Abstract 162: A Novel Nanotechnology Approach for Cholesterol Loading and Reverse Cholesterol Transport Assessment
Objective: Functional significance and efficiency of reverse cholesterol transport (RCT) pathways of cholesterol efflux is of major pathogenetic and therapeutic relevance. The assays used to test RCT however continue to remain fairly labor and time intensive with the “gold standard” approach in vitro employing acetylated low-density lipoprotein (acLDL)-mediated loading of cells with radioactively labeled cholesterol followed by exposure to a cholesterol acceptor (e.g. high-density lipoprotein, HDL). We tested the feasibility and efficacy of a synthetic CHolesterol Efflux Assay Probe (CHEAP) capable of Förster Resonance Energy Transfer (FRET) as an alternate strategy to quantify RCT that could theoretically replace more labor intensive methods via its advantages of high throughput screening, faster and economical synthesis, bulk fabrication, and real time cholesterol release monitoring.
Methods and results: CHEAP synthesis was accomplished through self-assembly methodologies from polystyrene core nanoparticles, lipids, and H3- or BODIPY- labeled cholesterol. Screening of 25 potential formulations for multiple physicochemicalproperties including stability, polydispersity, zeta potential and FRET efficiency upon cholesterol release resulted in identification of an optimized particle with mean size similar to LDL (32.3 nm). Fluorescence profiles of BODIPY-cholesterol loaded and cholesterol free CHEAP were characterized to allow real time determination of cholesterol release kinetics ex vivo and in vitro through changes in Förster Resonance Energy Transfer upon cholesterol release. Internalization was studied via confocal microscopy and the results showed that particles localized to early endosomal and lysosomal intracellular compartments. Assay efficacy of CHEAP was investigated in RCT with varying doses of human HDL as cholesterol acceptors. Figure 1 demonstrates that cholesterol loading with CHEAP resulted in dose-dependent cholesterol efflux (r2 = 0.83, p=0.011) as compared with acLDL loading (r2 = 0.67, p=0.048).
Conclusions: CHEAP has several advantages over lipoprotein cholesterol delivery probes and has reliable results in RCT assays.
- © 2012 by American Heart Association, Inc.