Degradable Amine-Reactive Coatings Fabricated by the Covalent

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Degradable Amine-Reactive Coatings Fabricated by the Covalent Layer-by-Layer Assembly of Poly(2-Vinyl-4,4Dimethylazlactone) with Degradable Polyamine Building Blocks Yashira M. Zayas-Gonzalez, and David M. Lynn Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00975 • Publication Date (Web): 15 Aug 2016 Downloaded from http://pubs.acs.org on August 16, 2016

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Degradable Amine-Reactive Coatings Fabricated by the Covalent Layer-by-Layer Assembly of Poly(2-Vinyl-4,4-Dimethylazlactone) with Degradable Polyamine Building Blocks Yashira M. Zayas-Gonzalez1 and David M. Lynn1,2,* 1

Department of Chemical and Biological Engineering, 1415 Engineering Drive, University of Wisconsin—Madison, Madison, Wisconsin 53706, and 2Department of Chemistry, 1101 University Avenue, University of Wisconsin—Madison, Madison, Wisconsin 53706. E-mail: [email protected] ABSTRACT: We report the fabrication of reactive and degradable crosslinked polymer multilayers by the reactive/covalent layer-by-layer assembly of a non-degradable azlactonefunctionalized polymer [poly(2-vinyl-4,4-dimethylazlactone), PVDMA] with hydrolytically or enzymatically degradable polyamine building blocks. Fabrication of multilayers using PVDMA and a hydrolytically degradable poly(β-amino ester) (PBAE) containing primary amine side chains yielded multilayers (~100 nm thick) that degraded over ~12 days in physiologically relevant media. Physicochemical characterization and studies on stable films fabricated using PVDMA and an analogous non-degradable poly(amidoamine) suggested that erosion occurred by chemical hydrolysis of backbone esters in the PBAE components of these assemblies. These degradable assemblies also contained residual amine-reactive azlactone functionality that could be used to impart new functionality to the coatings post-fabrication. Crosslinked multilayers fabricated using PVDMA and the enzymatically degradable polymer poly(L-lysine) were structurally stable for prolonged periods in physiological media, but degraded over ~24 h when the enzyme trypsin was added. Past studies demonstrate that multilayers fabricated using PVDMA and non-degradable polyamines [e.g., poly(ethyleneimine)] enable the design and patterning of useful nano/bio-interfaces and other materials that are structurally stable in physiological media. The introduction of degradable functionality into PVDMA-based multilayers creates opportunities to exploit the reactivity of azlactone groups for the design of reactive materials and functional coatings that degrade or erode in environments that are relevant in biomedical, biotechnological, and environmental contexts. This ‘degradable building block’ strategy should be general; we anticipate that this approach can also be extended to design of amine-reactive multilayers that degrade upon exposure to specific chemical triggers, selective enzymes, or contact with cells by judicious design of the degradable polyamine building blocks used to fabricate the coatings. For Table of Contents Use Only:

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Introduction Chemically reactive polymer coatings provide useful platforms for the design, functionalization, and patterning of surfaces and interfaces.1-5 Depending on the type of reactive polymer that is used,6-8 and the extent to which chemical functionality is present at the surface or in the bulk of a thin film, this ‘reactive’ approach can be used to define and modify important physical or interfacial properties of surface coatings (e.g., crosslink densities or wetting behaviors) or affect the post-fabrication immobilization or patterning of new chemical or biological motifs (e.g., peptides, proteins, etc.) of interest and potential utility in a broad range of fundamental and applied contexts.1-8 The work reported here was motivated by recent reports on the fabrication of polymerbased multilayer coatings using ‘reactive’ or ‘covalent’ layer-by-layer assembly.3,9-11 In contrast to conventional polyelectrolyte-based methods for layer-by-layer assembly—which typically exploit weak ionic interactions between oppositely charged polymers12-16 to drive the assembly of thin polymer ‘multilayers’—reactive layer-by-layer assembly is promoted by the formation of covalent bonds between mutually reactive polymers during each step of the fabrication process.3,9-11 As such, this approach yields thin multilayered films that are both (i) chemically crosslinked (and thus stable in chemically complex media) and (ii), chemically reactive (owing to the presence of residual reactive groups that were not consumed during assembly and can be exploited to immobilize other mutually reactive species). Many different classes of reactive polymers have been investigated for this purpose,3,17 with the extents to which the physical and interfacial properties of the resulting films can be manipulated, and the types of secondary functionality that can be installed post-fabrication, varying widely depending on the types of reactive polymers that are used.


