Employment of Targeted Nanoparticles for Imaging of Cellular Processes in Cardiovascular Disease

Abstract

Cardiovascular disease (CVD) is a leading cause of global mortality, accounting for pathologies that are primarily of atherosclerotic origin and driven by specific cell populations. A need exists for effective, non-invasive methods to assess the risk of potentially fatal major adverse cardiovascular events (MACE) before occurrence and to monitor post-interventional outcomes such as tissue regeneration. Molecular imaging has widespread applications in CVD diagnostic assessment, through modalities including magnetic resonance imaging (MRI), positron emission tomography (PET), and acoustic imaging methods. However, current gold-standard small molecule contrast agents are not cell-specific, relying on non-specific uptake to facilitate imaging of biologic processes. Nanomaterials can be engineered for targeted delivery to specific cell populations, and several nanomaterial systems have been developed for pre-clinical molecular imaging. Here, we review recent advances in nanoparticle-mediated approaches for imaging of cellular processes in cardiovascular disease, focusing on efforts to detect inflammation, assess lipid accumulation, and monitor tissue regeneration.

Introduction

Cardiovascular disease (CVD) is the primary cause of global mortality, accounting for an estimated 17.8 million deaths annually worldwide¹. CVD affects the heart and blood vessels and the vast majority of pathologies associated with it are of atherosclerotic origin. If unaddressed, these pathologies can lead to a potentially fatal major adverse cardiovascular event (MACE) involving hypoxic injury to cardiac tissue². Currently, there is a lack of non-invasive methods to determine risk of MACEs before they occur or to assess outcomes such as tissue regeneration following injury². Therefore, there is a need for non-invasive methods to determine this risk before occurrence and monitor tissue regeneration. Due to their critical roles in the pathology and treatment of CVD, specific single cell-mediated processes have emerged as key targets for diagnostic and therapeutic strategies, including vascular inflammation and lipid accumulation in atherosclerosis mediated by phagocytic immune cells³, as well as cardiac tissue regeneration via administration of stem cells⁴. Non-invasive assessment of these cellular processes would allow for better detection and assessment of CVD progression and therapeutic outcomes.

Molecular imaging has high utility in CVD diagnostics, allowing for the non-invasive detection, tracing, and quantification of single cell populations. Modalities for molecular imaging include magnetic resonance imaging (MRI), positron emission tomography (PET), acoustic imaging methods, and others. These methods differ in their spatial resolution, detection sensitivity, and imaging depth⁵. Of these, MRI provides numerous advantages including high imaging depth, spatial resolution, and soft tissue contrast, while being limited by low sensitivity⁵. Conversely, PET imaging offers high sensitivity and imaging depth, but is limited by lack of anatomic information⁶. Finally, acoustic imaging modalities including ultrasound (US) and photoacoustic imaging (PAI) have good clinical translatability and detection sensitivity, but relatively low penetration depth⁶. Although these imaging modalities can function independently, they are often used in strategic combinations for improved or custom application. Such multi-modal imaging approaches leverage the sensitivity and resolution of each technique⁷. For detection of specific biologic processes, such as inflammation or cell migration, the use of exogenous contrast agents capable of detecting or labeling the cells of interest is usually required. Current gold-standard small molecule contrast agents, such as fluorodeoxyglucose (FDG) for PET imaging, are not cell-specific and rely on other strategies such as increased cell metabolism to enhance uptake⁶.

