(W1130-06-36) Understanding Synthetic High-Density Lipoprotein Remodeling Ex Vivo Using Human Serum

Purpose: Nearly 17.9 million people die every year from cardiovascular-related diseases, which accounts for 31% of all deaths worldwide. Endogenous high-density lipoproteins (HDL) are natural cardioprotective particles that have been shown to reduce cardiovascular related risks, such as atherosclerotic disease progression, inflammation, and endothelial dysfunction. Therefore, synthetic high-density lipoproteins (sHDL) have been synthesized to mimic the natural cardioprotective behaviors of endogenous HDL. sHDLs can function as a therapeutic alone or be used as a delivery system. Endogenous HDL is remodeled by various transfer proteins and lipases, which play a role in HDL maturation, heterogeneity, and the exchange of lipids between HDL and other endogenous lipoproteins, such as very low-density lipoproteins (VLDL) and low-density lipoproteins (LDL). Due to this fact, we sought to investigate how our sHDLs remodel in the presence endogenous lipoproteins overtime to determine the stability of our particles in human serum. Remodeling can ultimately affect the ability of sHDL to reach its target site or to deliver cargo to the desired area of interest. Therefore, the purpose of this research is to (1) synthesize a library of sHDLs composed of different phosphatylcholine (PC) lipids and (2) test the stability of sHDLs in human serum ex vivo to better understand how lipid properties influence the stability and remodeling kinetics of sHDL in human serum.

Methods: sHDLs were prepared using the co-lyophilization method. Phosphatylcholine (PC) lipid and 22A, an ApoA-I mimetic peptide, were dissolved in acetic acid, freeze-dried overnight, and rehydrated with PBS. The resulting solution was heated above the PC lipid transition temperature and cooled below the transition temperature for three rounds to obtain the final sHDL product. Dynamic light scattering (DLS) and gel permeation chromatography (GPC) were used to determine the size and purity of the particles. sHDLs were incubated with human serum at 37°C at a final concentration of 1 mg/mL 22A for 0, 2 or 24 hours. The resulting mixture of sHDL and human serum were separated using a Waters HPLC system equipped with a Superose 6, 10/300 GL column (GE Healthcare, Piscataway, NJ). Post column elution fractions were collected between 5-30 minutes. A Waters UPLC-MS system was used to quantify 22A and PC lipid content in elution fractions.

Results: sHDLs were composed of 22A peptide and one PC lipid: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). All sHDLs presented homogenous particle distribution ranging from 9-12 nm in size. For sHDLs composed of POPC, DMPC and DPPC, after 2-hours and 24-hours incubation with human serum, 22A and PC lipid components were detected in LDL and VLDL elution fractions. The association of sHDL components with larger lipoproteins increased with 24-hour incubation of sHDL and human serum. We found that sHDLs composed of DSPC remodeled to a lesser extent than sHDLs composed of DPPC, DMPC and POPC. For sHDL composed of DSPC, after 0-hour and 2-hour incubation with human serum, both 22A and DSPC components were mainly detected in fractions that corresponded to sHDL elution times, showing limited remodeling. Only a small amount of 22A and DSPC were associated with larger lipoprotein after 24 hours, as shown by detection in earlier eluting fractions, while most of these components were detected within sHDL elution fractions, suggesting that DSPC-sHDL particles remained mostly intact.

Conclusion: Overall, the lipid composition of sHDLs affects its stability in human serum and the extent of remodeling during various incubation times. Remodeling and degradation of sHDL appeared to be the most rigorous with POPC-sHDL, followed by DMPC-sHDL and DPPC-sHDL, with DSPC-sHDL remodeling to the lowest extent. We predict this may be attributed to the fact that lipids with higher transition temperatures, such as DPPC and DSPC can cause the lipid structure to be less fluid and more ridged at physiological temperatures, leading to more stable particles and less remodeling. Overall, lipid and peptide transfer from sHDLs to larger endogenous lipoprotein populations in human serum occurred over longer incubation times. Further analysis is required to fully understand how lipid properties and various compositions of sHDL will affect the stability and drug delivery capabilities of the particle.