Nanoparticle Therapy for Vascular Diseases

Abstract

Nanoparticles (NPs) promise to advance strategies to treat vascular disease. Since being harnessed by the cancer field to deliver safer and more effective chemotherapeutics, nanoparticles have been translated into applications for cardiovascular disease. Systemic exposure and drug-drug interactions remain a concern for nearly all cardiovascular therapies, including statins, antithrombotic, and thrombolytic agents. Moreover, off-target effects and poor bioavailability have limited the development of completely new approaches to treat vascular disease. Through the rational design of nanoparticles, nano-based delivery systems enable more efficient delivery of a drug to its therapeutic target or even directly to the diseased site, overcoming biological barriers and enhancing a drug’s therapeutic index. In addition, advances in molecular imaging have led to the development of “theranostic” NPs that may simultaneously act as carriers of both therapeutic and imaging payloads. The following is a summary of nanoparticle therapy for atherosclerosis, thrombosis, and restenosis, and an overview of recent major advances in the targeted treatment of vascular disease.

Introduction

Cardiovascular disease is the number one cause of death globally.¹ The low delivery efficiency, poor target specificity and/or off-target activity of our therapies has contributed to the challenges we face in cardiovascular medicine.² Nanoparticles, on the scale of less than 0.1 microns in at least one dimension, have emerged as a powerful tool to increase the targeting selectivity of a drug and limit its distribution throughout the body. Their tunable shape, size, and surface chemistry enables nanoparticles to be “programmed” for site-specific delivery.³ A central goal of nanotherapy is to enhance the efficacy of a therapy and minimize side effects caused by freely delivered drug.⁴ Nanoparticles achieve this through rational design, frequently incorporating knowledge of normal vs. diseased biology to optimize residence time in the diseased tissue.

Oncology was the first field to leverage the properties of nanoparticles for drug delivery with Doxil, a liposome-encapsulated doxorubicin formulation approved by the FDA in 1995 for the treatment of Kaposi’s sarcoma.⁵ Now widely used for the treatment of multiple myeloma and other malignancies, the nanoformulation enables preferential uptake by cancer cells and limits exposure to the heart, reducing the risk of doxorubicin-induced cardiotoxicity and heart failure.⁶ Currently, more than 50 nanoparticle-based therapies are used for a variety of indications including infections, chronic kidney disease, and even psychiatric conditions.⁷

Nanotechnology has also expanded into the realm of cardiovascular disease. Currently marketed nanoformulations of fenofibrate are used in patients with hypertriglyceridemia to help overcome challenges with drug solubility and absorption. A number of delivery systems are under development to therapeutically target pathways of vascular disease (Figure 1). Additionally, multi-functional “theranostic” NPs hold promise for combined delivery of therapeutic and imaging agents. These theranostic NPs can serve to blend treatment with information from one or even multiple imaging modalities to more comprehensively assess disease. Prior work has highlighted the status of nanomaterials in cardiovascular imaging, including their potential to separately identify “vulnerable” plaques at risk for rupture.⁸ This review discusses advances in the application of nanoparticles for the treatment of vascular disease, their potential translation to the clinic, and challenges in their development. Greater emphasis is placed on nanoparticle-directed therapy for atherosclerosis and its associated complications, including thrombosis and restenosis, given their role in ischemic heart disease (Table 1).

Figure 1:

Targeted therapeutic strategies enabled by nanoparticles. In order to avoid adverse effects and toxicities due to systemic exposure, targeted nanoparticles have been developed to resolve inflammation specifically at the inflamed plaque (e.g. Collagen-IV targeted IL-10 NPs¹⁷), prevent plaque neovascularization (e.g. α_vβ₃-targed anti-angiogenic NPs⁶⁵), and deliver anti-proliferative or thrombolytic drugs to address restenosis and atherothrombotic events (e.g. endothelial-targeted NPs encapsulating paclitaxel⁷⁵, vWF-targeted NPs encapsulating tPA⁸¹). Nanoparticles have also enabled cell-specific modulation of molecules that drive atherosclerosis, such as CD40-induced TRAF6 signaling in macrophages⁴⁵ and regulation of PCSK9 in hepatocytes.⁵⁵

Table 1.

Examples of vascular nanotherapies in development.

