Photomediated post-fabrication modification of azlactone-functionalized gels for the development of hydrogel actuators.

We report an approach for the photomediated post-fabrication modification of reactive, azlactone-containing gels using light-initiated deprotection of amines caged with 2-(nitrophenyl)propyloxycarbonyl (NPPOC). Photomediated modification of these gels can be used to generate a gradient in chemical functionality. When functionalized with tertiary amine groups, these gradient gels exhibit rapid and reversible shape deformations in response to changes in pH.

Protein-Polymer Conjugates Synthesized Using Water-Soluble Azlactone-Functionalized Polymers Enable Receptor-Specific Cellular Uptake toward Targeted Drug Delivery.

Conjugation of proteins to drug-loaded polymeric structures is an attractive strategy for facilitating target-specific drug delivery for a variety of clinical needs. Polymers currently available for conjugation to proteins generally have limited chemical versatility for subsequent drug loading. Many polymers that do have chemical functionality useful for drug loading are often insoluble in water, making it difficult to synthesize functional protein-polymer conjugates for targeted drug delivery. In this work, we demonstrate that reactive, azlactone-functionalized polymers can be grafted to proteins, conjugated to a small-molecule fluorophore, and subsequently internalized into cells in a receptor-specific manner. Poly(2-vinyl-4,4-dimethylazlactone), synthesized using reversible addition-fragmentation chain transfer polymerization, was modified post-polymerization with substoichiometric equivalents of triethylene glycol monomethyl ether to yield reactive water-soluble, azlactone-functionalized copolymers. These reactive polymers were then conjugated to proteins holo-transferrin and ovotransferrin. Protein gel analysis verified successful conjugation of proteins to polymer, and protein-polymer conjugates were subsequently purified from unreacted proteins and polymers using size exclusion chromatography. Internalization experiments using a breast cancer cell line that overexpresses the transferrin receptor on its surface showed that the holo-transferrin-polymer conjugate was successfully internalized by cells in a mechanism consistent with receptor-mediated endocytosis. Internalization of protein-polymer conjugate demonstrated that the protein ligand maintained its overall structure and function following conjugation to polymer. Our approach to protein-polymer conjugate synthesis offers a simple, tailorable strategy for preparing bioconjugates of interest for a broad range of biomedical applications.

Analysis and Control of Chain Mobility in Protein Hydrogels.

Coiled-coil domains can direct the assembly of protein block copolymers into physically cross-linked, viscoelastic hydrogels. Here, we describe the use of fluorescence recovery after photobleaching (FRAP) to probe chain mobility in reversible hydrogels assembled from engineered proteins bearing terminal coiled-coil domains. We show that chain mobility can be related to the underlying dynamics of the coiled-coil domains by application of a three-state "hopping" model of chain migration. We further show that genetic programming allows the effective mobility of network chains to be varied 500-fold through modest changes in protein sequence. Destabilization of the coiled-coil domains by site-directed mutagenesis increases the effective diffusivity of probe chains. Conversely, probe mobility is reduced by expanding the hydrophobic surface area of the coiled-coil domains through introduction of the bulky leucine surrogate homoisoleucine. Predictions from the three-state model imply asymmetric sequential binding of the terminal domains. Brownian Dynamics simulations suggest that binding asymmetry is a general feature of reversible gels, arising from a loss in entropy as chains transition to a conformationally restricted bridged state.

Programming Molecular Association and Viscoelastic Behavior in Protein Networks.

A set of recombinant artificial proteins that can be cross-linked, by either covalent bonds or association of helical domains or both, is described. The designed proteins can be used to construct molecular networks in which the mechanism of crosslinking determines the time-dependent responses to mechanical deformation.

Multivalent alteration of quorum sensing in Staphylococcus aureus.

