**7. Gene eluting stents**

Stents represent an attractive alternative for targeted gene delivery, thanks to their permanent scaffolding structure. Polymer-coated stents are used as delivery devices for the elongated time release of small molecules. The greatest challenge with this delivery system lies in achieving a compatible relationship between the stent, coating matrix, and vessel wall. As a result of the long residence times of coatings on the stents, attention has been focused on using them as reservoirs for prolonged local drug administration. While there is much known about stent coatings for drug elution, less is known about the use of these substances for gene elution [12]. The polylactic-polyglycolic acid copolymer (PLGA) is an FDA-approved, biodegradable, and biocompatible polymer and is widely used in various drug release applications, as graft materials in tissue engineering studies.

Although the emergence of drug-eluting stents significantly reduced the rate of restenosis after the interventions, it is not completely eliminated especially in complex lesions. Beside, delayed endothelialization after drug-eluting stent implantation is reported and considered to be the cause of late thrombosis, which is a critical complication. Gene transfer can be an option to address these problems by inhibiting VSMCs proliferation, and with some genes, promoting endothelialization [6].

safety and delivery mechanisms, still have to be resolved before percutaneous gene therapy can be widely applied in clinic. With the aim of inhibition of restenosis, several new types of carriers and technology have been developed, and a great number of gene therapy methods

Vascular gene transfer is used to overexpress therapeutically important proteins and correct genetic defects. Promising therapeutic effects have been obtained in animal models of restenosis via transfer of genes, such as encoding vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), nitric-oxide synthase, thymidine kinase, tissue inhibitor of metalloproteinases, etc. [9]. In vascular gene therapy, it is required to combine a therapeutic gene or a therapeutic gene product with an appropriate vector. These complexes are delivered

Catheter delivery system is one of the devices used in vascular gene therapy. Several balloon catheters (porous and microporous catheters, hydrogel catheters, dispatch catheters, and infiltrator catheters) have been used for gene-based delivery. Rolland et al. [10] have investi‐ gated hydrogel-coated catheters for the delivery of interested drug and gene. In recent years, Saurer et al. [11] have designed ultrathin multilayered polyelectrolyte films fabricated on embolectomy catheter balloons by alternately adsorbing layers of a hydrolytically degradable poly (β-amino ester) for the localized delivery of plasmid DNA to vascular tissue. Although catheters seem to be a simple tool for gene delivery, several factors limit its efficiency. Most of the catheters cause localized vascular injury with increased inflammatory response and neointimal proliferation. Additionally, in direct injection method and catheter-based gene delivery, transgene expression is limited within the injection site and homogenous expression is not achieved. In point of fact, in stent-based gene delivery approach, a homogenous transgene expression is achieved in comparison to catheter-based gene delivery methods.

Stents represent an attractive alternative for targeted gene delivery, thanks to their permanent scaffolding structure. Polymer-coated stents are used as delivery devices for the elongated time release of small molecules. The greatest challenge with this delivery system lies in achieving a compatible relationship between the stent, coating matrix, and vessel wall. As a result of the long residence times of coatings on the stents, attention has been focused on using them as reservoirs for prolonged local drug administration. While there is much known about stent coatings for drug elution, less is known about the use of these substances for gene elution [12]. The polylactic-polyglycolic acid copolymer (PLGA) is an FDA-approved, biodegradable, and biocompatible polymer and is widely used in various drug release applications, as graft

have been studied.

420 Muscle Cell and Tissue

to target cells from a device.

**7. Gene eluting stents**

materials in tissue engineering studies.