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Our group has reported in a series of past studies on the design, assembly, and characterization of amine-reactive polymer multilayers fabricated using the azlactonefunctionalized polymer poly(2-vinyl-4,4-dimethylazlactone) (PVDMA; Figure 1).3,17-25 PVDMA contains azlactone groups that undergo rapid ring-opening reactions with primary amines under mild reaction conditions, without the need for a catalyst or the generation of reaction byproducts.17,26 When paired with polymers that contain primary amines, PVDMA thus provides a convenient and accessible platform for the reactive layer-by-layer assembly of polymer multilayers. In our past studies, we used branched poly(ethyleneimine) (PEI) as a model primary amine-containing polymer: this general approach yields PEI/PVDMA multilayers that are covalently crosslinked by chemically stable polyacrylamide-based ‘amide/amide’-type bonds17,19 (e.g., Figure 1B) and possess residual amine-reactive azlactone groups17,19 (Figure 2A) that can be functionalized, after fabrication, by simple treatment with a broad range of primary aminecontaining agents (including small molecules,17,18,20 peptides,27 and proteins;20,23,28 again, through the creation of stable ‘amide/amide’-type bonds; Figure 2B). When combined with many of the other practical advantages inherent to layer-by-layer assembly, including the ability to exert fine control over film thickness and composition and fabricate continuous and conformal films on the surfaces of topologically complex objects,29,30 this azlactone-based approach has the potential to be broadly useful for the development of new nano/bio-interfaces and other functional materials.3,17 In addition to past studies from our group on the design and application of azlactone-functionalized multilayers,3,17 several other groups have exploited the chemistry and reactivity of azlactone-functionalized polymers as platforms for the design of new materials and surfaces or interfaces of interest in these and other contexts.17,26,31-43 The PEI/PVDMA multilayers reported in past studies can be remarkably stable when


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exposed to harsh and chemically complex media (including organic solvents, biological media, and aqueous media of high or low pH, high ionic strength, or containing high concentrations of surface-active agents).18,20,24,25 The physical and chemical stability of these materials is likely to be useful and enabling in many applications,24,25 but it can serve as an obstacle to use in other applications where chemical degradation or physical erosion are either required or would be useful (e.g., in many potential biomedical or biotechnological contexts, including drug delivery and the design of new types of biointerfaces).3,18,20,22 The stability of these PEI/PVDMA-based materials results, in large measure, from the chemically stable nature of the ‘amide/amide’-type crosslinks that are formed during the reaction of PEI and PVDMA.17 One approach to designing degradable azlactone-containing multilayers would, thus, be to substitute PEI for a polymer bearing hydroxyl functionality (which would result in the formation of structurally similar ‘amide/ester’-type crosslinks that would degrade hydrolytically upon exposure to aqueous environments). Although such an approach is technically feasible, reactions between azlactones and hydroxyl groups proceed more slowly and generally require catalysts and higher operating temperatures than reactions between azlactones and primary amines.26,44,45 The overall stability of PEI/PVDMA-based materials is also derived, in part, from the fact that both PVDMA and PEI are inherently non-degradable polymers. This current study sought to explore the feasibility of fabricating chemically crosslinked and amine-reactive multilayer coatings using PVDMA and primary amine-containing polymers that are hydrolytically degradable. We demonstrate here that a model poly(β-amino ester) (polymer 1; Figure 1) containing primary amine side chains can be used as a degradable building block for the reactive/covalent assembly of chemically crosslinked multilayers. Thin films fabricated using PVDMA and this degradable polymer contain residual azlactone groups that can be


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functionalized by treatment with other amine-functionalized molecules and, in contrast to coatings fabricated using PVDMA and PEI, also degrade and erode gradually when incubated in aqueous environments (owing to the hydrolysis of ester bonds present in the polyamine components of these assemblies; as illustrated schematically in Figure 2B-C). This ‘degradable building block’ approach should be general, and can also be extended to the design of crosslinked multilayers that degrade upon exposure to enzymes by fabricating films using PVDMA and enzymatically degradable primary amine-containing polymers, such as poly(Llysine). The introduction of degradable functionality into chemically crosslinked, PVDMA-based multilayers creates new opportunities to exploit the unique reactivity of azlactone groups and design functional thin films and reactive coatings that degrade or erode in aqueous environments.

