Upper Critical Solution Temperature Layer-by-Layer Films of

Article Cite This: Chem. Mater. 2017, 29, 9084-9094

pubs.acs.org/cm

Upper Critical Solution Temperature Layer-by-Layer Films of Polyamino acid-Based Micelles with Rapid, On-Demand Release Capability Anbazhagan Palanisamy, Victoria Albright, and Svetlana A. Sukhishvili* Department of Materials Science and Engineering, Texas A&M University, College Station, Texas 77843, United States S Supporting Information *

ABSTRACT: Here, we report on upper critical solution temperature (UCST)-type polyamino acid-based block copolymer assembled in a layer-by-layer system with a natural polyphenol. The UCST-type block copolymer, polyvinylpyrrolidone-b-polyureido(ornithine-co-lysine) (PVP-b-PUOL), was synthesized via ring opening polymerization followed by postpolymerization functionalization with ureido groups. PVP-bPUOL exhibited UCST behavior that was controlled by both molecular weight and ornithine-to-lysine ratio. Importantly, PVP-b-PUOL with a UCST of 33 °C assembled into block copolymer micelles below UCST and dissociated above UCST, yet this behavior was not repeatable in solution due to β-sheet formation and large-scale aggregation. To overcome this limitation, UCST micelles (UCSTMs) were deposited within layer-by-layer (LbL) films via hydrogen bonding with tannic acid (TA). Significantly, the assembled TA/UCSTM films stabilized the micelles from desorption while maintaining their morphology and prevented β-sheet formation even after continuous exposure to 40 °C for 7 days. Moreover, these films demonstrated repeatable swelling−deswelling for up to five temperature cycles from 25 to 40 °C. The thermo-switchable hydrophobicity of micellar cores was used for trapping of a model active molecule, pyrene, and its on−off temperature-controlled release, demonstrating the potential of TA/UCSTM films for controlled delivery of active molecules.

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either by formation of inorganic shells 23 or through polyelectrolyte adsorption.20−22 An alternative approach of liposome stabilization through cross-linking their walls results in robust lipid nanocapsules24 but may compromise responsive behavior of the container walls. Another type of nanocontainers is assemblies of amphiphilic block copolymers, such as block copolymer vesicles25,26 and block copolymer micelles (BCMs),25,27−33 which are suitable for delivery of active molecules that are not readily soluble in water. BCMs containing responsive polymers can be manipulated using external stimuli such as pH, temperature, or light to exhibit switchable functionality.34 When incorporated within LbL surface films, these polymer micelles show good stability, yet preserve responsiveness to external stimuli.35 LbL assembly of temperature-responsive BCMs with lower critical solution temperature (LCST) behavior has previously been reported.33,36,37 This approach exploited a widely used LCST polymer, poly(N-isopropylacrylamide) (PNIPAM), which enabled film swelling and enhanced small molecule release upon temperature lowering. In this approach, poorly water-soluble functional molecules are trapped in the cores of

INTRODUCTION Confining responsive polymers to surfaces enables control over adhesive, mechanical, and adsorptive behavior at interfaces and thus is useful for biotechnological, food, catalysis, and cosmetics applications.1−4 The main feature of responsive surfaces is the capability to switch surface wettability, adhesion, and adsorption of chemicals or nanoparticles by external stimuli such as pH, temperature, light, or electric field. This switchability is enabled by the conformational adaption of polymers attached to surfaces as self-assembled monolayers,5 grafted polymer chains,6−9 thin polymer hydrogels, or assembled layer-by-layer (LbL) films10−13 to external stimuli. Among various architectures of thin polymer films, LbL coatings stand out as a facile and versatile way for surface functionalization.14,15 LbL films can be created to respond to a variety of external stimuli16,17 and can be used for loading and release of functional molecules from surfaces.18,19 One promising way to include functional cargo within the films is the use of nanocontainers (vesicles, micelles, or nanoparticles). Previous approaches have explored liposomes as vesicles that can be assembled within LbL films and used for controlled delivery of cargo.20−22 While availability of the inner aqueous compartment of liposomes affords encapsulation of water-soluble actives, the walls should be stabilized prior to their assembly © 2017 American Chemical Society

