Accessibility of Responsive End-Caps in Films Composed of Stimuli

Sep 12, 2013 - A microscale-pump made from films of depolymerizable .... depolymerizable poly(phthalaldehyde) (PPHA).12 The polymers are end-capped ...
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Accessibility of Responsive End-Caps in Films Composed of StimuliResponsive, Depolymerizable Poly(phthalaldehydes) Anthony M. DiLauro,† Hua Zhang,† Matthew S. Baker, Flory Wong, Ayusman Sen,* and Scott T. Phillips* Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, United States S Supporting Information *

ABSTRACT: Polymers that are capable of depolymerizing completely from head-to-tail upon cleavage of an end-cap from the terminus of the polymer have emerged recently as a new strategy for creating stimuli-responsive solid-state materials with amplified responses. In theory, solid-state materials made from these polymers will respond most efficiently to a stimulus in solution when the polymer end-caps are displayed into solution at the solid−liquid interface, rather than being buried in the solidstate material. This article defines two strategies for increasing the likelihood that end-caps are displayed at this interface. A microscale-pump made from films of depolymerizable poly(phthalaldehyde) serves as a test system for evaluating the location of end-caps in the films. By measuring the flow rate initiated by depolymerization of the polymers within the films, we determined that both the polymer length and hydrophilicity of the end-caps affect the density of end-caps at the solid−liquid interface.

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INTRODUCTION

amplified responses since a single detection event, in principle, leads to conversion of an entire polymer into monomers (or other small molecule products). When these types of polymers are used as solid-state materials, the end-cap on the polymer frequently must be displayed in solution to be accessible to a stimulus and to enable the solid-state material to undergo a selective and amplified response (Figure 1b).5 Strategies for defining, improving, and tuning the accessibility of the end-cap have not been identified in this emerging field, despite the importance of end-cap accessibility for initiating rapid responses. Consequently, in this article we demonstrate two design principles that begin to address this question of end-cap accessibility at the solid−liquid interface for these types of stimuli-responsive materials. Our specific test system is fluoride-responsive poly(phthalaldehyde)-based micrometer-scale pumps that operate by creating a gradient of 1,2-benzenedicarboxaldehyde monomers when a micrometer-scale film of the polymer is exposed to fluoride in water (Figure 2).8,12 This gradient of monomers induces movement of water from above the film toward the film and then away from the film through a presumed osmophoretic-driven flow.12−26 We used the pumping speed as a functional measure of the extent of depolymerization in the film and, ultimately, as a way of quantifying the accessibility of the end-caps. Design of the Test System. Our goal in this article is to provide initial guiding principles for increasing the accessibility of the reactive end-caps in solid-state materials such that the

An emerging strategy in designing stimuli-responsive materials involves the use of polymers that are capable of depolymerizing continuously and completely in a head-to-tail fashion when a stimulus reacts with and cleaves an end-cap from the terminus of the polymer (Figure 1a).1−3 This strategy offers the opportunity for selective responses via activity-based detection events when the stimulus cleaves the end-cap,4 as well as

Figure 1. (a) Illustration of the concept of head-to-tail depolymerization of end-capped polymers when the end-cap is cleaved from the polymer in response to a specific chemical or physical signal. The specific polymer in this illustration is end-capped poly(phthalaldehyde) (PPHA).8−12 (b) Nearly all end-caps at the solid− liquid interface must be accessible to the solution for optimal responses in solid-state materials that interact with stimuli in solution. © 2013 American Chemical Society

Received: July 11, 2013 Revised: August 29, 2013 Published: September 12, 2013 7257

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Figure 2. Fluoride-responsive micrometer-scale pumps created from depolymerizable poly(phthalaldehyde) (PPHA).12 The polymers are end-capped with tert-butyldimethylsilyl (TBS) groups.27 (a) The TBSend-capped PPHA film (dark gray) is drop-cast on a glass slide. Typical dimensions for the films are 0.5 mm × 0.5 mm × 25 μm. (b) The glass slide is placed in an enclosed chamber, and the stimulus is added in an aqueous solution (75 mM phosphate buffer, pH 7.4, 1 wt % BSA, 23 °C), causing the polymers on the surface of the film to depolymerize and create a gradient of monomers (i.e., 1,2benzenedicarboxaldehyde). (c) The gradient of monomers causes the fluid above the film to move toward the film (through a proposed osmophoretic-driven flow12−26) and then away from the film (due to fluid continuity), as indicated by the curved arrows. Polystyrene tracer particles (green) (6 μm diameter) are used to reveal the movement of the fluid. (d) The speed with which the fluid moves (i.e., is pumped) is calculated by tracking the distance that ∼30 tracer particles travel during ten 2.5 s time intervals at distances ranging from 100 to 200 μm from the edge of the PPHA film.