Therapeutic agent	Nanoparticle	Delivery	Model	In vivo findings	Reference
Resolving inflammation and defective efferocytosis
Annexin A1 mimetic peptide	Type IV collagen-targeted copolymers of PLGA-PEG	Systemic	Ldlr −/− mice	↓: plaque area, necrotic core, lesional oxidative stress ↑: fibrous cap thickness	16
IL-10	Type IV collagen-targeted copolymers of PLGA-PEG	Systemic	Ldlr −/− mice	↓: necrotic core and lesional oxidative stress ↑: lesional efferocytosis, fibrous cap thickness	17
Superoxide dismutase mimetic agent and hydrogen-peroxide-eliminating compound	Cyclodextrin-based polysaccharide	Systemic	apoE −/− mice	↓: ROS-induced inflammation, lesional cholesterol crystals, necrotic core	18
Prednisolone*	MRI detectable liposomes	Systemic	Hypercholesterolemic rabbits with repeated balloon aortic injury	↓: plaque inflammation, lesional macrophage content Delivery visualized by MRI Anti-inflammatory effect validated by 18F-FDG/PET-CT and MRI	19
VCAM1, ICAM1 and 2, E- and P-selectin siRNA	PEI polymer	Systemic	Coronary ligation of apoE −/− mice	↓: post-myocardial infarction leukocyte recruitment, plaque inflammation, necrotic core, lesion size ↑: fibrous cap thickness	23
Preventing plaque neovascularization
Fumagillin*	avb3 integrin–targeted paramagnetic NPs	Systemic	Hypercholesterolemic rabbits	↓: aortic plaque angiogenesis, expansion of the vasa vasorum Aortic neovascular delivery and treatment response assessed by MRI	32
Fumagillin*	avb3 integrin–targeted paramagnetic NPs	Systemic NP delivery, followed by oral statin treatment	Hypercholesterolemic rabbits	Marked anti-angiogenic effect of NPs sustained by statin therapy Angiogenesis of aortic wall assessed by MRI	33
Targeting macrophages
Pitavastatin	PLGA	Systemic	angiotensin II-infused apoE −/− mice	↓: plaque monocyte infiltration ↑: plaque stability and protection from rupture	37
CCR2 siRNA*	Lipid NPs	Systemic	apoE −/− mice	↓: monocyte migration and accumulation, lesion size Changes in biodistribution over time evaluated by FMT-CT	38
Simvastatin*	Reconstituted HDL	Systemic	apoE −/− mice	↓: lesion area due to reduced plaque macrophage content, mRNA expression levels of monocyte recruitment and pro-inflammatory genes No effect on serum lipid levels Biodistribution evaluated by ex vivo NIRF Plaque macrophage targeting and treatment efficacy assessed by MRI and FMT/CT	40
Simvastatin*	Reconstituted HDL	Short-term systemic NP delivery, followed by long-term oral statin treatment	apoE −/− mice	↓: plaque macrophage proliferation and subsequent plaque inflammation Rapid suppression of inflammation by NP intervention, with sustained effects by oral statin therapy Treatment efficacy assessed by MRI and ex vivo NIRF	41
Liver X receptor agonist	Type IV collaged-targeted PLA-PEG NPs	Systemic	Ldlr −/− mice	↓: plaque macrophage content and cholesterol efflux No change in plasma lipid levels and hepatic lipid metabolism	42
Macrophage scavenger receptor-blocking sugars	Amphiphilic NPs with PEG core	Systemic	apoE −/− mice	↓: oxidized LDL uptake by macrophages, lipid burden, and overall plaque development	43
Pioglitazone	PLGA	Systemic	angiotensin II-infused apoE −/− mice	↓: peripheral inflammatory monocytes, aortic M1 macrophage polarization, plaque rupture ↑: plaque stabilization No effect on plaque macrophage content or lesion area	44
TRAF6 inhibitor	Reconstituted HDL	Systemic	apoE −/− mice Cynomolgus monkeys	↓: monocyte recruitment, plaque macrophage content Lack of immune suppressive side effects Liver and spleen accumulation demonstrated by PET/MRI Short-term safety in mice and non-human primates	39, 45
Near-infrared fluorophore*	Light-activated iron-oxide NPs	Systemic, with local photodynamic therapy	apoE −/− mice	↓: lesional macrophage content following local light therapy ↑: plaque macrophage apoptosis Plaque localization demonstrated by intravital fluorescence microscopy	48
Photothermal ablation*	Single-walled carbon nanotubes	Systemic, with local activation by photoluminescence	Mice carotid artery ligation	In vivo delivery visualized by FMT Ex vivo near-infrared imaging and photothermal ablation of vascular macrophages	49
