Nanotherapeutic Shots through the Heart of Plaque

Abstract

The past several decades have brought significant advances in the application of clinical and preclinical nanoparticulate drugs in the field of cancer, but nanodrug development in cardiovascular disease has lagged in comparison. Improved understanding of the spatiotemporal kinetics of nanoparticle delivery to atherosclerotic plaques is required to optimize preclinical nanodrug delivery and to drive their clinical translation. Mechanistic studies using super-resolution and correlative light microscopy/electron microscopy permit a broad, ultra-high-resolution picture of how endothelial barrier integrity impacts the enhanced permeation and retention (EPR) effect for nanoparticles as a function of both atherosclerosis progression and metabolic therapy. Studies by Beldman et al. in the December issue of ACS Nano suggest atherosclerotic plaque progression supports endothelial junction stabilization, which can reduce nanoparticle entry into plaques, and metabolic therapy may induce similar effects. Herein, we examine the potential for advanced dynamic intravital microscopy-based mechanistic studies of nanoparticle entry into atherosclerotic plaques to shed light on the advantages of free extravasation versus immune-mediated nanoparticle uptake for effective clinical translation. We further explore the potential combination of metabolic therapy with another emerging cardiovascular disease treatment paradigm—efferocytosis stimulation—to enhance atherosclerotic plaque regression.

Graphical Abstract

Cardiovascular disease and cancer, the top two killers in the United States, together account for nearly 50% of U.S. deaths. These diseases appear starkly different at first blush. Atherosclerosis, the prototypical cardiovascular disease, results from lipid deposition and inflammation in the vessel wall.¹ Cancer, by contrast, most often involves somatic gene mutations that result in dysregulated cellular proliferation and may involve any organ in the body.²

Intriguingly, despite the apparent differences in pathogenesis and mechanism, new research results have shown that therapies designed for cancer can also have roles in treating atherosclerosis. Recent examples include: (i) Restimulation of intratumoral macrophage phagocytosis enables them to “eat” cancer cells and thereby to control tumor growth.^3,4 The same therapeutic process was also shown to be effective in clearing dead and dying cells in atherosclerotic plaques, stabilizing them and reducing plaque size.⁵ (ii) Antiglycolytic drugs have been used for several years to treat cancer, in part to induce vascular renormalization,⁶ which can be exerted as a potent therapeutic tool. Recent work by Beldman et al. in the December issue of ACS Nano⁷ suggests that metabolic antiglycolytic therapy can also induce vascular renormalization under atherosclerotic conditions and reverse inflammatory processes in plaques. Indeed, the parallels between the two diseases may have their roots in the role of inflammation in their pathogenesis.

Despite the apparent differences in pathogenesis and mechanism, new research results have shown that therapies designed for cancer may also have roles in treating atherosclerosis.

Although medical treatments for atherosclerosis, including lipid-lowering and antithrombotic drugs and invasive revascularization strategies, have lowered cardiovascular complications, there remains a need for therapeutic tools and delivery systems to address atherosclerotic vascular disease. Several classes of lipid-modulating drugs are currently approved by the U.S. Federal Drug Administration (FDA) to reduce cardiovascular events, including statins, fibrates, proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors, and ezetimibe.⁸ In addition to their effects on lipids, statins have pleiotropic vasculo-protective effects, independent of their effect on lipoproteins.⁹ The JUPITER trial corroborated these data in a large, randomized controlled trial of statins administered to patients with normal lipid levels, which demonstrated reduced cardiovascular events without lowering lipid levels.¹⁰ In spite of such vasculo-protective effects, statins are incompletely effective in protecting some high-risk patient populations.^11,12 Current treatment protocols for atherosclerotic vascular disease include multiple agents with different vascular targets to improve outcomes. Interestingly, sodium-glucose cotransporter-2 (SGLT2) inhibitors traditionally used to treat diabetes also provide significant protection from cardiovascular events,⁸ and SGLT2 inhibitors may also be used to treat cancer.¹³ Clinical trials of statins and SGLT2 inhibitors suggest closer overlap between inflammation, metabolism, and cardiovascular disease than previously appreciated and present new systemic and targeted therapeutic opportunities. Recent discoveries in cancer biology have lent further insight into immune and metabolic checkpoints with parallels in cardiovascular disease. Treatments to derepress immune activation in cancer are proving to reduce atherosclerotic plaque size in preclinical studies.^5,15,16

In the December issue of ACS Nano, Beldman et al. showed that both advanced atherosclerotic plaques and antiglycolytic therapy apparently induce vascular renormalization and endothelial junction stabilization, leading to decreased vascular leakiness and reduced uptake of hyaluronan nanomaterials in the case of advanced atherosclerosis.

