(K-437) Optimization of Polymeric Nanoparticles for siRNA Delivery

Small interfering RNA (siRNA) is a promising therapy for the knockdown of specific genes, but the endolysosomal escape presents a barrier against intracellular bioavailability and silencing potency. Here, siRNA-loaded nanoparticles (si-NPs) are conceptualized, synthesized, and screened for siRNA delivery. The si-NPs comprise a poly(dimethylaminoethylmethacrylate-co-butyl methacrylate) (PDB) and poly(lactide-co-glycolide) (PLGA) core to complex with and encapsulate the siRNA, as well as have pH-dependent membrane disruptive activity for endosome escape. The formulation also utilizes a 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (lipid-PEG) surfactant intended to provide colloidal stability and minimize NP surface charge and potential cytotoxicity. However, lipid-PEG does not effectively anchor to the si-NP core, leading to si-NP aggregation and inconsistent manufacturing and biological performance. Therefore, new polymeric surfactants have been developed with the goal of more stably anchoring to the si-NP core to promote stability while maintaining gene knockdown activity and cytocompatibility. The surfactants are diblock copolymers with an anchoring block with varied density and length of alkyl side groups, in order to investigate and optimize the contributions of hydrophobic interactions in anchoring to the si-NP core.

A library of diblock polymeric surfactants was synthesized with controlled reversible addition fragmentation chain-transfer (RAFT) polymerization. These surfactants have varied density and length of alkyl tails in order to anchor the surfactant to the NP core. An initial form of polymeric si-NPs was formulated via nanoprecipitation with a 50% octyl alkyl tail (50%O) polymeric surfactant and compared to those formulated with lipid-PEG. PLGA, PDB, and siRNA were dissolved in acetonitrile and added dropwise to the polymeric surfactant in deionized water. Dynamic light scattering (DLS) was performed to evaluate the size, polydispersity index (PDI), and zeta potential of the si-NPs. The viability and gene silencing activity of the si-NPs was then evaluated in luciferase-expressing MDA-MB-231 cells 48 hours after treatment.

The polymeric surfactant library was synthesized and verified using 1H NMR. The si-NPs made with the 50%O surfactant have a similar size, PDI, and zeta potential as si-NPs made with lipid-PEG. Notably, 25 nM and 50 nM doses of the 50%O si-NPs containing luciferase-targeting siRNA (siLuc) achieved around 80% knockdown of luciferase relative to si-NPs containing scrambled siRNA (siScr). As well, a 25 nM dose of the formulation with 50%O had significantly greater viability after 48 hours compared to the si-NPs made with lipid-PEG. Therefore, these si-NPs with novel polymeric surfactants show promise as an effective method of gene knockdown with improved cytocompatibility, in comparison to the previously used lipid-PEG surfactant. Further studies are underway to evaluate a broader library of si-NPs made with different custom surfactants.