Altering lipid metabolism
apoB siRNA	Liposomes	Systemic	Cynomolgus monkeys	↓: apoB mRNA expression in the liver, serum cholesterol, and LDL levels sustained for 11 days following single dose	52
apoB siRNA	Liposomes	Systemic	Ldlr CETP +/− mice	↓: apoB protein expression in the liver and serum LDL levels sustained for 3 weeks following single dose	53
PCSK9 siRNA	Lipidoid-PEG formulation	Systemic	Wild-type mice, rats, cynomolgus monkeys	↓: plasma PCKS9 protein and LDL levels sustained for 3 weeks following single bolus	55
TRPV1 antibodies*	Copper sulfide NPs	Systemic, with local photothermal activation	apoE −/− mice	↓: plaque lipid storage, VSMC foam cell formation, lesion formation ↑: autophagy and cholesterol efflux Vessel delivery monitored by photoacoustic imaging	61
Preventing neointimal growth
Rapamycin	Albumin NPs	Local catheter delivery	Porcine femoral artery balloon injury	↓: luminal stenosis at 28 days	64
Rapamycin*	avb3-targeted paramagnetic NPs	Local catheter delivery	Rabbit femoral artery balloon injury	↓: vascular stenosis, neointimal formation at 14 days No delay in endothelial healing Intramural delivery and luminal changes assessed by MRI	65
NOX2 siRNA	Lysine-based NPs	Open delivery onto adventitia	Rat carotid artery balloon injury	↓: ROS production, neointima formation at 14 days	66
Nitric oxide	Liposomes	Local catheter delivery	Rabbit carotid artery balloon injury	↓: neointimal proliferation at 14 days	67
Paclitaxel	Stent-targeted magnetic NPs	Local delivery, with targeting by magnetic exposure	Rat carotid artery stenting	↓: in-stent neointimal growth at 14 days	68
Imatinib (PDGF tyrosine kinase inhibitor)	PLGA NP-eluting stents	Coated on stent	Porcine coronary artery stenting	↓: in-stent restenosis at 28 days No effect on endothelial proliferation	70
Pitavastatin	PLGA NP-eluting stents	Coated on stent	Porcine coronary artery stenting	↓: in-stent restenosis at 28 days Lack of delay in endothelial healing	71
Paclitaxel	Albumin NPs	Systemic	Rabbit iliac artery stenting	↓: in-stent neointimal growth sustained for 28 days ↑: endothelialization and healing	74
Paclitaxel	Type IV collagen-targeted lipid-polymeric NPs	Systemic	Rat carotid artery balloon injury	↓: neointimal proliferation at 14 days	75
Alendronate (bisphosphonate)	Liposomes	Systemic	Rabbit iliac artery stenting	↓: blood monocyte count, arterial macrophage infiltration, in-stent neointimal formation at 28 days	76
Nitric oxide	Nanofibers	Systemic	Rat carotid artery balloon injury	↓: neointimal hyperplasia sustained for 7 months	78
Prednisolone	Subendothelial-targeted liposomes	Systemic	Rabbit iliac artery stenting	↓: in-stent stenosis at 42 days	79
Targeting thrombosis
tPA	PAA-coated iron oxide magnetic NPs	Local catheter delivery, with magnetic targeted thrombolysis	Rat iliac artery embolus	↑: aortoiliac blood flow, hind limb perfusion	80
tPA	vWF-targeted gelatin NP complex	Systemic, with drug release enhanced by local ultrasound	Porcine myocardial infarction	↑: coronary recanalization and left ventricular ejection fraction compared to free tPA	81
tPA*	Iron oxide PLGA NPs	Systemic	Rat aorta thrombosis	↑: thrombolytic efficiency Thrombus targeting and thrombolytic activity assessed by MRI	82
Streptokinase	GPIIb/IIIa and P-selectin-targeted liposomes	Systemic	Mouse carotid artery thrombosis	↑: delay in thrombus growth, time to vessel occlusion Minimal effect on tail bleeding time Targeting to activated platelets demonstrated by intravital microscopy	83
Urokinase	Iron oxide magnetic NPs	Local catheter delivery, with magnetic targeted thrombolysis	Rat carotid artery and jugular vein thrombosis	↑: thrombus dissolution Minimal changes in systemic plasminogen activity and tail bleeding time	85
Thrombin inhibitor*	PFC NPs	Systemic	apoE −/− mice carotid artery injury	↓: thrombin-induced inflammatory and pro-coagulant molecules ↑: time to carotid occlusion, restoration of endothelium No change in activated partial thromboplastin time Ex vivo assessment of delivery and endothelial damage by magnetic resonance spectroscopy	87

