Introduction

Epidemiological studies identified several independent risk factors for cardiovascular disease (CVD), including hypertension, age, smoking, insulin resistance, elevated low-density lipoprotein cholesterol (LDL-C) levels, and triglyceride levels [1]. The majority of people establish plaques during young adulthood, making plaque regression the optimal therapeutic strategy [2–5]. The most effective LDL lowering agent, PCSK9 inhibitor evolocumab, only regressed coronary atheroma volume as assessed by serial coronary intravascular ultrasound by 0.95%, although 78 weeks of treatment reduced the LDL-C to 36.6 mg/dL in humans [6,7]. Clinical studies confirm that apolipoprotein AI (apoAI) can largely promote the regression of atherosclerosis by increasing functional high-density lipoprotein (HDL) particles [8–12].

rHDL Nanoparticles as a Drug Delivery Vehicle

The application of rHDL nanoparticles for delivering therapeutic compounds for the treatment of cancer has been studied extensively [76–78]. Recent studies show that rHDL nanoparticle serve as a drug delivery system to deliver compounds efficiently into macrophages and atherosclerotic plaques [79]. To investigate the immunomodulatory drugs for atherosclerosis, several nanoparticles were developed to increase the specificity of the drug delivery. rHDLs were efficiently used to deliver a liver X receptors (LXR) agonist GW3965 to atherosclerotic plaques of Apoe^−/− mice [80]. Importantly, rHDLs loaded with GW3965 completely abolished the liver toxicity of GW3965 in a one-week intensive treatment regimen in atherosclerotic mice. The long-term treatment with rHDLs significantly reduced atherosclerotic plaques in Apoe^−/− mice [81].

Statins have potent anti-inflammatory functions, but these cannot be fully exploited with oral statin therapy owing to a low systemic bioavailability. Interestingly, an injectable rHDL nanoparticle was synthesized to deliver simvastatin, and the effect of simvastatin-rHDL on atherosclerotic plaques was examined in mice. This study demonstrates that statin-loaded reconstituted HDL nanoparticles improved inflammation in atherosclerotic plaque [82]. More interestingly, nanoparticle-based delivery of simvastatin inhibited plaque macrophage proliferation in Apoe^−/− mice with advanced atherosclerotic plaques [83]. rHDL nanoparticles increased the plasma half-life of statins to 20 h. In addition, a recent study showed that rHDL-mediated targeted delivery of the LXR agonist promoted atherosclerosis regression [84].

Arachidonic acid (AA) was engineered into the rHDL complex to increase the efficacy of statins. AA-LT-rHDL (arachidonic acid-lovastatin-rHDL) exhibited lower reactivity with LCAT and more potent inhibition effects on foam cell formation in the presence of LCAT because of less undesired LT leakage during the remodeling of rHDLs induced by LCAT and more cellular drug uptake [85]. In addition, increasing AA concentration in AA-LT-rHDL particles reduced intracellular lipid deposition, decreased intracellular cholesterol esters content, and DiI-oxLDL uptake, and inhibited the expressions of pro-inflammatory cytokines TNF-α and IL-6 [85]. Together, these results proved that AA modification prevented the reactivity of LT-rHDL with LCAT, thereby inhibiting the undesired drug leakage during rHDL remodeling induced by LCAT. To better fulfill the targeted-delivery of rHDL, it might be interesting to determine whether the efficacy of the incorporation of AA into LT-rHDL is better than LT-rHDL for the treatment of atherosclerosis in mice. It would also be intriguing to investigate whether the polyunsaturated fatty acids, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), have better efficacy than AA in preventing LCAT-induced degradation of rHDL.

Delivery of Oligonucleotides Using rHDL Nanoparticles

HDLs are highly heterogeneous and transport a large variety of lipids, proteins, and microRNAs [86]. Anti-sense nucleotides and siRNA(s) are widely used to modulate gene expression and are being considered for therapeutics of atherosclerosis [87–89]. One of the major issues is that the half-life of anti-sense nucleotides is usually low in the presence of serum nucleases [90]. In addition, the therapeutic efficiency of nucleic acids is relatively low owing to the non-specific bio-distribution and subsequent off-target effects of nucleotides. Recent studies demonstrate that HDLs are natural at carrying nucleotides and transporting nucleotides specifically to recipient cells [91]. Moreover, HDL-miRNA cargoes from atherosclerotic patients induced remarkable gene expression, with substantial loss of conserved mRNA targets in hepatocytes. Collectively, these results show that HDL is involved in a mechanism of intercellular communication by transporting and specific delivery of miRNAs to cells. Therefore, rHDLs are believed to be an efficient vehicle for the specific delivery of siRNA and other anti-sense nucleotides for therapeutic applications [76,92].