Received: July 1, 2017 Revised: October 6, 2017 Published: October 9, 2017 9084

DOI: 10.1021/acs.chemmater.7b02748 Chem. Mater. 2017, 29, 9084−9094

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Chemistry of Materials

Cbz-L-lysine amino acid monomers, (2) polymerization of peptide blocks from amino-terminated PVP and deprotection of polypeptide functional groups, and (3) ureido modification of polypeptide units of the block copolymer. The monomers Cbz-L-ornithine-NCA and CbzL-lysine-NCA were synthesized by phosgenation of protected peptides as described earlier45,46 with a slight modification in purification and recrystallization steps as described below. Specifically, in a 100 mL round-bottom flask, Cbz-L-ornithine (1.75 g, 6.57 mmol) was mixed with THF (25 mL) at 25 °C for 30 min. To the slurry, triphosgene (0.78 g, 2.62 mmol) was added and stirred under reflux at 50 °C in an oil bath until the slurry dissolved completely (∼4.5 h). Then the reaction flask was cooled to 10 °C, and 5 mL of ice water was added. The resultant solution was extracted with IPAc (3 × 25 mL) and washed with ice water (2 × 25 mL). The IPAc extract (∼75 mL) was first passed through a silica pad (50 g) and then reduced to 25 mL via rotary evaporator and dropped into hexane. The crude product was then recrystallized three times from toluene/hexane (1:1.5 v/v) mixture and dried to constant weight in the vacuum desiccator to yield white crystalline power (1.32 g, 75% yield). Cbz-L-lysine-NCA was synthesized from Cbz-L-lysine by following a similar procedure as described above. The polyamino acids of different compositions were synthesized from amino terminated PVP as a macroinitiator. In a typical PVP162-bPUOL513 synthesis, the monomers Cbz-L-ornithine-NCA (0.250 g, 0.858 mmol) and Cbz-L-lysine-NCA (0.112 g, 0.368 mmol) were first dissolved in 4 mL of anhydrous DMF, and then a DMF solution of initiator PVP (0.046 g, 0.002 mmol) was added. The reaction flask was subjected to three freeze−pump−thaw cycles, then filled with argon gas and stirred continuously for 4 days at 23 °C. The polymer was then precipitated into cold diethyl ether and further purified by reprecipitation. The completion of the polymerization reaction was monitored by the disappearance of the signal in 1H NMR (Mercury 300 MHz) for methine protons on NCA ring at 4.39 ppm. Finally, the polymer was dried in a vacuum oven and stored at −18 °C until further experiments. The Cbz protected block copolymers were then deprotected by reacting with hydrobromic acid solution. Typically, the polymer (0.1 g) was first dissolved in TFA (2.5 mL) at room temperature and then cooled in an ice bath. To the clear polymer solution, hydrobromic acid solution (0.5 mL) was added dropwise with vigorous stirring. The reaction mixture was stirred for 4 h in a sealed flask and was precipitated into cold diethyl ether to obtain the deprotected polymer with pale yellow color and 70% yield. The complete removal of Cbz protecting groups was confirmed by the disappearance of 1H NMR signals at 7.29 and 5.03 ppm (Figure S3). The primary amine groups in the deprotected lysine or ornithine were modified with urea groups by reacting the polymer with KOCN in aqueous imidazole buffer at 7 °C for 5 days. For this ureido modification reaction, the KOCN was used in a molar excess (1.5 times the amine groups) aiming for a complete ureido modification. At the end of the reaction, the cloudy polymer solution was dialyzed for 2 days with continuous flow of DI water and finally dialyzed with PBS buffer for 1 day yielding UCST responsive polymer. Characterization Methods. Chemical structures of monomers, intermediate, and final products were characterized using 1H NMR (Mercury 300 MHz), FTIR (Bruker, Tensor II), and gel permeation chromatography (GPC) (Agilent). GPC experiments were performed in DMF at a flow rate of 0.2 mL/min with two linear Styragel HR 4 columns at 30 °C and an Agilent 1260 infinity refractive index detector at 30 °C. The system was calibrated with a series of low polydispersity polystyrene standards. The chromatograms were analyzed using Agilent GPC/SEC Software version A.02.01. For FTIR spectroscopy measurements, either the polymer was either pressed into discs with potassium bromide or deposited as thin films (thickness greater than 150 nm) on IR transparent silicon wafers. For dynamic light scattering analysis, a home-built instrument was used to analyze 5 mg/mL solutions of PVP162-b-PUOL513 in PBS. Specifically, the DLS measurements were performed at a scattering angle of 90° with a Whisper Mini laser with a wavelength of 532 nm, power of 20 mW, and 0.5 mm beam diameter. To detect photon counts, a Fiber Optic Adapter for 8 mm photomultiplier tube module (Edmund Optics) and

the micelles when the micellar cores were collapsed, and they were expelled from the micelles when the temperature was lowered to render the BCM core hydrophilic. However, application of a lower temperature as a stimulus to release functional cargo has limited practical applicability. Instead, triggered delivery systems that are activated by heating (environmental heat or localized increase in temperature caused by inflammation) are highly desired. In addition, LCST-type LbL responsive films have another potential pitfall of premature degradation of encapsulated cargo due to prolonged exposure of films to temperatures above micellar LCST (typically >32−37 °C). Reversal of temperature response of BCMs, that is, generating micellar films with upper critical solution temperature (UCST) behavior, can enable interfacial films that are free of these shortcomings and release loaded cargo in a heated environment. However, polymers showing UCST-type response in aqueous solutions are rare, with the majority of work being focused on LCST-type polymers.38 Often, UCST behavior in water is achieved using zwitterionic polymers, which possess temperature response that can typically be realized only in lowionic-strength conditions.39 Recently, ureido-derivatized UCST polymers based on hydrogen bonding have been developed.40,41 More recently, UCST BCMs (UCSTMs) were reported utilizing ureido-derivatized42 or acrylamide-co-acrylonitrile moieties.43,44 Although degradability is an essential attribute of biomedical polymer materials, the reported UCSTMs contained nondegradable polymer chain backbones. Here, we address this challenge and explore the synthesis, LbL assembly, and responsiveness of films composed of UCSTMs with polypeptide-based micellar cores. Specifically, an UCST-type block copolymer, polyvinylpyrrolidone-b-polyureido (ornithine-co-lysine) (PVP-bPUOL), was synthesized via ring opening polymerization and ureido functionalization of the polymer pendant groups. Temperature-triggered assembly of these polymers yielded micelles with polypeptide PUOL core and PVP corona. The PVP block was judiciously selected to facilitate LbL deposition of micelles within LbL films using hydrogen bonding interactions with tannic acid (TA) and PVP.36 To the best of our knowledge, this is the first study that reports on UCSTMs of a polypeptide block copolymer and demonstrates ondemand release capability of UCST-type micellar LbL films.

