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We report the design and characterization of thin polymer-based coatings that promote the contact transfer of DNA to soft surfaces under mild and physiologically relevant conditions. Past studies reveal polymer multilayers fabricated using linear poly(ethylene imine) (LPEI), poly(acrylic acid) (PAA), and plasmid DNA promote contact transfer of DNA to vascular tissue. Here, we demonstrate that changes in the structure of the polyamine building blocks of these materials can have substantial impacts on rates and extents of contact transfer. We used two hydrogel-based substrate models that permit identification and manipulation of parameters that influence contact transfer. We used a planar gel model to characterize films having the structure (cationic polymer/PAA/cationic polymer/plasmid DNA) x fabricated using either LPEI or one of three poly(β-amino ester)s as polyamine building blocks. The structure of the polyamine influenced subsequent contact transfer of DNA significantly; in general, films fabricated using more hydrophilic polymers promoted transfer more effectively. This planar model also permitted characterization of the stabilities of films transferred onto secondary surfaces, revealing rates of DNA release to be slower than rates of release prior to transfer. We also used a three-dimensional hole-based hydrogel model to evaluate contact transfer of DNA from the surfaces of inflatable catheter balloons used in vascular interventions and selected a rapid-transfer coating for proof-of-concept studies to characterize balloon-mediated contact transfer of DNA to peripheral arterial tissue in swine. Our results reveal robust and largely circumferential transfer of DNA to the luminal walls of peripheral arteries using inflation times as short as 15 to 30 s. The materials and approaches reported here provide new and useful tools for promoting rapid, substrate-mediated contact transfer of plasmid DNA to soft surfaces in vitro and in vivo that could prove useful in a range of fundamental and applied contexts.

We report the design and characterization of pH-responsive polymer coatings that enable catheter balloon-mediated transfer of DNA to arterial tissue in short, clinically relevant inflation times. Our approach exploits the pH-dependent ionization of poly(acrylic acid) (PAA) to promote disassembly and release of plasmid DNA from polyelectrolyte multilayers. We characterized the contact transfer of multilayers composed of PAA, plasmid DNA, and linear poly(ethyleneimine) (LPEI) identified as promising in prior studies on the delivery of DNA to arterial tissue. In contrast to thinner films evaluated previously, we found thicker coatings composed of 32 repeating (LPEI/PAA/LPEI/DNA) x tetralayers to swell substantially in physiologically relevant media (in PBS; pH = 7.4). In some cases, these coatings also disintegrated or delaminated rapidly from their underlying substrates, suggesting the potential for enhanced balloon-mediated transfer. We developed a technically straightforward agarose gel-based hole-insertion model to characterize factors (inflation time, lumen size, etc.) that influence contact transfer of DNA when film-coated balloons are inflated into contact with soft surfaces. Those studies and the results of in vivo experiments using small animal (rat) and large animal (pig) models of peripheral arterial injury revealed catheters coated with these materials to promote robust contact transfer of DNA to soft hydrogel surfaces and the luminal surfaces of arterial tissue using inflation times as short as 30 s. These short inflation times are relevant in the context of clinical vascular interventions in peripheral arteries. Additional studies demonstrated that contact transfer of DNA using these short times can promote subsequent dissemination and transport of DNA to the medial tissue layer, suggesting the potential for use in therapeutically relevant applications of balloon-mediated gene transfer.

