Nanotherapies for Treatment of Cardiovascular Disease: A Case for Antioxidant Targeted Delivery

Abstract

Purpose of Review

Cardiovascular disease (CVD) involves a broad range of clinical manifestations resulting from a dysfunctional vascular system. Overproduction of reactive oxygen and nitrogen species are causally implicated in the severity of vascular dysfunction and CVD. Antioxidant therapy is an attractive avenue for treatment of CVD associated pathologies. Implementation of targeted nano-antioxidant therapies has the potential to overcome hurdles associated with systemic delivery of antioxidants. This review examines the currently available options for nanotherapeutic targeting CVD, and explores successful studies showcasing targeted nano-antioxidant therapy.

Recent Findings

Active targeting strategies in the context of CVD heavily focus on immunotargeting to inflammatory markers like cell adhesion molecules, or to exposed extracellular matrix components. Targeted antioxidant nanotherapies have found success in pre-clinical studies.

Summary

This review underscores the potential of targeted nanocarriers as means of finding success translating antioxidant therapies to the clinic, all with a focus on CVD.

Cardiovascular disease (CVD) encompasses a broad range of clinical manifestations that all involve a dysfunctional vascular system, for example hypertension, peripheral arterial disease, myocardial infarction (MI) and stroke[1]. In response to vascular injury incurred due to these cardiovascular diseases or physical therapeutic interventions like balloon angioplasty and stenting, the vasculature undergoes vessel remodeling involving significant alterations in cellular behavior. Vascular injury and the resultant vascular response remains a significant problem resulting in clinical complications of CVD [2].

A key early pathogenic event that underlies CVD is endothelial dysfunction [1, 3–8]. Endothelial dysfunction manifests as enhanced endothelial leukocyte recruitment, impairment of endothelial anti-thrombotic activity, increased endothelial permeability and impaired endothelial-dependent vasodilation. An inherent feature of endothelial dysfunction is impairment of nitric oxide (NO) bioavailability [1, 3]. NO is an important endothelial-derived vascular signaling molecule [3, 4, 9] necessary for the anti-inflammatory and anti-coagulant actions of the endothelium, regulating vascular tone [10] and arterial pressure [11], as well as inhibition of platelet aggregation [12], smooth muscle cell (SMC) proliferation and growth [13] and also leukocyte adhesion to the endothelium [14]. Finally, it is critical for endothelium-dependent vasodilation [1].

The extent of endothelial dysfunction directly correlates with an increased risk of CVD severity [15, 16] or cardiovascular clinical events [17–19] in humans. Additionally, endothelial dysfunction is prognostic for predicting the risk of future cardiovascular clinical events, such as MI, in coronary artery disease patients [19–21]. Due to the clinical relevance of impaired NO bioactivity and endothelial dysfunction there is great interest in understanding the underlying causes. A large body of evidence points to a major role for reactive oxygen and reactive nitrogen species (ROS, RNS) during cardiovascular disease, in the impairment of NO bioavailability, and damage to the endothelial glycocalyx, inter-endothelial junctions and the sub-endothelial matrix, ultimately causing endothelial dysfunction [1, 3, 8, 22]. Key ROS and RNS that are implicated are superoxide (O₂•⁻) [3, 5, 9], peroxynitrite (ONOO⁻), and hydrogen peroxide (H₂O₂). Furthermore, ROS and RNS are causally implicated in the pathogenesis of the vascular response to injury in multiple settings of cardiovascular disease including: atherosclerosis [23], abdominal aortic aneurysm [24, 25], restenosis [26], and ischemia reperfusion injury [27].

Due to the important pathogenic roles of ROS and RNS, the use of antioxidants and antioxidant enzymes as therapeutics in vascular injury is of substantial interest. However, one of the limitations facing the clinical translation of antioxidants and antioxidant enzymes is their accumulation in sufficient quantities at localized sites of vascular injury. Herein we will discuss the role of ROS/RNS in vascular injury and which antioxidants and antioxidant enzymes are being examined as therapeutics. Then we will discuss the current state of nanomedicine that is being investigated to target these therapies to sites of vascular injury.

Role of Rective Species in CVD

A critical role for O₂•⁻ as a cause of endothelial dysfunction has emerged through numerous clinical and experimental studies [1]. Diseased vessels retrieved from hypertensive, diabetic or atherosclerotic animals and humans are all characterized by elevated vascular O₂•⁻ levels [1, 23]. Acute intra-arterial administration of ascorbate, at high millimolar concentrations that prevent O2•⁻-mediated NO scavenging [28], into patients with diabetes, coronary artery disease, hypertension or chronic smokers, improves endothelium-dependent vasodilation [29–34]. In addition, Landmesser and co-workers [35] reported that the extent of reduction in the O₂•⁻-dismutating enzyme, vascular extracellular superoxide dismutase (SOD3), was closely related to the degree of impairment of endothelial function in humans. One mechanism by which O₂•⁻mediates injury is likely its rapid reaction with NO to simultaneously impair NO bioactivity and yield the oxidant, ONOO⁻. ONOO⁻ also plays an important role in the impairment of NO bioavailability through multiple actions [3, 4, 8, 36, 37]. Moreover, O₂•⁻ has been shown to be directly involved in several cardiovascular pathologies (e.g. atherosclerosis [23, 38], hypertension [39], myocardial infarction (MI) [40] and ischemia reperfusion [27], heart failure [41], and abdominal aortic aneurysm (AAA) [24, 25]) as well as following physical therapeutic interventions [26].

