Detection and Treatment of Atherosclerosis Using Nanoparticles

Abstract

Atherosclerosis is the key pathogenesis of cardiovascular disease, which is a silent killer and a leading cause of death in the United States. Atherosclerosis starts with the adhesion of inflammatory monocytes on the activated endothelial cells in response to inflammatory stimuli. These monocytes can further migrate into the intimal layer of the blood vessel where they are differentiate into macrophages, which take up oxidized low-density lipoproteins and release inflammatory factors to amplify the local inflammatory response. After accumulation of cholesterol, the lipid-laden macrophages are transformed into foam cells, the hallmark of the early stage of atherosclerosis. Foam cells can die from apoptosis or necrosis, the intracellular lipid is deposed in the artery wall forming lesions. The angiogenesis for nurturing cells is enhanced during lesion development. Proteases released from macrophages, foam cells and other cells degrade the fibrous cap of the lesion, resulting in rupture of the lesion and subsequent thrombus formation. Thrombi can block blood circulation, which represents a major cause of acute heart events and stroke. There are generally no symptoms in the early stages of atherosclerosis. Current detection techniques cannot easily, safely and effectively detect the lesions in the early stages, nor can they characterize the lesion feature such as the vulnerability. While the available therapeutic modalities cannot target specific molecules, cells, and processes in the lesions, nanoparticles appear to have a promising potential in improving atherosclerosis detection and treatment via targeting the intimal macrophages, foam cells, endothelial cells, angiogenesis, proteolysis, apoptosis, and thrombosis. Indeed, many nanoparticles have been developed in improving blood lipid profile and decreasing inflammatory response for enhancing therapeutic efficacy of drugs and decreasing their side effects.

INTRODUCTION

Atherosclerosis is a disease characterized by a process of building up of lipids, primarily cholesterol, in the artery wall ^{1, 2}. Atherosclerosis provides a pathological background for developing cardiovascular disease (CVD), the No. 1 killer in the United States. The structure of arteries from the inner cavity to the outermost layer is lumen, an intimal layer composed of an endothelial cell monolayer and underneath intima, a media layer composed of multiple layers of smooth muscle cells and connective tissues, and an adventitia layer composed of connective tissues³.

Cholesterol accumulation and deposition in the arterial wall and subsequent narrowing of the blood vessel lumen were considered as a sole cause of atherosclerosis in the past century¹. In the past two decades, research in both preclinical and clinical areas has suggested that inflammation integrated with dyslipidemia plays an important role in the development of atherosclerosis⁴. The endothelial cells are important in maintaining blood vessel integrity and permeability, adhesion molecule expression, leukocyte recruitment, and blood clotting⁵. Under normal circumstance, vascular endothelial cells resist the adhesion of circulating immune cells on them⁶. Atherogenic stimuli such as inflammation, hypertension, cigarette smoking, hyperlipidemia, especially hypercholesterolemia, and/or hyperglycemia increase their expression of adhesion molecules, disrupt the monolayer structure of endothelial cells, increase blood vessel wall permeability, and enhance their release of inflammatory factors¹. Although many immune cells contribute to atherosclerotic lesion formation, intimal macrophages play a critical role in the development of atherosclerosis^{4, 7}. After monocytes attach on the endothelial cells via binding to adhesion molecules, chemokines, especially monocyte chemoattractant protein 1 (MCP-1), direct monocytes migration into the intimal layer where they differentiate into macrophages. Lesion-resident macrophages recruit more monocytes into the evolving intimal lesion via secreting more MCP-1 and other inflammatory factors. When cholesterol influx is more than efflux, cholesterol is accumulated in the intimal macrophages. The lipid-laden macrophages are called foam cells, which are the hallmark of atherosclerosis. After foam cells die from apoptosis and necrosis, the cellular lipids are deposited in the artery wall leading to formation of atherosclerotic lesions. If the inflammatory condition and dyslipidemia persist, the advanced atherosclerotic lesion will be formed, which is characterized by a large lipid, primarily cholesterol, core, proliferated smooth muscle cells and remodeled extracellular matrix⁸.

Rupture of vulnerable lesions (plaques) followed by thrombi formation accounts for a majority of coronary events and/or sudden deaths^9–12. Vulnerable lesions are characterized by macrophage-dense inflammation, large lipid cores, thin fibrous caps and few smooth muscle cells^{11, 13}. Intimal macrophage accumulation promotes the development of vulnerable lesions by producing reactive oxygen species to increase the intimal levels of oxidized low density lipoproteins (oxLDL) and further foam cell formation; by producing matrix metalloproteinases and other proteases to degrade the extracellular matrix and fibrous caps; by releasing tissue factors to promote thrombus formation; by secreting pro-inflammatory cytokines to amplify the lesion inflammatory response^{6, 14, 15}. Current imaging and diagnostic techniques can detect stenotic lesions, but they cannot detect early-stage lesions and disclose the lesion biological aspects such as vulnerability¹⁶. Current preventive and therapeutic modalities focus on improving blood lipid profile, inhibiting thrombus formation, and decreasing blood pressure, but the treatment cannot directly target the atherosclerotic lesion¹⁷.

Since most biological processes, including atherogenesis, occur at the nanoscale, nanotechnology provides a promising opportunity for molecular imaging and targeted treatment of atherosclerosis¹⁸. Nanoparticles can increase the stability, aqueous solubility and absorption of diagnostic agents or therapeutic compounds, prolong their circulation time, enable high binding and uptake efficiency in the target cells (or tissue) over other cells (or tissue), protect them from degradation by enzymes in tissues and physiological fluids, reduce their side effects and toxicity¹⁹. Nanomedicine has gained tremendous attention in cancer therapy for more than 30 years. In contrast, however, its application in atherosclerosis is much less studied even given the fact that atherosclerosis is the key pathogenesis factor for developing CVD, a top cause of mortality worldwide. In the earliest studies published in 2000 and 2001, two studies reported that fibrin-targeted nanoparticles detected thrombi and perhaps vulnerable lesions^{20, 21}. Meanwhile, ultrasmall superparamagnetic particles of iron oxide were used for imaging atherosclerotic lesions in an animal model²². Shortly later, other investigators used iron oxide nanoparticles with anti-human E-selectin fragments conjugated on their surface to detect endothelial cells²³, or used alpha(v)beta3 (α_vβ₃) integrin-targeted nanoparticles to image angiogenesis in early-stage atherosclerosis²⁴. Last decade has seen a fast development in using nanoparticle technique as tool for molecular imaging of atherosclerotic lesion^{25, 26}. Since intimal macrophages are critical cells in atherosclerosis development, and can engulf nanoparticles by phagocytosis, they are the major nanoparticle targets in this research field^27–29. Currently, majority of studies are in the preclinical stage as we summarized in a chronological manner (Table 1–5), while only a limited number of clinical studies were conducted by using passive macrophage-targeted nanoparticles and listed in Table 1.

Table 1.

