Swelling Transitions in Layer-by-Layer Assemblies of UCST Block

Apr 26, 2018 - Department of Materials Science and Engineering, Texas A&M University ... of BCMs and reduced the film swelling degree in acidic soluti...
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Swelling Transitions in Layer-by-Layer Assemblies of UCST Block Copolymer Micelles Anbazhagan Palanisamy and Svetlana A. Sukhishvili* Department of Materials Science and Engineering, Texas A&M University, College Station, Texas 77843, United States S Supporting Information *

ABSTRACT: An upper critical solution temperature (UCST) block copolymer, poly(acrylamide-co-acrylonitrile)-b-polyvinylpyrrolidone (P(AAm-co-AN)-b-PVP), was synthesized by reversible addition−fragmentation chain transfer (RAFT) polymerization, assembled in solution, and deposited within functional layer-by-layer (LbL) temperature-responsive films. In aqueous solutions, the polymer formed well-defined block copolymer micelles (BCMs) at ambient temperature and exhibited a reversible micelle−unimer transition within an ∼40−50 °C temperature range in a wide range of pH and ionic strengths. Temperature-induced dissociation of BCMs to unimers was completely suppressed, however, when BCMs were assembled with tannic acid (TA) within LbL films. Instead, reversible changes in micellar sizes and film swelling occurred as a result of UCST-driven uptake/release of water within/from the micellar cores. The solution pH modulated strength of hydrogen bonding between TA and PVP in the micellar corona, thus strongly affecting film growth, micelle morphology, and film swelling. Spherical micellar morphology and high film swelling degrees were observed with films at neutral pH values, where hydrogen bonding was counteracted by the negative charge of partially ionized TA. In contrast, strong hydrogen bonding and absence of charge in TA caused crumpling of BCMs and reduced the film swelling degree in acidic solutions. Film swelling was also dependent on number of assembled micellar layers, with thicker films exhibiting larger swelling amplitudes and sharper temperature transitions. The LbL assemblies were stable in phosphate buffer saline up to pH 7.5 at 50 °C and preserved their response after 55 heating/cooling cycles. The robustness of the temperature transitions in these films taken together with their occurrence in an aqueous environment in a wide range of pH and ionic strengths makes these films potentially useful for controlling responses of soft interfaces in biological environments.

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the assembled films lacked well-defined compartments of responsive polymers but were able to preserve their response14 and/or disintegrate9 upon changing the temperature below LCST. In the second case, PNIPAM was used as part of a block copolymer, whose segregation within micellar cores above the LCST transition prevented direct participation of its units in binding within LbL films.15 These two types of LbL molecular architectures are achieved through assembly of PNIPAMcontaining polymers at temperatures below and above their LCST, respectively. The latter case is specifically attractive for engineering responsive films, as it enables creation of responsive domains which can be then addressed through an external stimulus to reversibly alter film morphology, swelling, and control delivery of small functional molecules. We have previously demonstrated that LCST-type response can be realized with micelle-containing films through cooperative transitions of PNIPAM chains confined within the filmassembled micellar cores and have used this responsive to support temperature-controlled release of drug molecules from surfaces.16−18

INTRODUCTION Layer-by-layer (LbL) assembly is a facile yet powerful technique for creating nanostructured functional polymer coatings based on noncovalent (such as electrostatic or hydrogen bonding) interactions.1−3 Highly functional nanocoatings useful in sensing, controlled delivery devices, or as selfrepairing materials4 can be achieved through the incorporation of responsive polymers within LbL films. Among several external stimuli such as pH,5,6 light,7 or electrochemical reactions,8 temperature has been used as an important physiologically relevant stimulus for designing functional polymer coatings.9 Temperature-responsive polymers which enable the application of this stimulus can also be coupled with plasmonic or magnetic moieties, allowing to additionally use light and magnetic field to stimulate material response.10 The most studied temperature-responsive polymerpoly(Nisopropylacrylamide) (PNIPAM)exhibits a lower critical solution temperature (LCST) and has been incorporated within ultrathin films via self-assembly to leverage temperaturetriggered film swelling response to control protein adsorption or drug release.11−13 Two methods to include temperatureresponsive PNIPAM moieties within LbL films have been explored. In the first method, temperature-responsive units were directly involved within hydrogen-bonding assembly as part of a homopolymer or a random copolymer.9 In this case, © XXXX American Chemical Society

Received: March 11, 2018 Revised: April 15, 2018

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DOI: 10.1021/acs.macromol.8b00519 Macromolecules XXXX, XXX, XXX−XXX

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the effect of solution pH (i.e., ionization of TA and the extent of hydrogen bonding) on film deposition and morphology as well as on UCST-triggered micellar expansion and film swelling. Moreover, we report on the role of the number of assembled micellar layers on the amplitude and sharpness of the UCST swelling transitions of the films.