Materials and Methods Materials. Reagent grade acetone, dimethylsulfoxide (DMSO), tetrahydrofuran (THF), acetonitrile, dichloromethane, toluene, (3-aminopropyl)triethoxysilane (APTES), trifluoroacetic acid (TFA), triethylamine (TEA), trypsin-EDTA solution (10X without phenol red), poly(Llysine hydrobromide) (PLL, MW = 15,000-30,000), propylamine, N-tert-butoxycarbonyl-1,2diaminoethane, and 1,4-butanediol diacrylate were purchased from Sigma-Aldrich Chemical Company (Milwaukee, WI), Acros Organics (Morris Plains, NJ), and Alfa Aesar Organics (Ward Hill, MA). Tetramethylrhodamine cadaverine (TMRcad) was purchased from AnaSpec, Inc. (San Jose, CA). D-Glucamine was purchased from TCI America (Portland, OR). Hank's Balanced Salt Solution (HBSS) was purchased from Invitrogen (Carlsbad, CA). 2-Vinyl-4,4-dimethylazlactone (VDMA) was a gift from Dr. Steven M. Heilmann (3M Corporation, Minneapolis, MN). Poly(2vinyl-4,4-dimethylazlactone) (PVDMA; MW=22,700; Ð = 3.3) was synthesized by conventional


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free-radical polymerization of VDMA as previously described.21 N,N’-Butylenebisacrylamide was synthesized as described previously.46 Fluorescently labeled PVDMA (PVDMATMR; approximately 0.5% labeled) was synthesized by the reaction of PVDMA with TMRcad as previously reported.21 Test-grade n-type silicon wafers were purchased from Silicon Inc. (Boise, ID). Commercial cotton and gauze substrates were purchased from Walmart, Inc. (Bentonville, AK). All materials were used without further purification unless otherwise noted. Compressed air used to dry films and coated substrates was filtered through a 0.2 μm membrane syringe filter.

General Considerations. 1H NMR spectra were recorded on Bruker AC+ 250 (250.133 MHz) and Bruker AC+ 300 (300.135 MHz) spectrometers. Chemical shift values are given in ppm and are referenced with respect to residual protons from solvent. Gel permeation chromatography (GPC) was performed using a GPC Max VE2001Solvent/Sample module (Viscotek Corp., Houston, TX) equipped with two PolyPore Organic GPC Columns (250 mm × 4.6 mm; Polymer Laboratories, Amherst, MA). For the characterization of PVDMA, THF was used as the eluent at a flow rate of 1.0 mL/min. For the characterization of degradable polyamines, THF with 0.1 M triethylamine was used as the eluent at a flow rate of 1.0 mL/min. Data were collected using the refractive index detector of a Viscotek TDA-302 triple detector array and processed using the OmniSEC 4.5 software package. Molecular weights are reported relative to monodisperse polystyrene standards. Silicon substrates (e.g., 1.0 × 5.0 cm) were cleaned with acetone, ethanol, methanol, and deionized water and dried under a stream of compressed air prior to the fabrication of multilayer films. Prior to film fabrication, silicon substrates were coated with an APTES coating to improve adhesion of the multilayers to their substrates during long-term incubation in aqueous media. Briefly, APTES was deposited on the silicon from a 1% solution


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(v/v) in anhydrous toluene over 1 h, followed by rinsing with toluene and ethanol. Silicon substrates used for reflective infrared (IR) spectroscopy experiments were prepared by depositing thin layers of titanium (10 nm) and gold (200 nm), sequentially, onto clean silicon wafers using an electron-beam evaporator (Tek-Vac Industries, Brentwood, NY). The optical thicknesses of films fabricated on silicon substrates were characterized using a Gaertner LSE ellipsometer (λ = 632.8 nm, incident angle = 70°). Data were processed using the Gaertner ellipsometer measurement program. Relative thicknesses were calculated assuming an average index of refraction of 1.577 for the multilayered films. Thicknesses were determined using at least three substrates in at least five different locations on each substrate and are presented as an average with standard deviation. Optical and fluorescence microscopy images were acquired using an Olympus IX70 microscope and analyzed using MetaMorph Advanced version 7.7.8.0 (Universal Imaging Corporation). Images were processed using NIH Image J software. Digital photographs were acquired using a Canon PowerShot SX130 IS digital camera. Solution fluorescence measurements were made using a Jobin Yvon FluoroMax-3 fluorometer. Polarization modulation infrared reflectance-absorbance spectroscopy (PM-IRRAS) was conducted in analogy to previously reported methods.47,48 Coated silicon substrates were placed at an incident angle of 83° in a Nicolet Magna-IR 860 Fourier transform infrared spectrophotometer equipped with a photoelastic modulator (PEM-90, Hinds Instruments, Hillsboro, OR), a synchronous sampling demodulator (SSD-100, GWC Technologies, Madison, WI), and a liquid-nitrogen-cooled mercury cadmium telluride detector.

Synthesis of Polymer 1BOC. Polymer 1Boc was synthesized using a variation of a previously reported procedure.49,50 Briefly, 1,4-butanediol diacrylate (495 mg, 2.50 mmol) and N-tert-


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butoxycarbonyl-1,2-diaminoethane (600 mg, 2.50 mmol) were mixed in a round-bottomed flask equipped with a magnetic stir bar. The flask was placed in an oil bath, raised to a temperature of 120 °C, and stirred overnight under an atmosphere of nitrogen to yield a viscous yellow product. The product was dissolved in dichloromethane, precipitated into diethyl ether, dried under vacuum, and used directly for the synthesis of polymer 1. Yield: 45%. 1H NMR (CDCl3, ppm) δ: 1.28 (s, 9H) 1.60-1.84 (m, 4H), 2.38-2.50 (m, 4H), 2.52-2.58 (m, 2H), 2.68-2.84 (m, 4H), 3.103.24 (m, 2H), 4.08-4.20 (m, 4H).

