(367b) Unravelling Cholesterol Precipitation in Biomimetic Solvents

Cholesterol is a ubiquitous molecule in human physiology, essential for various biological processes but also implicated in multiple pathological conditions. Its crystallization plays a key role in diseases such as atherosclerosis, where it contributes to plaque formation, and gallstone disease, where cholesterol supersaturation in bile leads to stone formation, causing complications like jaundice and Bouveret’s syndrome. Despite its clinical significance, the fundamental mechanisms governing cholesterol crystallization remain poorly understood. Developing strategies to control its precipitation could be highly valuable for therapeutic advancements.

While previous studies in literature have used lipid-based biomimetic solvents to investigate bulk cholesterol crystallization, these systems pose challenges for in situ characterization. To overcome this, we employed a binary water-alcohol mixture, where the alcohol serves as a lipid surrogate, enabling detailed mechanistic insights. In this presentation, we will show how different water-alcohol compositions lead to distinct crystalline forms, with some favoring cholesterol monohydrate while others promote solvate formation. Using a combination of oblique illumination microscopy (OIM) and dynamic light scattering (DLS), we demonstrated that cholesterol nucleation follows a two-step process involving the assembly of precursor clusters, which exist in both undersaturated and supersaturated solutions. Scattering measurements revealed that cluster size depends on temperature and water content but remains independent of cholesterol concentration. DLS data also showed that solvent composition significantly influences both the induction time and crystal growth rate. Using a water-isopropanol mixture, we observed that cholesterol monohydrate growth follows classical layer nucleation and spreading (1). Time-resolved imaging confirmed that layer generation originates at defect sites (dislocations), with monomer incorporation occurring primarily via surface diffusion rather than direct attachment at kinks. In situ atomic force microscopy (AFM) and microfluidic experiments further revealed that cholesterol monohydrate crystals exhibit macrostep formation, which induces a self-inhibition mechanism that slows crystal growth in contrast to the typical layer-by-layer growth observed in many crystalline systems. Finally, we will present dissolution studies highlighting a combination of classical and nonclassical mechanisms involved during cholesterol dissolution, presenting unique deviations from conventional crystallization systems reported in the literature.

Reference

1. D. Chakraborty et al., Direct observation of cholesterol monohydrate crystallization. Proceedings of the National Academy of Sciences 122, e2415719122 (2025).