Optimization of Synthetic High-Density Lipoprotein Nanostructures for Treatment of Inflammatory Diseases

Abstract

Biomimetic synthetic high-density lipoproteins (sHDLs) are nanoparticles that mimic the physical, chemical, and biological activity of endogenous HDL. A number of sHDL products have been investigated for the treatment of cardiovascular diseases (CVD) for the past three decades. sHDL is composed of apolipoprotein A-I (apoA-I) or apoA-I mimetic peptide complexed with phospholipids to form a discoidal nanoparticle. Infusions of sHDL in animals and humans have been shown to increase circulating HDL levels, improve plasma cholesterol efflux capacity, inhibit inflammation, and improve endothelial function. Phospholipid composition of sHDL appears to have a significant impact on its function by defining the plasma stability of and cholesterol and LPS binding to the nanoparticle. Thus, we proposed to optimize sHDL phospholipid composition to tailor nanoparticle functionality toward a specific therapeutic indication, such as atherosclerosis and sepsis. In the first chapter, we optimized sHDL for the treatment of sepsis by comparing different fluidities of sHDL based on various fatty acid chain lengths and saturation of phospholipid compositions (POPC, DMPC, DPPC, and DSPC). We hypothesized that sHDL with a fluid liquid crystalline phase would improve anti-inflammatory activities by accelerating the efflux of exogenous molecules and improving accessibility to phospholipids for potential signal transductions compared to sHDL in a rigid gel phase. We demonstrated that treating cells with 22A-DMPC, sHDL with a fluid liquid crystalline phase, resulted in the most effective inhibition of NF-κB signaling, TLR4 signaling through regulation of TLR4 recruitment into lipid rafts, and upregulation of activating transcription factor 3 (ATF-3) expression in vitro. Furthermore, 22A-DMPC effectively reduced mortality and protected organs in mice challenged with lethal doses of lipopolysaccharide (LPS) in a model of sepsis. In conclusion, we demonstrated that differences in sHDL phospholipid composition can impact sHDL’s anti-inflammatory signaling and the effectiveness of sepsis therapy. In the second study, we determined how phospholipid and peptide components of sHDL impacted the nanoparticle’s pharmacokinetic and pharmacodynamic properties. We synthesized two different sets of sHDL with either identical phospholipids with variable peptide sequences of different plasma stability or identical peptide sequences with variable fatty acid chain length and saturation. We observed that proteolytically stabilized 22A-P-sHDL increased nanoparticle half-life by 2-fold compared to less stable 22A-sHDL. Nevertheless, increased half-life did not translate to higher cholesterol mobilization. In contrast, varying phospholipids significantly impacted the nanoparticle’s pharmacokinetic profile whereby 22A-DSPC sHDL resulted in the longest half-life (6.0 h) compared to 22A-POPC sHDL (1.0 h). In addition, due to its increased half-life, 22A-DSPC sHDL notably impacted the nanoparticle’s cholesterol mobilization capability, resulting in a 6.5-fold increase compared to 22A-POPC sHDL. Thus, we observed that the phospholipid component of sHDL plays a critical role in cholesterol mobilization in vivo. In the final study, we formulated a new biomimetic nanomicelle called MiNano that is structurally similar to sHDL with a hydrophobic core, a hydrophilic exterior, and a particle size of 12 – 14 nm. We discovered that MiNano resulted in a similar functionality to sHDL, but with much more efficient suppression of the inflammatory response in vitro. Also, MiNano displayed robust inhibition of inflammatory cytokines in an LPS-induced endotoxin mice model. In summary, this thesis thoroughly investigates the importance of the phospholipid component of sHDL and MiNano in the regulation of inflammation and cholesterol mobilization, allowing us to optimize nanoparticle composition for the treatment of sepsis and atherosclerosis.