Nanomaterial-based strategies have been of great interest in recent years for contrast agent delivery due to their targeted accumulation in specific cells and tissues, particularly in immune cells⁸. Several nanomaterial systems exist for pre-clinical imaging, including metallic nanoparticles⁹, amphiphilic polymers¹⁰, solid core polymeric nanoparticles¹¹, and lipid-based nanoparticles¹². The molecular imaging signal provided by these nanoparticles arises from either their composition, as in the case of metallic nanoparticles like iron oxide nanoparticles (IONPs) for MRI¹³, or through encapsulation or conjugation of small molecules including Technetium-99 (⁹⁹Tc) for PET¹⁴. Single cell targeting via nanoparticles can be accomplished through numerous methods such as the use of targeting moieties like antibodies or peptides¹⁵, and ex vivo labeling¹⁶. The use of such nanoparticle platforms towards diagnostic, therapeutic, or combined (theranostic) applications in cardiovascular disease have been reviewed previously¹⁷, with some reviews focusing on a specific imaging modality^{18, 19} or nanoparticle design²⁰. However, with the rapid progression of pre-clinical research in the field of cardiovascular imaging and the emerging developments in nanoparticle targeting to specific cell populations, an updated overview of the field is of interest. Here, we review the latest breakthroughs in assessment of cellular processes in CVD via nanoparticle-mediated molecular imaging, with a focus on detecting inflammation, assessing lipid accumulation, and monitoring tissue regeneration (Figure 1).

Figure 1.

Schematic representation of strategies to utilize nanomaterials for imaging cellular processes in CVD. Assessing inflammation and lipid accumulation, and monitoring tissue regeneration are 3 key areas of recent advances. Specific references for the depicted nanoparticles and targeting strategies are listed in Table 1. HFn = heavy chain ferritin; HDL = high density lipoproteins; OPN = osteopontin; IONP = iron oxide nanoparticle; CPP = cell-penetrating peptide.

Targeting Macrophage-derived Vascular Inflammation

Cell-mediated inflammation is known to be a key factor in atherosclerosis, contributing to plaque instability and risk of rupture³. Targeting and assessing the immune cell burden within plaques using nanomaterials is a promising strategy for assessing plaque burden and development. While plaques contain a complex mixture of immune cells that include dendritic cells and T cells, macrophages have emerged as the primary cell of interest for molecular imaging-based plaque detection due to their major role in plaque progression²¹.

For this purpose, diverse ligands established for targeting macrophages have been utilized to decorate nanoparticles, including antibodies against markers of monocyte/macrophage lineage. Tarin et al. demonstrated macrophage targeting specificity of anti-CD163 and gold-coated iron oxide nanoparticles (NP-CD163) over non-targeted, IgG-coated control nanoparticles (NP-IgG), and found that NP-CD163 were more effectively able to target and allow T2-MRI visualization of plaques²². Similarly, anti-CD68 antibody-mediated targeting was shown to be effective at targeting Fe-doped hollow silica nanospheres (Fe-HSNs) to plaque macrophages (Figure 2). The multifunctional Fe-HSNs allowed for real-time plaque monitoring via US and follow-up examination of plaque macrophages using the 3D spatial resolution of MRI, indicating a robust tool for real-time, single-cell imaging in CVD²³.

Figure 2.

Fe-doped hollow silica nanospheres conjugated with anti-CD68 antibody for macrophage burden detection in atherosclerotic plaques via US and MRI²³. a) Schematic and transmission electron microscopy (TEM) of CD68-targeting, Fe-doped hollow silica nanospheres (CD68-Fe-HSNs) in macrophages. b) Illustration and images of CD8-Fe-HSN uptake by plaque macrophages and detection via contrast-enhanced ultrasound (CEUS), MRI, TEM, and immunofluorescence²³.

