Abstract 392: Nanoparticle-based Biosensors for Measuring Apolipoprotein A-I and HDL Function

Abstract

Background: High-density lipoproteins (HDL) are dynamic circulating nanoparticles that carry cholesterol and have numerous functions. Rapid measurement of HDL, the HDL-associated protein apolipoprotein A-I (apoAI), or HDL function(s) is not currently possible. Such technology may provide new opportunities for HDL monitoring. We utilized nanoparticles to develop a method of sequestering apoAI from solution, including human serum, and for measuring the activity of the HDL-maturing and apoAI-dependent enzyme, lecithin:cholesterol acyl transferase (LCAT). Methods: Gold nanoparticles were surface-functionalized with phospholipids. The synthesized particles were incubated with various amounts of apoAI or with purchased, LDL-depleted, human serum samples where the apoAI amount was measured using an ELISA. After incubation, the particles were easily isolated and apoAI sequestration by the nanoparticles was measured using ELISA. As apoAI is a cofactor that activates LCAT, nanoparticles were incubated with various amounts of apoA1, LCAT, and free cholesterol or with the LDL-depleted human serum samples (diluted) for 1 hour at 37°C to enable binding of apoAI, cholesterol, and LCAT activity. The nanoparticles were easily isolated and the particle-associated cholesteryl ester was measured using an Amplex Red Cholesterol Assay kit. Results: The nanoparticles sequester apoAI according to the concentration of apoAI in solution (R = 0.668, p = 0.035, N = 10). Also, data reveal that the LCAT binds the nanoparticle-apoAI structures and catalyzes the conversion of cholesterol to cholesteryl ester. The amount of cholesteryl ester bound to the nanoparticles is directly dependent on the amount of apoAI (R = 0.729, p = 0.016, N=10). Conclusion: These results demonstrate that specific, functionalized nanoparticles can be added to serum to sequester apoAI in dose-response. Also, the formed conjugate structures activate LCAT whereby the amount of cholesteryl ester formed on the nanoparticle directly correlates with apoAI. Overall, this technology provides a framework and new approach for developing next generation HDL- and HDL-function specific biosensors.