Atherosclerosis is a common underlying cause of a diverse range of cardiovascular clinical events. It is a chronic process involving progressive changes to arterial structure and function [38], characterized by vascular inflammation and endothelial dysfunction. Initial studies by Barry-lane et al. demonstrated a role for NADPH-oxidase (NOX)-derived O₂•⁻ in experimental atherosclerotic lesion development [42]. A life-threatening disease that commonly accompanies atherosclerosis in the aged population is AAA [43]. Previous reports have shown increased formation of O₂•⁻ as well as expression of NOX and the p47phox subunit of NOX1/2 in human aneurysmal aorta compared with adjacent healthy tissue from the same individuals [25]. More recently, genetic deletion of all vascular isoforms of NOX (NOX1, 2 and 4) as well as the p47phox subunit for NOX1 and 2[44], significantly reduced the incidence of experimental AAA[44, 45]. Taken together, these studies highlight an important role for O₂•⁻ in common cardiovascular pathologies. It has additionally been shown to be of importance to the pathogenesis of vessel remodeling following physical therapeutic interventions.

Vascular interventions such as percutaneous transluminal angioplasty and stenting are associated with endothelial denudation and SMC apoptosis, followed by re-endothelialization coupled with severe endothelial dysfunction[46], and increased rates of restenosis and thrombosis. Restenosis typically occurs due to the proliferation and migration of vascular SMCs and adventitial myofibroblasts: a process known as neointimal hyperplasia. NOX-derived O₂•⁻ is directly implicated in the pathogenesis of neointimal hyperplasia in multiple experimental models of restenosis [2, 47, 48]. Indeed, expression of NOX-1, NOX-4 and their subunits gp91phox and p22phox are increased in rat carotid arteries following balloon injury alongside enhanced O₂•⁻ levels[49]. NOX1 and NOX2 deficiency reduces experimental neointimal formation and leukocyte accumulation [50, 51]. These observations indicate that O₂•⁻ plays a critical role in vascular injury.

O₂•⁻ typically undergoes spontaneous or catalyzed dismutation to yield H₂O₂ [52, 53]. H₂O₂ is a non-radical, uncharged, weak oxidant that readily diffuse across cellular membranes[1]. Several antioxidant enzymes including catalase regulate cellular levels of H₂O₂. H₂O₂ that is not removed by these enzymes can oxidize or induce S-glutathionylation of critical protein cysteine residues of signaling proteins or effector enzymes to mediate both physiological and pathological cell signaling [1]. H₂O₂ is also an essential substrate of peroxidases including the innate immune cell-derived peroxidase, myeloperoxidase (MPO), which is capable of catalyzing a variety of deleterious oxidative reactions which play a significant role in vascular injury such as atherosclerosis and AAA[54]. For example, one of the main products of MPO is hypochlorous acid (HOCl), and HOCl-modified proteins and low-density lipoprotein have been found in ECs and human atherosclerotic lesions [55–58]. Additionally, protein tyrosine nitration is another hallmark of both the presence of ONOO⁻, or nitrating radicals (NO₂•) produced due to H₂O₂-MPO-mediated nitrite oxidation[54], which has been found in human atherosclerotic lesions[59]. Additionally, nitration is well-reported in vascular remodeling as a result of therapeutic interventions[60].

Antioxidants as therapeutics for CVD

Due to the ubiquitous role of O₂•⁻ and downstream oxidants such as H₂O₂ and ONOO⁻ in mediating endothelial dysfunction and pathogenesis of vascular injury; research efforts have identified the superoxide dismutase (SOD) enzymes and SOD mimics, as well as catalase as potential antioxidant therapeutics to limit cellular levels of O₂•⁻ and H₂O₂ respectively. In a murine experimental model of atherosclerosis, overexpression of catalase alone or overexpression of cytosolic SOD (SOD1) and catalase in combination reduced levels of plasma and aortic F2-isoprostane and attenuated the development of atherosclerosis [61]. Overexpression of both catalase and SOD3 individually has also been beneficial in experimental models of restenosis. For example, in a model of CaCl₂-induced AAA in mice, administration of riboflavin reportedly enhanced SOD activity and reduced vascular O₂•⁻ levels as well as prevented AAA formation [62]. SOD3 overexpression has proven beneficial in experimental models of restenosis [63–65]. Local catheter-mediated gene transfer of SOD3 to the arterial wall reduced restenosis and decreased the number of macrophages 2 and 4 weeks after the gene transfer compared with AdLacZ controls [64]. Additionally, SOD3 could attenuate tyrosine nitration in a rabbit balloon injury model [66]. With respect to catalase, overexpression of human catalase gene in vascular SMCs resulted in their resistance to Ang-II induced aortic wall remodeling thought to be involved in early AAA formation [67]. Additionally, in a CaCl₂-induced mouse model of AAA, both transgenic mice overexpressing catalase and mice administered PEG-Catalase were shown to exhibit decreased MMP activity, decreased vascular SMC apoptosis and decreased AAA formation [68].

