Tunable and Selective Degradation of Amine-Reactive Multilayers in

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Tunable and Selective Degradation of Amine-Reactive Multilayers in Acidic Media Xuanrong Guo, Matthew C. D. Carter, Visham Appadoo, and David M. Lynn Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.9b00756 • Publication Date (Web): 24 Jul 2019 Downloaded from pubs.acs.org on July 25, 2019

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Tunable and Selective Degradation of Amine-Reactive Multilayers in Acidic Media Xuanrong Guo,1 Matthew C. D. Carter,2 Visham Appadoo,2 and David M. Lynn1,2,* 1

Department of Chemical and Biological Engineering, 1415 Engineering Drive, and Department of Chemistry, 1101 University Avenue, University of Wisconsin Ð Madison, Madison, Wisconsin 53706. 2

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

Keywords: Stimuli responsive, reactive polymers, layer-by-layer, colloids, interfaces

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Introduction The layer-by-layer (LbL) assembly technique is a convenient and versatile approach to fabricate polymer coatings, thin films, and membranes with controlled structure and functional properties.1-5 This approach was first demonstrated using oppositely charged polyelectrolytes,6,7 and later extended to the assembly of various other types of polymers using a broad range of weak or strong molecular interactions,1-5 including hydrogen bonding,8,9 hydrophobic interactions,10,11 host-guest interactions,12 and covalent bonding.13,14 Among these different approaches, the alternating deposition of mutually reactive polymer building blocks on a surface, a process known as ÔreactiveÕ or ÔcovalentÕ LbL assembly, leads to formation of covalently crosslinked multilayer films.3-5 In contrast to multilayer films assembled using noncovalent interactions, these covalently assembled films are generally more stable and less susceptible to environmental degradation or erosion. More importantly, the residual reactivity contained within these filmsÑresulting from the incomplete consumption of reactive groups during assemblyÑ opens new opportunities to tailor material properties post-fabrication, providing a useful method for the design and assembly of functional interfaces for potential chemical and biological applications.3-5 Many pairs of mutually reactive polymers have been investigated for the covalent LbL assembly of thin films.3-5 The work reported here was motivated by previous studies on the fabrication of covalently crosslinked multilayer films using an azlactone-containing polymer building block, poly(2-vinyl-4,4-dimethylazlactone) (PVDMA, Figure 1A).5,15,16 Past studies have demonstrated that azlactone groups in PVDMA can undergo ring-opening reactions with a variety of nucleophiles, such as primary amine-, alcohol-, and thiol-containing molecules.15,17,18 Reactions of PVDMA and primary amines, in particular, proceed rapidly under mild conditions,


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leading to a broad range of nano/biointerfaces with unique surface properties (e.g., superhydrophobic/superhydrophilic surfaces,17,19,21 antifouling coatings,22-24 and biomolecular arrays20,25). The stability of these PEI/PVDMA films is useful in many fundamental and practical contexts, however, the lack of degradability in response to external chemical or biological cues, such as gradients in pH or redox potential, could limit their possible applications, particularly in biomedical contexts. Efforts to address this limitation and design azlactone-containing multilayer films that can undergo stimuli-responsive degradation have been reported in a series of past studies.26-28 One approach is to incorporate backbone degradable polymers as reactive building blocks for covalent LbL assembly. As demonstrated recently, non-degradable PVDMA or PEI polymer building blocks were paired with mutually reactive degradable polymers [for example, either hydrolytically degradable polyamines (poly(!-amino ester)s) or enzymatically degradable polyamines (poly(L-lysine))26 or, in other cases, a hydrolytically degradable azlactonefunctionalized copolymer27] to form crosslinked films. Upon exposure to changes in pH or the addition of enzymes, cleavage of degradable polymer backbones contributed to the disassembly of the crosslinked coatings.26,27 Alternatively, we also reported a strategy for the covalent LbL assembly of PVDMA with primary amine-containing, bifunctional small-molecule linkers (see Figure 1A for a general structure).28 When paired with the disulfide-containing diamine cystamine, PVDMA formed reactive films crosslinked by disulfide-containing linkages that are hydrolytically stable but chemically labile under reducing conditions. The addition of reducing agents that cleave disulfide bonds could therefore trigger disintegration or erosion of the multilayer films.28 This polymer/small-molecule-linker approach has also been exploited by other groups for the covalent