Other approaches have leveraged the overexpression of specific cell-surface markers by plaque macrophages as a targeting strategy. Scavenger receptor A (SR-A) has emerged as a plaque macrophage target, as it has been found to be overexpressed in atherosclerosis-activated macrophages and facilitates their uptake of oxidized LDL²⁴. With this rationale, a unique theranostic nanoparticle was developed by Ye et al. using dextran sulfate (DS)-labeled, chitosan-PLGA-iron oxide-perfluorohexane (PFH) nanoparticles to plaque macrophages via SR-A. The iron oxide facilitated MRI imaging to visualize plaques, while the loaded PFH allowed for subsequent ablation and apoptosis induction in targeted plaque macrophages via low intensity focused ultrasound (LIFU). This application demonstrated the importance of theranostics but will require further investigation into potential post-ablation consequences, such as unwanted plaque destabilization and rupture. A different, less specific approach was taken by Zheng et al., in which near infrared fluorescence (NIRF)-detectable, DNA-coated iron oxide nanoparticles (DNA-IONPs) targeted macrophages via binding to SR-A and more efficiently localized to aortic plaques than PEG-coated IONPs²⁵. However, the DNA-IONPs were also found to have high uptake into liver and splenic macrophages and dendritic cells, indicating that additional targeting agents may be necessary for plaque macrophage specificity. Another highly expressed receptor in infiltrated plaque macrophages is transferrin receptor 1 (TfR1). Liang et al. developed a PET- detectable nanomaterial that labeled the targeting compound heavy-chain ferritin with ^99mTc to enhance association with TfR1-positive plaque macrophages²⁶. This nanoconstruct produced a PET signal with increased strength, specificity, and diagnostic power relative to small molecule contrast agents that are currently proposed as gold standards for PET detection of plaque burden via passive targeting.

To specifically assess more vulnerable plaques, recent strategies have focused on identifying apoptotic macrophages in plaques via Annexin V-mediated targeting. Li et al. developed a single-photon emission computerized tomography (SPECT)/CT active construct of ^99mTc- and Annexin V-tagged gold nanoparticles (GNP) that localized signal to actively apoptotic cells prominent in atherosclerotic lesions in ApoE^−/− mice²⁷. Similar work from this group has demonstrated the use of ^99mTc- and Annexin V-tagged IONPs for SPECT/MRI-based assessment²⁸. By targeting such specific functional markers and employing multi-modality imaging, these strategies minimize the theoretical dose of solid metal nanoparticle contrast agents needed to effectively detect high-risk plaques.

Other inflammation-targeted strategies for CVD imaging employ nanoparticle composition for cell selectivity, utilizing natural nanoparticles such as high-density lipoproteins (HDL). Several studies have used HDL-mediated targeting to access macrophages in plaques. One notable study was conducted by Perez-Medina through the ⁸⁹Zr-labeling of HDL for PET-based assessment of the plaque²⁹. The ⁸⁹Zr-HDL nanoparticles showed a strong PET signal associated with damaged vessel walls in murine, rabbit, and porcine models, with enhanced uptake into plaque monocytes and macrophages. Subsequent work has built upon this HDL-mediated plaque targeting platform. Notably, Binderup et al. leveraged the use of MRI, PET, CT, and NIRF to assess the use of the ⁸⁹Zr-HDL nanoparticles to load and deliver simvastatin for atherosclerosis immunotherapy in rabbit and porcine models, demonstrating the efficacy of their treatment at targeting and inhibiting inflammatory macrophages in atherosclerotic plaques³⁰.

Targeting Non-Macrophage-Derived Inflammation

Aside from atherosclerosis, other areas of CVD are also marked by cell-mediated inflammation. Acute rejection (AR) following heart transplantation is a key complication and cause of graft disability and is known to be mediated by T lymphocyte infiltration³¹. As an alternative to the invasive, current gold standard of endomyocardial biopsy to detect AR, US-visible anti-CD3-labeled nanobubbles have been employed to detect AR after cardiac transplantation in vivo³². Targeted nanobubbles were able to noninvasively assess T lymphocyte infiltration in studies comparing isograft and allograft transplantation³².

Thrombosis is another CVD complication driven by single cell components (or in this case fragments), namely activated platelets, and can lead to severe complications if not detected. Bonnard et al. recently developed low-fouling, biodegradable NIRF stealth nanoparticles for noninvasive molecular imaging of thrombosis³³. The particles were functionalized with a single-chain variable fragment antibody specific to the activated form of the glycoprotein IIb/IIIa (anti—GPIIb/IIIa-scFv), the most prominent marker expressed by platelets upon activation. The functionalized particles facilitated targeting and non-invasive visualization of rat carotid thromboses. Fucoidan labeling of nanoparticles is another strategy used to achieve activated platelet targeting via binding to P-selectin, and was used by Li et al. to deliver fucoidanfunctionalized microbubbles (Fucoidan-MBs) for selective targeting and visualization of thrombi via US imaging³⁴. Interestingly, the authors showed that these microbubbles could also be burst using destructive US pulses, which they suggested could be used to trigger drug release in follow up studies as a theranostic approach. Other nanoparticle-based approaches to non-invasively visualize thromboses have focused on targeting non-cellular components such as fibrin or thrombin or responding to the microenvironment around a thrombus, and are summarized elsewhere³⁵.