Virulence in Staphylococcus aureus is strongly and positively correlated with local cell density. Here we present an effective approach to modulate this group behaviour using multivalent peptide-polymer conjugates. Our results show that by attaching multiple AIP-4' units to macromolecular scaffolds, the agr QS response in S. aureus was affected strongly, while displaying a clear multivalency effect.

In situ Synthesis of Oligonucleotide Arrays on Surfaces Coated with Crosslinked Polymer Multilayers.

We report an approach to the in situ synthesis of oligonucleotide arrays on surfaces coated with crosslinked polymer multilayers. Our approach makes use of methods for the 'reactive' layer-by-layer assembly of thin, amine-reactive multilayers using branched polyethyleneimine (PEI) and the azlactone-functionalized polymer poly(2-vinyl-4,4'-dimethylazlactone) (PVDMA). Post-fabrication treatment of film-coated glass substrates with d-glucamine or 4-amino-1-butanol yielded hydroxyl-functionalized films suitable for the Maskless Array Synthesis (MAS) of oligonucleotide arrays. Glucamine-functionalized films yielded arrays of oligonucleotides with fluorescence intensities and signal-to-noise ratios (after hybridization with fluorescently labeled complementary strands) comparable to those of arrays fabricated on conventional silanized glass substrates. These arrays could be exposed to multiple hybridization-dehybridization cycles with only moderate loss of hybridization density. The versatility of the layer-by-layer approach also permitted synthesis directly on thin sheets of film-coated poly(ethylene terephthalate) (PET) to yield flexible oligonucleotide arrays that could be readily manipulated (e.g., bent) and cut into smaller arrays. To our knowledge, this work presents the first use of polymer multilayers as a substrate for the multi-step synthesis of complex molecules. Our results demonstrate that these films are robust and able to withstand the ~450 individual chemical processing steps associated with MAS (as well as manipulations required to hybridize, image, and dehybridize the arrays) without large-scale cracking, peeling, or delamination of the thin films. The combination of layer-by-layer assembly and MAS provides a means of fabricating functional oligonucleotide arrays on a range of different materials and substrates. This approach may also prove useful for the fabrication of supports for the solid-phase synthesis and screening of other macromolecular or small-molecule agents.

Azlactone-Functionalized Polymers as Reactive Platforms for the Design of Advanced Materials: Progress in the Last Ten Years.

Polymers functionalized with azlactone (or oxazolone) functionality have become increasingly useful for the rapid and modular design of functional materials. Because azlactones can react via ring-opening reactions with a variety of different nucleophilic species (e.g., primary amines, hydroxyl groups, and thiol functionality), azlactone-functionalized materials can serve as convenient 'reactive' platforms for the post-synthesis or post-fabrication introduction of a broad range of chemical functionality to soluble polymers, insoluble supports, and surfaces/interfaces. The last decade has seen an increase in both the number and the variety of reports that exploit the properties and the reactivities of azlactone-functionalized polymers. Here, we highlight recent work from several different laboratories, including our own, toward the design and characterization of azlactone-functionalized polymers, with a particular emphasis on: (i) new synthetic approaches for the preparation of well-defined azlactone-functionalized polymers using living/controlled methods of polymerization, (ii) the design and modular synthesis of side-chain functionalized polymers and block copolymers via post-polymerization modification of azlactone-functionalized polymers, (iii) the development of reactive polymeric supports useful in the contexts of separations and catalysis, and (iv) methods for the fabrication of reactive thin films and other approaches to the immobilization of azlactone functionality on surfaces and interfaces. Examples discussed herein reveal a growing awareness of azlactone functionality as a useful tool for polymer chemists, and highlight several ways that the unique reactivity of these materials can both complement and provide useful alternatives to other reactive polymers currently used to design functional materials.

Fabrication and selective functionalization of amine-reactive polymer multilayers on topographically patterned microwell cell culture arrays.