Detection of atherosclerosis using macrophage-targeted nanoparticles

Molecular/ functional target	Nanoparticles	Imaging platforms	Animal model/patients, dose and administration route	Results	Year and reference
Macrophage phagocytosis	VSOPs electrostatically stabilized with malic acid, tartaric acid, etidronic acid, citric acid and dimercaptosuccinic acid (DMSA)	Magnetic particle spectroscopy (MPS), TEM, MRI	ApoE−/− mice; Fe 500 µmol/kg; I.V.	All four types of stabilized VSOPs accumulated in atherosclerotic lesions of apoE−/− mice, except that VSOPs coated by DMSA in myocardium; These four VSOPs accumulated in phagolysosomes of altered endothelial cells and macrophages in lesions; Citrate-coated VSOPs accumulation around 3-fold higher than malic, tartaric, etidronic acid-coated VSOPS in lesions.	2015162
Macrophage phagocytosis	USPIOs coated by carboxylated PEG and aminated PEG loaded with annexin V	MRI/SPECT	ApoE−/− mice; 18.5 MBq per mouse of 99mTc (Technetium) labeled USPIO−Annexin V; I.V.	Nanoparticle system (annexin V- hybrid) specifically targeted the vulnerable atherogenic lesions containing apoptotic macrophages.	2015163
CD36	Liposome-like nanoparticles modified with oxidized phospholipids	Near infrared in vivo IVIS® fluorescence imaging system	LDLr−/− mice; 0.5 µmol total phospholipids per mouse; I.V.	High binding affinity of targeted nanoparticles for the oxLDL binding sites of the CD36 receptor on macrophages (in vitro); Higher accumulation of targeted than non-targeted nanoparticles in aortic lesions (in vivo); Targeted nanoparticles co-localized with macrophages and their CD36 receptors in aortic lesions (in vivo).	201527
Macrophage phagocytosis	PEGylated dendrimer- entrapped Au nanoparticles (Au DENPs)	CT	ApoE −/− mice; Au 0.1 mol/L, 100 µL; I.V.	PEGylated Au DENPs accumulated in macrophages and dominantly in their lysosomes; PEGylated Au DENPs can be used to detect murine macrophages distribution by CT imaging in apoE−/− mice.	201425, 26
LOX-1	131I-labelled LOX-1- targeted USPIOs; ligand is LOX-1 antibody	MRI	ApoE−/− mice; 30 µCi of 131I- labelled LOX-1- targeted or untargeted USPIOs; I.V.	High uptake of targeted USPIOs in only activated RAW264.7 in vitro; Targeted USPIOs accumulated in carotid atherosclerotic lesions, co- localized with LOX-1 of macrophages and characterized vulnerable atherosclerotic lesions in vivo.	201436
CD44	Hyaluronan (HA) magnetic glyconanoparticles (HA-NPs)	MRI	Atherosclerotic rabbit; Fe 0.21 mg/kg of body weight; I.V.	Selectively high binding of HA-NPs to CD44; Low dose of HA-NPs was significantly effective.	2014164
Macrophage phagocytosis	Gd-gold nanorods (Gd-GNRs)	Photoacoustic imaging (PAI) and MRI	Mice	Precise morphology to quantify the infiltration area and invasion depth of macrophages in the arterial wall by intravascular PAI.	2013165
Macrophages in myocardial infarction	USPIOs	MRI	Patients with an acute myocardial infarction; 17 mL USPIOs containing 510 mg Fe; I.V.	USPIOs was used to characterize myocardial infarct pathology by detecting infiltrating macrophages.	2013166
MSR1	USPIOs modified with a peptidic ligand targeting MSR1	MRI	ApoE−/− mice; Fe 250 µmol/kg; I.V.	Higher accumulation of MSR1–targeted USPIOs in atherosclerotic lesions (3.5- fold, P=0.01) than non-targeted USPIOs; Detection of inflammatory lesions in situ by MSR1–targeted USPIOs.	201370
Macrophage phagocytosis	Dextran-coated USPIOs (D-USPIO) and Mannan–dextran- coated USPIOs (DM- USPIOs)	MRI, MRA	Watanabe heritable hyperlipidemic rabbits; Fe, 0.08, 0.4, 0.8 mmol/kg; I.V.	DM-USPIO was better than D-USPIO in targeting atherosclerotic lesions at all doses by reduction in the signal-noise ratio.	2012167
OxLDL	Lipid-coated USPIO nanoparticles (LUSPIOs) modified with oxLDL antibody as ligand	MRI	ApoE−/− mice; Fe 3.9 mg/kg; I.V.	LUSPIOs enabled the detection and characterization of atherosclerotic lesions by targeting oxLDL/oxidation- specific epitopes.	201140
Macrophage phagocytosis	Nanoparticles modified by the LyP-1 peptide as ligand labeled by Cy5.5	Maestro™ in vivo fluorescence imaging system	Carotid-ligated mice producing macrophage-rich vascular lesions; 8 nmol of Cy5.5 per mouse; I.V.	LyP-nanoparticles (protein cage) had high binding affinity to macrophages in vitro and in vivo; They detected macrophage-rich murine carotid lesions in situ and ex vivo	2011168
Macrophage phagocytosis	Human ferritin protein cages encapsulating magnetite nanoparticles or conjugated to Cy5.5	MRI and fluorescence imaging	FVB strain mice	Ferritin nanoparticles accumulated in macrophages in atherosclerotic carotids; They detected vulnerable atherosclerotic lesions.	2011169
Macrophage phagocytosis	Iron-cobalt (FeCo) core with a graphitic- carbon (GC) shell conjugated to Cy5.5 to form FeCo/GC nanocrystals	MRI and fluorescence imaging	Carotid-ligated (left) FVB strain mice to creat macrophage-rich atherosclerotic lesions; 8 nmol of Cy5.5 per mouse; I.V.	Strong signals of FeCo/GC-Cy5.5 were founded in the ligated left carotid arteries, but not in the right control carotid arteries (non-ligated); FeCo/GC nanocrystals were co- localized with macrophages in the ligated carotid.	2011170
Macrophage phagocytosis	Monocrystalline iron oxide nanoparticles-47 (MION-47)	MRI	New zealand white rabbits after balloon injury; Fe 10 mg/kg, 40 mg Fe per rabbit; I.V.	Iron was accumulated in immunoreactive macrophages in atheromatous lesions; MION-47 enabled macrophage burden estimation, inflamed lesion identification, therapy-mediated monitor in atheromatous lesions under MRI.	2010171
Macrophage phagocytosis	Gd phosphatidylserine enriched liposomes (Gd-PS-liposomes) with/without oxidized cholesterol ester derivative (cholesterol-9- carboxynonanoate [9- CCN]) as ligand	MRI	Watanabe heritable hyperlipidemic (WHHL) rabbits, I.V.	More Gd-PS-liposomes with 9-CCN (targeted) were accumulated in macrophages than liposomes without 9- CCN (non-targeted); Gd-PS-liposomes with 9-CCN (targeted) were co-localized with arterial macrophages.	2010172
Macrophage phagocytosis	Gd phosphatidylserine (PS) enriched liposomes (Gd-PS- liposomes) or Gd- liposomes	MRI	RAW264.7 macrophages, ApoE−/− mice	RAW264.7 showed PS dependent uptake of liposomes with PS composition (2, 6, 12, and 20%) compared to control liposomes without PS (0%); Gd-PS-liposomes were co-localized in macrophages and accumulated in atherosclerotic lesions.	2010173
Macrophage phagocytosis	Nanoparticles with a 23-merpoly-Guanine (polyG) oligonucleotide	Fluorescence imaging system	In vitro: RAW264.7 and THP-1 derived macrophages; ex vivo: tissue sections of atherosclerotic lesion from apoE− /− mice	Oligonucleotide nanoparticles had a high binding affinity to RAW264.7 and THP-1 derived macrophages, foam cells (in vitro) and sections of atherosclerotic lesions (ex vivo).	2010174
CD44	Iron oxide based magnetic nanoparticles bearing hyaluronic acid	MRI	THP-1 derived macrophages	The uptake of nanoparticles was dependent on both CD44 and hyaluronic acid.	2010175
CD36	Gd-lipid nanoparticles modified with CD36 antibody as a ligand	MRI	Human aortic specimens (ex vivo); 1 mM Gd nanoparticles	CD36-targeted nanoparticles had a high binding affinity to macrophages; They improved detection and characterization of vulnerable atherosclerotic lesions.	2009
Macrophages via targeting oxLDL extracellularl y	Micelles containing gadolinium and murine (MDA2 and E06) or human (IK17) antibodies that bound oxidation-specific epitopes	MRI	ApoE−/− mice; micelles, Gd 0.075 mmol/kg ; I.V.	Micelles with antibody of MDA2 or IK17 significantly accumulated in the arterial wall at 72 hours and E06 micelles at 96 hours than adjacent muscle IgG micelles; MDA2, IK17, and E06 micelles accumulated in macrophages in atherosclerotic lesions; Free MDA2 competed with MDA2 micelles and decreased its MRI signal.	200841
CD204	Gd immunomicelles modified with CD204 antibody as ligand	MRI and fluorescence imaging	ApoE−/− and wild type mice, I.V.	Targeted immunomicelles demonstrated a 79% increase in the signal intensity of aortic lesions compared with non- targeted micelles with only 34% increase in apoE −/− mice; They detected macrophage content in vulnerable lesions.	200771
Macrophage phagocytosis	Magnetofluorescent iron oxide nanoparticle modified with Cy5.5	Fluorescence tomography and MRI	C57BL/6 mice with infracted myocardium; Fe 3 to 20 mg/kg; I.V.	Nanoparticles enabled imaging of myocardial macrophage infiltration under both MRI and fluorescence tomography.	2007176
CD204	Micelles conjugated with CD204 antibodies modified with quantum dot, rhodamine and Gd	MRI	ApoE−/− mice; Gd 0.075 mmol/kg; I.V.	Micelles detected macrophages in apoE−/− mice effectively by optical methods and molecular MRI; CD204-targeted micelles enhanced signals up to 200% compared with non- targeted micelles.	200737
Macrophage phagocytosis	Dextran-coated near- infrared magnetofluorescent nanoparticles (MFNPs)	MRI and fluorescence imaging	ApoE−/− mice; Fe 15 mg/kg; I.V.	Dextranated MFNPs accumulated in macrophages; Detection of atheroma by MFNP deposition under MRI.	2006177
Macrophage phagocytosis	Near-infrared fluorescent (NIRF) magnetofluorescent nanoparticle (MFNP)	Laser scanning fluorescence microscopy (LSFM)	ApoE −/− mice	MFNP-enhanced carotid atheroma demonstrated a significant NIRF signal by multichannel LSFM; NIRF signals were co-localized with immunostained macrophages.	2006178
Macrophage phagocytosis	USPIOs	MRI	Patients; Fe 2.6 mg/kg; I.V.	USPIOs enabled detection of macrophages in predominantly rupture- prone and ruptured human atherosclerotic lesions by MRI.	2003

Notes:

Au, Aurum

ApoE−/−, apolipoprotein E null

Cy5.5, near-infrared (IR) fluorescence dye

CT, computed tomography

FVB, Friend leukemia virus B

Fe, iron

Gd, gadolinium (Gd)

I.V., intravenous injection

LDLr−/−: LDL receptor null

LOX-1: lectin-like oxidized LDL receptor-1

LyP-1, a nine residue peptide shown to target macrophages

MSR1, macrophage scavenger receptor 1

MBq, megabecquerel as unit of radioactivity

MRI, magnetic resonance imaging

PEG, polyethylene glycol

STEM, scanning transmission electron microscopy

TEM, transmission electron microscopy

USPIOs: ultrasmall superparamagnetic iron oxide nanoparticles

VSOPs, very small iron oxide particles

/kg: per kilogram body weight

Table 5.

