Coronary artery disease is the leading cause of death worldwide and can be life threatening especially when it develops into acute myocardial infarction (AMI), the most severe type of atherosclerotic cardiovascular disease. The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) are potent inhibitors of cholesterol synthesis and established therapies for the prevention of coronary artery disease. The first randomized trial to demonstrate that cholesterol-lowering therapy with statins improves prognosis in patients with high cholesterol and coronary artery disease, the Scandinavian Simvastatin Survival Study (4S), was reported in 1994 (1). Since then, lipid-lowering therapy with statins has become the mainstay for the prevention of coronary artery disease. The subsequent studies for the secondary prevention such as the CARE (Cholesterol and Recurrent Events) (2) and LIPID (Long Term Intervention with Pravastatin in Ischemic Disease) trials (3) further extended that the benefits of statins to the majority of patients whose cholesterol levels were in the normal range. The WOSCOPS (West of Scotland Coronary Prevention Study) (4) and the AFCAPS/TexCAPS (Air Force/Texas Coronary Atherosclerosis Prevention Study) (5) also extended the benefits for the primary prevention of atherosclerotic cardiovascular diseases. A decade after 4S, Ridker et al. demonstrated in the JUPITER (Justification for the Use of Statins in Primary Prevention: an Intervention Trial Evaluating Rosuvastatin) trial that high-intensity cholesterol-lowering therapy with statins reduced high-sensitivity C-reactive protein levels to lower than 2 mg/L and then achieved further risk reduction of cardiovascular events even among patients with normal cholesterol levels (6). Statins have a wide range of lipid-independent cardiovascular protective effects (so called “pleiotropic effects”), such as improvement of endothelial dysfunction, antioxidant properties, and inhibitory effects on inflammation (7) This landmark study suggested that anti-inflammatory therapy beyond lipid-lowering therapy is needed to further reduce cardiovascular events. At present, plaque erosion appears on the rise as a cause of acute coronary syndrome (ACS) in this statin era (8), but plaque rupture of unstable plaques with macrophage accumulation, fibrous cap thinning, and lipid deposition, followed by arterial occlusive thrombosis remains the primary mechanisms of ACS. Despite recent advances in lipid-lowering therapy by the emergence of Niemann-Pick C1-Like 1 inhibitor (ezetimibe) (9, 10) and proprotein convertase subtilisin/kexin type 9 inhibitors (11), there are still residual risks of cardiovascular events. One possible approach to the residual risks is to directly intervene in the inflammatory process during atherogenesis. Current medicinal therapy, including statins, has limitation of bioavailability in the diseased organs. Most small molecule drugs are absorbed from the intestines, metabolized in the liver, delivered to the bloodstream, and excreted from the kidneys, that is, drugs need to overcome the physiological barriers to achieve effective concentration in the blood and tissue. Adverse effects that any drugs possess may also limit their maximum dose in terms of safety. Recent innovation in nanomedicine can achieve effective drug delivery to targeted organs and cells, and spare undesirable adverse effects. In the first part of this review, we summarize recent advances in nanotechnology-based drug delivery system (nano-DDS). In the second part, we demonstrate the data of nano-DDS of statins in coronary artery disease for maximizing its anti-inflammatory effects.

Currently, various nano-sized materials (nanomaterials) have been tested and approved in clinical settings: lipid nanoparticles such as micelles or liposomes (12), polymeric nanoparticles (13), dendrimers (14), carbon nanotubes, and metallic nanoparticles. The characteristics of these materials and clinical applications are as follows (Table 1).

Although there are numerous determinants to affect in vivo kinetics of nano-DDS other than geometry as previously discussed, the size and surface modification of nanoparticles are the most important in the physiological behaviors of nano-DDS. Large-sized nanomaterials (>1,000 nm in diameter) tend to accumulate in the liver and lungs, and sometimes can be the cause of microemboli in capillaries, while small-sized nanomaterials (<10 nm) tend to be excreted from the kidneys. Middle-sized nanomaterials (10–1,000 nm) remain in circulation for longer time avoiding renal excretion. These circulating nanomaterials are generally incorporated by the mononuclear phagocytic system (MPS) in the liver, spleen, lymph nodes, and bone marrow (28). A surface modification with polyethylene glycol (PEG) serves as a hydrophilic shield that reduces protein absorption and undesirable non-specific interaction with MPS, which is preferable for nano-DDS in cancer. However, incorporation of nanomaterials into MPS itself is one of the intended mechanisms of drug delivery, especially when targeting inflammatory diseases including atherosclerosis. On the other hand, accumulation of nanomaterials depends on the permeability of target lesions. In tumor blood vessels, inflammatory atherosclerotic lesions, and ischemic myocardium, nanomaterials extravasate from blood vessels due to enhanced permeability. Tumors lack functional lymphatic vessels in their tumor microenvironment, which enhances the accumulation of nanomaterials. These phenomena are referred to as “enhanced permeability and retention effects” (29) or “passive-targeting” (Figure 1).