Figure 3. Reagents used to create enzyme-responsive micrometer-scale pumps12 as a platform for testing the accessibility of the fluorideresponsive end-caps in films of PPHA. (a) A small molecule reagent (1) that reacts with the model enzyme β-D-glucuronidase to release two molecules of fluoride per enzymatic reaction.12 (b) The released fluoride then reacts with accessible end-caps in films of 2 to cause depolymerization and to initiate the pumping response.

end-cap responds to specific stimuli at the solid−liquid interface and then translates that reaction into a response in the solid-state material. We used PPHA because it is the only polymer (within the small class of polymers that depolymerize from head-to-tail1−3) that depolymerizes quickly (i.e., seconds rather than days) in the solid state. We used pumps made from PPHA because they provide a functional output that we can measure and quantify to correlate end-cap accessibility with the performance of a stimuli-responsive material.12 Moreover, while the poly(phthalaldehyde) (PPHA) films in Figure 2 are responsive to fluoride, we were more interested in evaluating their response to enzymes, since a downstream goal of these pumps is autonomous pumping in response to trace levels of specific enzymes. Toward this goal, we employed β-Dglucuronidase (a marker for E. coli28) as a model enzyme and synthesized a small molecule reagent (reagent 1) that releases 2 equiv of fluoride in response to the enzyme stimulus (Figure 3a).12 The enzyme cleaves the glycosidic bond between the glucuronic acid functionality and the phenyl linker in 1, revealing a phenol that facilitates quinone methide formation. The released aniline eliminates 2 equiv of fluoride via azaquinone methide chemistry, and the fluoride then cleaves the silyl ether end-caps on PPHA (2, Figure 3b) to initiate depolymerization.8,12,29 When 2 is cast as a micrometer-scale film (Figure 2), the presence of β-D-glucuronidase causes the pump to turn on.12 In the absence of the enzyme, no fluoride is produced to initiate depolymerization, and the tracer particles move only by Brownian motion.

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through an Agilent oxygen trap BOT-4. Air- and moisture-sensitive liquids were transferred by syringe or stainless steel cannula. Organic solutions were concentrated by rotary evaporation (25−40 mmHg) at ambient temperature, unless otherwise noted. tert-Butyldimethysilanol, acetic anhydride, methanol, ethyl acetate, hexanes, β-D-glucuronidase, sodium fluoride, sodium thiocyanate, sodium chloride, sodium iodide, sodium bromide, sodium phosphate, sodium nitrate, sodium sulfate, dichloromethane, 2-(methyoxypoly(ethylenoxy) 6 − 9 propyl)dimethylchlorosilane, triacontyldimethylchlorosilane, and all other reagents were purchased commercially and were used as received unless otherwise noted. 1-tert-Butyl-2,2,4,4,4-pentakis(dimethylamino)-2λ5,4λ5-catenadi(phosphazene) (P2-t-Bu base) in THF was purchased commercially and stored in a glovebox before use. 1,2-Benzenedicarboxaldehyde was purified by recrystallizing 3× from 5:2 dichloromethanehexanes and dried under vacuum for 2 days as described by DiLauro, Robbins, and Phillips.30 Tetrahydrofuran was purified by the method developed by Pangborn et al.31 Dry pyridine was distilled over CaH2 at 760 mmHg. Dry isopropanol was distilled over CaH2 at 760 mmHg and stored over 3 Å molecular sieves. (2S,3S,4S,5R,6S)-6-(4-((4-(Difluoromethyl)phenylcarbamoxyloxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro2H-pyran-2-carboxylic acid (1) was synthesized as described by Zhang et al.12 Flash column chromatography was performed as described by Still et al.,32 employing silica gel (60 Å pore size, 32−63 μm, standard grade, Dynamic Adsorbents). Thin layer chromatography was carried out on Dynamic Adsorbents silica gel TLC (20 × 20 cm w/h, F-254, 250 μm). Deionized water was purified with a Millipore-purification system (Barnstead EASYpure II UV/UF). Instrumentation. GPC analyses were performed using an Agilent Technologies 1200 GPC equipped with a refractive index detector, a Malvern Viscotek model 270 Dual Detector with right and low-angle light scattering, and either a Viscotek T-column (300 mm × 7.8 mm, CLM3012) and Agilent Resipore column (300 mm × 7.5 mm) in series or a single Agilent Resipore column (300 mm × 7.5 mm) using THF as the mobile phase (flow rate: 1 mL/min, 25 °C). The GPC was

EXPERIMENTAL SECTION

Materials. The adhesive hybridization chamber (Cat. No. SA200.5) was purchased from Grace Biolabs; the 6 μm polystyrene tracer particles (Cat. No. 07312) were purchased from Polyscience Inc. All reactions were performed in flame-dried glassware under a positive pressure of argon. For polymerization reactions, glassware was flamedried and stored in a glovebox overnight, and argon was passed 7258

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μmol) in 500 μL of phosphate buffer (75 mM, pH 7.4, 1 wt % BSA), substrate 1 (11 mg, 0.023 mmol) in 1 mL of phosphate buffer (75 mM, pH 7.4, 1 wt % BSA), and polystyrene tracer particles in phosphate buffer (75 mM, pH 7.4, 1 wt % BSA). To vary the enzyme concentration, these solutions were mixed, along with phosphate buffer solutions containing no enzyme or substrate, as shown in Table 2, to give solutions with final volumes of 500 μL.