Detection and treatment of atherosclerosis using HDL nanoparticles

Molecular/ functional target	Nanoparticles	Imaging platforms	Animal model, dose and administration route	Results	Year and reference
Macrophages	PLGA-HDL nanoparticles: a lipid/apolipoprotein coating and a PLGA core	N/A	ApoE−/− mice	PLGA-HDL nanoparticles accumulated in atherosclerotic lesions; PLGA-HDL nanoparticles were co- localized with lesion macrophages.	2015184
Macrophages	Reconstituted HDL (rHDL) loaded with statin or Gd	MRI	ApoE−/− mice; Gd 50 mmol/kg in rHDL, statin 15 mg/kg as low dose; statin 60 mg/kg as high dose; I.V.	Statin-rHDL accumulated in macrophages in atherosclerotic lesions; Lesion inflammation was inhibited by 3-month low-dose of statin-rHDL; Inflammation in advanced atherosclerotic lesions was significantly reduced by 1 week high- dose of statin-rHDL.	2014149
Macrophages	Gd-based contrast agents-HDL (GBCA-HDL) modified by oxidized apoA-I or its peptides as ligands	N/A	ApoE−/− mice	The oxidation of apoA-I or its peptides increased uptake of GBCA- HDL (2–3 times) by macrophage in vitro; GBCA-HDL with oxidized apoA-I or its peptides accumulated in macrophages in the lesions in apoE−/− mice.	2014185
Macrophages	HDL mimic nanoparticles containing core, quantum dots (QDs) coated with PLGA, cholesteryl oleate, and a phospholipid bilayer decorated with cations triphenylphosphoniu m (TPP), apoA-I mimetic peptide and PEG	Optical imaging	Rats; TPP-HDL- apoA-I-QD NPs; 80 µg/kg with respect to QD, 10 mg/kg with respect to total nanoparticles; I.V.	Nanoparticles improved reverse cholesterol transport in in vitro studies; Nanoparticles decreased plasma triglyceride concentrations in rats.	2013129
Macrophages	Au-HDL	Multicolor CT	ApoE−/− mice; Au 500 mg/kg; I.V.	Multicolor CT identified lesion composition; Au-HDL accumulated in aortic macrophages.	2010186
Macrophages	HDL nanoparticles (peptide mimic and replace apoA-I) labeled with Gd and rhodamine	MRI	ApoE−/− mice; Gd-micelles, 50 µmol/kg; I.V.	HDL nanoparticles improved cholesterol efflux from macrophages and were taken up by cells in a receptor-dependent manner; HDL nanoparticles detected lesion macrophages in apoE−/− mice.	2009187
Macrophages	rHDL modified with a carboxyfluoresceine- labeled apoE- derived lipopeptide, P2A2 (rHDL-P2A2) nanoparticles	MRI	ApoE−/− mice; Gd-rHDL-P2A2, 50 µmol/kg; I.V.	rHDL-P2A2 nanoparticles significantly enhanced atherosclerotic signals; rHDL-P2A2 nanoparticles were co- localized with macrophages in lesions.	2008188

Notes:

ApoE−/−, apolipoprotein E null

Au, Aurum

ApoA-I, apolipoprotein A-I

CT, computed tomography

Gd, gadolinium

HDL, high-density lipoprotein

I.V., intravenous injection

MRI, magnetic resonance imaging

PLGA, poly(lactic-coglycolic) acid

PEG, polyethylene glycol

/kg, per kg body weight

rHDL, reconstituted HDL

In this review, we are focused on the nanoparticle-mediated detection and treatment of atherosclerosis via targeting intimal macrophages, foam cells, endothelial cells, and processes of neoangiogenesis, proteolysis, apoptosis, and thrombosis (Figure 1). Nanoparticle-mediated low density lipoproteins (LDL) and HDL metabolism and anti-inflammation will be addressed at the end of this review.

Figure 1. Potential lesion targets for detection and treatment of atherosclerosis.

Nanoparticles can target to the specific cells or processes in the atherosclerotic lesions. The molecular or functional targets include macrophage scavenger receptors, macrophage phagocytosis, reactive oxygen species, proteases, annexin V for apoptosis, αvβ3 for neoangiogenesis, adhesion molecules and others. (Figure adapted and reprinted with the permission from reference, page 35S).

(Libby P, DiCarli M, Weissleder R. The vascular biology of atherosclerosis and imaging targets. J Nucl Med. 2010 May 1;51 Suppl 1:33S-37S.)

STRUCTURAL AND FUNCTIONAL IMAGING OF ATHEROSCLEROSIS

Structural Imaging

Several imaging modalities have been used in visualizing the vascular structure of atherosclerosis including the lesion volume and fibrous cap thickness ³⁰. Magnetic resonance imaging/angiography (MRI/MRA) is a commonly used method, which utilizes gadolinium (Gd) chelates/nanoparticles, superparamagnetic iron oxide probes (SPIO), ultrasmall superparamagnetic iron oxide (USPIO) as contrast enhancement with resolution of 10–100 µm to visualize the structure of atherosclerotic lesions³¹. Computed tomography (CT) is a method utilizing iodinated molecules as imaging moieties and high-resolution X-ray as technology with resolution of 50 µm for clinical or preclinical imaging³². Positron emission tomography (PET)/Single-photon emission computed tomography (SPECT) as an approach is increasingly popular by using imaging moieties such as ¹⁸F, ⁶⁴Cu, ¹¹C Tracers/ ^99mTc,^{123/124/125/131}I, ¹¹¹In tracers and nuclear technology with resolution of ~2 µm³². Angiography (X-ray-based fluoroscopy and iodinated molecules as contrast agent), optical coherence tomography (OCT)/optical frequency domain imaging (OFDI), optical angioscopy, intravascular ultrasound are commonly used invasive approaches to detect atherosclerotic lesions³³.

Functional Imaging

Imaging of specific cells or components in lesions can disclose lesion biology and feature, especially vulnerability, which can help prevent major cardiovascular events³⁴. By incorporating peptides, antibodies or other ligands on its surface, a nanoparticle can target lesion components (i.e. collagen, proteinases, reactive oxygen species) and cells^{17, 35}. Diagnostic dyes or contrast agents are incorporated in the nanoparticles, which can be detected using modalities including MRI, PET/SPECT, CT, optical near infrared fluoroscopy (NIRF)^{36, 37}. Although fluorescence imaging cannot be used in clinical research because of short penetration, it is a good approach to image atherosclerosis in small animal models. Dysfunctional endothelial cells can be visualized by using nanoparticles, conjugated with specific ligands allowing to target adhesion molecules^{38, 39}. Macrophages and foam cells are the most abundant inflammatory cells in atherosclerotic lesions. Intimal macrophages and foam cells have phagocytic activities, express scavenger receptors (i.e., CD36, LOX-1, MSR1) and also release reactive oxygen species (oxidized epitopes) and matrix-degrading proteases (i.e., matrix metalloproteinases and cathepsins); thus all these features can serve as potential targets to visualize macrophages and foam cells and to estimate their oxidative and inflammatory activities ^{40, 41}. Fibrin and factor XIII can be used to target thrombosis⁴². The α_vβ₃ Integrin can be used to visualize lesion neoangiogenesis^{24, 43}. Abundance and distribution of those cells and the key active components in lesions provide valuable information beyond lesion volume^{34, 44}. The events such as inflammation, especially neoangiogenesis, fibrous cap degradation, oxidative stress, are critical for subsequent selection of preventive and therapeutic modalities.

NANOPARTICLES TARGET ATHEROSCLEROTIC LESIONS

When an atherosclerotic lesion is developing, the permeability of the endothelial layer of arterial wall increases, which allows more lipoproteins and small particles such as nanoparticles to migrate into the intimal layer^{45, 46}. Expanding atherosclerotic lesions requires oxygen and nutrients to allow neoangiogenesis occur⁴⁷. The neovessels are prone to be leaky and fragile⁴⁷ resulting in increased permeability and retention (EPR), further promoting lesion expansion. Nanoparticle migration into atherosclerotic lesions via the EPR effect is considered as a non-specific targeting process¹⁷. Recognition of nanoparticles by their binding to the specific cells or molecules in the lesions via their surface ligands are thought to be an active targeting process¹⁷.

Intimal Macrophages and Foam Cells

Macrophages and their derived lipid-laden foam cells are determinant cells of atherosclerotic lesions due to their ability to accumulate lipids and increase inflammatory responses². Recruitment and deposition of macrophages into the artery wall occur prior to lesion development⁴⁸. Additionally, accumulation and activation of intimal macrophages positively correlates with lesion size⁴⁹. The recruitment of blood monocytes followed by subsequent differentiation to intimal macrophages and their proliferation in situ increase lesion macrophage numbers, while macrophage emigration or death decreases their numbers^{2, 4} The content of intimal macrophages depends on the kinetic balance between the above processes ². Targeting intimal macrophages and foam cells is a promising avenue for detection and treatment of atherosclerosis.

