Patterning and Impregnation of Superhydrophobic Surfaces Using Aqueous Solutions

We report a solvent-assisted approach to the patterning and impregnation of porous superhydrophobic coatings that permits the use of entirely aqueous solutions. This approach permits immobilization of proteins and enzymes, creating opportunities to decorate superhydrophobic surfaces with hydrophilic domains and channels that can capture aliquots of aqueous media, guide and mix aqueous solutions, and chemically process streams of organic molecules. Because this approach does not require destruction of non-wetting features, it can also be used to transfer highly water-soluble polymers and small molecules without compromising superhydrophobicity, providing methods for post-fabrication loading of water-soluble agents into protective non-wetting coatings that are difficult to achieve using other approaches

Characterization of Degradable Polyelectrolyte Multilayers Fabricated Using DNA and a Fluorescently-Labeled Poly(β-amino ester): Shedding Light on the Role of the Cationic Polymer in Promoting Surface-Mediated Gene Delivery

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 <b>1</b>) 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 <b>1</b> that could not be obtained using other methods. LSCM and flow cytometry experiments revealed that polymer <b>1</b> 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 <b>1</b> and DNA in solution (e.g., after release from surfaces, but prior to internalization by cells). Our results support the view that polymer <b>1</b>, 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

Reactive Polymer Multilayers Fabricated by Covalent Layer-by-Layer Assembly: 1,4-Conjugate Addition-Based Approaches to the Design of Functional Biointerfaces

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 β-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 <i>in vitro</i>. 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

Covalently Crosslinked and Physically Stable Polymer Coatings with Chemically Labile and Dynamic Surface Features Fabricated by Treatment of Azlactone-Containing Multilayers with Alcohol‑, Thiol‑, and Hydrazine-Based Nucleophiles

We report approaches to the design of covalently crosslinked and physically stable surface coatings with chemically labile and dynamic surface features based on the functionalization of azlactone-containing materials with alcohol-, thiol-, and hydrazine-based nucleophiles. Past studies demonstrate that residual azlactone groups in polymer multilayers fabricated by the reactive layer-by-layer assembly of poly­(2-vinyl-4,4-dimethylazlactone) and branched poly­(ethylenimine) can react with amine-based nucleophiles to impart new surface and bulk properties through the creation of chemically stable amide/amide-type bonds. Here, we demonstrate that the azlactone groups in these covalently crosslinked materials can also be functionalized using less nucleophilic alcohol- or thiol-containing compounds, using an organic catalyst, or converted to reactive acylhydrazine groups by direct treatment with hydrazine. These methods (i) broaden the pool of molecules that can be used for post-fabrication functionalization to include compounds containing alcohol, thiol, or aldehyde groups and (ii) yield surface coatings with chemically labile amide/ester-, amide/thioester-, and amide/imine-type bonds that make possible the design of new dynamic and stimulus-responsive materials (e.g., surfaces that release covalently bound molecules or undergo changes in extreme wetting behaviors in response to specific chemical stimuli). Our results expand the range of functionality that can be installed in, and thus the range of new functions that can be imparted to, azlactone-containing coatings beyond those that can be accessed using primary amine-based nucleophiles. The chemical approaches demonstrated here using model polymer-based reactive multilayer coatings are general and should thus also prove useful for the design of new responsive surfaces based on other types of azlactone-functionalized materials

Covalent Layer-by-Layer Assembly of Water-Permeable and Water-Impermeable Polymer Multilayers on Highly Water-Soluble and Water-Sensitive Substrates

Aqueous methods for the layer-by-layer (LbL) assembly of polyelectrolyte multilayers (PEMs) are not, by their nature, well suited for the fabrication of thin films on substrates that are highly water-soluble or composed of water-sensitive materials. Here, we demonstrate that organic solvent-based processes for covalent (or “reactive”) LbL assembly can be used to fabricate multilayers directly on the surfaces of model water-soluble, water-reactive, and water-sensitive substrates that are either difficult or impossible to coat effectively using aqueous-based LbL methods. Our approach is based on methods for the reactive assembly of multilayers using poly­(ethyleneimine) (PEI) and an amine-reactive polymer containing azlactone functionality (PVMDA) in polar-aprotic solvents. The amine-reactive nature of the resulting PEI/PVDMA films facilitated subsequent modification of film-coated surfaces by reaction with primary amine-based nucleophiles. Functionalization of films with the hydrophobic small-molecule amine <i>n</i>-decylamine resulted in the prolonged dissolution and release of underlying water-soluble substrates, and could be used to tune interfacial properties (e.g., to render these water-permeable films more hydrophobic). Fabrication of thicker PEI/PVDMA films resulted in coatings with superhydrophobic properties (e.g., water contact angles of ∼160°) that resisted the penetration of water and enhanced considerably the stabilities of water-sensitive substrates. This approach can, therefore, also be used, in various ways, and to varying extents, to design barriers that protect, enhance the stabilities of, or control/pattern the responses of water-soluble/reactive substrates when they are exposed to aqueous environments. Finally, our results demonstrate that the solubility of PEI and PVDMA in a range of different polar-aprotic solvents can provide flexibility with respect to coating water-soluble/sensitive materials that may also be soluble in some other polar organic solvents. The versatility of this approach could prove useful for modifying the interfacial properties of water-soluble and water-sensitive materials of interest in biotechnology, catalysis, and many other fundamental and applied contexts

