RESUMO
We report a layer-by-layer approach to the fabrication of thin polymer-based multilayers that release DNA rapidly in physiologically relevant environments. This approach exploits the properties of a weak anionic polyelectrolyte [poly(acrylic acid); PAA] to disrupt ionic interactions and promote disassembly in coatings that otherwise erode slowly. We investigated this approach using multilayers fabricated from plasmid DNA and linear poly(ethylenimine) (LPEI), a model synthetic cationic polymer used widely for DNA delivery. LPEI/DNA multilayers erode and release DNA slowly over â¼4 days when incubated in PBS buffer. In contrast, substitution of every other layer of DNA with PAA lead to thin films that released DNA rapidly, with >60% being released in the first 5 min. These quick-release coatings release bioactive DNA and can be used to fabricate uniform coatings on a variety of objects, including the tips of inflatable balloon catheters. We demonstrate that these coatings can promote high levels of cell transfection in vitro and the robust contact transfer and expression of DNA in vascular tissue in vivo using a rat model of vascular injury. These materials provide useful alternatives to multilayers and other coatings that promote the prolonged release of DNA. More broadly, approaches that depart from the use of degradable polymers to promote film erosion create opportunities to design new gene delivery coatings using a broader range of polymer-based building blocks designed for other gene delivery applications. With further development, this approach could thus provide a new and useful platform for the rapid contact transfer of DNA to cells and tissues of interest in a range of fundamental and applied contexts.
Assuntos
Resinas Acrílicas/química , DNA/química , Portadores de Fármacos/química , Polietilenoimina/química , Transfecção/métodos , Animais , Células COS , Chlorocebus aethiops , RatosRESUMO
We report an approach to deliver DNA to vascular tissue in vivo using intravascular stents coated with degradable, DNA-containing polyelectrolyte multilayers (PEMs). Ionically cross-linked multilayers â¼120 nm thick were fabricated layer-by-layer on the surfaces of balloon-mounted stainless steel stents using plasmid DNA and a hydrolytically degradable poly(ß-amino ester) (polymer 1). Characterization of stents coated using a fluorescently end-labeled analog of polymer 1 revealed film erosion to be uniform across the surfaces of the stents; differential stresses experienced upon balloon expansion did not lead to faster film erosion or dose dumping of DNA in areas near stent joints when stents were incubated in physiologically relevant media. The ability of film-coated stents to transfer DNA and transfect arterial tissue in vivo was then investigated in pigs and rabbits. Stents coated with films fabricated using fluorescently labeled DNA resulted in uniform transfer of DNA to sub-endothelial tissue in the arteries of pigs in patterns corresponding to the locations and geometries of stent struts. Stents coated with films fabricated using polymer 1 and plasmid DNA encoding EGFP resulted in expression of EGFP in the medial layers of stented tissue in both pigs and rabbits two days after implantation. The results of this study, combined with the modular and versatile nature of layer-by-layer assembly, provide a polymer-based platform that is well suited for fundamental studies of stent-mediated gene transfer. With further development, this approach could also prove useful for the design of nonviral, gene-based approaches for prevention of complications that arise from the implantation of stents and other implantable interventional devices.
Assuntos
Artérias Carótidas/metabolismo , DNA/genética , Endotélio Vascular/metabolismo , Técnicas de Transferência de Genes , Polímeros/química , Stents , Animais , Artérias Carótidas/citologia , Materiais Revestidos Biocompatíveis , DNA/metabolismo , Eletrólitos , Endotélio Vascular/citologia , Genes Reporter , Proteínas de Fluorescência Verde/genética , Plasmídeos , Coelhos , SuínosRESUMO
New therapeutic approaches that eliminate or reduce the occurrence of intimal hyperplasia following balloon angioplasty could improve the efficacy of vascular interventions and improve the quality of life of patients suffering from vascular diseases. Here, we report that treatment of arteries using catheter balloons coated with thin polyelectrolyte-based films ('polyelectrolyte multilayers', PEMs) can substantially reduce intimal hyperplasia in an in vivo rat model of vascular injury. We used a layer-by-layer (LbL) process to coat the surfaces of inflatable catheter balloons with PEMs composed of nanolayers of a cationic poly(ß-amino ester) (polymer 1) and plasmid DNA (pPKCδ) encoding the δ isoform of protein kinase C (PKCδ), a regulator of apoptosis and other cell processes that has been demonstrated to reduce intimal hyperplasia in injured arterial tissue when administered via perfusion using viral vectors. Insertion of balloons coated with polymer 1/pPKCδ multilayers into injured arteries for 20 min resulted in local transfer of DNA and elevated levels of PKCδ expression in the media of treated tissue three days after delivery. IFC and IHC analysis revealed these levels of expression to promote downstream cellular processes associated with up-regulation of apoptosis. Analysis of arterial tissue 14 days after treatment revealed polymer 1/pPKCδ-coated balloons to reduce the occurrence of intimal hyperplasia by ~60% compared to balloons coated with films containing empty plasmid vectors. Our results demonstrate the potential therapeutic value of this nanotechnology-based approach to local gene delivery in the clinically important context of balloon-mediated vascular interventions. These PEM-based methods could also prove useful for other in vivo applications that require short-term, surface-mediated transfer of plasmid DNA.