Macrophages are phagocytic cells, and they eat up dying or dead cells and foreign particles or microbes. Iron oxide nanoparticles have been widely used to detect intimal macrophages by MRI, because like most other foreign particles, iron oxide particles can be taken up by macrophages through their phagocytic function of macrophages in the whole body^{28, 29} (Table 1). There are two major types of iron oxide nanoparticles are superparamagnetic iron oxide (SPIO) nanoparticles with size of more than 50 nm in diameter and ultrasmall SPIO (USPIO) nanoparticles with size of between 18 nm to 50 nm in diameter⁵¹. Magnetic nanoparticles used in MRI usually contain iron cores such as magnetite (Fe₃O₄) and maghemite (γ-Fe₂O₃), and their surface is modified by hydrophilic coating such as dextrans (most commonly), carboxydextran, carboxymethylated dextran, chitosan, starches, polyvinyl alcohol, poly(ethylene glycerol) (PEG), polylactic-co-glycolic acid, polymethyl methacrylate, polyacrylic acid and polyvinyl pyrrolidone³⁰.

Intimal macrophages bind and take up native LDL and oxLDL cholesterol via their scavenger receptors including the CD36 receptor, macrophage scavenger receptor 1 (MSR1/CD204/SR-A1), lectin-like oxidized LDL receptor-1 (LOX-1), SR-B1, CD68, macrophage receptor with collagenous structure (MARCO) among others^{33, 52}. CD36 is an 88-kDa transmembrane receptor belonging to the class B scavenger receptor family^{53, 54}. Studies performed in mice suggest that CD36 is more important than other macrophage scavenger receptors in the process of oxLDL uptake, foam cell formation, and atherosclerotic lesion development^55–57. Injection of CD36-null macrophages into atherosclerosis-prone mice profoundly reduced the atherosclerotic lesion formation, while reintroduction of macrophages with CD36 increased the lesion formation by 2-fold⁵⁸. Blockage of oxLDL binding site of CD36 using a peptide ligand reduced lesion size by more than 50% in apolipoprotein E null (apoE−/−) mice⁵⁹. Furthermore, CD36 correlates well with lesion severity^{56, 57, 60}. Since CD36 can recognize and bind to oxLDL, one or more components of oxLDL must be ligand(s) for CD36. Terpstra V and Bird DA et al. extracted the lipids from oxLDL exhaustively by using a chloroform and methanol mixture, and reconstituted these lipids into microemulsions. They found these microemulsions competed effectively for the binding of intact oxLDL to the macrophages. However, microemulsions containing lipids from native LDL did not show the effect^{61, 62}. Oxidized phospholipids naturally found on oxLDL are enriched in atherosclerotic lesions of animals^{63, 64}. Therefore, they seem to be the most likely ligands for binding oxLDL to CD36. On the surface of oxLDL, hydrophilic head and sn-2 acyl group of oxidized phosphatidylcholines protrude to the aqueous phase, resulting in a lipid whisker model⁶⁵. The protruded and oxidized sn-2 acyl group incorporating a terminal γ-hydroxy (or oxo)-α,β-unsaturated carbonyl is critical for its high binding affinity to CD36^{64, 66, 67}. Podrez EA et al. compared the binding affinity of different oxidized phosphatidylcholines to CD36⁶⁴. 1-(Palmitoyl)-2-(5-keto-6-octene-dioyl)phosphatidylcholine (KOdiA-PC), 1-palmitoyl-2-(4-keto-dodec-3-ene-dioyl)phosphatidylcholine (KDdiA-PC) and 9-keto-12-oxo-10-dodecenoic acid of 2-lysophosphatidylcholine (KODA-PC) have the highest binding affinity to CD36 among 14 tested oxidized phosphatidylcholines⁶⁴. We made liposome-like nanoparticles using phosphatidylcholine and KOdiA-PC²⁷. We intravenously injected those CD36-targeted nanoparticles carrying KOdiA-PC into LDL receptor null (LDLr−/−) mice, and found that those nanoparticles can target intimal macrophages via binding to their CD36 receptors²⁷. CD36-targeted nanoparticles had a higher binding affinity to mouse and human macrophages than non-targeted nanoparticles. When we knocked down CD36 using small interfering RNA (siRNA), the binding of CD36-targeted nanoparticles to macrophages was diminished²⁷. Lipinski MJ et al. incorporated CD36 antibody on the surface of gadolinium (Gd)-containing lipid-based nanoparticles. Phospholipids, Tween 80 and an aliphatic gadolinium complex were used to make the nanoparticles. They found that the CD36-targeted nanoparticles had high uptake by human macrophages in an in vitro experiment, increased signal intensity in human atherosclerotic lesions via binding to intimal macrophages in an ex vivo experiment⁶⁸.

LOX-1 is a 52 KDa type II membrane receptor. LOX-1 expression on intimal macrophages positively correlates with atherosclerotic lesion instability and vulnerability³⁶. Wen S et al conjugated LOX-1 antibody on the surface of USPIO nanoparticles³⁶. Those LOX-1 targeted nanoparticles had higher binding affinity to and uptake by RAW264.7 macrophages than non-targeted nanoparticles. After intravenous administration of nanoparticles into apoE−/− mice, targeted nanoparticles gave signal enhancement of atherosclerotic lesions, especially in the areas enriched with macrophages/foam cells³⁶. Besides imaging of the intimal macrophages and atherosclerotic lesions, this approach might also characterize vulnerable atherosclerotic lesions. MSR-1 is another important scavenger receptor involved in macrophage uptake of oxLDL and subsequent foam cell formation⁶⁹. After conjugating peptidic MSR1 ligands or MSR1 antibodies on the nanoparticles, those MSR1-targeted nanoparticles can target atherosclerotic lesions by binding to MSR-1 on intimal macrophages^{37, 70, 71}. Other macrophage targeting mechanisms include incorporating apolipoprotein A-1 peptides on high density lipoprotein (HDL)⁷²; incorporating phosphatidylserine on nanoparticles for targeting phosphatidylserine receptors on macrophages⁵². Table 1 lists detailed information about different types of macrophage-targeted nanoparticles, and their target mechanisms in published preclinical and clinical research studies.

Targeted delivery of therapeutic compounds, siRNA and others to intimal macrophages represents an innovative and efficient treatment to atherosclerosis (Table 2). Macrophage-targeted therapy can prevent or inhibit lesion development by decreasing lipid accumulation and inflammation. Most of intimal macrophages are differentiated from circulating monocytes of both bone marrow and spleen origin². There are two types of circulating monocytes: inflammatory and non-inflammatory monocytes². Inflammatory monocytes (Ly-6C^high in the mouse, CD14⁺⁺CD16⁻ in human) are differentiated to classical (M1 type) macrophages, which increase inflammatory response⁷³. Non-inflammatory monocytes (Ly-6C^low in the mouse, CD14^+/lowCD16⁺ in human) are differentiated to alternative (M2 type) macrophages, which decrease inflammatory response⁷³. The M2 macrophages are subdivided into three subtypes (M2a, M2b, and M2c), which have functions of Th2 responses, Th2 activation, and immunoregulation, respectively⁷⁴. Different phenotypes of macrophages have different functions^{74, 75}. Studies thus far have shown a lack of consensus in describing or defining their macrophage phenotypes⁷⁵. To our knowledge, none of nanoparticles has been developed to identify or target a specific phenotype of macrophages. Inflammatory, but not non-inflammatory, monocytes depend on the CC-chemokine receptor 2 (CCR2) for distribution to the blood vessel wall⁷³. Upon binding to CCR2 of inflammatory monocytes, MCP-1 directs their migration into the intimal layer. Increased invasion of inflammatory monocytes critically promote lesion formation, progression and its complications⁷⁶. In contrast, decreased invasion of inflammatory monocytes results in less foam formation and diminished local inflammatory response, which inhibit lesion formation and progression. Decreased expression of CCR2 prevents inflammatory monocyte migration to, and accumulation in the sites of inflammation⁷³. Leuschner F et al. developed CCR2 siRNA loaded lipid nanoparticles, which are composed of C12–200, disteroylphosphatidylcholine, cholesterol and PEG–dimyristolglycerol⁷³. After systemic administration of those nanoparticles, mRNA and protein expression of CCR2 in inflammatory monocytes were significantly decreased. The CCR2 siRNA loaded lipid nanoparticles decreased the number of inflammatory monocytes by more than 70%, and lowered the migratory capacity of inflammatory monocytes towards MCP-1 by more than 90%. After 3-week intravenous treatment to apoE−/− mice, the number of intimal macrophages was reduced by 82%, which correlated with a 38% reduction of aortic root lesion size⁷³. Majmudar MD et al. used polymeric nanoparticles to carry CCR2 siRNA⁷⁷. After administration of those CCR2 siRNA-loaded nanoparticles to apoE−/− mice, they found that more than 75% of nanoparticles were taken up by monocytes/macrophages. Mice treated with CCR2 siRNA-loaded nanoparticles had decreased monocyte invasion and subsequent decreased number of intimal macrophages, which are associated with decreased expression of inflammatory genes in the lesions⁷⁷. McCarthy JR et al. developed a light-activated nanoagent, which can be taken up by intimal macrophages in inflamed atherosclerotic lesions⁷⁸. They induced apoptosis of intimal macrophages using a therapeutic dose of light. Ablation of intimal macrophages might decrease lesion formation via decreasing foam cell formation, and stabilize lesions via lowering inflammation⁷⁸. Most of the above studies did not present deep underlying mechanisms, such as monocyte/macrophage population number, phenotype, their origins, or shift from inflammatory to inflammatory monocyte/macrophage. More intensive and deep investigation in the underlying mechanisms is required in this research field.