calibrated using monodisperse polystyrene standards from Malvern. SEM images of the films were acquired using a FEI Nova NanoSEM 630 using secondary electron detection at 1 kV. General Procedure for Synthesizing Polymer 2. Polymer 2 was synthesized by modifying our previously reported procedure.30 1,2-Benzenedicarboxaldehyde (2 g, 15 mmol, 1 equiv) was sealed in a round-bottom flask charged with a NdFeB magnetic stir bar in a glovebox under a N2 atmosphere. Outside of the glovebox, anhydrous THF (25 mL) was added to the round-bottom flask; the resulting solution was degassed via three freeze−pump−thaw cycles and subsequently backfilled with oxygen-free argon. tert-Butyldimethylsilanol (4.7 μL, 0.002 equiv, 0.03 mmol) was added, and the reaction mixture was cooled to −78 °C. After 2 min, a 2.0 M solution of 1-tertbutyl-2,2,4,4,4-pentakis(dimethylamino)-2λ 5 ,4λ 5 -catenadi(phosphazene) (P2-t-Bu base) in THF (30 μL, 0.06 mmol, 0.004 equiv) was added in one portion to the solution containing the monomer and initiating alcohol. The reaction mixture was stirred vigorously (∼350 rpm) at −78 °C. After 2 h, the polymer was endcapped via sequential addition of pyridine (0.6 mL, 7.5 mmol, 0.5 equiv) and acetic anhydride (140 μL, 1.5 mmol, 0.1 equiv) to the −78 °C solution. The solution containing the end-capped polymer was allowed to warm to rt over 2 h. The polymer was precipitated by adding the reaction mixture to a solution of cold methanol (60 mL). The resulting suspension was filtered, and the precipitate was washed using a solid phase washing vessel by adding solvent, bubbling N2 through the solution at a vigorous rate (see the Supporting Information of ref 8 for a video of the bubbling rate) for 15 min, and then draining the solvent. The washing steps included the following solvents in the indicated order: MeOH, EtOAc, MeOH, EtOAc, hexanes, EtOAc, and hexanes (2×). The resulting polymer was dried under reduced pressure (4.5 mmHg) overnight. Polymer 2 was obtained as a white solid (1.95 g, 98%). Mn = 65.1 kDa and Mw = 103 kDa. Spectral data matched those previously reported.30 To synthesize variants of polymer 2 with different molecular weights, the amount of initiating alcohol (tert-butyldimethylsilanol) added to the polymerization reaction was varied. The equivalents of tert-butyldimethylsilanol as well as the yield, molecular weight, and PDI of each resulting polymer are shown in Table 1.

Table 2. Volumes of Solutions Containing Enzyme, Substrate 1, Tracer Particles, and Phosphate Buffer (75 mM, pH 7.4, 1 wt % BSA) Mixed To Obtain Solutions with Varying Enzyme Concentrations for the Experiment [β-Dglucuronidase] (μM)

enzyme solution (μL)

solution of 1 (μL)

tracer particle solution (μL)

phosphate buffer solution (μL)

9 8 6 4 2 1

166 148 111 74 37 17

200 200 200 200 200 200

20 20 20 20 20 20

114 132 169 196 233 253

The solutions containing the enzyme and substrate were incubated for 30 min at 23 °C before being added to the hybridization chamber containing the film of polymer 2 (Mn = 8 kDa). While filling the chamber, care was taken to avoid forming air bubbles in the chamber. The chamber was sealed with adhesive tape to prevent evaporation. Immediately after adding the solution containing the enzyme and substrate, the movement of the tracer particles was captured using a Zeiss Axiovert 200 microscope (in reflectance/transmission mode) at 20× magnification. The video was processed at 5× or 20× speed before tracking the coordinates of the tracer particles using Physvis. For each experiment, 30 particles from three sides of the polymer films were selected. Their coordinates were acquired using Physvis every 2.5 s over a period of 25 s. These coordinates were converted into speed (Table S1). Evaluating the Selectivity of the Pumps for Fluoride. To hybridization chambers containing films of polymer 2 (Mn = 65 kDa), 0.1 M solutions of various sodium salts (e.g., thiocyanate, sulfate, iodide, bromide, chloride, nitrate, phosphate, and fluoride) were added. The solutions were added carefully to avoid forming air bubbles in the chamber. Immediately after adding the salt solution, the movement of the tracer particles was captured using a Zeiss Axiovert 200 microscope and then processed and analyzed as described above; the resulting data are summarized in Table S2. Measuring the Change in Film Thickness for Polymer 2 upon Exposure to Fluoride. A film of polymer 2 (Mn = 42 kDa) was evaluated by profilometry using a Tencor Alpha-Step 500, which gave a minimum height of 7.2 μm and a maximum height of 22 μm. The film was then placed in a hybridization chamber. To the hybridization chamber was added a 0.1 M solution of sodium fluoride. Thirty minutes after adding the fluoride solution, the film was removed and dried under vacuum. The film was then evaluated again by profilometry, which gave a minimum height of 6.2 μm (change = 1 μm) and a maximum height of 20 μm (change = 2 μm). Evaluating the Effect of Polymer Molecular Weight on Pumping Speed. Films of the variants of polymer 2 with different molecular weights (i.e., Mn = 8, 33, 45, 53, and 65 kDa) were prepared as described above and added to hybridization chambers. To each chamber was added a 9 μM enzyme solution that had been incubated for 30 min with substrate 1 (see Table 2). Immediately after adding the solution containing the enzyme and substrate, the movement of the tracer particles was captured using a Zeiss Axiovert 200 microscope and then processed and analyzed as described above; the resulting data are summarized in Table S3. General Procedure for Synthesizing Polymer 3. 1,2Benzenedicarboxaldehyde (3 g, 22 mmol, 1 equiv) was sealed in a round-bottom flask charged with a NdFeB magnetic stir bar in a

Table 1. (a) General Procedure for the Synthesis of Polymer 2; (b) Equivalents of tert-Butyldimethylsilanol Used To Initiate the Polymerization Reaction as Well as the Resulting Polymer Molecular Weights, PDI Values, and Yields for the Variants of Polymer 2

General Procedure for Preparing Polymer Films. A solution of polymer 2 (Mn = 8 kDa) (5 μL) in dichloromethane (50 mg/mL) was deposited on a glass microscope slide. The film was dried under vacuum for 12 h and, using a razor blade, was cut into a square with approximate dimensions of 0.5 mm × 0.5 mm (the thickness of the film was approximately 25 μm); the excess polymer was scraped off of the slide. The slide was placed into the hybridization chamber. Evaluating the Response of the Films to β-D-Glucuronidase. Stock solutions were prepared of β-D-glucuronidase (0.94 mg, 0.014 7259