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5 LbL assembly of stable, cross-linked multilayer films in other contexts.29-31 The above-mentioned strategies open the door to new designs of degradable, azlactonecontaining multilayer assemblies. In this study, we sought to further explore the PVDMA/smallmolecule-linker approach described above to design reactive, hydrolytically degradable thin films and hollow capsules that can undergo targeted pH-triggered destruction. In contrast to other (bio)chemical degradable systems that require the presence of specific stimuli, such as enzymes or reducing agents, pH-sensitive materials respond to changes in pH that are involved in a variety of chemical and biological processes, and are therefore used for a range of potential applications.32-34 To further explore this approach and demonstrate proof-of-concept, we fabricated and characterized reactive multilayer thin films and microcapsules via covalent LbL assembly of PVDMA and a model diamine linker containing acid-labile acetal groups. These crosslinked films and capsules remain relatively stable under slightly basic physiologically relevant conditions (pH = 7.4, in PBS buffer), but erode and degrade gradually in mildly acidic environments (pH = 5, in acetic buffer), driven by the acid-catalyzed hydrolysis of acetalcontaining linkages. Interestingly, our results reveal that the degradation of these materials can be tuned over a broad range (from ~4 hours to ~10 days) simply by post-fabrication modification of the parent reactive material. This approach differs from those typically used to tailor the degradation of polymer multilayers, and could thus facilitate the development of these degradable materials in a variety of fundamental and applied contexts. To demonstrate the potential to use acetal-crosslinked microcapsules for intracellular delivery of encapsulated cargo, we incubated microcapsules loaded with a model fluorescent payload, FITC-dextran, with mammalian cells. Cellular uptake and intracellular degradation of these acid-degradable capsules and delivery of the payload were confirmed using fluorescence microscopy. The concepts



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demonstrated here using PVDMA and acetal-containing linkers should be generalizable, and broaden the range of materials and stimuli that can be used to design and functionalize new types of coatings, capsules, and nano/biointerfaces.

Materials and Methods Materials. 2,2«-(Ethylenedioxy)bis(ethylamine) (linker 2), 2,2«-azobisisobutyronitrile (AIBN), decylamine (95%), propylamine (>95%), ethanol amine (>98%), FITC-dextran (average MW ~2,000,000), sodium hydroxide, sodium carbonate (>99.5%), calcium chloride (anhydrous, >97%), acetic acid (HAc), acetic anhydride (>99%), and tetrahydrofuran (THF) were obtained from Sigma-Aldrich (Milwaukee, WI). p-Toluenesulfonic acid (>98%) was recrystallized from toluene and was obtained from Sigma (Milwaukee, WI). 2-Methoxypropene (>95%) and Dglucamine were purchased from Scientific TCI (Portland, OR). Triethylamine was purchased from

Fisher

Scientific

(Pittsburgh,

PA).

N-(2-Hydroxyethyl)phthalimide

(98%),

3-

dimethylaminopropylamine (99%), and ethylenediaminetetraacetic acid trisodium salt hydrate (EDTANa3, >98%) were purchased from Acros Organics (Morris Plains, NJ). Ethanol was obtained from Decon labs (King of Prussia, PA). 2-Vinyl-4,4-dimethylazlactone (VDMA) was a kind gift from Dr. Steven M. Heilmann (3M Corporation, Minneapolis, MN). Poly(2-vinyl-4,4dimethylazlactone) (PVDMA, MW ~44,667; ! = 5.7, against narrow-dispersity polystyrene standards) was synthesized by the free-radical polymerization of VDMA, as described previously.20,35 Tetramethylrhodamine (TMR) cadaverine was obtained from Setareh Biotech (Eugene, OR). PVDMA labeled with TMR (1 mol%; referred to from hereon as PVDMATMR) was synthesized as described previously.35 Test grade n-type silicon wafers were purchased from Silicon Materials, Inc. (Glenshaw, PA). Deionization of a distilled water source was performed


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using a Milli-Q system (Millipore, Bedford, MA) yielding water with a resistivity of 18.2 M!. FluoroDishTM confocal dishes were obtained from World Precision Instruments (Sarasota, FL). Minimum Essential Medium (MEM), DulbeccoÕs phosphate-buffered saline (DPBS), fetal bovine serum (FBS), trypsin EDTA (0.25%, phenol red), and Trypan Blue were purchased from Thermo-Fisher Scientific. All materials were used as received without further purification unless otherwise noted.

