Key words

1 Introduction

Vascular diseases continue to be the number one cause of tissue morbidities and mortalities in the USA and globally [1, 2]. According to the recent statistical data reported by the American Heart Association, ~40 % of adult American adults suffer from vascular diseases and the number is over 80 % in the aging (80+ years) population. Mortalities from vascular diseases in the USA were reported to be close to 800,000 (male + female) in a recent statistical report in 2010 [1]. Consequently, significant research and clinical efforts are directed in prevention and treatment of these diseases. Vascular diseases can fall into many categories, for example, coronary heart disease leading to unstable angina and myocardial infarction, cerebrovascular disease leading to ischemia and stroke, atherosclerosis and peripheral arterial diseases, deep vein thrombosis, pulmonary and distal embolisms, restenosis following catheterized interventions, and congenital or acquired heart diseases or hemostatic dysfunctions. Many of these disease conditions have common spatiotemporal cellular and molecular mechanisms, the most predominant of which is the formation of intravascular occlusive clots (thrombi) that reduce antegrade blood flow to vital tissues and organs, often leading to tissue morbidities and mortalities. Therefore, many clinical strategies are focused on prophylactic, emergent and sustained prevention of thrombo-occlusive events to maintain normal blood flow to tissues and organs. The prophylactic strategies mainly involve oral or systemic administration of anticoagulant (e.g., heparin) and antiplatelet (e.g., Aspirin and Clopidogrel) agents, the emergent strategies mainly involve mechanical (e.g., catheter-mediated or aspiration-based thrombectomy, balloon angioplasty), surgical (e.g., aortic or coronary thrombus removal, bypass grafting) and fibrinolytic pharmacotherapy (e.g., intravascular bolus administration or infusion of plasminogen activators like streptokinase (SK) and tissue plasminogen activator (tPA)) procedures, while the sustained strategies mostly involve post-procedural prolonged oral administration of anticoagulant and antiplatelet drugs as well as drug-eluting stent (DES, releasing anticoagulant or antiproliferative drugs) placement during catheter-based interventional procedures like balloon angioplasty. As evident from these descriptions, systemic (oral and intravascular) administration of drug molecules that prevent platelet activation and aggregation (antiplatelet agents), block coagulation pathways (anticoagulant agents), degrade clot proteins (fibrinolytic agents), or downregulate unwanted cellular proliferation (antiproliferative agents) remain a major component of clinical regimen in treating occlusive vascular disease conditions. Systemic administration of these drugs presents several harmful issues [3–6]:

  1. (a)

    Rapid drug washout and clearance from the target site due to dynamic blood flow

  2. (b)

    Plasma-induced inactivation of the drugs and reduced circulation half-life

  3. (c)

    Systemic nonspecific distribution of the drugs resulting in suboptimal availability at target

  4. (d)

    Systemic nonspecific action of the drugs leading to harmful side effects like coagulopathy, neurotoxicity and nephrotoxicity, and hemorrhage

These issues can be potentially resolved by localizing the delivery (and action) of the drugs at the target clot sites. One way to achieve such site-selective delivery is by implanted devices like trans-arterial infusion catheters and DES. Implantation procedures like these are expensive, require specific expertise in terms personnel and facilities, and are not accessible by or amenable to many patients within required treatment windows [7–10]. Another way is to manufacture drug molecules that possess some target-specificity of binding (and action) by virtue of bioconjugation of antibodies and other ligands directly to the drug molecules or by recombinant modifications of the drug itself to impart target-specificity [11–15]. Direct antibody conjugation to drugs may affect drug activity and recombinant technologies make the resultant products quite expensive for global use especially in developing countries. Therefore, in recent years, alternative drug delivery strategies utilizing the “nanomedicine” approach have raised significant clinical interest [16]. The ideal “nanomedicine” design for site-selective delivery of drugs in vascular diseases should consist of a “carrier vehicle” that can encapsulate the drug in its core or embed it on the vehicle surface, protect the drug from plasma-induced inactivation while increasing its circulation half-life, localize via passive uptake and/or active molecular mechanisms to the vascular disease site to ensure site-specific delivery of the drug payload, enable controlled release of the payload via diffusion, dispersion, or stimuli-triggered mechanisms to allow site-selective therapeutic action while reducing systemic harmful side-effects, and biodegrade or get cleared from the body safely within a reasonable time frame so as to not render long term effects. The “payload” in such vehicles can not only be drug molecules, but also imaging probes that can allow detection and diagnosis of disease sites, and the combination of therapeutic and diagnostic payloads can potentially lead to “theranostic” nanomedicine systems targeted to vascular disease sites. The following sections review the various “nanomedicine” technologies that have been developed and are undergoing research currently in the context of the above-described design, followed by a discussion of the pros and cons and future endeavors.

