Synthetic High-Density Lipoprotein-Mediated Targeted Delivery of Liver X Receptors Agonist Promotes Atherosclerosis Regression

Abstract

Targeting at enhancing reverse cholesterol transport (RCT) is apromising strategy for treating atherosclerosis via infusion of reconstitute high density lipoprotein (HDL) as cholesterol acceptors or increase of cholesterol efflux by activation of macrophage liver X receptors (LXRs). However, systemic activation of LXRs triggers excessive lipogenesis in the liver and infusion of HDL downregulates cholesterol efflux from macrophages. Here we describe an enlightened strategy using phospholipid reconstituted apoA-I peptide (22A)-derived synthetic HDL (sHDL) to deliver LXR agonists to the atheroma and examine their effect on atherosclerosis regression in vivo. A synthetic LXR agonist, T0901317 (T1317) was encapsulated in sHDL nanoparticles (sHDL-T1317). Similar to the T1317 compound, the sHDL-T1317 nanoparticles upregulated the expression of ATP-binding cassette transporters and increased cholesterol efflux in macrophages in vitro and in vivo. The sHDL nanoparticles accumulated in the atherosclerotic plaques of ApoE-deficient mice. Moreover, a 6-week low-dose LXR agonist-sHDL treatment induced atherosclerosis regression while avoiding lipid accumulation in the liver. These findings identify LXR agonist loaded sHDL nanoparticles as a promising therapeutic approach to treat atherosclerosis by targeting RCT in a multifaceted manner: sHDL itself serving as both a drug carrier and cholesterol acceptor and the LXR agonist mediating upregulation of ABC transporters in the aorta.

Keywords: Atherosclerosis; High-density lipoprotein; Liver X receptors; Macrophage; Reverse cholesterol transport.

Fig. 1

Schematic representation of the sHDL nanoparticle formulations and efficacy assays. Schematic structure of T1317-encapsulated sHDL NPs (a). Representative TEM images of sHDL and the average size of sHDL and sHDLT1317 (b). The size bar corresponds to 20 μm. (c) In vitro release profile of T1317 from sHDL NPs. Quantification of the levels of Abca1 and Abcg1 in normal J774A.1 cells (d) and cholesterol loaded J774A.1 foam cells (e). sHDL at 50 μg/ml and T1317 at 0.1 μM. Cholesterol efflux was measured as the percentage of [³H]-cholesterol in the medium after 6 h of incubation with APOA-I at 10 μg/ml (f) or commercial HDL at 50 μg/ml (g). Data are mean ± S.E.M.; n = 3–5 per group; *P < 0.05, **P < 0.01 compared to control; #P < 0.05, ##P < 0.01 compared to sHDL.

Fig. 2

sHDL nanoparticles delivery cargos into the atherosclerotic plaques. (a) sHDL labeled with DiD (sHDL at 30 mg/kg and DiD at 200 μg/kg) was intraperitoneally injected to apoE-deficient mice, which had been placed on HFHC diet for 12 weeks to induce atherosclerotic plaque formation. Four hours later, the aorta were perfused and isolated from the body. Accumulation of DiD in the areas rich in atherosclerotic plaques was observed by IVIS image. DMSO, T1317, PBS, sHDL or sHDLT1317 was injected intraperitoneally in 20-week-old ApoE-deficient mice. Four hours later, the aortic arch was collected, RNA was isolated and gene expression of Abca1 (b) and Abcg1 (c) was analyzed by quantitative RT-PCR. Data are mean ± S.E.M.; n = 3–5 per group; *P < 0.05, **P < 0.01 compared to control; #P < 0.05, ##P < 0.01 compared to sHDL.

Fig. 3

Effects of combination of sHDL and T1317 on lipid biosynthesis in vivo. C57BL/6 mice were randomly divided into 6 groups and received one IP injection per day for continuous four days: (i) DMSO: control; (ii) T1317: T1317 compound in DMSO at 1.5 mg/kg; (iii) PBS: control group; (iv) sHDL: 30 mg/kg of sHDL NPs; (v) sHDLT1317: sHDLT1317 NPs at 30 mg/kg of sHDL and T1317 at 1.5 mg/kg; (vi) sHDL//T1317 (injections at two sites): sHDL NPs at 30 mg/Kg at the left abdomen site and T1317 at 1.5 mg/kg at the right abdomen site. Twenty-four hours after the last injection, the mice were sacrificed. T1317 and sHDL//T1317 treatment resulted in significant lipid accumulation in the liver tissues as assayed by oil red O-stained lesions (a) and increased liver weight over body weight ratio (b). The liver tissues were harvested, RNA was isolated and gene expression of LXR target genes Srebp1 (c), Cyp7a1 (d), Fasn (e) and Abcg1 (f) was analyzed by quantitative RT-PCR. Data are mean ± S.E.M.; n = 3–4 per group; *P < 0.05, **P < 0.01 compared to control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

sHDLT1317 treatment induced atherosclerosis regression by upregulation of ABC transporters. After 14 weeks HFHC diet challenge, ApoE^−/− mice were randomly divided into six groups and the baseline group animals were sacrificed. The others were switched to chow diet and received IP injection three times per week for 6 weeks: DMSO: control; T1317: T1317 compound in DMSO at 1.5 mg/kg; PBS: control; sHDL: 30 mg/kg; sHDLT1317: 30 mg/kg of sHDL and T1317 at 1.5 mg/kg. sHDLT1317-treated mice exhibited decreased oil red O-stained lesions in the whole aorta (a) and aortic root plaque area (c), corresponding quantitative analyses of the aortas (b) and the aortic root cross-sections (d).RNA was extracted from white blood cells isolated from the mice and gene expression of Abca1 (e) and Abcg1 (f) were analyzed by qRT-PCR. Data are mean ± S.E.M.; n = 5–9 per group; *P < 0.05, **P < 0.01 compared to control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Long term treatment with sHDLT1317 did not increase triglyceride level and induce hepatic lipogenesis. After 14 weeks HFHC diet challenge, ApoE^−/− mice were randomly divided into six groups and the baseline group animals were sacrificed. The others were switched to chow diet and received IP injection three times per week for 6 weeks: DMSO: control; T1317: T1317 compound in DMSO at 1.5 mg/kg; PBS: control; sHDL: 30 mg/kg; sHDLT1317: 30 mg/kg of sHDL and T1317 at 1.5 mg/kg. Plasma total cholesterol (a), HDL cholesterol (b), LDL cholesterol (c) and Triglycerides (d) were measured. The liver triglyceride (e) and cholesterol (f) were extracted and measured. g-j. The liver tissues were harvested from the above animals and gene expression analysis by qRT-PCR of the LXR target genes, Srebp1 (g), Fasn (h), Abca1 (i) and Scd1 (j) and. Data are mean ± S.E.M.; n = 5–9 per group; *P < 0.05, **P < 0.01 compared to control.

Fig. 6

A working model showing the sHDL NPs-mediated drug delivery system.