Finally, some approaches have aimed to develop nanoparticles for imaging that simultaneously target multiple inflammatory cells within atherosclerotic plaques, in addition to macrophages. Using an optimized antagonist to the integrin α4β1, which is a non-RGD binding integrin expressed on multiple immune cell types, Woodside et al. used Gd-loaded liposomes to target and non-invasively detect atheromas of ApoE^−/− mice via MRI³⁶. The rationale for this approach was to analyze the entire inflammatory cell burden within plaques. The authors discovered that their construct localized to mainly monocytes and macrophages in the subendothelium, along with neutrophils, with no uptake into nearby endothelial cells or cardiomyocytes.

Visualizing lipid accumulation

Lipid accumulation is another major factor in the development of atherosclerosis. Lipoprotein retention, modification, and internalization by macrophages and vascular smooth muscle cells (VSMCs) in the vessel subendothelium contributes to the formation of foam cells. These foam cells participate in further immune cell recruitment and promote plaque progression³⁷. Targeting foam cells is therefore another strategy for assessing lipid accumulation and plaque burden. The overexpression of osteopontin (OPN) by both macrophage- and VSMC-derived foam cells was targeted by Li et al. to deliver dual-modality nanoparticles for detection of vulnerable atherosclerotic plaques³⁸. These nanoparticles were composed of a perfluorooctyl bromide (PFOB) core to provide US contrast, a polylactic acid (PLA) surfactant coating, and decorated with fluorescent dye Cy5.5 for optical imaging and anti-OPN antibody for foam cell targeting.

A theranostic approach was taken by Gao et al., who utilized NIR- and acoustic imaging-responsive, copper sulfide nanoparticles labeled with antibodies to TRPV1 cation channels (CuS-TRPV1) to target VSMCs and inhibit foam cell formation via binding to TRPV1³⁹. Upon IV injection, CuS-TRPV1 nanoparticles were found to successfully target plaque VSMCs and facilitate PA imaging to visualize plaques in ApoE −/− mice, as compared with non-targeted CuS nanoparticles. Following PA-mediated plaque visualization, CuS-TRPV1 nanoparticles could be photothermally activated via NIR light, activating the thermosensitive TRPV1 cation channel and triggering autophagy to control lipid metabolism. After repeated photothermal treatments, this approach achieved inhibition of VSCM-derived foam cell formation and mitigated plaque progression.

As an alternative strategy for foam cell targeting, Wei et al. utilized platelet-membrane coated, Gd-loaded PLGA nanoparticles (PNP) to detect vulnerable plaques via MRI. In this strategy, the platelet membrane coating was employed for targeting multiple molecular and cellular targets, including foam cells. The group demonstrated that PNPs could target foam cells along with activated endothelial cells and collagen, labeling this approach “multifactored biologic targeting.” Further studies demonstrated the ability of the PNPs to allow live MRI imaging and to target plaques in various stages of development in ApoE −/− mice⁴⁰. Targeting foam cells is a promising method for non-invasive detection of vulnerable plaques in need of further assessment.

