Shape-Persistent, Thermoresponsive Polypeptide Brushes Prepared

Article pubs.acs.org/Macromolecules

Shape-Persistent, Thermoresponsive Polypeptide Brushes Prepared by Vapor Deposition Surface-Initiated Ring-Opening Polymerization of α‑Amino Acid N‑Carboxyanhydrides Yong Shen,† Solenne Desseaux,‡ Bethany Aden,§ Bradley S. Lokitz,∥ S. Michael Kilbey, II,§ Zhibo Li,*,† and Harm-Anton Klok*,‡ †

Beijing National Laboratory for Molecular Sciences (BNLMS), Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China ‡ Institut des Matériaux and Institut des Sciences et Ingénierie Chimiques, Laboratoire des Polymères, École Polytechnique Fédérale de Lausanne (EPFL), Bâtiment MXD, Station 12, CH-1015 Lausanne, Switzerland § Departments of Chemistry and Chemical & Biomolecular Engineering, University of Tennessee, Knoxville, Tennessee 37996-1600, United States ∥ Center for Nanophase Materials Sciences, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, United States S Supporting Information *

ABSTRACT: Surface-grafting thermoresponsive polymers allows the preparation of thin polymer brush coatings with surface properties that can be manipulated by variation of temperature. In most instances, thermoresponsive polymer brushes are produced using polymers that dehydrate and collapse above a certain temperature. This report presents the preparation and properties of polymer brushes that show thermoresponsive surface properties, yet are shape-persistent in that they do not undergo main chain collapse. The polymer brushes presented here are obtained via vapor deposition surfaceinitiated ring-opening polymerization (SI-ROP) of γ-di- or tri(ethylene glycol)-modified glutamic acid N-carboxyanhydrides. Vapor deposition SI-ROP of γ-di- or tri(ethylene glycol)-modified L- or D-glutamic acid N-carboxyanhydrides affords helical surface-tethered polymer chains that do not show any changes in secondary structure between 10 and 70 °C. QCM-D experiments, however, revealed significant dehydration of poly(γ-(2-(2-methoxyethoxy)ethyl)-L-glutamate) (poly(L-EG2-Glu)) brushes upon heating from 10 to 40 °C. At the same time, AFM and ellipsometry studies did not reveal significant variations in film thickness over this temperature range, which is consistent with the shape-persistent nature of these polypeptide brushes and indicates that the thermoresponsiveness of the films is primarily due to hydration and dehydration of the oligo(ethylene glycol) side chains. The results presented here illustrate the potential of surface-initiated NCA ring-opening polymerization to generate densely grafted assemblies of polymer chains that possess well-defined secondary structures and tunable surface properties. These polypeptide brushes complement their conformationally unordered counterparts that can be generated via surface-initiated polymerization of vinyl-type monomers and represent another step forward to biomimetic surfaces and interfaces.

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ture.11 Being limited to vinyl-type monomers, these techniques result in synthetic polymer-based surface coatings or interfaces that are composed of conformationally disordered polymer chains. In nature, however, macromolecules are composed of αamino acids, saccharides, or nucleotides and can adopt ordered secondary structures. The ability to produce thin polymerbased coatings from these natural building blocks and to present densely grafted arrays of chains with controlled secondary structure will add another tool to quest to engineer surface and interface properties, representing another step forward to biomimetic surfaces and interfaces.