Molecular Imaging of rHDL-Based Nanoparticles in Atherosclerosis

Mounting evidence shows that early stages of the lesion development is dominant by monocyte recruitment followed by monocyte differentiation into macrophages in mice, whereas macrophage proliferation is more predominant in advanced atherosclerotic plaques [43,93,94]. Molecular imaging approaches are developed to detect macrophage inflammation and lipid accumulation [95,96]. Immune cells such as neutrophils and monocytes are major sources of peroxidases because these enzymes are stored in granules, such as myeloperoxidase (MPO). MPO plays important roles in the inflammatory response and perpetuation of chronic inflammation in atherosclerosis [75]. Inactivation of MPO reduced reactive oxygen species (ROS)-mediated vascular inflammation and atherosclerosis [97–99]. Several imaging agents targeting myeloperoxidase were developed to monitor the inflammatory response and macrophage accumulation [75,100–102]. rHDLs were recently developed as imaging agents due to their ability of specific delivery to macrophages [51,103]. Interestingly, superparamagnetic rHDL nanoparticles were developed for magnetically-guided drug delivery and lipoprotein drug delivery through magnetic targeting which have shown to be effective chemotherapeutic approaches for prostate cancer [104]. Recent studies demonstrate that this nanomedicine-based delivery strategy based on rHDL nanoparticles also allows for the delivery of compounds to atherosclerotic plaque. Statin-rHDL ameliorates plaque inflammation and opens a new field for atherosclerosis nanotherapy [82]. S-rHDL labeled with Cy5.5 (lipid monolayer) and DiR (hydrophobic core) show that Cy5.5 and DiR were accumulated and detected in the atherosclerotic lesions [82]. Similarly, HDL mimetic CER-001 was radiolabeled with 89Zr to allow for imaging macrophage accumulation and positron emission tomography–computed tomography (PET/CT) imaging [105].

LXRs, oxysterol-activated nuclear receptors, play an important role in RCT through promoting ABCA1 and/or ABCG1-mediated cholesterol efflux. In vivo PET imaging probes radiolabeled with zirconium-89 (89Zr) on discoidal HDL nanoparticles were made by the reconstituting apoAI and the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine, the chelator deferoxamine B, and 89Zr [106]. It was demonstrated that the radioactivity in atherosclerotic aortas of rabbits was more than three-fold higher than the control animals after the injection with 89Zr-HDL nanoparticles. There was increased accumulation of radioactivity in lesions measured by the in vivo PET imaging [106]. Therefore, rHDLs demonstrated to be a reliable imaging probe and this allows us to study its in vivo properties to visualize the macrophage accumulation in advanced atherosclerotic lesions by using noninvasive PET imaging [106].

Concluding Remarks

HDL-targeted drug CETP inhibitors except anacetrapib did not decrease cardiovascular events in clinical trials. Convincing results demonstrate that increased HDL cholesterol levels do not always correlate with enhanced protective HDL properties [39–41], thus questioning its potential as a biomarker of HDL functionality. In addition, the association between low levels of HDL-C and CVD may be confounded by other factors, such as insulin resistance, inflammation, and/or metabolic derangements leading to altered plasma lipids. Importantly, current research is focused on both developing robust HDL functional assays and determining specific proteins or lipid molecules within the HDL complex to promote cholesterol efflux capacity for future translational and pre-clinical studies.

Although several rHDL nanoparticles failed to regress the atherosclerotic plaques in humans, it should be noted that these clinical trials are relatively short-term studies; the duration of these trials was only 4–6 weeks. There is solid evidence that HDL beneficial effects have to do more with the achievement of a continuous flux and steady export of cholesterol, rather than absolute levels of HDL cholesterol [36]. Whether rHDL nanoparticles would be more effective for the treatment of coronary artery disease over a longer period of time remains to be investigated. Furthermore, the field of rHDL nanoparticles has developed considerably and is poised for a big leap with the application of drug delivery systems and technologies that enable the specific delivery of new compounds to the biological system [107]. In conclusion, recent advances on rHDL nanoparticles have opened up a new avenue by which to ameliorate the inflammatory response for the treatment of CVD. Better understanding of the functional roles of HDL will likely lead to new approaches to battle and monitor the expanding burden of CVD.