467718 Microfluidic Production of Ternary Polyplexes for Non-Viral Gene Delivery
Gene therapy has the potential to treat a variety of diseases by delivering DNA that codes for a therapeutic protein into human cells. Recombinant viruses are the primary vector choice in the majority of gene therapy clinical trials due to their high gene delivery efficiency. However, viral vectors suffer from serious disadvantages including poor transduction of many human cell types, immunogenicity and lack of targetability. Non-viral polymeric gene delivery vectors (polyplexes) provide an alternative solution but are limited by poor efficiency and cytotoxicity.
Improvements in polyplex-mediated gene delivery can be achieved through control of the polyplex structure and composition. Microfluidic nano-precipitation is an emerging field in which researchers seek to tune the physicochemical properties of nanoparticles by controlling the mixing of particle components during fabrication. Using this approach, several groups have demonstrated the successful production of enhanced polymeric gene delivery vectors. Controlled mixing of polymer and DNA in the laminar flow environment resulted in higher transfection efficiency and lower cytotoxicity. Other groups have demonstrated that incorporating a polyanion to form ternary polyplexes improves transfection efficiency and serum stability.
To advance non-viral gene delivery towards clinical relevance, we are developing a microfluidic platform that provides more controlled and reproducible layer-by-layer assembly of ternary complexes. Our approach provides diffusion-controlled mixing of plasmid DNA and polycations to form polyplexes, separation of excess free polycation, and diffusion-controlled introduction of polyanions to form ternary polyplexes in a sequential manner on a single device. We will describe fabrication and characterization of polyglutamate (PGA)/polyethylenimine (PEI)/DNA ternary complexes. We will also compare these ternary polyplexes to conventional PEI/DNA and PGA/PEI/DNA polyplexes formed by conventional methods in terms of cytotoxicity and transfection in several human cell lines.