INTRODUCTION

Densely grafting polymers tethered by one of their chain ends to a surface results in thin “polymer brush” films, which have attracted attention for myriad (potential) applications, including stimuli-responsive surfaces,1,2 nonbiofouling surfaces,3−5 cell adhesive surfaces,6,7 and antibacterial coatings.8−10 Controlled/“living” radical polymerization (SI-CRP) techniques such as atom transfer radical polymerization (ATRP), reversible addition−fragmentation chain transfer (RAFT) polymerization, and nitroxide-mediated polymerization (NMP) provide many attractive opportunities for the “graf ting f rom” synthesis of polymer brushes. These polymerization methods are attractive since they provide a high tolerance toward a wide range of functional groups as well as excellent control over brush thickness, grafting density, and architec© XXXX American Chemical Society

Received: January 4, 2015 Revised: April 5, 2015

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DOI: 10.1021/acs.macromol.5b00017 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules

properties of poly(L-EGx-Glu) provide access to a conceptually novel class of temperature-responsive polymer brushes that allow temperature-induced switching of surface properties yet are shape-persistent in that they do not undergo main chain collapse, as is the case for brushes based on PPEGMEMA and PNIPAM. This finding is derived from investigation of the secondary structural as well as the thickness and viscoelastic properties of poly(L-EGx-Glu) brush films made by SI-ROP of the corresponding EGx-Glu NCA monomers.

Polypeptide brushes represent an interesting alternative platform to generate functional surfaces. Such polymer brushes can be obtained via surface-initiated ring-opening polymerization (SI-ROP) of the appropriate α-amino acid Ncarboxyanhydride (NCA) monomers. In addition to the fact that they are potentially biodegradable, polypeptide brushes are also attractive because the constituent polymer chains can adopt ordered secondary structures such as α-helices or βsheets. This is in contrast to polymer brushes prepared from vinyl-type monomers, which typically adopt a random coil conformation. The ability to switch the conformation of a surface-grafted polypeptide chain using variations in e.g. pH, temperature, or ion strength provides opportunities for the development of stimuli-responsive surface coatings.12−15 Poly(L-glutamic acid) brushes, for example, transition from an αhelical to a random coil conformation when the pH is increased from 4 to 8. These pH-induced conformational changes have been used to develop polypeptide brush-coated membranes that show pH-dependent water permeabilities.16 In addition, αhelical polypeptides possess a dipole moment, and surfaceinitiated polymerization allows to generate dense arrays of parallel aligned helical polypeptides that display interesting electric17 and piezoelectric properties.18 Furthermore, surfaceinitiated NCA polymerization has been used to generate polymer brush based platforms for bioconjugation,19,20 for the formation of templates to mimic biosilification,21 and for the modification of poly(vinylidene fluoride) membranes with a coating that enables chiral separation.22 Most of the efforts that focus on polypeptide brushes, including those cited above, consist of systems composed of natural amino acids or well-established and commercially available side chain protected precursors such as e.g. γ-benzylL-glutamate and ε-benzyloxycarbonyl-L-lysine. Side chain modification of amino acids, however, offers a broad range of possibilities to add further functionality to synthetic polypeptides prepared via NCA ring-opening polymerization. While this strategy has found widespread application for the synthesis of soluble polypeptides, it has only found limited use so far for the preparation of surface-grafted polypeptide brushes.23 One interesting example of the use of side chain functionalization to enhance the functionality of synthetic polypeptides are thermoresponsive polypeptides. Recently, it was demonstrated that poly(L-glutamate) modified with short oligo(ethylene glycol) chains (poly(L-EGx-Glu)) shows lower critical solution temperature (LCST) behavior similar to that of poly(poly(ethylene glycol) methyl ether methacrylate) (PPEGMEMA).24 However, in contrast to vinyl polymers such as PPEGMEMA and poly(N-isopropylacrylamide) (PNIPAM) where the polymer main chain collapses upon increasing the temperature above the LCST, poly(L-EGx-Glu) retains an α-helical secondary structure also above the LCST. In the case of poly(L-EGx-Glu), the aggregation in dilute solution is proposed to be due to collapse and dehydration of the EGx side chains. Thermoresponsive polymers also provide manifold opportunities to develop thin polymer coatings that allow surface properties such as wettability and adhesion as well as capture and release to be reversibly switched by cycling above and below the LCST.25,26 Polymer brushes generated from conventional thermoresponsive monomers such as NIPAM and PEGMEMA collapse and dehydrate and concomitantly show a decrease in film thickness upon increasing the temperature above the LCST. In this contribution, we will show that the peculiar LCST behavior and secondary structural

