Nanotechnology-Based Gene-Eluting Stents - Molecular

Review pubs.acs.org/molecularpharmaceutics

Nanotechnology-Based Gene-Eluting Stents Debbie Goh,†,‡ Aaron Tan,†,‡ Yasmin Farhatnia,† Jayakumar Rajadas,§ Mohammad S. Alavijeh,∥ and Alexander M. Seifalian*,†,⊥ †

Centre for Nanotechnology & Regenerative Medicine, UCL Division of Surgery & Interventional Science, University College London, London NW3 2QG, United Kingdom ‡ UCL Medical School, University College London, London WC1E 6BT, United Kingdom § Biomaterials & Advanced Drug Delivery Laboratory, School of Medicine, Stanford University, California 94305, United States ∥ Pharmidex Pharmaceutical Services Ltd., London W1S 1YH, United Kingdom ⊥ Royal Free London NHS Foundation Trust, London NW3 2QG, United Kingdom ABSTRACT: Cardiovascular disease is one of the major causes of death in the world. Coronary stenting in percutaneous coronary intervention (PCI) has revolutionized the field of cardiology. Coronary stenting is seen as a less invasive procedure compared to coronary artery bypass graft (CABG) surgery. Two main types of stents currently exist in the market: bare-metal stents (BMS) and drug-eluting stents (DES). DES were developed in response to problems associated with BMS use, like neointimal hyperplasia leading to restenosis. However, the use of DES engendered other problems as well, like late stent thrombosis (ST), which is a serious and lethal complication. Gene-eluting stents (GES) have recently been proposed as a novel method of circumventing problems seen in BMS and DES. Utilizing nanotechnology, sustained and localized delivery of genes can mitigate problems of restenosis and late ST by accelerating the regenerative capacity of re-endothelialization. Therefore this review seeks to explore the realm of GES as a novel alternative to BMS and DES, and its potential implications in the field of nanotechnology and regenerative medicine. KEYWORDS: gene-eluting stents, nanotechnology, interventional cardiology, regenerative medicine

1. INTRODUCTION Coronary artery disease is one of the major causes of death in the world. Atherosclerosis is a systemic disease where plaque is gradually deposited on the arterial vessel wall, eventually developing into a flow-limiting lesion that results in impaired oxygen delivery to the heart and clinical symptoms such as angina pectoris.1 Unstable lesions can rupture and trigger thrombosis, which may result in myocardial infarction if there is obstruction of blood flow in the coronary artery.2 Removal of atherosclerotic plaque commonly involves the use of invasive techniques such as percutaneous transluminal coronary angioplasty (PTCA), otherwise known as balloon angioplasty, and/or coronary artery bypass graft (CABG). PTCA involves the insertion of a balloon fitted on a catheter into the occluded vessel and subsequent inflation of the balloon to compress the plaque. On the other hand, CABG involves the removal of the occluded artery and replacement with a peripheral vein, usually the saphenous vein. PTCA is usually favored over CABG because it is less invasive and achieves similar results at a lower cost. Despite immediate improvements, PTCA is complicated in 30−40% of patients by reobstruction of the vessel due to the formation of a neointima in the lumen of the coronary artery and subsequent narrowing of the lumen, a process known as restenosis.3 Restenosis is primarily induced by a complex © 2013 American Chemical Society

interaction of multiple mitogenic, chemotactic, and/or proinflammatory factors activated by inevitable injury to the arterial wall during PTCA, which consequently stimulate vascular smooth muscle cell (VSMC) and leukocyte proliferation and migration, as well as excessive extracellular matrix (ECM) production.4 Constrictive vessel remodelling and vessel elastic recoil have also been found to play a role in postangioplasty restenosis.5,6 To counteract the problem of post-angioplasty restenosis, balloon-expandable bare metal stents (BMS) were introduced in the early 1990s to act as a permanent scaffolding structure by propping open the arterial wall upon insertion after angioplasty, thus preventing reocclusion and vessel recoil.7,8 The advent of BMS reduced the incidence of post-angioplasty restenosis to 20−53%, depending on the type of stent used.2 However, the success of BMS was limited by significant rates (15−30%) of instent restenosis (ISR).1 ISR is primarily a result of neointimal hyperplasia in response to local mechanical vascular injury caused by stenting. The radial force employed on the occluded vessel during balloon inflation and stent deployment often Received: Revised: Accepted: Published: 1279

November 19, 2012 January 30, 2013 February 10, 2013 February 10, 2013 dx.doi.org/10.1021/mp3006616 | Mol. Pharmaceutics 2013, 10, 1279−1298

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Figure 1. Different catheters and GES for gene delivery to the vessel wall. (a) Double balloon catheter. Two inflatable balloons separated by an intermediate space, into which therapeutic gene vectors can be infused. (b) Single lumen porous balloon. Single balloon containing multiple microscopic perforations, through which therapeutic gene vectors can exit lumen. (c) Dispatch coil balloon. Autoperfusion multichamber catheter which allows blood flow through central lumen while infusing therapeutic gene vector between arterial wall and catheter. (d) Infiltrator nipple balloon. Balloon catheter with micro injection needles attached to balloon to inject vectors into vessel wall, especially media and adventitia. (e) Gene eluting stent. Reproduced with permissions from ref 18. Copyright 2012 Nature Publishing Group.

late stent thrombosis (ST), where blood clotting inside the stent occurs more than 1 year post-stenting.13 As a result, longterm use of dual antiplatelet therapy is required post-DES implantation to decrease the persistent risk of late ST. This in itself may bring about adverse effects, such as hemorrhagic complications and thrombotic thrombocytopenic purpura in a minority of cases.14,15 Furthermore, polymer coatings on DES have been shown to induce a more distinct inflammatory reaction than bare metal surfaces, and are thus only able to delay, rather than prevent, neointimal formation. Taken together, these bring into serious question the long-term durability and efficacy of DES.3 There is therefore an urgent need to develop novel and efficacious methods of circumventing the problems seen in BMS and DES, for which gene eluting stents hold much promise.

causes tearing of atherosclerotic plaque, as well as denudation of endothelial and medial layers.1 Additionally, contact of BMS with vessel intima has been shown to elicit undesirable inflammatory and thrombogenic cascades. These induce abnormal VSMC proliferation and neointimal hyperplasia, resulting in luminal narrowing and vessel reocclusion once again.8 The problem of ISR with BMS led to the development of polymer-coated drug eluting stents (DES), which allow for the localized delivery of small molecular weight antiproliferative and anti-inflammatory therapeutic agents, such as paclitaxel and rapamycin, to the neointimal area.8 In initial clinical trials, the use of DES saw a marked reduction in percentage restenosis to less than 10%, accompanied by a diminished need for revascularization procedures.2,9 These encouraging results led to the use of DES in more than 85% of all coronary interventions, with DES being the current standard of care for restenosis.2 Despite definite short- to midterm improvements with the usage of DES post-angioplasty, long-term follow-up studies of DES have raised concerns about the long-term safety of DES.10 Although DES have indeed been proven successful in reducing neointimal formation, this is accompanied by delayed arterial healing, characterized by persistent fibrin deposition and impaired re-endothelialization.11,12 This observation may be explained by the nonselective inhibition of both endothelial cell (EC) and SMC proliferation by the antiproliferative drugs eluted from DES. Although primarily aimed at preventing VSMC proliferation, which is central to the pathogenesis of ISR, antiproliferative drugs also perturb endothelial recovery. Evidence shows that paclitaxel directly inhibits re-endothelialization while rapamycin inhibits EC proliferation.11,12 Reendothelialization is a key step in the long-term treatment of restenosis, and the impairment of re-endothelialization in stented vessels delays vessel healing.7 This consequently increases the risk of subacute in-stent thrombosis and even