2 Nanomedicine Systems Without Ligand-Based Site-Specific Active Binding Mechanisms

Direct systemic delivery of therapeutic (and diagnostic) agents often leads to inactivation of the agents by various plasma components, rapid washout from target site, and rapid clearance from circulation via organs like liver and kidney. Resolving these issues require increasing the circulation residence time of the agents in active form. This is where “packaging” of the agents within carrier vehicles can provide a solution. The concept is derived originally from the “Ringsdorf Model” in the application of macromolecular modifications of cancer drugs (Fig. 1), where the drug molecules are conjugated to polymers that prevent rapid plasma clearance of the small drug molecules due to enhancement of overall hydrodynamic radius by virtue of the drug–polymer conjugates [17–19]. The conjugation of the drugs to the various polymers are mediated by chemical bonds like amide, orthoester, ester, anhydride, carbonate, and urethane that can be cleaved by enzymatic and/or pH-sensitive reaction mechanisms to release the active drug for subsequent action. For cardiovascular drugs, this design has been tried by polyethylene glycol (PEG )-based modification (PEG-ylation) of fibrinolytic agents like tPA, SK, urokinase (uPA), and staphylokinase (Sak) [20–24]. The antiproliferative drug Paclitaxel (clinicaly used in DES for treatment of restenosis and intimal hyperplasia) has also been conjugated to polymers like polyglutamic acid (PGA) to result in products like Xyotax that are undergoing clinical study for cancer treatment but may also find cardiovascular applications. Besides drug–polymer conjugates, the other strategy to protect the drugs and increase circulation stability and residence time, is to package them in microparticulate and nanoparticulate vehicles. To this end, extensive research has been carried out using vehicles like liposomes, polymeric particles, lipoprotein particles, micelles, engineered red blood cells (RBCs), quantum dots, gold particles, dendrimers , ultrasound-sensitive bubbles and iron oxide particles (Fig. 1).

Fig. 1
figure 1

The Ringsdorf Model of drug–macromolecule conjugates and some common nanoparticle systems utilized for vascular nanomedicine technologies

Full size image

Vesicular liposomal structures, originally reported by Sir Alec Bangham [25, 26], have a lipidic (hydrophobic) shell and an aqueous core, thereby providing potential volume fractions for encapsulating both hydrophobic and hydrophilic drugs. Liposomes are formed by thermodynamically driven self-assembly of lipid-based amphiphilic molecules when exposed to an aqueous environment. Specifically, these molecules would need to have a packing fraction (v × a −1 × l −1 where “v” is the hydrophobic volume, “a” is the hydrophilic surface area, “l” is hydrophobic length) equal to 1, such that when exposed in an aqueous environment, they would form planar lamellar bilayer structures that ultimately fold into spherical vesicles with a bilayer lipidic shell and aqueous core. This kind of self-assembled vesicular structure can be unilamellar (single lamellar shell) or multilamellar (multiple concentric bilayer shells), and their size can range from about 50 nm to a few microns in diameter. Usually by extrusion through nanoporous polycarbonate membranes or by exposing to high frequency ultrasound, larger multilamellar vesicles can be reduced to nanoscale (50–200 nm diameter) unilamellar vesicles. Furthermore, modification of the liposome lamella outer surface with hydrophilic polymers like PEG imparts a steric hindrance to opsonization (blood protein adsorption) and macrophagic uptake, and thereby renders a “stealth” property to avoid rapid clearance from circulation [27], that in effect enhances the circulation residence time of the encapsulated drug payload. The most significant clinical application of liposomes is in the formulation of cancer drugs like Doxil®, Daunosome®, and Myocet® [28], which have made this class of vehicles a popular choice in studying encapsulation and delivery of drugs to other diseases including cardiovascular diseases. To this end, various antithrombotic agents have been encapsulated in liposomes and these formulations have shown enhanced circulation half-life of the drugs and increased therapeutic efficacy in vitro as well as in vivo in small animal models [29–33]. Liposomes, especially with cationic lipid shells, have been also used to complex DNA for gene delivery in cardiovascular diseases [34–39]. Besides drugs and DNA, liposomes have also been reported to encapsulate imaging agents like the MRI contrast agent gadolinium (Gd), either by direct loading of Gd salts or by lipid conjugation of Gd chelates, for imaging of vascular diseases [40–43]. Instead of lipidic systems, amphiphilic block co-polymeric systems with packing fraction equal to 1 can also be used to assemble similar vesicular structures called polymersomes [44–46]. Potentially such structures can also be used to package and deliver a wide variety of therapeutic agents in cardiovascular pathologies. Similar to liposomes, micelles are also self-assembled colloidal nanostructures with a hydrophobic core and a hydrophilic shell formed from amphiphilic molecules with packing parameter of ~1/3 when exposed to aqueous environment, and can be formed from lipid-based or polymer -based amphiphilic systems. These vehicles also have been extensively investigated in formulation of cancer drugs, but only a limited number of reports are available regarding their drug delivery applications in the cardiovascular area. PEG-polycation micelles have been utilized for gene delivery to arterial disease lesions in rabbit models [47]. Potential micelle-based strategies that could be directed toward diseased or dysregulated endothelial components of atherosclerotic and thrombotic sites in vascular diseases have been recently reviewed [48, 49]. To this end, several micelle-based strategies have been studied by incorporating ligand-based active targeting, which will be discussed in the next section.