In addition to antioxidant enzyme several small molecules have been tested in clinical trials to treat CVD. The use of alpha lipoic acid for the prevention of heart disease trials (NCT00765310 and NCT00765310) are expected to report results this year. The use of N-acetyl cysteine for preventing atrial fibrillation trial (NCT00765310) is expected to conclude this year as well. Additionally, a wide range of small molecule antioxidants ranging from flavonoids, quinones, and electrophiles have been tested for different CVD with various degrees of success. For a review on antioxidant treatment see Buglak et al., 2018 [69]. Despite some ongoing promising trials, the majority of trials looking to correct redox dysfunction to treat CVD have been largely unsuccessful. One variable that may explain early failures of redox-active molecules in clinical trials is the delivery method, as most clinical trials assess the effect of systemic delivery of a drug. We propose that nanotechnology-assisted targeted delivery may be a critical approach to translate new cardiovascular therapies to the clinic. Cardiovascular events with localized redox balance disruption would benefit tremendously from the development of targeted therapeutic strategies that aim to deliver redox-modulating molecules directly and preferentially to the diseased site [69]. Nanotechnology has the potential to revolutionize redox based therapeutics by maximizing the effects of the drug in the site of interest, potentially overcoming the disappointment of clinical trials and advancing a new generation of therapies for CVD. While still a field in its infancy, pre-clinical studies making use of targeted nanocarriers and molecules with broad antioxidant effect are promising. In the context of CVD three modalities of such therapeutics have been employed: 1) ROS-responsive moieties for targeting where the released therapeutic is NOT an antioxidant; 2) ROS-responsive moieties for targeting where the released therapeutic IS an antioxidant (including those where the antioxidant is both the probe and therapeutic); and 3) targeted delivery of an antioxidant as therapeutic.

Nanoparticles as delivery vehicles

Therapeutic targeting oftentimes involves the use of nano sized delivery vehicles. Nanoparticles (NPs) in specific are attractive for the task based on their intrinsic properties including, the ability to tailor their size, high surface to volume ratio, and tunable physicochemical properties [70]. NPs transport therapeutics on their surface or their interior, achieved through, non-covalent interactions, conjugation or encapsulation [70, 71]. Common scaffolds for the design of drug delivery NPs include liposomes, polymeric micelles, drug conjugated polymers, and dendrimers, among others [70]. The ideal drug vehicle NP formulation should be stable, biocompatible, non-immunogenic, and amenable to modifications for adaptable site-specific delivery of therapeutics [71]. Importantly, NPs can be functionalized to actively target accumulation and drug delivery at a specific site. This functionalization of the particle with a ligand often involves a peptide, protein, antibody, or otherwise interacting partner to a receptor or epitope overexpressed at the diseased site. Physical targeting uses external forces such as electromagnetic fields to control localization and release of cargo, namely magnetic particles [70]. For the purpose of this review, we will focus on active targeting. Reviews that illustrate the use of a wide array of targeting strategies with NPs in the context of vascular disease can be found here [71], and here [72].

Current State of Nanotherapeutics in Cardiovascular Disease Treatment

Nanotherapeutic development for disease prevention and treatment is still in its early stages. It is estimated since 2013 the FDA has approved a modest yet significant number of nano-based therapies, a close to threefold increase from previous years. The large majority of approved nanotherapies are marketed towards cancer treatment, where drug toxicity is of great concern. Approved nanotherapeutics such as the liposomal Irinotecan (Onivyde®) for the treatment of pancreatic cancer allow for increased drug bioavailability and circulation time, decreasing accumulation in unrelated areas and associated off target effects [73]. It is undeniable that the recent push for approval of nanotherapeutics is a testament to their potential in the treatment of disease. It is certain that future endeavors in CVD medicine will also see a shift towards the use of these attractive therapeutic modalities.

A brief search on PUBMED for studies related to targeted nanotherapies in cancer and CVD in the past five years (figure 1) reveals the substantial disparity between research in these two disease groups, with CVD related publications making up 11% of the cancer related publications in average. However, the field of targeted nanotherapeutics for treatment of CVD has been in steady development. Table 1 summarizes the targeted nanotherapies published in the context of CVD while Table 2 focuses on active targeted nano-antioxidant therapies.

Figure 1.

Number of PUBMED publications in the past five years (2014–2019) focused on cancer versus CVD targeted nanotherapies. Keywords: targeted delivery (disease) nanomedicine.

Table 1.