2. GENE ELUTING STENTS Gene eluting stents employ the use of stents as permanent scaffolds to achieve localized and sustained delivery of therapeutic genes to the affected vessel wall and, in so doing, target both the structural and biological basis of restenosis.9 Initial studies in the field of vascular gene therapy explored the use of catheters, such as the Dispatch and Infiltrator catheters, to deliver both viral and nonviral gene vectors (Figure 1). Although these were able to achieve localized delivery of therapeutic genes in conjunction with the balloon angioplasty procedure,4,16 several major limitations have dampened further advancement in the field. To name a few, most catheters require prolonged total occlusion of the target vessel for effective vector delivery, which can potentially induce myocardial ischemia. They also inevitably damage the vessel wall, inducing inflammatory responses and neointimal hyperplasia. In addition, they have shown overall low levels of gene transfer efficiency, which may be attributed to necessary dilution of the gene in vector solutions, as well as frequent side branches of the coronary vasculature that permit runoff of 1280

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Figure 2. Summary of therapeutic genes, their mode of action, and molecular target.

neointimal hyperplasia; acceleration of re-endothelialization; inhibition of thrombosis; and reduction of inflammation (Figure 2) . Of these, the inhibition of restenosis via reduction of neointimal hyperplasia has been the most investigated method. 2.1.1. Antiproliferative. The pathogenesis of restenosis is multifactorial, although, as previously mentioned, a key element is increased VSMC proliferation and migration. It has been shown that vascular injury causes contractile SMCs to assume a synthetic phenotype which is much more proliferative and ECM-producing than the contractile phenotype. Excessive ECM production and SMC migration toward the vessel lumen consequently results in restenosis.4 SMC proliferation is a good process to target for the inhibition of restenosis, over other processes such as migration and ECM synthesis, for several reasons − it is one of the better-defined, early processes in restenosis, and blocking a single molecular target in the proliferation cascade can halt the entire proliferative process.22 As SMC proliferation is dependent on cell cycle activation, most antiproliferative approaches to inhibiting or reducing neointimal hyperplasia include cytostatic and/or cytotoxic modes of action.17 The cytotoxic antiproliferative approach involves transfecting target cells with genes encoding for proteins known to selectively destroy cells as they enter the S phase of the cell cycle,6 such as thymidine kinase,23−28 cytosine deaminase,27,29 and FasL.30 Several studies have demonstrated that transfer of the herpes simplex virus thymidine kinase (HSV-tk) transgene, followed by administration of the prodrug ganciclovir, is able to significantly inhibit SMC proliferation in both nonatherosclerotic and atherosclerotic balloon-injured vessels, thereby reducing neointimal hyperplasia. Thymidine kinase phosphorylates ganciclovir to a toxic nucleoside analogue that disrupts DNA replication in transfected cells and results in cell apoptosis. Moreover, as this nucleoside analogue is diffusible, adjacent nontransfected cells can also be affected, thus amplifying the antiproliferation action of thymidine kinase.23−25 In addition, later studies exploring the coexpression of HSV-tk with guanylate kinase26 or cytosine deaminase27 were able to show higher levels of VSMC apoptosis and enhanced inhibition of neointimal formation at lower prodrug doses in vitro and in vivo. On the other hand, the cytostatic antiproliferative approach involves transfecting target cells with genes encoding for regulatory proteins that take part in the cell cycle. Modulation of these genes can therefore potentially arrest the cell cycle.6,31−33 Specifically, proteins called cyclins control the

the delivered volume. This suboptimal localization of vector suspension carries the risk of distal spread.17,18 In recent years, there has thus been a surge of interest in and preference for balloon-expandable gene eluting stents (GES) over catheters as a platform for gene delivery. GES represent a more appealing method for gene delivery to atherosclerotic coronary vessels for the following reasons: first, vector immobilization to stent struts allows for increased local concentration of therapeutic agent at the targeted arterial segment without distal spread to nontarget tissue, thereby avoiding systemic toxicity and increasing the chance of effective gene transfection to adjacent cells.4,8,19,20 Second, the therapeutic effect is targeted to the anticipated site of pathophysiological processes such as mural thrombosis and VSMC proliferation.19,21 Third, there is already extensive clinical experience in coronary stenting procedures, making it extremely convenient to combine revascularization with gene delivery in a single procedure.11 Fourth, stents are able to act as permanent scaffolding structures and reservoirs for prolonged vector release. Last, stent-tethered vectors can better persist in tissues as they are physically protected from the shearing effect of blood flow.3 Nevertheless, there are a number of technical and biological challenges with stent-based vascular gene delivery. Technical challenges include the following: preservation of the stent’s mechanical properties; limited surface area for vector immobilization and gene loading; residence time of delivery system for sustained release;2,20 cellular uptake and intracellular stability of the gene construct; and efficient transcription and translation of the encoded protein.2 Biological challenges include the following: potential inflammatory and thrombogenic responses; impaired re-endothelialization; and toxicity of delivery vector and coating material.2 Gene eluting stents, like most gene therapy strategies, require a combination of three main components. First, a therapeutic gene product directed to an appropriate molecular target; second, a safe and efficient gene vector for introduction of the gene into cells; and third, a suitable gene delivery system for mechanical delivery of the vector to the target cells.1,17 The aim of this review is therefore to review and summarize the currently available therapeutic genes, vectors, and delivery systems for stent-based vascular gene therapy, with a special focus on the incorporation of nanotechnology-based methods. 2.1. Molecular Targets and Therapeutic Genes. Stentbased gene therapy strategies can be categorized by their molecular target and mode of action. There are four main molecular targets that researchers have focused on: reduction of 1281



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initiation of DNA synthesis. In a balloon-injured rat carotid artery model, p53 transfection on its own was able to achieve approximately 80% reduction in neointimal hyperplasia, a significantly high level than that achieved by p21 transfection (approximately 50%). This potentially superior antiproliferative property of p53 may be due to p53 acting upstream of p21, thus affecting cell cycle progression via other genes apart from p21.45 Moreover, studies have reported that p53 exerts its therapeutic effect not only by cytostatic antiproliferative mechanisms but also via paracrine mechanisms such as the repression of urokinase and tissue plasminogen activators to suppress plasmin generation, which is crucial for VSMC migration.45 There are, however, conflicting results regarding the role of p53 in VSMC apoptosis. This may be due to differences in the expression levels of exogenous p53 across different studies, which may affect its biological properties, as well as differences in the species and cell lines used.45,46 While p53 is mainly involved in controlling the expression of genes required in G1 phase, the transcription factor E2F is mainly involved in the S phase. In quiescent SMCs, E2F is associated with retinoblastoma protein (Rb) to form an inactive complex. However, under growth factor stimulation, Rb is hyperphosphorylated by cyclin D−CDK complex, causing dissociation of E2F, which in turn activates the expression of cyclins A and B, and CDK-1.31 Several strategies targeting E2F (or Rb) have been explored to inhibit growth factor-stimulated SMC proliferation and thereby reduce neointima formation. For example, Chang et al. (1995)47 transfected SMCs with a nonphosphorylatable form of Rb, and was able to achieve significantly reduced SMC proliferation and neointima formation in both balloon-injured rat carotid and pig femoral artery restenosis models.47 As E2F is also known to play a crucial role in activating genes mediating neointima formation after vascular injury, namely, c-myc, cdc2, and the proliferatingcell nuclear antigen (PCNA) gene, Isobe’s group explored the use of dsDNA with high affinity for E2F as a decoy to bind E2F, thereby blocking the activation of the above-mentioned genes. This strategy was successful in inhibiting c-myc, cdc2, and PCNA gene expression both in vitro and in vivo. Furthermore, neointima formation was significantly inhibited for up to 8 weeks in a balloon-injured rat carotid artery model.48,49 Wills et al. (2001)50 explored an alternative chimeric transgene (SMAE2F/p56) formed from the fusion of fragments of Rb, E2F, and smooth muscle alpha-actin (SMA). This demonstrated SMCspecific G0/G1 cell cycle arrest, and effectively reduced neointimal hyperplasia in a balloon-injured rat carotid artery model.50 Apart from p53, GAX and GATA-6 are also transcription factors which induce p21 expression and cause G1 cell cycle arrest. Conversely, GAX and GATA-6 are rapidly downregulated when VSMC proliferation is induced.51 Overexpression of these SMC-specific transcription factors has thus expectedly yielded promising results in terms of inhibiting SMC proliferation in various animal models of restenosis.51−55 Finally, in addition to cyclin−CDK complexes, CDKIs, and transcription factors, nitric oxide (NO) is also involved in SMC cell cycle regulation. Evidence exists to show that NO is a potent downregulator of CDK2 and cyclin A, as well as an upregulator of p21, and is therefore able to induce cell cycle arrest.31 As NO has many other modes of action apart from antiproliferation, it will be discussed in greater detail in the following sections.

