Role of Mesoscopic Solute-Rich Clusters in Cholesterol Crystallization

The underlying mechanisms of cholesterol crystallization are generally not well understood, despite its critical role in physiological and pathological processes of the human body. One challenge associated with studying cholesterol crystallization in vitro is the identification of a biomimetic solvent that allows for in situ characterization. In this study, we explore various alcohol–water solvents and show that the selection of alcohol is critical to achieving physiologically relevant cholesterol monohydrate crystals, whereas other alcohols may give rise to cholesterol solvates. In particular, we focus on ethanol–water solvents and show that these systems produce a combination of monohydrate and hemiethanolate crystals, depending on the water content. A combination of several scattering techniques reveals the formation of mesoscopic solute-rich clusters over a broad range of cholesterol concentrations spanning from undersaturated to supersaturated solutions. The presence of clusters is highly suggestive of a nonclassical (two-step) nucleation pathway. We explore the impact of three growth conditions, water content, cholesterol concentration, and temperature, on the size and population of clusters. We observe that cluster size varies only with temperature, whereas the number of clusters increases with increasing water content and cholesterol concentration. In situ atomic force microscopy measurements reveal a classical mechanism of cholesterol hemiethanolate crystal growth based on monomer addition via a surface diffusion pathway without the involvement of mesoscopic solute-rich clusters. The incorporation of the solute by surface diffusion invokes strong competition for supply between adjacent steps on crystal surfaces, which leads to a unique self-inhibition pathway that has the potential to induce surface growth cessation. Collectively, these findings highlight the importance of solvent selection in studies of cholesterol crystallization and the ability of these systems to have disparate nonclassical and classical pathways of nucleation and growth, respectively.