Table 2.

Treatment of atherosclerosis using macrophage-targeted nanoparticles.

Molecular/ functional target	Nanoparticles	Imaging platforms	Animal model/patients, dose and administration route	Results	Year and reference
Macrophage phagocytosis	PLGA- b –PEG nanoparticles encapsulating liver X receptor (LXR) agonist GW3965 (NP-LXR)	N/A	LDLr−/− mice; GW3965: 10 mg/kg; I.P.	NP-LXR significantly decreased inflammatory factor expression and increased LXR-target gene expression in macrophages compared to free GW3965 in vitro and in vivo; NP-LXR were co-localized with lesion macrophages and reduced the CD68-positive macrophage content in lesions (by 50%) without increasing triglycerides or total cholesterol in the plasma and liver.	2015
Monocytes/ macrophages	Immune-modifying nanoparticles (IMPs), derived from polystyrene, microdiamonds and biodegradable PLGA	N/A	C57BL/6 mice with myocardial infarction; nanoparticles 1.4 mg/kg; I.V.	IMPs were taken up by inflammatory monocytes; Negatively charged IMPs bound to inflammatory monocytes, directed them to the spleen for apoptosis.	2014179
Macrophage phagocytosis	Liposomal dexamethasone	N/A	In vitro: primary human macrophages	Dexamethasone-loaded liposomes Inhibited monocyte and macrophage migration, reduced proinflammatory cytokine secretion (specifically TNF, IL-1β, IL-6).	2014180
Macrophage phagocytosis	89Zirconium- radiolabeling dextran nanoparticle (89Zr- DNP) modified with a near-infrared fluorochrome for microscopy; DNP loaded with CCR 2 siRNA	PET/MRI/Flu orescence imaging	ApoE−/− mice; I.V.	DNP were taken up predominantly by monocytes/macrophages (76.7%) and other leukocytes (12.5%); High uptake of 89Zr-DNP in the aortic root of apoE−/− mice; 89Zr-DNP lesion signals were decreased by therapeutic silencing of CCR2; Hybrid MRI/PET DNP demonstrated assessment of atherosclerotic inflammation.	201377
Macrophage phagocytosis	Single-walled carbon nanotubes (SWNT) modified by Cy5.5	N/A	Carotid-ligated mice to produce macrophage-rich atherosclerotic lesions; 8 nmol of Cy5.5 per mouse or 0.6 nmol of SWNT per mouse; I.V.	High signal intensity of SWNT was found in ligated carotids; SWNT-Cy5.5 were co-localized with atherosclerotic macrophages; Light (808 nm) induced apoptosis of macrophages in ligated carotid arteries containing SWNTs, but not in control arteries without light exposure or without SWNTs.	2012181
Phosphatidyl serine (PS) receptor	PS-presenting liposomes containing iron oxide	MRI	Rats; 150 µL of 0.06 M PS- presenting liposomes or PS-lacking liposomes; I.V.	PS-presenting liposomes upregulated macrophage mannose receptor— CD206, increased secretion of anti- inflammatory cytokines, downregulated proinflammatory cytokines.	201152
Monocytes/ macrophages	Lipid nanoparticle- encapsulated CCR2- siRNA	CT	ApoE−/− mice; 0.5 mg/kg/day of CCR2 siRNA, twice a week for 3 weeks; I.V.	Nanoparticles were co-localized with monocytes, and prevented monocytes accumulation in inflammatory sites by degrading CCR2 mRNA; CCR2 RNA silencing decreased monocytes/macrophages number in atherosclerotic lesions, reduced infarct size after coronary artery occlusion.	201173
MSR1	Nanoscale amphiphilic macromolecules composed of a sugar backbone and PEG loaded with liver X receptor agonist GW3965	N/A	Sprague Dawley rats	The nanoscale macromolecules decreased intimal cholesterol levels (macromolecule alone 50%; macromolecule-encapsulated GW3965 70%) and prevented retention of macrophage (macromolecule 92%; macromolecule-encapsulated GW3965 96%) compared to non-treated controls.	201189
Macrophage phagocytosis	Liposomal prednisolone phosphate (L-PLP)	PET/CT/MRI	Rabbits; PLP, 15 mg/kg; I.V.	L-PLP demonstrated significant higher anti-inflammatory effects than free PLP;	2010157
Macrophage phagocytosis	Dextran coated iron oxide nanoparticles modified with a near infrared fluorescence dye (detection) and a potent chlorin-based photosensitizer (treatment)	MRI	ApoE−/− mice; Fe 10 mg/kg (detection); chlorin 65 mg/kg (treatment); I.V.	Nanoparticles were accumulated in macrophage-rich atherosclerotic lesions; Photosensitizer nanoparticles eradicated inflammatory macrophages, subsequently stabilized lesions.	2010
Macrophage phagocytosis	L-PLP	PET/CT/MRI	Rats and rabbits; Toxicokinetic and pharmacokinetics study in rats: weekly L-PLP 0.5, 2 or 8 mg/kg; daily PLP 15 or 60 mg/kg for 28 days; I.V. Pharmacokinetics and anti- inflammatory effects in rabbits: 1 mg/kg or 10 mg/kg; I.V.	L-PLP had improved pharmacokinetics in rats and rabbits; No toxic effects of L-PLP were observed in rats; L-PLP decreased inflammatory response on the blood vessel wall in atherosclerotic rabbits.	2015159
Macrophage phagocytosis	L-PLP	PET/CT/MRI	Human subjects Pharmacokinetics : 0.375 mg/kg, 0.75 mg/kg or1.5 mg/kg; I.V. Lesion macrophage delivery: 1.5 mg/kg; I.V. Therapeutic efficacy: 1.5 mg/kg; I.V.	Nanoencapsulation increased plasma half-life of PLP; PLP was accumulated in 75% of the lesion macrophages; No anti-inflammatory effects on the arterial wall.	2015160

Notes:

ApoE−/−, apolipoprotein E null

CT, computed tomography

CCR 2, C-C chemokine receptor type 2

Cy5.5, near-infrared (IR) fluorescence dye

IL-1β, interleukin-1 beta

IL-6, interleukin 6

I.V., intravenous injection

MRI, magnetic resonance imaging

MSR1, macrophage scavenger receptor 1

N/A, not applicable

PLGA, poly(lactic-coglycolic) acid

PEG, polyethylene glycol

PET, positron emission tomography

PLP, prednisolone phosphate

L-PLP, liposomal prednisolone phosphate

siRNA, small (short) interfering RNA

TNF, tumor necrosis factors

/kg: per kilogram body weight

Technically, specificity is still not satisfactory as most of targeted nanoparticles target not only intimal macrophages, but other types of cells in the body are also impacted. For example, many “intimal macrophage specific” target molecules including CD36, LOX-1, SR-B1 and other scavenger receptors are also present in other cells, and even the most advanced nanoparticles cannot target a specific monocyte or macrophage phenotype, which render the danger of off-target effects. Future studies are expected to provide more mechanistic insight as to how nanoparticles function to decrease inflammation in the atherosclerotic lesion, which at least involves abundance, phenotype, origins, and transformation of monocytes/macrophages.

Important macrophage membrane proteins involved in cholesterol efflux are ATP-transporter cassette A1 (ABCA1), ATP-transporter cassette G1 (ABCG1) and scavenger receptor B class 1 (SR-B1)⁷⁹. Ligand activation of liver X receptors (LXR), cholesterol-sensing nuclear receptors, reverses atherosclerosis through regulating lipid absorption, transport and metabolism and suppressing inflammatory response⁸⁰. Both LXRα and LXRβ are expressed in macrophages ⁸¹. GW3965 is one of LXR agonists^{81, 82}. Activation of LXR in lesion macrophages can enhance cholesterol efflux and inhibit inflammatory response^{80, 83, 84}. ABCA1 promotes free cholesterol efflux from macrophages or foam cells to pre-beta-HDL (pre-β-HDL), which is composed of apolipoprotein AI (apoA-1) and phospholipids^{83, 85, 86}. Lecithin cholesterol acyltransferase (LCAT) esterifies free cholesterol on pre-β-HDL into cholesteryl ester, which is then sequestered into the hydrophobic core of HDL⁸⁷. After picking up more cholesterol from peripheral cells, increased cholesteryl ester accumulation enlarges the HDL size and converts it into a mature HDL⁸⁷. Cholesteryl ester in the mature HDL is selectively taken up by liver cells through apoA-1-mediated binding to SR-B1 of hepatocytes⁸⁸. Cholesteryl ester in hepatocytes can be used to synthesize bile acids, and cholesterol and bile acids can be excreted into the bile. If cholesterol and bile acid are not reabsorbed in the intestine, they are eliminated into feces. This process is called reverse cholesterol transport⁸⁸. Even though LXR agonists can increase cholesterol efflux by upregulating ABCA1 and ABCG1 expression on intimal macrophages, they increase liver fat content resulting in a fatty liver disease, which limits the application of LXR agonists including free GW3965 in clinics. Iverson N et al. made a polymeric micelle, which surface amphiphilic macromolecules targeted to macrophage MSR1, resulting in less oxLDL binding and uptake by macrophages⁸⁹. They also encapsulated GW3965 into the micelles, resulting in decreased inflammation and increased cholesterol efflux in macrophages, which was correlated with increased expression of ABCA1, apoA-1 and LXRα⁸⁹. After administering them to Sprague Dawley rats with injured carotid arteries, they found significantly decreased intimal cholesterol content, and inhibited macrophage retention in the inflamed lesion⁸⁹. Another research group encapsulated GW3965 into poly(lactide-co-glycolide)-b-poly(ethylene glycol) (PLGA-b-PEG) nanoparticles⁹⁰. Nanoencapsulated GW3965 had does advantage in inhibiting inflammatory factor expression in macrophages both in vitro and in vivo. After intravenous injection of those GW3965-encapsulated PLGA-b-PEG nanoparticles into LDLr−/− mice for 2 weeks, the macrophage content in atherosclerotic lesions was dramatically decreased, but liver fat content and blood lipid profile were not changed. Therefore, nanoencapsulation decreased the side effects of free GW3965, and enhanced its therapeutic efficacy⁹⁰.