By contrast, “active-targeting” strategy employs target-specific structures, such as antibodies and proteins on nanomaterials, which bind to the target molecule that is specific for a certain disease process. “Active-targeting” strategy is being developed in cancer therapeutics targeting molecules associated with angiogenesis and cell proliferation (30). Adhesion molecules are one of the candidates for cardiovascular imaging as described later in this review, and innovative cardiovascular imaging using “active-targeting” strategy is being investigated. Regardless of the benefits of “active targeting,” only a few clinically validated nanomaterials utilize this strategy. Engineered protein combining IL-2 and diphtheria toxin (Ontak^®) is the only FDA-approved “active-targeting” nanomaterial to date (15).

Monocytes/macrophages play a key role in the inflammatory hypothesis of atherothrombosis (31). Atherogenesis starts with endothelial dysfunction caused by oxidative, hemodynamic, or biochemical stimuli (from smoking, hypertension, or dyslipidemia) and inflammatory factors. Endothelial dysfunction leads to enhanced permeability for cholesterol-containing low-density lipoprotein (LDL) particles and the expression of adhesion molecules to promote adhesion of circulating monocytes. Adhesive monocytes migrate into subendothelial space by chemoattractant proteins including monocyte chemoattractant protein-1 (MCP-1), where monocytes are differentiated into macrophages by macrophage-colony stimulating factor and activated through phagocytosis of oxidized lipid components to become foam cells. Activated macrophages secrete inflammatory cytokines to trigger positive feedback loop between cytokines and immune cells and matrix metalloproteinases (MMPs) leading to fibrous cap thinning. Rupture-prone plaques are characterized by the abundant accumulation of innate immune cells (mainly monocytes/macrophages), lipid core formation, and induction of several proteinases that catabolize the extracellular matrix (32).

