Binary Copolymer Blending Enhances pDNA Delivery Performance and Colloidal Shelf Stability of Quinine-Based Polyplexes

Successful gene therapies require the efficient delivery of the therapeutic nucleic acids in the target cells, which is a major bottleneck. Our group has demonstrated that quinine-based polymers are effective and promising carriers for delivering nucleic acids, such as plasmid DNA (pDNA). However, the inherent hydrophobicity of quinine-based polymers makes the polymer-pDNA complexes (polyplexes) colloidally unstable leading to aggregation, which is relevant in clinical scenarios as larger particles (diameter >1000 nm) tend to perform poorly when administered systemically in vivo. Herein, we overcome the hydrophobicity-induced aggregation by using two types of quinine-based polymer systems to form polyplexes via a facile blending approach. We balanced desirable properties using quinine-based copolymers (HQ-X) as the pDNA binding component along with a quinine-based diblock copolymer (PHQ), having a polyethylene glycol chain, to provide colloidal stability to the particles. Using 5 polymer pairs, 5 mixing ratios, and 3 mixing sequences, we screened 66 formulations out of which 37 resulted in nonaggregating small polyplexes (diameter <300 nm) with colloidal stability tested up to 7 days at 4 °C. Furthermore, 18 out of these 37 colloidally stable formulations showed transfection performance better than or comparable to the commercial control, jetPEI. Our results clearly indicated that while the three mixing sequences generate polyplexes of similar characteristics, the best balance of transfection efficiency, toxicity, and colloidal stability is achieved at moderate PHQ % in the mixture when colloidal stability does not compromise payload binding. Our results showcase that polymer blending, in a manner similar to lipids, is an effective and parallel approach to achieving desirable polyplex characteristics, such as particle size, colloidal stability, and performance.