Plasmid DNA (pDNA) nanoparticles synthesized by complexation with linear polyethylenimine (lPEI) are one of the most effective non-viral gene delivery vehicles. However, the lack of scalable and reproducible production methods and the high toxicity have hindered their clinical translation. Previously, we have developed a scalable flash nanocomplexation (FNC) technique to formulate pDNA/lPEI nanoparticles using a continuous flow process. Here, we report a tangential flow filtration (TFF)-based scalable purification method to reduce the uncomplexed lPEI concentration in the nanoparticle formulation and improve its biocompatibility. The optimized procedures achieved a 60% reduction of the uncomplexed lPEI with preservation of the nanoparticle size and morphology. Both in vitro and in vivo studies showed that the purified nanoparticles significantly reduced toxicity while maintaining transfection efficiency. TFF also allows for gradual exchange of solvents to isotonic solutions and further concentrating the nanoparticles for injection. Combining FNC production and TFF purification, we validated the purified pDNA/lPEI nanoparticles for future clinical translation of this gene nanomedicine.
Keywords: DNA nanoparticles; biocompatibility; gene delivery; linear PEI; tangential flow filtration.