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EXPERIMENTAL SECTION

Materials. Nε-Z-L-lysine (≥98%) and Nd-Z-L-ornithine (≥98%) were purchased from Chem-Impex International, Inc. Triphosgene, pyrene, potassium cyanate (KOCN), imidazole, trifluoracetic acid-99% (TFA), isopropyl acetate (IPAc), tetrahydrofuran (THF), diethyl ether, and dialysis tubing (12 000 Da) were purchased from Alfa Aesar chemicals. Hexane, dimethylformamide (DMF), branched polyethylenimine (BPEI, 750 000 g mol−1), hexyl amine-99%, deuterated chloroform, acetone, sodium phosphate monobasic dehydrate, polyvinylpyrrolidone (PVP) (MW 55 g mol−1), and hydrobromic acid solution (33 wt % in acetic acid) were purchased from SigmaAldrich. Amino terminated PVP with Mn = 18 000 g mol−1 was obtained from Polymer Source Inc. Distilled DMF was used for polymerization reactions. All other solvents were obtained in anhydrous form and used without further purification. Both borondoped and undoped silicon wafers were purchased from University Wafer, Inc. Synthesis of PVP-b-PUOL. The UCST block copolymers were composed of a PVP block and a ureido modified polypeptide block. The typical polymer synthesis involved (1) synthesis of Ncarboxyanhydride (NCA) of carbobenzyloxy (Cbz)-L-ornithine and 9085


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Figure 1. Block copolymer chemical structure and UCST-driven assembly in solution. (a) Synthesis scheme of PVP-b-PUOL block copolymer consisting of a PVP hydrophilic block (blue) and a temperature-responsive ureido-functionalized polypeptide block (red). (b) Hydrogen bonding between ureido groups brings about UCST behavior and micellization of PVP-b-PUOL polymers. (c) UCST determination of PVP162-b-PUOL216, PVP162-b-PUOL168, and PVP162-b-PUOL513 block copolymer solutions in PBS as measured by transmittance at 700 nm. Inset shows 5 mg/mL PVP162-b-PUOL513 solution in PBS buffer, showing transition from cloudy to clear upon heating. (d) Hydrodynamic diameter of PVP162-b-PUOL513 in PBS buffer at 10 and 35 °C as measured by DLS. inelastic scattering peak of phosphate buffer has a clear distinct maximum at 358 nm. All further data was collected with λex = 320 nm and the peak of phosphate buffer at 358 nm was used to correct all data (in other words, all data were shifted up or down until the inelastic scattering peak had the same intensity). For release studies, 1 × 1 cm2 silicon wafers coated with either 2.5 bilayers of UCST films or PVP/TA layers of matched thickness were soaked in 10 mL of PBS at pH 7.4. To collect spectra, at each time point, a 2 mL aliquot of solution was taken, measured, and quickly put back. To quantify the release of pyrene over time, the intensity of the peak at 371 nm was measured at 2.5 or 2 min intervals.

two Hamamatsu photon counters (H10682−210) were used. Furthermore, temperature was controlled using a Luma 40 Temperature-Controlled Cuvette Holder (Quantum Northwest). Turbidity measurements were performed with a Shimadzu 2600 UV−vis at a wavelength of 700 nm with 5 mg/mL polymer solutions in PBS. The UCST polymer assemblies were characterized using TEM (JEOL JEM-2010 at 100 kV). LbL Film Assembly and Characterization. The UCSTMs were deposited on silicon wafers using the well-known LbL deposition technique. Specifically, silicon wafers were cleaned and primed with a precursor layer of BPEI, which was deposited from an aqueous, pH 9, 0.2 mg/mL solution for 30 min. The primed wafers were then alternatively exposed to 0.2 mg/mL TA solution and 0.5 mg/mL UCSTM solution for 30 min with a 1 min rinse of 10 mM phosphate buffer between each layer and after the final TA top layer. All the deposition steps and rinsing were carried out at 10 °C in pH 4 aqueous solutions. Dry and in situ thickness measurements were taken by a spectroscopic ellipsometer (J.A. Woollam Co. M-2000). To measure the swelling response of the films, the spectroscopic ellipsometer with a temperature-controlled liquid cell was used with a heating/cooling rate of 0.33 °C/min. The surface morphology of the micellar films was characterized using SEM (JEOL JSM-7500F) and AFM (BrukerDimension Icon). For SEM imaging, the samples were sputter-coated with 3 nm of platinum/palladium (Pt/Pd) alloy prior to imaging. Pyrene Loading and Release from UCST Films. To load (and reload samples), 1 × 1 cm2 samples were soaked in 0.2 mg/mL pyrene in acetone overnight. Afterward, samples were washed three times with cold PBS adjusted to pH 7.4 and dried. All spectra were collected using a Photon Technology International QuantaMaster series spectrofluorometer. To determine the ideal λex at which no scattering peaks overlap with the characteristic peaks of pyrene (λex: 371, 392 nm), preliminary tests were run in PBS at excitation wavelengths ranging from 264 to 340 nm. It was determined that at λex = 320 nm, the

