Rapid Release of Plasmid DNA from Surfaces Coated with Polyelectrolyte Multilayers Promoted by the Application of Electrochemical Potentials

摘要

We report an approach to the rapid release of DNA based on the application of electrochemical potentials to surfaces coated with polyelectrolyte-based thin films. We fabricated multilayered polyelectrolyte films (or ‘polyelectrolyte multilayers’, PEMs) using plasmid DNA and a model hydrolytically degradable cationic poly(β-amino ester) () on stainless steel substrates using a layer-by-layer approach. The application of continuous reduction potentials in the range of -1.1 to -0.7 V (vs. a Ag/AgCl electrode) to film-coated electrodes in PBS at 37 °C resulted in the complete release of DNA over a period of 1-2 minutes. Film-coated electrodes incubated under identical conditions in the absence of applied potentials required 1-2 days for complete release. Control over the magnitude of the applied potential provided control over the rate at which DNA was released. The results of these and additional physical characterization experiments are consistent with a mechanism of film disruption that is promoted by local increases in pH at the film/electrode interface (resulting from electrochemical reduction of water or dissolved oxygen) that disrupt ionic interactions in these materials. The results of cell-based experiments demonstrated that DNA was released in a form that remains intact and able to promote transgene expression in mammalian cells. Finally, we demonstrate that short-term (i.e., non-continuous) electrochemical treatments can also be used to promote faster film erosion (e.g., over 1-2 h) once the potential is removed. Past studies demonstrate that PEMs fabricated using can promote surface-mediated transfection of cells and tissues in vitro and in vivo. With further development, the electrochemical approaches reported here could thus provide new methods for the rapid, triggered, or spatially patterned transfer of DNA (or other agents) from surfaces of interest in a variety of fundamental and applied contexts. class="kwd-title">Keywords: Layer-by-Layer, Thin Films, DNA, Rapid Release, Electrochemical Methods class="head no_bottom_margin" id="S1title">IntroductionMaterials that provide control over the release of DNA from surfaces are important in a variety of contexts, ranging from the development of new research tools to the development of gene-based therapies. Methods for the immobilization of DNA on surfaces provide straightforward ‘physical’ approaches to defining the locations at which DNA is made available (e.g., by local delivery to cells residing in the vicinity of a film-coated implant or to cells growing on patterned features in an assay plate, etc.).^- Many different approaches to the immobilization of DNA on surfaces^- and the encapsulation of DNA in polymer-based films, coatings, and matrices^,,- have been developed for this purpose. Much of the success with which these approaches can be translated for use in many applied contexts, however, will also depend upon the ability to exert simultaneous (and often variable) levels of control over the timing with which DNA is released (that is, to develop mechanisms that also allow for temporal control). For example, although sustained release may be desirable for some applications, others may require methods that can be used to promote rapid transfer or the triggered/on-demand release of DNA and other agents. Although many materials can promote sustained release of DNA from the surfaces of implants and interventional devices, the development of materials platforms that permit rapid or triggered release remains a general challenge. The work reported here takes a step toward addressing this broader goal by developing electrochemical approaches that can be used to promote the rapid release of plasmid DNA from surfaces coated with ultrathin polyelectrolyte-based films.The approach reported here exploits methods for the ‘layer-by-layer’ fabrication of thin polyelectrolyte-based films (called ‘polyelectrolyte multilayers’, or PEMs) on surfaces.^, This aqueous-based approach can be used to design films using a broad range of different polyelectrolytes, including charged proteins, viruses, and DNA.^- In the context of drug delivery, these methods offer several practical advantages for the encapsulation and release of therapeutic agents. These advantages include: (i) precise, nanometer-scale control over film thickness and drug loading (by control over the number of layers incorporated), (ii) control over the relative locations of individual layers in a film (permitting the design of hierarchical films and films that can be used to release multiple agents),^{href="#R23" rid="R23" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_306046129">23-href="#R27" rid="R27" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_306046142">27} and (iii) the ability to fabricate thin films on surfaces with complex shapes.