**6. Catheter-mediated local gene transfer**

Klugherz et al. [13] have developed stents coated with polylactic-polyglycolic acid copolymer (PLGA), and they incorporated green fluorescent protein (GFP) plasmid DNA via emulsion coating. They reported the first successful in vivo transfection using a DNA controlled-release stent. GFP-encoding plasmid was efficiently expressed in rat aortic SMCs with 1% percent efficiency. In later years, the same group developed an intravascular stent with a denatured collagen-polylactic-polyglycolic acid for controlled release of GFP-encoding plasmid. Target protein expression was determined with 10.8% efficiency. The level of expression was significantly higher than previous study. They have concluded that denatured collagen incorporated into plasmid DNA-stent coating formulation increased the target protein expression via integrin-related mechanisms and associated changes in the arterial smooth muscle cell actin cytoskeleton [14]. In another study, Takahashi et al. have developed metallic stent-coated polyurethane emulsion containing plasmid DNA. They have evaluated in vivo transgene expression levels, and they have reported that transgene expression has occurred only in vessel segments in contact with the stent. Moreover, analysis of the GFP expression pattern revealed a high frequency of marker protein-positive cells occurring at or near the luminal surface. They have concluded that polymer-coated stents provide a new capability for transgene delivery to immune cells that are believed to contribute to the development of instent restenosis [15].

Walter et al. [16] have evaluated the delivery of human vascular endothelial growth factor (hVEGF-2)-encoding plasmid delivery from a gene-eluting stent. They did not use a vehicle to encapsulate the plasmid DNA, encoding for could achieve similar reductions in neointima formation while accelerating, rather than inhibiting, re-endothelialization. They have found that the lumen cross-sectional area (4.2 ± 0.4 versus 2.27 ± 0.3 mm2, *P* < 0.001) was significantly greater and the percentage of cross-sectional narrowing was significantly lower (23.4 ± 6 versus 51.2 ± 10, *P* < 0.001) in VEGF stents compared with control stents implanted in hypercholes‐ terolemic rabbits [16]. In another study, Walsh et al. developed metallic stents coated with a polyurethane emulsion containing plasmid DNA (plasmid-encoded marker genes, b-galacto‐ sidase, luciferase, and GFP), which were implanted by Takahashi et al. [15] in rabbit iliac arteries to evaluate transgene delivery. They have observed transgene expressions only in vessel segments in contact with the stent, and they have also emphasized that the extent of transgene expression was dependent upon the quantity of DNA loaded onto the stent [15].

Nitric oxide (NO) is an important regulator of vascular cellular proliferation. NO promotes EC growth and inhibits proliferation of SMCs in the vessel. Additionally, NO reduces platelet adhesion and aggregation. ECs produce NO via nitric oxide synthase (NOS). Accordingly, Bohl Masters et al. [17] have evaluated the effects of localized delivery of NO from hydrogels covalently modified with S-nitrosocysteine (Cys-NO) on neoinitma formation in a rat balloon injury model. They have reported that localized the delivery of NO from hydrogels inhibited neointima formation by approximately 75% at 14 days. Recently, Sharif et al. [18] have studied therapeutic gene delivery from a stent. They have developed lipoplexes composed of lipofectin and therapeutic eNOS gene. They have coated lipoplexes directly onto the surface of stents and have demonstrated efficient gene delivery for 28 days via liposome-mediated gene delivery.

In another study, Zhu et al. [19] have developed stent-coated dodecylated chitosan-plasmid DNA nanoparticles (DCDNPs) and used them as scaffolds for localized and prolonged delivery of reporter genes into the diseased blood vessel wall. As prepared DCDNPs were spray coated on stents, and a thin layer of dense DCDNPs was successfully distributed onto the metal struts of the endovascular stents. Both in vitro and in vivo expression levels of plasmid DNA-encoding GFP were evaluated. In cell culture, DCDNP stents containing plasmid EGFP-C1 exhibited high level of GFP expression in cells grown on the stent surface and along the adjacent area. In animal studies, reporter gene activity was observed in the region of the artery in contact with the DCDNP stents, but not in adjacent arterial segments or distal organs. Thus, they have concluded that the DCDNP stent provides a very promising strategy for cardiovascular gene therapy [19].

