(T1330-10-56) Effect of Lipid Composition on the Atheroprotective Properties of HDL-Mimicking Micelles

Purpose: Cardiovascular-related diseases are the number one cause of death worldwide. Heart attack and stroke contribute to a large number of these deaths, and atherosclerosis plays a substantial role in the occurrence of these diseases. Current treatment options, such as statins and other cholesterol-lowering drugs still leave patients at high risk of major coronary events, therefore, new therapeutics are being designed to target atherosclerosis pathology. Two factors that contribute to atherosclerosis are cholesterol imbalance and unresolved local inflammation in the arteries. Synthetic high-density lipoproteins (sHDL) have been synthesized to mimic the natural cardioprotective behaviors of endogenous HDL, and have been shown to enhance cholesterol efflux and improve inflammation. Though sHDL have shown promise in preclinical and clinical trials, difficulties in purification and production of ApoA-I, HDL’s main protein component, makes sHDL costly and challenging to scale. Therefore, phospholipid-based micelles have been created to mimic the function of HDL nanoparticles. Preliminary data shows that micelles are capable of enhancing cholesterol efflux and reducing inflammation, but the effect of micelle lipid composition on these anti-atherogenic activities is yet to be determined. Therefore, the purpose of this research is to investigate the effect of lipid composition on the cholesterol crystal dissolution ability, cholesterol efflux capacity, anti-inflammatory effects, as well as the in vivo PK/PD profiles of micelles to better understand how lipid properties influence the therapeutic potential of micelles for the treatment of atherosclerosis.

Methods: Micelles were composed of 1 of 4 phosphatylcholine (PC) lipids (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine -N-[azido(polyethylene glycol)-2000] (DSPE-PEG2k) at various molar lipid ratios (1:0.5. 1:1, 1:1.5, 1:2, 1:2.5, 1:3). Micelles were prepared using the co-lyophilization method. Lipids were dissolved in acetic acid and mixed at specified molar ratios, flash frozen in liquid nitrogen, lyophilized overnight, and rehydrated with PBS. The resulting solution was heated above and cooled below PC transition temperature to obtain the final product. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) were used to determine the size and morphology of micelles. To test the effect of composition on the anti-inflammatory activity of micelles, RAW 264.7 mouse macrophage cells were treated with lipopolysaccharide (LPS) endotoxin and micelles simultaneously. After 18 hours of co-incubation, TNF-α and IL-6 levels were measured using ELISA kits. Cholesterol crystals were formed on the bottom of a 96-well plate and incubated with micelles for 7 days to test the effect of composition on the cholesterol crystal dissolution capacity of micelles. Cholesterol E kit was used to quantify the amount of cholesterol dissolved. To test cholesterol efflux abilities of the micelles, J774A.1 macrophage cells were radiolabeled with 1 µCi/ml [³H] cholesterol in media containing 3% BSA and 5 ug/mL ACAT inhibitor. After a 4 hour incubation with micelles, media and cell lysates were collected and cholesterol context was measured by liquid scintillation counter. For in vivo studies, Sprague Dawley rats were given a 136 umol/ml dose of micelles via IV tail vein injection and blood was collected at various time points between 0 and 48 hours. Total cholesterol, free cholesterol, and phospholipid kits (Wako) were used to analyze isolated serum and a non-compartmental analysis (NCA) was used to derive basic pharmacokinetic and pharmacodynamic parameters from the serum concentration versus time profiles.

Results: Micelles ranged from 14-28 nm in diameter. Changing the type of PC lipid at a fixed ratio of PC:DSPE-PEG2k lipid resulted in similar micelle sizes, while an increase in DSPE-PEG2k percentage resulted in a reduction of particle size. The effect of PC lipid type on micelle activity was accessed first. Micelles composed of DMPC significantly dissolved more cholesterol crystals and reduced TNF-α and IL-6 cytokine levels to a greater extent as compared to POPC, DPPC and DSPC micelles. There was no significant differences in cholesterol efflux capabilities or PK/PD parameters between micelles containing different PC lipids at a fixed PC:DSPE-PEG2k ratio. Next, the effect of PEGylation on micelles activity was observed. Micelles with a 1:2 ratio of DMPC:DSPE-PEG2k dissolved the greatest amount of cholesterol crystals and facilitated the most cholesterol efflux as compared to the other micelles. Micelles composed of smaller PEGylation ratios had a slightly greater ability to reduce TNF-α and IL-6 cytokine levels. Lastly, 1:3 ratio micelles had a significantly higher phospholipid AUC and C_max, and a two-fold increase in total cholesterol AUEC as compared to 1:0.5 micelles, suggesting an increased circulation and cholesterol mobilized for the more PEGylated micelles.

Conclusion: Overall, HDL-mimetic micelles seem to possess anti-inflammatory properties, as well as cholesterol crystal dissolution abilities and cholesterol mobilization capacity, highlighting its therapeutic potential for atherosclerosis. The lipid composition affects micelle’s anti-atherogenic activity. Further analysis using in vivo atherosclerosis models need to conducted to fully understand how lipid properties and compositions of micelles will affect it’s efficacy in order to determine an optimal composition of micelles for the treatment of atherosclerosis.