Biology subjects

Sudarsan, A.

Publications and source records attributed to Sudarsan, A..

3 recordsLinked to original sources

bioRxiv2026

Thermodynamic Model and Experiments Indicate Kinetically Trapped States in Ionisable Lipid Nanoparticles

Lipid nanoparticles (LNPs) are the leading delivery vehicles for RNA-based therapies, with pH-dependent mesophase transitions of their ionizable lipids proposed to play a crucial role in LNP function and endosomal escape. However, a consistent quantitative framework linking molecular lipid properties to the thermodynamics of these transitions is still lacking. Here, we establish such a framework by combining X-ray scattering, fluorescence spectroscopy, all-atom molecular dynamics simulations, and continuum modeling. Using small-angle X-ray scattering (SAXS) and Laurdan fluorescence spectroscopy, we characterize the pH- and temperature-dependent phase behavior of both bulk lipid systems and LNPs containing the ionisable lipids MC3 or ALC-0315. Laurdan fluorescence measurements reveal the same pH- and temperature-dependent transitions in bulk and LNP systems, which closely follow the inverse micellar (LII) - inverse hexagonal (HII) transitions identified by SAXS, establishing Laurdan as a reporter of these mesophase transitions in LNPs. We combine experimental and simulation data to parameterize a continuum model of the LII - HII transition, providing a quantitative description of mesophase stability. The model reveals that geometric frustration arising from packing constraints is a key determinant of mesophase stability. By combining experimental and simulation data with theoretical analysis, we determine the relevant membrane parameters, including mean and Gaussian curvature bending rigidities, and identify the parameter ranges that govern phase stability. Together, these results establish a quantitative continuum theory for pH-dependent mesophase transitions of ionisable lipid and suggest metastable states in response to pH changes as an inherent feature of LNP core phase.

biophysics↗

bioRxiv2025

Buffer specificity of ionizable lipid nanoparticle transfection efficiency and bulk phase transition

Lipid nanoparticles (LNPs) are efficient and safe carriers for mRNA vaccines based on advanced ionizable lipids. It is understood that the pH dependent structural transition of the mesoscopic LNP core phase plays a key role in mRNA transfer. However, buffer specific variations in transfection efficiency remain obscure. Here we analyze the effect of buffer type on the structure and transfection efficiency of LNPs. We find that LNPs formulated with the cationic ionizable lipid DLin-MC3-DMA (MC3) in citrate compared to phosphate and acetate buffers exhibit earlier onset and stronger mRNA-GFP expression in-vitro. Using synchrotron small angle X-ray scattering (SAXS) we determine the pH dependent mesophases of ionizable lipid MC3/cholesterol/water dialyzed against the various buffers. The results show that the phase transition with decreasing pH from inverse micellar cubic to inverse hexagonal (Fd3m-HII) is shifted by one unit to a lower transition pH for acetate and phosphate compared to citrate buffer. Based on continuum theory and ion specific adsorption obtained from all-atom MD simulations, we propose a mechanism for buffer specificity. Citrate stabilizes the inverse hexagonal phase thus shifting the formation of HII to a higher pH. By contrast, phosphate and acetate stabilize LII. We propose that the Fd3m-to-HII transition, which is facilitated in citrate buffer, is responsible for a sensitized pH-response of the LNP core phase. This, in turn, enhances endosomal release efficiency and accounts for the earlier onset of gene expression observed in LNPs prepared with citrate buffer.

biophysics↗

bioRxiv2023

Combining molecular dynamics simulations and X-ray scattering techniques for the accurate treatment of protonation degree and packing of ionizable lipids in monolayers

The pH-dependent change in protonation of ionizable lipids is crucial for the success of lipid-based nanoparticles as mRNA delivery systems. Despite their widespread application in vaccines, the structural changes upon acidification are not well understood. Molecular dynamics simulations support structure prediction but require an a-priori knowledge of the lipid packing and protonation degree. The presetting of the protonation degree is a challenging task in the case of ionizable lipids since it depends on pH and on the local lipid environment and often lacks experimental validation. Here, we introduce a methodology of combining all-atom molecular dynamics simulations with experimental total-reflection X-ray fluorescence and scattering measurements for the ionizable lipid Dlin-MC3-DMA (MC3) in POPC monolayers. This joint approach allows us to simultaneously determine the lipid packing and the protonation degree of MC3. The consistent parameterization is expected to be useful for further predictive modeling of the action of MC3-based lipid nanoparticles.

biophysics↗