Polymer based microparticles and nanoparticles have been of great interest in vascular drug delivery for past two decades [7, 8, 50]. Polymer-based drug-carrier particles can be formed by a wide variety of methods like oil/water or water/oil/water emulsion based solvent evaporation technique, solvent diffusion technique, solvent displacement technique, salting out technique, interfacial polymerization technique, and supercritical fluid technologies [51]. Polymeric nanoparticle carriers based on co-polymerized systems of biocompatible polymers like poly-lactic-co-glycolic acid (PLGA ), polyethylene glycol (PEG ), polyvinyl alcohol (PVA), etc. have been utilized to encapsulate and deliver anticoagulant agents like heparin [52], fibrinolytic agents like tPA and SK for clot dissolution [31, 53], and antiproliferative agents like probucol, rapamycin and paclitaxel for reducing restenosis [54–61]. Some polymer nanoparticle-based anti-restenotic formulations have also been evaluated in clinical studies [62]. Ultrasound imaging modalities are well established in diagnosis of physiological and pathological tissues , and ultrasound contrast agent bubbles like Definity (Bristol Meyers Squibb), a perfluoropropane/air filled lipid-shelled microbubble, have been approved in the clinic for cardiac imaging [63]. Consequently, such bubble systems have also been investigated for vascular therapeutic and diagnostic applications, where the bubble not only acts as a carrier for drugs but also allows focused ultrasound-mediated cavitation for site-selective release of the drugs. For example, poly(vinyl alcohol) (PVA)-based bubble structures were reported that can be loaded with the vasodilatory and antithrombotic bioactive gas nitric oxide (NO), for simultaneous imaging and NO delivery to vascular disease tissues [64]. Ultrasound-sensitive bubbles that allow cavitation-induced payload release have been reported for delivery of DNA, double stranded RNA and oligonucleotides, recombinant proteins , growth factors and thrombolytic agents (e.g., SK, tPA, etc.) [65–69]. Terminologies like “sonothrombolysis” has been coined to emphasize the combined effect of ultrasound-induced mechanical cavitation and site-specific thrombolytic drug release to enhance clot dissolution properties. Dendrimers are another important class of highly branched polymeric globular nanosystems originally developed in the 1980s by “convergent” or “divergent” chemical techniques [70, 71], that have undergone extensive studies in the delivery of genes, drugs and imaging agents utilizing the dendrimer core, the branching zone and the branch extremities [72]. Most dendrimeric applications in vascular drug delivery have involved ligand-based active targeting and will be discussed in the next section.