Monocytes are functionally heterogeneous and are classified into at least two major subsets in mice: inflammatory monocytes (Ly-6C^highCCR2⁺CX3CR1^low) and non-inflammatory monocytes (Ly-6C^lowCCR2⁻CX3CR1^high) (33). Previously, we developed a mouse model of “plaque rupture” utilizing a high-fat diet and angiotensin II infusion in apolipoprotein E (ApoE)-deficient mice and reported that adoptive transfer of Ly-6C^highCCR2⁺ inflammatory monocytes increases buried fibrous caps in the brachiocephalic arteries (34), suggesting a critical role for inflammatory monocytes in plaque destabilization. Although their functional similarities have not been fully determined, CD14⁺⁺CD16⁻ classical monocytes and CD14⁺CD16⁺⁺ non-classical monocytes are described as their respective counterparts of Ly-6C^high and Ly-6C^low monocytes according to their chemokine receptor expression in humans, and intermediate monocytes (CD14⁺⁺CD16⁺) have been recently identified as not only a transitory state but also likely to possess unique features (35). There were not any significant differences in the peak levels of circulating CD14⁺⁺CD16⁻ monocytes among the patients with AMI, unstable angina pectoris (UAP), and stable angina pectoris (SAP), but that of circulating CD14⁺CD16⁺ monocytes (defined as CD14⁺⁺CD16⁺ plus CD14⁺CD16⁺⁺ monocytes) were significantly decreased in AMI patients compared with those in patients with UAP or SAP (36). A conventional paradigm proposed two subpopulations in macrophages corresponding to the monocyte heterogeneity: “classically activated or inflammatory M1 macrophages” and “alternatively activated or anti-inflammatory M2 macrophages.” Although the balance between “M1” and “M2” subpopulations may contribute to the development of cardiovascular disease, emerging evidence suggests that heterogeneity of macrophage subpopulation seems much more complex than “M1” and “M2” dichotomy (37). “M2” macrophages are divided into at least three subpopulations according to the stimulus they are activated; “M2a,” “M2b,” and “M2c.” All “M2” macrophages have anti-inflammatory properties to secrete IL-10 and TGF-β. “M2b” is an exception because they additionally secrete pro-inflammatory cytokines, such as IL-1β, IL-6, and TNFα. The tissue microenvironment in atherosclerotic plaques can affect macrophage population (38). Oxidized phospholipids induce “Mox” and intraplaque hemorrhage induces “Mhem (HA-mac)” or “M(Hb).” Although localization of “Mox” in human atherosclerotic lesions remains to be elucidated, the population was detected in approximately 30% of macrophages in advanced atherosclerotic plaque of LDL receptor-deficient mice. “Mox” not only shows an pro-inflammatory phenotype by producing cyclooxygenase-2 and IL-1β but also control redox status by inducing redox-regulating genes such as heme oxygenase-1 (HO-1), sufiredoxin-1 (Srnx1), and thioredoxin reductase 1 via nuclear factor erythroid 2-like 2 (NFE2L2). They also display reduced chemotactic and phagocytic capacities, which might contribute to perpetuation of inflammation and tissue damage (39). “Mhem” (previously named as “HA-mac” by the same investigators), originally defined as CD163^high human leukocyte antigen-DR^low, was detected in human hemorrhaged atherosclerotic plaque. “Mhem” is an atheroprotective subpopulation that drives adaptation to intraplaque hemorrhage through activation transcription factor 1-mediated induction of HO-1 by heme (40). Another investigators named this population “M(Hb)” and further demonstrated that “M(Hb)” has high expression of “M2 markers” mannose receptor and CD163, and reduces ROS production and promotes LXRα-mediated reverse cholesterol transport (41). Platelet chemokine CXCL4 induces “M4” macrophages. They lack CD163 and are unable to increase HO-1 production in response to hemoglobin or hemoglobin–haptoglobin complexes. Co-expression of MMP7 and S100 calcium-binding protein A8 (S100A8) in “M4” in human atherosclerotic plaques might lead to plaque destabilization by increased expression of MMP7 and affect lesion progression through decreased macrophage proliferation (42). In this way, the description of macrophage activation has been expanded and confusing, and then a group of scientists proposed the updated nomenclature to define the activator, i.e., M(IFNγ), M(LPS), M(IL-4), and M(IL-10), instead of the current complex nomenclature in in vitro experiments. This helps researchers to avoid using different definitions of activated macrophages. They also provide the framework within which researchers place a given population for in vivo experiments (37).

Macrophage is also a protagonist in myocardial ischemia-reperfusion (IR) injury and wound healing process of ischemic heart disease following AMI. In patients with ST-segment elevation acute MI (STEMI), early reperfusion is a standard therapy to salvage viable myocardium and limit MI size. Regardless of significant reductions in door-to-balloon time in the last decade, the mortality of patients with MI has not improved as shown in recent cohort studies. It is well recognized that the reperfusion of coronary arteries can paradoxically induce cardiomyocyte death, called “myocardial IR injury.” The major contributing factors in myocardial IR include ROS, calcium overload, and the rapid restoration of physiological PH at reperfusion, which induces the opening of the mitochondrial permeability transition pore that leads to the necrosis and apoptosis of cardiomyocytes in the first several minutes of reperfusion. Over several hours after reperfusion, myocardial inflammation contributes to cardiomyocyte apoptosis and the healing of infarcted myocardium (43, 44). The recruitment of neutrophils and inflammatory monocytes is an established phenomenon after myocardial injury. Uncontrolled macrophage infiltration following MI results in ischemic heart failure. We and another group demonstrated that infarcted myocardium sequentially recruits Ly-6C^hi monocytes and Ly-6C^lo monocytes in murine MI model (45) and IR injury model (46); Ly-6C^hi monocyte dominates in early phase to exhibit inflammatory functions including phagocytic and proteolytic activity, while Ly-6C^low monocyte dominates in later phase to resolute inflammation and promote angiogenesis. In consistent with this preclinical data, circulating CD14⁺⁺CD16⁻ and CD14⁺CD16⁺ monocytes sequentially increase in patients with AMI. The peak levels of circulating CD14⁺⁺CD16⁻ monocytes were negatively correlated with the degree of myocardial salvage, suggesting that manipulating classical inflammatory monocyte could be a therapeutic target for salvaging for ischemic myocardial damage after MI (36).