General Considerations. Planar silicon substrates (~10 mm ! 50 mm) were cleaned with acetone, ethanol, methanol, and deionized water and dried under a stream of filtered, compressed air prior to the fabrication of multilayered films. Silicon substrates used for reflective IR spectroscopy experiments were prepared by depositing thin layers of Ti (10 nm) and Au (200 nm) sequentially onto silicon wafers using an electron-beam evaporator (Tek-Vac Industries, Brentwood, NY). The optical thicknesses of films were determined using a Gaertner LSE ellipsometer (632.8 nm, incident angle = 70¡). Data were processed using the Gaertner Ellipsometer Measurement Program. Average refractive indices of film-coated substrates were calculated using the Gaertner Ellipsometer Measurement Program from at least five different standardized locations for three replicate films. Pre-determined refractive indices were assumed to remain constant during film fabrication and degradation, and were used for determination of optical thicknesses. Characterization of multilayered films by polarization-modulation infrared reflectance-absorbance spectroscopy (PM-IRRAS) was conducted in analogy to methods reported previously.16 Contact angle measurements were performed using a Dataphysics OCA 15 Plus instrument with an automatic liquid dispenser at ambient temperature. Advancing contact angles were measured using a 5 "L droplet of deionized water (18 M!) in at least three different



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locations on each film. Solution fluorescence intensity was measured using an Infinite 200Pro plate reader. Bright-field, phase contrast and fluorescence microscopy images were acquired using an Olympus IX70 inverted microscope (Center Valley, PA) equipped with a Lumen Dynamics XCite 120PC-Q fluorescence source and a Q Imaging EXi Aqua camera. An RFP filter set was used for TMR and a GFP filter set was used for fluorescein (FITC-dextran). Images were analyzed and false-colored using the Metavue version 4.6 software package (Universal Imaging Corporation).

Synthesis of Linker 1. Linker 1 was synthesized according to a modified literature36-39 procedure as follows: N-(2-hydroxyethyl)phthalimide (11.01 g, 57.6 mmol) and ptoluenesulfonic acid (0.176 mg, 0.923 mmol) were added to an oven-dried 250 mL three-neck round-bottomed flask outfitted with a vacuum adapter and placed under nitrogen. Anhydrous DMF (100 mL) was added to the flask via cannula transfer to give a light yellow solution which was stirred on an ice/water bath. 2-Methoxypropene (6.73 g, 93.3 mmol) was added dropwise via a glass syringe through a rubber septum, and the solution was allowed to stir for 1 hour and 15 minutes. The solution was warmed to room temperature and then heated to 45 ¡C and opened to high vacuum. Over a six hour period, a clear liquid was distilled from the reaction mixture and a significant amount of white solid precipitate formed. The reaction mixture was cooled to room temperature, opened to atmosphere, and triethylamine (20 mL) and acetic anhydride (3.1 mL) were added to the flask. After stirring overnight, DMF was removed under reduced pressure to give a light brown slurry. The reaction mixture was filtered through a coarse fritted funnel and washed with cold ethyl acetate (~3 x 5 mL) to yield a white powder. Recrystallization from ~50 mL of ethyl acetate gave the desired intermediate as a brilliant white solid with 44.3% yield. This


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intermediate product (5.34 g) was weighed into a 100 mL round-bottomed flask and deprotection was achieved by refluxing in 6 M NaOH (30 mL) overnight. (The intermediate product was insoluble at room temperature, but solubilized at reflux). The crude reaction mixture was cooled to room temperature, transferred to a separatory funnel, and extracted with 1:1 (v/v) iPrOH:CHCl3 (3 x 100 mL). The organic extractions were collected, dried over magnesium sulfate, and then gravity filtered. Solvent was removed under reduced pressure to give the desired product as a light yellow oil with 76.4% yield. 1H NMR (500.022 MHz, CDCl3, " ppm): 1.36 (s, 6H, C-(CH3)2, 1.49 (s, broad, 4H, -NH2), 2.84 (t, 4H, -CH2-NH2), 3.36 (t, 4H, -O-CH2).

Fabrication of PVDMA/Linker Multilayer Films. Solutions of linker 1, linker 2, and PVDMA were prepared in dichloromethane (20 mM; for PVDMA, the concentration was calculated with respect to the molecular weight of the polymer repeat unit). Multilayered films were deposited on silicon substrates or gold-coated silicon substrates manually according to the following general protocol: (1) Substrates were submerged in a solution of PVDMA for 20 s, (2) substrates were removed and immersed in succession into two dichloromethane rinse solutions for 20 s, (3) substrates were submerged in a solution of linker 1 or 2 for 20 s, and (4) substrates were rinsed in the manner described above. This cycle was repeated until the desired number of PVDMA/linker layers (typically 25, terminated with PVDMA as the outermost layer) was reached. Films were either characterized and used in subsequent experiments immediately, or were dried under a stream of filtered, compressed air and stored in a vacuum desiccator until use. For experiments designed to post-functionalize multilayer films, film-coated substrates were immersed in solutions of small molecule amines (40 mM; propylamine, decylamine, ethanolamine, DMAPA in THF, glucamine in DMSO) for 2 hr, followed by rinsing with fresh solvents. All films were



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fabricated and functionalized at ambient room temperature.