Targeted Nanotherapies for Treatment of Cardiovascular Disease

Disease	Target	Targeting Method	References
Endothelial Dysfunction	PECAM-1 (CD31) or ICAM-1 (CD54)	Targeting antibody	[75]
	PECAM-1 (CD31)	Targeting antibody	[74]
	ICAM-1 (CD54)	Targeting antibody	[76]
	P-selectin	Targeting peptide, sequence LVSVLDLEPLDAAWL	[107]
Atherosclerosis	Collagen IV	Targeting peptide, sequence KLWVLPKGGGC	[108], [88], [109], [110]
	LDL receptors	Previous studies suggest that liposomes comprised of at least 75% anionic phospholipids can combine with LDL-Cholesterol, leading to the formation of complexes that are cleared via the LDL receptors	[93]
	E-selectin	E-selectin-targeting multistage vector (ESTA-MSV) N-(2-hydroxypropyl)methacrylamide (HPMA)-based E-selectin binding copolymer	[83], [77]
	VCAM-1	Targeting peptide, sequence VHPKQHR (homologous to very late antigen-4)	[84], [90], [111], [86], [85], [87]
	Macrophage Scavenger Receptor 1 (MSR1) and CD36 scavenger receptor	Oxidized LDL mimic	[94]
	Stabilin-2	Targeting peptide, sequence CRTLTVRKC	[112]
	αvβ3 integrin	Peptidomimetic αvβ3-integrin antagonist Cyclo RGD (cRGD) peptide	[113], [88]
	Phosphatidyl serine on apoptotic cells	Annexin V conjugation	[92], [91]
	Apolipoprotein A1	Apolipoprotein A1-mimetic peptide 4F	[89]
	Fibrin clot	Targeting peptide, sequence CREKA	[114], [115]
	p32	LyP-1	[115]
Restenosis	Lectin-like oxidized low-density lipoprotein receptor-1 (LOX1)	Targeting antibody	[95]
	Inflammatory microenvironment responsive	pH responsive acetalated β-cyclodextrin (β-CD) material and ROS responsive through hydrophobic functionalization of β-CD with an oxidation-labile group	[98]
	Collagen IV	Targeting peptide, sequence KLWVLPKGGGC Platelet derived membrane (GPVI, α2β1)	[96] [97]
Aneurysm	Elastin	Targeting antibody	[116]
	Collagen	CNA-35, a natural collagen binding protein	[117]
	P-selectin	Fucoidan	[118]
	αvβ3 integrin	Targeting peptide, sequence Arg-Gly-Asp (RGD)	[119], [120]
	Fibrin	DMAB, a cationic amphiphile	[121]
Ischemia Reperfusion Injury	EC-Neutrophil interaction	Neutrophil-derived nanovesicles	[122]
	P-selectin	Targeting Antibody	[103]
	Ischemic myocardium	Ischemic targeting peptide, sequence CSTSMLKAC decorated cardiosphere-derived cell exosome vesicles	[104]
	Cardiac troponin I	Modified Vascular Endothelial Growth Factor	[123]

Terms of interest: Endothelial cells (EC), Reactive oxygen species (ROS), Immunoliposomes (ILP), Immunoparticles (IP), NADPH oxidase (NOX), Lipopolysaccharide (LPS), Ischemia-reperfusion (I/R)

Table 2.

Antioxidant Mediated Targeted Nanotherapies for Treatment of Cardiovascular Disease

