Synergistic Enhancement of siRNA Transfection by Hybrid Lipid−Trimethyl Chitosan Nanoparticles

Abstract The clinical potential of RNA interference (RNAi) technologies, particularly siRNA-based therapeutics, remains limited by the need for safe, stable, and efficient delivery systems. Both lipid nanoparticles (LNPs) and chitosan-based carriers have demonstrated significant potential for nucleic acid delivery; however, each platform presents distinct limitations. LNPs may show limited mucosal retention and formulation instability, whereas trimethyl chitosan (TMC)-based systems often exhibit lower transfection efficiency despite their favorable stability and mucoadhesive properties. In this study, we developed a hybrid delivery system combining LNPs with a polymeric matrix composed of N,N,N-trimethyl chitosan (TMC) and the helper components dextran sulfate (DS), tripolyphosphate (TPP), and hyaluronic acid (HA). The resulting hybrid nanoparticles exhibited reduced hydrodynamic diameters relative to TMC-based nanoparticles, low polydispersity, and composition-dependent changes in surface charge and structural organization. Fluorescence-based analysis further demonstrated high apparent siRNA association and increased resistance to decomplexation in the complete hybrid formulations. Electron microscopy revealed a more complex hybrid architecture and interparticle organization compared with the parent LNPs. Using a stable 293T-eGFP cell model, the hybrid system achieved high gene silencing (∼87%) at optimized TMC/siRNA weight ratios, outperforming polymer-only and lipid-only formulations under selected conditions. Confocal and live-cell fluorescence imaging confirmed efficient intracellular uptake and trafficking through acidic endolysosomal compartments, while cytotoxicity assays indicated high biocompatibility at moderate TMC concentrations. Short-term stability studies further showed that incorporation of the TMC-based matrix preserved transfection performance after 5 days of storage. Overall, the hybrid formulation combines the efficient intracellular delivery characteristics of LNPs with the structural and cargo-retention advantages of TMC-based systems, supporting its potential as a versatile platform for siRNA delivery and gene-silencing applications.