passage of a cell through the various phases of the cell cycle by binding to and activating specific cyclin-dependent kinases (CDKs), which in turn phosphorylate specific protein substrates modulating the cell cycle. The G1 phase is predominantly controlled by cyclin D and CDK4, the S phase by cyclins A and E and CDK2, and the mitotic phase by cyclins A and B and CDK1.31,33 Downregulation of cyclin expression to induce cell cycle arrest is therefore a logical antiproliferative approach to inhibit neointimal formation. One method of downregulating endogenous cyclin expression is by using antisense techniques, which block specific processes in the proliferation pathway, such as DNA uncoiling, DNA transcription, DNA splicing, RNA stabilization, RNA translocation, or RNA translation. Many antisense agents have been developed; however, antisense oligodeoxynucleotides (ODNs) are the most commonly used antisense agents, and also the most extensively investigated in vitro and in vivo.22 Antisense ODNs targeted to mRNA in a sequence-specific manner can reduce target gene mRNA and/or protein product formation, thereby exerting a biological effect.22,31 For a thorough explanation of the mechanism of action of antisense agents, readers are referred to Bennett and Schwartz (1995).22 Numerous studies have demonstrated that ODNs targeted against DNA binding proteins and transcription factors (e.g., c-myc, c-myb, c-fos, cjun) and cell-cycle regulators (e.g., cdc-2, CDK 1 and 2, cyclins B1 and G1) inhibit SMC proliferation and migration in vitro and/or inhibit in vivo neointimal hyperplasia after balloon injury in animal restenosis models when delivered to the vessel lumen.34−39 Several studies, however, have contended that the observed inhibitory effects may be a result of suppression of SMC migration rather than proliferation.40 While cyclins and CDKs are positive regulators of the cell cycle, CDK inhibitors (CDKIs) such as p16, p21, and p27 are critical negative regulators.31 Overexpression of CDKIs is thus a logical alternative approach to arrest SMC proliferation. Tanner et al. (2000)43 demonstrated that p27 and p21 are potent inhibitors of VSMC proliferation in vitro and in vivo, and are effective in inhibiting neointimal formation in balloon-injured pig femoral arteries. This confirms the results of earlier studies.41,42 This therapeutic effect was shown to be due to the inactivation of CDK2 and CDK4 by both p27 and p21. In contrast, p16 appeared to be less potent in its inhibition of CDK2, which may explain its inability to reduce VSMC proliferation or neointimal formation.43 However, a chimeric p27-p16 fusion gene proved to be a more potent antiproliferative agent than p27 or p16 alone, inhibiting SMC proliferation in vitro to a greater extent. In a balloon-injured pig coronary artery model, catheter-mediated transfer of adenoviral vectors encoding for the p27-p16 fusion gene was able to prevent 50− 62% of injury-induced neointimal hyperplasia despite a low transduction level (5%). In vitro studies revealed that this was due to inactivation of cyclin D1-CDK4, cyclin E-CDK2, and cyclin B-CDK2 complexes, as well as the inhibition of cell cycle progression at several points in the cell cycle.44 Apart from cyclin−CDK complexes and CDKIs, progression through the cell cycle is also regulated by transcription factors, such as p53, E2F, GAX, and GATA-6, which activate CDK or CDKI expression.31 Wild-type p53 (wt-p53) is known to indirectly inhibit CDK−cyclin A, D, and E complexes by directly activating p21, and is therefore able to bring about G1 arrest. It has also been reported to be involved in several other phases of the cell cycle: transit through G1/S and G2/M, and 1282



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on neointimal formation, as assessed by an enlarged luminal area in TIMP-3-treated vessels. Finally, TIMP-3 appears to be superior to TIMP-1 as TIMP-3 has the unique ability to bind tightly to the local ECM for an extended period of time, thereby allowing for prolonged, localized expression.59 TIMP-3 is therefore a promising therapeutic gene that warrants further investigations of its long-term therapeutic effects. Alternative antimigratory approaches investigated and shown to be effective in inhibiting restenosis include downregulation of platelet-derived growth factor (PDGF)-β receptor expression64 and upregulation of plasminogen activator inhibitor (PAI)-1.65 2.1.3. Re-Endothelialization. In recent years, interest has grown in the use of vascular gene therapy targeting multiple pathways simultaneously to elicit pleiotropic therapeutic effects: specifically, accelerating re-endothelialization in addition to reducing intimal hyperplasia. As PTCA and stenting unavoidably causes denudation of the endothelium, accelerated reendothelialization is therefore a desirable post-intervention event that encourages long-term vessel recovery and decreases the risk of stent thrombosis. Furthermore, apart from acting as a protective antithrombotic and anticoagulant barrier between blood and the vessel wall, an intact confluent endothelial monolayer on the luminal surface can also reduce neointimal hyperplasia after angioplasty or stent deployment by limiting SMC proliferation and migration.9 Regrowth of the endothelium is therefore critical to prevention of luminal narrowing through neointimal thickening, and thrombosis. This concept has led to studies exploring the potential of using vascular endothelial growth factors (VEGFs), a group of potent endothelial mitogens, to enhance anatomical and functional endothelial recovery after vascular injury.1,66−69 Early preclinical studies employing catheter-based gene delivery systems used VEGF to accelerate endothelial regrowth and simultaneously reduce SMC proliferation with some success. Isner’s group was the first to report enhanced endothelialization (95% re-endothelialization in 1 week vs incomplete reendothelialization after 4 weeks) along with decreased neointimal thickening and frequency of thrombotic occlusion, when naked plasmid DNA encoding for the 165 amino acid isoform of VEGF (phVEGF165) was concurrently delivered to rabbit femoral arteries during balloon injury via a hydrogelcoated balloon catheter.66 A later study by the same group once again showed that recombinant human VEGF (rhVEGF) delivered to balloon-injured rabbit iliac arteries via a channel balloon catheter was able to significantly reduce in-stent intimal formation (VEGF, 0.87 ± 0.06 mm2, vs control, 1.47 ± 0.12 mm2; p < 0.001), as well as reduce mural thrombus within the stented arterial segment.67 These findings are supported by later studies done by Yla-Herttuala’s group, which reported similar reductions in neointimal thickening using cathetermediated adenoviral gene transfer of VEGF-C and VEGF-D in a hypercholesteromic rabbit aorta restenosis model.68,69 The group also demonstrated that the therapeutic effect of VEGF was nitric oxide-dependent, as the addition of L-NAME, a nitric oxide synthase-inhibitor, blocked the reduction in intimal thickening.69 Extending the findings of earlier studies, in 2004, Isner’s group was able to demonstrate, for the first time, successful stent-based VEGF gene delivery that was safe, effective, and feasible. The study demonstrated that local delivery of naked plasmid encoding for human VEGF-2 (phVEGF-2) via phosphorylcholine (PC)-coated stents was able to achieve