Although nanoparticle drug formulations entered the clinic in other disciplines as early as 1990, only one cardiovascular drug, a nanoparticulate formulation of fenofibrate, is FDA-approved for clinical use.¹⁴ The successes and promise of nanotherapeutics in medicine have fueled interest in developing new approaches to cardiovascular treatments to improve drug efficacy, precise targeting, stability, and limiting off-target effects.¹⁵

In this Perspective, we outline the role of delivery in atherosclerosis nanotherapy, highlight the potential for antiglycolytic combination therapy with nanomaterials, explore the potential to examine mechanistic insights using advanced imaging, and evaluate the future clinical potential of these treatments. In the December issue of ACS Nano, Beldman et al. showed that both advanced atherosclerotic plaques and antiglycolytic therapy apparently induce vascular renormalization and endothelial junction stabilization, leading to decreased vascular leakiness and reduced uptake of hyaluronan nanomaterials in the case of advanced atherosclerosis, but antiglycolytics can also reduce the burden of inflammatory plaque.⁷

Nanoparticle Treatment of Atherosclerosis

Nanomaterials have been used as imaging and therapeutic agents to assess and to treat atherosclerosis preclinically for decades^17–19 and clinically for several years.²⁰ These nanoparticles typically enter plaque via the enhanced permeation and retention (EPR) effect, by which nanomaterials traverse openings between or within endothelial cells lining the blood vessels and extravasate into the interstitial space—much like the EPR effect in cancer.^21–24

The EPR effect, first discovered as a feature in cancer,²⁵ has also shown promise in cardiovascular disease²⁶ because of similarities in pathogenesis between the two diseases, including rampant neoangiogenesis.²⁷ Entry of nanomaterials into the blood vessel/plaque sites was thought to correspond roughly to a plaque stage.²⁸ However, recent work from Beldman et al.⁷ using super-resolution and correlative light microscopy and electron microscopy (also previously applied to explore the EPR effect of nanoparticles in the context of cancer²⁹) suggests that endothelial junctions stabilize in advanced plaque, decreasing extravasation of nanoparticles. This finding casts doubt on the utility of the EPR effect to deliver therapeutics in atherosclerosis reliably. Almost mockingly, drug-bearing nanoparticle entry is most restricted when plaques most require treatment. Moreover, for imaging diagnostics, this finding suggests that the extent of atherosclerosis cannot be measured as a function of extravasation-dominant plaque nanoparticle uptake because the imaging signal may decrease with plaque progression.

Another recent discovery in the context of cancer showed that some nanomaterials, such as single-walled carbon nanotubes (SWNTs), may enter immune cells in the circulatory system and be taken up into tumors as a Trojan Horse.³⁰ This delivery mechanism could help overcome key issues that continue to plague EPR-based delivery, including heterogeneous uptake within a lesion, across different lesions, across different people, and now, as described by Beldman et al., across stages of atherosclerosis. These SWNTs selectively enter inflammatory monocytes,³⁰ which, due to the inflammatory nature of cancer, natively home to tumors.³¹ By again exploiting a discovery made in cancer for atherosclerosis due to the inflammatory similarity of the disease, SWNT-loaded immune cells might be used as delivery vehicles in atherosclerosis for treating and imaging disease as inflammatory monocytes are well-established to home to atherosclerotic plaques.^32,33 Given the key role of inflammatory monocytes and their descendant macrophages in plaque pathogenesis, use of SWNTs as an immunotherapeutic tool to treat atherosclerosis shows high potential that could fill major gaps in the clinical cardiovascular treatment paradigm.⁴⁰ In addition, SWNTs can be loaded with many types of therapeutic small molecules via a noncovalent chemistry termed π–π stacking, while restricting intracellular delivery specifically to monocytes/macrophages, thereby potentially amplifying therapeutic effects while minimizing off-target effects. These strengths suggest the potential for SWNTs to become a platform immunotherapeutic tool in atherosclerosis and also in cancer, neurodegenerative and gastrointestinal diseases, and other inflammatory disorders involving these monocytes.