^*Nanoparticles designed as theranostic agents.

PLGA: poly lactide-co-glycolide. PEG: polyethylene glycol. IL-10: interleukin-10. ROS: reactive oxygen species. VCAM1: vascular cell adhesion molecule 1. ICAM1: intercellular adhesion molecule 1. PEI: polyethyleneimine. MRI: magnetic resonance imaging. 18F-FDG/PET-CT: 2-deoxy-2-[fluorine-18]fluoro-D-glucose positron emission tomography-computed tomography. PLA: polylactic acid. LDL: low density lipoprotein. HDL: high density lipoprotein. NIRF: near-infrared fluorescence. FMT-CT: fluorescence molecular tomography-computed tomography. CCR2: C-C chemokine receptor type 2. TRAF6: tumor necrosis factor receptor-associated factor 6. apoB: apolipoprotein B. PCSK9: proprotein convertase subtilisin/kexin type 9. TRPV1: transient receptor potential cation channel subfamily V member 1. VSMC: vascular smooth muscle cell. NOX2: NADPH oxidase 2. PDGF: platelet-derived growth factor. GPIIb/IIIa: glycoprotein IIb/IIIa. vWF: von Willebrand factor. tPA: tissue plasminogen activator. PFC: perfluorocarbon.

Resolving inflammation and defective efferocytosis

Atherosclerosis is an inflammatory disease characterized by the accumulation of lipids, diseased cells, and necrotic debris. Pro-inflammatory leukocytes and cytokines act at different stages during the formation of the atherosclerotic plaque.⁹ Heightened inflammation is driven, in part, by the failure to clear apoptotic tissue from the diseased vessel wall due to a defect in efferocytosis (programmed cell removal), such that apoptotic cells accumulate, become secondarily necrotic, and release their pro-inflammatory intracellular contents.^{10, 11} Importantly, this non-resolving inflammation drives clinically dangerous lesions that are at increased risk of rupture and thrombosis. The recent CANTOS (Canakinumab Anti-inflammatory Therapy Outcomes Study) trial demonstrated the benefit of suppressing inflammation on cardiovascular disease in high-risk patients.^{12, 13} However, targeting inflammation systemically also has significant potential to inhibit innate immunity and compromise host defense against infections.¹⁴ Indeed, deaths due to infection or sepsis were more common among CANTOS trial patients who received the systemic anti-inflammatory treatment. Because of their ability to achieve local delivery, atherosclerosis-targeted nanoparticles may be able to address these risks.

Type IV collagen is a major sub-endothelial basement membrane protein that is exposed upon vascular injury and inflammation.¹⁵ When combining a Type IV-collagen targeting peptide and pro-resolving peptide derived from Annexin A1, there was a 70% increase in selectivity of the NPs for atherosclerotic lesions, relative to the spleen and liver.¹⁶ The targeted, inflammation-resolving NPs enhanced resolution to a much greater extent than the free-resolving peptide, where NP treatment effectively suppressed plaque oxidative stress, necrosis, and fibrous cap thinning. In another study using a similar Type IV-collagen targeting system, NPs that incorporate the anti-inflammatory cytokine IL-10 were engineered.¹⁷ IL-10 nanotherapy had similar protective effects on advanced atherosclerosis in Ldlr^−/− mice, in addition to enhancing macrophage-mediated clearance of apoptotic debris. Resolving local inflammation thus also appeared to have a pro-efferocytic effect. Short-term toxicity studies revealed no alterations in blood cytokine levels, suggesting the IL-10 nanotherapy was specific to sites of inflammation and may not compromise host defense. In a study using an NP designed to attenuate inflammation due to the production of reactive oxygen species (ROS), delivery of a free-radical scavenging payload led to a decrease in cell apoptosis within the plaques of apoE^−/− mice. Following internalization by macrophages and vascular smooth muscle cells (VSMCs), the “ROS-scavenging” NPs decreased cellular oxLDL uptake and subsequent transformation to foam cells. NPs were thus able to overcome the rapid elimination and short retention time of the free therapeutic agent in atherosclerotic plaques. Additionally, their benefit on plaque progression and stability was importantly observed without side effects, indicated by normal clinical chemistry, hematology, and viability of mice following treatment.¹⁸

Inflammation-targeting nanoparticles have also been formulated as theranostic NPs. In a rabbit model of atherosclerosis, magnetic resonance imaging (MRI)-detectable liposomes were developed for delivery of prednisolone to the inflamed vessel wall.¹⁹ Liposomal encapsulation improved the pharmacokinetics of prednisolone and prolonged its circulating half-life, without systemic toxicity. After a single dose, rapid and sustained decreases in plaque inflammation were observed by MRI and correlated with ¹⁸F-FDG-positron emission tomography/computed tomography (PET/CT), a validated method of tracking inflammation in atherosclerosis imaging.²⁰ Decreases in plaque inflammation were attributable to a decrease in monocyte chemoattracts and lesional macrophage density, effects that were observed to a much lesser degree in rabbits treated with the free corticosteroid. Investigators then executed a pharmaceutical development program in which they optimized a scaled up synthesis method and formed a purified and storage-stable good manufacturing practice (GMP)-grade product.²¹ Following pharmacokinetic and toxicologic evaluation in healthy rats and rabbits, the prednisolone-containing liposomes failed to induce measurable effects on arterial wall inflammation in Phase I/II trials.²² Optimizing the dose and treatment schedule in larger animal models may have led to a more thorough understanding of the therapeutic margin and dose required to achieve efficient target engagement. Despite the lack of treatment benefit, multi-modal imaging demonstrated that the nanoparticles accumulated in plaque macrophages without adverse effects, thus serving as a guide for imaging-based efficacy measures and demonstrating the feasibility of targeting nanoparticles to human atherosclerotic areas.

In a study specifically aiming to interfere with leukocyte recruitment into the atherosclerotic plaque, Sager and colleagues combined small interfering RNA (siRNA) targeting multiple cell adhesion molecules into a polymer-based NP.²³ Made up of a variety of synthetic or natural polymers, polymeric NPs are more resistant to degradation and offer a more tunable architecture than liposomes.²⁴ In apoE^−/− mice that underwent coronary ligation, treatment with NPs encapsulating five siRNAs targeting leukocyte adhesion molecules significantly reduced vascular inflammation after myocardial infarction.²³ The resultant decrease in leukocyte accumulation led to a decrease in tissue injury and necrotic core formation following ischemic insult. Altogether, these studies exemplify the exciting possibility that plaque inflammation and apoptotic cell accumulation can be directly addressed using targeted NPs.