Monocyte chemoattractant protein-1 belongs to the CC chemokine subfamily and its primary receptor CCR2 is highly expressed on a subpopulation of monocytes (Ly-6C^high monocytes in mice and CD14⁺⁺CD16⁻ monocytes in humans). These inflammatory monocytes depend on CCR2 to migrate into the injured tissue. We previously demonstrated that systemic gene therapy with plasmids encoding 7ND, a deletion mutant of MCP-1, limits atherogenesis associated with increased lesional extracellular matrix content, one of characteristics of stable plaques, without any effects on serum lipid concentration (47). Furthermore, this gene therapy not only limits progression of established preexisting atheroma but also leads to plaque stabilization (48). We then utilized the 7ND-incorporated PLGA nanoparticle for interfering with MCP-1/CCR2 signaling and demonstrated inhibition of macrophage accumulation in the atherosclerotic plaque, followed by amelioration of morphological characteristics similar to human destabilized/ruptured plaque in the brachiocephalic artery of ApoE-deficient mice (34). Leuschner et al. demonstrated that inhibition of monocyte CCR2 with siCCR2-incorporated lipid nanoparticle inhibits macrophage accumulation in sites of inflammation and reduces atherosclerotic formation and infarct size in myocardial IR injury (49). Another group reported that blockade of CCR2 markedly reduced macrophage infiltration in ischemic lesions that results in attenuation of myocardial IR injury in mice via inhibition of macrophage-related oxidative stress and MMPs (50) They also demonstrated that macrophage infiltration into infarcted tissue was impaired in CCR2^−/− mice after coronary ligation and the CCR2 deficiency ameliorates post-MI left ventricular (LV) remodeling via inhibition of macrophage-related MMPs (51). These data suggested that inflammatory subpopulations of monocytes/macrophages are feasible therapeutic targets for coronary artery disease.

Other immune cells, including dendritic cells (DCs), T cells, and B cells, also play important roles in atherosclerosis. A predominant subpopulation of T cells in atherosclerotic lesion is CD4⁺ T cells. CD8⁺ T cells and natural killer T cells are minor T cell subpopulations. Antigen-presenting cells such as macrophages and DCs process the disease-related antigens including oxidized LDL, heat-shock proteins and microbes, and present them to CD4⁺ T cells as fragments loaded onto major-histocompatibility-complex class II molecules, which translates innate to adaptive immunity and is one of the therapeutic targets in adaptive immunity. In fact, vaccination targeting oxidized LDL-immuned DCs ameliorates atherosclerosis in LDL receptor-deficient mice (52). Activated T cells subsequently produce the type 1 helper T (Th1) cytokines (e.g., interferon γ) to initiate pro-inflammatory responses. Atherosclerotic lesions usually contain Th1 cytokines rather than the type 2 helper T (Th2) cytokines. In contrast, regulatory T cells modulate the process by producing anti-inflammatory cytokines, such as IL-10 and transforming growth factor-β (TGF-β) (53). The role of B cells in the progression of atherosclerosis still remains poorly understood, but it has recently gained more attention. B cells have two main subsets in mice based on their origin; B1 and B2. The role of the majority subset B2 cells in atherosclerotic lesion is controversial, while B1 cells seem to be atheroprotective (54). The corresponding B cell subsets in humans have not been clearly demonstrated. Nanomedicine can be applied to those atherosclerosis-regulating immune cells. However, the application of nanomedicine to atherosclerotic disease is extremely limited so far. A previous study demonstrated that intranasal immunization with chitosan/pCETP nanoparticles inhibits atherosclerosis in rabbit, in which the nanoparticulation enabled an efficient delivery of the antigen peptide to antigen presenting cells while sparing mucociliary clearance (55). Further studies are needed for the development of nanomedicine targeting adaptive immunity by utilizing nanoparticles as antigen delivery carriers of vaccination.

