Molecular Imaging and prevention of Atherosclerosis using CD36-targeted nanoparticles and Epigallocatechin gallate

Intimal macrophages are lesion-determinant cells by releasing inflammatory factors and taking up oxidized low density lipoprotein (oxLDL) via scavenger receptors, primarily the CD36. The presence of macrophages is positively associated with lesion progression, severity and destabilization. Hence, intimal macrophages are an important diagnostic and therapeutic target. Epigallocatechin gallate (EGCG) has a potential to prevent atherosclerosis through decreasing inflammatory responses and cholesterol accumulation in macrophages. \nWe have successfully synthesized EGCG encapsulated nanostructured lipid carriers (NLCE) and chitosan coated NLCE (CSNLCE) using natural triglyceride, surfactant, chitosan and EGCG, and measured their anti-atherogenic bioactivities in macrophages in our first study (Chapter III). Nanoencapsulation dramatically improved EGCG stability. CSNLCE significantly increased EGCG content in THP-1 derived macrophages compared with native EGCG. As compared to 10 µM of native EGCG, both NLCE and CSNLCE at the same concentration significantly decreased macrophage cholesteryl ester content. NLCE and CSNLCE significantly decreased mRNA levels and protein secretion of monocyte chemoattractant protein-1 (MCP-1) in THP-1 derived macrophages, respectively. \n1-(Palmitoyl)-2-(5-keto-6-octene-dioyl) phosphatidylcholine (KOdiA-PC), a major type of oxidized phosphatidylcholines (PC) found on oxLDL, has a high binding affinity to oxLDL binding site of the CD36 receptor and participates in CD36-mediated recognition and uptake of oxLDL by intimal macrophages. Therefore, KOdiA-PC is a natural ligand to target macrophage CD36 receptors and could be incorporated on the surface of nanoparticles to form CD36-targeted nanoparticles, which could noninvasively assess the distribution and accumulation of intimal macrophages. Additionally, targeted delivery of EGCG to intimal macrophages using CD36-targeted nanoparticles may prevent atherosclerosis development. \nSince KOdiA-PC is difficult to be incorporated on the surface of nanostructured lipid carriers (NLC), we made KOdiA-PC incorporated liposomes, investigated their targeting mechanisms to macrophages in vitro, and determined their target specificity to intimal macrophages in vivo in the second study (Chapter IV). All in vitro data demonstrated that these targeted liposomes had a high binding affinity for the oxLDL binding site of the macrophage CD36 receptor. Intravenous administration into LDL receptor null (LDLr-/-) mice of targeted compared to non-targeted liposomes resulted in higher uptake in aortic lesions. The liposomes co-localized with macrophages and their CD36 receptors in aortic lesions. \nSince chitosan coated nanostructured lipid carriers (CSNLC) developed in chapter III contained a large amount of chitosan (carbohydrate) and triglyceride, CSNLC might increase blood glucose and triglyceride concentrations. Liposomes developed in chapter IV are not as stable as NLC and CSNLC. Therefore, we removed chitosan and replaced triglyceride with (+) alpha-tocopherol acetate to synthesize new CD36-targeted nanoparticles, which are composed of PC, surfactant, (+)-alpha-tocopherol acetate and KOdiA-PC. We optimized their formulation, measured their characteristics, confirmed their CD36-mediated binding mechanism to macrophages, loaded EGCG into these ligand (KOdiA-PC) nanoparticles (L-Enano) and measured their anti-atherogenic bioactivities in THP-1 derived macrophages in our third study (Chapter IV). Compared to non-targeted EGCG nanoparticles (Enano), CD36-targeted L-Enano had significantly higher binding affinity to and uptake by THP-1 derived macrophages at the same pattern as oxLDL. CD36-targeted L-Enano dramatically improved EGCG stability, increased macrophage EGCG content, delivered EGCG to macrophage cytosol and avoided lysosomes. L-Enano significantly decreased macrophage mRNA levels and protein secretion of MCP-1, but did not significantly change macrophage cholesterol content. \nWe measured binding affinity of L-Enano to mouse peritoneal macrophages, and determined their anti-atherogenic bioactivities in LDLr-/- mice in the last study (Chapter VI). L-Enano compared to Enano had a higher binding affinity to mouse peritoneal macrophages. LDLr-/- mice were treated intravenously with 1XPBS, void nanoparticles, native EGCG, Enano and L-Enano at EGCG dose of 25 mg per kg body weight weekly for 22 weeks. L-Enano compared to 1XPBS significantly lowered lesion surface areas of aortic arches, lowered liver EGCG content, but did not change blood lipid profile, body weight and food intake. \nIn summary, our KOdiA-PC nanoparticles can noninvasively assess intimal macrophages accumulation and distribution via targeting their CD36 receptors and facilitate targeted delivery of EGCG to intimal macrophages to prevent atherosclerosis development. \n