Among inorganic nanovehicle systems, carrier particles made from gold and iron oxide have been extensively studied in delivery of therapeutic and imaging agents in cancer [73–78]. Colloidal gold nanoparticle s can be prepared by an array of methods that are mainly based on reduction of chloroauric acid in presence of a colloidal suspension stabilizing agent, and the methods vary mostly in terms of the reducing agents and reaction conditions [79–82]. Galvanic replacement methods have also been utilized to synthesize hollow gold nanostructures from gold salts [83, 84]. Gold nanoparticles and nanostructures have been studied not only as carrier vehicles for drug delivery and imaging, but also because of their plasmonic activity and near infra-red (NIR) wavelength sensitivity, they have been used to render NIR-induced targeted photothermal phenomena and photoacoustic imaging. To this end, gold nanoparticles have been recently reported in the context of cell-specific imaging as well as image -guided targeted drug delivery in the cardiovascular disease area [85, 86]. In a recent report, novel Au-based lipoprotein-coated nanoparticles (Au core coated with Apolipoprotein A-1 and phospholipids) were shown to be taken up by atherosclerosis-relevant macrophages in ApoE−/− mice in vivo and hence provided a way for enhanced multispectral and multimodality imaging of the lesions for characterization of macrophage burden, calcification and stenosis [87]. In another report, NIR-fluorescence-quenched gold nanoparticle based imaging probes were used where the particles were surface-modified by a peptide sequence that can be specifically degraded by matrix metalloproteases (MMPs) and also surface-modified with an NIR fluorescence dye, Cy5.5 [88]. MMP activity is prevalent in matrix remodeling and lesion progression processes in atherosclerosis, and therefore such nanosystems may become useful in detection and evaluation of vascular lesion progression. In another interesting work, the photothermal ablative effects of gold nanoparticles were used to render disruption and recanalization of atherosclerotic plaques in coronary arteries in human postmortem ex vivo specimens [89]. Iron oxide particles are usually prepared by co-precipitation methods involving addition of alkali to iron salts [90]. Superparamagnetic iron oxide (SPIO) particles are categorized mostly by their hydrodynamic diameter, e.g., Oral-SPIO (300 nm–3.5 μm), Standard-SPIO (SSPIO, 60–150 nm), Ultrasmall-SPIO (USPIO, 5–40 nm), and a subset of USPIO called monocrystalline iron oxide NPs (MION). Furthermore, MIONs with a chemically cross-linked polysaccharide shell are termed Cross Linked Iron Oxide (CLIO) [91, 92]. Iron oxide nanoparticles have been extensively reported in magnetic resonance based cellular and molecular imaging of cardiovascular diseases [93–102]. Incorporating therapeutic molecules in such iron oxide systems can provide efficient theranostic systems for cardiovascular therapies, as has been recently demonstrated regarding delivery of antithrombotic and anticoagulant agents using such particles [103, 104]. In another interesting work, iron oxide nanoparticles were incorporated within PLGA particles and co-loaded with paclitaxel to form drug-loaded magnetic nanoconstructs, which were guided by an induced magnetic field to carotid artery sites in vivo in animal models for sustained-release vascular antiproliferative therapy [105]. In recent years, there has been significant interest in engineering of multicomponent nanoparticle systems for theranostic use or multimodal targeted imaging applications, by combining different types of imaging probes and therapeutic agents on an iron oxide nanoparticle platform [106–109]. Another class of inorganic nanostructures that have been extensively researched in targeted drug delivery and imaging is Quantum dots (QDs), that are semiconductor nanocrystals (e.g., QD with a cadmium selenide core with a zinc sulfide shell) with unique size - and composition-dependent fluorescent properties and are also sufficiently electron dense to facilitate electron microscopy [110]. The in vivo distribution, residence, and safety of QDs remain a matter of debate [111–114]. Nonetheless, QDs have been investigated in vascular delivery and imaging applications, for example, by incorporating them in high density lipoprotein (HDL)-based plaque-targeting for optical imaging of plaques [115]. The same HDL particles, incorporated with MRI probes, have been further investigated for targeted imaging of atherosclerotic plaques [115–117]. Several QD-based nanosystems have also been investigated in ligand-mediated active targeting to vascular lesions, which will be discussed in the next section.

3 Nanomedicine Systems with Ligand-Based Site-Specific Active Binding Mechanisms

Many of the nanovehicle systems described in the previous section have also been utilized to developed “actively targeted” delivery devices where the particles can bind to disease sites and diseased cells by virtue of specific ligand–receptor interactions. The ligands in such cases can be antibodies, antibody fragments, proteins and peptides, while, the receptors are antigens and proteins either uniquely expressed or quantitatively upregulated at the disease site cells and matrix. Such active targeting is thought to help with selectivity and specificity of targeting as well as with receptor-mediated internalization of the vehicles within diseased cells for intracellular delivery in some cases [118–120]. The ligands can be decorated on the nanoparticulate vehicles via non-covalent methods as well as a variety of covalent bioconjugation techniques.