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RESULTS AND DISCUSSION Synthesis of UCSTMs and Their Solution Behavior. Here, with the goal of controlling the UCST and resultant micellization of diblock copolymers of polyvinylpyrrolidone-bpolyureido(ornithine-co-lysine) (PVP-b-PUOL) (Figure 1a), a series of PVP-b-PUOL block copolymers with different ornithine-to-lysine ratios, r, and molecular weights were synthesized. Ureido modification of polyornithine was previously reported to yield a hydrogen-bonding homopolymer that exhibited UCST behavior in aqueous solution;40 here, we additionally introduced lysine as a more hydrophobic moeity,47,48 with the goal of controlling the UCST response of PVP-b-PUOL block copolymers. Hydrophobicity of polymers has been shown earlier to increase the immiscibility region and therefore increase UCST of hydrogen-bonding polymers in aqueous solutions,39,49 most likely due to the disruption of polymer−solvent hydrogen bonds and formation of lower-entropy hydration shell around the phase-separated domains. The series of block copolymers was obtained through several steps involving (1) preparation of monomers, (2) ring 9086


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Figure 2. Temperature response of UCSTMs in solution: (a, b, d, e, g, h) TEM images, (c, f, i) DLS data, and (insets between the panels) pictures of 5 mg/mL PVP162-b-PUOL513 solutions illustrating aggregation of UCSTM solutions upon repeated heating cycles between 10 and 35 °C. (j) Drastic decrease in DLS photon count of micellar solutions from cycle 1 to 3 agrees with precipitation of aggregated micelles. (k) The band at 1623 cm−1 in the FTIR spectra confirms the formation of intermolecular polypeptide β-sheets in the precipitates collected after three heating cycles.

polypeptide-core micelles at low temperatures in both Milli-Q water and phosphate buffered saline (PBS, 10 mM phosphate buffer with 150 mM NaCl). The UCST of PVP-b-PUOL block copolymers was defined as the temperature at which solutions became 100% transparent (at 700 nm) when slowly heated from 10 to 40 °C. UCST of PVP-b-PUOL polymers was found to depend on both ornithine and lysine ratio and well as length of PUOL block (with a constant ratio of ornithine to lysine). Figure 1c shows that when comparing PVP162-b-PUOL216 and PVP162-b-PUOL513, which have a constant ratio between ornithine and lysine (r = 70:30), as the molecular weight of the PUOL block increased, so did the UCST (compare UCSTs of 14 and 33 °C for PVP162-b-PUOL216 and PVP162-b-PUOL513, respectively). The inset in Figure 1c illustrates how a cloudy solution of PVP162-b-PUOL513 at 10 °C turned clear upon heating above the UCST of the polypeptide block. On the other hand, when the overall molecular weights of the block copolymers were comparable but r changed from 70:30 to 30:70 (for PVP162-b-PUOL216 and PVP162-b-PUOL168, respectively), the UCST increased as a result of enhanced hydrophobicity of the core-forming PUOL block. The latter result illustrates that hydrophobicity of the polypeptide chains

opening polymerization (ROP) initiated by an aminoterminated homopolymer polyvinylpyrrolidone (PVP) and deprotection of amino groups on ornithine and lysine, followed by (3) side-chain modification with ureido moieties as is shown in Figure 1a, detailed in the Experimental Section, and shown in detail in the scheme in Figure S1. In brief, N-carboxyanhydrides (NCA) of carbobenzyloxy (Cbz)-L-ornithine and Cbz-L-lysine amino acid monomers were synthesized and success confirmed by 1H NMR (Figure S2). Then ROP of NCA-monomers was performed as initiated by an amino terminated PVP block. Cbz protecting groups were then removed by treating the precursor polymer with a hydrobromic acid solution as confirmed by studies with 1H NMR (Figure S3). Finally, the primary amino groups (−NH2) of the polypeptide side chains were converted to ureido groups (−NH−CO-NH2)40 to yield PVP-b-PUOL. Synthesized polymers included PVP162-b-PUOL216 (Mn = 145 000 g mol−1, PDI 1.23, r = 70:30), PVP162-b-PUOL168 (Mn = 132 000 g mol−1, PDI 1.27, r = 30:70), and PVP162-bPUOL513 (Mn = 233 000 g mol−1 PDI 1.17, r = 70:30) as can be seen in Figure S4. Hydrogen bonding between the pendant ureido groups (Figure 1b) drives assembly of this block copolymer within 9087


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Figure 3. Strategy, thickness, and surface morphology of LbL assembly of UCSTMs. (a) Schematics of LbL assembly of UCSTMs and TA on a silicon wafer. (b) Dry thickness of multilayer films of TA and PVP162-b-PUOL513 micelles as measured by ellipsometry. The inset shows that a 2.5bilayer film remained stable up to pH 8.5. (c, d) SEM and AFM images of a 2.5-bilayer film showing dense coverage of the surface with micelles of the average diameter of 36 ± 14 and 32 ± 9 nm, as determined from SEM and AFM images, respectively.