Preventing plaque neovascularization

Advanced atherosclerotic plaques frequently display extensive adventitial and neointimal neovascularization. In humans, increased plaque vascularity has been observed in lesions from patients with acute coronary syndrome and symptomatic carotid stenosis, relative to individuals with stable or asymptomatic disease.^{25, 26} These data suggest that plaque neovascularization may have an important role in atherogenesis and intraplaque hemorrhage.²⁷ Angiogenesis is coordinated by a number of cytokines, including vascular endothelial growth factor (VEGF) and platelet-derived growth factor.²⁸ Anti-VEGF therapies and other anti-angiogenic agents have successfully been used to promote regression of tumor vessels and prolong survival in cancer patients in some studies^{29, 30}, but come with an increased risk of arterial thromboembolic events that is further compounded in patients with a history of cardiovascular disease.³¹ Based on prior work demonstrating that high dose anti-angiogenic therapy reduces plaque development in apoE^−/− mice, investigators developed a targeted theranostic NP in efforts to avoid the drug’s neurocognitive effects and combine an imaging agent for serial monitoring of neovessel formation.³² Using a ligand for the α_vβ₃-integrins that are up-regulated during angiogenesis, hyperlipidemic rabbits treated with the MRI-detectable NPs exhibited a reduction in α_vβ₃-related signal enhancement in the aorta. T₁-weighted MRI signal in the aorta correlated with the degree of neovessel formation in the atherosclerotic aorta. Interestingly, this benefit occurred at a dosage of 50,000 times less when the anti-angiogenic agent was encapsulated as a nanoformulation than when the therapy was delivered alone. In a follow-up study, combining their α_vβ₃-targeted anti-angiogenic treatment with atorvastatin achieved a greater and more sustained decrease in MR signal and plaque neovessel count of hyperlipidemic rabbits.³³ This finding is in line with work suggesting that statins inhibit endothelial proliferation and VEGF production, potentially explaining the synergistic effect on plaque neovessel formation.³⁴ By leveraging plaque biology, these studies highlight the potential of NPs to feature an imaging/therapeutic payload and prevent disease in ways that were previously inaccessible due to off-target effects.

Targeting macrophages

Macrophages have a key role in atherosclerosis, from lesion initiation, foam cell formation, and by contributing to the pool of apoptotic cells that affect plaque size and vulnerability.^{35, 36} Importantly, in advanced lesions, defective efferocytic activity by lesional macrophages is what causes the build-up of toxic intracellular material and subsequent plaque necrosis. Several nanotherapies have been described that target plaque monocyte recruitment and infiltration^37–39, macrophage proliferation^{40, 41}, cholesterol metabolism^{42, 43}, and polarization to a less inflammatory M2 phenotype.⁴⁴ In a study by Lewis and colleagues, sugar-based NPs were designed to block oxidized LDL uptake from macrophage scavenger receptors (SR) by both direct inhibition and long-term downregulation of SR expression on the cell surface.⁴³ Binding to macrophage SRs was shown to directly correlate with targeting of NPs to established atherosclerotic plaques. They reported that treatment resulted in markedly reduced lipid burden and overall plaque occlusion in the aorta of apoE^−/− mice. In another recent study, a targeted nanoimmunotherapy was developed to block CD40-induced tumor necrosis receptor-associated factor 6 (TRAF6) in monocytes and macrophages, thereby preventing monocyte recruitment into the arterial wall.^{39, 45} While systemic inhibition of the CD40-TRAF6 axis results in serious complications such as thromboembolic events and immune suppression, investigators selectively targeted TRAF6 in monocytes by incorporating TRAF6 inhibitors into recombinant HDL NPs (TRAF6i-HDL). As an extension of the group’s experience with HDL particles in atherosclerosis nanotherapy⁴⁰ and in vivo imaging⁴⁶, TRAF6i-HDL was shown to both hamper the initiation of disease in young apoE−/− mice with no atherosclerosis and induce a more stable plaque phenotype in animals with established disease. Following incorporation with fluorescent dyes or radiolabeled molecules, TRAF6i-HDL was shown to accumulate primarily in the liver and spleen of apoE−/− mice and non-human primates. Investigators also provided evidence of the short-term safety of the nanoimmunotherapy in mice and cynomolgus monkeys.⁴⁵ These safety experiments revealed that TRAF6i-HDL did not elicit adverse immune responses, alter major serological parameters, or cause any organ toxicity, although an acute increase in alkaline phosphatase levels and reticulocyte count was noted in mice treated for 1 week. In mice that received long-term treatment, these changes were not observed, although interestingly, cholesterol levels and white blood cell counts were both elevated in mice treated for 6 weeks.³⁹ Because the HDL NPs primarily accumulate in the liver and spleen, further toxicological studies are needed for this promising nanoimmunotherapy.