Non-covalent adsorption methods to surface-decorate nanovehicles with ligands mostly involve physical (e.g., hydrophobic, affinity-based, charge-based) interactions of ligand molecules with the surface material of the particles. For example, polystyrene particles have been reported to be coated with P-selectin and E-selectin targeting antibodies using adsorbed bacterial protein A molecules as spacers [121]. These selectins are often expressed on activated platelets, stimulated endothelial cells and monocytes at the site of vascular injuries and lesions, and therefore are relevant systems for active targeting of drug delivery system s to such vascular disease sites. Similar techniques have also been reported for coating chitosan particles with anti-amyloid monoclonal antibodies to target amyloid beta-protein deposits in cerebral vasculature of mice [122]. In other work, liposomes, latex beads and albumin particles have been non-covalently surface-modified with recombinant glycoprotein Ib-alpha (rGPIbα) and recombinant glycoprotein Ia-IIa (rGPIa-IIa) to actively bind to von Willebrand Factor (vWF) and collagen respectively [123–125]. vWF is secreted and deposited from injured endothelial cells and activated platelets, while collagen is often exposed as the major sub-endothelial matrix protein at vascular injury sites due to endothelial denudation. Therefore such vWF- and collagen-targeting systems can have potential application in targeted delivery to vascular injury sites. Another interesting non-covalent approach to decorate nanoparticles with targeting motifs is the use of avidin-biotin affinity interaction . Avidin (and analogous Streptavidin) is a highly glycosylated positively charged protein that is uniquely stable against heat, denaturants, pH and proteolytic enzymes, and has high affinity towards Biotin (Vitamin B6) with a dissociation constant (K d) of 10−15 M [126, 127]. Consequently particles can be surface-modified with avidin and incubated with biotinylated ligand motifs, or vice versa, to create ligand-decorated actively targeted delivery systems. This approach has been used extensively in decorating particle surfaces with antibodies and antibody fragments for targeting to cancer . In the area of targeting cardiovascular diseases, this technique has been employed to surface-decorate RBCs with antithrombotic molecules (e.g., tPA) as well as to decorate various nanovehicle systems with antibodies directed to a variety of vascularly relevant cell adhesion molecules (CAMs) [128–131]. Covalent bioconjugation techniques involve specific chemical reactions of reactive groups on ligand motifs to complimentary reactive groups on the nanovehicle surface. The most common chemical bioconjugation methods are amide linkages (reaction between amine and carboxyl termini), hydrazine-based linkages (reaction between hydrazide and aldehyde termini), sulfhydryl-mediated linkages (reaction between sulfhydryl group and maleimide, sulfone, acetamide or pyridyl groups) and alkyne-azide based “click” chemistry [132]. Figure 2 shows schematic of some common bioconjugation strategies for decorating nanoparticle surfaces with targeting motifs. These methods can be utilized to conjugate antibodies, antibody fragments, aptamers, proteins , and peptides to a wide variety of nanovehicle systems either by reacting to appropriate functional groups on the surface of preformed particles (solid polymer particles, QDs, dendrimers , etc.), or by reacting to the termini of constituent molecules first and then assembling the modified molecules into particles (e.g., liposomes, micelles). Figure 3 shows the commonly studied cellular and noncellular targets for vascular nanomedicine technologies.

Fig. 2
figure 2

Common bioconjugation strategies for decorating nanoparticle surfaces with targeting motifs; the targeting motifs can be antibodies, antibody fragments, peptides, aptamers, etc.

Full size image
Fig. 3
figure 3

Relevant cellular and noncellular targets utilized for active targeting of vascular nanomedicine systems. The targets can be cell-surface antigens as well as a variety of substrate proteins relevant to the vascular disease site. CAMs cell adhesion molecules, ADP adenosine diphosphate, PDGF platelet derived growth factor, FGF fibroblast growth factor, LDL low density lipoproteins