Synthesis of Polymer 1. A solution of polymer 1Boc (201.1 mg) was dissolved in 1 mL of cold dichloromethane and 1 mL of TFA was added drop-wise with vigorous stirring. The mixture was stirred for four hours and then concentrated under vacuum to yield a viscous product. Yield: 89%. 1H NMR (D2O, ppm) δ: 1.56-1.68 (m, 4H), 2.64-2.88 (m, 4H), 3.14-3.58 (m, 8H), 3.944.20 (m, 4H). GPC: Mn = 4300 g/mol, Mw = 6600 g/mol, Ð = 1.53.

Synthesis of Polymer 2BOC and Polymer 2. Polymer 2BOC was synthesized by mixing equimolar amounts of N,N'-butylenebisacrylamide and N-tert-butoxycarbonyl-1,2-diaminoethane in analogy to procedures reported previously for the synthesis of poly(amidoamines),51,52 following the general protocol described above for the synthesis of polymer 1Boc. Yield: 42%. Polymer 2 was synthesized by the deprotection of polymer 2Boc using TFA and methods described above for the synthesis of polymer 1.51 Yield: 83%. 1H NMR (D2O, ppm) δ: 2.28 (m, 4H, 2CH2), 2.50-2.80 (m, 8H, 4CH2), 4.40 (m, 2H, CH2). GPC: Mn = 4100 g/mol, Mw = 6200 g/mol, Ð = 1.51.


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Fabrication of Amine-Reactive Multilayers. Solutions of polymer 1, polymer 2, and azlactonefunctionalized polymers (either PVDMA or PVDMATMR) were prepared in acetonitrile (20 mM with respect to the molecular weight of the polymer repeat unit). Films were fabricated by reactive/covalent layer-by-layer assembly on aminosilane treated silicon substrates, gold-coated silicon substrates or cotton gauze using the following general protocol: (i) Substrates were submerged in a solution of PVDMA for one minute, (ii) substrates were then removed and submerged in succession into two acetonitrile rinse solutions for 30 seconds, (iii) substrates were then immersed in a solution of polyamine (polymer 1 or polymer 2) for one minute, and (iv) substrates were removed and rinsed again as described in step 2. This cycle was repeated until the desired number of PVDMA/polyamine bilayers (typically eight) had been achieved. Films fabricated in this manner are denoted using the notation (PVDMA/polyamine)x, where x is the number of ‘bilayers’, or PVDMA/polyamine layer pairs, deposited. PVDMA/PLL films were fabricated using the general protocol above, using DMSO as the solvent; an equimolar quantity of TEA was added to the PLL solution relative to the polymer repeat unit. Following final rinse steps, substrates were dried under a stream of filtered compressed air and stored in a vacuum desiccator prior to characterization or use in other experiments. For experiments aimed at characterization of film growth profiles by ellipsometry or PM-IRRAS, films were dried after every two cycles of the above procedure using filtered compressed air. Films used in stability and erosion experiments were either used immediately or were dried under a stream of filtered compressed air and stored in a vacuum desiccator until use. All film fabrication procedures were performed at ambient room temperature.


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Post-Fabrication Functionalization of Reactive Polymer Multilayers. Azlactone-containing multilayers were covalently functionalized by treatment with solutions of primary aminecontaining nucleophiles. For functionalization with the fluorophore TMRcad, film-coated substrates were either (i) immersed in DMSO solutions of TMRcad (0.5 mg/mL) for two hours, followed by rinsing with fresh DMSO and EtOH, or (ii) patterned in small circular regions by treatment with small droplets (1 μL) of a TMRcad solution (1 mg/mL in DMSO). For functionalization with propylamine, film-coated substrates were immersed in THF solutions of propylamine (50 mM) for two hours followed by rinsing with THF. For PM-IRRAS experiments, controls were functionalized with D-glucamine (50 mM in DMSO) for two hours followed by rinsing with fresh DMSO and EtOH. Substrates were dried with filtered air prior to imaging or use in other experiments.

