(700d) In Situ Characterization of Cholesterol Crystallization in Biomimetic Solvents

There are multiple pathological diseases involving the crystallization of cholesterol, which is a ubiquitous molecule in human physiology. Cholesterol is a key component in heart plaque (atherosclerosis) and the imbalance of cholesterol supersaturation promotes the formation of gallstones from bile, leading to several diseases such as jaundice and Bouveret’s syndrome. Cholesterol is both essential to physiological processes and a key component of severe pathological diseases. Despite its relevance to worldwide healthcare problems, few studies have examined fundamental mechanisms of cholesterol crystallization. It is recognized, however, that elucidating methods to control cholesterol precipitation can be highly valuable for the development of new drugs to treat these diseases.

Prior studies have used lipids as biomimetic solvents to study bulk cholesterol crystallization; however, these systems are not amenable to facile in situ characterization techniques. In this presentation, we will discuss how we selected a binary mixture of water and alcohol with the latter being a lipid surrogate to examine cholesterol crystallization. We have demonstrated that some water-alcohol media lead to the formation of cholesterol monohydrates whereas others lead to formation of solvates. We used a combination of oblique illumination microscopy (OIM) and dynamic light scattering (DLS) to prove that cholesterol nucleation involves a two-step process involving the assembly of clusters, which are present in both undersaturated and supersaturated media. Scattering measurements confirmed that cluster size is a function of temperature and water content but is independent of cholesterol concentration. DLS data also revealed that solvent composition has a notable impact on the induction time and rate of crystal growth. Using a binary mixture of water and isopropanol, we showed that cholesterol monohydrate growth involves classical layer nucleation and spreading. Time-resolved imaging confirms that layer generation originates from defects (dislocations) where monomer incorporation into advancing steps occurs by a surface diffusion pathway over direct incorporation of monomers to kinks from solution. In situ atomic force microscopy (AFM) and microfluidics measurements both confirmed that cholesterol monohydrate crystals are prone to the formation of macrosteps, which engender a self-inhibition mechanism that reduces the rate of crystal growth, counter to a majority of crystalline systems where classical mechanisms of growth lead to single unfinished layers. Here we will present the results of these studies along with dissolution measurements where we observe a combination of classical and nonclassical mechanisms that differ from many conventional crystallization systems reported in literature.