Synergistic Polymer Blending Informs Efficient Terpolymer Design and Machine Learning Discerns Performance Trends for pDNA Delivery

Cationic polymers offer an alternative to viral vectors in nucleic acid delivery. However, the development of polymer vehicles capable of high transfection efficiency and minimal toxicity has remained elusive, and continued exploration of the vast design space is required. Traditional single polymer syntheses with large monomer bases are very time-intensive, limiting the speed at which new formulations are identified. In this work, we present an experimental method for the quick probing of the design space, utilizing a combinatorial set of 90 polymer blends, derived from 6 statistical copolymers, to deliver pDNA. This workflow facilitated rapid screening of polyplex compositions, successfully tailoring polyplex hydrophobicity, particle size, and payload binding affinity. This workflow identified blended polyplexes with high levels of transfection efficiency and cell viability relative to single copolymer controls and commercial JetPEI, indicating synergistic benefits from copolymer blending. Polyplex composition was coupled with biological outputs to guide the synthesis of single terpolymer vehicles, with high-performing polymers P10 and M20, providing superior transfection of HEK293T cells in serum-free and serum-containing media, respectively. Machine learning coupled with SHapley Additive exPlanations (SHAP) was used to identify polymer/polyplex attributes that most impact transfection efficiency, viability, and overall effective efficiency. Subsequent transfections on ARPE-19 and HDFn cells found that P10 and M20 were surpassed in performance by M10, contrasting with results in HEK293T cells. This cell type dependency reinforced the need to evaluate transfection conditions with multiple cell models to potentially identify moieties more beneficial to delivery in certain tissues. Overall, the workflow employed can be used to expedite the exploration of the polymer design space, bypassing extensive synthesis, and to develop improved polymer delivery vehicles more readily for nucleic acid therapies.