Fabrication of Multilayer Microcapsules. Multilayer-coated particles and hollow capsules were prepared in analogy to previous reports.28 Polydisperse CaCO3 microparticles containing FITC-dextran were fabricated using a co-precipitation method as described previously.40 By varying the stirring rate during precipitation, FITC-dextran-loaded CaCO3 microparticles with different average diameters (typically ~8 "m) were obtained. CaCO3 microparticles (8 mg) were weighed, placed into plastic microcentrifuge tubes, and rinsed using 1 mL of dichloromethane prior to the fabrication of multilayers. Films were fabricated on CaCO3 microparticles via layerby-layer assembly of PVDMA (or PVDMATMR; 20 mM with respect to the molecular weight of the polymer repeat unit in dichloromethane) and the diamine linker 1 or 2 (20 mM in dichloromethane). Briefly, the first layer of PVDMA was deposited onto the CaCO3 microparticles by adding 1 mL of the PVDMA solution to the particle suspension and manually shaking the particles for 1 minute to allow sufficient time for the polymer to adsorb to the particle surface. The particles were then centrifuged for 1 minute at 1500 rpm. The supernatant was then carefully removed by pipette and the particles were rinsed two times by resuspending them in 1 mL of dichloromethane and vortexing. After each rinse in dichloromethane, the particles were centrifuged for 1 minute at 1500 rpm and the supernatant was removed. The second layer of the multilayered film was then deposited by adding 1 mL of a diamine linker solution to the particle suspension and shaking manually for 1 minute. The particles were then rinsed in the manner described above. Subsequent layers were fabricated by repeating this process (by alternately depositing PVDMA or linker solutions and allowing each layer to react for 1 minute) until the desired number of polymer PVDMA/linker layers (typically five,


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terminated with PVDMA as the outermost layer) were deposited onto the particle surface. After every two bilayers, particles were placed into a new micro-centrifuge tube to minimize aggregation. After film fabrication, the coated particles were placed into a 1 mL solution of a primary amine (decylamine, ethanolamine, or DMAPA; 40 mM in THF) and shaken on an automated shaker plate for 1 hour to exhaustively react with all remaining azlactone functional groups and install different desired functionality. To dissolve the CaCO3 templates, coated particles were rinsed with THF and deionized water, and then treated with 0.2 M EDTA (pH = 9.5) for 30 minutes. The resulting hollow polymer capsules were washed three times with deionized water and characterized using optical microscopy.

Characterization of Film Stability and Degradation of Multilayer Assemblies. Experiments to investigate the stability and degradation behaviors of multilayer films in various environments were performed in the following general manner. Degradation was characterized under acidic (200 mM NaAc/HAc buffer, pH = 5) and slightly basic (10 mM PBS buffer, pH = 7.4) conditions. For experiments using planar substrates, film-coated silicon substrates were placed in a glass vial and 2 mL of the corresponding buffer solution was added to cover the substrate. The samples were removed at predetermined time intervals for measurement of optical thicknesses using ellipsometry, and returned immediately to the buffer solution. For experiments involving films fabricated using PVDMATMR, 1 mL of the buffer solution covering the substrate was removed from the vial and transferred into a 48 well plate at predetermined time intervals, and the fluorescence of PVDMATMR released into solution as a function of time was characterized using a plate reader (excitation, 543 nm; emission, 571 nm). The solution in the vial was replenished with 1 mL of the corresponding fresh buffer solution after each measurement. For



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experiments designed to investigate the degradation of multilayer microcapsules, capsules were centrifuged for 3 minutes at 4500 rpm and the supernatant was removed, and then 1 mL of the corresponding buffer solution was added. At specified time intervals, aliquots of the dispersion were characterized using fluorescence microscopy.

