Substitution of Hydrophobic Tails on Hydrophilic Polyethyleneimine via Urethane Linkages Yields Cleavable Lipopolymers with Effective mRNA Delivery

Background/Objectives: Messenger RNA (mRNA) delivery to circulating, suspension-growing cells remains challenging due to their inherent barriers to mRNA uptake. This study aimed to develop a novel lipopolymer-based nanoparticle (NP) platform to improve mRNA delivery and gene expression in suspension-growing cells and investigate the influence of NP physicochemical properties on delivery efficiency. Methods: Polyethyleneimine (PEI) was functionalized with lipidic moieties through degradable urethane linkages to generate lipopolymers with varying lipid modifications. mRNA delivery and gene expression were evaluated in suspension-growing myeloid leukemia cells in vitro and following systemic administration in mice. The effects of incorporating the anionic additive Trans-Booster (TB) into binary and ternary NPs were investigated. Machine learning analysis using SHapley Additive exPlanations (SHAP) was performed across 125 formulations to identify key physicochemical determinants of delivery performance. Results: Geraniol-conjugated PEI demonstrated the highest gene expression among the lipid modifications investigated. Incorporation of TB further enhanced transfection efficiency by modulating NP surface charge. In mice, binary NPs preferentially enhanced mRNA delivery to the spleen and lungs, whereas ternary NPs promoted gene expression primarily in the liver and intestine. SHAP analysis identified TB–charge interactions, TB presence, steric hindrance, lipid double bonds, and effective hydrophobicity as key determinants of mRNA delivery efficacy. Conclusions: The developed lipopolymer-based NP platform demonstrates potential for improving mRNA delivery to suspension-growing cells. Lipid functionalization and TB incorporation influenced transfection efficiency and tissue distribution, while SHAP analysis provided insights into the physicochemical factors governing delivery performance. These findings support further development of engineered lipopolymer NPs for mRNA-based applications targeting circulating cells.