Layer-by-Layer Assembly of Amine-Reactive Multilayers Using an Azlactone-Functionalized Polymer and Small-Molecule Diamine Linkers

We report the reactive layer-by-layer assembly of amine-reactive polymer multilayers using an azlactone-functionalized polymer and small-molecule diamine linkers. This approach yields cross-linked polymer/linker-type films that can be further functionalized, after fabrication, by treatment with functional primary amines, and provides opportunities to incorporate other useful functionality that can be difficult to introduce using other polyamine building blocks. Films fabricated using poly­(2-vinyl-4,4-dimethylazlactone) (PVDMA) and three model nondegradable aliphatic diamine linkers yielded reactive thin films that were stable upon incubation in physiologically relevant media. By contrast, films fabricated using PVDMA and varying amounts of the model disulfide-containing diamine linker cystamine were stable in normal physiological media, but were unstable and eroded rapidly upon exposure to chemical reducing agents. We demonstrate that this approach can be used to fabricate functionalized polymer microcapsules that degrade in reducing environments, and that rates of erosion, extents of capsule swelling, and capsule degradation can be tuned by control over the relative concentration of cystamine linker used during fabrication. The polymer/linker approach used here expands the range of properties and functions that can be designed into reactive PVDMA-based coatings, including functionality that can degrade, erode, and undergo triggered destruction in aqueous environments. We therefore anticipate that these approaches will be useful for the functionalization, patterning, and customization of coatings, membranes, capsules, and interfaces of potential utility in biotechnical or biomedical contexts and other areas where degradation and transience are desired. The proof of concept strategies reported here are likely to be general, and should prove useful for the design of amine-reactive coatings containing other functional structures by judicious control of the structures of the linkers used during assembly

Covalent Immobilization of Caged Liquid Crystal Microdroplets on Surfaces

Microscale droplets of thermotropic liquid crystals (LCs) suspended in aqueous media (e.g., LC-in-water emulsions) respond sensitively to the presence of contaminating amphiphiles and, thus, provide promising platforms for the development of new classes of droplet-based environmental sensors. Here, we report polymer-based approaches to the immobilization of LC droplets on surfaces; these approaches introduce several new properties and droplet behaviors and thus also expand the potential utility of LC droplet-based sensors. Our approach exploits the properties of microscale droplets of LCs contained within polymer-based microcapsule cages (so-called “caged” LCs). We demonstrate that caged LCs functionalized with primary amine groups can be immobilized on model surfaces through both weak/reversible ionic interactions and stronger reactive/covalent interactions. We demonstrate using polarized light microscopy that caged LCs that are covalently immobilized on surfaces can undergo rapid and diagnostic changes in shape, rotational mobility, and optical appearance upon the addition of amphiphiles to surrounding aqueous media, including many useful changes in these features that cannot be attained using freely suspended or surface-adsorbed LC droplets. Our results reveal these amphiphile-triggered orientational transitions to be reversible and that arrays of immobilized caged LCs can be used (and reused) to detect both increases and decreases in the concentrations of model contaminants. Finally, we report changes in the shapes and optical appearances of LC droplets that occur when immobilized caged LCs are removed from aqueous environments and dried, and we demonstrate that dried arrays can be stored for months without losing the ability to respond to the presence of analytes upon rehydration. Our results address practical issues associated with the preparation, characterization, storage, and point-of-use application of conventional LC-in-water emulsions and provide a basis for approaches that could enable the development of new “off-the-shelf” LC droplet-based sensing platforms

Nonwoven Polymer Nanofiber Coatings That Inhibit Quorum Sensing in Staphylococcus aureus: Toward New Nonbactericidal Approaches to Infection Control

