Elsevier

Biomaterials

Volume 32, Issue 2, January 2011, Pages 610-618
Biomaterials

Delivery of plasmid DNA to vascular tissue in vivo using catheter balloons coated with polyelectrolyte multilayers

https://doi.org/10.1016/j.biomaterials.2010.09.009Get rights and content

Abstract

We report an approach for the localized delivery of plasmid DNA to vascular tissue from the surfaces of inflatable embolectomy catheter balloons. Using a layer-by-layer approach, ultrathin multilayered polyelectrolyte films were fabricated on embolectomy catheter balloons by alternately adsorbing layers of a hydrolytically degradable poly(β-amino ester) and plasmid DNA. Fluorescence microscopy revealed that the films coated the surfaces of the balloons uniformly. Coated balloons that were incubated in phosphate-buffered saline at 37 °C released ∼25 μg DNA/cm2 over 24 h. Analysis of the DNA by gel electrophoresis showed that the DNA was released in open-circular (‘nicked’) and supercoiled conformations, and in vitro cell transfection assays confirmed that the released DNA was transcriptionally active. Arterial injury was induced in the left common, carotid arteries of Sprague–Dawley rats using uncoated balloons, followed by treatment with film-coated balloons for 20 min. X-gal, immunohistochemical, and immunofluorescence staining of sectioned arteries indicated high levels of β-galactosidase or enhanced green fluorescent protein (EGFP) expression in arteries treated with film-coated balloons. β-galactosidase and EGFP expression were observed throughout the medial layers of arterial tissue, and around approximately two-thirds of the circumference of the treated arteries. The layer-by-layer approach reported here provides a general platform for the balloon-mediated delivery of DNA to vascular tissue. Our results suggest the potential of this approach to deliver therapeutically relevant DNA to prevent complications such as intimal hyperplasia that arise after vascular interventions.

Introduction

Advances in the fields of interventional cardiology and vascular surgery have led to the development of less invasive approaches for the treatment of atherosclerosis, including angioplasty and the implantation of bare metal or drug-eluting intravascular stents. These and other new techniques have saved or improved the lives of millions of patients suffering from coronary artery disease or other vascular diseases [1]. Unfortunately, however, the implantation of stents is not appropriate for the treatment of all atherosclerotic lesions [2], and restenosis caused by complications such as thrombosis and intimal hyperplasia can impact the success of many vascular interventions [3], [4], [5]. In total, it is estimated that intimal hyperplasia resulting from interventional procedures leads to the failure of 5–30% of all vascular reconstructions. Therefore, the development of new, targeted approaches to prevent intimal hyperplasia after vascular surgery would represent a significant step toward the clinical treatment of atherosclerosis and contribute significantly to the well-being of patients requiring vascular interventions [2].

Intimal hyperplasia is characterized in part by the migration and proliferation of vascular smooth muscle cells, leading to a narrowing of the lumen of an affected artery [6]. Recent animal studies suggest that bone marrow-derived progenitor cells also contribute to intimal lesions [7]. Activation of residential smooth muscle cells and mobilization of progenitor cells are triggered by signals (e.g., growth factors, cytokines, chemokines, and other factors) released at the site of an injured vascular wall during or after interventional procedures. While cell proliferation can be prevented to varying extents by the localized delivery of anti-proliferative small-molecule drugs coated on stents [[8], [9]], the development of protein and gene-based therapies has the potential to target a wider range of underlying cellular mechanisms responsible for triggering activation of smooth muscle cells and progenitor cells and could subsequently provide more effective control of intimal hyperplasia [10]. Experimental approaches based on the delivery of macromolecular therapeutics (e.g., antibodies, soluble receptors, DNA, siRNA, etc.) that can modulate the expression of key regulatory genes and proteins have demonstrated the potential of these approaches to prevent intimal hyperplasia in animal models [2], [11], [12], [13]. The development of clinical therapies based on these approaches, however, has been limited by the lack of materials and approaches that can be used to locally deliver these macromolecular agents to the vascular wall safely and effectively [4], [14].