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(4 mL) to the −78 °C solution. The solution containing the endcapped polymer was allowed to warm to rt over 2 h. The polymer was precipitated by adding the reaction mixture to a solution of cold methanol (30 mL). The resulting suspension was filtered, and the precipitate was washed using a solid phase washing vessel by adding solvent, bubbling N2 through the solution at a vigorous rate for 15 min, and then draining the solvent. The washing steps included the following solvents in the indicated order MeOH, EtOAc, MeOH, EtOAc, hexanes, EtOAc, and hexanes (2×). The resulting solid was redissolved in dichloromethane (10 mL) and then filtered through a syringe filter into cold methanol (30 mL). The precipitate was washed again with MeOH. The resulting polymer was dried under reduced pressure (4.5 mmHg) overnight. Polymer 4 was obtained as a white solid (0.70 g, 70%). Mn = 49.2 kDa and Mw = 89.8 kDa. To synthesize variants of polymer 4 with different molecular weights, the amount of initiating alcohol (isopropanol) added to the polymerization reaction was varied. The equivalents of isopropanol and the yield, molecular weight, and PDI of each resulting polymer are shown in Table 4.

glovebox under a N2 atmosphere. Outside of the glovebox, anhydrous THF (37 mL) was added to the round-bottom flask; the resulting solution was degassed via three freeze−pump−thaw cycles and subsequently backfilled with oxygen-free argon. Dry isopropanol (3.4 μL, 0.002 equiv, 0.044 mmol) was added, and the reaction mixture was cooled to −78 °C. After 2 min, a 2.0 M solution of 1-tert-butyl2,2,4,4,4-pentakis(dimethylamino)-2λ 5,4λ5-catenadi(phosphazene) (P2-t-Bu base) in THF (45 μL, 0.09 mmol, 0.004 equiv) was added in one portion to the solution containing the monomer and initiating alcohol. The reaction mixture was stirred vigorously (∼350 rpm) at −78 °C. After 2 h, the polymer was end-capped via sequential addition of pyridine (0.9 mL, 11 mmol, 0.5 equiv) and 2-(methyoxypoly(ethylenoxy)6−9propyl)dimethylchlorosilane (1.1 mL, 2.2 mmol, 0.1 equiv) to the −78 °C solution. The solution containing the endcapped polymer was allowed to warm to rt over 2 h. The polymer was precipitated by adding the reaction mixture to a solution of cold methanol (90 mL). The resulting suspension was filtered, and the precipitate was washed using a solid phase washing vessel by adding solvent, bubbling N2 through the solution at a vigorous rate for 15 min, and then draining the solvent. The washing steps included the following solvents in the indicated order: MeOH, EtOAc, MeOH, EtOAc, hexanes, EtOAc, and hexanes (2×). The resulting polymer was dried under reduced pressure (4.5 mmHg) overnight. Polymer 3 was obtained as a white solid (1.88 g, 63%). Mn = 46.8 kDa and Mw = 73.7 kDa. To synthesize variants of polymer 3 with different molecular weights, the amount of initiating alcohol (isopropanol) added to the polymerization reaction was varied. The equivalents of isopropanol and the yield, molecular weight, and PDI of each resulting polymer are shown in Table 3.

Table 4. (a) General Procedure for the Synthesis of Polymer 4; (b) Equivalents of Isopropanol Used To Initiate the Polymerization Reaction along with the Molecular Weights, PDI Values, and Yields for the Variants of Polymer 4

Table 3. (a) General Procedure for the Synthesis of Polymer 3; (b) Equivalents of Isopropanol Used To Initiate the Polymerization Reaction along with the Molecular Weights, PDI Values, and Yields for the Variants of Polymer 3

Measuring the Effect of End-Cap Polarity on Pumping Speed. Films of polymer 3 (Mn = 32, 37, 47, 54, and 61 kDa) and polymer 4 (Mn = 20, 32, 43, 49, and 54 kDa) were prepared as described above and added to hybridization chambers. To each chamber was added a 9 μM enzyme solution that had been incubated for 30 min with substrate 1 (see Table 2). Immediately after adding the solution containing the substrate and enzyme, the movement of the tracer particles was captured using a Zeiss Axiovert 200 microscope and then processed and analyzed as described above; the resulting data are summarized in Tables S4 and S5. XPS Analysis. Glass slides (1 mm thick) with dimensions of 1 cm × 1 cm were cut using an Epilog Mini CO2 laser cutter. Solutions of polymer 2 (Mn = 33 kDa), 3 (Mn = 32 and 47 kDa), and 4 (Mn = 32 kDa) in dichloromethane (50 mg/mL) were deposited on separate slides so that the resulting films completely covered the surfaces of the slides. The films were dried under vacuum for 12 h and then analyzed for elemental composition by XPS (Figures S1−S4). The XPS is a Kratos Axis Ultra XPS with a monochromatic Al Kα X-ray source operated at 14 kV, 20 mA in hybrid slot mode. The survey scans are accomplished using a pass energy of 80 eV, with a 0.5 eV step size and 150 ms dwell time. The resulting spectra were analyzed using CasaXPS. Measuring the Effect of Film Area and Thickness on Pumping Speed. Films with 2× and 3× Area. Three films of polymer 3 (Mn = 43 kDa) were prepared as described above, with the exception that one film was cut to give a film with dimensions of 0.7 mm wide × 0.7 mm long and another was cut to give a film with dimensions of 0.9 mm wide × 0.9 mm long. These two films along