Full size image

By utilizing the various non-covalent or covalent surface-modification techniques stated above, a large number of actively targeted nanoparticle systems have been reported for site-specific delivery of drugs and imaging probes in vascular diseases. To this end, echogenic liposomes have been reported that can target fibrinogen, fibrin or intercellular adhesion molecule-1 (ICAM-1) by virtue of anti-fibrinogen, anti-fibrin, and anti-ICAM-1 antibodies and allow ultrasound-induced cavitation mediated delivery of thrombolytic agents [133–135]. In another work, Gadolinium (Gd)-based MRI contrast agent delivery to atherosclerotic tissue was demonstrated by using liposomes modified with Gd-lipid conjugates and phosphatidylserine (PS) to enable preferential uptake by atherosclerotic site-relevant macrophages [41]. Similar strategy in macrophage-targeting of liposomes was also demonstrated with liposomes modified by the ligand decadeoxyguanine, which has high affinity to macrophagic scavenger receptor class A (SRA) [42]. Liposomes surface-decorated with antibodies directed to low density lipoprotein receptors LOX-1 have been reported to enable atherosclerotic lesion-targeted delivery of radioimaging and MR imaging agents [136]. Another liposomal formulation, named LipoCardium, was reported for targeted delivery of anti-inflammatory prostaglandins to atheroslecrotic sites using liposomes surface decorated with antibodies directed to Vascular Cell Adhesion Molecule-1 (VCAM-1) [137]. Besides surface-decoration of antibodies, liposomes have also been reported to be surface-decorated with small peptides having targeting ability to vWF, collagen, activated platelet glycoprotein IIb-IIIa (GPIIb-IIIa) and P-selectin, all of which are suitable target molecules in the context of endothelial injury, endothelial denudation, platelet activation, and thrombosis in vascular pathologies [138–145]. Therefore these liposomal systems can have potential application in targeted delivery of drugs and imaging agents to various spatiotemporal phases of vascular injury and vascular disease. Similar to liposomes, micelles (both lipidic and block co-polymeric) have been studied for actively targeted delivery to vascular disease sites. Micelles surface-decorated with antibodies specific for macrophage scavenger receptors (MSR) and loaded with Gd chelates or fluorescent probes were shown to selectively target and accumulate at atherosclerotic arterial sites in ApoE−/− mice for molecular imaging of the disease [146, 147]. Gd-loaded PEG -lipid micelles surface-modified by antibodies that bind to oxidized LDL lipoproteins in atherosclerotic plaques, have also been reported [148]. Similar Gd-loaded micelles surface-decorated with anti-CD36 antibodies were shown to target macrophages in atherosclerotic vessels [149]. Recently, lipid-polymer hybrid particles (polymer core with lipid shell) decorated with a phage library-identified peptide sequence KZWXLPX (Z: hydrophobic amino acid, X: any amino acid) were reported as “nanoburrs” that can actively target exposed collagen IV at arterial injury (i.e., endothelial denudation) sites and deliver antiproliferative agents to modulate smooth muscle cell activity [150, 151]. In another recent work, micelles were surface decorated with a 9-amino acid sequence CGNKRTRGC (also known as Lyp-1) that binds to p32 receptors in atherosclerotic plaques as well as with CREKA peptides that bind to fibrin-fibronectin clots, and these micelles showed enhanced homing to atherosclerotic plaques in vivo [152, 153].

Similar to liposomes and micelles, solid polymeric particles have also been studied for surface-modification with ligands to enable targeted binding to vascular injury or vascular disease sites. For example, PLGA nanoparticles have been loaded with thrombolytic drugs like tPA and coated with Arginine-Glycine-Aspartic Acid (RGD)-peptide modified chitosan to render targeted binding to clots for enhanced thrombolytic efficacy [53]. PLGA nanoparticles have also been reported to be surface-modified with anti-ICAM-1 antibodies for specific immunotargeting to inflamed vascular endothelium in vitro and in vivo, which has relevance to targeting atherosclerotic plaques [154]. Similarly, poly(sebacic acid)-co-PEG (PSAPEG) microparticles and nanoparticles surface-modified with anti-VCAM-1 antibodies have been reported to undergo enhanced adhesion, binding and accumulation at atherosclerotic lesion sites in ApoE−/− mice [155]. Nanoparticles made from poly-l-lysine-co-poly-lactic acid copolymer (PLL-PLA), surface-decorated with RGD peptides have been reported to be able to aggregate with active platelets at the site of traumatic vascular injury [156]. Similar designs of RGD-decorated or the fibrinogen-derived peptide sequence HHLGGAKQAGDV-decorated particles have also been reported using RBCs, latex beads or albumin particles as the carrier vehicle [157–162]. The HDL nanoparticles described in the previous section were designed to be naturally taken up into atherosclerotic lesions via lipoprotein transport mechanisms; however these same particles have also been reported to be modified with RGD peptides to enable active targeting ability to vasculature [163]. Ligand-based active targeting strategies have also been reported for ultrasound-sensitive bubbles where the bubbles were surface-decorated with antibodies directed against inflammation and atherosclerosis relevant upregulated cell-surface markers like various CAMs and integrins (e.g., αVβ3), on leukocytes and injured endothelium, in vitro and in vivo, for targeted drug delivery to and molecular imaging of vascular disease [164–166]. In similar work, ultrasound-sensitive bubbles were developed with shells bearing maleimido-4(p-phenylbutyrate)-phospholipid, which were then surface-conjugated with platelet integrin GPIIb-IIIa-specific therapeutic antibody Abciximab (ReoPro by Eli Lilly, Indianapolis, Indiana), that enabled enhanced targeting to activated platelet-rich thrombi for molecular imaging applications in vitro and in vivo [167]. Dendrimers have also been studied for active targeting to vascular pathology sites, where biodegradable dendritic structures surface-modified with endothelial αVβ3 integrin-targeting cyclic RGD peptides and loaded with radioactive Bromine (76Br) for positron emission tomography (PET), were capable of targeted molecular imaging of hindlimb ischemia in a mouse model [168]. Similar targeted molecular imaging of vascular disease-specific biomolecules and cellular phenotypes have also been demonstrated with dendrimers modified by a variety of other ligands [169–171].

