Reactive Polymer-Engineered Dual-Targeting Fluorescent Lipid Nanoparticles for Enhanced saRNA Delivery

Abstract Lipid nanoparticles (LNPs) are widely explored as delivery vehicles for self-amplifying RNA (saRNA); however, incorporation of surface functionalities, such as fluorescent dyes for imaging and ligands for targeted delivery, remains challenging. In this study, we introduce a reactive poly(pentafluorophenyl acrylate) (PPFPA)-based copolymer platform containing a terminal C12 alkyl chain for LNP incorporation and reactive sites for modular conjugation of near-infrared dye IR775, targeting peptides, and polyethylene glycol chains. The resulting copolymers are successfully incorporated into LNPs, enabling fluorescence imaging and targeted delivery while maintaining colloidal stability and low cytotoxicity. Systematic variation of peptide loading and composition identifies an optimal loading of 8 peptides per chain for cellular targeting. Additionally, dual-peptide-functionalized LNPs exhibit higher cellular uptake than single-peptide-functionalized LNPs, with 7-fold and 5-fold increases relative to nonpeptide-functionalized LNPs, respectively. This enhanced uptake translates into increased functional saRNA delivery, with the dual-peptide-functionalized LNPs showing a 128% enhancement in protein expression. These findings establish PPFPA-C12-based copolymers as a versatile platform for designing multifunctional LNP systems for RNA delivery.