Disease	Target	Ligand	Antioxidant Mediator	Outcome	Reference
Endothelial Dysfunction	PECAM-1 (CD31)	Anti-PECAM-1 Antibody	MJ33, an indirect suppressor of NOX2activity	PECAM-1 targeted ILPs specifically bound to ECs and attenuated angiotensin II-induced ROS production in vitro and in vivo with dose dependent VCAM-1 inhibition. Treatment alleviated pathological endothelial permeability in ECs exposed to VEGF and in a mouse model of acute lung injury induced by bacterial LPS.	[78]
	PECAM-1 (CD31)	Anti-PECAM-1 Antibody	EUK-134, a synthetic SOD and catalase mimetic	PECAM-1 targeted PEGylated ILPs loaded with EUK-134 inhibited cytokine-mediated inflammatory activation in vitro, and accumulated in lungs after intravascular injection, providing <60% protection against pulmonary edema in bacterial endotoxin-challenged mice	[79]
	PECAM-1 (CD31)	Anti-PECAM-1 Antibody	SOD or catalase antioxidant enzymes	PECAM-1 targeted F127 pluronic tri-block copolymer based IPs loaded with CuZnSOD or catalase antioxidant enzymes localized to the pulmonary vasculature, delivering about 33% of the injected therapeutic dose within 30 minutes. Treatment of mouse model of endotoxin-induced lung injury with catalase IPs exhibits a protective effect from hydrogen peroxide induced damage, diminishing pulmonary edema and leukocyte infiltration; whereas treatment with SOD IPs reduces cytokine induced EC inflammatory activation and lung inflammation in the same mouse model.	[80]
	PECAM-1 (CD31)	Anti-PECAM-1 Antibody	Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), a synthesic SOD mimetic and tocopherol phosphate	PECAM-1 targeted F127 pluronic tri-block copolymer based IPs loaded with MnTMPyP and tocopherol phosphate bound to ECs in cell culture with nearly 60% protection against stimulation of VCAM-1 synthesis.	[81]
	PECAM-1 (CD31) or ICAM-1 (CD54)	Anti-PECAM-1 or ICAM-1 Antibodies	SOD antioxidant enzyme	Intravenously injected ICAM-1 IPs accumulated in the pulmonary vasculature to a lesser extent than PECAM-1 IPs. Even so, ICAM-1 IPs more effectively inhibit cytokine-induced VCAM-1 expression in the lungs, resulting in a 3.5 fold increase in anti-inflammatory potency compared to PECAM-1 IPs.	[82]
	PIvap (PV1)	Anti-PIvap Antibody	SOD antioxidant enzyme	PV1 conjugated SOD enzyme IPs bound to the endothelium to a lower degree that PECAM-1 targeted particles, however at the same concentration PV1, but not PECAM-1 IPs inhibited bacterial LPS induced VCAM-1 expression in the lung endothelium of mice.	[124]
	PIvap (PV1)	Anti-PIvap Antibody	SOD antioxidant enzyme	Tri-molecular conjugates of ferritin with an PIvap antibody ligand and loaded with antioxidant enzyme cargo, SOD, localized to caveolar endosomes of pulmonary ECs, alleviating LPS induced inflammation in mouse model.	[125]
Atherosclerosis	Oxidized LDL	Apolipoprotein A1-mimetic peptide 4F	ROS responsive release of therapeutic Ac-2-26	In vitro macrophage LPS and IFN-γ mediated activation was significantly reduced after 24 hour treatment with the targeted ROS cleavable peptide amphiphiles.	[126]
	Scavenger receptor type B-1 (SR-B1) and other putative receptors	HDL mimic	Nitric oxide	SNO loaded HDL NPs mimic HDL functionality with no toxicity to ECs, VSMCs and macrophages. NPs ameliorate I/R injury in a mouse kidney transplant model, and atherosclerotic plaque burden in a mouse model of atherosclerosis.	[127]
	CD36 scavenger receptor	1-(Palmitoyl)-2-(5-keto-6-octene-dioyl) phosphatidylcholine (KOdiA-PC) a CD36-targeted ligand found on oxLDL	Epigallocatechin-3-gallate	NPs present high binding affinity to macrophages and increase internal epigallocatechin-3-gallate content while decreasing mRNA levels and secretion of the inflammatory chemokine MCP-1.	[128]
Restenosis	Collagen IV	Targeting peptide, sequence KLWVLPK	Nitric oxide	Single bolus intravenous injection of NO releasing nanofibers at the time of injury continues to inhibit neointimal hyperplasia up to 7 months post-balloon angioplasty surgery in rat model.	[99]
	Laminin	Epigallocatechin-3-gallate	Epigallocatechin-3-gallate	Gold NPs functionalized with the green tea polyphenol are internalized in vitro by VSMCs and ECs. NP formulations are less cytotoxic to both cell groups compared to free drug. NP treatment show inhibition of migration of VSMCs without impairment of EC proliferation.	[129]
Aortic Aneurysms	Integrin targeting, and macrophage-mimetic	Targeting peptide, sequence cRGDfK coupled with use of macrophage cell membrane	ROS responsive release of rapamycin	After iv treatment in AAA rat model, NPs showed great antianeurysmal activity by attenuating the expansion of the aortic diameter, preventing calcification, decreasing elastin degradation, and maintaining endothelial integrity, as well as dampening inflammation and lowering generation of ROS in aneurysmal tissues.	[102]
	Elastin	Targeting antibody	Pentagalloyl glucose (PGG), a tannic acid derivative	PGG-loaded albumin NPs with a surface-conjugated elastin-specific antibody reduced AAA development, a consequence of reduction in macrophage recruitment, matrix metalloproteinase activity, and elastin degradation and calcification	[100]
	Elastin	Targeting antibody	Pentagalloyl glucose (PGG), a tannic acid derivative	Treatment with EDTA loaded NPs with a surface-conjugated elastin-specific antibody, followed by PGG loaded NPs of the same formulation led to reduction in macrophage recruitment, MMP activity, elastin degradation and calcification in the aorta of advanced AAA mouse model.	[101]
Ischemia Reperfusion Injury	Sites of increased H2O2production	H2O2-responsive antioxidant polymer vanillyl alcohol-incorporating polyoxalate (PVAX)	Vanillyl alcohol	PVAX NPs significantly upregulated the expression of angiogenic VEGF and PECAM-1 and enhanced the blood perfusion into ischemic tissues in a mouse model of hind limb I/R. At 7 days post-ischemia, the PVAX NPs-treated hindlimb showed remarkable regenerated muscle tissues with minimal inflammatory responses.	[105]
	Sites of increased H2O2 production	H2O2-responsive antioxidant polymer vanillyl alcohol-incorporating polyoxalate (PVAX)	Vanillyl alcohol	PVAX effectively suppressed the generation of ROS caused by I/R, significantly reduced the level of NOX2and NOX4 expression, which favors the reduction in ROS generation after I/R in a mouse model of hind limb I/R.	[106]