Characterization of Film Stability and Erosion Profiles. Experiments designed to characterize the stability of multilayers in various environments were performed in the following general manner. Film-coated substrates were placed in a plastic UV-transparent cuvette and 1.0 mL of PBS (pH 7.4, 137 mM NaCl) was added to completely cover the film-coated portion of the substrate. For experiments using PLL-containing films, film-coated substrates were submerged in HBSS in the presence or absence of trypsin-EDTA (0.5%). The samples were incubated at 37 °C and removed at pre-determined intervals for analysis by ellipsometry or infrared spectroscopy. Film-coated substrates were returned immediately to buffer solutions after characterization. For experiments involving films fabricated using PVDMATMR, the concentration of PVDMATMR released into solution as a function of time was also characterized by fluorometry (excitation: 545 nm; emission: 576 nm). The presence or absence of changes in


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gross film morphology during and after these experiments were characterized visually and by fluorescence microscopy.

Results and Discussion Synthesis of Degradable and Non-Degradable Polyamines with Primary Amine Side Chains Our approach to the covalent assembly of hydrolytically degradable polymer multilayers exploits rapid reactions between the azlactone groups in PVDMA and the primary amine groups of a degradable poly(β-aminoester) building block. This overall approach yields reactive multilayers that are crosslinked by hydrolytically stable ‘amide/amide’ crosslinks, yet capable of physical erosion upon chemical hydrolysis of backbone ester groups. To explore the feasibility of this approach, we synthesized poly(β-aminoester) 1 (Figure 1), a hydrolytically degradable polyester that contains tertiary amines in its backbone and primary amine-functionalized side chains that should be capable of reacting with the azlactone groups of PVDMA. Polymer 1 was synthesized by the step growth polyaddition of N-Boc protected ethylenediamine and 1,4-butanediol diacrylate to yield polymer 1Boc (neat; 120 °C), followed by treatment with acid (TFA; at ambient temperature) to remove the N-Boc protecting groups (Figure 3; top).49,50 Successful and complete removal of N-Boc groups was confirmed by 1

H NMR spectroscopy. Characterization of polymer 1 by GPC revealed a molecular weight of

6600 g/mol relative to polystyrene standards, with dispersity Ð = 1.53. We also synthesized a non-degradable poly(amidoamine) analog of polymer 1 containing hydrolytically stable amide linkages (rather than hydrolyzable ester groups) in the backbone (polymer 2) by the polyaddition of N,N’-butylenebisacrylamide and N-Boc protected ethylenediamine, followed by treatment


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with acid (Figure 3; bottom). These polymers were used directly for the layer-by-layer fabrication of degradable and non-degradable crosslinked/reactive multilayers.

Fabrication, Characterization, and Chemical Functionalization of Reactive Polymer Multilayers Figure 4A shows a plot of optical film thickness versus the number of layer pairs (or ‘bilayers’) of PVDMA and polymer 1 (closed circles) deposited layer-by-layer on planar silicon substrates. For these and all other silicon-based experiments described below, we used substrates treated with (3-aminopropyl)triethoxysilane (APTES) to promote stronger adhesion at the multilayer/substrate interface during long-term incubation in aqueous media (by reaction with PVDMA; vide infra).21 Inspection of these results reveals film growth to occur in a manner that is approximately linear to a thickness of ~90 nm after the deposition of eight PVDMA/polymer 1 bilayers. Figure 4A also shows the film growth profile for otherwise equivalent films fabricated using PVDMA and non-degradable polymer 2 under similar conditions (open circles; final thickness ~60 nm). These growth profiles are consistent with layer-by-layer assembly and the linear film growth profiles observed during the fabrication of PVDMA/PEI multilayers reported in past studies.19 Although both polymer 1 and PVDMA are soluble in a wide range of organic solvents, the use of solutions of each polymer in acetonitrile yielded coatings that were more uniform (as determined by measurements of film thickness, visual inspection, and characterization of films using fluorescence microscopy, as described below) than those fabricated using solutions in acetone used in past studies to fabricate PVDMA/PEI films.19 Fabrication of PVDMA/polymer 1 films on gold-coated silicon substrates facilitated characterization of film growth and composition using polarization-modulation infrared reflectance-absorbance spectroscopy (PM-IRRAS). Figure 4B shows PM-IRRAS spectra for a


