Reconfiguring Nature's Cholesterol Accepting Lipoproteins as Nanoparticle Platforms for Transport and Delivery of Therapeutic and Imaging Agents

Abstract

Apolipoproteins are critical structural and functional components of lipoproteins, which are large supramolecular assemblies composed predominantly of lipids and proteins, and other biomolecules such as nucleic acids. A signature feature of apolipoproteins is the preponderance of amphipathic α-helical motifs that dictate their ability to make extensive non-covalent inter- or intra-molecular helix-helix interactions in lipid-free states or helix-lipid interactions with hydrophobic biomolecules in lipid-associated states. This review focuses on the latter ability of apolipoproteins, which has been capitalized on to reconstitute synthetic nanoscale binary/ternary lipoprotein complexes composed of apolipoproteins/peptides and lipids that mimic native high-density lipoproteins (HDLs) with the goal to transport drugs. It traces the historical development of our understanding of these nanostructures and how the cholesterol accepting property of HDL has been reconfigured to develop them as drug-loading platforms. The review provides the structural perspective of these platforms with different types of apolipoproteins and an overview of their synthesis. It also examines the cargo that have been loaded into the core for therapeutic and imaging purposes. Finally, it lays out the merits and challenges associated with apolipoprotein-based nanostructures with a future perspective calling for a need to develop "zip-code"-based delivery for therapeutic and diagnostic applications.

Keywords: apolipoprotein AI; apolipoprotein E; bioflavonoids; cancer therapy; diagnostics; drug delivery; gold nanoparticles; lipoproteins; nanodiscs; reconstituted HDL.

Figure 1

Schematic depiction of discoidal and spherical nanolipoproteins with embedded cargo. (A) Discoidal apolipoprotein-based nanostructures (nanodiscs) are composed of a bilayer of phospholipids (grey) with amphipathic α-helices of apolipoproteins (blue) circumscribing the bilayer. (B) Cross-section of a spherical lipoprotein composed of a monolayer of phospholipids and small apo-based helical peptides. The phospholipid interior offers an ideal environment to embed hydrophobic and/or amphipathic biomolecules (yellow).

Figure 2

Apolipoprotein-based nanostructures targeting LDLr family of proteins. Nanoparticles bearing ligands, such as apoB-100 or apoE3 or their derivatives, target the LDLr family of proteins. (A) ApoB-100-based peptides with a hydrophobic tail to promote lipid binding. (B) Plasma-derived LDL bearing apoB-100 on the surface and a core of neutral lipids such as CE or triglycerides with hydrophobic molecules incorporated into the core. (C) The core of the LDL particle has been substituted with hydrophobic agents of interest with a surrounding monolayer of amphipathic lipids and apoB-100. (D) ApoB-100 on LDL modified with dextran or dendrimers. (E) ApoE crosslinked to human serum albumin (HSA) nanoparticles. Particle sizes are not to scale.

Figure 3

Entry mechanism(s) for various types of nanoparticles (NPs) in cells. HDL reconstituted with apoAI and various types of inorganic nanocrystals are taken up by macrophages, a process that is mediated by SR-B1. HDL-AuNP have been shown to promote cholesterol efflux not only in THP-1 macrophages, but also in other cells that express SR-B1 such as B lymphoma cells and myeloid derived suppressor cells (MDSCs). HDL reconstituted with apoE3, either as discoidal or spherical NPs, could gain entry through the LDLr family of proteins in GBM and possibly also through heparan sulfate proteoglycans (HSPG). In endothelial cells, rHDL reconstituted with acrolein-modified apoE showed entry through both SR-B1 and LOX1, but not through conventional oxidized LDL receptors such as CD36. Interestingly, HSA-crosslinked-apoE3 NP without lipid content were shown to enter brain endothelial cells mainly through LRP-1, although other LDLr family receptors could also contribute to the entry.

Figure 4

Incorporation of nonpolar molecules into apolipoprotein-based nanostructures. Nonpolar molecules’ incorporation into apolipoprotein-based nanostructures can be achieved by different approaches. (A) Co-sonication method; (B) detergent dialysis method; (C) incorporate hydrophobic molecules into pre-formed empty nanodiscs; (D) co-sonicate 3, 10 or 17 nm gold nanoparticles (AuNP) with phospholipid vesicles and apoE3; (E) build HDL-like lipoproteins around AuNP as a core template with apoAI and phospholipids.

Figure 5

Applications of rHDLs as transport vehicles. rHDLs can serve as nanovehicles for transporting hydrophobic, amphipathic, and hydrophilic substances. The surface of rHDLs can be conjugated with antibodies or aptamers/nucleic acids for enhancing targeted delivery or modified with coating materials and polymers for extending circulation time inside the body.

Figure 6

Chemical structures of select hydrophobic agents that have been incorporated into lipoproteins.

Figure 7

Lysosomal targeting of rHDL/res/NBD by apoE3 in glioblastoma cells. (A) Uptake of individual components of rHDL/res was monitored by direct or indirect fluorescence: lipid (a–c), apoE3 (d–f) and resveratrol (g–i). Following exposure to rHDL/res/DiI at 37 °C for 3 h (a–c), the cells were visualized under a confocal laser scanning microscope: (a) DAPI; (b) DiI; (c) merge of (a) and (b). Following exposure to rHDL/res under the same conditions (d–f), the cells were visualized by: (d) DAPI; (e) apoE3 monoclonal antibody, 1D7, and Alexa555-conjugated secondary antibody; (f) merge of (d) and (e). Following exposure to rHDL/res/NBD (5 μg) as described above (g–i), the cells were visualized by: (g) propidium iodide; (h) resveratrol conjugated to 4-chloro-7-nitrobenz-2-oxa-1,3-diazole (res/NBD); (i) merge of (g) and (h). Panel (j) shows that uptake of res/NBD is minimal in the absence of rHDL. (B) Co-localization of res/NBD with apoE3, or with LAMP1 in late endosomal/lysosomal vesicles following cellular uptake of rHDL/res/NBD. Following exposure to rHDL/res/NBD, the cells were visualized by fluorescence associated with: (a) NBD to detect res, (b) Alexa555-conjugated secondary antibody to detect apoE3; (c) merge of (a) and (b); (d) NBD to detect res; (e) Alexa 594-conjugated secondary antibody to detect LAMP1; (f) merge of (d) and (e). Reproduced with permission from [63]. Copyright Public Library of Science, 2015.

Figure 8

Nanolipoproteins reconstituted with 3 nm and 10 nm AuNP. TEM image (Left) and schematic representation (Right) of 3 nm (A) and 10 nm (B) AuNP incorporated into rHDL/apoE3. The scale bar represents 100 nm in TEM images. TEM image of rHDL-AuNP prepared with 3 nm AuNP (A) revealed 60–80 nm spheroid structures with several AuNP (Inset, A), while that prepared with 10 nm AuNP revealed ~23 nm spherical structures with a single AuNP (Inset, B). The light area around the 10 nm AuNP likely represents the lipoprotein shell. Reproduced with permission from [64]. Copyright Dove Medical Press, 2017.