^{href="#R3" rid="R3" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_339267468">3,href="#R20" rid="R20" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_306046087">20-href="#R22" rid="R22" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_306046088">22} Provided that they can be constructed in ways that permit subsequent disassembly, PEMs also provide a unique platform for control over the release of macromolecular agents that are otherwise too large to be released by diffusion. Different approaches to promoting film disruption have been reviewed recently,^{href="#R3" rid="R3" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_339267456">3,href="#R4" rid="R4" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_306046149">4,href="#R18" rid="R18" class=" bibr popnode">18,href="#R20" rid="R20" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_306046124">20-href="#R22" rid="R22" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_306046090">22} and include (i) films that respond to changes in environmental conditions (e.g., pH or ionic strength),^{href="#R28" rid="R28" class=" bibr popnode">28-href="#R31" rid="R31" class=" bibr popnode">31} (ii) films fabricated using hydrolytically-,^{href="#R23" rid="R23" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_306046098">23,href="#R32" rid="R32" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_306046148">32,href="#R33" rid="R33" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_306046117">33} enzymatically-,^{href="#R34" rid="R34" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_339267460">34-href="#R36" rid="R36" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_339267465">36} and reductively-degradable^{href="#R37" rid="R37" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_306046150">37-href="#R39" rid="R39" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_306046106">39} polyelectrolytes, and (iii) films that respond to the application of external stimuli (e.g., light, electrochemical potentials, etc.).^{href="#R40" rid="R40" class=" bibr popnode">40-href="#R45" rid="R45" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_306046113">45}Several groups have demonstrated that the approaches outlined above can be used to design PEMs that promote the release of DNA.^{href="#R3" rid="R3" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_339267461">3} Work in our group has focused on the design of PEMs fabricated using plasmid DNA and hydrolytically degradable cationic poly(β-amino ester)s such as href="/pmc/articles/PMC3359390/figure/F6/" target="figure" class="fig-table-link figpopup" rid-figpopup="F6" rid-ob="ob-F6" co-legend-rid="lgnd_F6">polymer 1.^{href="#R33" rid="R33" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_306046132">33,href="#R46" rid="R46" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_339267467">46,href="#R47" rid="R47" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_306046104">47} We and others have demonstrated that films fabricated using DNA and href="/pmc/articles/PMC3359390/figure/F6/" target="figure" class="fig-table-link figpopup" rid-figpopup="F6" rid-ob="ob-F6" co-legend-rid="lgnd_F6">polymer 1 (referred to hereafter as ‘polymer >1/DNA films’) erode in aqueous environments and promote the release of DNA.^{href="#R33" rid="R33" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_306046103">33,href="#R46" rid="R46" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_339267464">46-href="#R52" rid="R52" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_306046123">52} These studies have also demonstrated that these materials can be used to promote the localized and surface-mediated delivery of DNA to cells and tissues in vitro^{href="#R46" rid="R46" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_339267459">46,href="#R47" rid="R47" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_306046111">47} and in vivo.^{href="#R50" rid="R50" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_306046118">50,href="#R52" rid="R52" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_306046114">52}In general, polymer >1/DNA films erode and release DNA gradually when incubated in physiologically relevant media [e.g., over a period of ∼2-4 days when incubated in phosphate-buffered saline (pH= 7.4) at 37 °C].^{href="#R33" rid="R33" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_306046101">33,href="#R47" rid="R47" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_306046094">47} We have also reported the design of films that exhibit more extended release profiles (e.g., over weeks or months),^{href="#R25" rid="R25" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_306046146">25,href="#R26" rid="R26" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_306046099">26,href="#R53" rid="R53" class=" bibr popnode">53,href="#R54" rid="R54" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_339267470">54} but we have, in general, found it difficult to design PEMs that erode and release DNA more rapidly (e.g., over seconds or minutes). As a first step toward the design of faster-releasing films, we recently reported that incorporation of layers of poly(acrylic acid) (a pH-dependent weak polyelectrolyte) into polymer >1/DNA films leads to films that release DNA over 3-6 hours in physiologically relevant media.^{href="#R55" rid="R55" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_339267458">55} Other groups have reported PEMs that can be induced to undergo rapid, triggered disassembly to release DNA or oligonucleotides upon exposure to chemical reducing agents (e.g., by contact of films containing disulfide functionality with thiol-based reducing agents).