General Procedure for Synthesizing Polymer 4. 1,2Benzenedicarboxaldehyde (1 g, 7.5 mmol, 1 equiv) was sealed in a round-bottom flask charged with a NdFeB magnetic stir bar in a glovebox under a N2 atmosphere. Outside of the glovebox, anhydrous THF (12 mL) was added to the round-bottom flask; the resulting solution was degassed via three freeze−pump−thaw cycles and subsequently backfilled with oxygen-free argon. Dry isopropanol (1.1 μL, 0.002 equiv, 0.015 mmol) was added, and the reaction mixture was cooled to −78 °C. After 2 min, a 2.0 M solution of 1-tert-butyl2,2,4,4,4-pentakis(dimethylamino)-2λ 5,4λ5-catenadi(phosphazene) (P2-t-Bu base) in THF (15 μL, 0.03 mmol, 0.004 equiv) was added in one portion to the solution containing the monomer and initiating alcohol. The reaction mixture was stirred vigorously (∼350 rpm) at −78 °C. After 2 h, the polymer was end-capped via sequential addition of pyridine (0.9 mL, 11 mmol, 0.5 equiv) and a solution of triacontyldimethylchlorosilane (0.4 g, 0.75 mmol, 0.1 equiv) in THF 7260

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with a film with the standard area and thickness (0.5 mm wide × 0.5 mm long × 25 μm) were evaluated for their response to β-Dglucuronidase as described above (Table S6). Films with 2× and 3× Thickness. Two films of polymer 3 (Mn = 43 kDa) were prepared as described above, with the exception that after the initial deposition of polymer solution both films were allowed to dry before another drop of polymer solution was deposited, thus giving films 2× as thick as the standard size film. For one of these films, this process was repeated again to give a film 3× as thick as the standard size film. These two films were evaluated for their response to β-D-glucuronidase as described above (Table S6). Measuring the Change in Film Thickness for Polymers 3 and 4 upon Exposure to F−. Films of polymers 3 and 4 (both polymers had Mn = 43 kDa) were evaluated by profilometry before and after being exposed to 0.1 M aqueous sodium fluoride for 30 min as described above for the film of polymer 2 (Mn = 42 kDa). The data for all three polymers are shown in Table S7. SEM Imaging of Films before and after Exposure to F−. Films of polymers 2 (Mn = 42 kDa), 3 (Mn = 43 kDa), and 4 (Mn = 43 kDa) were prepared on silicon wafers and imaged by SEM; the full images are shown in Figures S5−S7. The films were then placed in hybridization chambers. To each hybridization chamber was added a 0.1 M solution of sodium fluoride. Thirty minutes after adding the fluoride solution, the films were removed and dried under vacuum. The films were imaged again by SEM; the full images are shown in Figures S8−S10. Determining Film Wettability. On a smooth celluloid plastic surface, films of polymers 2 (Mn = 42 kDa), 3 (Mn = 43 kDa), and 4 (Mn = 43 kDa) were formed by depositing solutions of the polymers and drying under reduced pressure for 3 h. The surface contact angle of each film was measured in triplicate using a Ramé-Hart Model 590 advanced automated goniometer (Table S8).

Figure 4. Pumping response of a film of polymer 2. (a) Films made from 8 kDa (Mn) polymer 2 provide predictable dose-dependent pumping when exposed to the model stimulus β-D-glucuronidase. (b) The pumping response of films of 65 kDa (Mn) polymer 2 is selective for fluoride. The concentration of the anions was 0.1 M. In both graphs, the data points represent the averages of ∼30 particles, each measured 10 times over the 25 s window of the experiment. The error bars reflect the standard deviations from these averages.

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1,2-benzenedicarboxaldehyde to drive the pumping response. On the other hand, films composed of longer polymers will have fewer end-caps per volume of film (when the size of the film is constant) than films composed of shorter polymers. It stands to reason that films with a higher density of end-caps are more likely to have more end-caps displayed at the solid−liquid interface than films with a lower density of end-caps. To test these considerations, we prepared five different length versions of polymer 2 (Table 1), with number-average molecular weights ranging from 8 to 65 kDa. Films (approximately 0.5 mm wide × 0.5 mm long × 25 μm thick) of these polymers each were exposed to 9 μM β-Dglucuronidase under identical conditions (i.e., 9.3 mM 1, 75 mM phosphate buffer, pH 7.4, 1 wt % BSA, 23 °C). As shown in Figure 5, shorter versions of 2 provide faster pumping speeds than longer versions. This result is consistent with the hypothesis that shorter polymers increase the density of endcaps in films of 2 and thus increases the likelihood that endcaps are available for reaction with fluoride at the solid−liquid interface. In fact, the 8 kDa version of 2 provides pumping speeds that are 3× faster than the pumping speed for the 65 kDa version of 2. On a per atom basis (excluding hydrogen atoms), the end-cap for the 8 kDa version of 2 constitutes 1.2% of a single polymer, whereas in the 65 kDa version of 2, the end-cap makes up only 0.1% of the polymer. Modulating the Accessibility of the End-Cap. An ideal stimuli-responsive solid-state material would enable substantially faster responses to an applied stimulus than was achieved in Figure 5. Thus, we altered the polarity of the silyl ether endcap on PPHA to further improve the accessibility of the endcap and to test whether this variable could be used to further tune the response of a solid-state material made from these types of polymers. In polymer 2, the silyl ether is the hydrophobic TBS group, which presumably associates more