Assuntos
Angioplastia Coronária com Balão/efeitos adversos , Lesões das Artérias Carótidas/etiologia , Lesões das Artérias Carótidas/prevenção & controle , Materiais Revestidos Biocompatíveis/farmacologia , DNA/metabolismo , Proteína Quinase C-delta/metabolismo , Túnica Íntima/patologia , Animais , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/patologia , Lesões das Artérias Carótidas/patologia , Eletrólitos/química , Imunofluorescência , Técnicas de Transferência de Genes , Hiperplasia , Marcação In Situ das Extremidades Cortadas , Masculino , Microscopia de Fluorescência , Plasmídeos/metabolismo , Polímeros/química , Ratos , Ratos Sprague-Dawley , Túnica Íntima/efeitos dos fármacosRESUMO
We report on conjugate addition-based approaches to the covalent layer-by-layer assembly of thin films and the post-fabrication functionalization of biointerfaces. Our approach is based on a recently reported approach to the "reactive" assembly of covalently cross-linked polymer multilayers driven by the 1,4-conjugate addition of amine functionality in poly(ethyleneimine) (PEI) to the acrylate groups in a small-molecule pentacrylate species (5-Ac). This process results in films containing degradable ß-amino ester cross-links and residual acrylate and amine functionality that can be used as reactive handles for the subsequent immobilization of new functionality. Layer-by-layer growth of films fabricated on silicon substrates occurred in a supra-linear manner to yield films ≈ 750 nm thick after the deposition of 80 PEI/5-Ac layers. Characterization by atomic force microscopy (AFM) suggested a mechanism of growth that involves the reactive deposition of nanometer-scale aggregates of PEI and 5-Ac during assembly. Infrared (IR) spectroscopy studies revealed covalent assembly to occur by 1,4-conjugate addition without formation of amide functionality. Additional experiments demonstrated that acrylate-containing films could be postfunctionalized via conjugate addition reactions with small-molecule amines that influence important biointerfacial properties, including water contact angles and the ability of film-coated surfaces to prevent or promote the attachment of cells in vitro. For example, whereas conjugation of the hydrophobic molecule decylamine resulted in films that supported cell adhesion and growth, films treated with the carbohydrate-based motif D-glucamine resisted cell attachment and growth almost completely for up to 7 days in serum-containing media. We demonstrate that this conjugate addition-based approach also provides a means of immobilizing functionality through labile ester linkages that can be used to promote the long-term, surface-mediated release of conjugated species and promote gradual changes in interfacial properties upon incubation in physiological media (e.g., over a period of at least 1 month). These covalently cross-linked films are relatively stable in biological media for prolonged periods, but they begin to physically disintegrate after ≈ 30 days, suggesting opportunities to use this covalent layer-by-layer approach to design functional biointerfaces that ultimately erode or degrade to facilitate elimination.