In summary, the efficacy of both cytotoxic and cytostatic antiproliferative therapies as elucidated above provides a compelling reason for further studies utilizing this approach in a primarily proliferative problem such as restenosis. A major challenge in this approach is ensuring precision and specificity of cell cycle-targeting agents for SMCs only, as major events of the cell cycle are common to almost all cell types. GAX, GATA6, and NO are therefore attractive therapeutic targets due to their SMC-specific effects. Delivery of the therapeutic agent in a localized manner will also ensure greater specificity of this strategy.31 One caveat to be acknowledged is that if SMC proliferation only plays a small role in restenosis, then an antiproliferative approach alone may be insufficient.22 2.1.2. Antimigratory. Apart from the antiproliferative approach to reducing neointimal hyperplasia, the antimigratory approach has also been investigated. Without SMC migration to the vessel lumen, neointimal formation would be impossible. SMC migration is dependent on ECM degradation, and this process is effected by matrix metalloproteinases (MMPs). Overexpression of endogenous inhibitors of MMPs, tissue inhibitors of MMPs (TIMPs), has therefore been explored in several studies.1,56−60 TIMP-1 has been the gene of choice in many of these studies as it inhibits MMP-2 and MMP-9. Overexpression of MMP-2 and MMP-9 has been observed in atherosclerotic plaques, and has also been shown to be associated with balloon injury in vessels and neointimal formation. Early studies on TIMP-1 have shown that adenoviral-mediated transfer of TIMP-1 is able to significantly reduce neointimal hyperplasia and inhibit restenosis in various models.58,61−63 The potency of TIMP-1 as a therapeutic gene was further demonstrated in a study by Puhakka et al. (2005),57 which compared the therapeutic effect of TIMP-1 alone, TIMP1 and VEGF-C in combination, and VEGF-A and -C in combination in inhibiting restenosis in a balloon-denuded hypercholesteromic rabbit aorta model. VEGFs are potent endothelial mitogens which have been shown to enhance endothelial recovery and inhibit SMC proliferation in various restenosis models. It was thus hypothesized that combination gene therapy directed to different pathogenic processes may potentially be more efficient than single-agent gene therapy. However, the study showed that adenoviral-mediated TIMP-1 transfection alone was sufficient to reduce restenosis, and combination therapy with VEGF-C did not bring about any significant advantages. Furthermore, TIMP-1 gene transfer (with or without VEGF-C) was able to achieve a prolonged therapeutic effect as compared to VEGF-A and VEGF-C in combination. This may be because VEGFs are unable to inhibit inflammatory and proliferative processes, which also are initiated after balloon injury. The observation that TIMP-1 was as equally effective as TIMP-1 and VEGF-C together suggests that inhibiting SMC migration may possibly be a more efficacious strategy against restenosis compared to enhancing endothelial recovery.57 Other studies, however, have contended that TIMP-1 is only effective in inhibiting early SMC migration in the short run, and may therefore only be suitable for early restenosis treatment.56,60 Studies investigating TIMP-3 have yielded more promising results in inhibiting neointimal hyperplasia, and this has been explained by in vitro and in vivo evidence that TIMP-3 not only inhibits SMC migration but also promotes apoptosis of SMCs without affecting endothelial cells.59,60 Furthermore, although SMC proliferation is not inhibited by TIMP-3, increased SMC proliferation after vascular injury did not negate the therapeutic effect of TIMP-3 1283



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investigated, employing the same method of iNOS gene delivery. Stent deployment in the coronary arteries resulted in more extensive neointima formation than in femoral arteries, hence the dose of iNOS lipoplexes was doubled. Again, this achieved a significant inhibition of neointima formation, as assessed by neointima-to-media ratio (iNOS, 2.2 ± 0.3, vs control, 4.0 ± 0.3; p < 0.01). Importantly, this study demonstrated that the effects seen in a peripheral femoral artery model were reproducible in the coronary artery model of the same species, despite vessel-specific degrees of response to stent-induced injury.4 In a similar study by Wang et al. (2003),16 luminal delivery of adenoviral vectors encoding for human iNOS (AdiNOS) using the Infiltrator catheter in a porcine coronary stent model also achieved a significant reduction in neointima-to-media ratio (AdiNOS, 1.41 ± 0.51, vs control, 2.41 ± 0.84; p < 0.05), resulting in a significantly larger luminal area (p < 0.01).16 Stent-based iNOS gene therapy has also yielded very promising results. Levy’s group has investigated different ways of tethering AdiNOS to stent surfaces for sustained release of vectors. The first method, which made use of bisphosphonatemediated metal stents in conjunction with a recombinant Adreceptor protein, was able to achieve a significant therapeutic effect with reduced in-stent restenosis in a rat carotid stent angioplasty model, as devidenced by the 51% decrease in neointima-to-media ratio and difference in percentage luminal stenosis (AdiNOS, 23.1 ± 3.4, vs BMS control, 40.7 ± 4.2; p < 0.05).3 To further improve vector stability and delivery kinetics, the same group developed a synthetic complex, enabling AdiNOS vector particles to bind reversibly to the stent surface via a hydrolyzable bond. Once again, they achieved a significant 53% reduction of neointimal formation as assessed by neointima-to-media ratio (p < 0.001), among other morphometric parameters.21 These studies, however, have yet to elucidate the effect of iNOS on re-endothelialization. Similarly, eNOS-gene eluting stents have demonstrated the ability to exert a therapeutic effect by inhibiting restenosis and enhancing endothelial regeneration in vivo. The first study to show this was by Sharif et al. (2008),9 who investigated adenoviral-mediated eNOS gene delivery to the vessel wall in normocholesteromic and hypercholesteromic rabbit iliac artery restenosis models. A marked reduction in neointimal formation seen across all parameters assessed was only observed in the hypercholesteromic rabbit model at 4 weeks follow-up. These animals showed a significantly larger luminal area (AdeNOS, 2.73 ± 1.18 mm2, vs AdβGal control, 0.98 ± 0.98 mm2; p < 0.05), as well as markedly reduced percentage restenosis (AdeNOS, 45.23 ± 20.81, vs AdβGal control, 79.6 ± 20.31; p < 0.05) and neointimal area (AdeNOS, 2.32 ± 1.13 mm2, vs AdβGal control, 3.73 ± 0.95 mm2; p < 0.05).9 In a similar study by Brito et al. (2010),7 the use of eNOS lipopolyplexes (LPP) coated onto a stent in a normocholesteromic rabbit iliac artery restenosis model showed that local eNOS production was able to suppress SMC proliferation and macrophage activity, leading to a significant reduction in the neointima-to-media ratio of eNOS LPP-coated groups (1.68 ± 0.35) compared to empty vector-LPP-coated controls (2.44 ± 0.37) at 2 weeks follow-up, indicating decreased neointimal growth. In both of the above-mentioned studies, the reduction in neointima formation in the eNOS-stented vessels was also accompanied by a significant acceleration of endothelial regeneration in the completely balloon-denuded rabbit iliac artery lumen, as early as 2 weeks,7,9,15 and persisted for at least

