Kinetic Control in Assembly of Plasmid DNA/Polycation Complex Nanoparticles

Abstract

Polyelectrolyte complex (PEC) nanoparticles assembled from plasmid DNA (<i>p</i>DNA) and polycations such as linear polyethylenimine (<i>l</i>PEI) represent a major nonviral delivery vehicle for gene therapy tested thus far. Efforts to control the size, shape, and surface properties of <i>p</i>DNA/polycation nanoparticles have been primarily focused on fine-tuning the molecular structures of the polycationic carriers and on assembly conditions such as medium polarity, pH, and temperature. However, reproducible production of these nanoparticles hinges on the ability to control the assembly kinetics, given the nonequilibrium nature of the assembly process and nanoparticle composition. Here we adopt a kinetically controlled mixing process, termed flash nanocomplexation (FNC), that accelerates the mixing of <i>p</i>DNA solution with polycation <i>l</i>PEI solution to match the PEC assembly kinetics through turbulent mixing in a microchamber. This achieves explicit control of the kinetic conditions for <i>p</i>DNA/<i>l</i>PEI nanoparticle assembly, as demonstrated by the tunability of nanoparticle size, composition, and <i>p</i>DNA payload. Through a combined experimental and simulation approach, we prepared <i>p</i>DNA/<i>l</i>PEI nanoparticles having an average of 1.3 to 21.8 copies of <i>p</i>DNA per nanoparticle and average size of 35 to 130 nm in a more uniform and scalable manner than bulk mixing methods. Using these nanoparticles with defined compositions and sizes, we showed the correlation of <i>p</i>DNA payload and nanoparticle formulation composition with the transfection efficiencies and toxicity <i>in vivo</i>. These nanoparticles exhibited long-term stability at -20 °C for at least 9 months in a lyophilized formulation, validating scalable manufacture of an off-the-shelf nanoparticle product with well-defined characteristics as a gene medicine.