Assuntos
Membranas Artificiais , Polietilenoimina/química , Estrutura MolecularRESUMO
Polyelectrolyte multilayers (PEMs) fabricated from cationic polymers and DNA have been investigated broadly as materials for surface-mediated DNA delivery. One attractive aspect of this "multilayered" approach is the potential to exploit the presence of cationic polymer "layers" in these films to deliver DNA to cells more effectively. Past studies demonstrate that these films can promote transgene expression in vitro and in vivo, but significant questions remain regarding roles that the cationic polymers could play in promoting the internalization and processing of DNA. Here, we report physicochemical and in vitro cell-based characterization of DNA-containing PEMs fabricated using fluorescently end-labeled derivatives of a degradable polycation (polymer 1) used in past studies of surface-mediated transfection. This approach permitted simultaneous characterization of polymer and DNA in solution and in cells using fluorescence-based techniques, and provided information about the locations and behaviors of polymer 1 that could not be obtained using other methods. LSCM and flow cytometry experiments revealed that polymer 1 and DNA released from film-coated objects were both internalized extensively by cells and that they were colocalized to a significant extent inside cells (e.g., ~58% of DNA was colocalized with polymer). Fluorescence anisotropy measurements of solutions containing partially eroded films were also consistent with the presence of aggregates of polymer 1 and DNA in solution (e.g., after release from surfaces, but prior to internalization by cells). Our results support the view that polymer 1, which is incorporated into these materials as "layers" rather than as part of optimized, preformed "polyplexes", can act to promote or enhance surface-mediated DNA delivery. More broadly, our results suggest opportunities to improve the delivery properties of DNA-containing PEMs by incorporation of additional "layers" of other conventional cationic polymers designed to address specific intracellular barriers to transfection, such as endosomal escape, more effectively.
Assuntos
DNA/química , Corantes Fluorescentes/química , Técnicas de Transferência de Genes , Poliaminas/química , Polímeros/química , Animais , Células COS , Chlorocebus aethiops , DNA/metabolismo , Endocitose , Ésteres/química , Citometria de Fluxo , Polarização de Fluorescência , PolieletrólitosRESUMO
We report the synthesis of a fluorescently end-labeled analog of a synthetic and degradable cationic poly(ß-amino ester) (PBAE; polymer 1) used in past studies for the delivery of DNA and the layer-by-layer assembly of erodible polyelectrolyte multilayers (PEMs). The synthesis of an analog of polymer 1 having acrylate functionalized end groups provided a platform for the introduction of fluorescent labels by post-polymerization conjugate addition of amine-functionalized fluorophores. This approach enabled the synthesis of fluorescently end-labeled polymer (polymer 1(FL)) with molecular weights and polydispersities (M(n) = 18,000; PDI ~1.8) similar to those used in past studies for the fabrication of PEMs using polymer 1. Layer-by-layer assembly of PEMs using polymer 1(FL) and poly(styrene sulfonate) enabled characterization of film erosion and, for the first time, direct observation of the release of cationic polymer from these assemblies using fluorescence microscopy and fluorometry. Our results shed new light on the behaviors of the cationic components of these PEMs and could prove useful for the design of thin films for a range of different controlled release applications. Our results also provide new fluorescent cationic polymer probes that could be useful for characterization of the behaviors of PBAEs in other fundamental or applied biotechnological contexts.
RESUMO
Laser scanning confocal microscopy (LSCM) and atomic force microscopy (AFM) were used to characterize changes in nanoscale structure that occur when ultrathin polyelectrolyte multilayers (PEMs) are incubated in aqueous media. The PEMs investigated here were fabricated by the deposition of alternating layers of plasmid DNA and a hydrolytically degradable polyamine onto a precursor film composed of alternating layers of linear poly(ethylene imine) (LPEI) and sodium poly(styrene sulfonate) (SPS). Past studies of these materials in the context of gene delivery revealed transformations from a morphology that is smooth and uniform to one characterized by the formation of nanometer-scale particulate structures. We demonstrate that in-plane registration of LSCM and AFM images acquired from the same locations of films fabricated using fluorescently labeled polyelectrolytes allows the spatial distribution of individual polyelectrolyte species to be determined relative to the locations of topographic features that form during this transformation. Our results suggest that this physical transformation leads to a morphology consisting of a relatively less disturbed portion of film composed of polyamine and DNA juxtaposed over an array of particulate structures composed predominantly of LPEI and SPS. Characterization by scanning electron microscopy and energy-dispersive X-ray microanalysis provides additional support for this interpretation. The combination of these different microscopy techniques provides insight into the structures and dynamics of these multicomponent thin films that cannot be achieved using any one method alone, and could prove useful for the further development of these assemblies as platforms for the surface-mediated delivery of DNA.