Terms of interest: Endothelial cells (EC), Reactive oxygen species (ROS), Immunoliposomes (ILP), Immunoparticles (IP), NADPH oxidase (NOX), Lipopolysaccharide (LPS), Ischemia-reperfusion (I/R)

Targeting the activated endothelium

Studies focused on targeting the activated endothelium are primarily through conjugation of monoclonal antibodies or their light chain variable fragments to imaging or drug-loaded liposomes, (immunoliposomes - ILPs) or polymeric nanoparticles, (immunoparticles - IPs). Main targets include platelet-endothelial cell adhesion molecule (PECAM-1) [74–75], and intracellular adhesion molecule (ICAM-1) [75,76]. PECAM-1 and ICAM-1 are transmembrane glycoproteins expressed in the membrane of endothelial cells. PECAM-1 is involved in vascular endothelial permeability and leukocyte transmigration in response to inflammatory insult [77]. ICAM-1 contributes to adhesion of activated leukocytes to endothelial cells (ECs) at sites of inflammation. ICAM-1 is expressed constitutively in quiescent vascular endothelial cells; however its expression is promptly upregulated in response to NFκB-mediated inflammatory activation of the endothelium through the actions of cytokines, oxidants, and aberrant flow [76]. PECAM-1 and ICAM-1 targeting provides the advantage of targeting carriers for both preventative and therapeutic treatment [76].

Coupling antioxidant targeted delivery to the activated endothelium

Endothelium exposure to circulating pro-inflammatory signals lead to NOX activation and production of high levels of O₂•⁻, which in turn activates NFκB-driven inflammation. Acute endothelial dysfunction is further aggravated through recruitment of leukocytes, which become active to produce further reactive species in the area. Recently, two separate studies coming from the same laboratory successfully breached the gap between antioxidant therapy and targeting with promising results. In the first study, Hood et al. synthesized liposomes decorated with anti-PECAM-1 antibody loaded with the therapeutic MJ33. While not a traditional antioxidant, MJ33 inhibits reactive species production through indirect inhibition of NOX2 activation. Analysis of MJ33 concentration in lung tissue via mass spectroscopy after intravenous injection of targeted vs non-targeted liposomes reveals a 200-fold concentration increase of MJ33 compared to non-targeted liposomes. Moreover, angiotensin II mediated production of ROS was effectively inhibited by targeted immunoliposomes (ILs) in cell culture and in mouse lungs. Importantly, injection of targeted ILs prior to tracheal lipopolysaccharide exposure suppresses vascular cell adhesion molecule 1 (VCAM-1) expression, a marker of inflammation. Lastly, prophylactic and therapeutic modes of PECAM-1-targeted MJ33 intervention attenuates pulmonary edema by approximately 50% [78]. In the more recent study by Howard et al. PECAM-1 targeted PEGylated ILs were loaded with the synthetic manganese-porphyrin complex EUK-134, a therapeutic that has a dual role as a catalytic scavenger of O₂•⁻ and H₂O₂, imitating the function of SOD and catalase. Preliminary results in this study conclude that prophylactic IL therapy mitigates pulmonary edema [79].

Hood et al. reported synthesis of PECAM-1-targeted Protective Antioxidant Carriers (PACkET) with efficient loading of SOD1 and catalase antioxidant enzymes. The particles exhibit specific EC binding and internalization both in vitro and in vivo, validating previous PECAM-1 targeted studies[74–76]. Targeted catalase PACkETs treatment of ECs exposed to toxic levels of H₂O₂ provided protection against cell death. Treatment of LPS challenged mice with catalase PACkET particles reduced pathological elevation of protein levels as well as leukocyte infiltration. Treatment with SOD but not catalase PACkETs leads to a marked reduction in VCAM-1 expression. Analysis of lung homogenates of SOD PACkETs treated EPS challenged mice confirms the particles as inhibitors of VCAM-1 expression, further validated by the decrease in inflammatory cytokines like TNF and MIP2 [80].

In another exciting article Howard et al. report the successful synthesis of a PECAM-1-targeted pluronic based copolymer particle loaded with dual bioactive compounds, an SOD mimetic Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP) and tocopherol phosphate, a derivative of the α-tocopherol form of vitamin E which is thought to function primarily as an anti-inflammatory signaling agent. In vitro results show EC uptake with protection against upregulation of inflammatory markers observed at both the RNA and protein levels [81].

Shuvaev et al. report SOD-loaded particles conjugated to ICAM-1 results in limited EC endocytosis compared to the more studied PECAM-1 conjugated particles. At comparable doses, however, ICAM-1 targeted particles result in more potent anti-inflammatory effect assessed by VCAM-1 expression after cytokine exposure in a mouse model [82].