We report the fabrication and biological evaluation of nonwoven polymer nanofiber coatings that inhibit quorum sensing (QS) and virulence in the human pathogen Staphylococcus aureus. Our results demonstrate that macrocyclic peptide <b>1</b>, a potent and synthetic nonbactericidal quorum sensing inhibitor (QSI) in S. aureus, can be loaded into degradable polymer nanofibers by electrospinning and that this approach can deposit QSI-loaded nanofiber coatings onto model nonwoven mesh substrates. The QSI was released over ∼3 weeks when these materials were incubated in physiological buffer, retained its biological activity, and strongly inhibited agr-based QS in a GFP reporter strain of S. aureus for at least 14 days without promoting cell death. These materials also inhibited production of hemolysins, a QS-controlled virulence phenotype, and reduced the lysis of erythrocytes when placed in contact with wild-type S. aureus growing on surfaces. This approach is modular and can be used with many different polymers, active agents, and processing parameters to fabricate nanofiber coatings on surfaces important in healthcare contexts. S. aureus is one of the most common causative agents of bacterial infections in humans, and strains of this pathogen have developed significant resistance to conventional antibiotics. The QSI-based strategies reported here thus provide springboards for the development of new anti-infective materials and novel treatment strategies that target virulence as opposed to growth in S. aureus. This approach also provides porous scaffolds for cell culture that could prove useful in future studies on the influence of QS modulation on the development and structure of bacterial communities

Generation of Gaseous ClO₂ from Thin Films of Solid NaClO₂ by Sequential Exposure to Ultraviolet Light and Moisture

We report that thin films of solid sodium chlorite (NaClO₂) can be photochemically activated by irradiation with ultraviolet (UV) light to generate gaseous chlorine dioxide (ClO₂) upon subsequent exposure to moisture. The limiting role of water in the reaction is evidenced by an increase in yield of ClO₂ with relative humidity of the gas stream passed over the UV-activated salt. The UV-activated state of the NaClO₂ was found to possess a half-life of 48 h, revealing the presence of long-lived UV activated species that subsequently react with water to produce gaseous ClO₂. The yield of ClO₂ was determined to be proportional to the surface area of NaClO₂ particles projected to the incident illumination, consistent with activation of a ∼10 nm-thick layer of NaClO₂ at the surface of the micrometer-sized salt crystals (for an activation wavelength of 254 nm). We also found that the quantity of ClO₂ released can be tuned ∼10-fold by varying wavelength of UV irradiation and relative humidity of the gas stream passed over the UV-activated NaClO₂. The UV-activated species were not detectable by electron paramagnetic resonance spectroscopy, indicating that the activated intermediate is not an excited triplet state of ClO₂^–. Additionally, neither X-ray photoelectron spectroscopy, nor Raman spectroscopy, nor attenuated total reflection infrared spectroscopy revealed the identity of the activated intermediate species. The ability to preactivate solid phase chlorite salt for subsequent generation of ClO₂ upon exposure to moisture suggests the basis of new materials and methods that permit triggered release of ClO₂ in contexts that use its disinfectant properties

Nanoporous Superhydrophobic Coatings that Promote the Extended Release of Water-Labile Quorum Sensing Inhibitors and Enable Long-Term Modulation of Quorum Sensing in <i>Staphylococcus aureus</i>

Materials and coatings that inhibit bacterial colonization are of interest in a broad range of biomedical, environmental, and industrial applications. In view of the rapid increase in bacterial resistance to conventional antibiotics, the development of new strategies that target nonessential pathways in bacterial pathogensand that thereby limit growth and reduce virulence through nonbiocidal meanshas attracted considerable attention. Bacterial quorum sensing (QS) represents one such target, and is intimately connected to virulence in many human pathogens. Here, we demonstrate that the properties of nanoporous, polymer-based superhydrophobic coatings can be exploited to host and subsequently sustain the extended release of potent and water-labile peptide-based inhibitors of QS (QSIs) in <i>Staphylococcus aureus</i>. Our results demonstrate that these peptidic QSIs can be released into surrounding media for periods of at least 8 months, and that they strongly inhibit agr-based QS in <i>S. aureus</i> for at least 40 days. These results also suggest that these extremely nonwetting coatings can confer protection against the rapid hydrolysis of these water-labile peptides, thereby extending their useful lifetimes. Finally, we demonstrate that these peptide-loaded superhydrophobic coatings can strongly modulate the QS-controlled formation of biofilm in wild-type <i>S. aureus</i>. These nanoporous superhydrophobic films provide a new, useful, and nonbiocidal approach to the design of coatings that attenuate bacterial virulence. This approach has the potential to be general, and could prove suitable for the encapsulation, protection, and release of other classes of water-sensitive agents. We anticipate that the materials, strategies, and concepts reported here will enable new approaches to the long-term attenuation of QS and associated bacterial phenotypes in a range of basic research and applied contexts