Yet, several applications require the opposite, i.e., upper critical solution temperature (UCST) type of film response. For instance, protein-repellant surfaces19 or release at high temperatures cannot be easily achieved with LCST films. In addition, one disadvantage of LCST-based micellar LbL films is the need to use elevated temperatures20 for film assembly and storage. Nevertheless, replacing LCST with UCST-type polymers in functional assemblies is challenging because of the scarcity of the UCST-type polymers, especially those which can function at physiologically relevant temperatures, pHs, and ionic strengths. Until now, several polymers and polymer systems exhibiting UCST in aqueous solutions or water−organic solvent mixtures have been developed, which are based on hydrogen bonding,21−26 Coulombic interactions,27,28 and/or achieved via redox/electrochemical response.29,30 Among them, neutral UCST polymers with temperature-induced dissociation of interchain hydrogen bonding are particularly attractive because their response is barely affected by solution pH or ionic strength.31,32 One example of such polymers includes random copolymers of poly(acrylamide-co-acrylonitrile), P(AAm-coAN), which exhibits UCST response at neutral pH.26 This copolymer has been used as part of a block/graft copolymer with poly(ethylene glycol) to yield amphiphilic solution assemblies which supported UCST-driven drug release.33,34 Another example includes homo- and copolymers of Nacryloylglycinamide, which showed UCST behavior in aqueous solutions that could be controlled between 10 and 85 °C via polymer composition.26 As compared to UCST homo- and copolymers, block copolymers with UCST response in aqueous media are poorly explored. UCST behavior was reported, however, for an amphiphilic poly(2-methacryloyloxyethylphosphorylcholine)-bpoly(2-ureidoethyl methacrylate) block copolymer in solution.35 At surfaces, temperature-triggered film decomposition was achieved with LbL assemblies of BCMs with UCST ionically aggregated cores.36 In our recent work, for the first time we reported on a hydrogen bonding based UCST block copolymer that contained a temperature-responsive polypeptide and studied its assembly in solution and within LbL films.25 However, block copolymer micelles (BCMs) of this copolymer were highly prone to aggregation in solution due to the irreversible formation of intra- and intermicellar polypeptide βsheets, which could compromise UCST response. Therefore, fresh synthesis of the copolymers, the use of low temperatures for film assembly, and working within a limited shelf life of assembled LbL films were required to observe UCST transitions. In this work, we aimed to develop a new micellarcontaining LbL system which could be assembled at ambient temperature and would exhibit robust, repeatable UCST transitions. Such films not only are advantageous for applications but also enable exploring fundamental features of such assemblies, such as correlations between film assembly conditions, film morphology, and response. To that end, here we report on synthesis of a novel block copolymer, P(AAm-co-AN)-b-polyvinylpyrrolidone (P(AAmco-AN)-b-PVP), via RAFT polymerization using a switchable RAFT agent in a two-step process, its micellization in solution, and assembly within UCST LbL films. The film assembly could be readily performed at room temperature using solutions of preassembled BCMs. The judicious selection of PVP as coronal block enabled depositing these micelles within LbL films through hydrogen bonding with tannic acid (TA). We explore

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EXPERIMENTAL SECTION

Materials. Solvents and reagents were purchased from SigmaAldrich Co. Acrylamide (AAm) (>99%, electrophoresis grade), cyanomethyl methyl(4-pyridyl)carbamodithioate (CMPC, 98%), branched polyethylenimine (BPEI, ∼750 000 g mol−1), and ptoluenesulfonic acid (p-TsOH, ≥98.5%) were used as received. Acrylonitrile (AN, ≥99%), 1-vinyl-2-pyrrolidinone (VP, ≥99%), and dimethyl sulfoxide (DMSO, ≥99%) were purified by distillation under vacuum. 2,2′-Azobis(2-methylpropionitrile) (AIBN, 98%) was recrystallized from ethanol and dried under high vacuum before use. Ultrapure water (Milli-Q Plus, Millipore) was used for the preparation of polymer solutions. Polymer Synthesis. To synthesize P(AAm-co-AN)-b-PVP block copolymer, a switchable RAFT37,38 agent, CMPC, was chosen to enable polymerization of “more activated” AAm and AN monomers and a “less activated” VP monomer. The P(AAm-co-AN) macro-RAFT agent was first synthesized to contain 19% of AN in order to achieve a targeted UCST range in the final polymer, P(AAm-co-AN)-b-PVP. The second block (PVP) was then polymerized by chain extension from the living end of the P(AAm-co-AN). Scheme 1 shows chemical structures and the steps involved in the polymer synthesis, while specific experimental procedures are presented in the following sections. Synthesis of P(AAm-co-AN) Macro-RAFT Agent. The P(AAm-coAN) macro-RAFT agent was synthesized using CMPC as a chain transfer agent mixed with stoichiometric amount of p-TsOH. A 25 mL round-bottom flask was charged with AN (0.3 g), AAm (1.7 g), CMPC (13.20 mg), p-TsOH (10.18 mg), AIBN (1.94 mg), and DMSO (6 mL). After the reactants were completely dissolved, the mixture was degassed by three freeze−pump−thaw cycles and placed in an oil bath preheated to 70 °C. The polymerization was continued for 24 h in a vacuum-sealed flask. Afterward, the reaction was quenched by rapid cooling in ice and exposing to air. The DMSO solution containing was passed through a column of sodium bicarbonate to deprotonate the RAFT chain end and finally purified by precipitation in cold methanol twice. To completely remove DMSO for UV−vis measurements, the polymer was dialyzed against deionized water in a continuous flow setup for 12 h at 40 °C and freeze-dried. For block extension, P(AAm-co-AN) macro-RAFT agent precipitated in methanol was used after drying under vacuum overnight. Synthesis of P(AAm-co-AN)-b-PVP Block Copolymer. A 25 mL round-bottom flask was charged with P(AAm-co-AN) macro-RAFT agent (0.5 g), VP (0.92 g), AIBN (0.54 mg), and DMSO (3 mL). The reagents were dissolved, subjected to three freeze−pump−thaw cycles, and transferred to an oil bath preheated to 70 °C. After 36 h, the reaction flask was cooled in an ice bath and the polymer recovered by precipitation into cold methanol. Prior to characterization, P(AAm-coAN)-b-PVP block copolymer (BCP) was dialyzed against deionized water in a continuous flow setup for 12 h at 40 °C to remove residual DMSO and freeze-dried. Characterization of Block Copolymers. All polymers were characterized using GPC and 1H NMR (Figures S1−S3). 1H NMR spectra were recorded using an Inova 300 MHz spectrometer in D2O solutions at 70 °C. Size exclusion chromatography (SEC) measurements were carried out at 45 °C using an Agilent GPC system, equipped with a Phenogel 5 μm column (300 × 4.6 mm) and an Agilent 1260 Infinity refractive index detector at 45 °C. DMSO was used as an eluent at a flow rate 0.1 mL/min, and samples of poly(ethylene oxide) (PEO) were used as standards. The chromatograms were analyzed using Agilent GPC/SEC Software, version A.02.01. B