Vascular Endothelial Cells

Endothelium is a continuous monolayer lining in the blood vessel wall⁹¹. The activation and dysfunction of endothelial cells can be triggered by oxidative stress, dyslipidemia, viral or bacterial infection, inflammation, turbulent blood flow and low shear stress, amongothers^{92, 93}. The dysfunctional endothelial cells impact leukocyte adhesion and recruitment, platelet activation, and thrombus formation^{91, 94}. Endothelium-targeted nanoparticles in combination of medical imaging modalities including MRI, PET, and multiple-row detector computed tomography (MDCT) have been developed to visualize atherosclerotic endothelium wall structures and activities^{39, 95}. Those nanoparticles can also prevent or treat atherosclerosis via targeted delivery of preventive or therapeutic agents to the activated or dysfunctional endothelial cells^{94, 96} (Table 3).

Table 3.

Detection and treatment of atherosclerosis using endothelial cell- and angiogensis-targeted nanoparticles

Molecular/ functional target	Nanoparticles	Imaging platforms	Animal model/patients, dose and administration route	Results	Year and reference
VCAM-1	Plant viral nanoparticle with tobacco mosaic virus (VCAM-TMV)	MRI	ApoE−/− mice,10 mg/kg VCAM- TMV and PEG- TMV; I.V.	VCAM-TMV targeted atherosclerotic lesions and increased the detection limitation of MRI.	2014182
VCAM-1	PEG-USPIO-VCAM- 1 peptide nanoparticles with P03011 as a contrast agent	MRI and surface- enhanced coherent anti- Stokes Raman scattering (SECARS)	ApoE−/− mice; in vivo MRI measurements injected with contrast agent P03011 and control PEG- USPIO; ex vivo SECARS conducted on sections of aortic root.	SECARS microscopy and high magnetic field MRI combined with nanoencapsulated P03011 as a contrast agent improved visualization of atherosclerotic lesions.	201238
VCAM-1 and apoptosis	USPIOs-R832 ( VCAM-1 targeting) USPIO-R826 (apoptosis targeting)	MRI	ApoE−/− mice; Fe 0.1 mmol /kg USPIO-R832 and USPIO- R826; I.V.	Targeting apoptosis and VCAM-1 could be achieved in 30 minutes after treatment.	201299
Transmembr ane protein: stabilin-2	Peptide CRTLTVRKC (S2P)-HGC-Cy5.5 nanoparticles (S2P- HGC-Cy5.5-NP)	Fluorescence molecular imaging	ApoE−/− mice;10 mg/kg S2P–HGC- Cy5.5-NP; I.V.	S2P–HGC-Cy5.5-NP specifically targeted stabilin-2 expressed on endothelial cells.	2011103
VCAM-1	VCAM-1 internalizing peptide-28 (VHPKQHR) nanoparticles (VINP28-NPs )	High resolution MRI	ApoE−/−mice; I.V.	Detection of endothelial activation and damage was achieved by VINP28-NPs- enhanced ex vivo MRI.	2007109
αvβ3- integrin	αvβ3-fumagillin nanoparticles	MRI	Rabbits, αvβ3- Fumagillin NP 30 µg/kg	Angiogenesis was inhibited by αvβ3- fumagillin-nanoparticles in rabbits.	2006183
VCAM-1	VCAM-1 internalizing peptide-28 (VHPKQHR) nanoparticles (VINP28-NPs )	MRI	Murine cardiac endothelial cells	VINP28 modified multimodal nanoparticles had a high binding affinity to endothelial cells expressing VCAM-1, but not smooth muscle cells and macrophages.	2006102
VCAM-1	VCAM-1 internalizing peptide-28 (VHPKQHR) nanoparticles (VINP28-NPs )	MRI and optical imaging	ApoE−/− mice; 5 nmol/L fluorochrome of VINP28-NPs; I.V.	VINP28-NPs detected endothelial cells and macrophages by targeting VCAM-1; VINP28-NPs enabled spatial monitoring of anti–VCAM-1 pharmacotherapy in vivo.	2006101
VCAM-1	Peptide VHSPNKK- modified magnetofluorescent nanoparticles (VNP)	MRI	ApoE−/− mice; 5 ng/50 µL mTNF- α to induce inflammatory model;10 nmol/L VCAM-1 peptide, VNP or control nanoparticles.	VNP detected VCAM-1 expressed endothelial cells; VNP were co-localized with VCAM-1 expressed cells in atherosclerotic lesions in apoE−/− mice.	2005100

Notes:

MCP-1, monocyte chemoattractant protein-1

IL-8, interleukin 8

VCAM-1, endothelial vascular adhesion molecule-1

MRI, magnetic resonance imaging

I.V., intravenous injection

BAECs, bovine aortic endothelial cells

TMV, tobacco mosaic virus

AP, atherosclerotic plaque-homing peptide

HGC, hydrophobically modified glycol chitosan

Cy5.5, near-infrared (IR) fluorescence dye

HA, hyaluronan

USPIOs, ultrasmall superparamagnetic iron oxide

PEG, polyethylene glycol

SECARS, surface-enhanced coherent anti-Stokes Raman scattering

FMT, fluorescence molecular tomography

PET, positron emission tomography

SPECT, single photon emission computed tomography

Adhesion molecules contribute to recruitment of inflammatory monocytes into the intimal layer where they differentiate into macrophages, and transform into lipid-laden foam cells, which features the early stage of atherosclerosis. Vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), P- and E-selectin are major adhesion molecules expressed on endothelial cells⁹⁷. VCAM-1 expression is increased on endothelial cells in both early and advanced atherosclerotic lesions, but it is also expressed on activated macrophages and smooth muscle cells⁹⁸, VCAM-1 is a potential marker for vascular inflammation and dysfunctional endothelial cells. Tsourkas A et al. conjugated anti-VCAM-1 antibodies on the magneto-optical nanoparticles⁹⁹. The VCAM-1-targeted nanoparticles could detect VCAM-1 expression on the endothelial cells, and label the activated endothelium⁹⁹. Non-targeted nanoparticles had low target specificity to the endothelium⁹⁹. Many VCAM-1 targeting peptides have been selected using the phage display or other approaches^{100, 101}. VHSPNKK-modified nanoparticles had 12-fold higher binding affinity to VCAM-1 than VCAM-1 antibodies¹⁰⁰. Importantly, they had low binding affinity to macrophages¹⁰⁰. The same research group identified another peptide VHPKQHR, which was used to develop VCAM-1 internalizing nanoparticles (VINP-28)¹⁰¹. In vitro experiments revealed a 20-fold higher cellular binding and internalization of VINP-28 by VCAM-1 expressing cells than the previous nanoparticles¹⁰¹. VINP-28 had high binding affinity to endothelial cells, but low binding affinity to macrophages and smooth muscle cells¹⁰². After intravenous injection into apoE−/− mice, VINP-28 co-localized with endothelial cells in atherosclerotic lesions, and they detected decreases in VCAM-1 expression in the aortic root in statin-treated mice¹⁰¹. VINP-28 also detected endothelial cells and other VCAM-1 expression cells in resected human carotid artery lesion ex vivo¹⁰¹. Other VCAM-1 ligands have been conjugated to nanoparticles for imaging endothelial cells^{38, 39}. Beside VCAM-1, ICAM-1, selectins, stabilin-2, interleukine-4 receptor and other membrane proteins on activated or dysfunctional endothelial cells have been used as targets for designing endothelium-targeted nanoparticles^{39, 103, 104}.