We report an approach to the fabrication and selective functionalization of amine-reactive polymer multilayers on the surfaces of 3-D polyurethane-based microwell cell culture arrays. "Reactive" layer-by-layer assembly of multilayers using branched polyethyleneimine (BPEI) and the azlactone-functionalized polymer poly(2-vinyl-4,4'-dimethylazlactone) (PVDMA) yielded film-coated microwell arrays that could be chemically functionalized postfabrication by treatment with different amine-functionalized macromolecules or small molecule primary amines. Treatment of film-coated arrays with the small molecule amine d-glucamine resulted in microwell surfaces that resisted the adhesion and proliferation of mammalian fibroblast cells in vitro. These and other experiments demonstrated that it was possible to functionalize different structural features of these arrays in a spatially resolved manner to create dual-functionalized substrates (e.g., to create arrays having either (i) azlactone-functionalized wells, with regions between the wells functionalized with glucamine or (ii) substrates with spatially resolved regions of two different cationic polymers). In particular, spatial control over glucamine functionalization yielded 3-D substrates that could be used to confine cell attachment and growth to microwells for periods of up to 28 days and support the 3-D culture of arrays of cuboidal cell clusters. These approaches to dual functionalization could prove useful for the long-term culture and maintenance of cell types for which the presentation of specific and chemically well-defined 3-D culture environments is required for control over cell growth, differentiation, and other important behaviors. More generally, our approach provides methods for the straightforward chemical functionalization of otherwise unreactive topographically patterned substrates that could prove to be useful in a range of other fundamental and applied contexts.

Fabrication of Covalently Crosslinked and Amine-Reactive Microcapsules by Reactive Layer-by-Layer Assembly of Azlactone-Containing Polymer Multilayers on Sacrificial Microparticle Templates.

We report on the fabrication of covalently crosslinked and amine-reactive hollow microcapsules using 'reactive' layer-by-layer assembly to deposit thin polymer films on sacrificial microparticle templates. Our approach is based on the alternating deposition of layers of a synthetic polyamine and a polymer containing reactive azlactone functionality. Multilayered films composed of branched poly(ethylene imine) (BPEI) and poly(2-vinyl-4,4-dimethylazlactone) (PVDMA) were fabricated layer-by-layer on the surfaces of calcium carbonate and glass microparticle templates. After fabrication, these films contained residual azlactone functionality that was accessible for reaction with amine-containing molecules. Dissolution of the calcium carbonate or glass cores using aqueous ethylenediamine tetraacetic acid (EDTA) or hydrofluoric acid (HF), respectively, led to the formation of hollow polymer microcapsules. These microcapsules were robust enough to encapsulate and retain a model macromolecule (FITC-dextran) and were stable for at least 22 hours in high ionic strength environments, in low and high pH solutions, and in several common organic solvents. Significant differences in the behaviors of capsules fabricated on CaCO(3) and glass cores were observed and characterized using scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS). Whereas capsules fabricated on CaCO(3) templates collapsed upon drying, capsules fabricated on glass templates remained rigid and spherical. Characterization using EDS suggested that this latter behavior results, at least in part, from the presence of insoluble metal fluoride salts that are trapped or precipitate within the walls of capsules after etching of the glass cores using HF. Our results demonstrate that the assembly of BPEI/PVDMA films on sacrificial templates can be used to fabricate reactive microcapsules of potential use in a wide range of fields, including catalysis, drug and gene delivery, imaging, and biomedical research.

Layer-by-Layer Fabrication of Covalently Crosslinked and Reactive Polymer Multilayers Using Azlactone-Functionalized Copolymers: A Platform for the Design of Functional Biointerfaces.