In general, materials and approaches that have been developed for the localized delivery of conventional small-molecule drugs to the vascular wall (e.g., thin polymer coatings that permit gradual, diffusion-controlled release of small-molecule agents from the surfaces of drug-eluting stents) are not well suited for the release of larger macromolecular drugs such as DNA. As a result, the development of new materials that can be used to localize the delivery of nucleic acid-based therapeutics to the vascular wall will be critical to unlocking the clinical potential of these agents. In addition, approaches that permit delivery of macromolecular drugs directly and locally from the surfaces of interventional devices, such as angioplasty balloons or intravascular stents, could eliminate the need for systemic therapies with these drugs (thus reducing required dosages and the potential for undesired side effects) without increasing the complexity or the number of interventional procedures required [4], [14], [15], [16]. In this paper, we report a first step toward addressing several of these broader goals by developing methods for the localized delivery of plasmid DNA to vascular tissue using catheter balloons coated with ultrathin polyelectrolyte-based films.

The approach reported here is based on methods developed for the layer-by-layer deposition of multilayered polyelectrolyte films (or ‘polyelectrolyte multilayers’, PEMs) on surfaces [17], [18]. These methods are entirely aqueous and can be used to fabricate ultrathin films (e.g., from ∼10 nm to several hundred nm thick) using a broad range of positively and negatively charged polymers, including biomacromolecules such as proteins, viruses, and DNA. Numerous past studies have demonstrated that these materials can be used as platforms for the release of small-molecule drugs and macromolecular therapeutics. Examples of these approaches and other biomedical applications of these thin film materials, including the general design of PEMs for the delivery of DNA [19], have been reviewed comprehensively and are not discussed in detail here [19], [20], [21], [22]. Of particular relevance to the work reported here, however, we note that several recent studies by our group and others have demonstrated that PEMs fabricated using plasmid DNA can be used to promote the surface-mediated delivery of DNA to cells [19], [23], [24], [25], [26], [27], [28], [29], [30]. In addition, we note that other groups have reported the design of PEMs that promote virus-mediated cell transfection [31] and assemblies that promote the internalization of other nucleic acid-based agents (e.g., siRNA) to cells [32].

In the context of DNA delivery, layer-by-layer approaches to the fabrication of PEMs offer several potential practical advantages as compared to conventional approaches to the immobilization and release of DNA from thin films of bulk polymer. First, DNA can be directly incorporated into multilayered assemblies (e.g., as a ‘layer’), providing opportunities to precisely control the loading of DNA within a film (e.g., by changing the number of layers of DNA deposited during fabrication). The modular nature of layer-by-layer assembly can also be exploited to incorporate multiple layers of different therapeutic agents to design thin films and coatings that release multiple agents simultaneously or sequentially [23], [26], [29], [33], [34], [35], and to incorporate auxiliary agents (e.g., cationic polymers [36], [37], [38]) that could facilitate the delivery and cellular internalization of DNA. In addition, the aqueous procedures used for film fabrication do not require the use of organic solvents that could compromise the biological function of the DNA or harm the surfaces of devices on which they are deposited. Finally, layer-by-layer assembly leads to uniform surface coatings that conform faithfully to surfaces with complex or irregular shapes and microscale dimensions, such as those typical of interventional devices and implants [25], [39], [40], [41], [42], [43], [44].

The work described here builds on past reports from our group demonstrating that PEMs fabricated using plasmid DNA and a hydrolytically degradable cationic polymer (polymer 1) can be used to promote the release [45], [46], [47] and surface-mediated delivery [23], [25], [48] of DNA to cells in vitro. These past studies have also demonstrated that the layer-by-layer approach used to assemble these materials can be used to fabricate ultrathin DNA-containing films (e.g., 100 nm thick) on the surfaces of interventional devices, such as intravascular stents [25]. This current study sought to determine whether this approach could be used to coat the surfaces of inflatable embolectomy catheter balloons with thin, DNA-containing PEMs and promote localized transgene expression in the vascular wall in vivo using a rat model of balloon-induced arterial injury used previously for studies of intimal hyperplasia.