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Scheme 1. Synthesis of UCST P(AAm-co-AN)-b-PVP Block Copolymer, BCP, by RAFT Polymerization Using a Switchable RAFT Agent, Cyanomethyl Methyl(4-pyridyl)carbamodithioate (CMPC)

A two-layer model was used to fit the ellipsometric data from the dry films, where first layer was a native oxide layer on the silicon substrate and the second polymer layer was treated as a Cauchy material having a wavelength-dependent refractive index n(λ) = A + B/ λ2 + C/λ3 (where A, B, and C are fitting coefficients and λ is the wavelength). The thickness and the three coefficients A, B, and C were fitted simultaneously at all the four angles of incidence. For thickness measurements in liquid, the cell was filled with ∼10 mL of PBS adjusted to designed pH and equilibrated for 15 min prior to measurements. To fit the ellipsometric data for wet films, a three-layer model was used with an additional layer representing the semi-infinite buffer solution which was treated as a transparent Cauchy medium. After data collection, the films were dried in gentle flow of nitrogen gas for measurements of dry film thicknesses to ensure that film retained their mass after swelling experiments. The swelling ratio was determined from the ratio of the ellipsometric thickness of films exposed to a buffer to that measured with dry films. Atomic Force Microscopy (AFM). Dry and in situ surface morphology of the micellar films were characterized using a BrukerDimension Icon AFM system. For imaging in dry conditions at room temperature (∼22 °C, ∼50% relative humidity), tapping mode was used to scan a 1 μm × 1 μm area at a scan rate of 1 Hz with a resolution of 512 scan lines. A sharp silicon tip (Nanosensors PPPNCSTR probe) with the normal stiffness of Kn = 7.4 N/m and a resonance frequency of 160 kHz was used for dry measurements. To measure film thickness, a razor-blade scratch was made in the micellar films deposited on a silicon wafer and imaged with AFM. Slope corrections were made with the plane fitting function using the NanoScope image analysis 1.5 software. For in situ measurements, tapping mode in liquid was used to scan a 1 μm × 1 μm area at a scan rate of 0.5 Hz with a resolution of 256 scan lines. A silicon nitride tip (Bruker-ScanAsyst Fluid+ probe) with the normal stiffness of Kn = 1.4 N/m and a resonance frequency of 200 kHz was used. For imaging in liquid, the silicon wafer containing polymer films were immersed in PBS adjusted to desired pH values (3 to 7) and mounted on the temperature-controlled sample holder. After 15 min immersion in liquid at 25 °C, the tip was engaged for scanning. Before switching the temperature to 50 °C, the tip was withdrawn and re-engaged for scanning after equilibration for 5 min at 50 °C.