Notably, if the injected nanoparticles (e.g., hyaluronan nanoparticles⁷) are not targeted to circulating immune cells, they tend to be primarily taken up within the plaque via extravasation,⁷ a relatively inefficient and heterogeneous homing mechanism to target sites.^22,30,34 Nanoparticle delivery using immune-cell-mediated uptake may thus be considerably more robust than freely circulating nanoparticles for entry into atherosclerotic plaques for imaging and treatment. However, standardized assessments of this hypothesis remain to be performed.

Glycolysis Inhibition Therapy

Lipid-lowering agents, anti-inflammatory drugs, and drug-eluting stents have shown clinical promise in treating atherosclerosis over the past several decades.^8,35 However, atherosclerosis remains the leading killer in the United States, and improved treatments alone and in combination with standard therapeutics are energetically sought because the high rate of recurrent cardiovascular events in patients treated for atherosclerosis suggests that these standard treatments are incomplete.³⁶ Recent clinical trials suggest metabolic targets in diabetes and cancer offer therapeutic promise to reduce cardiovascular disease further.^13,37,38 In a parallel preclinical approach, Beldman and colleagues followed intriguing data on antiglycolytic effects on macrophages³⁹ to test the effects of glycolysis inhibitors as an atherosclerosis therapy.

Antiglycolytic therapies normalize vasculature in cancer, which improves treatment by enhancing chemotherapeutic efficacy and reducing metastasis.⁶ In contrast, antiglycolytics given to atherosclerotic mice stabilized plaques based on measures of collagen and smooth muscle content.⁷ Despite its apparent stabilizing impact on plaque, the effects of antiglycolytic therapy on enhancing cell death with postapoptotic necrosis are not well-understood,³⁹ stoking concerns of pro-atherogenic apoptotic cell debris accumulation.⁵ The recent development of pro-efferocytic therapies that stimulate plaque-dwelling macrophages to consume apoptotic cell debris^5,40 may offer resolution. A combination therapy of antiglycolytics and pro-efferocytics might act synergistically upon plaques to reduce plaque size more globally and to stabilize the remainder of the plaque (Figure 1).^7,40 Mice showed no significant change in plaque nanoparticle uptake following antiglycolytic therapy, a surprising result considering that the therapy resulted in vascular normalization and the consequent dissipation of vascular pores.⁷ The investigators attributed this finding to vascular heterogeneity, but why their observation is in contrast to those made in cancer remains unknown. It is plausible that glycolytic silencing of atherosclerotic endothelium may modulate other endothelial functions, such as immune cell recruitment, which remains to be investigated. Advanced tools and approaches, such as intravital microscopy (IVM), which enables quantitative microscopic examination at the subcellular scale in living animals in real-time, typically via fluorescence approaches (Figure 1),^21,41–45 may shed critical light on mechanisms underlying these observations, described below.

Figure 1.

Intravital microscopy schematic: investigating the mechanisms of atherosclerosis progression and therapy. In vivo fluorescent image of arterial blood vessels in a mouse using intravital microscopy (IVM). Single-walled carbon nanotubes (SWNTs) were injected and circulating monocytes within the vasculature took up SWNTs (grayscale, blue arrows). An atherosclerotic plaque schematic drawn in yellow (white arrow) illustrates a blockage in the artery with a necrotic core containing apoptotic cellular debris and macrophages (white arrow). New antiglycolytic therapy used to treat plaques can increase cellular debris but if combined with novel pro-efferocytic therapy might be used to enhance therapy synergistically by inducing macrophages to clear excess debris. Thus, IVM may be valuable to track, to quantify, and to optimize these therapeutic processes, in addition to other mechanistic questions troubling the field, by visualizing the microscopic dynamics in real-time in living subjects.