RESULTS AND DISCUSSION Establishing Predictable Behavior for the Solid-State Test System. Before quantifying the accessibility of the endcap, we first established that the solid-state pump is a predictable test system. For example, we incubated reagent 1 with different concentrations of β-D-glucuronidase for 30 min to enable the release of fluoride from 1 and then added the solution to an enclosed chamber containing a 0.5 mm × 0.5 mm × 25 μm film of 2. By tracking the distance that ∼30 tracer particles traveled during ten 2.5 s time intervals (i.e., for 25 s total) for each concentration of enzyme, we obtained a direct relationship between the average pumping speed and the concentration of β-D-glucuronidase (Figure 4a). Figure 4b further shows that the pumping response is selective for fluoride over other anions that might be in solution. Furthermore, over the 25 s duration of these experiments, no change was observed in the size or thickness of the films. However, exposure of the films of 2 (Mn = 42 kDa) to 0.1 M fluoride for 30 min (rather than only 25 s) did cause a decrease in the maximum thickness of the film by 2 μm (a 7% change). This small decrease in film thickness over extended durations reveals that few polymer chains must depolymerize to induce the pumping effect. Overall, these combined results indicate that the pump is a reliable model system for evaluating the accessibility of the end-caps. Establishing a Baseline Level of Accessibility for the End-Cap. Having established that the pumps are a suitable model system, we questioned whether the test system would show faster pumping for films made from longer or shorter derivatives of polymer 2. On one hand, more monomer should emanate from films of longer polymers upon depolymerization than films of shorter polymers per reaction with the stimulus and therefore should create a greater concentration gradient of 7261

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Figure 6. Effect of end-cap polarity on the average pumping speed of silyl ether-end-capped PPHA films when exposed to 9 μM β-Dglucuronidase in a 75 mM phosphate buffer solution (pH 7.4, 1 wt % BSA) containing 9.3 mM 1. The data points represent the averages of 30 particles, each measured 10 times over the 25 s window of the experiment. The error bars reflect the standard deviations from these averages. The colors of the data points correspond to the color-coded structure of the silyl ether end-cap. The open circles correspond to data from control experiments in which the PPHA films were not exposed to β-D-glucuronidase.

Figure 5. Effect of polymer length on the accessibility of the end-cap at the solid−liquid interface as illustrated by the relationship between the Mn for polymer 2 and the pumping speed when the films were exposed to 9 μM β-D-glucuronidase. The line is included as a visual aid. The data points represent the averages of 30 particles, each measured 10 times over the 25 s window of the experiment. The error bars reflect the standard deviations from these averages.

with the hydrophobic PPHA film than with water at the solid− liquid interface when the film is exposed to water. To overcome this likelihood of the end-cap burying itself in the film, we synthesized PPHA polymer 3 (Table 3), which replaces the tert-butyl group in the end-cap of polymer 2 with a short poly(ethylene glycol) oligomer. We reasoned that the hydrophilic poly(ethylene glycol) oligomer on the end-cap would facilitate local reorganization of the polymer films to optimize exposure of the end-cap to water at the solid−liquid interface rather than burying the end-cap in the hydrophobic PPHA backbone. We also prepared PPHA polymer 4 (Table 4), which replaces the tert-butyl group with a 30-carbon-long hydrocarbon group that is essentially equal in length to the poly(ethylene glycol) oligomer. This hydrocarbon silyl ether should be more hydrophobic than the tert-butyl group and should have the opposite effect on the accessibility of the endcap than the poly(ethylene glycol)-modified end-cap. Moreover, polymer 4 serves as a control for the poly(ethylene glycol)-modified end-cap since a reduced steric environment around the silyl ether is known to accelerate the rate of reaction of the silyl ether with fluoride33 (presumably, the poly(ethylene glycol) and hydrocarbon silyl ethers are less sterically encumbered than the tert-butyldimethylsilyl ether). Figure 6 reveals that 3 (the poly(ethylene glycol)-modified silyl end-capped polymer) indeed improves the pumping speed compared to 2 when films of different length versions of 3 are exposed to 9 μM β-D-glucuronidase under identical conditions to those described previously for 2. For example, 47 kDa (Mn) polymer 3, in theory, provides approximately equal pumping speeds as 13 kDa (Mn) polymer 2, indicating that polymers with ∼250 more repeating units can be used with equal pumping efficiency when the tert-butyl group on the end-cap is exchanged for the poly(ethylene glycol) group. This result is significant because longer polymers tend to form more robust solid-state materials than shorter polymers; in this case, longer polymers provide pumping speeds that are equal in magnitude to shorter polymers as well by simply improving the accessibility of the polymer end-cap. Alternatively, if shorter polymers are preferred for a particular application, then extrapolation of the line for 3 suggests that films made from an 8 kDa version of 3 would provide pumping speeds that are 2.6× greater than films made from 8 kDa 2. In

