Combinatorial Discovery of RAFT Cationic Polymers for mRNA Delivery: Structure–Function Insights from High-Throughput Screening and Machine Learning

Cationic polymers are considered promising delivery systems for mRNA, offering potential advantages over lipid nanoparticles. Here a library of tertiary amine-containing, methacrylate-based cationic polymers with diverse molecular characteristics and properties were prepared by combinatorial RAFT polymerization for mRNA delivery. The ability of the synthesized cationic polymers to complex with mRNA was thoroughly investigated. The biological responses, including cellular uptake, cytotoxicity, and mRNA transfection efficiency, of the formed mRNA-polymer polyplexes were systemically investigated. Through high-throughput screening assays, we identified several lead polymers that showed superior effectiveness in delivering mRNA, with performance significantly outperforming other synthesized cationic polymers as well as polyethylenimine (PEI) and Lipofectamine, two benchmark gene delivery materials. To unravel the complex structure–function relationships between the chemical and physical properties of cationic polymers/mRNA polyplexes and their biological responses, machine learning analyses were conducted. These in silico studies identified several key attributes that are predictive of cellular uptake, cytotoxicity, and mRNA transfection efficiency, providing valuable insights for the future design of more potent cationic polymers for mRNA delivery.