Studying the Mechanisms of Inflammatory Pathogenesis and Therapy via Imaging

Intravital microscopy has become a widely utilized tool to study pathogenesis and therapeutic impact across many normal and diseased physiologies, including in atherosclerosis, despite difficulties in tissue access.⁴⁶ Studies leveraging IVM suggest that leukocytes are recruited into plaques through microvessels and venules, corroborating some histological work¹⁸ but contrasting with other histological studies that suggest that inflammatory monocytes infiltrate from the luminal side of the plaque.³² It is likely that both microvascular and luminal extravasation occur but depend upon plaque model, stage, time point of visualization, and other factors. Indeed, Beldman et al. show that more advanced lesions generally permit less extravasation of injected nanoparticles compared with nascent lesions, and their work also suggests both microvascular (minor uptake) and luminal vessels (major) as extravasation contributors across lesion stages. The emergence of deeper IVM strategies using multiphoton, optical coherence tomography, and near-infrared (NIR) reporter (potentially including the NIR-II (second window of the NIR), with excellent tissue penetration) techniques may help resolve such questions on immune cell and nanoparticle access points, distributions, and behavior via dynamic, real-time, in vivo imaging of the interactions between immune cells,⁴⁷ between immune cells and nanomaterials, and between both nanomaterials and immune cells and the microvascular and large vessel endothelium.

Multicolor imaging of several immune cell subsets (up to at least 5) deeply within plaque tissues sets the stage to peel back the mechanisms of initiation and maintenance of the dysregulated immune state, leading to atherosclerosis. Unfortunately, however, much of the mechanism of nanoparticle delivery to plaques has not been studied; indeed, “the trafficking of systemically administered nanoparticles to atherosclerotic regions is poorly understood”,⁷ unlike in cancer, which has been well studied for multiple decades.^21,22,24,43 Use of nanoparticles to interrogate plaque behavior has at least two potential consequences, with the ability to provide evidence of (1) the location, magnitude, dynamics, and persistence of plaque vessel leakiness and molecular retention (as has been done in cancer studies^48,49) and (2) the dynamic spatiotemporal patterns of nanoparticle localization within plaques. Such data will bear strongly upon the diagnostic power of nanomaterials by supporting an understanding of what reporter signal in the plaque actually represents (e.g., nanoparticles within cells, within the necrotic core, within cellular interstitium, or on the endothelium; these nanoparticles may ultimately report their presence and location clinically via magnetic resonance imaging, positron emission tomography, computed tomography, photoacoustic imaging, and many other imaging modalities⁴¹) and their therapeutic potential. Given that many emerging nanotherapies are designed to function by modulating specific cell type(s), such as macrophages or T cells, over time, it follows that a dynamic analysis of the in vivo subcellular localization of the nanomaterials would provide a wealth of information that can inform selection of appropriate drugs and dosage regimens/frequency and, just as importantly, may hint at the drugs to avoid, based on the specific compartmentalization of the drug carriers. We envision that IVM of injected nanomaterials could resolve questions such as (1) above, stimulated by Beldman et al.’s study, as well as (i) clarify the dynamics of plaque progression’s negative effect on nanoparticle extravasation and how it relates to the increased collagen production across time and space (e.g., by analyzing collagen formation using second harmonic generation (SHG) strategies in connection with multiphoton IVM^50,51); (ii) offer insights to explain why endothelial junctional architecture improves as the plaque advances (e.g., based on visualizing key immune cells and molecules near the endothelium); (iii) inform the in vivo mechanisms of antiglycolytic therapy, including when and where the increased collagen (a marker of “stabilized” plaque) is deposited and the relationship between metabolism and inflammation in vivo (e.g., via use of fluorescent sugar analogues,^7,52 macrophage and activation markers, and other inflammatory markers^53,54); (iv) report the effect of glycolytic inhibition therapy on immune cell recruitment to the endothelium; and (v) reveal potential synergies of antiglycolytic and pro-efferocytic therapies as an effective combination antiatherosclerotic treatment regimen (Figure 1).

Fortunately, despite the lack of many dynamic IVM studies of atherosclerosis to date, enhancements in surgical techniques, access sites, and murine anesthesia indicate that novel observations highly relevant to atherosclerotic pathogenesis and therapeutic dissolution will emerge in the coming years. These insights, integrated with advanced flow cytometry, transcriptomic, and metabolomic methods,⁵⁴ will help researchers develop new ways to image/diagnose and to treat atherosclerotic plaques.