We report the design of reactive and hydrolytically degradable multilayers by the covalent layer-by-layer assembly of an azlactone-containing polymer, poly(2-vinyl-4,4-dimethylazlactone), with an acid-degradable, acetal-containing, small-molecule diamine linker. This approach yields cross-linked multilayers that contain (i) residual azlactone reactivity that can be used for further functionalization after fabrication and (ii) acid-labile cross-links that can undergo pH-triggered degradation. Thin films and hollow capsules fabricated using this approach were relatively stable in slightly basic media (pH = 7.4) but eroded and degraded gradually in mildly acidic environments (pH = 5). The residual azlactones in these materials could be functionalized by reaction with hydrophilic or hydrophobic amines to tune physicochemical properties, including surface wetting and rates of degradation/erosion. Interestingly, our results reveal that rates of degradation could be tuned over a broad range (from ∼4 h to ∼10 days) simply by post-fabrication modification of the parent reactive material. We further demonstrate the potential of acetal-containing microcapsules to be used for the acid-triggered release of encapsulated cargo. The results of in vitro experiments reveal that microcapsules loaded with fluorescently labeled dextran can be internalized by mammalian cells and that cell uptake and intracellular degradation were also influenced by the types of functional groups installed post-fabrication. The introduction of acid degradability expands the range of stimuli that can be used to trigger the destruction of these reactive materials to include changes in pH relevant to chemical and biological processes. Our results also introduce an approach to tuning degradation profiles that differs from past strategies used to design degradable multilayers. We conclude that this approach provides a new, useful, and modular platform for the design of stimuli-responsive nano/biointerfaces with transient environmental stability.

In the presence of zinc, the protein tubulin assembles into two-dimensional sheets that are a useful model system for the study of both tubulin and microtubule structure. Tubulin sheets present an ideal protein structure for study with atomic force microscopy because they contain a two-dimensional crystalline protein lattice and retain many of the structural features of tubulin and microtubules. However, high-resolution imaging requires nonperturbative immobilization onto an appropriate imaging substrate. In this report, several substrates commonly used for scanning probe microscopy are evaluated for their ability to effectively immobilize tubulin sheets: mica, gold, highly ordered pyrolytic graphite, and carbon-coated electron microscopy grids. We hypothesize that the different intermolecular interactions presented by these substrates will affect the morphology of adsorbed tubulin sheets as well as the amount of other contaminating adsorbates. Tubulin sheets were successfully imaged on all of these substrates and structural characterization is reported. The most consistent results were obtained on carbon-coated electron microscopy grids, which preserved fine structural features of the sheets and had the least amount of contamination from the adsorption of unpolymerized tubulin. Images of tubulin sheets obtained with atomic force microscopy also compare favorably with published electron micrographs of sheets produced using similar procedures. This work demonstrates the importance of assessing substrate effects when studying two-dimensional protein crystals and identifies suitable substrates for immobilizing tubulin sheets., Competing Interests: The authors declare no competing financial interest.

We report the design of erodible 'mixed multilayer' coatings fabricated using plasmid DNA and combinations of both hydrolytically degradable and charge-shifting cationic polymer building blocks. Films fabricated layer-by-layer using combinations of a model poly(β-amino ester) (polymer 1 ) and a model charge-shifting polymer (polymer 2 ) exhibited DNA release profiles that were substantially different than those assembled using DNA and either polymer 1 or polymer 2 alone. In addition, the order in which layers of these two cationic polymers were deposited during assembly had a profound impact on DNA release profiles when these materials were incubated in physiological buffer. Mixed multilayers ∼225 nm thick fabricated by depositing layers of polymer 1 /DNA onto films composed of polymer 2 /DNA released DNA into solution over ∼60 days, with multi-phase release profiles intermediate to and exhibiting some general features of polymer 1 /DNA or polymer 2 /DNA films (e.g., a period of rapid release, followed by a more extended phase). In sharp contrast, 'inverted' mixed multilayers fabricated by depositing layers of polymer 2 /DNA onto films composed of polymer 1 /DNA exhibited release profiles that were almost completely linear over ∼60-80 days. These and other results are consistent with substantial interdiffusion and commingling (or mixing) among the individual components of these compound materials. Our results reveal this mixing to lead to new, unanticipated, and useful release profiles and provide guidance for the design of polymer-based coatings for the local, surface-mediated delivery of DNA from the surfaces of topologically complex interventional devices, such as intravascular stents, with predictable long-term release profiles.