contrast, polymer 4 shows reduced pumping speeds compared to 2. Extrapolation of the line for 4 indicates that an 8 kDa version of 4 would provide 1.4× slower pumping speeds than 8 kDa 2 and 3.7× slower than 8 kDa 3. In all cases, the background pumping speed (i.e., in the absence of enzyme) is equally small among all polymers, thus confirming that the increase in pumping speed when 3 is used is not a result of competitive hydrolysis reactions. Further extrapolation of the data in Figure 6 reveals equally interesting trends. For example, when the length of the PPHA chain reaches ∼60 kDa (Mn), the pumping speeds for polymers 2, 3, and 4 are nearly equal within the error of the experiments. At this length, the silyl ether end-caps constitute only 0.2% of the number of atoms (excluding hydrogen) within a single polymer, which suggests that there may be an upper limit in polymer length to which increasing the polarity of the end-cap will improve the response of the solid-state material when it interacts with stimuli in aqueous solution. At this limit, we propose that the reduced number of end-caps dominates the kinetics of the reaction with fluoride in solution more than the accessibility of the end-cap. At the other end of the spectrum (i.e., in 8 kDa polymers where the silyl ether end-caps constitute 1.2% of the number of atoms within a polymer), the density of the end-caps in the film is 6× higher than for films composed of 60 kDa polymers, and therefore maximizing exposure of the end-caps at the solid−liquid interface appears to dominate the response. While these macroscopic pumping results are compelling, factors other than end-cap accessibility also may affect the rate of pumping. In particular, changes in film morphology due to the different chemistry of the end-caps may give rise to the observed effects. Thus, as described later in this article in the section entitled Characterizing the Physical Properties of the Films, we investigated the effect of film surface area and volume on pumping speed and the effect of the polarity of the end-caps on the film morphology and the wettability of the films before drawing conclusions about the relationships between end-cap polarity, end-cap accessibility at the solid−liquid interface, and the observed pumping speeds. Quantification of Silicon in the Top Few Nanometers of the Polymer Films. The dependence of the solid-state 7262

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Table 5. Surface Elemental Composition and Average Pumping Speed in the Presence of 9 μM β-D-Glucuronidase for films of Polymers 2 (Mn = 33 kDa), 3 (Mn = 32 and 47 kDa), and 4 (Mn = 32 kDa) polymer

polymer Mn (kDa)

2 3 3 4

33 32 47 32

%O 21.4 22.0 20.7 21.8

± ± ± ±

0.14 0.14 0.14 0.18

%C 74.3 69.7 75.6 73.1

± ± ± ±

0.16 0.17 0.16 0.20

% Si 4.22 8.30 3.65 5.12

± ± ± ±

0.10 0.11 0.11 0.12

av pumping speed (μm/s) 1.08 2.55 1.24 0.58

± ± ± ±

0.25 0.34 0.28 0.17

Figure 7. SEM images of films of polymers 2 (Mn = 42 kDa) (a, b), 3 (Mn = 43 kDa) (c, d), and 4 (Mn = 43 kDa) (e, f) showing the effect of the end-cap on film morphology before and after exposure to 0.1 M aqueous NaF for 30 min. The before and after images were not acquired on the same locations on the films. The full images are shown in Figures S5−S10.

Characterizing the Physical Properties of the Films. The functional responses (i.e., pumping speed) and the results obtained from XPS analysis are suggestive that there is a direct relationship between the polarity of the end-cap and its accessibility at the solid−liquid interface, at least in the context of solid-state plastics interacting with analytes in aqueous solution. However, before drawing definitive conclusions about this relationship, we characterized the structures of the films made from polymers 2, 3, and 4 to ensure that the physical properties of the films are nearly equal and thus comparable. Our characterization focused on investigating other factors that may lead to the observed differences in the average pumping speed. Specifically, we examined the effects of the size and thickness of the film on pumping speed as well as differences in surface morphology and wettability that may arise from altering the polarity of the end-caps in 2, 3, and 4. a. Effect of the Surface Area and Volume of Films on Pumping Speed. We prepared films of polymer 3 (Mn = 43 kDa) that were (i) 2× and 3× as large in area and (ii) 2× and 3× as thick as those used in previous experiments.35 When these films were exposed to 9 μM β-D-glucuronidase under identical conditions to those described previously, less than 11% deviation in pumping speed was observed in comparison to the standard size film (Table S6). This degree of error is within the typical error for the experiments (Figure 6). Given that variations in film thickness and area are better controlled in typical experiments than these extreme examples, small differences in size or thickness of the films used in the experiments shown in Figure 6 are unlikely to significantly affect the pumping speed of the tracer particles.

response on the polarity of the end-cap is further supported by X-ray photoelectron spectroscopy (XPS) analysis of films of polymers 2, 3, and 4 (Table 5).34 XPS analysis enables quantification of the percentage of silicon (which is present only in the polymer end-cap and not in the polymer backbone) in relation to the other atoms in the top 10 nm of the polymer films.34 This analysis reveals that the surfaces of films composed of 33 kDa (Mn) polymer 2 (the polymer containing the TBS end-cap) or 32 kDa (Mn) polymer 4 (the polymer containing the alkyl silyl-modified end-cap) contain 4.22 ± 0.10% and 5.1 ± 0.12% silicon, respectively, whereas the surfaces of films composed of 32 kDa (Mn) polymer 3 (the polymer containing the poly(ethylene glycol)-modified end-cap) contain 8.30 ± 0.11% silicon, which suggests that films made from 3 display the silyl ether end-caps toward the surface of the film at least 1.6× more than 2 or 4. This result is consistent (within the error of the experiment) with the ∼2.4× increase in the pumping speed for films composed of 32 kDa (Mn) 3 compared to films composed of nearly equal number-average molecular weight 2 and 4. Equally telling is the extent of silicon near the surface of films composed of polymers with identical chemical composition, but different molecular weights. For example, approximately 2.3× more silicon is near the surface of a film composed of 32 kDa (Mn) 3 compared with a film made from 45 kDa (Mn) 3 (i.e., 8.30 ± 0.11% silicon vs 3.65 ± 0.11%, respectively). This difference in silicon content near the surface of the film reflects, in part, the density of the silyl ether end-cap and explains why films made from 45 kDa (Mn) 3 exhibit pumping speeds when exposed to 9 μM β-D-glucuronidase that are 2.1× slower than films made from 32 kDa (Mn) 3. 7263

