Immune Implications of Cholesterol-Containing Lipid Nanoparticles

Abstract

The majority of clinically approved nanoparticle-mediated therapeutics are lipid nanoparticles (LNPs), and most of these LNPs are liposomes containing cholesterol. LNP formulations significantly alter the drug pharmacokinetics (PK) due to the propensity of nanoparticles for uptake by macrophages. In addition to readily engulfing LNPs, the high expression of cholesterol hydroxylases and reactive oxygen species (ROS) in macrophages suggests that they will readily produce oxysterols from LNP-associated cholesterol. Oxysterols are a heterogeneous group of cholesterol oxidation products that have potent immune modulatory effects. Oxysterols are implicated in the pathogenesis of atherosclerosis and certain malignancies; they have also been found in commercial liposome preparations. Yet, the in vivo metabolic fate of LNP-associated cholesterol remains unclear. We review herein the mechanisms of cellular uptake, trafficking, metabolism, and immune modulation of endogenous nanometer-sized cholesterol particles (i.e., lipoproteins) that are also relevant for cholesterol-containing nanoparticles. We believe that it would be imperative to better understand the in vivo metabolic fate of LNP-associated cholesterol and the immune implications for LNP-therapeutics. We highlight critical knowledge gaps that we believe need to be addressed in order to develop safer and more efficacious lipid nanoparticle delivery systems.

Keywords: cholesterol; cholesterol oxidation; immune modulation; lipid metabolism; lipid nanoparticle; liposome; macrophage; oxysterol.

Figure 1

Common structure and components of lipid nanoparticles. A) Liposome, B) nanoemulsions, C) solid lipid nanoparticles, D) nanostructured lipid carriers, E) polymer–lipid hybrid nanoparticles, and F) protein–lipid nanoparticles (sHDL).

Figure 2

Summary of LNP and lipoprotein trafficking in macrophages showing uptake, cholesterol homeostasis, and immune regulation. ABC, ATP-binding cassette transporter; ACAT1, acetyl-coenzyme A acetyltransferase-1; ApoER, apolipoprotein E receptor; Arg 1, arginase 1; CEHs, cholesterol ester hydrolases; COX, cyclo-oxygenase; CXCL, chemokine (C-X-C motif) ligand; ER, endoplasmic reticulum; IL, interleukin; iNOS, inducible nitric oxide synthase; LDLR, low-density lipoprotein receptor; LNP, lipid nanoparticle; LOX-1, lectin-like oxidized low-density lipoprotein receptor-1; LXR, liver X receptor; MCP, monocyte chemoattractant protein; PPAR, peroxisome proliferator-activated receptor; RXR, retinoid X receptor; SR, scavenger receptor; TLR, toll-like receptor; TNF, tumor necrosis factor; VLDLR, very low-density lipoprotein receptor.

Figure 3

Cholesterol transport and elimination. A) Cholesterol from exogenous (e.g., LNPs and food) or endogenous sources (e.g., liver and peripheral tissues) is combined with apolipoproteins (APOs) and triglycerides (TGs) to form VLDL particles that are secreted into the circulation. Through VLDLR, VLDL enters the adipose tissue, skeletal muscle, heart, and kidneys. VLDL can be converted to intermediate-density lipoproteins (IDLs) by lipoprotein lipase. IDL can be directly taken up by the liver or converted to LDL through the action of hepatic lipases. B) The reverse cholesterol transport (RCT) pathway is where cholesterol is transported in high-density lipoproteins (HDLs) from extrahepatic tissues to the liver. HDL-associated cholesterol ester may be transferred to LDL or VLDL through the cholesteryl ester transfer protein (CETP). C) Cholesterol is excreted through the hepatobiliary pathway as bile or through the transintestinal cholesterol efflux (TICE) pathway.

Figure 4

Major cholesterol metabolite production pathways.

Figure 5

Primary pathways linking lipid metabolism and immune responses promoted by oxysterols. PPAR, peroxisome proliferator-activated receptors; LXR, liver X receptor; RXR, retinoid X receptor; ROR, retinoic acid receptor-related orphan receptor; EBI2, Epstein–Barr virus induced gene 2; NK cells, natural killer cells; MDSCs, myeloid-derived suppressor cells; Tregs, regulatory T cells; DC, dendritic cells.