Targeting atherosclerosis by using modular, multifunctional micelles

Abstract

Subtle clotting that occurs on the luminal surface of atherosclerotic plaques presents a novel target for nanoparticle-based diagnostics and therapeutics. We have developed modular multifunctional micelles that contain a targeting element, a fluorophore, and, when desired, a drug component in the same particle. Targeting atherosclerotic plaques in ApoE-null mice fed a high-fat diet was accomplished with the pentapeptide cysteine-arginine-glutamic acid-lysine-alanine, which binds to clotted plasma proteins. The fluorescent micelles bind to the entire surface of the plaque, and notably, concentrate at the shoulders of the plaque, a location that is prone to rupture. We also show that the targeted micelles deliver an increased concentration of the anticoagulant drug hirulog to the plaque compared with untargeted micelles.

Fig. 1.

Construction of modular multifunctional micelles. (A) Individual lipopeptide monomers are made up of a DSPE tail, a poly(ethylene glycol) (PEG₂₀₀₀) spacer, and a variable polar head group (X) of CREKA, FAM-CREKA, FAM, N-acetylcysteine, Cy7, or hirulog. The monomers were combined to form various mixed micelles. (B) The 3D structure of FAM-CREKA/Cy7/hirulog mixed micelle.

Fig. 2.

Ex vivo imaging of the aortic tree of atherosclerotic mice. Micelles were injected intravenously and allowed to circulate for 3 h. The aortic tree was excised after perfusion and imaged ex vivo. (A) (Images correspond to the bars directly below them in B). Increased fluorescence was observed in the aortic tree of ApoE-null mice after injection with FAM-CREKA targeted micelles, but not with nontargeted fluorescent micelles. When an excess of unlabeled CREKA micelles was injected before the FAM-CREKA micelles, fluorescence in the aortic tree was decreased. A preinjection of an excess of nontargeted unlabeled micelles did not cause a significant decrease in fluorescence. (B) Fluorescence in the aortic tree was quantified by measuring the intensity (au, arbitrary units) of fluorescent pixels (n = 3 per group).

Fig. 3.

Localization of CREKA micelles in atherosclerotic plaques. (A) Serial cross-sections (5 μm thick) were stained with antibodies against CD31 (endothelial cells; Top), CD68 (macrophages and other lymphocytes; Middle), and fibrin(ogen) (Bottom). Representative microscopic fields are shown to illustrate the localization of micelle nanoparticles in the atherosclerotic plaque. Micelles are bound to the entire surface of the plaque with no apparent binding to the healthy portion of the vessel. CREKA targeted micelles also penetrate under the endothelial layer (CD31 staining) in the shoulder of the plaque (Inset) where there is high inflammation (CD68 staining) and the plaque is prone to rupture. Clotted plasma proteins are seen throughout the plaque and its surface [fibrin(ogen) staining]. (Left) Images were taken at a 10× magnification. (Scale bar, 200 μm.) (Right) Images were taken at a 150× magnification. (Scale bar, 20 μm.) (B) Fluorescence was not observed in the heart or lung, and only a small amount was seen in the kidney, spleen, and liver. Images were taken at a 20× magnification. (Scale bar, 100 μm.)

Fig. 4.

Targeting of hirulog to atherosclerotic plaques. (A) Equal molar concentrations of hirulog peptide and hirulog micelles were tested for antithrombin activity to ensure that potency did not decrease when hirulog was in micellar form. Hirulog peptide and micelles showed similar activity in a chromogenic assay. (B) CREKA targeted or nontargeted and hirulog mixed micelles were injected intravenously into mice and allowed to circulate for 3 h. The aortic tree was excised and analyzed for bound hirulog. Significantly higher levels of antithrombin activity were observed in the aortic tree of ApoE-null mice after injection of CREKA targeted hirulog micelles than nontargeted micelles (1.8 and 1.2 μg/mg of tissue; P ≤ 0.05; n = 3). Antithrombin activity generated by CREKA targeted hirulog micelles in ApoE-null mice was also significantly higher than that in wild-type mice (0.8 μg/mg of tissue; P ≤ 0.05; n = 3).