significant reductions in neointima formation while accelerating re-endothelialization in a hypercholesteromic rabbit iliac artery restenosis model. At 10 days follow-up, re-endothelialization was almost complete (98.7 ± 1%) in balloon-denuded vessels treated with phVEGF-2-eluting stents, whereas PC control stents were only able to achieve 79.0 ± 6% re-endothelialization. A 2.4-fold increase in NO production was also observed in VEGF-2-treated arteries as compared to controls, revealing that anatomical endothelial recovery induced by VEGF-2 gene transfer was also accompanied by enhanced functional recovery.11 Local delivery of VEGF-2 to stented vessels also reduced neointimal proliferation, as seen by significantly increased lumen cross sectional area (VEGF-2, 4.2 ± 0.4 mm2, vs control, 2.27 ± 0.3 mm2; p < 0.001) and reduced percentage cross sectional area narrowing (VEGF-2, 23.4 ± 6%, vs control, 51.2 ± 10%; p < 0.001) at 3 month follow-up. These results once again support the idea that a functional endothelium is critical to the prevention of luminal narrowing by neointimal hyperplasia.11 Another such pleiotropic agent is nitric oxide (NO), which is produced in the vasculature by enzymes called nitric oxide synthases (NOS). NO is a critical molecule as it is responsible for many different functions, including inhibition of platelet activation, inhibition of VSMC proliferation and migration, induction of VSMC death, inhibition of leukocyte chemotaxis, vasodilation, and re-endothelialization.3,4,7,9 The therapeutic potential of NO is therefore much broader than that of any of the drugs currently used with DES.3 Not surprisingly, a number of NO-releasing systems were thus explored initially for clinical application.7 However, these systems were largely limited by several factors: first, the limited bioavailability of NO due to the short half-life (∼2−5 s) resulting from rapid inactivation by circulating hemoglobin, and second, unwanted systemic circulatory effects (e.g., vasodilation) distal to the target vessel.4,7 Systemic administration of NO donors is thus not a feasible option.4 On the other hand, NOS poses an attractive alternative due to its potential for sustained local production.7 Furthermore, NO generated by the NOS transgene can easily diffuse to neighboring cells. This “bystander” effect allows the therapeutic effect to be exerted not only in the transfected cells but also in adjacent cells.4 To date, studies have focused on 2 main types of NOS to reduce neointimal hyperplasia and accelerate reendothelialization: inducible NOS (iNOS) and endothelial NOS (eNOS), both of which have yielded impressive results in vivo. eNOS is one of the constitutive NO synthases, which continuously synthesize low amounts of NO, and is predominantly responsible for vasodilation and regulation of physiological NO homeostasis. In contrast, iNOS is only induced by cytokines such as interferon gamma or tumor necrosis factor alpha in an inflammatory response, and produces high amounts of NO.70 Both catheter-based and stent-based delivery techniques employing iNOS as a therapeutic gene have met with considerable success in reducing neointimal hyperplasia and accelerating re-endothelialization. Using the Infiltrator catheter to deliver 1 μg of iNOS lipoplexes in a porcine femoral artery stent model, Muhs et al. (2003)4 achieved a significant reduction of in-stent plaque area (39.6% decrease; p < 0.05) and plaque ratio (41.8% decrease; p < 0.05), as well as a significantly lower neointima-to-media ratio (iNOS, 1.3 ± 0.27, vs control, 2.0 ± 0.14; p < 0.05) as compared to controls. In addition, a porcine coronary artery stent model was also 1284



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4 weeks after balloon injury and stent implantation.9 In Brito et al. (2010),7 45% re-endothelialization was seen in eNOS-LPP stented vessels, compared to 28% in the control group at 2 weeks follow-up. Likewise, in Sharif et al. (2008),9 85% reendothelialization was seen in eNOS-stented vessels of normocholesteromic animals, compared to 63% in the controls at 2 weeks follow-up. A more pronounced difference was seen in both normocholesteromic (AdeNOS, 91.1 ± 10%, vs AdβGal control, 63.1 ± 2.2%) and hypercholesteromic (AdeNOS, 96.97 ± 3.2%, vs AdβGal control, 28.33 ± 38.76%) animals alike at 4 weeks follow-up.9 In a more recent study by the same group, eNOS delivery via a nonviral, liposome-mediated method also achieved significantly enhanced endothelial regeneration at 2 weeks post-stenting in vessels deployed with lipoeNOS-coated stents (lipoeNOS, 82.48 ± 12.86%, vs lipo-null, 49.58 ± 8.16%; p < 0.05). The results of these 3 studies are explained by the increased bioavailability of NO, which results in enhanced EC migration and/or mobilization of endothelial progenitor cells (EPCs).15 However, in the most recent study by Sharif et al. (2012),15 a therapeutic effect on decreasing neointima formation was not seen. This may be accounted for by the differing cell population targeted by LPPs in vivo: in contrast to adenoviral-mediated gene delivery in vitro and in vivo, which targeted SMCs, LPPs were preferentially taken up by macrophages rather than SMCs in vivo. Macrophages, unlike SMCs, produce endogenous NOS in the form of iNOS. LPP-mediated expression of eNOS in macrophages may have depleted cofactors required for NO production by iNOS, and subsequently led to uncoupling of endogenous macrophage iNOS. This uncoupling could have led to increased superoxide formation, which may account for the raised inflammatory score as seen for lipoeNOS versus liponull (13.61 ± 40.52 vs 7.85 ± 20; p < 0.05). An enhanced inflammatory response may have nullified potential beneficial effects on neointimal formation by LPP-delivered eNOS, which may then account for why, in contrast to AdeNOS-eluting stents, lipoeNOS-eluting stents were able to enhance reendothelialization but not inhibit restenosis.15 Taken together, there is strong evidence reporting the therapeutic benefit of stent-based eNOS gene delivery to the vessel wall. The small differences in therapeutic efficiency seen between groups using the same rabbit model may be attributed to the varying degrees of restenosis upon balloon denudation between the normo- and hypercholesteromic model.7 It is, however, important for groups to test their gene delivery system in atherosclerotic vessels, as transfection efficiency and efficacy of the system may vary in diseased vessel.7 Finally, one caveat must be acknowledged: while the above-mentioned studies have shown that iNOS and eNOS do indeed improve endothelial regeneration, none have demonstrated an inhibition of in-stent thrombosis. 2.1.4. Antithrombotic. Apart from reducing neointimal hyperplasia and/or accelerating re-endothelialization, antithrombotic and anti-inflammatory approaches have also been investigated for use in vascular gene therapy, albeit to a smaller extent. The problem of in-stent thrombosis and late stent thrombosis after percutaneous coronary intervention (PCI) has largely been dealt with by aggressive long-term treatment with antiplatelet agents such as aspirin, clopidogrel, heparin, and abciximab. This has somewhat impeded further work in the field of antithrombotic gene therapy in the context of PCI.17 There are, however, several pertinent reasons for continued