After intravenous administration, nanoparticles contact endothelial cells of the blood vessel wall. The effects of nanoparticle exposure on endothelium structure, function, activity are gaining considerable attentions. It is crucial to understand endothelial cell functional changes and toxicity and underlying mechanisms upon nanoparticle exposure. Many metal nanoparticles including cobalt, titanium oxide¹⁰⁵, silica¹⁰⁶, zinc oxide¹⁰⁷ and iron oxide¹⁰⁸ nanoparticles significantly upregulated the expression of MCP-1, IL-8 and adhesion molecules including ICAM-1, VCAM-1 and E-selectin on endothelial cells, which can increase endothelial inflammatory responses, result in endothelial activation and dysfunction, and induce atherosclerosis development^{108, 109}. Superparamagnetic iron oxide nanoparticles change endothelial cell morphology by dramatically increasing intracellular reactive oxygen species concentrations¹¹⁰. These results suggest that some metal nanoparticles could potentially enhance endothelial inflammation and atherosclerosis.

Angiogenesis

Neovascularization is a key feature of atherosclerosis development¹¹¹. New microvessels developed in vasa vasorum, the adventitial layer, nurture the cells in atherosclerotic lesions, contribute to the lesion progression, and play an important role in lesion destabilization and rupture^111–113. Integrin is composed of two transmembrane subunits (α and β) via noncovalent bonds, and plays an important role in interaction of cell to cell, and cell to extracellular matrix¹¹⁴. The α_vβ₃ integrin is widely expressed by monocytes, endothelial cells, vascular smooth muscle cells, and fibroblasts, and it involves in the regulation of many intracellular signaling pathways to modulate cell migration, recruitment and invasion during angiogenesis^115–117. The α_vβ₃ integrin is upregulated in those cells, especially endothelial cells, when they are induced by the angiogenic stimuli¹¹². Therefore, it becomes a common target for imaging neoangiogenesis (Table 3).

Winter et al. has developed an α_vβ₃ integrin-targeted paramagnetic nanoparticles. After intravenous injection of those nanoparticles to New Zealand White rabbits fed with high cholesterol diet, nanoparticles targeted new angiogenic vessels and detected neoangiogenesis in the early-stage of atherosclerotic lesions²⁴. This group later developed theranostic nanoparticles, the previous α_vβ₃ integrin-targeted paramagnetic nanoparticles carrying fumagillin and atorvastatin¹¹⁸. Fumagillin can inhibit blood vessel formation¹¹⁹. Atorvastatin (Lipitor), a type of statin drugs, can decrease cholesterol biosynthesis via inhibiting the key enzyme, 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase)¹²⁰. The theranostic nanoparticles allowed them to treat and visualize the improvement of atherosclerosis simultaneously. After the nanoparticles was administered to hyperlipidemic rabbits, the α_vβ₃ integrin-targeted fumagillin nanoparticles significantly decreased the neovascular signals by more than 50%, while the α_vβ₃ integrin-targeted fumagillin and atorvastatin nanoparticles exhibited higher and sustainable antianogenic effects¹¹⁸. The α_vβ₃ integrin-targeted nanoparticles can also be used for evaluating anti-angiogenic therapeutic responses in patients with the peripheral vascular disease⁴³.

Proteolysis, Apoptosis and Thrombosis

Proteases, mainly capsineses and matrix metalloproteinases (MMPs), are excreted from intimal macrophages and foam cells¹²¹. Increased expression of MMPs is associated with decreased thickness of the fibrous cap and increased lesion vulnerability. MMPs expression is induced by inflammatory factors, such as IL-1β and TNF-α. Hence, it is a functional marker of active inflammation and lesion vulnerability in atherosclerotic lesions⁶. Schellenberger E et al. synthesized a protease-specific iron oxide nanosensor that can berapidly switched to a high-relaxivity aggregated particle from a stable low-relaxivity stealth state after cleaved by proteases like MMP9¹²². The nanoparticles detected MMP9 activity in vitro. Nahrendorf M et al. synthesized protease-specific polymeric nanosensors, and these polymers were cleavable by proteases¹²³. After administering them to apoE−/− mice, they imaged the mice using combined fluorescence molecular tomography (FMT) and CT. Results indicated that these nanoparticles imaged protease activity in the atherosclerotic lesions, and robustly detected the therapeutic effects of the anti-inflammatory drug¹²³ (Table 4).

Table 4.

Detection and treatment of atherosclerosis using targeted nanoparticles via proteolysis, apoptosis and thrombosis processes

Molecular/ functional target	Nanoparticles	Imaging platforms	Animal model, dose and administration route	Results	Year and reference
Activated platelets	Versatile ultrasmall super paramagnetic iron oxide (VUSPIO) nanoparticles with recombinant human IgG4 antibody	MRI	In vitro human platelets; ex vivo human aorta samples; ApoE −/− mice; 10 µg/mL of antibody; I.V.	Nanoparticles bound to activated platelets.	2015136
Fibrin	Fibrin-binding peptides conjugated iron oxide nanoparticle–micelles (FibPep-ION- Micelles)	Magnetic particle imaging (MPI)	Human clots incubated with FibPep-ION- Micelles in vitro	Nanoparticles bound to the blood clot and gave a high signal.	2013134
Collapse of mitochondria l membrane potential during apoptosis	Synthetic biodegradable HDL- nanoparticles quantum dots	Optical imaging- fluorescence microscopy	RAW 264.7 macrophages, Male Sprague– Dawley rats; I.V.	Both in vivo and in vitro data revealed the increased detection of apoptotic cells. Synthetic biodegradable HDL- nanoparticles quantum dots targeted the collapse of mitochondrial membrane potential during apoptosis.	2013129
Activated platelets	Versatile ultrasmall superparamagnetic iron oxide (VUSPIO) particles, coupled to an anti-human P- selectin antibody (VH10)	MRI	Human platelets and endothelial cells; ApoE−/− mice. I.V.	VH10 bound to human activated platelets and endothelial cells.	2011135
Phosphatidyl serine on apoptotic cells and macrophages	Annexin A5- conjugated micellar nanoparticles carrying multiple Gd- labeled lipids and fluorescent lipids	MRI Fluorescence imaging	In vitro T- lymphoma cell line (Jurkat cells) at a concentration of 1 mM total lipid of nanoparticles; ApoE−/− mice, 2.5 µmol lipids per mouse; I.V.	Annexin A5-conjugated micellar nanoparticles targeted to apoptotic cells, and they detected PS-presenting cells in atherosclerotic lesions in apoE−/− mice.	2010128
Fibrin	Clot-binding peptide cysteine-arginine- glutamic acid-lysine- alanine (CREKA) bound micelles loaded with an anticoagulant drug hirulog	Fluorescence imaging	ApoE−/− mice; 100 µL of 1 mM micelles; I.V.	Micelles bound to unstable lesions in apoE−/− mice; Hirulog-loaded micelles had high hirulog concentrations and anti- thrombin activity in lesions.	2009133
Protease	Polymeric nanoparticles containing a protease sensor	Fluorescence molecular tomography (FMT) and CT	ApoE−/− mice; 5 nmol of protease sensor; I.V.	High signal intensity of nanoparticles was detected in the artery wall.	2009123
Phosphatidyl serine on apoptotic cells	Annexin V conjugated superparmagnetic iron oxide particles (SPIONs)	MRI	Watanabe heritable hyperlipidemic rabbits; SPIONs containing 0.05 mg of iron, I.V.	Annexin V conjugated SPIONs targeted to atherosclerotic lesions, but not healthy blood vessels.	2007127
Fibrin	Fibrin targeted (via anti-fibrin antibodies) Gd-DTPA-PE nanoparticles	MRI	Blood clots incubation	Gd-DTPA-PE nanoparticles modified with anti-fibrin antibodies had a high binding affinity to thrombi.	2003131

Notes:

ApoE−/−, apolipoprotein E null

CD44, cell surface adhesion molecule

Cy5.5, near-infrared (IR) fluorescence dye

CT, computed tomography

DTPA, diethylene-triamine-pentaacetic acid

Fe, iron

Gd, gadolinium

HDL, high-density lipoprotein

I.V., intravenous injection

MBq, megabecquerel as unit of radioactivity

MRI, magnetic resonance imaging

PE, phosphatidylethanolamine

PEG, polyethylene glycol

SPIONs , superparmagnetic iron oxide particles

Foam cells can die from apoptosis, a programmed cell death. Phosphatidylserine is located in the inner leaflet of the cell membrane in normal and healthy cells, but it is translocated to the outer leaflet of the cell membrane in apoptotic cells¹²⁴. It has been used as a target to detect apoptotic cells in atherosclerotic lesions. Annexin A5 (Annexin V) is a 36 kDa protein with high binding affinity to phosphatidylserine¹²⁵. Technetium-99m–labeled annexin A5 successfully detected apoptotic cells in atherosclerotic lesions in 11 human subjects using SPECT, and this modality may open the door to the detection of lesion vulnerability and to identify high risk patients¹²⁶. Superparmagnetic iron oxide particles (SPIONs) conjugated with annexin A5 targeted to apoptotic foamy macrophages in atherosclerotic lesions of Watanabe heritable hyperlipidemic rabbits, and their target specificity was much higher than non-targeted SPIONs¹²⁷. Annexin A5-conjugated micelles also targeted to apoptotic cells in atherosclerotic lesions of apoE−/− mice, and the targeted micelles had more than 100-fold dose advantage than non-targeted micelles¹²⁸. Apoptotic cells have mitochondrial membrane potential collapse. In another study, synthetic HDL nanoparticles carrying quantum dots were decorated with apoA1 and triphenylphosphonium (TPP) cations, which were used for detecting mitochondrial membrane potential collapse and identifying apoptotic cells ¹²⁹.