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EXPERIMENTAL SECTION

Materials. (3-Aminopropyl)triethoxysilane (APS) was obtained from Acros. L-Glutamic acid was obtained from Sigma. D-Glutamic acid was obtained from Bachem. Diethylene glycol monomethyl ether was obtained from Alfa Aesar. Other reagents were purchased from Aldrich. All chemicals were used as received unless otherwise noted. Tetrahydrofuran (THF) and dichloromethane (DCM) were purified by purging with dry nitrogen, followed by passing through active alumina columns. Toluene was dried by passing through an alumina and a copper column. Dimethylformamide (DMF) was dried by passing through two columns of molecular sieves on a Pure Solv 400 solvent purification apparatus. Ultrapure water was obtained from a Millipore Milli-Q gradient system fitted with a 0.22 μm filter. Silicon wafers were provided by the Center of Micronanotechnology of EPFL and were cut into pieces of size 1 × 0.8 cm2. γ-(2-(2-Methoxyethoxy)ethyl)-L-glutamate (L-EG2-Glu), γ-(2-(2-methoxyethoxy)ethyl)-D-glutamate (D-EG2-Glu), γ-(2-(2-methoxyethoxy)ethyl)-racglutamate (rac-EG2-Glu), γ-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)-Lglutamate (L-EG3-Glu), and their corresponding NCAs were synthesized as reported previously.24 The synthesis and immobilization of 6-(2-(2-bromo-2-methyl)propionyloxy)hexyldimethylchlorosilane, which was used for the surface-initiated ATRP of poly(diethylene glycol methyl ether methacrylate) (PPEG2MEMA), have also been described before.27 Methods. Grazing angle attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy was performed on a nitrogen-purged Nicolet 6700 FT-IR spectrometer equipped with a VariGATR grazing angle ATR accessory (Harrick Scientific Products Inc., Pleasantville, NY) with a fixed incident angle of 60°. The spectrum of a clean silicon wafer was used as a reference. X-ray photoelectron spectroscopy (XPS) was carried out using an Axis Ultra instrument from Kratos Analytical equipped with a conventional hemispheric analyzer. Brush thicknesses were measured using a Sopra GES 5E spectroscopic ellipsometer. All calculations were done with a three-layer silicon/polymer brush/ambient model, assuming the polymer brushes to be homogeneous and isotropic. A second set of ellipsometry measurements designed to measure swelling behavior was made using a variable-angle Beaglehole picometer ellipsometer that uses a He−Ne laser light source (λ) 632.8 nm. Each brush-modified surface was measured in air and in a cylindrical fluid cell at multiple angles ranging from 80° to 60° using 1° steps using previously published protocols.28 Three different locations were measured on each sample, and four replicate samples were used. To fit the data acquired for the brushes measured in air, a Cauchy model was used assuming a single layer with a refractive index of 1.5. Measurements in water were made at 25 and 50 °C. To fit data acquired from the swollen brushes, both the thickness and refractive index are allowed to vary with the refractive index constrained between water (η = 1.33) and polymer (η = 1.5).25 It should be noted that ellipsometry is not sensitive to the detailed shape of the segment density profile, and in each case (dry and solvated) the brushes are modeled as if they have a boxlike segment density profile. While a boxlike density profile is a suitable model for a collapsed brush, the sharpness of the segment density profile diminishes as the degree of swelling of the chains increases. As a result, the discrepancy between the thickness as the first moment of the segment density profile (boxlike model), and the true swollen extent of the chains grows as the degree of swelling increases. Water contact angles were recorded on a Dataphysics OCA30 B