Characterization of Microcapsules Internalized by Cells. HeLa cells were seeded in confocal dishes (# = 35 mm) at an initial density of 150,000 cells/mL in 2 mL of growth medium (MEM supplemented with 10% v/v fetal bovine serum, 100 units/mL penicillin and 100 µg/mL streptomycin) and allowed to grow to 70-80 % confluency prior to the start of experiments. A dispersion of capsules (75 µL, ~10,000 capsules/µL) was added to the cells. The cells were imaged at time points of 1 hr, 6 hr, 18 hr, and 72 hr using a fluorescence microscope. For the three later time points, Trypan Blue was added to the cells according to the following general protocol: cells were first rinsed with 1 mL of DPBS; 0.1 mL of Trypan Blue was subsequently added to cells containing 0.9 mL of fresh growth medium. Cells were then imaged by phase contrast and fluorescence microscopy.

Results and Discussion Fabrication and Characterization of PVDMA/Linker Reactive Multilayer Films To explore designs of multilayers containing PVDMA and that could degrade selectively in acidic environments, we selected as small-molecule building blocks a small diaminefunctionalized linker containing acid-labile acetal functionality (linker 1, Figure 1B) and a nondegradable diamine analogue (linker 2, Figure 1B). We hypothesized that acetal-containing linkages in films fabricated using PVDMA and linker 1 would be stable in near-neutral or basic


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media, but would cleave upon exposure to mildly acidic conditions (Figure 1C), leading to film degradation and disassembly. In our initial studies, we fabricated planar thin films by the iterative deposition of PVDMA and degradable linker 1 or non-degradable linker 2 on reflective silicon substrates. Figure 2A shows a plot of the optical thicknesses of films versus the number of PVDMA/linker deposition cycles. Inspection of these results reveals films fabricated using both linkers to grow in an approximately linear manner. The optical thicknesses of PVDMA/1 and PVDMA/2 films were ~50 nm and ~30 nm, respectively, after 24 deposition cycles, followed by the deposition of a final layer of PVDMA (strictly speaking, these films consisted of 24 and a half deposition cycles, see Materials and Methods; however, for simplicity, these films are referred to from hereon as having 25 polymer layers using the notation described below). Films and multilayer assemblies fabricated in this manner are denoted as (PVDMA/1)X and (PVDMA/2)X, respectively, where x denotes the number of deposition cycles. We further characterized the chemical composition and reactivity of (PVDMA/1)25 films fabricated on gold-coated silicon substrates using polarization-modulation infrared reflectanceabsorbance spectroscopy (PM-IRRAS). Figure 2B shows representative spectra of (PVDMA/1)25 films before and after treatment with, propylamine, a model primary amine-containing small molecule. The PM-IRRAS spectrum of untreated (PVDMA/1)25 films reveals a characteristic absorbance peak at ~1826 cm-1 corresponding to the carbonyl stretch of residual azlactone groups in PVDMA.16 Further inspection reveals an absorbance band with a peak centered at ~1652 cm-1, characteristic of both the C=N functionality in the azlactone ring and the C=O of amide bonds that form when azlactone groups react with primary amines.16 After treatment with propylamine, the peak at ~1826 cm-1 was fully consumed, suggesting the complete reaction of the residual azlactone groups in these films.




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assess the extent to which the residual azlactone functionality in (PVDMA/1)25 films could be further exploited for the post-modification of interfacial properties (e.g. wetting behaviors), we treated these reactive films with a series of primary amine-containing molecules and measured their advancing water contact angles (WCAs). Inspection of Figure 3A reveals the WCA of untreated, azlactone-containing films to be 78.4¡ ± 1.0¡. The treatment of (PVDMA/1)25 films with n-decylamine led to an increase in WCA to 91.5¡ ± 2.1¡ (Figure 3B). In contrast, films treated with hydrophilic dimethylaminopropylamine and ethanolamine exhibited lower WCAs (57.6¡ ± 0.3¡ and 55.6¡ ± 1.8¡, respectively; Figure 3C-D). The results of these experiments demonstrate successful installation of hydrophilic or hydrophobic functional groups on (PVDMA/1)25 films by treatment with small-molecule amines, demonstrating that the residual azlactone groups in these PVDMA/linker 1 films can serve as reactive handles for postfabrication introduction of additional functionality. We anticipate that the strategy demonstrated here could also be extended to the post-fabrication functionalization of films with other primary amine-containing reagents, providing opportunities for the design of more specifically-tailored and ligand-targeted degradable thin films and membranes.15,17