The inorganic nanosystems described in the previous section have also undergone extensive investigation for actively targeted delivery to vascular disease and injury sites. Cross-linked dextran-coated iron oxide (CLIO) nanoparticles have also been surface-decorated with peptides and small molecules that can target CAMs and clot-associated fibrin to enable active targeting of the particles to inflammatory, angiogenic and thrombotic cellular phenotypes and biomarkers of atherosclerosis for contrast enhanced targeted molecular imaging [92]. Iron oxide particles have also been reported to be surface-decorated with ligands directed towards VCAM-1, P-selectin and platelet integrin GPIIb-IIIa for targeted contrast-enhanced MR imaging of atherosclerosis and thrombosis in animal models [172]. In another interesting work, SPIOs were surface-modified by Annexin V that can specifically interact with lipoproteins on the outer membrane leaflet of apoptotic cells and hence enabled interaction and selective targeting of “foam cells” in atheromatous plaque in rabbit models for T2-weighted MR imaging [173]. QDs have also been utilized to actively bind a variety of CAMs (e.g., VCAM, ICAM, PECAM) using QD surface-decoration with anti-CAM antibodies [174, 175] and these facilitated in vivo optical imaging of atherosclerotic lesions. Other approaches to ligand-directed vascular disease-specific targeting of QDs for optical imaging include targeting to oxidized LDL receptor CD36, phosphatidylserine-exposing cells, and plaque-relevant MMPs [176, 177]. Instead of directly targeting QDs to the vascular disease site, they have also been used as “payloads” in other actively targeted nanosystems to allow for concurrent optical imaging modality for vascular diseases. This strategy has been utilized by loading QDs within paramagnetic micelles immunotargeted to macrophagic scavenger receptors [178] as well as within HDL nanoparticles [115]. In an interesting work, gold nanoparticle s were conjugated to QDs via a proetolytically degradable peptide sequence such that in the “bound” state the QD luminescence was non-radiatively suppressed, and enzymatic cleavage of the conjugate links significantly restored luminescence [179]. Such unique strategies can be envisioned to be applicable in probing proteolytic activities (e.g., MMP activity) in atherosclerotic lesions.

4 Other Miscellaneous Applications of Nanomaterials in Cardiovascular Disease Treatment

As evident from the descriptions and examples provided in the previous sections, “nanotechnology” has provided an efficient way to render localized or site-selective delivery of various therapeutic agents and imaging probes in vascular diseases and injuries. Such localized delivery can potentially overcome the issues of potency and narrow therapeutic window of many drug molecules by achieving greater local concentrations with lower overall dose, to maximize the effects in target tissue while avoiding systemic indiscriminate distribution and harmful side-effects. The idea of local delivery in the cardiovascular arena emerged about two decades ago in the context of using perivascular delivery systems successfully in animal models [180, 181]. In these systems, heparin-releasing polymeric matrix devices were placed around rat carotid arteries at the time of balloon angioplasty, to allow continuous local release of the drug for predetermined periods of time. These approaches were found to reduce post-procedural arterial occlusion more effectively compared to systemic heparin infusion from pumps or from drug-releasing polymer matrices placed subcutaneously distant from the target artery site. Similar local polymeric systems bearing endothelial cells as a source of endogenous vasoregulatory agents were also shown to have enhanced efficiency in reducing neointimal hyperplasia in rat and pig models of vascular injury [182, 183]. Over the past two decades, other “local delivery” systems have been developed for cardiovascular applications, including intraluminal, intramural and stent-based systems [184], all of which have proved to be much more efficient in rendering therapeutic effect at the target tissue while avoiding poor distribution and harmful side-effect issues of systemic delivery. Nanotechnology has contributed to refinement such devices. For example, silver nanoparticles have been used to modify implantable and intravascular devices to prevent bacterial adhesion, growth and biofilm development [185]. Carbon nanotubes have been incorporated in catheters to provide mechanical versatility as well as impart antithrombotic and drug delivery functions [186]. Such carbon nanotubes have also been incorporated in stents [187]. Other application of nanotechnology in stents include refined nanofabrication and nanomorphological texturing techniques to allow for enhanced drug loading, tissue-material interactions and drug release [188], as well as incorporating drug- or gene-loaded nanoparticles within stent coatings for sustained local delivery following angiopalstic procedures and stent placement [189, 190]. Nanotechnology has also been used in fabrication of artificial arterial grafts and conduits [191], although it is too early to conclude on long-term success of these designs. In the context of artificial vascular grafts, chemical nanocomposites have also been incorporated to release nitric oxide and to impart infection resistance [192].