We have described that macrophage-mediated inflammation plays a crucial role in coronary artery disease. Hence, imaging of macrophages provides insight for future therapeutic options for cardiovascular diseases, in which several nanomaterials are being investigated as new imaging modalities. Developing those modalities for imaging macrophages is also indispensable in terms of quantitative analysis of therapeutic effects of anti-inflammatory drugs. Computed tomography (CT) is widely used for imaging of coronary artery in clinical setting. Nanomaterials for CT are limited because it requires high concentrations of absorbent nanomaterials to detect macrophages with X-ray. N1177, a crystalline iodined aroyloxy ester covered with a polymer, can detect macrophage-rich arterial walls in rabbits by CT (56). Macrophage-targeted gold high-density lipoprotein (HDL) nanoparticle can be localized in the macrophages of atherosclerotic plaque in the aorta of ApoE-deficient mice (57). MRI has high soft tissue contrast resolution and can detect plaque morphology non-invasively without radiation exposure. SPIO nanoparticle and ultrasmall superparamagnetic iron oxide (USPIO) are nanomaterial developed as the negative contrast for MRI. SPIO- and USPIO-based contrast agents consist of iron oxide core with hydrophilic polymeric coating, such as dextran-coated monocrystalline iron oxide nanoparticle-47 (MION-47) and dextran-crosslinked iron oxides. High-resolution MRI after administration of MION-47 can assess macrophage burden in atheromata induced by balloon injury of cholesterol-fed New Zealand White rabbits (58). In clinical setting, ATHEROMA trial has been conducted to examine USPIO-related signal change in patients with carotid stenosis >40%, but USPIO-enhanced MRI did not predict cardiovascular events significantly (59). On the other hand, USPIO-based contrast, ferumoxytol, has succeeded in characterizing infarcted myocardium mainly by detecting infiltrating macrophages (60). There are some reagents that adopt “active-targeting” strategy to image lesional macrophages. One good example is that iron oxide nanoparticle conjugated with ligand of vascular cell adhesion molecule 1 (VCAM-1) visualized VCAM-1-expressing endothelial cells and macrophages in ApoE-deficient mice (61). Nano-sized probes for near infrared fluorescence (NIRF) are used in animal studies. The excitation and emission wavelengths of NIRF probes range from 600 to 900 nm. In that range, the absorbance and scattering of biological tissues are relatively low. These probes are designed to be activated when target protease cleaves protease-specific peptide substrates linked to quenched fluorescent dyes. MMP (62, 63) and cathepsin are used as the substrates to image macrophage burden (64).

In this review, we have summarized a series of evidence including (1) the anti-inflammatory therapeutics for coronary artery disease, (2) a variety of nanomaterials for drug delivery systems, (3) the role and imaging of monocytes/macrophages in coronary artery disease, and (4) application of nano-DDS based on PLGA nanoparticle to coronary artery disease, such as plaque destabilization, IR injury, and post-MI LV remodeling. Applications of nano-DDS for coronary artery disease is a feasible strategy by utilizing increased incorporation of nanomaterials by MPS and enhanced permeability of inflammatory vasculature (Figure 1). In addition to the therapeutic effects of nano-DDS on coronary artery disease, there are a wide variety of opportunities to combine nano-DDS and various therapeutic agents, including chemical, nucleotide, peptide, and others, which may extend the possibility of current pharmacotherapy for several cardiovascular diseases. Possible application of nano-DDS in other cardiovascular diseases may include, pulmonary hypertension (79, 80), vein graft disease (81), and coronary artery stents (82). The clinical application of nano-DDS utilizing Good Manufacturing Practice-compliant Pitava-NPs in this review is in progress. We have already completed phase I/IIa investigators’ initiated clinical trial testing the efficacy of Pitava-NP in patients with critical limb ischemia in the Kyushu University Hospital (UMIN000008011). We have also completed a phase I clinical trial testing the safety of an intravenous administration of Pitava-NP in healthy volunteers (UMIN000014940). Recently, phase III CANTOS trial targeting interleukin-1β with human anti-IL-1β monoclonal antibody, canakinumab, has demonstrated that the inhibition of systemic inflammation may reduce cardiovascular risk even in the absence of concomitant lipid lowering (83). This trial has proved the concept that anti-inflammatory therapeutics targeting innate immunity pathway can be applicable in humans, and our novel DDS utilizing PLGA polymer-based nanotechnology might be one of promising therapeutic strategies for various cardiovascular diseases in the near future.

SK, TM, J-iK, KN, and KE make substantial contributions to conception and design and acquisition, analysis, and interpretation of data.

KE is the inventor of an issued patent on part of the results reported in the present study (Pharmaceutical composition containing statin-encapsulated nanoparticle, WO 2008/026702). Applicants for this patent include Kyushu University (https://airimaq.kyushu-u.ac.jp/), KOWA Inc. (http://www.kowa.co.jp), and Sentan Medical Inc. (http://sentaniryou.co.jp). Sentan Medical Inc. is a drug discovery venture company from Kyushu University. KE is a founder of Sentan Medical Inc., possesses stocks, serves as one of Directors of the company, and reports personal fees from the company outside the submitted work. The intellectual property division of Kyushu University is reviewing that Sentan Medical Inc. did not play a direct role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript in KE’s Laboratory.