The preferential uptake of NPs by inflammatory monocytes and macrophages has also served as a means for focal therapy of inflamed lesions. Iron-oxide NPs undergo uptake by macrophages (>75%) and to a lesser degree by neutrophils and other vascular cells.⁴⁷ When modified with a near-infrared fluorophore, irradiation of the atheroma resulted in focal ablation of the macrophage-rich plaques of mice following carotid ligation.⁴⁸ However, the authors note the phototoxicity also may affect other cell types with an affinity for the NPs, including VSMCs and endothelial cells. Subjecting these cell types to photothermal therapy could potentially make the plaques more dangerous and prone to rupture. Nanoparticles that may be more cell-specific are under development, including single-walled carbon nanotubes (SWNTs, Figure 2A).⁴⁹ SWNTs undergo highly selective uptake by inflammatory Ly-6C^hi monocytes.⁵⁰ Within the diseased vessels of carotid-ligated mice, SWNTs co-localized with lesional macrophages, with negligible amounts observed in VSMCs.⁴⁹ Further exploration of in vivo efficacy and therapy-loading ability is needed. Given that inflammatory monocytes represent the majority of circulating cells recruited to the atherosclerotic plaque, there is compelling rationale for the use of SWNTs and other NPs that afford similar cell-specific drug delivery. Moreover, SWNTs and other carbon nanomaterials exhibit a natural photoacoustic contrast and near-infrared fluorescence signal.^{49, 51} These intrinsic imaging capabilities make carbon nanomaterials particularly useful for theranostic strategies to detect and treat the inflamed “vulnerable” plaque.

Figure 2:

Schematic of nanoparticles functionalized with agents that control their stability and interactions with the biological environment. A. Single-walled carbon nanotube (SWNT) tailored for vessel delivery of a therapeutic payload by coating the SWNT with polyethylene glycol (PEG) chains linked to arginine-glycine-aspartic acid (RGD), a potent α_vβ₃ integrin-binding peptide. B. Lipidoid nanoparticles that are formulated with phospholipids and cholesterol to facilitate intracellular delivery of siRNA for potent gene knockdown. Figures modified from [50] and [60].

Altering lipid metabolism

Nanoparticle delivery systems have particularly impacted the ability to target liver cholesterol metabolism using RNA interference (RNAi). A liposomal formulation of apolipoprotein B (apoB) siRNA resulted in specific silencing of apoB in hepatocytes and reductions in apoB, LDL, and total cholesterol levels in rodents and monkeys.^{52, 53} The opportunity to target apoB is particularly exciting given genetic and epidemiological studies that suggest the clinical benefit of lowering LDL may depend on a corresponding reduction in apoB levels.⁵⁴

Most recently, siRNA silencing of proprotein convertase subtilisin/kexin type 9 (PCSK9) shows promise as a future strategy for reducing LDL cholesterol in a potent and convenient manner. The field of RNAi therapy has historically been impeded by the instability of naked siRNA in the bloodstream and their inability to cross the cell membrane. RNA silencing of PCKS9 using lipid-based nanoparticles, termed “lipidoids” (Figure 2B), has recently been shown to efficiently target and suppress PCSK9 synthesis in the liver. Unlike the currently available PCSK9 antibodies (i.e. Evolucumab) that require bi-weekly injections, the novel nanoformulations of PCSK9 siRNA (Inclisiran) caused rapid and durable effects after a single dose. First shown in rodents and monkeys⁵⁵, Inclisiran effectively reduced levels of PCSK9 and LDL for at least 6 months in Phase 1/2 trials.^56–59 Further evaluation is ongoing in Phase III trials (NCT03399370). This development is a major advance in cardiovascular medicine, and was driven by nanotechnology that enabled stable delivery of RNAi therapeutics.⁶⁰

In another recent study, antibodies against the VSMC-expressed ion channel, transient receptor potential vanilloid subfamily 1 (TRPV1), were conjugated to copper sulfide (CuS) NPs.⁶¹ TRPV1 induces autophagy in VSMCs, reduces lipid accumulation, and prevents foam cell formation. Upon irradiation of the aortic arch of apoE^−/− mice, the local increase of temperature opened TRPV1 channels and allowed an influx of calcium ions to activate autophagy. Excitingly, NPs provided highly precise, non-invasive treatment under image-guidance due to the characteristic near infrared absorption of CuS NPs that generates a strong photoacoustic signal.

Preventing neointimal growth

High restenosis and reintervention rates are still a significant limitation of revascularization procedures, particularly among patients with peripheral arterial disease. A number of NPs have been identified that enhance retention of anti-restenotic agents in the local vascular bed^{62, 63}. These include NPs that are delivered locally via catheter, implanted in stents, and even systemically administered surrounding the time of revascularization.