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film thickness was observed for (PVDMA/1)25-EA films incubated at pH 7.4 (open squares, in 10 mM PBS) for up to 55 hr. These results, when combined, are consistent with the faster hydrolysis of acetal-containing linkages at acidic pH than at slightly basic pH, and the resulting erosion and disassembly of multilayer films. The results of additional experiments performed using otherwise identical films fabricated via assembly of PVDMA and non-degradable linker 2 provide additional support for this view. Figure 4A shows that the thicknesses of ethanolaminefunctionalized (PVDMA/2)25 films (referred to as (PVDMA/2)25-EA; closed circles) remained stable upon incubation in acidic buffer for up to 55 hr. We note that the film thicknesses of both types of degradable films (hydrophilic (PVDMA/1)25-EA and hydrophobic (PVDMA/1)25-DA) decreased in a manner that was approximately linear during the first 7 hr of incubation in these experiments, but remained constant thereafter (these residual films also did not demonstrate a further decrease in thickness when exposed to strong acid, e.g., HCl, or organic solvents, e.g., acetone, dichloromethane, and THF, etc; data not shown). The reasons for this behavior are not clear, but it could result, at least in part, from the rearrangement or adsorption of insoluble polymer fragments within the film or on the silicon substrates during these experiments.44 We return to a discussion of the degradation behaviors of unsupported multilayer membranes (e.g. hollow capsules) in the discussion below.




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functionalized

them

with

ethanolamine

as

described

above

(referred

to

here

as

(PVDMATMR/1)40-EA and (PVDMATMR/2)40-EA, respectively). Films were then incubated in buffer solutions and the release of PVDMATMR as a function of time was then characterized by measuring the fluorescence intensity in solution (excitation, 543 nm; emission, 571 nm). As shown in Figure 4B, PVDMATMR was not released from (PVDMATMR/1)40-EA (closed squares) in substantial amounts over a period of 48 hr when incubated in PBS (10 mM, pH = 7.4). However, after transferring these films into acidic buffer (200 mM HAc/NaAc, pH = 5) at 48 hr, PVDMATMR was released into solution, over a period of ~ 7 hr, consistent with the results of thickness reduction profiles observed in Figure 4A. In contrast, the amount of PVDMATMR released from non-degradable (PVDMATMR/2)40-EA films (closed circles) was negligible under identical experimental conditions. The results of these experiments demonstrate stimuliresponsive nature of these acetal-containing films upon exposure to different environmental pHs, with chemical degradation of the acetal groups leading to the release of the incorporated polymer.

Fabrication of Reactive, Acid-Degradable Microcapsules Degradable polymer microcapsules have been widely studied as promising candidates for the encapsulation and delivery of chemical and biological functional payloads.4,45-51 Building on the results above demonstrating the degradability and stimuli-responsiveness of (PVDMA/1) films fabricated on solid substrates, we explored the feasibility of this assembly approach to fabricate reactive, acetal-containing microcapsules with tunable degradation behaviors, using a strategy involving the alternating deposition of mutually reactive polymers on sacrificial colloidal templates, followed by template removal.28,40,45-50 Using this approach, spherical




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Inspection of the fluorescence image in Figure 5B reveals two channels of fluorescence signals: green fluorescence corresponding to the FITC-dextran loaded in the core of the CaCO3 microparticles, and surrounding red fluorescence arising from PVDMATMR used to fabricate the coatings, localized within the capsule walls. The CaCO3 cores were subsequently dissolved using 0.2 M EDTA (pH = 9.5), yielding hollow, FITC-dextran-loaded microcapsules (Figure 5C-D). The majority of the capsules appeared spherical, showing strong localized green fluorescence within well-defined red fluorescent shells. The observation of green fluorescence inside the capsules is consistent with the confinement of macromolecular FITC-dextran inside otherwise hollow multilayer polymer shells. These results, when combined, suggest that these capsules remained intact after EDTA treatment; no pores or defects of a size that allowed substantial leakage of encapsulated FTIC-dextran were observed in these covalently crosslinked (PVDMATMR/1)5 microcapsules. We further investigated the stability of these reactive, acetal-containing microcapsules when suspended in different aqueous solutions. To explore the influence of capsule functionality on degradation behavior, we treated (PVDMATMR/1)5 films with hydrophilic ethanolamine or hydrophobic decylamine (referred to as (PVDMATMR/1)5-EA or (PVDMATMR/1)5-DA, respectively) prior to EDTA treatment. Panels A, B, and G, H, of Figure 6 show representative fluorescence microscopy images acquired immediately after suspending (PVDMATMR/1)5-EA and (PVDMATMR/1)5-DA microcapsules in acidic buffer (200 mM HAc/NaAc, pH = 5). Inspection of these two groups of images reveals a large population of intact capsules in solution, as evidenced by the spherical capsule wall and green fluorescence of FITC-dextran within these capsules. Interestingly, we observed capsules with different post-fabrication functionality to degrade at substantially different rates under these conditions. For example, hydrophilic



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(PVDMATMR/1)5-EA capsules degraded completely after only 4 hr of incubation at pH 5 (Figure 6E-F), as determined by the complete disappearance of spherical capsules, the absence of observable capsule fragments, and an increase in background fluorescence intensity resulting from the release of FITC-dextran. In contrast, hydrophobic (PVDMATMR/1)5-DA capsules degraded much more slowly (Figure 6 I-L); complete degradation of (PVDMATMR/1)5-DA capsules was not observed until 10 d of incubation at pH 5 (Figure 6K-L).


