High-Density Lipoproteins: Nature's Multifunctional Nanoparticles

Abstract

High-density lipoproteins (HDL) are endogenous nanoparticles involved in the transport and metabolism of cholesterol, phospholipids, and triglycerides. HDL is well-known as the "good" cholesterol because it not only removes excess cholesterol from atherosclerotic plaques but also has anti-inflammatory and antioxidative properties, which protect the cardiovascular system. Circulating HDL also transports endogenous proteins, vitamins, hormones, and microRNA to various organs. Compared with other synthetic nanocarriers, such as liposomes, micelles, and inorganic and polymeric nanoparticles, HDL has unique features that allow them to deliver cargo to specific targets more efficiently. These attributes include their ultrasmall size (8-12 nm in diameter), high tolerability in humans (up to 8 g of protein per infusion), long circulating half-life (12-24 h), and intrinsic targeting properties to different recipient cells. Various recombinant ApoA proteins and ApoA mimetic peptides have been recently developed for the preparation of reconstituted HDL that exhibits properties similar to those of endogenous HDL and has a potential for industrial scale-up. In this review, we will summarize (a) clinical pharmacokinetics and safety of reconstituted HDL products, (b) comparison of HDL with inorganic and other organic nanoparticles,

Keywords: apolipoprotein mimetic peptides; apolipoproteins; delivery; high-density lipoproteins; imaging reagents; multifunctional nanoparticles; nucleic acids; peptides; proteins; small molecules.

Figure 1. Metabolic fate of HDL in vivo

The major protein component of HDL, lipid-free ApoA1, is produced in the liver and intestine. ApoA1 can associate with lipids effluxed by ABCA1 to form nascent pre-β HDL. The lipid layers of pre-β HDL can be interspersed with free cholesterol, which is converted to cholesterol ester by LCAT. Cholesterol ester, which is more hydrophobic than cholesterol, is internalized into the HDL core to form spherical HDL3. Additional cholesterol from peripheral tissues can be loaded into spherical HDL3 and subsequently converted to cholesterol ester with the help of LCAT to form HDL2. Mature HDL can also exchange cholesterol ester for triglycerides from other lipoproteins such as LDL and VLDL in a process mediated by CETP. Mature HDL delivers cargo molecules to hepatocytes in the liver for metabolism through a SR-BI-mediated process. Reproduced with permission from [1].

Figure 2. Delivery of different types of molecules to various target organs/tissues by HDL

HDL nanoparticles have been used to deliver small molecules, peptides/proteins, and nucleic acids to different target organs/tissues.

Figure 3. Delivery of statin to the atherosclerotic plaque using HDL

(A) Schematic of Simvastatin-loaded rHDL [S]-rHDL and blank rHDL (rHDL). (B) Thickness of the vessel wall of apoE-KO mice receiving 12 weeks of biweekly low-dose [S]-rHDL (15 mg/kg statin, 10 mg/kg ApoA1), blank rHDL, statin (15 mg/kg statin) and placebo. The thickness of the vessel wall is defined as the ratio between the mean wall area and the outer wall area, which is expressed as normalized wall index (NWI). (C) Plaque area of mice receiving different formulations. High dose [S]-rHDL (60 mg/kg statin, 40 mg/kg ApoA1) led to smaller plaque area than Placebo, blank rHDL, and low dose [S]-rHDL (15 mg/kg statin, 10 mg/kg ApoA1). (D). Plaque macrophage content (CD68 positive area) of mice receiving different formulations. High dose [S]-rHDL significantly decreased the macrophages in the plaque compared to placebo, blank rHDL, and low dose [S]-rHDL. Figures combined and reproduced from [102].

Figure 4. Increased delivery of cytotoxic cytochrome C to tumor cells using HDL

(A) Schematic for the preparation of protein (cytC)-loaded nanoparticle. (B) Biodistribution profile of different formulations of Alexa-488 labeled cytC after intravenous injection into H460 xenograft mice. (C) Tumor growth inhibition of different formulations of cytC (40 ug/kg) or MPS-cytC (160 ug/kg) in an H460 xenograft mouse model. Figures combined and reproduced from [115].