UV−Vis and FTIR Spectroscopy. Transmittance of solutions of P(AAm-co-AN) macro-RAFT agent and BCP in phosphate buffer saline (PBS, 10 mM phosphate buffer, 150 mM NaCl) (pH 7) at varied temperatures was recorded using a Shimadzu UV-2600 spectrophotometer at a wavelength of 670 nm. The temperature of the cuvettes was controlled using a Julabo CORIO CD heating immersion circulator. The heating and cooling rates were 1.0 °C/min and 0.5 °C/min, respectively. For FTIR spectroscopy measurements, freeze-dried powders of PVP, P(AAm-co-AN), and P(AAm-co-AN)-bPVP were used in ATR mode with high-pressure clamp attachment. Dynamic Light Scattering (DLS). DLS measurements were performed at a scattering angle of 90° using a homemade instrument equipped with a 532 nm Whisper Mini laser with a power of 20 mW and a 0.5 mm beam diameter. To detect photon counts, a fiber-optic adapter for an 8 mm photomultiplier tube module (Edmund Optics) and two Hamamatsu photon counters (H10682-210) were used. Furthermore, the temperature was controlled using a Luma 40 temperature-controlled cuvette holder (Quantum Northwest). The data analysis was performed using the Corcle_v.0.18 software. Transmission Electron Microscopy. Micellization of BCP was examined using a JEOL JEM-2010 transmission electron microscope (TEM) operating at 100 kV. The samples for TEM were prepared by casting a drop of dilute polymer solution (0.01 wt %) on a carboncoated copper grid (CF400-Cu-UL 400 mesh) supported by a filter paper. Excess solution was then removed by a piece of filter paper, and samples dried either at room temperature using nitrogen gas (for preserving micellar assemblies below UCST) or by blowing hot air (to achieve dissociation of micelles at temperatures above UCST). Assembly of Micellar LbL Films. Block copolymer micelles (BCMs) used for LbL assembly were prepared by incubating 2 mg/ mL solutions of P(AAm-co-AN)-b-PVP in PBS at pH 7 at 70 °C for 30 min, followed by adjusting the solution pH to a desired pH ranging from 2.0 to 7 and cooling down to 22 °C before assembly. The micellar films were built on silicon wafers which were cleaned by exposing them to UV light, followed by immersion in concentrated sulfuric acid and rinsing with DI water. The clean wafers were first primed with BPEI to improve adhesion between TA and silicon wafer substrate by exposing them to an aqueous solution of BPEI (pH 9, 0.2 mg/mL) for 30 min. After rinsing with PBS at pH 7, the BPEI-primed silicon wafers were alternately exposed to 0.2 mg/mL TA and BCM solutions in PBS adjusted to the desired pH for 10 min using 1 min rinsing steps of PBS at the same pH between each layer. Spectroscopic Ellipsometry. Dry and in situ film thicknesses of the micellar LbL films were measured using a J.A. Woollam Co. M2000 spectroscopic ellipsometer equipped with a temperaturecontrolled liquid cell. In dry measurements, the thickness was measured at four angles of incidence 45°, 55°, 65°, and 75° at room temperature (∼22 °C). The nominal angle of incidence for wet measurements was 75°. The swelling response of the films in the liquid was measured at different heating/cooling rates from 0.3 to 1 °C/min by applying a continuous temperature ramp, or a step-hold temperature profile (as shown in Figure S6), between 25 and 50 °C, was used to study the swelling response. To avoid absorption by the buffer solution in the ultraviolet and near-infrared region, the working wavelength range was set to 370.5−999 nm.

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RESULTS AND DISCUSSION Synthesis and Temperature-Triggered Micellization of P(AAm-co-AN)-b-PVP in Solution. Because conventional RAFT agents are incompatible with “more activated” AAm and AN and “less activated” VP monomers,37 a switchable RAFT agent, CMPC, was used for synthesis of P(AAm-co-AN)-b-PVP (BCP). This enabled synthesis of BCP in a two-step polymerization process. In contrast, previous studies explored a multistep synthesis of PEG-based P(AAm-co-AN) block/graft copolymers.33,34 Here, BCP was prepared via polymerization of VP using P(AAm-co-AN) macroRAFT agent, which contained 89% of AAm units and had a number-average molecular weight (M̅ n) of 22 500 g/mol and a polydispersity index (PDI) of 1.37 as determined by GPC. The resultant P(AAm-co-AN)-b-PVP C

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Macromolecules had M̅ n of 51 000 g/mol and PDI of 1.25, i.e., was composed of approximetely 260 AAm and 80 AN units in the temperatureresponsive block and 260 VP units in the PVP block. In FTIR spectra of P(AAm-co-AN)-b-PVP, the characteristic band associated with C−N stretching vibrations in PVP was observed at 1291 cm−1, indicating successful inclusion of PVP within the block copolymer. The polymerization conditions and polymer structures are shown in Scheme 1. To explore temperature response of P(AAm-co-AN) single block and P(AAm-co-AN)-b-PVP BCP, UV−vis spectroscopy was first used. Panels a and b in Figure 1 show the temperature

observed in this work were reproducible after multiple heating/ cooling cycles. Figure 2a presents DLS and TEM results on temperaturecontrolled self-assembly in 2 mg/mL BCP solutions, which

Figure 2. (a) Temperature-dependent hydrodynamic diameters (Dh) in 2 mg/mL P(AAm-co-AN)-b-PVP solutions in PBS at pH 7 as determined by DLS and the TEM images for BCP in cold and hot solutions (insets in panel a). (b) Stability of BCMs formed in 2 mg/ mL solutions at 20 °C and pH 7 in 0.01 M phosphate buffer containing various concentrations of sodium chloride (above) or in PBS adjusted to the different pH (below). Prior to size measurements at 20 °C, polymer solutions were exposed to temperatures above 50 °C. All experiments were repeated twice, and the average values are shown.

