(337aj) Unique Mechanism of Cholesterol Crystallization in Biomimetic Systems

• Studying Polymorphism using Powder XRD, Single Crystal XRD, UV-vis spectroscopy
• In-Vitro testing of potential drug molecules using techniques like DLS (Dynamic Light Scattering), Microfluidics, AFM (Atomic Force Microscopy) and OIM (Oblique Illumination Microscopy)
• Characterizing crystal structures using TEM, SEM, FTIR etc.

Numerous pathological conditions are characterized by the excessive crystallization of cholesterol, a molecule that plays a crucial role in human physiology. Cholesterol is prominently involved in the development of atherosclerotic plaque in the arteries, and the formation of gallstones from bile, resulting in various ailments such as jaundice and Bouveret's syndrome. Despite these global healthcare challenges, there is a paucity of research exploring the underlying mechanisms of cholesterol crystallization. Nevertheless, it is widely recognized that unraveling approaches to control the precipitation of cholesterol holds immense potential in the development of novel pharmaceutical interventions for the treatment of these conditions.

Previous studies have employed lipid-based systems as biomimetic models to investigate the crystallization of cholesterol on a larger scale having limitations for in situ characterization techniques. This led us to use a binary mixture of water and ethanol as a surrogate for lipid media to explore the crystallization of cholesterol. A combination of oblique illumination microscopy (OIM) and dynamic light scattering (DLS) revealed that the cholesterol crystallization process involves two-step nucleation, involving the formation of clusters. Notably, these clusters were present in both undersaturated and supersaturated environments and played a direct role in surface growth. We also ascertained that the cluster size is influenced by temperature and the water content of the mixture, rather than the concentration of cholesterol itself. The DLS data further revealed the significant impact of solvent composition on both the induction time and the rate of crystal growth. Moreover, we conducted in situ atomic force microscopy measurements to validate our findings regarding surface growth mechanisms. These experiments confirmed that the growth of cholesterol monohydrate crystals involves a combination of classical layer-by-layer mechanisms, regulated by surface diffusion, as well as nonclassical pathways that entail cluster attachment. Presence of these clusters introduced unique dynamics to the propagation of layers during surface growth, which deviated from any previously reported crystal growth mechanisms in the literature. Overall, our study reveals a very complex and unique mechanism of cholesterol monohydrate crystallization that contribute to a deeper understanding of cholesterol-related pathological conditions and offer potential avenues for the development of targeted therapeutic interventions.