Microfluidic-assisted synthesis and development of lipid-polymer hybrid nanoparticles for nucleic acid delivery

Gene medicines hold great promise for the treatment and prevention of diverse medical conditions including cancer, genetic disorders and infectious diseases. However, the gene therapeutic requires site-specific delivery in target cells to induce therapeutic effect. This is typically achieved using carriers in the form of modified viruses or nanoparticles. The latter vehicle is preferred due to ease of synthesis, reduced immunogenic responses and lower production costs. Despite these benefits, improvements are needed for nanoparticle systems in several aspects including storage stability, cytotoxicity and transfection efficiency. This thesis describes research conducted on the development of a novel lipid-polymer hybrid nanoparticles for plasmid DNA (pDNA) delivery. The nanoparticles were capable of production using microfluidics, the leading platform for industrial-scale manufacture of nanomedicines. The first section of this thesis concentrates on nanoparticle production using poly (lactic-co-glycolic acid) (PLGA), a biodegradable and biocompatible polymer of neutral charge. The second part describes nanoparticle production based on the cationic polymer Eudragit, with varying pH-sensitive dissolution properties. The polymer, formulation composition and production conditions significantly impacted nanoparticle formation, colloidal stability, in vitro cytotoxicity and transfection efficiency. Importantly, the Eudragit-based hybrid nanoparticles obtained highly reproducible in vitro transfection in both nanoprecipitation and microfluidics platform and retained transfection activity following storage at 4oC for 3 months. Those prepared using nanoprecipitation achieved targeted pDNA transfection in the mouse testes, which could serve as a model for nanoparticle production using microfluidics. This thesis demonstrates the utility of lipid-polymer hybrid nanoparticles based on Eudragit as a facile tool for in vitro and in vivo assessment of experimental gene nanomedicines. The work also confirms the capability of hybrid nanoparticle production using microfluidics, thereby helping facilitate the transition of gene medicines from bench to clinic.