Tracking tissue regeneration

A third, unique area of single cell-mediated activity in CVD is tissue regeneration. Following cardiac injury, endogenous regenerative mechanisms are not always sufficient to replace injured tissue. To address this, stem cell administration is of interest for replacing damaged myocardium, requiring a non-invasive method to assess cell migration and activity post-administration⁴. IONPs are well-established for cellular tracking as they can efficiently label stem cells without transfection agents and allow for non-invasive MRI monitoring. This approach was used by Hua et al. to track bone marrow mesenchymal stem cells (BMSCs) in rat models of myocardial infarction( MI)⁴¹. While the negative contrast signal of the BMSCs was significant one day post-administration, by day 21 the signal intensity had decreased and was indistinct from surrounding tissue. Unfortunately, this study did not present sufficient timepoints to provide insight into the signal variance between day 1 and day 21 post-administration. Other MRI-based cell tracking approaches such as ¹⁹F-MRI avoid this issue of low signal intensity, given that ¹⁹F has no background tissue signal. Perfluorocarbon nanoparticles (PFCE-NPs) suitable for ¹⁹F-MRI detection have been explored for labeling and tracking of stem cells. Constantinides et al. used the transfection agent FuGENE to facilitate labeling of two different murine cardiac progenitor cells with PFCE-NPs, allowing for in vivo tracking up to 8 days post-injection⁴². Other strategies to minimize background signal in cell tracking applications include photoacoustic (PA) imaging via semiconducting polymers (SP), which have specific and narrow PA spectra leading to low background signal. SPs-based PAI nanoprobes with cell-penetrating peptides as a surface modification have been developed to track the delivery and engraftment of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) in living mouse hearts for the treatment of ischemic heart disease⁴³ (Figure 3). The most effective approach to increase cell-tracking sensitivity while minimizing background signal may be through strategic combinations of imaging modalities. Lemaster et al. designed a trimodal contrast agent “nanobubble” for US, magnetic particle imaging (MPI), and photoacoustic imaging via assembly of PLGA, iron-oxide, and the fluorescent dye DiR⁴⁴. These nanobubbles were used to label and track cardiac stem cells following intramyocardial injection, and demonstrated high temporal resolution, imaging depth, and contrast due to the unique combination of modalities employed in the study.

Figure 3.

Semiconducting polymer (SP) based photoacoustic nanoparticles (PANP) for labeling and tracking of human embryonic stem cell-derived cardiomyocytes (hESC-CMs). a) Structure and b) TEM of SP PANPs. c) Brightfield microscopy of a sectioned slide of the heart to investigate hESC-Cm engraftment. d) PAI detection of engrafted hESC-CMs (red) in a fixed mouse heart. e) Detection of engrafted hESC-CMs (blue) in host myocardium via fluorescence imaging using intrinsic fluorescence of PANPs. f) Confocal microscopy of engrafted hESC-CMs within host myocardium. Both DAPI (blue) and cardiac troponin T (cTnT) (green) indicate host and engrafted cardiomyocytes. The engrafted hESC-CMs are distinguished via staining with human mitochondria antibody (purple) and via PANP fluorescence (red)⁴³.

Future directions and conclusions

Here, we have reviewed the recent use of nanomaterials for imaging of cellular processes in CVD, with a focus on monitoring and visualizing inflammation, lipid accumulation, and tissue regeneration. We found that while these recent studies employed a range of modalities, MRI and PET were by far the most commonly used. The minimal use of CT was surprising considering its widespread clinical utility. Studies mainly employed CT to provide anatomic context in combination with plaque detection via other modalities^{27, 30}. Delivery of nanoparticles to single cell targets was primarily achieved via specific antibodies to known cell-surface receptors, such as CD163²² or transferrin receptor-targeting²⁶ for targeting plaque macrophages, or through ex vivo labeling in the case of cell tracking. These studies illustrated how cell-selective active targeting is superior to current conventional molecular imaging contrast agents, primarily through enhanced uptake by and specificity for atherosclerotic plaques or other biologic processes of interest. Furthermore, active targeting of nanoparticles to specific cells known to correlate with vulnerable or early active plaques provides more powerful diagnostic assessment of plaque burden and risk stratification. In comparison, passive targeting strategies that mainly prolong diagnostic nanoparticle circulation time⁴⁵ and aim to enhance general plaque localization fail to provide details on cellular composition that provide further insight of the underlying pathology.

