Evaluating pathological levels of intracellular cholesterol through Raman and surface-enhanced Raman spectroscopies.

Abstract

Versatile methods for the quantification of intracellular cholesterol are essential for understanding cellular physiology and for diagnosing disorders linked to cholesterol metabolism. Here we used Raman spectroscopy (RS) and surface-enhanced Raman spectroscopy (SERS) to measure changes in cholesterol after incubating human fibroblasts with increasing concentrations of cholesterol-methyl-β-cyclodextrin. RS and SERS were sensitive and accurate enough to detect high levels of cholesterol in fibroblasts from patients affected by type C Niemann-Pick disease (NPC), a lysosomal storage disorder characterized by the primary accumulation of cholesterol. Moreover, SERS was able to distinguish between fibroblasts from different NPC patients, demonstrating higher accuracy than RS and standard fluorescent labeling of cholesterol with filipin III. We show that the type of gold nanoparticles used as signal enhancer surfaces in our SERS measurements are internalized by the cells and are eventually found in lysosomes, the main site of accumulation of cholesterol in NPC fibroblasts. The higher sensitivity of SERS can thus be attributed to the specific trafficking of our gold nanoparticles into these organelles. Our results indicate that RS and SERS can be used as sensitive and accurate methods for the evaluation of intracellular cholesterol content, allowing for the potential development of an optical detection tool for the ex-vivo screening and monitoring of those diseases characterized by abnormal modification in cholesterol levels.