In recent years, another facet of nanotechnology that is raising significant interest, especially in the context of drug delivery vehicles in the vascular compartment, is the role of “physical” design parameters like shape, size , modulus, etc. Several recent reports have established that particles of anisotropic shapes (spheroids, rods, disks, etc.) have a higher probability of margination from flowing blood volume towards the vascular wall [193–195]. Parallel studies have also shown that size of particles play an important role in their extent of margination to the vascular wall [196–198]. In fact a natural example of this is seen in blood platelets which can marginate better to the vascular wall through the RBC volume of flowing blood owing to their biconvex discoid shape and their quiescent ~2 μm size [199–201]. Based on such studies, recent research has focused on development of particles with tailored shapes and sizes to facilitate margination to the vascular wall [202–207]. Such margination-facilitating geometric parameters can be potentially integrated with ligand-based active targeting functionalities on particle platforms to create drug delivery and nanomedicine systems with increased site-selective localization and delivery efficiency in the vascular compartment. Another important design parameter for drug delivery system s is the mechanism of drug release. Traditionally most particulate delivery systems depend upon diffusion and degradation/dissolution mediated mechanisms for payload release [208–210]. Beyond such mechanisms, certain stimuli-triggered mechanisms have been investigated for drug delivery systems, where the payload release is induced by chemical and/or physical changes in the drug delivery system in response to internal stimuli like pH, enzyme action, temperature, etc., or, external triggers like NIR irradiation (e.g., for gold nanoparticle s ), electromagnetic wave (e.g., for iron oxide particles), high frequency focused ultrasound (e.g., for ultrasound bubbles), etc. [211–216]. A few recent studies have utilized “shear forces” as a physical release trigger because of its relevance to vascular thrombo-occlusive sites. In these studies using polymeric or lipidic particles, the increased shear forces caused by thrombo-occlusion have resulted in the disintegration of the carrier particles to release drugs like thrombolytic agents, site-selectively [217, 218]. Furthermore, an interesting aspect in the context of ligand-mediated active targeting of drug delivery vehicles is the utilization of concurrent binding to multiple receptor/antigen types pertinent to the disease site (also known as heteromultivalent binding) instead of targeting to only one type of receptor. Such targeting approaches have shown enhanced efficacy of anchorage of the vehicles to the target site under hemodynamic flow environment [142, 219], and this can potentially allow for increased target specificity as well as retention for enhanced therapeutic release. These newer design parameters are continuing to add exciting properties to vascular nanomedicine systems, that can be tailored to act selectively at disease sites by virtue of enhanced margination, enhanced anchorage, and enhanced drug release.

5 Discussion

Localized delivery of therapeutic molecules and imaging probes at the sites of vascular disease results in enhanced treatment and detection efficacy. Nanotechnology provides an efficient way to achieve such localized delivery in the context of packaging therapeutic payloads within nanoparticulate vehicles that can be intravenously injected and can accumulate passively or bind actively at the target vascular sites. The success of such approaches depend upon efficient encapsulation of the payload within the nanoparticles to protect from plasma-induced deactivation, minimize pre-target leakage or release of the payload, maintain circulation for sufficient periods of time to reach the target site, render efficient passive or active binding to the target site under hemodynamic flow, and enable efficient release of the payload by internal or external triggers. Because of the need to stay retained at the target site under hemodynamic flow environment, active binding strategies may be more effective in vascular drug delivery, compared to passive accumulation mechanisms. The delivery systems must be biocompatible, in terms of minimal immunogenicity, minimal complement activation, minimal toxicity, and minimal carcinogenicity. The drug delivery system s must also be either easily cleared from the body within a reasonable period of time, or easily biodegradable to resorbable or metabolizable products in the body. Additional design parameters to consider for the particulate vehicles are their margination-influencing shape, size , and morphology, their active targeting and anchorage-influencing ligand decoration chemistry and density, and their response to internal and external triggers. For efficient clinical translation, research should also be focused on cost and convenience of manufacture and quality control of vascular nanomedicine technologies.