Figure 1. Temperature dependence of transmittance of 5 mg/mL P(AAm-co-AN) (a) and P(AAm-co-AN)-b-PVP (b) solutions in PBS (pH 7) during heating and cooling cycles measured at 670 nm. The insets show polymer solutions at 22 and 70 °C. The heating and cooling rates were 1 and 0.5 °C/min in (a) and (b), respectively.

confirm UCST-type micelle−unimer transition. At 20 °C, the average hydrodynamic diameter (Dh) of micelles was 122 ± 12 nm, i.e., ∼3.3-fold smaller than the double contour length of BCP chains. The hydrodynamic size of the micelles remained constant within a concentration range from 0.5 to 5 mg/mL. TEM image in the inset shows that the block copolymer micelles (BCMs) were of spherical shape. The average micellar diameter calculated through the analysis of TEM images was 95 ± 11 nm, i.e., ∼25% smaller than that determined by DLS, reflecting differences between “dry” and “wet” measurements of micellar sizes. Interestingly, DLS indicated nonmonotonic dependence of hydrodynamic size of BCMs on temperature. Specifically, Dh initially increased from 122 ± 12 to 163 ± 18 nm upon heating from 20 to 40 °C and then sharply decreased to 6 ± 2 nm between 40 and 50 °C. The initial increase in micellar sizes reflects gradual weakening of the polymer− polymer and polymer−water hydrogen bonds prior to complete disintegration of micelles. The average hydrodynamic diameter detected in BCP solutions above 50 °C is in good agreement with the expected size of individual P(AAm-co-AN)-b-PVP chains, when they are approximated as Gaussian coils with a persistence length of 15 Å. Dissociation of BCMs to unimers at

dependence of transmittance of P(AAm-co-AN) and BCP solutions, respectively. While the upper inflection point in the turbidity curves was previously used to define UCST of polymer solutions,39 Figure 1 indicates that the UCST transition occurred within wide temperature ranges of ΔT ≈ 20 °C for P(AAm-co-AN) and even a broader transition window of ΔT ≈ 30 °C for BCP. Remarkably, unlike P(AAmco-AN), which exhibited aggregation (Figure S5) resulting in hysteresis in turbidity during cooling and heating, P(AAm-coAN)-b-PVP showed negligible hysteresis upon heating and cooling. Continuous exposure to lower temperatures for 4 days caused macroscopic precipitation in P(AAm-co-AN) solutions (Figure 1a, inset) due to strong inter-/intramolecular hydrogen bonding between AAm and AN units. Progressive aggregation was also observed with the UCST homopolymer.39 In contrast, BCP solutions remained colloidally stable during long-term incubation at T < 40 °C (Figure 1b, inset) for at least 40 days. These results suggest self-assembly in BCP solutions at lower temperatures and solubilization of assembled structures by PVP chains. Unlike our previous UCST system, where polypeptidecontaining block copolymer micelles underwent macrophase separation after two heating/cooling cycles,25 UCST transitions D

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terminated with a layer of TA to preserve the integrity of the micelles during temperature variations. For example, a 5.5-bl BCM/TA film contained five alternating bilayers of TA and BCMs and a top layer of TA. The average bilayer thickness of BCM/TA dry films determined by ellipsometry decreased from 12.9 nm to 10.6 and 5.6 nm as deposition pH increased from 3 to 5 and 7, respectively. This trend is assigned to changes in ionization of TA (pKa of TA is 8.5).43 Suppressed ionization of TA at low pH favors the attachment of TA to the micellar corona and results in a considerable increase in surface coverage, giving thicker films. At the same time, partial ionization of TA at pH 7 introduces negative charge within the film, which opposes hydrogen bonding between TA and BCM corona. At pH higher than 7.5 films could not be constructed because of the further increased electrostatic repulsions between ionized groups of TA. Decomposition of BCM/TA films was highly cooperative, with a dramatic film mass loss in a narrow pH range. Figure 3b shows that films assembled at pH 5 and 7 both retained their thickness up to pH 7 and abruptly dissolved at higher pH values. The inset in Figure 3b shows that 5.5-bl films exposed to buffer solutions within pH range from 3 to 7 remained stable and retained their mass for at least 3 days. Interestingly, films assembled in the acidic environment (pH 3) were of improved pH stability and did not decompose until pH 7.5 because of more extensive multisite hydrogen bonding between TA and PVP in BCM corona. We then aimed to explore whether ionization of TA, controlled by pH in assembly solutions, affected morphology and functionality of micellar LbL films. Figure 4 shows AFM images of dry 2.5-bl BCM/TA films deposited at pH 3, 5, and 7. While assembled structures could be seen in all the films, micellar shapes and sizes differed significantly for the film deposited at various pH (Figure 4a). Specifically, films assembled at pH 5 and 7 contained more round-shaped micelles with relatively narrow distribution of their diameters (98 ± 5 and 102 ± 6 nm for pH 5 and 7, Figures 4b and 4c, respectively). In contrast, for films deposited at pH 3, the spherical shape of the micelles was largely distorted, and the size of surface assemblies was significantly smaller (85 ± 9 nm, as estimated by averaging diameters of the round-shaped aggregates with the transverse dimensions of the elongated aggregates). The morphological differences between BCMs deposited within the films at various pH were also reflected in variations in the root-mean-square (rms) roughness determined from the AFM images, which were 3.8, 15.4, and 14.3 nm for the films deposited at pH 3, 5, and 7, respectively. The pHdependent morphology of micellar films correlated to ionization of TA. Based on the pKa of TA of ∼8.5,43 the acid carried a small amount of charge at pH 5 and 7 (0.03% and 3% of phenol groups ionized, respectively), and this had a dramatic effect on micellar morphology. Specifically, the presence of charge in the film was critical for sustaining the round shape of polymer micelles after their assembly within BCM/TA films and drying. This phenomenon is somewhat reminiscent of the well-known effect of charge on the bending rigidity of lipid membranes,44 which was studied both theoretically45−47 and experimentally.48,49 An enhanced bending rigidity of charged membranes is usually attributed to increased repulsion in the membrane plane that efficiently suppresses the membrane undulations and resists formation of defects. While similar arguments can be made for the experimental system studied in this work, the packing and structure within micellar LbL films