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PVDMA/polymer 1 film as a function of the number of bilayers deposited, and reveals absorbance peaks at ~1826 cm-1 corresponding to the carbonyl group of the azlactone ring in PVDMA. The signal intensity of this peak increased with the number of bilayers of PVDMA and polymer 1 deposited, consistent with the increase in the optical film thickness shown in Figure 4A. Further inspection reveals peaks centered at ~1735 and ~1652 cm-1 characteristic of the ester functionality of polymer 149,50 and both the C=N group in the azlactone ring and the carbonyl group of the amide-type bonds that form when PVDMA reacts with primary amines,19,26 respectively. These results, when combined, are consistent with a reactive assembly process that leads to (i) covalently crosslinked films and (ii) films that contain residual amine-reactive azlactone functionality at the surface and in the bulk of the material that are available for further reaction. Figure 5A shows a fluorescence micrograph of a silicon substrate coated with a PVDMA/polymer 1 film after treatment with a small droplet of DMSO (~1 μL) containing the primary amine-containing fluorophore tetramethylrhodamine cadaverine (TMRcad; see Materials and Methods for additional details). This image reveals uniform red fluorescence in the area of the film treated with the droplet, consistent with the covalent attachment of the fluorophore. This red fluorescence did not diminish after rigorous washing with DMSO, and control experiments performed using otherwise identical films that were pre-treated with a solution of propylamine (to react with and consume any remaining azlactone functionality) prior to treatment with TMRcad revealed negligible fluorescence (Figure 5B), suggesting that the signal observed in Figure 5A was not the result of physisorption of the fluorophore. Figure 5E shows PM-IRRAS spectra of a PVDMA/polymer 1 film before treatment (blue line) and after treatment (red line) with a model (non-fluorescent) primary amine-containing small molecule (D-glucamine; a model


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carbohydrate-based hydrophilic amine used in past studies20 to create hydrophilic PEI/PVDMA multilayers), and reveals a substantial reduction in the azlactone peak at ~1826 cm-1 upon treatment. This result is also consistent with covalent or reactive immobilization. The results of these experiments, when combined, demonstrate that these reactive PVDMA/polymer 1 films can be functionalized and patterned by treatment with primary amine-based nucleophiles, suggesting opportunities to design new degradable bio-interfaces presenting a range of different chemical and biological motifs. Finally, the images shown in Figure 5C-D demonstrate that these reactive PVDMA/polymer 1 multilayers can be fabricated on the surfaces of topologically complex objects, including the fibers that comprise commercial woven cotton gauze. Figure 5C shows two small swatches of gauze coated with either reactive (azlactone-containing) PVDMA/polymer 1 multilayers (left) or otherwise identical films pre-treated with propylamine (as described above) after immersion into a DMSO solution of TMRcad. We observed a bright and uniform pink color over large areas of the substrate coated with the reactive films (Figure 5C) as well as uniform red fluorescence on individual cotton fibers (Figure 5D), consistent with covalent chemical functionalization of the reactive film-coated fibers.19,21

Characterization of Film Stability and Degradation in Aqueous Environments Polymer 1 belongs to a class of polyamines known as poly(β-aminoester)s that can degrade by hydrolysis of their backbone ester units when dissolved in aqueous media or exposed to aqueous environments.49,50,53-55 We conducted a series of studies to characterize the stability of covalently crosslinked PVDMA/polymer 1 films in physiologically relevant media and determine whether the hydrolyzable ester bonds in polymer 1 could be exploited to promote the


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degradation of these covalently crosslinked assemblies. These experiments were performed using multilayers fabricated using PVDMA labeled with TMR (PVDMATMR; ~0.5% labeled; see Materials and Methods and past publications for details) to facilitate characterization of film erosion and the release of film components into solution using fluorometry. Films used in these studies were also functionalized, post-fabrication, by treatment with D-glucamine (see above) to consume remaining azlactone groups and impart hydrophilic character. Figure 6A shows a plot of the optical film thicknesses of glucamine-functionalized PVDMATMR/polymer 1 films (closed circles) as a function of time upon incubation in PBS (pH 7.4) at 37 °C. Inspection of these results reveals film thickness to decrease gradually and nearly completely, and in a manner that is approximately linear, over a period of 12 days. During these experiments, we also characterized the release of PVDMATMR as a function of time by monitoring the fluorescence (excitation: 545 nm; emission: 576 nm) of the PBS solutions used to incubate these materials. As shown in Figure 6B (closed circles), PVDMATMR was released gradually into solution, again in a manner that was approximately linear, over a period of 12 days, consistent with reduction in the thicknesses of the films observed in Figure 6A. These results are consistent with the gradual hydrolysis of the backbone esters in the polymer 1 components of these crosslinked materials and the resulting physical disassembly and disintegration of the films. Additional evidence in support of this view was provided by the results of experiments using otherwise identical films fabricated using PVDMA and nondegradable polymer 2. Figures 6A and B (open circles) show that the thicknesses of glucaminefunctionalized PVDMATMR/polymer 2 films remained stable upon incubation in PBS for up to 21 days (Figure 6A) and that PVDMATMR was not released into solution in substantial amounts over this time period (Figure 6B). This general conclusion is also supported by the results of PM-


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IRRAS characterization of glucamine-functionalized PVDMA/polymer 1 and PVDMA/polymer 2 films incubated in PBS (see Supporting Information; Figure S1). The results of those studies reveal a gradual reduction in the intensity of the carbonyl ester stretch at 1730 cm-1 in the PVDMA/polymer 1 multilayers, consistent with the gradual loss of ester functionality; no significant changes in the IR spectra of PVDMA/polymer 2 films were observed under similar conditions.

