-PEI and intravenously injected in the mice carrying a subcutaneously established eGFP-expressing tumor. Reduced expression of the target eGFP gene was quantified by flow cytometry of the recovered individual tumor cells, histological studies on eGFP expression in tumor tissue, and reduced circulatory biomarkers in blood. The polyplexes formulated in the same manner but labeled with a red fluorescent dye afterwards were also directly injected into upper cervical spinal cord of eGFP transgenic mice. Preliminary results indicate that KL-PEI/siRNA polyplexes were able to diffuse into the dorsal column and taken up by cells in the spinal cord, silencing eGFP expression. Results from in vitro and in vivo studies clearly demonstrate that KL-PEI could be a novel platform for delivering siRNA to treat cancer as well as chronic orofacial pain.