Endothelial Dysfunction Markers as a Strategy to Target Atherosclerosis

The atherosclerotic plaque milieu is characterized by endothelial dysfunction coupled with chronic inflammation. Hence, targeting strategies include IP conjugation of small peptides of known affinity to proteins overexpressed in response to inflammation like is the case with E-selectin [83, 77] and VCAM-1 [84]. E-selectin is upregulated threefold in the aorta of ApoE ^−/− mice under high fat diet [77]. A recently developed drug-free E-selectin-targeted copolymer decreases atherosclerotic burden by significantly decreasing the necrotic core area in the plaque, reducing leukocyte recruitment, and modifying plaque macrophages towards an anti-inflammatory phenotype [77]. VCAM-1-targeted agents have used for successful optical and magnetic resonance imaging of atherosclerotic plaque in atherosclerotic ApoE ^−/− mice [84, 85] and successful delivery of mi-RNA treatment [86, 87]. Effective targeting of activated endothelium using VCAM-1 conjugated IPs has been reported in vitro [86, 87]. Kheirolomoom et al. report drug loaded carrier was able to inhibit atherosclerosis development in ApoE^−/− mice without major off-target effects. [87]

An angiogenic marker called αvβ3 integrin has also been used as a target as it is expressed in vascular ECs and macrophages present in atherosclerotic regions. Atherosclerotic targeting capacity of αvβ3 integrin was studied using targeted IPs loaded with iron oxide NPs, and functionalized with a fluorophore for dual magnetic resonance and optical imaging of atherosclerotic ApoE^−/− mice. Efficient plaque targeting was shown after iv injection in mice. [88]

Other Targets for Drug Delivery to the Atherosclerotic Plaque

Apolipoprotein A1 (ApoA1) is a major component of HDL particles, with lipid binding, transport, and metabolism heavily dependent on its secondary structure. Targeting ApoA1 mimetic peptide localizes to regions of atherosclerotic plaque based on its high affinity binding to oxidized lipids like oxLDL. So et al. report ApoA1 targeted peptide amphiphile nanofibers reach atherosclerotic sites in low-density lipoprotein receptor-deficient (Ldlr^−/−) mice with high specificity [89].

Another interesting approach is targeting phosphatidyl serine (PS) by conjugating annexin V to NP [90]. PS is normally sequestered in the inner leaflet of the cell membrane and it is translocated and exposed in apoptotic cells like is the case with foam cells in the atherosclerotic lesion. PS targeting has been used in the context of atherosclerosis for fluorescent [91] and magnetic resonant imaging (MRI) [91, 92]. Van Tilborg et al. report in vivo MRI and ex vivo fluorescence imaging of excised aortas showing dual fluorescent and MRI Annexin V conjugated NPs localized mainly to the aortic arch, principal branches of the abdominal aorta, and at the aortic bifurcation into the iliac arteries of ApoE^−/− mice [91]. Annexin V-conjugated magnetoradioisotopic NPs injected into Watanabe heritable hyperlipidemic and Watanabe Heritable Hyperlipidemic Myocardial Infarction rabbits localized to atheroma rich areas as confirmed by MRI and histology [92].

Based on previous studies highlighting the interaction of anionic phospholipids with receptors involved in lipid metabolism, Krishna et al. designed and studied a heavily anionic liposomal nanoformulation thought to intermingle with LDL cholesterol, forming complexes that are cleared by LDL receptors in hepatocytes and macrophages. Intravenous injection of (Ldlr^−/−) mice with liposomes over a period of four weeks resulted in decreased atherosclerosis severity in this mouse model [93].

Lewis et al. report synthesis of an innovative nanoformulation for treatment of lipid enriched atherosclerotic plaque through the use of sugar-based amphiphilic macromolecules that mimic the charge and hydrophobicity of oxidized lipoproteins and bind both scavenger receptors MSR1 and CD36 with high affinity. When administered to ApoE^−/− mice, the NPs show increased binding to atherosclerotic lesion and reducing lipid accumulation at the site [94].

Targeting Arterial Injury to Inhibit Restenosis

Immunotargeting through the use of IPs has been successfully used with conjugation of lectin-like oxidized low-density lipoprotein receptor-1 (LOX1) antibodies [95]. LOX1 expression is significantly upregulated in endothelial and vascular SMCs of restenotic vessels with peak expression seven days post carotid balloon injury in rat model. Single intravenous injection 72 hours post-surgery of the LOX1 ILPs carrying a potent rho kinase inhibitor lead to significant inhibition of neointimal hyperplasia [95].

Surgical intervention for treatment of severe atherosclerosis leads to endothelial denudation and exposure of collagen in the vascular bed membrane. Collagen IV-targeted polymeric NPs loaded with the anti-proliferative drug paclitaxel localizes to injured vessels and effectively diminishes neointimal growth and restenosis after rat carotid intervention [96]. Another study focused on targeting collagen IV for prevention of restenosis took an interesting approach by using platelet membrane-derived NPs which interact with known putative targets of platelets. These particles exhibit platelet mimicry with low immunogenicity and selective adhesion to damaged human and rodent vasculature. Docetaxel loaded platelet membrane-derived NPs injected into rats undergoing carotid balloon angioplasty show selective particle binding to the artery denuded of endothelium two hours after administration. Treatment leads to significant inhibition of restenosis compared to control [97].