While these studies represent progress in this area, there are specific areas in need of improvement and future investigation. Most of the current strategies for assessing plaque burden through single cell targeting focus on targeting macrophages and monocytes. Although these cells play a major role in plaque development, other cells such as neutrophils and dendritic cells (DCs) are also known to facilitate atherogenesis and should be a focus of future non-invasive plaque assessment strategies⁴⁶. Furthermore, the inability to evade the mononuclear phagocyte system (MPS) resulting in clearance through the liver and spleen is a key limitation of many current approaches. Though the studies discussed here illustrate accumulation of intravenously injected nanoparticles in aortic plaques, those that assessed nanoparticle biodistribution found that the majority of the injected nanoparticles are cleared by the liver or spleen^{25, 29, 30}. For the purposes of CVD diagnosis and intervention, improved targeting methods will be required to enhance site- and cell-specific uptake, while lowering MPS clearance.

Emerging methods to achieve enhanced active targeting include combining nanoparticle shape-based targeting with receptor-targeted methods⁴⁷. Yi et al. demonstrated nanostructure enhanced targeting (NSET), wherein vesicular nanoparticles achieved significantly higher uptake by DCs within atherosclerotic lesions compared to smaller spherical micelles or high aspect ratio filamentous structures⁸. In subsequent work, they improved upon this NSET strategy by engineering the nanoparticle surfaces for optimal display of targeting peptides specific for DC surface receptors, demonstrating significantly higher selectivity when the two targeting methods were combined⁴⁸. A trimodal PET/CT/PA example of this dual-targeting approach was developed recently by Guo et al., who functionalized 2D Pd/Au nanosheets with ^125/131I and folic acid for targeting of activated macrophages⁴⁹. The 2D architecture was determined to be essential for enhanced targeting and allowed high uptake in aortic plaque macrophages in ApoE^−/− mice that correlated with disease progression and severity.

Other emerging strategies for active cellular targeting in CVD include smart or responsive nanomaterial probes that are detectable with molecular imaging only upon activation by a biological process. For example, instead of targeting specific cell-surface receptors, Ikeda et al. developed an activatable NIRF probe using iron oxide nanoparticles (IONP) conjugated with indocyanine green (IONP-ICG)⁵⁰. The self-quenched IONP-ICG could be activated only upon lysosomal degradation following internalization into macrophages via scavenger receptor-mediated uptake. Similarly, a responsive PAI nanoprobe containing two types of NIRF agents significantly enhanced absorbance at 765 nm and 680 nm in response to glutathione (GSH)/hydrogen peroxide (H₂O₂) redox in macrophages for early identification of rupture-prone plaques with high sensitivity⁵¹.

Finally, the field is currently building upon the use of nanomaterials for CVD diagnostic applications, such as detecting various stages of atherosclerosis by targeting unique cell populations and focusing more on theranostic approaches. In such cases, following initial plaque detection and assessment, a therapeutic functionality of the nanoparticle can be employed such as target cell ablation via LIFU²⁴ or reactive oxygen species (ROS) scavenging to limit inflammation⁵². While the majority of nanoparticle-based systems are still in pre-clinical studies, the field of nanomaterials is progressing rapidly. We expect that nanoparticles for diagnostic, therapeutic, and theranostic uses towards cellular processes in CVD have immense potential and will continue to increase in utility and start to transition to clinical studies in the years to come.

Table 1.

Recent nanoparticle-mediated approaches for single-cell imaging in CVD.