exploration of this therapeutic avenue. First, localized, sitespecific delivery of antithrombotic genes can reduce systemic toxicity and bleeding complications brought about by the use of intravenous antiplatelet drugs. Second, certain antithrombotic genes have been shown to reduce neointima formation in addition to preventing thrombosis. Third, not all patients are suitable for antiplatelet therapy due to antiplatelet drug resistance. The pathophysiological process of in-stent thrombosis is relatively well-understood. Atherosclerotic plaque rupture during balloon angioplasty and/or vascular injury resulting from angioplasty and stent implantation can trigger platelet activation and aggregation, the production of coagulation factors on the platelet surface, formation of a fibrin clot and eventually, a stable, occlusive thrombus.14 Events such as platelet aggregation and fibrin clot formation are therefore intuitive molecular targets in antithrombotic gene therapy. Along this line of reasoning, Takemoto et al. (2009)14 explored the possibility of targeting adenosine diphosphate (ADP), which is a key agonist required to trigger platelet aggregation. Using a gene encoding for human placental ectonucleoside triphosphate diphosphohydrolase (pENTPDase), an enzyme that rapidly hydrolyzes ATP and ADP to AMP, they successfully demonstrated that human pENTPDase gene transfer via a stent was able to effectively inhibit in-stent thrombosis, suppress neointimal hyperplasia and inflammation, and accelerate re-endothelialization in a rabbit femoral artery restenosis model. E-NTPDase is an endogenous enzyme primarily expressed by endothelial cells in the vasculature, and it is critical for inhibiting platelet aggregation. It carries out this role in 2 ways: apart from reducing ADP binding to the platelet surface by hydrolyzing ADP, the resultant AMP is further converted by 5′-nucleotidase to adenosine, a known antithrombotic and anti-inflammatory mediator. However, levels of this critical enzyme are decreased after vessel injury. Restoring vascular E-NTPDase to normal physiological levels was shown in this study to be sufficient for preventing in-stent thrombosis after stenting. Furthermore, human pE-HTPDase gene transfer was able to accelerate reendothelialization, as seen from early recovery of eNOS mRNA expression in injured vessels, as well as inhibit SMC proliferation and macrophage infiltration. However, the mechanisms by which pE-NTPDase exerts these effects were not elucidated in the study.14 More established antithrombotic genes have been investigated for their potential in post-angioplasty intervention, and have been reviewed in detail in ref 71. To name a few, prostacyclin synthase (PGIS),72−75 cyclooxygenase (COX)1,76,77 thrombin inhibitor hiridun,78 and tissue factor pathway inhibitor (TFPI) 79,80 have all demonstrated successful suppression of thrombosis and/or restenosis in various animal models. Prostacyclin (PGI2) is an endogenous inhibitor of platelet aggregation and also a potent vasodilator that, together with the vasoconstrictor thromboxane A2 (TXA2), contributes to the maintenance of vascular tone.74 In addition, PGI2 directly inhibits VSMC proliferation, and also elevates VEGF expression via elevation of cAMP levels. VEGF is then able to accelerate re-endothelialization and endothelial recovery, which can potentially inhibit thrombosis and restenosis.75 As PGI2 is synthesized through a series of enzymatic reactions involving COX-1 and PGIS, overexpression of PGIS and/or COX-1 has hence been explored as a way to increase PGI2 1285



Review

synthesis and subsequently inhibit thrombosis.72,74−77 Zoldhelyi et al. (1996)72 was the first to show that transfection of balloon-injured porcine carotid arteries with a sufficiently high dose of adenoviral vector encoding COX-1 was able to restore COX-1 expression and induce a 4-fold increase in PGI2 expression, thus preventing angioplasty-induced thrombosis. A later study by Liu et al. (2005)77 showed that COX-1 gene transfer was able to induce prolonged vasodilation and reduce thrombus formation in a normocholesteromic porcine artery model. These effects were also observed in a hypercholesteromic rabbit carotid model. It was noted, however, that COX-1 gene transfer prevents restenosis by vasodilation via production of cAMP, PGI2, PGE1, and PGE2, rather than by inhibition of neointimal proliferation.77 The overexpression of PGIS has likewise shown similar therapeutic potential in various animal models.74,75 Numaguchi et al. (2004)75 showed that PGIS gene transfer is able to accelerate re-endothelialization, suppress VSMC proliferation, and prevent restenosis via inhibition of neointimal hyperplasia in a balloon-injured hypercholesteromic rabbit iliac artery model. Compared to previously discussed therapeutic genes, PGI2 may be considered superior in several aspects: First, like NO, PGI2 is secreted from transfected cells and is therefore able to exert a bystander effect on adjacent, nontransfected cells. This means that a lower dose of gene vector and/or rate of transfection can still induce a therapeutic effect, unlike antiproliferative cell cycle regulators.75 Second, cAMP-elevating agents such as PGI2 may be more potent in inhibiting VSMC proliferation than cGMP-elevating agents such as NO.74,75 Despite these encouraging results, there are a couple of limitations to the overexpression of PGIS or COX-1 on their own to increase PGI2 synthesis. First, overexpression of COX-1 leads to the concurrent production of PGE2 together with PGI2: while PGE2 is a vasodilator, it is also a pro-inflammatory prostaglandin, which may bring about undesirable side effects or negate the effects of increased PGI2 production. On the other hand, while studies suggest that PGIS may be superior to COX-1 in this respect as it is able to selectively increase PGI2 synthesis and decrease PGE2 synthesis without affecting TXA2 production,75,77 the degree to which PGIS overexpression can increase PGI2 synthesis is limited by low endogenous levels of COX-1.76 One way of addressing this problem may be to combine COX-1 gene transfer with specific fatty acids, such as dihomo-gamma(γ)-linolenic acid (DGLA), to promote a favorable prostaglandin production profile. DLGA can potentially drive PGE1 synthesis and reduce PGE2 synthesis, without affecting levels of PGI2 synthesis and TXA2 generation. This would augment the strong vasodilative and anti-inflammatory effects of PGE1, and downplay the pro-inflammatory effects of PGE2.75 Alternatively, cotransfection of endothelial cells with COX-1 and PGIS genes at an optimized ratio has been shown to selectively augment PGI2 synthesis by shunting its precursor through the PGIS pathway.76 2.1.5. Anti-Inflammatory. Inflammation is increasingly being acknowledged as one of the key elements leading to neointimal hyperplasia and restenosis.12,69,81 As inflammation after angioplasty and stenting is unavoidable, therapies targeting post-intervention inflammation are therefore a reasonable and appropriate approach to the problem of restenosis. There is a growing body of evidence to show that, in addition to mechanical injury incurred during PCI, stents can induce acute and chronic inflammation in the vessel wall. Recruitment and activation of chronic inflammatory cells such as monocytes

and macrophages have been observed in both early and late stages after arterial injury due to PCI.15,81 This is associated with greater neointimal formation, a likely result of inflammatory cytokines and growth factors (e.g., IL-6, IL-1b, VEGF) produced by activated lesional monocytes, which in turn induce VSMC proliferation and migration.81 As monocyte chemoattractant protein-1 (MCP-1) is a potent chemokine specific for monocytes and is shown to be present at increased levels after vascular injury, an anti-inflammatory approach targeting MCP-1 or its receptor CCR2 would be a rational one. Sunagawa’s group has investigated this approach and achieved much success in the suppression of neointimal hyperplasia with the use of 7ND, a mutant MCP-1 that functions as a dominant negative inhibitor of MCP-1. A 2007 study by the group investigating stent-based 7ND gene delivery in rabbit femoral and monkey iliac arteries demonstrated that blockade of MCP-1 by 7ND not only was able to attenuate monocyte chemotaxis and infiltration, thereby suppressing inflammation, but also was able to directly reduce the number of proliferating SMCs in the neointima, of which proliferation was originally induced by MCP-1.12 Rutanen et al. (2005)69 also demonstrated a decrease in inflammation and neointimal formation by indirectly modulating MCP-1 via NO. NO is a known regulator of MCP-1 in ECs and SMCs, as well as an inhibitor of several cytokine-mediated processes involved in the inflammatory response to vascular injury. Intravascular gene transfer of VEGF-DΔNΔC, which binds to VEGFR-2 and induces endothelial NO production, was shown to reduce neointima formation in balloon-denuded rabbit aortas via a NO-dependent mechanism involving a reduction of macrophage influx into the denuded vessel wall after VEGF-D transfer.69 In summary, an enormous amount of research has been conducted to find appropriate molecular targets and respective therapeutic gene products (Table 1). It is necessary to then define an ideal vector and design an effective and efficient delivery system. 2.2. Gene Delivery Vectors. A large array of gene delivery vectors has been evaluated for their ability to introduce therapeutic genes into vascular tissue. An ideal vector should be able to exhibit several characteristics: cell specificity, exceptional potency (to achieve high transfection/transduction of target cells), biocompatibility with low toxicity and immunogenicity, high efficiency (rapid uptake and incorporation), high retention, and prolonged expression.17 It also needs to overcome the anatomical barriers of the vasculature (i.e., tunica intima and basement membrane, external and internal elastic lamina, and tunica adventitia) to reach the SMCs of the tunica media, which is the major targeted site of action in most vascular gene therapy studies.7,18 Vectors for gene delivery can be broadly classified into 2 categoriesviral and nonviral both of which will be reviewed here. 2.2.1. Nonviral Plasmid-Mediated Gene Transfer. Many different forms of nonviral vectors have been investigated, including naked plasmid DNA, plasmid DNA-containing cationic liposomes, and DNA−polycation complexes (Table 2). Compared to viral vectors, nonviral vectors can deliver a larger insert size, and also hold more promise for clinical application as they have a high safety profile and are less immunogenic.15 However, they are generally limited by low efficiency of cell transfection and quick intracellular degradation.1 In spite of this, inefficient transduction can still exert a significant therapeutic effect if used in combination with a 1286