In recent years, Paul et al. [20] have developed a really functional nanobiohybrid hydrogelbased endovascular stent device. The hydrogel was comprised of fibrin matrices, assembled layer by layer on stent surface with alternate layers carrying endosomolytic Tat peptide/DNA nanoparticles or nanoparticles hybridized to polyacrylic acid wrapped single-walled carbon nanotubes. In vitro studies have demonstrated that CNTs incorporated in the hydrogel layers play a major role in tuning the bioactivity of the stent. In addition, the developed stent formulation can significantly reduce the loss of therapeutics while traversing through the vessel and during deployment. In addition to all these, they have demonstrated that the hydrogel-based scaffold carrying therapeutic gene significantly enhances the re-endotheliali‐ zation of injured artery via in vivo experiments compared to controls. In conclusion, they have declared that this new technology is going to be very useful for controlled delivery of multiple biotherapeutics from stent and other biomedical devices [21].

Since the long-term clinical studies of DES have reported high incidence of late thrombosis, Yang et al. [22] have developed a drug and a gene containing system. They have coated the stent with bilayered PLGA nanoparticles containing VEGF plasmid in the outer layer and paclitaxel in the inner core. They have suggested that while re-endothelialization is going to promote by early release of VEGF gene, slow release of Paclitaxel is going to suppress smooth muscle cell proliferation. They have demonstrated that VEGF/Paclitaxel containing NP-coated stents showed complete re-endothelialization and significantly suppressed in-stent restenosis after 1 month compared to commercial DES [22].

Evidence about restenosis suggests that vascular injury during stent placement and angio‐ plasty procedure activates medial VSMCs, changing them from a quiescent to a proliferative phenotype, and leads them to migrate from the media into the intima. Matrix metalloprotei‐ nases (MMPs) are zinc-dependent endopeptidases. They digest extracellular matrix compo‐ nents, and they play a major role in the formation of restenosis. It is well known that following angioplasty, MMPs are secreted increasingly. MMPs are secreted as zymogens. In normal physiological vascular remodeling, the activity of MMPs is tightly regulated at the transcrip‐ tion level, the activation of their pro-form or zymogens, the interaction with specific ECM components, and the inhibition by endogenous inhibitors. Tissue inhibitor of matrix metallo‐ proteinases (TIMPs) are the inhibitors of MMPs. Many MMPs and TIMPs are regulated at the level of transcription by a variety of growth factors, cytokines, and chemokines [23]. The interruption of MMPs activity by tissue inhibitor metalloproteinase infection has been shown to limit SMC proliferation and migration through various models by researchers [24]. Local gene transfer of tissue inhibitor of metalloproteinase-2 (TIMP-2) has been studied on a mouse model. TIMP-2 recombinant adenoviruses overexpressing human TIMP-2 gene have been transferred to SMCs, and the findings demonstrated significant decrease in vein graft diameter [25]. Thus, VSMCs seem to be the most promising cell type to be targeted for inhibition of restenosis. Recently, Laçin et al. [26] have used PEGylated nanoparticles poly(St/PEG-EEM/ DMAPM) monosized nanoparticles with significantly high cationic charge for the transfection of TIMP-2-encoding plasmid to SMCs. Increased TIMP-2 protein expression in SMCs accord‐ ing to nontransfected SMCs confirmed the successful delivery and expression of the tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) gene via a nonviral transfection gene therapy approach. This PEG-lated monosized, nontoxic, and highly positively charged nanoparticle poly(St/PEG-EEM/DMAPM) was successfully used in SMCs transfection studies.

injury model. They have reported that localized the delivery of NO from hydrogels inhibited neointima formation by approximately 75% at 14 days. Recently, Sharif et al. [18] have studied therapeutic gene delivery from a stent. They have developed lipoplexes composed of lipofectin and therapeutic eNOS gene. They have coated lipoplexes directly onto the surface of stents and have demonstrated efficient gene delivery for 28 days via liposome-mediated gene

In another study, Zhu et al. [19] have developed stent-coated dodecylated chitosan-plasmid DNA nanoparticles (DCDNPs) and used them as scaffolds for localized and prolonged delivery of reporter genes into the diseased blood vessel wall. As prepared DCDNPs were spray coated on stents, and a thin layer of dense DCDNPs was successfully distributed onto the metal struts of the endovascular stents. Both in vitro and in vivo expression levels of plasmid DNA-encoding GFP were evaluated. In cell culture, DCDNP stents containing plasmid EGFP-C1 exhibited high level of GFP expression in cells grown on the stent surface and along the adjacent area. In animal studies, reporter gene activity was observed in the region of the artery in contact with the DCDNP stents, but not in adjacent arterial segments or distal organs. Thus, they have concluded that the DCDNP stent provides a very promising strategy