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The reasons for these large differences in degradation rates are not completely understood, but likely arise from differences in the hydrophobicity of these capsules, as suggested by the results described above in Figure 3, that are imparted by post-fabrication functionalization. The ability to affect the degradation profiles of these materials over such a broad range of time simply by post-fabrication modification of the reactive parent material is an unexpected and potentially useful feature of this system that differs from strategies (such as varying crosslink density or changing the structure of the polymer or linker building blocks) used in past studies on degradable multilayers. These results thus suggest the basis of facile and modular template-based approaches to the further development of these degradable materials. Additional experiments revealed hydrophilic (PVDMATMR/1)5-EA capsules to retain their spherical shapes and FITC-dextran payloads for up to 96 hr in PBS (10 mM, pH = 7.4, Figure S1), consistent with the greater stability of acetal-containing linkages at neutral pH, with previous reports in the literature,36-39,43 and with results described above for experiments using planar (PVDMA/1) films on silicon substrates. We note that the degradation profiles for polymeric microcapsules observed here have demonstrated important features that are not observed in the PVDMA/1 planar films fabricated on silicon discussed above. For example, in acidic buffer, the degradation of multilayer assemblies appears to proceed completely for microcapsules, while thin films on silicon substrates do not appear to erode completely under conditions described above. The influence of post-functionalization on degradation rate, as discussed above, is also more significant for polymeric capsules than for substrate-mounted films. To provide further insights into the mechanism of capsule degradation, we also fabricated ethanolamine-functionalized microcapsules using PVDMATMR and the hydrolytically stable,


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non-degradable linker 2 (referred to as (PVDMATMR/2)5-EA). Fluorescence images in Figure 6M-R reveal the capsules containing non-degradable linkers to remain stable in acidic buffer (200 mM HAc/NaAc, pH = 5) for up to 11 d, in sharp contrast to acetal-containing (PVDMATMR/1)5-EA capsules, which degraded completely in 4 hr under identical conditions. These results, when combined, further support the view that degradation of acetal-containing capsules in acidic environments is caused by the gradual cleavage of their acid-labile linkages.

Cellular Uptake and Intracellular Degradation of Microcapsules Acid-labile, reactive microcapsules with tunable degradation behaviors are useful for the design and targeting of drug delivery systems that can respond to changes in environmental or physiological pH.32,38,52-55 Motivated by the results described above and the results of past studies demonstrating the utility of other degradable micro/nano capsules for intracellular drug delivery,52-55 we conducted a series of experiments to characterize the internalization of our acetal-containing microcapsules by mammalian cells. For these experiments, we fabricated FITC-dextran-loaded, acid-degradable (PVDMATMR/1)5-EA and (PVDMATMR/1)5-DA capsules and non-degradable (PVDMATMR/2)5-EA analogues with average diameters of ~3 "m, and selected HeLa cells as a model because this cell line is known to be able to internalize micrometer-scale particles.56 In our initial experiments, we added microcapsules to cell culture media and incubated them with HeLa cells for a period of 18 hr (Figure S3A-F). Capsules that were loosely bound to cells were subsequently removed by manual rinsing (Figure S3G-L). The locations of capsules (e.g., whether they were inside or outside of the cells) were then determined using a Trypan blue fluorescence quenching assay.57,58 As shown in Figure S2, Trypan blue can quench the red fluorescence signal of TMR-



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labeled capsule walls with which it comes into immediate contact, but does not completely quench the green fluorescence of FITC-dextran that is encapsulated in the polymer shells.57,58 Since Trypan blue cannot penetrate intact lipid membranes of cells, addition of Trypan blue into cell culture media can help distinguish internalized capsules, which exhibit fluorescence in both red and green channels, from those that are not internalized, which exhibit only green fluorescence. Inspection of the images in Figure S3M-R reveals qualitatively higher levels of attachment (green channel only) and internalization (red and green channels) for capsules functionalized with decylamine ((PVDMATMR/1)5-DA) than for those functionalized with ethanolamine ((PVDMATMR/1)5-EA and (PVDMATMR/2)5-EA), presumably due to differences in the hydrophobicity of these capsules.