high temperatures was also confirmed by TEM imaging, which showed the absence of micellar assemblies in BCP solutions above 50 °C (inset in Figure 2a). The broad range of UCST transition is also reflected in the transmittance measurements of BCP solutions (Figure 1). In general, UCST-driven transitions occur in a wider temperature range in comparison to LCST ones, such as those observed in solutions of PNIPAM within 2−3 °C of PNIPAM’s LCST.40 The reason for such a behavior is the different nature of intermolecular interactions involved in LCST and UCST transitions in aqueous assemblies. Specifically, while LCST-driven transitions are mostly determined by polymer−water interactions, UCST transitions largely involve polymer−polymer binding and its evolution with temperature.41 In Figure 2a, the changes in the micellar hydrodynamic sizes in the temperature range between 20 and 60 °C were reproducible upon heating and cooling and could be repeated for at least 55 temperature cycles (data not shown). Moreover, BCM solutions remained colloidally stable and showed no signs of precipitation when kept at ambient temperature for more than a month, additionally indicating stability of BCP (and specifically of the P(AAm-co-AN) core)42 against hydrolysis. Moreover, micellization persisted within in a wide range of salt concentrations and solution pHs (Figure 2b). This behavior is due to the overall low sensitivity of interpolymer hydrogen bonds to solution salinity as well as to the absence of charged or ionizable groups in BCP. Surface assembly of P(AAm-co-AN)-b-PVP micelles was then explored in order to engineer films with assembled responsive UCST containers. Figure 3 shows that BCMs preassembled in solutions at 20 °C could be deposited within LbL films using TA as a partner molecule. In analogy with LbL construction of hydrogen-bonded films containing linear PVP,43 micellar multilayers exhibited robust linear growth at three different solution pH values (Figure 3a). BCM deposition was always

Figure 3. Ellipsometric dry thickness of BCM/TA films as a function of bilayer number during film deposition in PBS at pH 3, 5, or 7 (a) and stability of 5.5-bl BCM/TA films upon 30 min exposure to PBS solutions at increasing pH ranging from 2 to 9 (b). The inset shows long-term (3-day) stability of LbL films deposited at pH 5 and exposed to PBS at pH 3, 5, or 7. All experiments were conducted at 22 °C. E

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Figure 4. AFM topography images of dry 2.5-bl BCM/TA films deposited at pH 3 (a), 5 (b), and 7 (c). Images on the right illustrate the crosssectional analysis of the razor-cut films. When measuring the film height, the film area within ∼0.5−1 μm from the razor cut has been disregarded because of the distortions to the film caused by cutting.

We then explored whether different film morphology, achieved by assembly at varied pH, affected film swelling and temperature response. In these experiments, in situ spectroscopic ellipsometry was used to determine the average degree of film swelling, while in situ AFM was employed to study the effect of pH and temperature on film morphology. Figure 5a shows that when a 2.5-bl dry film deposited at pH 3 was immersed in PBS at the same pH value whose temperature was controlled at 25 °C, the micellar aggregates remained crumpled due to strong hydrogen bonding between TA and micellar corona. At the same time, the average ellipsometric film thickness increased from 41 ± 2 to 69 ± 4 nm (swelling ratio of 1.7) due to uptake of water. Upon an increase in temperature to 50 °C, the micellar aggregates appeared further swollen in the AFM images, with the average film thickness increasing to 98 ± 3 nm as a result of additional temperature-triggered water uptake (swelling ratio of 2.4) (Figure 5a, right). While oscillations in the swelling ratio of the films at pH 3 were highly repeatable, it is clear that the micellar morphology was largely distorted in the acidic environment. In contrast, film assembled at neutral pH values (5 and 7) had well-defined micellar morphology both in dry (Figure 4b,c) and wet states (Figure 5b). When a dry 2.5-bl film assembled at pH 5 was immersed in PBS at 25 °C, the average size of the