formation of hydrogen bonds between relatively hydrophilic core-forming polymers. Note that even in the case of LCST micelles, whose core composed of hydrophobic chains above their LCST transition, the micellar cores contained as much as 80% of water.52,53 In this work, upon the first heating cycle of UCSTMs above the UCST (33 °C), the cloudy solution became clear as spherical micelles dissociated into individual polymer chains, as the hydrogen bonds between the ureido groups of PUOL dissociated (Figure 2a,b). This is supported by dissolution of polymer assemblies seen in TEM image (Figure 2a), disappearance of the 139 nm DLS peak (Figure 2c), and a drastic decrease in photon count (Figure 2j). When PVP162-bPUOL513 solutions were exposed to 35 °C for a short time, even as little as 15 min, and then cooled to 10 °C, spherical micelles did not reform, but instead UCSTMs assembled into larger-size interconnected micelles (Figure 2d). Correspondingly, a drastic decrease in photon counts and broadening of the DLS profile were observed (Figure 2f and j), all suggesting the onset of aggregation triggered after the first heating cycle. With repeated heating and cooling, the micellar aggregates showed a substantial increase in hydrodynamic diameter (>200 nm) and size distribution as a result of micellar coalescence and aggregation. The aggregation further progressed during the third heating/cooling cycle (Figure 2g−i), yielding precipitates and a dramatic decrease in photon count detected from solutions above precipitated aggregates (Figure 2j). Taken together, the data above demonstrate that solutions of PVP-bPUOL micelles possess sufficient molecular mobility to form larger, irreversibly assembled structures upon repeated temperature cycling. Note that while the exposure to elevated temperatures shortened the time scale to aggregation of micelles, aggregation had also proceeded when micelles were kept at lower temperature for longer periods of time (data not

plays a significant role in their UCST behavior. These data are consistent with prior studies of ureido-based polymers, such as poly(2-ureidoethyl methacrylate) and poly(L-ornithine)-copoly(L-citrulline), which demonstrated that UCST of these polymers was strongly dependent on ureido group content, polymer molecular weight, and concentration.40−42 Figure 1d shows dynamic light scattering (DLS) data of PVP162-bPUOL513 in PBS buffer, which suggest the presence of assembled structures with the average diameter of 139 ± 11 nm at a temperature below the UCST. Moreover, above the UCST, these structures dissociate, which agrees with UCSTtype behavior of polymer assemblies in solution. The hydrodynamic diameter of individual polymer chains after disassembly (Figure 1d, 35 °C) could not be determined because of their low scattering contrast and the additional masking by scattering from a small number of ∼700 nm-sized aggregates which emerged even in the first heating cycle. With the polymer that exhibited the highest UCST (PVP162b-PUOL513; UCST of 33 °C), reversibility of solution assembly was explored. To that end, the block copolymer solution was subjected to multiple heating/cooling cycles, and the size distribution and morphology of assembled structures were studied using transmission electron microscopy (TEM) and DLS. With fresh block copolymer solutions at 10 °C, spherical micelles with a diameter of 35 ± 15 nm and 139 ± 11 nm were observed with TEM and DLS, respectively. The difference between micellar sizes in “dry” TEM and “wet” DLS measurements were significantly larger than those previously observed with traditional amphiphilic block copolymer micelles, which are driven by pure hydrophobic interaction of the core forming blocks,50,51 probably due to hydration of polypeptide core. High hydration of UCSTMs is expected since UCST transition, unlike LCST, is mostly driven enthalpically by 9088


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Figure 4. Reversible swelling−deswelling of TA/UCSTM films: AFM topology images of a 2.5-bilayer TA/UCSTM films exposed to PBS buffer at (a) 25 °C and (b) 40 °C. Upon heating, the micellar size increased from 56 ± 14 nm to 117 ± 22 nm. (c) Schematics of the temperature-induced size changes of the micelles assembled within TA/UCSTM films. (d) In situ spectroscopic ellipsometry measurements of reversible swelling− deswelling of a 2.5-bilayer TA/UCSTM film exposed to PBS at pH 7.4 upon temperature cycling between 25 and 40 °C. (e) FTIR spectra illustrating complete suppression of β-sheet formation in micelles assembled within a 7.5-bilayer TA/UCSTM film (above) during long-term exposure to PBS solution at 40 °C, as compared to β-sheet formation in UCSTM precipitates (below).

solutions after multiple exposure to temperature cycling. Thus, micellar aggregation in solution is related to the occurrence of β-sheets in the PVP-b-PUOL polymer. Polypeptide aggregation is often linked to β-sheet-rich amyloid-like insoluble structures,59−61 whose formation is enhanced at elevated temperatures and pressures.60,62 Freshly prepared UCSTMs are highly hydrated, as suggested by the comparison of TEM and DLS data in Figure 1, with PUOL adopting α-helix or disordered conformation within the micellar core. The high hydration of micellar cores enables micellar coalescence and transformation of the PUOL block to β-sheets, which stabilize micellar precipitates. In all further experiments described below, micellar aggregation was avoided by using only freshly synthesized micelles which were kept refrigerated at 4 °C for not longer than 2 weeks. LbL Assembly of UCSTMs. We then aimed to explore whether UCSTMs can be assembled into films that prevent intermolecular aggregation which compromises micellar functionality. In earlier work, LCST-type BCMs with PNIPAM responsive cores were bound with a coassembly partner within a LbL film, which prevented dissociation of LCST polymer micelles to individual polymer molecules.36,37,63 Here, we are searching instead to prevent the opposite process, that is, micellar aggregation via molecular binding and immobilization within a film. Figure 3a illustrates our strategy of taking advantage of hydrogen bonding between TA and PVP to from TA/UCSTM films at the surface of silicon wafers, a model substrate. Binding between TA and PVP persists within a wide

