Efficient endosomal escape remains a critical bottleneck in lipid nanoparticle (LNP)-mediated delivery of mRNA and other nucleic acid therapeutics. While ionizable lipids (ILs) are known to promote escape, the mechanisms by which they alter host membranes remain poorly defined. We hypothesize that ILs redistribute from LNPs into endosomal membranes, where they modulate biophysical properties such as fluidity, order, and bending rigidity, thereby lowering the energetic barrier for escape. To test this hypothesis, we performed fluctuation analysis on giant unilamellar vesicles (GUVs) under acidic conditions that mimic the endosomal environment. In control membranes, thermal undulations followed expected spectra consistent with purely thermally driven fluctuations. However, in the presence of ionizable lipids at low pH, we observed a marked deviation from thermal behavior. Fluctuation amplitudes were enhanced and relaxation dynamics altered, consistent with active-like perturbations arising from lipid redistribution and charge-driven bilayer destabilization. Quantitative analysis revealed modifications in bending rigidity and variability in membrane tension under conditions where ionizable lipids partitioned most efficiently. These observations were complemented by small-angle X-ray scattering (SAXS) measurements, which showed increased negative spontaneous curvature upon incorporation of ionizable lipids, and by micropipette aspiration experiments, which independently confirmed reductions in area compressibility modulus. Together, these three orthogonal approaches converge on a consistent picture: ionizable lipids accumulate in endosomal-like membranes at acidic pH, driving mechanical softening and curvature frustration that facilitate local deformation and escape. By linking lipid chemistry to measurable changes in fluctuation spectra, curvature, and mechanical response, our work provides direct biophysical evidence for how ionizable lipids promote endosomal escape. This multi-pronged strategy establishes a framework for quantifying membrane remodeling by delivery lipids and offers mechanistic insight to guide the rational design of next-generation LNP formulations.
Kumarage et al. (Sun,) studied this question.