differ drastically from that in the lipid bilayers. Unlike in lipid bilayers, the charge is likely distributed within a 3D space of the micellar corona rather than concentrated in a 2D bilayer plane, and electrostatic effects on the bending rigidity and deformability of assembled micelles are further affected by intermixing within LbL films. The above result suggests, however, that the presence of charge in the micellar corona which is brought in by TA decreased deformability of the assembled micelles. On the other hand, similarly to perturbation and disruption of lipid vesicles by extensive electrostatic pairing with polyelectrolytes,50 extensive hydrogen bonding of uncharged TA with PVP corona at pH 3 caused micellar crumpling. AFM images of dry micellar films were also used to assess the average film thickness (Figure 4, right) in order to corroborate the ellipsometry data in Figure 3. To that end, the average height of razor-cut steps in the LbL films were measured from the top of the film to the silicon wafer substrate while the top film thickness was averaged over the rough, micelle-containing surface. The AFM measurements with 2.5-bl films assembled at pH 3, 5, and 7 gave thicknesses of 42 ± 3, 23 ± 5, and 21 ± 4 nm, respectively, which were in good agreement with those determined by ellipsometry (i.e., 41 ± 2, 24 ± 3, and 23 ± 2 nm, respectively, Figure 3). F

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Figure 5. AFM topography images (left) of 2.5-bl BCM/TA films exposed to different temperatures PBS at pH 3 (a) and pH 7 (b) and corresponding film swelling ratios (right) as measured by in situ spectroscopic ellipsometry during alternating exposure of the film to PBS solutions at 25 and 50 °C. The change in refractive indices upon film swelling is presented in the Supporting Information (Figure S7). (c) Schematic representation of temperature-induced changes in micellar sizes and film swelling.

Figure 6. Swelling ratios for BCM/TA films of various thicknesses exposed to PBS solutions at pH 3 (a) and pH 7 (b) as determined by in situ ellipsometry. The films were subjected to stepwise heating/cooling profiles shown in Figure S6.

assembled micelles as measured by AFM increased from 98 ± 5 to 140 ± 33 nm (∼50% increase) due to uptake of water (Figure 5b, left). Water uptake also resulted in an increase in the rms roughness of BCM/TA films from 13.2 nm for dry films to 35.4 nm. The swelling ratio of 1.9 was larger than an increase in the micellar sizes (factor of 1.4), suggesting that at 25 °C, when micellar cores are below their UCST and collapsed, water uptake preferably occurred within intermicellar spaces in the film. Raising the temperature to 50 °C caused an ∼2-fold increase in the micellar sizes from 140 ± 33 to 255 ± 45 nm as well as the corresponding 2-fold increase in the average thickness of the film up to swelling ration of ∼3.9, indicating uptake of large amounts of water within micellar cores. A decrease in the surface roughness from 35.4 to 26.8 nm upon temperature increase can be due to a higher degree of surface occupancy by highly swollen micelles at 50 °C.

Interestingly, perfectly spherical micellar morphology was retained in highly swollen film, with no changes in micellar morphology, film swelling degree, or total film mass upon exposure to a buffer solution at 50 °C for as long as 6 h (data not shown). This is in dramatic contrast with micellar behavior in solution, where BCMs were molecularly dissociated to unimers at this temperature. While hydrogen bonding between TA and PVP in BCM corona was sufficiently strong to provide stability of micellar morphology at increased temperature, when AN and AAm units within the micellar cores were dissociated, at the same time it was sufficiently dynamic to accommodate larger micellar sizes at an increased temperature. Figure 5 shows that the temperature-induced swelling was completely reversible and that both micellar sizes and the average film thicknesses returned to their original values when the temperature was decreased and the hydrogen bonding G