shown). It was necessary, therefore, to work with freshly synthesized micelles whose solutions were kept refrigerated at 4 °C for not longer than for 2 weeks. The relatively high degree of hydration within polypeptide cores and the ease of restructuring of polypeptide chains54 are likely to contribute to the temperature-assisted loss of responsiveness of UCSTMs in solution. We then were seeking to provide spectroscopic evidence for irreversible changes in conformations of polypeptide chains composing micellar cores. Figure 2k shows a comparison of Fourier transform infrared spectroscopy (FTIR) spectra of freshly prepared and aggregated UCSTMs, collected from micellar solutions at 10 °C before heating or taken from the precipitates after the third heating cycle. Infrared absorbance in the amide I region at ∼1650 cm−1 are mostly associated with CO stretching vibrations, with minor contributions from CN stretching, CCN deformation, and NH bending modes. Spectral features in this region are largely dependent on the secondary structure of the backbone and are commonly used for conformational analysis of polypeptides and proteins.55,56 The freshly prepared UCSTMs (Figure 2k, top) primarily exhibited a band with a maxima at ∼1655 cm−1 and a shoulder ∼1720 cm−1, with no absorption peak in the 1615−1637 cm−1 region, which is characteristic of β-sheet conformation.57,58 The 1655 cm−1 band is consistent with the α-helix conformation of the polypeptide block, but random coil conformations cannot be ruled out.54 In contrast, a new 1623 cm−1 band indicative of βsheet structures emerged in the precipitates collected from 9089


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show individual micelles assembled within the film. The lateral size of the assembled micelles exposed to PBS at 25 °C (Figure 4a) was 56 ± 14 nm, that is, 1.7-fold larger than the AFMmeasured diameter of assembled micelles in their dry state (Figure 3d), indicating water uptake within micellar cores even at temperatures below micellar UCST. Importantly, exposure of the film to temperatures above the micellar UCST did not deteriorate individual micelle morphology, but caused an increase in the micellar diameter from 56 ± 14 nm at 25 °C to 117 ± 22 nm at 40 °C, reflecting ∼2-fold temperatureinduced micellar swelling. The corresponding root−mean− square roughness of the film at 25 and 40 °C was 5.0 and 2.7 nm, respectively. Note that to retain the morphology and prevent disintegration and desorption of micelles from the surface, it was important to cover the micelles with TA, which was assembled as the top layer of the film.63 The above result is in contrast to temperature-response behavior of UCSTMs in solution, where micelles completely disintegrated during the first cycle of temperature increase (Figure 2a,b). Within the LbL film, hydrogen bonding between TA and PVP preserved micellar morphology as shown schematically in Figure 4c. Note that temperature-induced micellar swelling observed with AFM was completely reversible, that is, individual micelles were “breathing”, reversing their sizes to lower ones upon a temperature decrease. Spectroscopic ellipsometry was then used to study in situ temperature response TA/UCSTM film in the direction perpendicular to the substrate using a temperature controlled liquid-cell. Figure 4d shows changes in wet thickness of a 2.5 bilayer film immersed in PBS buffer during multiple heating/ cooling cycles between 25 and 40 °C. Exposure of the TA/ UCSTM film to PBS at 25 °C resulted in an increase of the film thickness from its original dry thickness of 11 ± 0.9 nm (also shown in the graph) to a steady value of 17 ± 0.8 nm (Figure 4d). The corresponding changes in the refractive indices of the film confirm that water uptake/temperature-induced film swelling cause the observed change in thickness measurements (shown in Figure S6). Because of its nm-scale thickness, film swelling was rapid and was completed within the first minute required to start ellipsometry measurements in the liquid cell. The film swelling ratio of 1.5 calculated from these values is in good agreement with an AFM-measured increase in the micellar size due to micellar hydration when films are exposed to PBS at 25 °C (swelling ratio 1.7, discussed above). At the same time, the difference between “wet” and “dry” measurements of film swelling and free micelles in solution was large (the swelling ratios of 1.5−1.7 for micellar assemblies vs. 3.5 for free micelles in solution). The lower degree of swelling for micelles within the film was expected because of increased hydrophobicity and swelling constrains brought about by complexation of micellar PVP chains with TA. When the solution temperature was increased to 40 °C, the film thickness increased to 31 ± 1.2 nm (to swelling ratio of ∼2.9) because of the uptake of water within the micellar polyamino acid cores. This swelling was accompanied by a decrease in the refractive index of the film (Figure S6). The increase of the film swelling ratio from 1.7 at 25 °C to 2.9 at 40 °C correlates well with the temperature-induced increase in micellar sizes observed in situ AFM measurements (Figure 4a,b), suggesting isotropic expansion of micelles within the film. Figure 4d also shows remarkable reversibility and repeatability of the TA/UCSTM film deswelling/swelling when the temperature of PBS buffer was cycled between 25 and 40 °C.