DOTAP-PbAE-pDNA lipopolyplexes

adenovirus

pDNA complexed with lipofectin (1:1 mixture of DOTMA and DOPE) to form lipoplexes naked pDNA naked pDNA

eNOS

eNOS

eNOS β-gal

1287

adenovirus

GFP

dodecylated chitosanpDNA nanoparticles adenovirus

GFP

adenovirus

cationic pDNA/PEI polyplexes

GFP

GFP

DOTAP-PbAE-pDNA lipopolyplexes

GFP

pDNA

pDNA

7ND

β-Gal TIMP-3 GFP

pDNA

pENTPDase

GFP VEGF-2

HL-modified adenovirus

GFP Luc iNOS

delivery vector

adenovirus (Ad)

GFP iNOS

gene

Table 1. Gene Eluting Stentsa gene delivery platform

in vitro

in vivo

stents coated with denatured collagen-PLGA coating conjugated to antiadenoviral antibodies

rat aortic SMCs (A10)

rat aortic SMCs (A10), bovine aortic endothelial cells (BAEC), and murine endothelioma cells (H 5 V) rat arterial SMCs (A10)


porcine coronary artery stent model

n/a

porcine coronary artery stent model rabbit carotid artery stent model

rabbit carotid artery stent model

COS-7 cells

stents coated with dopamine-derivatized hyaluronic acid (HA-DA); pDNA/PEI polyplexes ionically adsorbed onto stent surface stents spray-coated with dodecylated chitosan gene carrier BiodivYsio HI matrix PC-coated premounted stents stents coated with collagen conjugated to anti-pDNA antibody via SPDP stents coated with polylactide (PLA)-based nanoparticles conjugated to Ad-binding proteins porcine coronary tissue

rabbit iliac artery restenosis model with balloon denudation of endothelium n/a

human aortic SMCs and endothelial cells

stents coated with type B gelatin containing dispersed lipopolyplexes

rabbit iliac artery restenosis model with balloon denudation of endothelium rabbit iliac artery model with balloon denudation of endothelium; normocholesterolemic and hypercholesterolemic rabbit iliac artery model with balloon denudation of endothelium; normocholesterolemic and hypercholesterolemic rabbits pig stent angioplasty model rabbit iliac artery angioplasty with balloon denudation of endothelium; hypercholesterolemic and normocholesterolemic rabbits rabbit femoral artery model with repeated balloon injury to induce platelet-rich thrombus rabbit femoral artery and monkey iliac artery models

rat carotid stent angioplasty model

rat carotid stent angioplasty model

THP-1 cells and human coronary artery VSMC

n/a

rat aortic SMCs (A10) n/a

n/a

n/a

n/a



stents coated with PVOH polymer solution containing 7ND cDNA plasmid

stents coated with biodegradable cationic gelatin hydrogel

PLGA-coated stents BiodivYsio HI matrix PC-coated premounted stents

BiodivYsio HI matrix PC-coated premounted stents

PAA-BP-treated stents; covalent conjugation of surface-bound PAA-BP with either antiAd antibody or D1, a recombinant Adreceptor protein, to enable Ad tethering PABT/PEI(PDT)-treated steel stents; covalent attachment of HL-modified Ad vectors to the PABT/PEI(PDT)-treated metal surfaces via HL cross-linker stents coated by depositing LPP-containing type B gelatin solution and then adding PLGA uniformly over gelatin coating BiodivYsio HI matrix PC-coated premounted stents

results

efficient and highly localized gene delivery

increased gene transduction via coxsackie-adenovirus receptor (CAR)independent cellular uptake of Ad vector

significant reduction of neointimal area via increase in apoptosis and decrease in neointimal cell density; no increase in inflammation or proliferation high efficiency and site-specific gene delivery

localized and prolonged delivery of reporter genes

inhibition of in-stent thrombosis by preserving local NTPDase activity; inhibition of restenosis by acceleration of re-endothelialization, suppression of neointimal hyperplasia and reduction of inflammation inhibition of mononuclear leukocytes chemotaxis and VSMC proliferation/ migration; attenuation of stent-associated monocyte infiltration; long-term reduction of neointima formation successful immobilization of LPP onto stents using gelatin; high cellular uptake and transfection efficiency in vitro and in vivo; however, poorly sustained LPP delivery in vivo due to rapid dissolution of gelatin coating successful deposition of DNA/PEI polyplexes onto HA-DA-coated stents via electrostatic assembly; sustained and controlled pDNA release; high gene transfection efficiency and effective biocompatibility

successful arterial transfection using DNA-eluting stent increase in NO production and acceleration of re-endothelialization; inhibition of restenosis via reduction of neointima formation

acceleration of re-endothelialization but no reduction in neointimal formation (i.e., does not reduce restenosis); prolonged and localized gene expression

successful immobilization of adenovirus onto PAA-BP coated stents via D1 and anti-Ad antibody; site-specific and localized transduction of Ad-GFP in vitro and in vivo; inhibition of restenosis with Ad-iNOS; greater Ad-binding capacity of D1 vs anti-Ad antibody successful immobilization of adenovirus onto stent using 3-component gene vector binding complex: PABT, PEI(PDT), HL; controlled and sustained release of Ad via hydrolyzable HL; localized transgene expression in vitro and in vivo; increase in NO production; inhibition of restenosis successful immobilization of LPP using PLGA and type B gelatin coatings; increase in NO production and acceleration of re-endothelialization; reduction of restenosis via inhibition of neointimal hyperplasia acceleration of re-endothelialization but no inhibition of in-stent thrombosis demonstrated; reduction of restenosis via inhibition of neointimal formation

83

84

89

59

13

8

2

12

14

82 11

15

9

7

21

3

ref

Molecular Pharmaceutics Review


gene delivery platform

stents coated with PAA-BP conjugated to anti-pDNA antibody via SPDP

denatured collagen

in vitro

pig arterial SMCs

rat arterial SMCs (A10)

in vivo porcine coronary artery stent model n/a

results denatured collagen incorporated into pDNA-stent coating increases the level of gene expression in vitro and in vivo high localization and efficient gene delivery

87

86

ref

1288

effective and rapid cell uptake; high efficiency of gene transfection; high levels of transgene expression within 24 h; prolonged transgene expression (3−42 days); low dose of pDNA required for in vivo expression (∼25.5 μg); localized delivery to area of increased cellular proliferation (e.g., areas of neointimal formation); LPPs predominantly target macrophages rather than SMCs in vivo, and therefore should not be used to carry eNOS to avoid uncoupling of endogenous macrophage iNOS and subsequent increased inflammation