Using an endovascular microinfusion catheter for local delivery, albumin-bound rapamycin NPs were concentrated in the adventitial and medial layers of the arterial wall, and reduced luminal stenosis in a porcine femoral artery balloon angioplasty model.⁶⁴ Rapamycin levels in the femoral artery remained >100 times higher in the perivascular tissues than in the blood for 8 days. Importantly, this period was when cell proliferation rates were the highest in control animals, suggesting that the NPs promote drug retention during a particularly critical period of neointima formation. In another study, Cyrus and colleagues developed α_vβ₃-targeted paramagnetic NPs for delivery of rapamycin to balloon injured femoral arteries of rabbits.⁶⁵ Local infusion of vessel wall-targeted NPs resulted in a decrease in neointimal formation, and their retention in the injured walls was amenable to MRI due to the high contrast potential of paramagnetic nanomaterials. Highlighting the tunable properties of NPs, nitric oxide gas and RNAi components have also been encapsulated within NPs and delivered locally to the diseased arterial wall.^{66, 67}

The surfaces of stents may also be loaded or targeted with NPs. In a study by Chorny et al., “stent-targeted” magnetic NPs were designed.⁶⁸ Following stent placement in the carotid artery of rats, these paclitaxel-loaded NPs were infused in the isolated artery and were targeted to the stent by applying a magnetic field to the body surface. Treatment effectively inhibited in-stent stenosis at drug doses below those provided by paclitaxel-eluting stents. Innovative NP-eluting stents have also been developed to prevent stenosis after stent implantation.^69–71 Importantly, these stents were found to prevent in-stent restenosis without delayed endothelial healing, which is a central reason for late stent thrombosis following percutaneous interventions.

Rather than requiring stent placement or advancement of a catheter for local delivery, targeted NPs may enable systemic therapy for restenosis. Although a promising albumin-stabilized nanoformulation of paclitaxel failed to show efficacy in early clinical trials^72–74, this work highlighted the existing space for more targeted or “precision medicine” approaches to restenosis nanotherapy. Using lipid-polymeric NPs that were surface modified with Type IV-collagen targeting peptides, Chan and colleagues reported the efficacy of paclitaxel-encapsulated NPs that preferentially localized to the denuded vessel wall.⁷⁵ Systemically administered NPs led to a reduction in arterial stenosis in a rat carotid injury model. While 15 mg/kg doses of free paclitaxel induced signs of toxicity, NPs enabled higher 35 mg/kg doses that were well-tolerated in animals. Another group described a liposomal formulation of the bisphosphonate alendronate that reduced neointimal formation by transiently suppressing circulating monocyte levels in rabbits that had undergone iliac artery stenting.⁷⁶ Early-phase clinical trials supported the safety of liposomal alendronate for infusion at the time of percutaneous coronary intervention. Although there was no difference in restenosis rates between the treatment and placebo groups, the anti-inflammatory NP led to a significant reduction in in-stent late loss in an “inflammatory patient” subgroup with elevated baseline monocyte counts (NCT02645799).⁷⁷ Other systemic nanoformulations that have prevented restenosis include nitric-oxide containing nanofibers⁷⁸ and glucocorticoid-encapsulated NPs that are targeted to subendothelial matrix proteins⁷⁹. Taken together, NP-based delivery may be a useful adjunct to revascularization procedures, particularly for patient-personalized treatment and for those with diffuse disease and other challenging lesion patterns.

Targeting thrombosis

Platelet activation, the coagulation cascade, and fresh thrombus include unique factors that enable targeted delivery of therapeutic agents. Thrombus-targeted NPs have been developed for delivery of thrombolytic agents and anti-coagulants, including tissue plasminogen activator (tPA)^80–82 streptokinase^{83, 84}, urokinase^{85, 86}, and direct thrombin inhibitors.⁸⁷ By encapsulating a von Willebrand factor-binding protein within NPs, investigators effectively targeted tPA to thrombi induced in swine coronary arteries.⁸¹ Intravenous delivery of NPs resulted in reperfusion and vessel recanalization in 90% of animals. Interestingly, NPs were designed for controlled release of tPA using transthoracic ultrasound, where application of ultrasound led to greater tPA off-loading and thrombolytic activity at the affected artery. In another study, anti-thrombin theranostic NPs directly attenuated plaque coagulant activity within the injured arteries of apoE^−/− mice.⁸⁷ Detectable by magnetic resonance spectroscopy, systemically administered NPs were retained within the plaques and exerted rapid inactivation of any locally produced thrombin. These effects were observed without altering activated partial thromboplastin time or other systemic effects on coagulation. Moreover, focal inhibition of plaque thrombin reduced the expression of plaque inflammatory molecules and enhanced restoration of the disrupted vascular endothelium, suggesting the anti-thrombin NPs promoted plaque stability. Taken together, these studies illustrate the broad potential that NPs have for reperfusion therapy and anticoagulation with decreased bleeding consequences.