In recent years, Paul et al. [20] have developed a really functional nanobiohybrid hydrogelbased endovascular stent device. The hydrogel was comprised of fibrin matrices, assembled layer by layer on stent surface with alternate layers carrying endosomolytic Tat peptide/DNA nanoparticles or nanoparticles hybridized to polyacrylic acid wrapped single-walled carbon nanotubes. In vitro studies have demonstrated that CNTs incorporated in the hydrogel layers play a major role in tuning the bioactivity of the stent. In addition, the developed stent formulation can significantly reduce the loss of therapeutics while traversing through the vessel and during deployment. In addition to all these, they have demonstrated that the hydrogel-based scaffold carrying therapeutic gene significantly enhances the re-endotheliali‐ zation of injured artery via in vivo experiments compared to controls. In conclusion, they have declared that this new technology is going to be very useful for controlled delivery of multiple

Since the long-term clinical studies of DES have reported high incidence of late thrombosis, Yang et al. [22] have developed a drug and a gene containing system. They have coated the stent with bilayered PLGA nanoparticles containing VEGF plasmid in the outer layer and paclitaxel in the inner core. They have suggested that while re-endothelialization is going to promote by early release of VEGF gene, slow release of Paclitaxel is going to suppress smooth muscle cell proliferation. They have demonstrated that VEGF/Paclitaxel containing NP-coated stents showed complete re-endothelialization and significantly suppressed in-stent restenosis

Evidence about restenosis suggests that vascular injury during stent placement and angio‐ plasty procedure activates medial VSMCs, changing them from a quiescent to a proliferative phenotype, and leads them to migrate from the media into the intima. Matrix metalloprotei‐ nases (MMPs) are zinc-dependent endopeptidases. They digest extracellular matrix compo‐ nents, and they play a major role in the formation of restenosis. It is well known that following

delivery.

422 Muscle Cell and Tissue

for cardiovascular gene therapy [19].

biotherapeutics from stent and other biomedical devices [21].

after 1 month compared to commercial DES [22].

Polyethylene glycol (PEG) is the polymer of choice for nonviral vector systems because it possesses several favorable properties such as the lack of immunogenicity, antigenicity, and toxicity and a high solubility in water and in many organic solvents. The cytotoxicity of the polycationic carriers used in gene therapy is an important consideration, especially when polycations with high positive charge were used. Thus, to overcome the cationic polymeric vector-induced toxicity, many researchers encapsulate genetic materials or drugs into a PEG shielded cationic liposomal bilayer [27]. PEG is also approved by the FDA for human use. PEGylation of a drug or a material helps to reduce its excretion by the kidneys and avoids its degradation by proteolytic enzyme. Additionally, PEGylation prevents molecule from reticuloendothelial (RES) clearance by enhancing the water solubility of the molecule and to reduce its immunogenicity and antigenicity [28-30].

Cardiovascular gene therapy is the third most popular application for gene therapy. Although preclinical studies of gene therapy studies for restenosis have shown promising results for the potential application of the gene delivery methods in cardiovascular disease, numerous cardiovascular gene therapy clinical trials have not demonstrated substantially positive results for effective gene transfer. A major disappointing feature of the trials is that while preclinical and uncontrolled phase-I gene therapy trials have been continued in a positive matter, none of the randomized controlled phase-II/III cardiovascular gene therapy trials have shown clinically relevant positive effects [31]. Low gene transfer efficiencies were observed with most of trials. A sophisticated efficient delivery method for cardiovascular applications is still not existing, and only low gene expression levels could be detected in target tissues [31]. Recently, several delivery approaches have been designed for the treatment of restenosis, but a number of challenging obstacles must be solved. For example, for different types of biomolecules (miRNA, siRNA, plasmid, peptide, etc.), different types of materials and different types of vector systems are used. Therefore, it is important to develop unique gene delivery systems that have enhanced transgene efficacy, are safe, and are clinically reliable.