range of pHs64,65 and enables robust LbL assembly. Freshly prepared UCSTMs of PVP162-b-PUOL513 were assembled within TA/UCSTM films using the LbL technique and a surface priming procedure similar to that described elsewhere,63 which is detailed in the Experimental Section. Importantly, the deposition was carried out at 10 °C, temperature well below the UCST of micelles, to ensure that PVP162-b-PUOL513 assembled within the film in its micellar form. Figure 3b illustrates growth via dry thickness measurements of TA/UCSTM films as measured by spectroscopic ellipsometry. In all cases, film deposition on the prime layer (see Experimental Section) started from TA and was terminated by the deposition of TA on the outermost micellar layer to provide stabilization of the micellar corona. Consequently, films denoted as 1.5−5.5-bilayer films contained 1−5 layers of micelles. Film growth became linear after the deposition of two bilayers with an average bilayer thickness of 15.7 ± 1 nm, which is 2.5-times smaller than the average diameter of dry micelles (39 ± 11 nm) as observed by TEM in Figure 2a. This difference is likely due to a combination of incomplete coverage of micelles per single deposition cycle and possible flattening of micelles within the film in the dry state. While LbL assembly was performed at pH 4 to ensure optimal hydrogen bonding between TA and PVP, films are required to withstand varied environmental conditions in their applications. For example, near-neutral pH and elevated ionic strength are relevant for biotechnological uses of these films. To that end, TA/UCSTM films were exposed to a range of pHs from 4.0 to 13.0 in PBS at 22 °C. The inset in Figure 3b shows that TA/UCSTM films were stable up to pH 8.5 and disintegrated at higher pH values because of enhanced TA ionization.64 Importantly, when a 2.5-bilayer TA/UCSTM film was exposed to pH 7.4 in PBS at 40 °C for as long as 7 days, no loss of film mass has occurred (Figure S5). Scanning electron microscopy (SEM) and atomic force microscopy (AFM) images in Figure 3c and d show the surface morphology of dry 2.5 bilayer TA/UCSTM films. The silicon wafer was covered with spherical micelles indicating that hydrogen bonding between micellar corona and TA within the film preserved micellar structure during film assembly. The average diameters of the micelles of 36 ± 14 nm and 32 ± 9 nm calculated from SEM and TEM images in Figure 3c and d, respectively, are in good agreement with the average diameter of solution-dried micelles of 39 ± 11 nm as determined by TEM (Figure 2a). The root−mean−square roughness of the film determined from the AFM image in Figure 3d was 4.2 nm. Taken together, these results demonstrate that when assembled within LbL films with TA, UCST micelles preserve their individual morphology and neither aggregate nor disintegrate after binding within the film. Temperature Response of UCSTM Films. A critical question is whether LbL-assembled UCSTMs preserve their response, and whether robustness of this response is affected by the new environment of micelles within the films. Earlier, LCST-type BCMs with PNIPAM responsive cores included within LbL films were demonstrated to provide LCST type film swelling/deswelling,36,37,63 as well as temperature controlled “on−off” release of cargo.36 To that end, film morphology and swelling of TA/UCSTM films were explored using in situ AFM and spectroscopic ellipsometry, respectively, when films were exposed to aqueous solutions kept at various temperatures. Figure 4a and b show AFM images of a 2.5 bilayer film exposed to PBS at 25 and 40 °C, respectively. The AFM data clearly 9090


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Figure 5. Controlled release of pyrene from UCSTM films. (a) Cumulative release of pyrene from 2.5-bilayer TA/UCSTM films. Rapid release was observed at 38 °C when the micelles swelled above the UCST of PUOL cores. (b) Controlled release of pyrene achieved by switching the temperature above and below the UCST of micelles, demonstrating on-demand release. Control films, composed of 2.5 bilayers of TA/PVP, released much less pyrene, and did not demonstrate temperature sensitivity. (c) Schematics of LbL assembled micelles showing temperature-triggered UCSTtype pyrene release from the films.

The swelling response was highly repeatable for at least five heating/cooling cycles, and the films did not demonstrate any mass loss after completion of several swelling cycles, as demonstrated by ellipsometric measurements of dry film thicknesses (Figure S7a). In situ spectroscopic ellipsometry was used to explore long-term stability of the swollen film exposed to 40 °C, that is, conditions in which free micelles disintegrate. The only driving force that prevents block copolymer disintegration in these conditions is binding of TA with PVP in micellar corona. Figure S5 shows dry thickness of a 2.5-bilayer film during continuous exposure to PBS at 40 °C, with no dry mass loss persisting for at least 7 days. The demonstration of a robust and repeatable response of TA/UCSTM films to temperature is surprising, considering that polypeptide cores of PVP-b-PUOL micelles free in solution were highly prone to cross-micellar β-sheet formation and precipitation. Unlike micelles in solution, TA-bound, filmassembled UCSTMs were capable of sustaining their response and maintained their spherical morphology in multiple heating/ cooling cycles (Figure S7b). We then hypothesized that stabilization of individual micellar morphology within TA/ UCSTM films is related to inhibition of formation of β-sheet structures. Figure 4e shows the FTIR data of a 7.5-bilayer TA/UCSTM films exposed to PBS solutions at 40 °C for 1, 3, and 7 days. For these experiments, 7.5-bilayer instead of 2.5-bilayer films were used to achieve sufficient signal-to-noise ratio during FTIR measurements of the LbL films deposited on the IRtransparent silicon wafers. Remarkably, even after continuous incubation of the assembled films at a temperature above PUOL’s UCST for as long as 7 days, there were no signs of βsheet formation by the polyamino acid included within the cores. This is obvious from the absence of the characteristic absorption band of β-sheet around 1623 cm−1 in Figure 4e. In polypeptides, the formation of secondary structures, such as βsheets, is defined by the side chain steric restrictions and the