We report a method for modulating the physicochemical properties of surfaces that is based on the reactive layer-by-layer fabrication of covalently crosslinked thin films using azlactone-functionalized copolymers. We demonstrate that copolymers containing different molar ratios of methylmethacrylate (MMA) and 2-vinyl-4,4-dimethylazlactone (VDMA) can be alternately deposited with poly(ethyleneimine) to assemble covalently crosslinked thin films. Characterization using ellipsometry demonstrates that, in general, film growth and thickness decrease as the content of reactive, azlactone functionality in the copolymer used to assemble the film decreases. Reflective infrared spectroscopy experiments demonstrate that films fabricated from MMA:VDMA copolymers contain residual azlactone functionality and that these reactive groups can be exploited to modify film-coated surfaces. Fabricating films from MMA:VDMA copolymers containing different compositions permitted modulation of the density of reactive groups within the films and, thus, the extent to which the films are functionalized by exposure to small molecule amines. For example, functionalization of MMA:VDMA copolymer films with the small molecule D-glucamine resulted in films with water contact angles that varied with the composition of the copolymer used to fabricate the film (e.g., as the azlactone content in the film increased, glucamine-modified films became more hydrophilic). We demonstrate further that treatment of copolymer-containing films with glucamine resulted in changes in the numbers of mammalian cells that grow on the surfaces of the films. Our results suggest the basis of methods that could be used to modulate or tune the density of chemical and biological functionality presented on surfaces of interest in a variety of fundamental and applied contexts.

Superhydrophobic Thin Films Fabricated by Reactive Layer-by-Layer Assembly of Azlactone-Functionalized Polymers.

We report an approach to the fabrication of superhydrophobic thin films that is based on the 'reactive' layer-by-layer assembly of azlactone-containing polymer multilayers. We demonstrate that films fabricated from alternating layers of the azlactone functionalized polymer poly(2-vinyl-4,4-dimethylazlactone) (PVDMA) and poly(ethyleneimine) (PEI) exhibit micro- and nanoscale surface features that result in water contact angles in excess of 150º. Our results reveal that the formation of these surface features is (i) dependent upon film thickness (i.e., the number of layers of PEI and PVDMA deposited) and (ii) that it is influenced strongly by the presence (or absence) of cyclic azlactone-functionalized oligomers that can form upon storage of the 2-vinyl-4,4-dimethylazlactone (VDMA) used to synthesize PVDMA. For example, films fabricated using polymers synthesized in the presence of these oligomers exhibited rough, textured surfaces and superhydrophobic behavior (i.e., advancing contact angles in excess of 150º). In contrast, films fabricated from PVDMA polymerized in the absence of this oligomer (e.g., using freshly distilled monomer) were smooth and only moderately hydrophobic (i.e., advancing contact angles of ~75º). The addition of authentic, independently synthesized oligomer to samples of distilled VDMA at specified and controlled concentrations permitted reproducible fabrication of superhydrophobic thin films on the surfaces of a variety of different substrates. The surfaces of these films were demonstrated to be superhydrophobic immediately after fabrication, but they became hydrophilic after exposure to water for six days. Additional experiments demonstrated that it was possible to stabilize and prolong the superhydrophobic properties of these films (e.g., advancing contact angles in excess of 150° even after complete submersion in water for at least six weeks) by exploiting the reactivity of residual azlactones to functionalize the surfaces of the films using hydrophobic amines (e.g., aliphatic or semi-fluorinated aliphatic amines). Our results demonstrate a straightforward and substrate-independent approach to the design of superhydrophobic and reactive polymer-based coatings of potential use in a broad range of fundamental and applied contexts.

Free-standing and reactive thin films fabricated by covalent layer-by-layer assembly and subsequent lift-off of azlactone-containing polymer multilayers.