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Macromolecules semiautomatic contact angle measurement instrument using Milli-Q water. Quartz crystal microbalance with dissipation (QCM-D) measurements were conducted on a Q-Sense E4 system using SiO2coated quartz crystals purchased from Q-Sense. The quartz sensor was mounted in a flow module with one side exposed to Milli-Q water. The temperature was increased step by step and equilibrated for at least 40 min at each step. A clean quartz sensor was used as reference, and data were background corrected by subtracting the signal from the reference crystal. Circular dichroism (CD) spectra were recorded on a Jasco J-715 spectropolarimeter equipped with a Jasco PTC-348 WI temperature controller. For these experiments, polymer brushes were grown from quartz wafers. To record the spectra, the substrates were placed in a 1 cm path length quartz cuvette and immersed in Milli-Q water. Each spectrum was recorded after equilibrating the sample at the desired temperature for 25 min. AFM measurements were performed on a Multimode Nanoscope IIIa (Bruker) equipped with a liquid cell and a thermal application controller. The cantilever spring constants were determined using their thermal spectra. Tapping mode was used to obtain topography images. The measurements were carried out in air or in Milli-Q water. To measure the thickness of PPEG2MEMA brushes, a patterned brush was prepared using the method reported before.29 To determine the thickness of poly(EGxGlu) brushes, a needle was used to make scratch on the polypeptide film. While the scratch method is suitable for measuring the thickness of dry brushes, the measurement and interpretation of swollen brush thickness are complicated by the physical situation. The dilution (swelling) of the chains coupled with their favorable interactions with the solvent likely requires that the probe tip penetrate into the layer before a sufficient number of segments interact with the tip to produce a meaningful deflection as it is rastered through the brush. Procedures. Initiator Immobilization. First, silicon wafers were cleaned by sonication in water for 15 min and then in acetone for 15 min, followed by drying under a stream of nitrogen. The silicon wafers were subsequently exposed to oxygen plasma for 15 min. The clean wafers were then immersed in a 2% v/v solution of (3-aminopropyl)triethoxysilane (APS) in toluene for 1.5 h at room temperature. After this time, the wafers were removed and extensively rinsed with toluene and then methanol and dried under a stream of nitrogen. Silicon oxidecoated QCM chips and quartz wafers were modified in a similar way. Vapor Deposition Surface-Initiated Ring-Opening Polymerization. Vapor deposition surface-initiated ring-opening polymerizations were performed following a procedure similar to that described by Chang and Frank.30 Typically, 15 μL of the NCA monomer was spread on the bottom of a Schlenk tube, and an APS modified wafer was placed horizontally above the monomer. The distance between the wafer and the monomers was about 6 mm. The Schlenk tube was evacuated down to 0.5 mbar and then immersed in an oil bath that was preheated at 65 °C. The Schlenk tube was continuously evacuated to keep the vacuum of the system the same during the reaction. After the polymerization, the silicon wafers were sonicated for 5 min in DMF (3 times) and then immersed in DMF for 12 h. Finally, the wafers were thoroughly rinsed with THF and dried under nitrogen. SI-ATRP of PPEG2MEMA. Poly(diethylene glycol methyl ether methacrylate) (PPEG2MEMA) brushes were prepared via SI-ATRP following a published procedure and therefore are not repeated here.31