Design of Enzymatically Degradable Crosslinked Multilayers using PVDMA and Poly(L-Lysine) The degradable building block approach used above is likely to be general and can, in principle, be used with many other types of degradable polyamines, including polymers that can be degraded enzymatically or upon exposure to other specific chemical triggers. To explore the potential of this approach for the design of crosslinked and reactive multilayers that can be degraded upon exposure to enzymes, we fabricated films using PVDMA and poly(L-lysine) (PLL; Figure 1) as a model enzymatically degradable polymer. PLL contains backbone amide bonds that do not hydrolyze readily by chemical hydrolysis, as well as primary aminefunctionalized side chains that should react readily with the azlactone groups of PVDMA. Figure 7A shows a plot of film thickness versus the number of PVDMA and PLL layers deposited onto APTES-treated silicon substrates. These results reveal PVDMA/PLL films to grow in a linear manner to yield films ~90 nm thick after the deposition of 15.5 bilayers. These PLL-containing films also contained residual azlactone functionality as observed by PM-IRRAS (Figure 7B; C=O carbonyl, 1826 cm-1), and these reactive films could be functionalized further by treatment with primary amine-based nucleophiles, including TMRcad as described above for films fabricated using polymer 1 (see Figure S2A-B).


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Glucamine-functionalized PVDMA/PLL multilayers remained stable when incubated in PBS or other aqueous or physiological saline solutions (e.g., HBSS or water) at 37 °C, and did not physically erode or exhibit substantial decreases in film thickness for up to two weeks (see Figure 7C, closed and open squares, for time points up to 30 hours, and Figure S2C for the results of longer-term experiments). However, the thicknesses of these films decreased gradually over a period of ~1 day when incubated in saline solutions containing trypsin-EDTA (0.5% in HBSS, 37 °C; Figure 7C, closed circles; trypsin is a serine protease56 that can degrade PLL and other polypeptides or proteins in many different contexts.57,58 Our results demonstrate that this reactive approach can be used to design crosslinked and functionalizable multilayer coatings that are physically and chemically stable in aqueous environments, but that can be induced to degrade and erode rapidly and completely by the addition of enzymes that cleave the backbone amides of the PLL building blocks. While the studies reported here were designed to demonstrate proof of concept, we anticipate that this general approach can be used to design crosslinked and aminereactive coatings that degrade selectively in response to specific chemical triggers (e.g., reducing agents), the presence of specific enzymes, or upon contact with cells by using polyamine building blocks that are reductively degradable or that contain polypeptides with sequences that are cleaved selectively by specific proteases.

Summary and Conclusions We have reported the fabrication and characterization of crosslinked and chemically reactive degradable polymer multilayer coatings by the reactive layer-by-layer assembly of an azlactone-functionalized polymer (PVDMA) with polyamine building blocks that are hydrolytically or enzymatically degradable. This approach exploits the rapid reaction of


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azlactone groups with primary amine functionality, and leads to thin polymer films that are (ii) covalently crosslinked (owing to the formation of hydrolytically stable amide/amide-type bonds during fabrication), but (ii) degradable by virtue of labile bonds contained in the backbones of the polyamine building blocks. These coatings also contain residual amine-reactive azlactone functionality that can be used to immobilize additional chemical or biological functionality by treatment with amine-functionalized nucleophiles. Our results demonstrate that the properties of the degradable polyamine building block can be manipulated to influence the rates at which, and the conditions under which, these degradable coatings erode in physiological media. For example, crosslinked multilayers fabricated using a model hydrolytically degradable PBAE degraded gradually over a period of 12 days in physiologically relevant media, through a mechanism that is driven, at least in part, by the chemical hydrolysis of backbone esters in the PBAE components of the films. In contrast, multilayers fabricated using the enzymatically degradable polymer poly(L-lysine) are structurally stable for prolonged periods in physiological media, but degrade over a period of 24 hours in the presence of an enzyme that can degrade the polyamide component. Several past studies demonstrate that crosslinked and reactive multilayers fabricated using PVDMA and non-degradable polyamine building blocks such as poly(ethyleneimine) can enable the design and patterning of useful nano/bio-interfaces and other materials that are structurally stable in physiological media.17 The introduction of degradable functionality into PVDMA-based multilayers reported here creates opportunities to leverage the unique reactivity of azlactone functionality to design new functional coatings and interfaces that erode in aqueous environments that are relevant in many biomedical, biotechnological, and environmental contexts. This ‘degradable building block’ approach to reactive/covalent assembly is likely to be


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general. We anticipate that this strategy will also prove useful for the design of new aminereactive and functionalizable multilayers that degrade upon exposure to specific chemical triggers, selective enzymes, or upon contact with cells through the design and reactive incorporation of polyamine building blocks that degrade in response to other environmental cues.