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(Figure 7A-B). These patterns of fluorescence are consistent with observations reported in past studies of pH-sensitive capsules designed to degrade in endosomal/lysosomal environments,52-55 and suggest the intracellular degradation of (PVDMATMR/1)5-EA capsules. In contrast, internalized (PVDMATMR/1)5-DA and (PVDMATMR/2)5-EA capsules appeared to be largely intact (marked by orange arrowheads in Figure 7D-E, G-H), though their shapes were often crumpled in comparison with capsules attached to cell surfaces (marked by yellow arrowheads in Figure 7D-E). Inspection of Figure 7E and 7H reveals capsules exhibiting strong fluorescence from loaded FITC-dextran and TMR-labeled capsule walls, but no evidence of substantial degradation after 18 hr. However, after longer incubation periods of 72 hr, (PVDMATMR/1)5-DA capsules did exhibit some hallmarks of capsule degradation (Figure 7F), suggesting that these capsules do degrade, albeit more slowly than (PVDMATMR/1)5-EA capsules. (PVDMATMR/2)5EA capsules did not demonstrate evidence of degradation for up to 72 hr of incubation (Figure 7I). These results, when combined, are generally consistent with the behaviors of capsules containing degradable or non-degradable linkages suspended in acidic buffer as reported above. Overall, these results demonstrate the intracellular degradability of acetal-containing microcapsules and provide a platform that could, with further development, be used to design capsules decorated with chemical and biological motifs or filled with active agents to promote more targeted or customized delivery of cargo to cells.47-55

Conclusions We have reported the design, fabrication, and characterization of reactive, aciddegradable multilayer films by the covalent layer-by-layer assembly of an azlactone-containing polymer with an acetal-containing small-molecule diamine linker. Thin supported films and hollow microcapsules crosslinked by acetal-containing linkages remained relatively stable under


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slightly basic physiologically relevant conditions (pH = 7.4), but eroded or degraded gradually in mildly acidic environments (pH = 5), driven by the acid-catalyzed hydrolysis of acetal groups. The residual azlactone reactivity in these films and capsules could be functionalized postfabrication to tune physical and chemical properties of the assemblies, including surface wetting properties and rates of degradation/erosion. Interestingly, the combination of chemical reactivity and hydrolytic instability introduced here permits the degradation of these materials to be tuned over a broad range, from four hours to 10 days, simply by post-fabrication modification of the reactive parent material. This approach obviates the need to manipulate crosslinking density or the chemical structures of the polymer or linker components to achieve new degradation profiles, and introduces a basis for modular design that could facilitate or expedite the development of these materials for a variety of fundamental and applied contexts. Our results demonstrate that FITC-dextran-loaded, acetal-containing microcapsules can be internalized by mammalian cells in culture, and that the cellular uptake and intracellular degradation of these capsules are also influenced strongly by the types of functional groups that are installed post-fabrication. Overall, our results broaden the range of materials and stimuli that can be used to design and functionalize these reactive materials and introduce new post-functionalization approaches to affecting rates of hydrolytic degradation in acidic media. This work also expands the potential utility of this class of reactive materials to a variety of potential bio-oriented applications, including the targeted delivery of drugs or active agents, which require transient environmental stability or triggered stimuli-responsive hydrolytic degradability.



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Supporting

Information.

Additional

fluorescence

and

phase

contrast

microscopy

characterization of multilayer capsules in aqueous solutions and in contact with mammalian cells (PDF). This material is available free of charge via the Internet at: DOI:

Acknowledgments. Financial support for this work was provided by the National Science Foundation through a grant provided to the UW-Madison Materials Research Science and Engineering Center (MRSEC; DMR-1121288), the Office of Naval Research (N00014-07-10255), and the University of Wisconsin Vice ChancellorsÕ Office for Research and Graduate Education (VCRGE). The authors acknowledge the use of instrumentation supported by the NSF through the UW MRSEC and the UW Nanoscale Research and Engineering Center (NSEC; DMR-1121288). X. G. was supported in part by a 3M Graduate Fellowship. V.A. acknowledges the American Heart Association for a graduate fellowship and the UW-Madison Biotechnology Center for a Morgridge Biotechnology Fellowship. M.C.D.C. acknowledges the Natural Sciences Engineering Research Council of Canada for a graduate fellowship.

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Tunable and Selective Degradation of Amine-Reactive Multilayers in

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