Messenger RNA (mRNA) therapeutics are promising tools for vaccines, protein replacement, and cancer immunotherapy, but effective delivery systems are crucial to protect mRNA and ensure cellular uptake. Cationic polymers have gained attention for their tunable architectures and ability to form nanoscale complexes with nucleic acids, called polyplexes. This study investigates the use of bottlebrush polymers (BBs), a class of branched polymers, with different structures for mRNA delivery. A library of poly(2-(dimethylaminoethyl)methacrylate) (PDMAEMA) BBs is synthesized with varying backbone lengths and charge density to evaluate their impact on polyplex formation and delivery efficiency. After characterization to assess size distribution and encapsulation efficiency, in vitro assays are performed to examine cytotoxicity, protein expression, and cellular uptake. In vivo administration further investigates their performance. Quaternized PDMAEMA BBs, positively charged at any pH, demonstrate superior encapsulation efficiency at lower charge ratios, achieving optimal particle size and reduced cytotoxicity. Notably, the presence of permanent positive charges significantly diminishes lung accumulation, a challenge in systemically administered cationic formulations. Additionally, longer backbone improves encapsulation efficiency and cellular uptake rate. This comprehensive evaluation underscores the potential of quaternized BBs as a promising platform for mRNA delivery and provides key insights for designing next-generation bottlebrush polymers with improved transfection efficiency and in vivo applicability. STATEMENT OF SIGNIFICANCE: Messenger RNA medicines need carriers that protect the fragile genetic strands and reach the right tissues without harming cells. Lipid nanoparticles do the job today but require high doses of positively charged lipids that can trigger toxicity and tend to accumulate in the lungs. We present the first systematic in-vitro and in-vivo evaluation of "bottlebrush" polymers, which are dense, comb-shaped chains tailored for mRNA delivery. By adjusting the backbone length and introducing permanent, evenly spaced charges, we create nanocomplexes that fully encapsulate mRNA at one-tenth of the charge used previously, remain stable in blood, minimise lung uptake, and sustain protein production in liver cells with low toxicity. These structure-function rules open a versatile, scalable platform for next-generation vaccines and gene replacement therapies.
