: RNA interference (RNAi) is a promising strategy for mitigating diseases at the molecular level. However, RNAi is limited by its instability in biological fluids and impermeability to cellular membranes. In response, our lab has previously patented a non-ionizable lipid nanoparticle (LNP) platform (R8-PLP) for RNAi therapeutic delivery. This formulation incorporates 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG) to improve particle stability and drug retention. However, long-anchored PEGylated lipids like DSPE-PEG may impair internalization and stimulate immune responses. The literature suggests substituting short-anchored PEGylated-lipids like 1,2-dimyristoyl-rac-glycero-3-[methoxy(polyethylene glycol)-2000] (DMG-PEG) to attenuate these effects. Here, we evaluated whether substituting DMG-PEG for DSPE-PEG in our R8-PLP would improve in vitro cellular delivery and gene transfection without compromising in vitro critical quality attributes (CQAs) or increasing cytotoxicity.: CQAs [encapsulation efficiency (EE%), particle size (nm), homogeneity (polydispersity index; PDI), and membrane zeta-potential] were assessed at assembly and after storage for up to 28 days at 4 °C. Additionally, in-serum stability at 4 °C and serum release kinetics at 37 °C were assessed. Human aortic smooth muscle cells (HASMCs) were treated with R8-PLPs and analyzed for cellular uptake (fluorometry), cytotoxicity (LIVE/DEAD stain), and gene modulation (qPCR).: DMG-PEG incorporation at variable mol% did not alter R8-PLP size, homogeneity, or siRNA EE% at assembly or after long-term storage, but did accelerate siRNA release kinetic profiles compared to DSPE-PEG controls. DMG-PEG substitution enhanced cellular uptake compared to DSPE-PEG R8-PLPs without increasing cytotoxicity. DMG-PEG incorporation also achieved significant silencing versus non-treated controls but did not improve gene silencing compared to DSPE-PEG R8-PLPs.: Thus, DMG-PEG substitution did not enhance R8-PLP in vitro gene modulation efficacy despite improving cellular uptake and maintaining CQAs. Background/Objectives Methods Results Conclusions