Figure 5. Efficient delivery of cytolytic peptide, melittin, to tumor cells using HDL

(A) Schematic of loading α-melittin to HDL nanoparticles (α-melittin-NP). (B) Real time imaging of the release of FITC-α-melittin (green) from HDL nanoparticles attacking tumor cells expressing KatushkaS158A (red) with confocal microscopy. KatushkaS158A (red) inside the cells decreased over time, while FITC-α-melittin (green) increased inside the cells, indicating FITC-R-melittin was released from the HDL nanoparticles and made pores on the cell membrane, which allowed KatushkaS158A to leak out of the cells. (C) Tumor growth inhibition of different formulations in a B16F10 tumor model. Figures combined and reproduced from [116].

Figure 6. Efficient delivery of siRNA molecules to hepatocytes using HDL

(A) Schematic of incorporation of cholesterol modified siRNA (chol-siRNA) into an HDL nanoparticle. (B) In vivo apoB mRNA silencing effect of A-lip-chol-siApoB-1 and A-lip-cholsiApoB-2 48 h after intravenous injection of 50 mg/kg siRNA to C57BL/6 mice. ApoB mRNA silencing with chol-siRNA alone and chol-siApoB complexed with purified mouse HDL (mHDL) were used as control groups. (C) In vivo ApoB mRNA silencing effect of E-lip-siApoB-1 and E-lip-siApoB-2 48 hours after intravenous injection of 30 mg/kg siRNA to C57BL/6 mice. Data represent mean ± SD. *P < 0.05. Figures combined and reproduced from [119].

Figure 7. Efficient delivery of STAT3 siRNA molecules to tumor cells using HDL

(A) Schematic of encapsulation of siRNA into HDL after complexation with poly-lysine. (B) A TEM of siRNA-loaded HDL. (C) In vivo antitumor effect of STAT3 siRNA/rHDL in chemosensitive (HeyA8 and SKOV3ip1) and chemoresistant (HeyA8-MDR) mouse models of ovarian carcinoma. Figures combined and reproduced from [120].

Figure 8. The use of HDL to deliver a MRI contrast agent for atherosclerotic plaque imaging

(A) Schematic of Gd-loaded HDL (37pA-Gd), Gd-loaded micelles (Gd-micelles), and blank HDL (37pA-Gd). (B) MRI images of aorta of an apoE-KO mouse. Top panel: images acquired before injection of 37pA-Gd; Bottom panel: images acquired 24 h after injection of 37pA-Gd. The images on the right of each panel are enlargements of the white box area, where aorta is indicated by the white arrow. (C) Confocal microscopy images of aortic tissue from an apoE-KO mouse intravenously injected with 37pA-Gd 24 h prior to excision. Blue: nuclei stained by DAPI; Red: Rhodamine lipid in HDL; Green: Macrophages labeled by Alexa 647-labeled CD68 antibody. The yellow color in the merged images indicate the HDL is colocalized with the macrophages. Figures combined and reproduced from [88].

Figure 9. The use of HDL to deliver a near-infrared fluorescent dye for tumor imaging

(A) Confocal microscopy imaging of uptake of DiR-BOA-loaded HDL (DNC) by ldlA transfected with SR-BI [ldl(mSR-BI)] or not transfected with SR-BI (ldlA7) in the absence of excess HDL (left panel) and in the presence of excess HDL (right panel). (B) Schematic depiction of the uptake of HDL cargoes via SR-BI mediated pathway. (C) Confocal microscopy images of ldlA (mSR-BI) (SR-BI+) cells showing the fluorescein-labeled lipid (green color in top panel) and peptide (green color in bottom panel) localized on the cell surface and the DiR-BOA cargo (red) in the cytosol. (D) Optical imaging of the tumor targeting of DiR-BOA-loaded HDL in tumor-bearing mice using the whole-body optical imaging system. Figures combined and reproduced from [73].