We report an approach to the fabrication of free-standing and amine-reactive thin films that is based on the reactive layer-by-layer assembly and subsequent lift-off of azlactone-containing polymer multilayers. We demonstrate that covalently cross-linked multilayers fabricated using the azlactone-functionalized polymer poly(2-vinyl-4,4-dimethylazlactone) (PVDMA) and a primary amine-containing polymer [poly(ethyleneimine) (PEI)] can be delaminated from planar glass and silicon surfaces by immersion in mildly acidic aqueous environments to yield flexible freestanding membranes. These free-standing membranes are robust and can withstand exposure to strong acid, strong base, or incubation in high ionic strength solutions that typically lead to the disruption and erosion of polymer multilayers assembled by reversible weak interactions (e.g., "polyelectrolyte multilayers" assembled by electrostatic interactions or hydrogen bonding). We demonstrate further that these PEI/PVDMA assemblies contain residual reactive azlactone functionality that can be exploited to chemically modify the films (either directly after fabrication or after they have been lifted off of the substrates on which they were fabricated) using a variety of amine-functionalized small molecules. These free-standing membranes can also be transferred readily onto other objects (for example, onto the surfaces of planar substrates containing holes or pores) to fabricate suspended polymer membranes and other film-functionalized interfaces. In addition to planar, two-dimensional free-standing films, this approach can be used to fabricate and isolate three-dimensional free-standing membranes (e.g., curved films or tubes) by layer-by-layer assembly on, and subsequent lift-off from, the surfaces of topologically complex substrates (e.g., the curved ends of glass tubing, etc.). The results of this investigation, when combined, suggest the basis of methods for the fabrication of stable, chemically reactive, and flexible polymer thin films and membranes of potential utility in a variety of fundamental and applied contexts.

Immobilization of polymer-decorated liquid crystal droplets on chemically tailored surfaces.

We demonstrate that the assembly of an amphiphilic polyamine on the interfaces of micrometer-sized droplets of a thermotropic liquid crystal (LC) dispersed in aqueous solutions can be used to facilitate the immobilization of LC droplets on chemically functionalized surfaces. Polymer 1 was designed to contain both hydrophobic (alkyl-functionalized) and hydrophilic (primary and tertiary amine-functionalized) side chain functionality. The assembly of this polymer at the interfaces of aqueous dispersions of LC droplets was achieved by the spontaneous adsorption of polymer from aqueous solution. Polymer adsorption triggered transitions in the orientational ordering of the LCs, as observed by polarized light and bright-field microscopy. We demonstrate that the presence of polymer 1 on the interfaces of these droplets can be exploited to immobilize LC droplets on planar solid surfaces through covalent bond formation (e.g., for surfaces coated with polymer multilayers containing reactive azlactone functionality) or through electrostatic interactions (e.g., for surfaces coated with multilayers containing hydrolyzed azlactone functionality). The characterization of immobilized LC droplets by polarized, fluorescence, and laser scanning confocal microscopy revealed the general spherical shape of the polymer-coated LC droplets to be maintained after immobilization, and that immobilization led to additional ordering transitions within the droplets that were dependent on the nature of the surfaces with which they were in contact. Polymer 1-functionalized LC droplets were not immobilized on polymer multilayers treated with poly(ethylene imine) (PEI). We demonstrate that the ability to design surfaces that promote or prevent the immobilization of polymer-functionalized LC droplets can be exploited to pattern the immobilization of LC droplets on surfaces. The results of this investigation provide the basis of an approach that could be used to tailor the properties of dispersed LC emulsions and to immobilize these droplets on functional surfaces of interest in a broad range of fundamental and applied contexts.

Functionalization of fibers using azlactone-containing polymers: layer-by-layer fabrication of reactive thin films on the surfaces of hair and cellulose-based materials.