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Macromolecules within the micellar cores was restored. A 2-fold difference in the swelling ratios of BCM/TA assemblies at pH 3 and pH 7 (swelling ratios 2.3 and 3.7, respectively) reflects the effect of TA ionization on strength of binding within micellar corona, with slight ionization of TA favoring more dynamic hydrogen bonding and allowing larger swelling amplitudes. The films swelling was also reversible when cycled between various pH (data not shown). However, the pH dependence of film swelling maintained their crumpled morphology and relatively low swelling ratios of ∼2.5 regardless the pH of PBS solutions to which the films were exposed (Figures S8 and S9). While 2.5-bl BCM/TA films showed reversible swelling transitions and indicated distinct behavior for the films assembled from acidic and neutral solutions, the small thickness of these films (24 ± 3 nm for film deposited at pH 5) suggested a possibility that binding with a solid Si substrate and a precursor BPEI layer additionally affected their behavior. Therefore, studies with thicker films were performed in order to explore swelling in a quasi-3D matrix in which the effects of proximity of a substrate were largely avoided. Figure 6 shows temperature-induced swelling of films with varied thicknesses in acidic and neutral solutions. In these experiments, the temperature was varied stepwise, in order to ensure equilibrated film swelling, and films were incubated at each temperature for 10 min prior to measurements. In both heating and cooling cycles, all the films demonstrated UCST-induced increase in swelling ratios when temperature was changed between 25 and 50 °C. However, the amplitude and the temperature profile of the swelling response were strongly dependent on film thickness and solution pH. In 2.5-bl films, the micelles were largely confined within the region close to the substrate and pinned to a solid surface, which restricted their swelling amplitude. Specifically, the swelling ratio of 2.5-bl films linearly increased with temperature and did not exceed 2.5 and 4.0 at 50 °C for pH 3 and 7, respectively. These results agree with the distorted morphology and restricted swelling enforced by stronger hydrogen bonding of uncharged TA with BCMs at acidic pH, also seen in Figure 5. With thicker films, the diminished substrate restrictions and more 3D-like environment enabled higher swelling amplitudes for BCM/TA assemblies. For films exposed to pH 3, this effect was relatively weak, and 5.5-bl films reached the swelling ratio of 3 at the highest temperature. In contrast, with weaker bound 5.5-bl BCM/TA films exposed to pH 7, the swelling ratio was dramatically increased at high temperatures. Moreover, films composed of 3.5, 4.5, and 5.5 bilayers of BCM/TA at pH 7 had two distinct regions in the swelling profiles, with a stronger dependence of film swelling on temperature at elevated temperatures. The transition temperatures of 34, 35, and 41 °C between the weaker and stronger dependences on temperature were observed for 3.5, 4.5, and 5.5-bl BCM/TA films, respectively (Figure 6b). It is interesting that the transition temperatures of 35 and 41 °C were close to the onset of micellar disintegration in solution seen in Figure 2a, suggesting that in thicker films swelling of assembled BCMs is driven by their UCST behavior as in solution. However, in contrast to solution assemblies, binding of BCM with TA within the films prevented micellar dissociation to unimers, and the films retained their original thickness after cooling to 22 °C without any loss of material. Finally, repeatability of temperature-induced swelling transitions in BCM/TA films was explored. Figure 7a shows that when 2.5-bl films were subjected to multiple cycles of heating

Figure 7. Reversible changes in swelling ratios in 2.5-bl (a) and 5.5-bl (b) BCM/TA films in PBS (pH 7) upon multiple heating and cooling cycles between 25 and 50 °C. Insets in (a) show AFM topography images of dry as prepared 2.5-bl film (left) and the same films after 45 heating and cooling cycles (right).

and cooling, the swelling ratios varied between 1.4 and 3.7 in the first six cycles, and retained similar swelling amplitude after as many as 45 cycles, with some increase in the film swelling ratio at 25 °C due to possible film restructuring. AFM morphology studies of the film before and after multiple heating and cooling cycles (insets in Figure 7a) clearly shows retention of micellar structures, though some distortions of the micellar shape and a decrease in film roughness (from 13.5 to 3.2 nm) were observed. Similarly, the 5.5-bl films retained their swelling amplitude up to 55 temperature cycles (Figure 7b). After the first ten temperature cycles, 2.5-bl and 5-bl films did not show any mass loss. Upon further temperature variations, the mass retention was dependent on film thickness. While 2.5bl films continued to retain all their mass, 5-bl films lost ∼14% of dry film material after 55 temperature cycles. Overall, the micellar films composed of P(AAm-co-AN)-b-PVP micelles with hydrolytically stable P(AAm-co-AN) core block and PVP chains in corona which were capable of strong yet dynamic hydrogen bonding with TA51 preserved their robust temperature-responsive swelling behavior during multiple temperature cycles. In summary, we have demonstrated how a robust UCSTtype response can be endowed to surface coatings via hydrogen-bonded LbL assembly of temperature-responsive block copolymer micelles. To that end, a non-peptide UCST block was introduced in a copolymer to support reversible, highly repeatable temperature transitions in solution and within self-assembled film. pH of film assembly was critical for controlling morphology and temperature response of LbL films. In an acidic environment, the lack of charge and excessive hydrogen bonding between polymer micelles and TA compromised micellar morphology and reduced the amplitude of temperature response. In contrast, the presence of small amount of charge in films exposed to neutral pH stabilized spherical morphology of assembled micelles and enabled large changes in temperature-triggered film swelling. Moreover, swelling response of the films was thickness-dependent, with thicker films, where assembled micelles experienced more substrate-independent 3D environment, exhibiting larger swelling amplitudes. The versatility and ease of controlling UCST response of the assembled films at the step of synthesis of the temperature-responsive polymer block as well as during film assembly via solution pH and number of deposition steps H

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makes this system attractive for designing functional surfaces with controlled response characteristics.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.8b00519. GPC, 1H NMR characterization, DLS profiles, change in refractive indices with temperature cycling, and the stepwise heating/cooling profile used in the ellipsometric measurements (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (S.A.S.). ORCID

Anbazhagan Palanisamy: 0000-0002-8366-3330 Svetlana A. Sukhishvili: 0000-0002-2328-4494 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Use of the TAMU Materials Characterization Facility is acknowledged. This project was supported by the Texas A&M Engineering Experiment Station (TEES), and in part by NSF-DMR, Award No. 1610725.

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