Acknowledgments. This work was supported by the National Science Foundation (DMR1121288), the Office of Naval Research (N00014-16-1-2185), and made use of NSF-supported facilities (DMR-1121288, DMR-0832760, and CHE-1048642). Y. Z. acknowledges the Graduate Research Scholars (GERS) program at UW-Madison for a graduate fellowship. We thank Maren Buck, Adam Broderick, and Matthew Carter for technical advice and many helpful discussions.

Supporting Information. Supporting information including the results of additional characterization of multilayers fabricated using polymer 1 and poly(lysine) can be found in online version at doi:

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Figure 1. (A) Chemical structures of the primary amine-containing polymers [polymer 1, polymer 2, and poly(L-lysine)] and the azlactone-containing amine-reactive polymer (PVDMA) used for the covalent/reactive assembly of polymer multilayers. (B) The ringopening reaction of PVDMA with a generalized primary amine-functionalized nucleophile yields a poly(acrylamide)-based ‘amide/amide’-type bond.


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Figure 2. (A) Schematic illustration depicting a covalently-crosslinked and azlactonefunctionalized polymer multilayer (shown in grey) fabricated by reactive/covalent layer-bylayer assembly using an azlactone-functionalized polymer and a hydrolytically degradable polymer containing primary amine groups. (A-B) Subsequent chemical functionalization by treatment with a primary amine-functionalized nucleophile results in the ring-opening of residual azlactone groups and a covalently functionalized film (B; shown in green). (C) Gradual hydrolysis of the hydrolytically degradable polymer component of the covalently crosslinked film results in gradual film erosion.


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Figure 3. Reaction schemes showing the step growth approaches used to synthesize primary amine-containing poly(β-aminoester)s 1 and 2. The approach is based on the conjugate addition of N-tert-butoxycarbonyl-1,2-diaminoethane to 1,4-butanediol diacrylate (top) or N,N’butylenebisacrylamide (bottom). Conditions: (i) 120 °C, neat, overnight, (ii) DCM, TFA at room temperature for four hours, (iii) 120 °C, neat, overnight, (iv) DCM, TFA at room temperature.


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Figure 4. (A) Plot of ellipsometric thickness versus the number of PVDMA/polymer 1 (●) and PVDMA/polymer 2 (○) bilayers deposited on silicon substrates. (B) Plot showing PMIRRAS spectra for a PVDMA/polymer 1 film as a function of the number of bilayers deposited on a gold-coated silicon substrate. Error bars represent the standard deviation of measurements obtained using three identically prepared films.


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Figure 5. (A-B) Fluorescence microscopy images of (A) a native (azlactone-containing) PVDMA/polymer 1 film and (B) a propylamine-treated PVDMA/polymer 1 film, both fabricated on silicon substrates, after treatment with the amine-functionalized fluorophore TMRcad. (C) Digital photograph of two samples of film-coated commercial woven gauze after treatment with TMRcad: (left) gauze coated with a native (azlactone-containing) PVDMA/polymer 1 film; (right) gauze coated with a PVDMA/polymer 1 film and subsequently pre-treated with propylamine prior to treatment with TMRcad. (D) Highmagnification fluorescence microscopy image of the left-most sample of gauze shown in panel (C). (E) PM-IRRAS spectra of a (PVDMA/polymer 1)8 film before functionalization (blue), showing a distinctive azlactone carbonyl stretch at ~1826 cm-1, and after treatment with D-glucamine (red). Scale bars represent 500 µm (A, B, D) and 1 cm (C).


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Figure 6. (A) Plot of normalized ellipsometric thickness versus time for (PVDMATMR/polymer 1)8 films (●) and (PVDMATMR/polymer 2)8 films (○) incubated in PBS buffer at 37 °C. (B) Plot of normalized solution fluorescence in the incubation buffer versus time measured for samples used to collect the data shown in panel (A). Error bars represent the standard deviation of measurements obtained using three identically prepared films.


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Figure 7. (A) Plot of ellipsometric thickness versus the number of bilayers deposited during the fabrication of films fabricated using PVDMA and PLL on silicon substrates. (B) PMIRRAS spectra of a (PVDMA/PLL)16 film. (C) Plot of ellipsometric thickness versus time for (PVDMA/PLL)16 films incubated in (○) HBSS, (▲) PBS, and (●) trypsin-EDTA (0.5%) solution at 37 °C. Error bars represent the standard deviation of measurements obtained using three identically prepared films.


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Degradable Amine-Reactive Coatings Fabricated by the Covalent

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