Abstract B32: Impact of Lipid-Substitution on Assembly and Delivery of siRNA by Cationic Polymers

Abstract

Abstract Downregulation of target protein expression by small interfering RNA (siRNA) has been one of the most promising strategies in medicinal therapy since the discovery of this revolutionary process in late 1990s. Many obstacles, however, have to be overcome to achieve safe and efficient siRNA delivery to target cells. Short in vivo half-life of siRNA and safety concerns involved with viral vectors used for DNA transfection are among the main reasons for the necessity of a better delivery system to take full advantage of this strategy. Polycationic polymers have been studied extensively for this purpose and high molecular weight polyethylenimines (PEIs) have led to promising results; however, unacceptable toxicity profile of these polymers has been a hurdle for their clinical application. In this study, we report characterization of a library of polymers synthesized by hydrophobic modification of a low molecular weight (2 kDa) PEI (PEI2) with a wide range of different fatty acids, engineered to enhance their ability to protect and deliver their nucleotide cargo to the cells, while still being water soluble. A general increase in lipid substitution was observed as the lipid:PEI ratio was increased during the synthesis. Among the polymers derived from lipid:PEI ratio of 0.066, caprylic acid-substituted polymer showed a lower binding affinity, while all other polymers performed similarly. Complete siRNA binding was typically achieved at polymer:siRNA ratio of < 0.5. The BC50 (polymer:siRNA ratio needed for 50% binding) was generally increased with the extent of lipid substitution, indicating an adverse effect of lipid substitution on siRNA complexation. Particle size analysis showed a range of 300 to 600 nm for all of the polymer/siRNA complexes, and for all lipid-substituted polymers, a continuous increase in the ζ-potential was observed with increasing polymer:siRNA weight ratio, and all polymers showed positive ζ-potential at the ratio of 10:1. The ζ-potentials of complexes formed with lipid-modified polymers were higher than complexes of PEI2 at all ratios. With the polymer:siRNA ratio of 1:1, all lipid-substituted polymers showed complete protection against degradation, while naked siRNA was readily degraded (<5% intact siRNA remaining) and only ∼68% of siRNA bound with PEI2 remained intact under the experimental conditions. The cytotoxicity of the synthesized polymers was slightly higher than PEI2, but significantly lower than larger MW PEI. Polymer/siRNA complexes formed with lipid-modified polymers increased the cellular uptake of siRNA significantly. Down-regulation of two different target proteins was also evaluated using the modified polymers: a housekeeping enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and an efflux protein involved in multi-drug resistance (MDR), P-glycoprotein (P-gp). Optimum silencing for the target proteins was achieved by using polymer:siRNA ratio of 4:1 for GAPDH and 8:1 for P-gp. A maximum of 66% and 67% downregulation was observed for GAPDH and P-gp, respectively. Our results indicate that hydrophobic modification of low molecular PEI could render this otherwise ineffective polymer to a safe effective delivery system for intracellular siRNA delivery and silencing of protein expression.