Development of lipid nanoparticles for delivery of siRNA to neural cells

Abstract

Lipid nanoparticles (LNPs) are a clinically approved platform to deliver siRNA to the liver. We are optimizing this platform to deliver siRNA for the treatment of pain. siRNA against TRPV1 (siTRPV1) was chosen as a proof of concept. siTRPV1-LNPs were formulated using C12-200, an ionizable cationic lipid and helper lipids. Their particle size and surface charge was characterized using dynamic light scattering and siRNA encapsulation efficiency was determined using a Ribogreen assay. A panel of breast cancer cell lines (MCF-7, MDA-MB-231 and BT-549) that express TRPV1 were selected for in vitro screening of LNPs to determine their cytocompatibility and gene knockdown effects using an ATP assay, qPCR and western blotting. The effects of mixing speeds during LNP formulation, slow vs. fast, were compared for determining their effects on %siRNA encapsulation. The values increased from 74.4% at slow mixing to 83.8% at faster mixing speeds. siRNA-LNPs showed a particle diameter of ca. 251 nm compared to 434 nm for empty LNPs. A lower polydispersity index of 0.15 was observed for siRNA-LNPs compared to 0.41 for empty LNPs, suggesting narrower particle size distribution upon siRNA loading. Zeta potential of siRNA-LNPs (+0.15 mV) was slightly lower compared to empty LNPs (+0.41 mV). siRNA-LNPs had a negligible impact on cell viability, which was >95% in all three cell lines and primary rat dorsal root ganglion neurons four hours post-exposure. We are currently optimizing transfection parameters to determine TRPV1 knockdown in primary DRG cultures. LNPs were well-tolerated by TRPV1-expressing cell lines and primary DRG cultures, allowing their further exploration for TRPV1 silencing.