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b. Physical Changes in the Films When Exposed to β-DGlucuronidase. Because of the depolymerization-based response, films that show the fastest pumping speeds might be expected to show faster degradation of the film than films that provide slow pumping speeds. We minimized the impact (if any) of this relationship on the analysis of end-cap accessibility by evaluating the responses of the films for short periods of time (i.e., 25 s), over which we observed no measurable change in the size or thickness of the films. In fact, the change in film thickness is sufficiently small during these experiments that exposure of films made from 2 (Mn = 42 kDa), 3 (Mn = 43 kDa), and 4 (Mn = 43 kDa) to 0.1 M fluoride for 30 min (i.e., 72× longer than the duration of measurement of average pumping speed) provided a change in the maximum film thickness of no more than 33% (Table S7). We also noticed that films made from 2 or 4 decreased in thickness less than films made from 3 (7−9% vs 33%, respectively). This increased extent of erosion for the film made from 3 (the poly(ethylene glycol)-modified silyl endcapped polymer) is consistent with an increase in end-cap accessibility that presumably gives rise to the 1.2× and 1.6× faster pumping speeds when films of 3 (Mn = 47 kDa) are compared with films of 2 (Mn = 45 kDa) and 4 (Mn = 43 kDa), respectively. c. Characterization of the Surface Morphology of the Films. SEM images of the films of polymers 2 (Mn = 42 kDa), 3 (Mn = 43 kDa), and 4 (Mn = 43 kDa) before exposure to fluoride show that each film has a smooth and uniform surface, indicating that the chemical composition of the end-cap does not affect the initial film morphology (Figures 7a,c,e). When the films were exposed to 0.1 M NaF for 30 min (72× longer than the duration of a 25 s pumping experiment), however, the differences in the responses of the films are reflected in observable changes in roughness of the films (Figures 7b,d,f). For example, films made from polymer 4 (the polymer with the slowest pumping speed) show no observable change over the course of the experiment (Figures 7e,f). Comparatively, films made from polymers 2 and 3, both of which provide faster pumping speeds than 4, show observable changes in film morphology, with the surface of the film of polymer 3 (the fastest pump) showing the most significant change in roughness (Figures 7c,d). These types of changes in morphology, particularly the formation of dimples in the surface (e.g., Figure 7d), are consistent with morphology changes observed in the context of core−shell microcapsules made from depolymerizable PPHA.36 d. Effect of the End-Cap on the Wettability of the Films. The concentration of hydrophilic functionality on the surface of a polymer film is known to affect the wettability of the film, which in turn alters the rate of reaction with the functionality.37 Contact angles of films made from polymers 2 (Mn = 42 kDa), 3 (Mn = 43 kDa), and 4 (Mn = 43 kDa) are essentially equal (i.e., 83.27 ± 2.38°, 82.97 ± 1.02°, and 83.31 ± 1.12°, respectively), suggesting that the increases in end-cap accessibility (as verified by XPS analysis) between films containing polar versus nonpolar end-caps are too small to affect the global wettability of the films. e. Summary of the Results for Characterizing the Physical Properties of the Polymer Films. Overall, these combined results suggest that differences in the hydrophilicity of the polymer end-caps do not affect the morphology or wettability of films of 2, 3, and 4 in a manner that would significantly influence the observed trends in pumping speeds in the time

frame of the experiments. Small differences in the size and thickness from film to film also do not appear to change the functional responses of the pumps. Thus, the variations in pumping speed appear to be a direct consequence of the density of the end-cap at the surface of the film, which is controlled by the polarity of the end-cap and the length of the polymer used to make the film.

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CONCLUSION In conclusion, this article offers a first step in understanding and, ultimately, improving and tuning the accessibility of endcaps at the solid−liquid interface when head-to-tail depolymerizable polymers are used to create solid-state stimuli-responsive materials. The results of these studies suggest that the fastest responses to stimuli in water occur when (i) the end-caps are hydrophilic and (ii) when short polymers are used instead of long polymers (short polymers increase the density of the endcaps that are accessible at the solid−liquid interface to a stimulus in water). There are certainly other ways to consider increasing the accessibility of the end-caps,38 but changing the polarity of the polymer end-caps and/or using different length polymers represent facile methods for modulating how endcaps are displayed at the solid−liquid interface. We anticipate that these results will be useful for designing improved stimuliresponsive capsules,36,39 nanoparticles,6,40 micrometer-scale pumps,12 shape-changing materials,8,30 phase-switching materials for point-of-care diagnostics,41 and polymer networks42 as well as other types of stimuli-responsive materials made from polymers that depolymerize from head-to-tail when an end-cap is cleaved from the polymer. It is also likely that these concepts will apply to solid-state materials made from other existing classes of polymers that depolymerize from head-to-tail in response to a specific stimulus.43

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ASSOCIATED CONTENT

S Supporting Information *

Polymer GPC traces and tables of primary data. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail [email protected] (S.T.P.). *E-mail [email protected] (A.S.). Author Contributions †

A.M.D. and H.Z. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project received support from the Defense Threat Reduction AgencyJoint Science and Technology Office for Chemical and Biological Defense (Grant HDTRA1-13-1-0039). Additional support was provided by the Penn State MRSEC (DMR-0820404), the Arnold and Mabel Beckman Foundation, the Camille and Henry Dreyfus Foundation, and Louis Martarano. S.T.P. acknowledges support from the Alfred P. Sloan Research Fellows Program. The XPS instrument was funded by NSF DMR-0114104. The authors thank Vince Bojan and Jennifer Gray for their assistance with XPS analysis. 7264

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