conformational freedom to chain rearrangements.66 Assembly of UCSTMs within the film, enabled by binding of TA with micellar corona, restricted the conformational mobility of the polyamino acid blocks, confined the micellar cores, and inhibited cross-micellar aggregation of the PUOL blocks. Suppression of the β-sheets formation in the confined environment of the film enabled highly repeatable temperature response of the micellar film−behavior not attainable with free micelles in solution. Temperature-Controlled Release of Pyrene from TA/ UCSTM Films. The highly repeatable temperature-triggered swelling transitions in 2.5-bilayer TA/UCSTM films were then explored with regard to their applicability to control the release of active compounds. Pyrene was used as a model for a small hydrophobic molecule, which mimics drugs such as antibacterial or antifungal agents. The partitioning of this molecule in the micellar PUOL cores was expected to be high when the cores were collapsed at T < UCST. Temperature was then explored as a trigger to release the loaded pyrene as the micellar cores become more hydrophilic and expand at temperatures above the micellar UCST. 2.5-bilayer TA/UCSTM films were loaded with pyrene at 10 °C using a solution of pyrene in acetone. Figure 5a shows cumulative continuous release of the pyrene dye from TA/UCSTM films. Fluorescence emission intensity at 371 nm was used to monitor pyrene release from the films (details in Methods Section), which were immersed in PBS buffer (pH 7.4) at different temperatures. At 5 °C, less than 10% of pyrene was released over a period of 30 min. When the temperature of the solution was increased to 20 °C, ∼ 30% of pyrene was released in 5 min, while the maximum of 80% release was reached at 30 min. Even at higher temperatures, such as 38 °C (T > UCST of micelles), twice the amount of pyrene (∼60%) was released within 5 min when compared to the release at 20 °C, and up to ∼95% was released over a period of 30 min. The difference in the release rate at various temperatures was clearly due to UCST responsive behavior of 9091


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synthetic methods,70 polyamino acid blocks can be programmed to respond to multiple stimuli such as light, pH, enzymes, etc. and then retain that functionality in surface assemblies. Furthermore, the choice of polyamino acids for preparing UCST-type micelles may enable biocompatibility and enzymatic degradability of the coatings in future biomedical applications.

the polyamino acid-cores, while partial release of pyrene at 20 °C (a temperature below UCST of micelles) was due to the breadth of the UCST transition. At lower temperatures, the pyrene molecules were trapped within the hydrophobic micellar cores, exhibiting slow release kinetics. In contrast, at temperatures above the UCST of micelles, the polyamino acid chains in the cores were hydrated and pyrene was released rapidly, driven by the large difference pyrene concentration gradient between the film and the release medium. In addition to the continuous release observed at temperatures above UCST, on demand pulsated release is highly desirable to fully take advantage of the temperature responsive films. To achieve a pulsated release, the temperature of the solution was switched between 5 and 38 °C, which were below and above the micellar UCST, respectively. Pyrene release was observed in a step-like fashion (Figure 5b), with release occurring exclusively at 38 °C. The step-like release profile agrees well with the sharp swelling transition of the film shown in Figure 4d. We also made an attempt to reuse the TA/ UCSTM films after extracting pyrene completely at 38 °C. The reloaded films were also efficient in trapping pyrene, and released above the UCST of micelles, making them reusable if needed (Figure S8). To differentiate the loading capacity between the micellar cores and the TA/PVP film matrix, 2.5bilayer LbL films of PVP homopolymer (MW 55 g mol−1) and TA of comparable thicknesses to TA/UCSTM films (∼18 nm for TA/PVP films and ∼22 nm for TA/UCSTM films) were assembled in the same conditions and loaded with pyrene for control experiments. The control samples showed no temperature responsive release, with an overall small amount of dye being continuously released from the TA/PVP film. The temperature-induced pulsated release of functional molecules in a UCST-type fashion described here is first of its kind as opposed to the conventional LCST-type films. Because of the potential degradability of the UCSTMs reported here,67,68 taken together with the low toxicity of the film components69 and the robust response of the film to the practically useful increase, rather than a decrease in the surrounding temperature, these assemblies are promising candidates for controlled delivery of actives from surfaces in several applications including biotechnology, biomedicine, agricultural, and food industries.

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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.chemmater.7b02748. Polymer synthesis reaction scheme; GPC and 1H NMR characterizations; refractive index of films during temperature cycling; film stability during continuous exposure to 40 °C; reusability of the UCST films to load and release pyrene (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

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

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Use of the TAMU Materials Characterization Facility is acknowledged. We acknowledge financial support from Texas A&M University/Association of Former Students Graduate Merit Fellowship, Texas A&M Engineering Experiment Station (TEES), and partial support by NSF-DMR Award No. 1610725.

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CONCLUSIONS In summary, this work reports the synthesis of UCST-type polymers and demonstrates a versatile method of preparing novel functional UCST-type thin films. Reversible hydrogen bonding between ureido groups with an additional contribution from the polypeptide backbone imparts temperature response to the micelles. UCST can be controlled via ornithine to lysine ratio and molecular weight of PVP-b-PUOL with a range of 14−33 °C. Yet, in solution, β-sheet formation and large-scale aggregation prevents reversibility of the transition. This limitation can be overcome via hydrogen-bonding assembly with tannic acid within LbL films. Hydration of UCST-type micelles at T > UCST brings in large and reversible transitions in film swelling and small molecule retention. Here, opening of assembled micellar cores for on-demand temperature-controlled release of small molecules is shown in response to mild heating at ambient or physiologically relevant temperatures. We believe our results open new avenues for the use of a wide range of polypeptide-based polymers in responsive functional assemblies for a variety of applications. Through advanced

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Upper Critical Solution Temperature Layer-by-Layer Films of

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