Conclusions and future directions

Target-driven NPs have opened the door to improved and even novel treatment options for patients with vascular disease. Those that have advanced into early clinical trials provide important lessons, namely the need for (1) a well-defined patient population most likely to benefit from therapy, and (2) a thorough understanding of a formulation’s therapeutic margin. In addition to implementing clinically relevant animal models, these considerations will greatly draw from the use of biomarker and molecular imaging based-strategies to enable patient selection and assessment of drug accumulation and response.

Nonetheless, there are important limitations and challenges in cardiovascular nanomedicine that should be noted (Table 2). First, NPs are inherently heterogeneous in composition and can present challenges in synthesis of large volumes that adhere to pharmaceutical GMP guidelines (e.g. sterility, stability, and purity). It will be important to develop methods to scale-up production with high reliability and reproducibility, reasonable cost, and time-efficiency for successful translation from bench-to-bedside. Moreover, stability in circulation and during storage is a common pitfall of nanotechnology. Although conventional methods for quality control exist, each nanoformulation is a specialized unit and requires unique measurements of physicochemical properties and drug release rates. In particular, there is a need to develop a drug release profile under both physiological and storage conditions, such that the drug is not released too slowly, prematurely in circulation, or even during storage. In addition, although some nanoformulations use disease-targeting ligands, several NPs have been described to have an intrinsic affinity for certain cell types (e.g. intraplaque macrophages). The distinct mechanisms by which such NPs are taken up by cells are poorly understood and is an intriguing area for future study. Lastly, there is a major gap in the evaluation of the long-term safety of nanoformulations in vivo. Many currently available nanoformulations were developed to reduce the side effects of a loaded drug, but some nanoparticles undergoing preclinical development have retroactively been discovered to be cytotoxic and/or immunogenic.⁸⁸ In an ideal situation these studies will be pursued in parallel as a means of informing decisions earlier in the development process. Coating NPs with polyethylene glycol (PEG), or “PEGylation” is a commonly used approach for reducing their systemic toxicity and interaction with plasma proteins and circulating cells.⁸⁹ Further optimization of methods to reduce unwanted nano-bio interactions may be necessary.

Table 2.

Advantages and limitations of nanotherapy for vascular disease.

Advantages		Limitations
Targeted delivery to site of disease (e.g. atherosclerotic plaque) or therapeutic target (e.g. PCSK9 in the liver)		Challenges and cost of scaled-up production
Increased efficacy per drug dose		Gap in knowledge of clinical safety
Avoidance of systemic exposure and/or off-target effects		Requirement for formulation-specific methods for characterization of composition and purity
Potential for combining treatment, diagnosis, and monitoring disease in a single formulation		Long-term stability of loaded agents
Ability to be loaded with molecular recognition agents for “precision” therapy

While there has been substantial progress, the global epidemic of vascular disease and resultant deaths are predicted to increase over the next decades, affecting developed and developing nations alike.⁹⁰ Lipid-lowering therapies are the dominant treatment for atherosclerosis, but there is much room for improvement with complementary therapies. Our understanding of plaque biology has evolved to include inflammatory mechanisms and impaired efferocytosis as causal drivers of lesion progression and instability. However, anti-inflammatory therapies are known to cause immunosuppression. Additionally, reactivating efferocytosis has the drawback of inducing the off-target clearance of healthy tissues under some conditions. For example, in addition to efficiently restoring phagocytosis and preventing atherosclerosis, pro-efferocytic therapy also caused a clinically relevant anemia due to clearance of red blood cells in the spleen.^{91, 92} To overcome the toxicities associated with anti-inflammatory and pro-efferocytic approaches, it is likely that nanoparticles will serve as the innovative delivery systems that will enable targeting of these important pathways specifically within the vulnerable plaque.

Highlights.

• Nanoparticles have uniquely appealing features that enable them to be programmed as cell- and tissue-specific delivery systems, thus overcoming the low drug delivery and off-target effects which commonly impede developments in cardiovascular medicine.

• Nanotechnology is driving efforts to develop novel and more effective treatments for vascular disease, namely by targeting chronic inflammation, resolving defective efferocytosis, and producing more potent lipid-lowering therapies.

• This review discusses advances in the application of nanoparticles for the treatment of vascular disease, their potential translation to the clinic, and challenges in their development.

Abbreviations

NPs

Nanoparticles

ROS

Reactive oxygen species

VSMCs

Vascular smooth muscle cells

MRI

Magnetic resonance imaging

PET

Positron emission tomography

CT

Computed tomography

GMP

Good manufacturing practice

siRNA

small interfering RNA

VEGF

Vascular endothelial growth factor

SR

Scavenger receptor

TRAF6

Tumor necrosis receptor-associated factor 6

HDL

high-density lipoprotein

SWNTs

Single-walled carbon nanotubes

RNA

RNA interference

PCSK9

Proprotein convertase subtilisin/kexin type 9

TRPV1

Transient receptor potential vanilloid subfamily 1

CuS

Copper sulfide

tPA

Tissue plasminogen activator

PEG

Polyethylene glycol