Thrombus formation and its subsequent blockage of blood circulation cause most of myocardial infarction or stroke. Thrombosis is the formation of a blood clot after activation of platelets and the clotting cascade¹³⁰. Fibrin, platelets, erythrocytes, and leukocytes are major components of thrombi¹³⁰. Many fibrin-targeted nanoparticles have been developed to detect thrombi by modifying the surface of nanoparticles with fibrin antibodies or binding peptides^{21, 131–134}. Peter D et al. loaded anticoagulant drug hirulog into the fibrin-targeted micelles. The targeted micelles increased hirulog concentrations in the rupture-prone lesion areas and significantly decreased thrombin activity in the lesions¹³³. Platelet-targeted nanoparticles were also developed by conjugating platelet antibodies on their surface, which may have a potential to inhibit thrombus formation via decreasing platelet activities^{135, 136}.

LIPID LOWERING AND ANTI-INFLAMMATORY THERAPY

Lipoprotein-mediated Treatment

LDL, the cholesterol-rich lipoproteins, are derived from very low-density lipoproteins (VLDL). VLDL are triglyceride-rich lipoproteins. Triglyceride in VLDL is hydrolyzed by lipases and removed, making VLDL to turn into intermediate-density lipoproteins (IDL), which are in turn converted to LDL after triglyceride hydrolysis and removal. LDL can deposit cholesterol to peripheral tissues including the blood vessel wall. LDL can be taken up by the liver via binding to LDL receptor and LDL receptor-related protein (LRP) completing a process called the endogenous pathway of lipoprotein transport. ApoB100 is a signature apolipoprotein on VLDL, IDL and LDL, and is required for assembling VLDL in the liver. Decreasing apoB100 expression in the liver can reduce VLDL production, further decrease circulating LDL particle concentrations. ApoB-specific siRNA has been encapsulated into liposomes¹³⁷. After intravenous administration of those liposomes into cynomolgus monkeys, they found significantly decreased liver apoB gene expression, lower serum concentrations of apoB100, total cholesterol and LDL-cholesterol in those non-human primates. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secretory serine protease, and this enzyme can bind to LDL receptor to prevent it from being recycled back to the cell surface, and thus enhancing LDL receptor destruction in the cells, especially hepatocytes¹³⁸. Decreased liver LDL receptor levels are associated with increased circulating LDL-cholesterol concentrations. Mutation or decreased expression of PCSK9 correlates with lowered circulating LDL-cholesterol concentrations, and has vascular benefits¹³⁹. Intravenous administration of the PCSK9 siRNA-loaded nanoparticles into different animal models including mouse, rat, non-human primate decreased levels of PCSK9 transcripts in the liver¹⁴⁰. These nanoparticles also lowered plasma concentrations of PCSK9 protein and LDL-cholesterol, but had little effect on plasma concentrations of HDL-cholesterol and triglyceride¹⁴⁰.

HDL pick up cholesterol from intimal macrophages and other peripheral cells, and send it back to the liver for cholesterol elimination completing a process termed reverse cholesterol transport^{141, 142}. ApoA1 is a signature apolipoprotein on HDL. Increased circulating HDL or apoA1 concentrations correlate with decreased risks of developing atherosclerosis¹⁴³. Many rHDL or HDL-mimic nanoparticles are developed by using lipids and apoA-1 or its derived peptides¹⁴⁴ (Table 5). ApoA1_milano, a molecular variant of apoA-1, has many cardiovascular benefits including anti-atherogenic, anti-thrombotic, anti-platelet effects. Kaul S et al. made reconstituted HDL nanoparticles (rHDL) using ApoA1_milano and phospholipid complex¹⁴⁵. After intravenous administration of those nanoparticles into apoE−/− mice, the aortic cholesterol content was decreased, and the function of endothelial cells was improved¹⁴⁵. Luthi AJ et al. made a functional mimic of HDL (fmHDL) using a gold nanoparticle coating with a phospholipid bilayer and apoA-I¹⁴⁶. They demonstrated that fmHDL accepted cholesterol from macrophages via ABCA1, ABCG1 and SR-B1¹⁴⁶. Direct administration of rHDL can increase reverse cholesterol transport and subsequently decrease atherosclerosis risk¹⁴⁷. Shaw JA et al. found that infusion of rHDL increased reverse cholesterol transport capacity, decreased macrophage number and lipid content in lesions, and reduced lesion volume in humans¹⁴⁸. Duivenvoorden R et al. intravenously administered statin-loaded rHDL to apoE−/− mice and found that these nanoparticles delivered statin to the atherosclerotic lesions, decreased macrophage content in the lesions, lowered lesion inflammatory response. One-week of high dose treatment significantly decreased inflammation in advanced lesions, while three-month low dose treatment inhibited lesion inflammation progression¹⁴⁹.

Anti-inflammatory Treatment

Atherosclerosis is a lipid-driven slowly progressing chronic inflammatory disorder of the arteries¹⁵⁰. Treatment of atherosclerosis is still mainly focused on lowering blood lipid concentrations, which partially reduces the risk for cardiovascular disease^{151, 152}. To further improve treatment of patients, targeting of inflammatory pathways is now believed to offer an additional benefit¹⁵³. Dexamethasone (DXM), an anti-inflammatory steroid drug, can inhibit atherosclerosis development via decreasing intimal macrophage recruitment and foam cell formation^{154, 155}. However, long-term administration of DXM has side effects including hypertension, weight gain and depression¹⁵⁶. Chono S et al made DXM-loaded liposomes with different particle sizes (70, 200 and 500 nm), and intravenously administered them into atherogenic mice¹⁵⁶. As compared to free DXM and liposomes with other sizes, L200 (DXM-loaded liposomes with the size of 200 nm in diameter) significantly decreased aortic cholesterol content, which correlated with increased aortic uptake of DXM. L200 had a potent dose advantage as indicated by higher anti-atherogenic effects at 55 µg/kg body weight than free DXM at 550 µg/kg body weight¹⁵⁶. Glucocorticoid is a potent anti-inflammatory steroid drug, and has been studied for atherosclerosis treatment¹⁵⁷. Due to its side effects and poor pharmacokinetic profile, glucocorticoid has not been used for atherosclerosis treatment in the clinic¹⁵⁸. After giving a single intravenous administration of glucocorticoid-loaded liposomes at dose of 15 mg/kg into a rabbits model with atherosclerosis, Lobatto ME found a significant decrease in inflammatory response at day 2, and this inhibitory effect lasted for additional 5 days¹⁵⁷. Importantly, the lowered inflammation correlated with decreased intimal macrophage content in the animals¹⁵⁷. This group also developed a good manufacturing practice (GMP)-grade prednisolone phosphate (PLP)-loaded liposomes (L-PLP)¹⁵⁹. Data from pharmacokinetics and toxicokinetics studies indicated that these liposomes had longer circulation half-life and less side effects than free PLP in rats¹⁵⁹. Intravenous administration of these liposomes into hyperlipidemic New Zealand white rabbits decreased the inflammatory response in the artery wall¹⁵⁹. Van der Valk FM et al. intravenously administered L-PLP to patients with iliofemoral atherosclerosis¹⁶⁰. Compared to free PLP, L-PLP increased the drug’s half-life by 7- to 15-fold, which was partially contributed to its increased accumulation in atherosclerotic lesion macrophages¹⁶⁰. Although the long-circulating L-PLP have been successfully delivered to lesion macrophages, they did not decrease inflammatory responses in the artery walls of patients, who had atherosclerotic CVD¹⁶⁰. The inconsistency between animal studies and the human trial could be due to insufficient dose of L-PLP, or a short treatment duration in the human trial^{157, 159, 160}. Additionally, their effects on host defense in acute inflammatory situations are yet to be investigated¹⁶¹.

CONCLUDING REMARKS

Atherosclerosis is a silent, progressive disease, and it cannot be easily detected by the current imaging methods at its early stage. Current therapeutic approaches treat atherosclerosis systemically, not locally, which is often associated with decreased efficacy and increased side effects. Nanoparticle-mediated, targeted delivery of diagnostic agents or therapeutic compounds to specific molecules, cells, or tissues represents an innovative approach for the diagnosis and treatment of atherosclerosis. Nanoencapsulation in combination with targeted delivery may enhance stability and bioavailability of agents and drugs, improve their pharmacokinetics, increase detection sensitivity and therapeutic efficacy, and decrease unintended effects directed to the normal tissues. However, it should be pointed that the efficacy of nanoparticles is largely proved in the in vitro and animal model studies, and their movement to clinical phases still faces substantial challenges. Future studies are expected to not only address the translational value, but also further elucidate the working modes for more specifically targeted application. Another emerging direction is to develop multifunctional nanoparticles allowing multimodal imaging and targeted delivery of the therapeutic compounds, which are expected to have broader clinical application. Despite being still in the early stage, the steady progress has been made in both basic research and application study in the field, which makes the diagnostic and therapeutic values of nanoparticle technology in atherosclerosis increasingly promising. We are optimistic in anticipating more breakthroughs to come along in a near future.