Application	Nanocarrier	Target cells/molecule	Targeting strategy	Modality	Model	Ref.
Inflammation	Gold-coated lONPs with anti-CD163 antibody	CD163+ macrophages	Anti-CD163 antibody	MRI	ApoE−/− mice	[22]
	Anti-CD68 receptor-targeted Fe-doped hollow silica nanoparticles (CD68-Fe-HSNs)	Macrophages	Anti-CD68 antibody	MRI + US	ApoE−/− mice	[23]
	IONP-PLGA-perfluorohexane-chitosan-dextran sulfate NPs	Activated macrophages	Dextran sulfate targeting to scavenger receptor A	MRI + US	ApoE−/− mice	[24]
	DNA oligonucleotide- and PEG-coated IONPs	Macrophages	Oligonucleotide binding to class A scavenger receptors	NIRF	ApoE−/− mice	[25]
	99mTc-HFn	TfR1-positive plaque macrophages	HFn/TfR1	PET	ApoE−/− mice	[26]
	99mTC-G NPs-Annexin V	Apoptotic macrophages	Annexin V/phosphatidylserine	SPECT + CT	ApoE−/− mice	[27]
	PEGylated + 99mTc-DTPA -IONPs	Apoptotic macrophages	Annexin V-labeling	MRI + SPECT	ApoE−/− mice	[28]
	89Zr-HDL	Macrophages and monocytes	HDL uptake	PET	ApoE−/− mice, double-balloon-injury-induced rabbit model, familial hypercholesterolemia porcine model	[29]
	89Zr- and DiO labelled HDL	Macrophages	HDL-mediated plaque targeting	MRI + PET + CT + NIRF	Double-balloon-injury-induced rabbit model, familial hypercholesterolemia porcine model	[30]
	Anti-CD3 antibody-labeled lipid nanobubbles	T lymphocytes	Anti-CD3 antibody	US	Heart transplant model using Brown Norway and Lewis rats	[32]
	NIR molecules and single-chain variable fragment antibody functionalized PASKE mesoporous silica nanoparticles	Activated platelets	Anti-glycoprotein IIb/IIIa single-chain antibody	NIRF	Carotid artery thrombosis mouse model (C57BLl6 mice)	[33]
	Fucoidan-functionalized microbubbles	Activated platelets	Fucoidan-mediated targeting fo P-selectin	US	FeCl3-induced thrombus rat model	[34]
	Liposomal Gd with α4β1-integrin targeting	Monocytes / Macrophages	α4β1-integrin antagonist (THI0567)	MRI	ApoE−/− mice	[36]
Lipid accumulation	Cy5.5-anti-OPN-PEG-PLA-PFOB	Foam cells	Osteopontin (OPN) antibody	US + NIRF	ApoE−/− mice	[38]
	Anti-TRPV1-labeled copper sulfide nanoparticles (CuS-TRPV1)	VSMCs and VSMC-derived foam cells	TRPV1 antibody	PA and NIRF	ApoE−/− mice	[39]
	Platelet membrane-coated, Gd-loaded PLGA	Foam cells, endothelium, collage	Platelet-membrane surface coating	MRI	ApoE−/− mice	[40]
Tissue regeneration	IONPs	Mesenchymal stem cells	Passive uptake	MRI	Sprague-Dawley rat myocardial infarction model	[41]
	Perfluorocarbon-NPs	Cardio-progenitor cells	FuGENE-enhanced NP uptake	MRI	C57BLl6 mice	[42]
	Semiconducting polymer nanoparticles	Human embryonic stem cell-derived cardiomyocytes (hESC-CMs)	Cell penetrating peptides for in vitro cellular uptake	PA	NOD SCID mice	[43]
	PLGA-IONP nanobubble with DiR	Cardiac stem cells (human MSCs)	Passive uptake	US + PA + Magnetic Particle Imaging (MPI)	nu/nu mice	[44]

IONPs = iron oxide nanoparticles; PLGA = poly(lactic-co-glycolic acid); DNA = deoxyribonucleic acid; PEG = polyethylene glycol; HFn = H-ferritin; TfR1 = transferrin receptor 1; GNP = gold nanoparticles; DTPA = Diethylenetriamine pentaacetate; HDL = high density lipoproteins; DiO = 3,3’-Dioctadecyloxacarbocyanine Perchlorate; PASKE = proline, alanine, serine, lysine, and polyglutamic acid; OPN = osteopontin; PLA = polylactic acid; PFOB = perfluorooctyl bromide; VSMCs = vascular smooth muscle cells; NOD = non-obese diabetic; SCID = severe combined immune deficiency; DiR = 1,1’-Dioctadecyl-3,3,3’,3’-Tetramethylindotricarbocyanine Iodide.