^{href="#R38" rid="R38" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_306046115">38,href="#R39" rid="R39" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_306046091">39,href="#R56" rid="R56" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_339267469">56} This approach is well-suited to affecting the intracellular release of DNA or in other situations where chemical reducing agents (e.g., glutathione) are present. This current investigation sought to develop approaches that could be used to promote the rapid release of DNA in response to an externally applied stimulus (e.g., the application of an electrochemical potential) that would not otherwise be present in biological environments and would be unlikely to cause adverse effects to neighboring cells or tissues.This investigation builds upon the results of past studies demonstrating that the application of electrochemical potentials can be used to disrupt or deconstruct PEMs. In general, these approaches fall into one of two categories. The first approach involves the fabrication of multilayers using electro-active molecules as film components; the application of oxidation or reduction potentials to these films changes the redox-states of these components and, as a result, leads to changes in intramolecular interactions in the films.^{href="#R42" rid="R42" class=" bibr popnode">42,href="#R45" rid="R45" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_306046093">45,href="#R57" rid="R57" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_306046110">57,href="#R58" rid="R58" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_306046136">58} The second approach is based on the creation of local changes in pH near the surfaces of film-coated electrodes by (i) electrochemical oxidation of water (which results in the production of hydronium ions)^{href="#R43" rid="R43" class=" bibr popnode">43,href="#R44" rid="R44" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_306046143">44,href="#R59" rid="R59" class=" bibr popnode">59-href="#R62" rid="R62" class=" bibr popnode">62} or (ii) electrochemical reduction of water or dissolved oxygen (which results in the production of hydroxide ions).^{href="#R63" rid="R63" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_339267450">63} This second approach does not require the use of electro-active film components, and it is useful for promoting the electrochemically-triggered dissolution of PEMs with components that respond to increases or decreases in pH. For example, electrochemically-induced decreases in local pH near electrodes have been used to promote the dissolution or disruption of PEMs fabricated from poly(lysine)/heparin,^{href="#R43" rid="R43" class=" bibr popnode">43} as well as films fabricated from poly(lysine) and fish sperm DNA,^{href="#R60" rid="R60" class=" bibr popnode">60} and avidin and an imminobiotin-labeled polymer.^{href="#R44" rid="R44" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_306046116">44} Other studies have demonstrated that electrochemically-induced increases in local pH can be used to dissolve or deconstruct hydrogen-bonded multilayers fabricated from poly(vinylpyrrolidone) and tannic acid.^{href="#R63" rid="R63" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_339267457">63}Here, we report that electrochemically induced changes in pH can be used to accelerate dramatically the erosion of PEMs fabricated using transcriptionally active plasmid DNA. We demonstrate that the application of reduction potentials to stainless steel electrodes coated with polymer >1/DNA films results in the complete release of DNA over periods ranging from seconds to several minutes (as opposed to several days in the absence of applied potentials). We demonstrate further that the rate of release can be tuned by adjusting the magnitude of the reduction potential applied to the electrode, and that the DNA that is released under these conditions remains transcriptionally active and able to promote transgene expression in mammalian cells. Our results are consistent with a mechanism of release that involves the localized generation of elevated pH near electrode surfaces. We note that a recent study reported the electron transfer-mediated release of DNA from multilayers fabricated from redox-active zirconium ions.^{href="#R45" rid="R45" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_306046108">45} Our current approach provides a method for the release of DNA that (i) does not involve electron-transfer to the film itself (or, ultimately, even direct contact of the film with an electrode), and (ii) exploits multilayer structures that promote the simultaneous release^{href="#R64" rid="R64" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_306046109">64} of DNA and a cationic polymer (href="/pmc/articles/PMC3359390/figure/F6/" target="figure" class="fig-table-link figpopup" rid-figpopup="F6" rid-ob="ob-F6" co-legend-rid="lgnd_F6">polymer 1) used in several past studies to deliver DNA to cells.^{href="#R46" rid="R46" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_339267471">46,href="#R52" rid="R52" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_306046151">52,href="#R65" rid="R65" class=" bibr popnode">65,href="#R66" rid="R66" class=" bibr popnode">66} With further development, this approach could therefore lead to new methods for the rapid transfer or patterned delivery of DNA to cells and tissues of interest in a range of fundamental and applied contexts.