Optimizing Drug Release: Bilayer to Inverted Hexagonal Phase Transition of Cationic XTC2 and Anionic DSPS Lipid System is Influenced by pH, Temperature, and Salt Concentration

Abstract

The effectiveness of a macromolecular drug delivered in lipid nanoparticles (LNP) depends upon the biophysical properties of the delivery vehicle. Recent research has shown that the design of the cationic lipid component of LNPs improves the intracellular delivery of therapeutic siRNA[1]. Even in these optimized LNPs, only 1% of the siRNA taken up by the cell via endocytosis is actually released into the cell cytosol. We proposed a mechanism of endosome disruption that relies on the formation of non-bilayer phases in the presence of anionic endosomal lipid and synthetic cationic lipids. A model system using prototypical anionic lipid 1,2-distearoyl(d70)-sn-glycero-3-[phospho-L-serine] (DSPS-d70) in 1:1 molar ratio to the cationic lipid DLin-KC2-DMA (XTC2) (pKa∼6.7) was characterized by 2H and 31P NMR spectroscopy. Through spectral analysis, we determined that at physiological pH (∼7.4) the XTC2/DSPS system exhibits a stable gel phase for temperatures below 45oC while an isotropic signal emerges at higher temperatures - no inverted hexagonal (HII) phase is observed. At low pH (∼4.75), the XTC2/DSPS system is principally in a bilayer gel phase at low temperatures with a non-bilayer HII phase predominating at higher temperatures. The transition from gel to HII phase is dependent on salt concentration and is most evident in the range of 15-25oC for 0.25M [Na+], 20-30oC for 0.5M [Na+] and 35-45oC for 1M [Na+]. Through depaking the spectra, order parameter profiles SCD have been obtained and compared for DSPS-d70 chains in bilayer and HII phases. These will be useful for computational simulation and eventually to design in vivo animal model experiments.[1] Semple, S.C., et al., Rational design of cationic lipids for siRNA delivery. Nat Biotechnol, 2010. 28(2): p. 172-6.