Intravital microscopy can be exploited to answer key questions about the effects of antiglycolytic therapy on tissues such as endothelium. For example, it is likely that the endothelial cell surface protein landscape is modulated in response to antiglycolytic therapy. To study this effect further, instead of using freely circulating nanoparticles, which support examination of pores in the vasculature, one could observe immune cell-mediated nanoparticle uptake and trafficking to monitor antiglycolytic therapeutic response of the plaque. To uncover mechanistic features of immune cell interaction with endothelium, blocking antibodies could also be used to implicate or to rule out specific receptors. Beldman and colleagues suggest that the endothelium is the target of the glycolytic inhibitor, despite the fact that the vascular permeability is not significantly changed in response to therapy using hyaluronan nanoparticles. Indeed, the evidence supporting the endothelium as a target is limited (e.g., antiglycolytic therapy showed selectivity for endothelial cells in vitro, despite the fact that metabolic imaging suggested in vivo uptake localized primarily in macrophages and smooth muscle cells). Perhaps there is a dose-dependent antiglycolytic effect in atherosclerosis similar to the effect in cancer, wherein high-dose antiglycolytics induce tumor endothelial cell death, whereas low doses stimulate vascular normalization.⁵⁵ Thus, such dose-dependent responses would need to be dynamically characterized under atherosclerotic conditions. Intravital microscopy constitutes an ideal centerpiece for a strategy to monitor endothelial cell health in real-time to assess the cell death/dose–response time course and uncover whether other factors (such as other cell types) may also be involved.

Intravital microscopy can be exploited to answer key questions about the effects of antiglycolytic therapy on tissues such as endothelium.

Clinical Translation of Plaque-Targeted Nanoparticles: Conclusions and Future Perspectives

Ultimately, despite their differences, atherosclerosis and cancer display certain striking similarities that support use of similar therapeutic strategies and analogous methods to reveal mechanistic insights. First, for example, the heterogeneity between and within plaques may favor a “Trojan Horse” approach of using nanoparticle-loaded immune cells mediating uptake into plaques for therapeutics and diagnostics, rather than freely circulating nanoparticles or drug molecules. Such strategies may enable more reliable delivery of therapeutic/diagnostic agents to plaques. Second, glycolysis inhibition therapy may help normalize atherosclerotic endothelial barriers and increase collagen production to help stabilize atherosclerotic plaques. Based on the mechanism of action of glycolysis inhibition, novel approaches for combination therapy with pro-efferocytic treatments should be tested in the future. Toward that end, the effects of antiglycolytic therapy on macrophage and endothelial function and viability require further study. Third, IVM can be used to tease out answers to a number of key preclinical questions mechanistically: (i) How does nanoparticle delivery to atherosclerotic plaques differ between immune-mediated and free nanoparticle strategies? Can immune-mediated strategies help reduce the effects of heterogeneous endothelial barrier function? (ii) What are the mechanisms underlying advanced plaques and glycolysis inhibition therapy driving endothelial junction stability? What bearing does endothelial continuity have on freely circulating and immune cell-mediated nanoparticle uptake into plaques, both in the microvasculature and in large vessels? (iii) How does combined antiglycolytic and pro-efferocytic therapy function, and what are the appropriate frequency and time points for optimal treatment? Insights gleaned from IVM data are thus expected to accelerate and, perhaps, to help identify clinically actionable, new, nanoparticle-based diagnostics and therapeutic strategies.^41,56,57

The overlapping molecular pathways in cancer and cardiovascular disease have important regulatory implications—although the cost of moving a new nanodrug from development into human studies is substantial, the potential to cross-utilize the same nanoparticle in two distinct patient populations provides a significant advantage to incentivize pharmaceutical companies to advance it. In addition, the distinct pathologies and temporal paradigms between atherosclerosis and cancer mean that a failure of the drug in one disease does not necessarily predict failure in the other—an important downside protection to investment risk.

Insights gleaned from intravital microscopy data are thus expected to accelerate and, perhaps, to help identify clinically actionable, new, nanoparticle-based diagnostics and therapeutic strategies.