64

13

8

34

2, 4, 7, 15

refs 11, 82

advantages/limitations

effective and efficient delivery to stented site; no systemic expression; transgene expression detectable for up to 10 days after implantation; high dose of pDNA required for in vivo expression (100 μg to 1 mg)

superior to conventional liposomal carriers; enhanced efficiency of cell uptake, nuclear localization and intracellular stability of encapsulated gene; increased efficiency of gene transfection; high transgene expression; nontoxicenvelope protein of inactivated virus cannot replicate and integrate into genome polymeric nanoparticle-based gene delivery cationic pDNA/PEI poly2-fold increase in gene transfection efficiency and prolonged gene expression compared to solution-state DNA delivery; spherical morphology and nanoscale size increase rate of cellular uptake and internalization of DNA plexes dodecylated chitosan-plasmid large packaging capacity; excellent biocompatibility; low immunogenicity, no inflammatory response, high safety profile; easy preparation; high gene transfection efficiency and expression; DNA nanoparticles dodecylated chitosan protects DNA from DNase degradation and enhances thermal stability of DNA; chitosan nanoparticles often lead to liver aggregationcrucial that DCDNPs are (DCDNPs) covalently tethered onto stent in order to prevent systemic spread; mechanisms of DCDNP release from stent and DNA release from complex still unknown ODN-encapsulated PLGA high encapsulation efficiency; addition of Ca2+ to the PLGA formulation minimizes escape of negatively charged ODNs to the exterior; increased resistance of ODNs to nuclease degradation; spherical morphology and nanoscale size increase efficacy of cellular uptake; sustained and controlled release of ODNs over a period of 1 month nanoparticles

lipid nanoparticle-based gene delivery DOTAP-PbAE-plasmid DNA lipopolyplexes, lipofectin (DOTMA/DOPE)-plasmid DNA lipoplexes HVJ-liposomes

naked plasmid DNA

method of nonviral gene transfer

Table 2. Summary of Methods for Nonviral Gene Transfer

a Key: GFP, green fluorescent protein; iNOS, inducible nitric oxide synthase; PAA-BP, polyallylamine bisphosphonate; NO, nitric oxide; PABT, polyallylamine bisphosphonate with latent thiol groups; PEI(PDT), polyethyleneimine with pyridyldithio groups; HL, bifunctional (amine- and thiol-reactive) cross-linker with a labile ester bond; DOTAP, cationic 1-oleoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4yl)amino] hexanoyl]-3-trimethylammonium propane; PbAE, poly(beta-amino ester; pDNA, plasmid DNA; PLGA, poly(D,L-lactide-co-glycolide; PC, phosphorylcholine; DOTMA cationic lipid, N-[1-(2,3dioleyloxy)propyl]-N,N,N-trimethylammonium chloride; DOPE, dioleoyl phosphatidylethanolamine; VEGF-2, vascular endothelial growth factor-2; pE-NTPDase, human placental ectonucleoside triphosphate diphosphohydrolase gene; 7ND, anti-monocyte chemoattractant protein-1 gene; PVOH, polyvinyl alcohol; PEI, polyethyleneimine; TIMP-3, tissue inhibitor of metalloproteinase-3; SPDP, Nsuccinimidyl 3-(2-pyridyldithio)propionate.

pDNA

GFP

delivery vector

pDNA

GFP

gene

Table 1. continued

Molecular Pharmaceutics Review



Review

potent secretory gene product with effects on adjacent cells, for example, VEGF. This is especially so if there is sustained release of the vector from the stent. Furthermore, recent studies have shown that small vector size in the nanoscale range bestows significant advantages in numerous aspects: increased cellular uptake, decreased inflammatory response (which is heightened in larger-sized particles due to uptake by macrophages rather than the target SMCs or ECs), increased cellular incorporation, and decreased accumulation in the liver, spleen, and lungs.6,13 In addition, although high shear arterial pressure allows for only a very short residence time of the vector at the target site, the use of nanosized vectors allows for rapid cell uptake during this short window of time. Nanoparticle-based nonviral vectors will therefore be reviewed in greater detail in the following sections. 2.2.1.1. Naked Plasmid DNA. Naked plasmid DNA comprises DNA encoding for the recombinant gene joined with DNA sequences permitting replication as a plasmid in bacterial hosts.17 Klugherz et al. (2000) was the first group to report successful use of a GFP plasmid DNA-coated stent to transfect rat aortic SMCs in vitro (7.9% vs 0.6% in controls) as well as stent-treated coronaries in a pig coronary angioplasty model in vivo. This approach was able to demonstrate successful transfection of 500 μg to 1 mg of plasmid DNA per stent.82 A later study by Walter et al. (2004)11 was able to demonstrate, for the first time, successful delivery of a therapeutic gene, VEGF-2, also using plasmid DNA-coated stent, though at a lower DNA load per stent (100−200 μg). The plasmid was effectively delivered to only the stented site within 24 h of stent implantation, with no evidence of systemic expression. Transgene expression was detectable in the stented site up to 10 days after implantation, which was sufficient to exert a therapeutic effect.11 2.2.1.2. Lipid Nanoparticle-Based Gene Delivery. An early study by Muhs et al. (2003)4 examined iNOS plasmid delivery to porcine femoral and coronary stent injury models using a lipid−DNA complex (lipoplex) vector, which was produced by complexing iNOS plasmid with the cationic lipid DAC-30 (a mixture of the monocationic lipid 3β-(N,N′-dimethylaminoethane)-carbamoylcholesterol (DAC) at 30% w/w and the neutral colipid dioleoyl phosphatidylethanolamine (DOPE) at 70%, w/w). Cationic lipid-mediated iNOS gene transfer combined with local delivery by an Infiltrator infusion catheter was shown to enable sufficiently high expression of iNOS protein to bring about therapeutic efficacy in diminishing neointimal formation. The group demonstrated proof of successful transfection and dose-dependent iNOS transgene expression (up to 2 μg of iNOS lipoplexes), and also confirmed correct transgene processing. Transgene expression was seen within 24 h of stenting, reached a maximum at 3 days with 2 μg of iNOS lipoplexes (44.3 ± 4.2% iNOS-positive area, p < 0.001 compared to controls), and was sustained for at least 7 days after stenting.4 Using a similar liposomal system, Brito et al. (2010)2 examined gene delivery in a balloon-injured rabbit iliac artery restenosis model via GFP plasmid DNA-containing cationic liposomes (lipopolyplexes). GFP plasmid DNA was precondensed with poly(beta-amino ester) (PbAE) and encapsulated in cationic 1-oleoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-3-trimethylammonium propane (DOTAP) liposomes to form DOTAP-PbAE-plasmid DNA lipopolyplexes (LPP) with an approximate particle diameter of 236.2 ± 73.5 nm (Figure 3). In vitro studies showed LPP-mediated SMC uptake within 2 h and GFP transgene expression within 24 h of

Figure 3. LPPs of spherical morphology and size 200−400 nm were prepared by plasmid DNA complexation with PbAE followed by encapsulation in cationic liposomes. Reproduced with permission from ref 7. Copyright 2010 BioMed Central.

stent implantation. Expression efficiency was sustained for 48 h and then decreased by 72 h, although GFP expression in LPPtreated cells was still significantly higher than in controls. In vivo studies likewise demonstrated GFP expression in rabbit iliac arterial tissues after 24 h of LPP-containing type B gelatincoated stent implantation, with the medial layer having the highest level of GFP expression. These results, achieved with a dose of approximately 25.5 μg total plasmid DNA upon in vivo implantation, were apparently comparable to previous studies using plasmid DNA at 50- to 100-fold higher concentrations and viral-based delivery systems.2 In a follow-up study by the same group using the same LPP vector for eNOS-encoding plasmid DNA this time, in combination with a modified stent coating to allow sustained LPP delivery in vivo, the group was able to show sufficiently high eNOS expression locally (125 pg eNOS vs 10 nm), stably self-crosslinked hydrogel layer, which was firmly coated onto the stent surface by direct DA−metal interactions. Finally, the highly water-absorbing HA layer was shown to confer protection from nonspecific protein adsorption upon contact with platelet-poor

Figure 4. Illustration of pDNA-PBAE-DOTAP liposome-based lipopolyplexes (LPPs) embedded within type B gelatin matrix. Reproduced with permission from ref 2. Copyright 2010 John Wiley & Sons, Inc.

limited stent surface area (

Nanotechnology-Based Gene-Eluting Stents - Molecular

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