We report an approach to the functionalization of fibers and fiber-based materials that is based on the deposition of reactive azlactone-functionalized polymers and the "reactive" layer-by-layer assembly of azlactone-containing thin films. We demonstrate (i) that the azlactone-functionalized polymer poly(2-vinyl-4,4-dimethylazlactone) (PVDMA) can be used to modify the surfaces of a model protein-based fiber (horsehair) and cellulose-based materials (e.g., cotton and paper), and (ii) that fibers functionalized in this manner can be used to support the fabrication of covalently cross-linked and reactive polymer multilayers assembled using PVDMA and poly(ethyleneimine) (PEI). The growth, chemical reactivity, and uniformity of films deposited on these substrates were characterized using fluorescence microscopy, confocal microscopy, and scanning electron microscopy (SEM). In addition to the direct functionalization of fibers, we demonstrate that the residual azlactone functionality in PVDMA-treated or film-coated fibers can be exploited to chemically modify the surface chemistry and physicochemical properties of fiber-based materials postfabrication using amine functionalized molecules. For example, we demonstrate that this approach permits control over the surface properties of paper (e.g., absorption of water) by simple postfabrication treatment of film-coated paper with the hydrophobic amine n-decylamine. The azlactone functionality present in these materials provides a platform for the modification of polymer-treated and film-coated fibers with a broad range of other chemical and biological species (e.g., enzymes, peptides, catalysts, etc.). The results of this investigation thus provide a basis for the functionalization of fibers and fiber-based materials (e.g., textile fabrics or nonwoven mats) of potential utility in a broad range of consumer, industrial, and biomedical contexts.

Azlactone-functionalized polymers as reactive templates for parallel polymer synthesis: synthesis and screening of a small library of cationic polymers in the context of DNA delivery.

Azlactone-functionalized polymers are used as reactive templates for the synthesis of a library of amine-functionalized polymers of interest in the context of DNA delivery and other applications.

Characterization of nanoscale transformations in polyelectrolyte multilayers fabricated from plasmid DNA using laser scanning confocal microscopy in combination with atomic force microscopy.

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.

Langmuir films of flexible polymers transferred to aqueous/liquid crystal interfaces induce uniform azimuthal alignment of the liquid crystal.

We reported recently that amphiphilic polymers can be assembled at interfaces created between aqueous phases and thermotropic liquid crystals (LCs) in ways that: (i) couple the organization of the polymer to the order of the LC and (ii) respond to changes in the properties of aqueous phases that can be characterized as changes in the optical appearance of the LC. This investigation sought to characterize the behavior of aqueous-LC interfaces decorated with uniaxially compressed thin films of polymers transferred by Langmuir-Schaefer (LS) transfer. Here, we report physicochemical characterization of interfaces created between aqueous phases and the thermotropic LC 4-cyano-4'-pentylbiphenyl (5CB) decorated with Langmuir films of a novel amphiphilic polymer (polymer 1), synthesized by the addition of hydrophobic and hydrophilic side chains to poly(2-vinyl-4,4'-dimethylazlactone). Initial characterization of this system resulted in the unexpected observation of uniform azimuthal alignment of 5CB after LS transfer of the polymer films to aqueous-5CB interfaces. This paper describes characterization of Langmuir films of polymer 1 hosted at aqueous-5CB interfaces as well as the results of our investigations into the origins of the uniform ordering of the LC observed upon LS transfer. Our results, when combined, support the conclusion that uniform azimuthal alignment of 5CB is the result of long-range ordering of polymer chains in the Langmuir films (in a preferred direction orthogonal to the direction of compression) that is generated during uniaxial compression of the films prior to LS transfer. Although past studies of Langmuir films of polymers at aqueous-air interfaces have demonstrated that in-plane alignment of polymer backbones can be induced by uniaxial compression, these past reports have generally made use of polymers with rigid backbones. One important outcome of this current study is thus the observation of anisotropy and long-range order in Langmuir films of a novel flexible polymer. A second important outcome is the observation that the existence, extent, and dynamics of this order can be identified and characterized optically by transfer of the Langmuir film to a thin film of LC. Additional characterization of Langmuir films of two other flexible polymers [poly(methyl methacrylate) and poly(vinyl stearate)] using this method also resulted in uniform azimuthal alignment of 5CB, suggesting that the generation of long-range order in uniaxially compressed Langmuir films of polymers may also occur more generally over a broader range of polymers with flexible backbones.