Redox-based targeted therapies to inhibit Restenosis

Since, the inflammatory microenvironment in the diseased vasculature is characterized by the development of mild acidity and high production of ROS, local changes in the environment present a strategy for targeting and drug delivery. As such Feng et al. created pH-, and ROS-responsive NPs loaded with rapamycin that effectively attenuated neointimal hyperplasia in a rat model of arterial stenosis [98]. Additionally, Bahnson et al. report successful collagen-targeting of NO releasing peptide amphiphiles nanofibers to sites of injury arterial injury. The nanofibers promote a sustained effect of long term neointimal hyperplasia inhibition in a rat model of restenosis [99].

Targeted Drug Delivery to Aortic Aneurysm

Destruction of the extracellular matrix through the action of matrix metalloproteinases is the major contributing event to progression of AAA. Elastin-conjugated IPs have been a popular option for targeting drug carriers to sites of AAA. In two different studies coming from the same investigator, targeting of elastin antibody conjugated IPs to a rat model of AAA has found success. After a previous study determined local application of pentagalloyl glucose (PGG), a tannic acid derivative, to an AAA rat model hinder the development of AAA, the investigator produced PGG-loaded albumin NPs with a surface-conjugated elastin-specific antibody. After induction of AAA, single bolus injection of IPs shows enhanced targeting to the site of elastin damage with persistent residence for two weeks after injection. Importantly, IP treatment lead to attenuation of AAA development through reduction in macrophage recruitment, matrix metalloproteinase activity, and elastin degradation [100]. In a follow-up study, the investigators tested their delivery system in a more clinically relevant model of aortic aneurysm presenting elastin damage and calcification. Similar IPs were loaded with PGG or with EDTA, to promote de-calcification. Treatment of AAA with EDTA IPs followed by PGG IPs reverses calcification and offers protection from further calcification and damage, which led to improvement in tissue biomechanical parameters and reversal of aneurysmal dilation [101].

Coupling Redox Functionality in AAA-Targeted Therapeutics

In an interesting approach to AAA targeting, Cheng et al. report synthesis of a macrophage cell membrane-based IP encapsulating rapamycin drug and decorated with a ligand to target integrins. In addition to the targeted and bio-mimetic stealth features, the particle features a ROS- responsive delivery of drug. Effectiveness of therapy is shown as the intravenously injected ROS-responsive IPs more effectively prevented aneurysm expansion in AAA rats compared to unresponsive control [102].

Targeting Sites of Ischemia Reperfusion Injury

Ischemia refers to deficient blood supply to a tissue resulting in tissue damage. Paradoxically, reperfusion of the ischemic tissue results in further damage hence the term ischemia/reperfusion (I/R) injury. In myocardial infarction (MI) there is irreversible tissue damage caused by I/R injury. Additionally, there is an increase in inflammation and expression of several cell adhesion molecules such as P-selectin [103]. Scott et al. report that single injection of P-selectin ILP carriers of VEGF results in significant improvements in cardiac function compared to controls 4 weeks post MI in rat model [103].

Inflammation in I/R injury is partially initiated by neutrophil adhesion to ECs, having this into account recent publication reports synthesis of neutrophil membrane-derived nanovesicles loaded with drug which dramatically decreased inflammation in ischemic stroke. Following this idea of NP “cloacking” Antes et al. report use of extracellular vesicles of cardiosphere-derived cells decorated with an ischemic myocardium targeted peptide allowing targeting to the heart in vivo [104].

Coupling Redox Functionality to Target I/R Injury

Two studies published by the same investigators, explore the use of ROS-dependent antioxidant release polymeric particles. The investigators report the use of the H₂O₂-responsive antioxidant polymer vanillyl alcohol-incorporated polyoxalate (PVAX). In response to H₂O₂, this polymer releases the antioxidant, anti-inflammatory molecule vanillyl alcohol. Results of one study in a mouse model of hindlimb ischemia conclude that PVAX NP promote revascularization and restoration of blood perfusion into high ROS-producing ischemic tissues by upregulating angiogenic VEGF and PECAM-1 [105]. Another study focus on the therapeutic effect of NPs during cardiac I/R as evaluated in mouse model of MI. PVAX treatment suppressed ROS generation, through reduction of NOX2 and NOX4 expression levels. A single dose of PVAX NPs showed a significant improvement in both cardiac output and fraction shortening compared to non-ROS responsive NP [106].

Concluding Remarks

Nanotechnology-assisted drug delivery for CVD applications is a growing field still in its infancy. However, there is increasing evidence suggesting that it is a promising approach to advance the development of therapeutics to treat a variety of CVD. Additionally, nanotechnology approaches hold promise to overcome challenges associated with systemic delivery of antioxidant therapies, and lead to successful translation of novel therapeutic approaches to the clinic.