Section snippets

Materials and general considerations

Linear poly(ethylene imine) (LPEI, MW = 25,000) was purchased from Polysciences, Inc. (Warrington, PA). Sodium poly(4-styrenesulfonate) (SPS, MW = 70,000) was purchased from Aldrich Chemical Co. (Milwaukee, WI). Commercial polyelectrolytes were used as received without further purification. Concentrated sodium acetate buffer was purchased from Lonza (Rockland, ME). Polymer 1 (Mn = 16,000) was synthesized as described previously [49]. Plasmid DNA encoding enhanced green fluorescent protein

Layer-by-layer assembly of DNA-containing films on the surfaces of embolectomy catheter balloons

We have demonstrated in several past studies that hydrolytically degradable cationic polymers (such as polymer 1) can be used to fabricate ultrathin PEMs that erode and promote the surface-mediated release of DNA [23], [25], [34], [45], [47], [48], [51], [52], [53]. These past studies have demonstrated that the hydrolytically degradable ester functionality in polymer 1 plays an important role in promoting film disassembly [47], [53], and that changes in the side-chain or backbone structures of

Conclusions

We have demonstrated that polyelectrolyte multilayers (PEMs) fabricated using plasmid DNA and a hydrolytically degradable cationic polymer can be used to promote the localized transfection of arterial tissue in vivo. Inflatable catheter balloons coated layer-by-layer with thin films fabricated using polymer 1 and DNA encoding either EGFP or β-galactosidase promoted localized tissue transfection in the carotid arteries of rats using a rat model of balloon-induced arterial injury used previously

Acknowledgments

Financial support to D.M.L. was provided in part by the National Institutes of Health (R01 EB006820) and the Alfred P. Sloan Foundation. Financial support to B.L. was provided by NIH (R01 HL081424) and the American Heart Association. E.M.S. was supported in part by a 3M Foundation Graduate Research Fellowship.

References (54)

  • O. Etienne et al.

    Antifungal coating by biofunctionalized polyelectrolyte multilayered films

    Biomaterials

    (2005)
  • P. Schultz et al.

    Polyelectrolyte multilayers functionalized by a synthetic analogue of an anti-inflammatory peptide, alpha-MSH, for coating a tracheal prosthesis

    Biomaterials

    (2005)
  • W. He et al.

    Nanoscale neuro-integrative coatings for neural implants

    Biomaterials

    (2005)
  • P.V. Pavoor et al.

    Wear reduction of orthopaedic bearing surfaces using polyelectrolyte multilayer nanocoatings

    Biomaterials

    (2006)
  • E.M. Saurer et al.

    Assembly of erodible, DNA-containing thin films on the surfaces of polymer microparticles: toward a layer-by-layer approach to the delivery of DNA to antigen-presenting cells

    Acta Biomater

    (2009)
  • M.W. Liu et al.

    Restenosis after coronary angioplasty. Potential biologic determinants and role of intimal hyperplasia

    Circulation

    (1989)
  • F. Sharif et al.

    Current status of catheter- and stent-based gene therapy

    Cardiovasc Res

    (2004)
  • E.L. Eisenstein et al.

    Clopidogrel use and long-term clinical outcomes after drug-eluting stent implantation

    JAMA

    (2007)
  • M.G. Davies et al.

    Pathobiology of intimal hyperplasia

    Br J Surg

    (1994)
  • J.W. Moses et al.

    Sirolimus-eluting stents versus standard stents in patients with stenosis in a native coronary artery

    N Engl J Med

    (2003)
  • G.W. Stone et al.

    A polymer-based, paclitaxel-eluting stent in patients with coronary artery disease

    N Engl J Med

    (2004)
  • D.H. Walter et al.

    Local gene transfer of phVEGF-2 plasmid by gene-eluting stents: an alternative strategy for inhibition of restenosis

    Circulation

    (2004)
  • R. Kundi et al.

    Arterial gene transfer of the TGF-beta signalling protein Smad3 induces adaptive remodelling following angioplasty: a role for CTGF

    Cardiovasc Res

    (2009)
  • D. Yamanouchi et al.