Chemical modification of reactive multilayered films fabricated from poly(2-alkenyl azlactone)s: design of surfaces that prevent or promote mammalian cell adhesion and bacterial biofilm growth.

We report an approach to the design of reactive polymer films that can be functionalized post-fabrication to either prevent or promote the attachment and growth of cells. Our approach is based on the reactive layer-by-layer assembly of covalently crosslinked thin films using a synthetic polyamine and a polymer containing reactive azlactone functionality. Our results demonstrate (i) that the residual azlactone functionality in these films can be exploited to immobilize amine-functionalized chemical motifs similar to those that promote or prevent cell and protein adhesion when assembled as self-assembled monolayers on gold-coated surfaces and (ii) that the immobilization of these motifs changes significantly the behaviors and interactions of cells with the surfaces of these polymer films. We demonstrate that films treated with the hydrophobic molecule decylamine support the attachment and growth of mammalian cells in vitro. In contrast, films treated with the hydrophilic carbohydrate d-glucamine prevent cell adhesion and growth almost completely. The results of additional experiments suggest that these large differences in cell behavior can be understood, at least in part, in terms of differences in the abilities of these two different chemical motifs to promote or prevent the adsorption of protein onto film-coated surfaces. We demonstrate further that this approach can be used to pattern regions of these reactive films that resist the initial attachment and subsequent invasion of mammalian cells for periods of at least one month in the presence of serum-containing cell culture media. Finally, we report that films that prevent the adhesion and growth of mammalian cells also prevent the initial formation of bacterial biofilms when incubated in the presence of the clinically relevant pathogen Pseudomonas aeruginosa . The results of these studies, collectively, suggest the basis of general approaches to the fabrication and functionalization of thin films that prevent, promote, or pattern cell growth or the formation of biofilms on surfaces of interest in the contexts of both fundamental biological studies and a broad range of other practical applications.

Nanoimprinted thin films of reactive, azlactone-containing polymers: combining methods for the topographic patterning of cell substrates with opportunities for facile post-fabrication chemical functionalization.

Approaches to the fabrication of surfaces that combine methods for the topographic patterning of soft materials with opportunities for facile, post-fabrication chemical functionalization could contribute significantly to advances in biotechnology and a broad range of other areas. Here, we report methods that can be used to introduce well-defined nano- and microscale topographic features to thin films of reactive polymers containing azlactone functionality using nanoimprint lithography (NIL). We demonstrate that NIL can be used to imprint topographic patterns into thin films of poly(2-vinyl-4,4-dimethylazlactone) and a copolymer of methyl methacrylate and 2-vinyl-4,4-dimethylazlactone using silicon masters having patterns of grooves and ridges ranging in width from 400 nm to 2 microm, demonstrating the potential of this method to transfer patterns to films of these reactive polymers over a range of feature sizes and densities. We demonstrate further that the azlactone functionality of these polymers survives temperatures and pressures associated with NIL, and that topographically patterned films can be readily functionalized post-fabrication by treatment of surface-accessible azlactone functionality with small molecules and polymers containing primary amines. The results of experiments in which NIH-3T3 cells were seeded onto films imprinted with lined patterns having a pitch of 4 microm demonstrated that cells attach and proliferate on these azlactone-containing films and that they align in the direction of the imprinted pattern. Finally, we demonstrate that the treatment of these materials with amine-functionalized poly(ethylene glycol) (PEG) can be used to create regions of topographically patterned films that prevent cell adhesion. The results of this study suggest approaches to the functionalization of topographically patterned surfaces with a broad range of chemical functionality (e.g., peptides, proteins, carbohydrates, etc.) of biotechnological interest. The ability to manipulate and define both the physical topography and chemical functionality of these reactive materials could provide opportunities to investigate the combined effects of substrate topography and chemical functionality on cell behavior and may also be useful in a broad range of other applications.