    Protein kinase C delta mediates arterial injury responses through regulation of vascular smooth muscle cell apoptosis

    Cardiovasc Res

    (2010)
  • S. Nikol

    Gene therapy of cardiovascular disease

    Curr Opin Mol Ther

    (2008)
  • R. Riessen et al.

    Arterial gene transfer using pure DNA applied directly to a hydrogel-coated angioplasty balloon

    Hum Gene Ther

    (1993)
  • T. Asahara et al.

    Accelerated restitution of endothelial integrity and endothelium-dependent function after phVEGF165 gene transfer

    Circulation

    (1996)
  • Cited by (34)

    • Poly(β-amino ester)-based gene delivery systems: From discovery to therapeutic applications

      2019, Journal of Controlled Release
      Citation Excerpt :

      The in vitro release of pDNA in PBS at 37 °C revealed a sustainable profile up to four days, and the transfection activity in COS-7 cell line was confirmed without the aid of additional transfection agents. These results were then explored in vivo assays using surface-modified inflatable embolectomy catheter balloons with PEMs in the carotid arteries in a rat model of ballon-induce arterial injury [74]. Ultrathin PEMs based on polymer 1 and pDNA encoding either eGFP or β-galactosidase were used to promote the localized tissue transfection.

    • Three-dimensional biomaterial degradation - Material choice, design and extrinsic factor considerations

      2014, Biotechnology Advances
      Citation Excerpt :

      As expected, nanospheres with larger surface area to volume ratio were found to have significantly accelerated release kinetics compared to larger carrier spheres. A different approach to molecular delivery involves layer-by-layer (LBL) assembly where sequential deposition of positively and negatively charged polyelectrolytes onto material surfaces can be utilized to construct drug delivery depots by incorporating drugs of interest into self-assembled nanoscale thin films (Ma et al., 2007; Macdonald et al., 2008, 2011; Mao et al., 2005; Saurer et al., 2011). LBL technique benefits from mild fabrication conditions which given the fragile nature of incorporated elements including biomolecules or cells, render more aggressive incorporation techniques (e.g. ones using organic solvents) obsolete.

    • Water soluble polymer films for intravascular drug delivery of antithrombotic biomolecules

      2013, European Journal of Pharmaceutics and Biopharmaceutics
    • Hyaluronic acid / chitosan multilayer coatings on neuronal implants for localized delivery of siRNA nanoplexes

      2013, Journal of Controlled Release
      Citation Excerpt :

      Moreover, chitosan (Ch) can be combined with polyanions, such as hyaluronic acid (HA) to build up polyelectrolyte multilayers (PEMs) [11–13]. This approach represents a promising technique for coating different biomaterials and offers a new opportunity for generation of sustained release systems, applicable in regenerative medicine and for the preparation of supramolecular nano-architectures [14]. The layer-by-layer (LbL) assemblies are built up by alternately adsorbed positively and negatively charged polyelectrolytes [11].

    • Reduction of intimal hyperplasia in injured rat arteries promoted by catheter balloons coated with polyelectrolyte multilayers that contain plasmid DNA encoding PKCδ

      2013, Biomaterials
      Citation Excerpt :

      Other studies of particular relevance to this current investigation are described briefly below. We have reported in several past studies on the design and characterization of PEMs using plasmid DNA and polymer 1 [23,34,40–45], a model hydrolytically degradable poly(β-amino ester) originally developed for the polyplex-mediated delivery of DNA [46,47]. That past work demonstrated (i) that these materials (referred to hereafter as ‘polymer 1/DNA films’) erode and release DNA gradually in physiologically relevant media (e.g., over several days) [34,40,43,45], (ii) that surfaces coated with films ∼100 nm thick can be used to promote the localized and surface-mediated delivery of DNA to cells in vitro [34,40,41,45], and (iii) that, in addition to promoting the release of DNA, polymer 1 can also play a role in promoting the internalization and trafficking of DNA by cells [45].

    View all citing articles on Scopus
    1

    These authors contributed equally to this work.

    View full text