Chart 1. Chemical Structures of the Polymer Brushes Used in This Study

The PPEG2MEMA brushes are used as a reference to elucidate the influence of polymer backbone conformation on the properties of polymer brushes. Scheme 1 outlines the synthesis of the poly(EGx-Glu) brushes. In a first step, APS was reacted with a clean wafer to produce an amine-functionalized surface. The polypeptide brushes were subsequently grown from these surfaces via a vapor deposition protocol developed earlier by Chang and Frank.30 This vapor deposition protocol was used, since attempts to grow polypeptide brushes by immersing APSmodified substrates in solutions of the corresponding NCA monomers were not successful. The difficulties with the solution-based SI-ROP of EGx-Glu NCA may be attributed to impurities present in the monomers. The EGx-Glu NCA monomers are viscous oils at room temperature and hard to purify by recrystallization. In addition, traces of moisture introduced by the solvent and atmosphere can also terminate the reaction prematurely. The use of the vapor deposition SIROP protocol allowed poly(L-EG2-Glu) brushes with thicknesses up to 80 nm to be successfully prepared. As an example, Figure 1 illustrates the growth of the poly(L-EG2-Glu) and poly(L-EG3-Glu) brushes as a function of time. In both cases, an almost linear dependence of the polypeptide film thickness as a function of polymerization time is observed for the first 15 min. Compared to poly(L-EG2-Glu), the final thickness of the poly(L-EG3-Glu) brushes is much smaller. The difference in growth profiles reflects a lower reactivity of EG3-Glu NCA, which may be due to a larger steric hindrance resulting from the increased length of the ethylene glycol side chain as compared to the EG2-Glu-based monomer. Furthermore, it is worth pointing out that the final thickness of the poly(rac-EG2-Glu) brushes is about half of that of the poly(L-EG2-Glu) brushes, even after prolonged reaction times, i.e., 43 nm after 1 h. In contrast to the poly(L-EG2-Glu) brushes where the α-helical secondary structure (vide inf ra) enforces the presentation of the N-terminal amine groups that mediate chain growth at the brush interface, the unordered secondary structure of the poly(rac-EG2-Glu) chains may result in some degree of burying/trapping of part of these amine groups in the polymer brush, thereby reducing their availability to mediate further chain growth. Figure S1 in the Supporting Information shows AFM topography scans as well as cross-sectional profiles of poly(rac-EG2-Glu) and poly(L-EG3-Glu) brush films, which indicate that these polypeptide brushes form relatively smooth films in water. The polypeptide brushes were characterized by XPS and FTIR spectroscopy. Figure 2 shows XPS survey spectra and high-resolution C1s scans of APS as well as poly(L-EG2-Glu) and poly(L-EG3-Glu) brush modified silicon substrates. The survey spectra show the presence of O1s, N1s, and C1s signals,

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RESULTS AND DISCUSSION Chart 1 illustrates the chemical structure of the polymer brushes prepared and investigated in this study. The poly(LEGx-Glu) and poly(D-EGx-Glu) brushes have identical chemical composition, but as they are composed of the Land D-isomers of EGx-Glu, they are anticipated to form helical secondary structures of opposite handedness. Poly(rac-EGxGlu) brushes in contrast are not expected to possess an ordered secondary structure. The PPEG2MEMA brushes have the same ethylene glycol methyl ether side chain as the poly(EGx-Glu)based polypeptide brushes but are based on a polymethacrylate backbone that does not adopt a regular secondary structure. C


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Scheme 1. Preparation of Poly(EGx-Glu) Brushes via Vapor Deposition, Surface-Initiated Ring-Opening Polymerization: (a) Silanization of the Silicon Wafer with APS; (b) Ring-Opening Polymerization of NCA Monomers To Form Poly(EGx-Glu) Brushes

Figure 1. Evolution of film thickness, as measured by ellipsometry, as a function of time for the surface-initiated vapor deposition ring-opening polymerization of (■) L-EG2-Glu NCA and (●) L-EG3-Glu NCA. The line in the figure is used to guide the eye for the first 15 min.

which is in agreement with the structures of APS as well as the poly(L-EG2-Glu) and poly(L-EG3-Glu) brushes. As shown in Figure 2A, the high-resolution C1s scan of the APS-modified silicon wafer can be deconvoluted in two signals with relative areas of 1:2, which is in agreement with the chemical composition of the APS-modified surface. The C1s highresolution scans of the poly(L-EG2-Glu) and poly(L-EG3-Glu) brushes that are presented in Figures 2B and 2C are more complex, but they can be deconvoluted in four signals with the expected peaks areas. The polypeptide brush C1s scans are characterized by an ester CO contribution at 289.3 eV (signal “d”), an amide CO peak at 288.0 eV (signal “a”), and a signal (indicated as peak “b”) that contains the contribution of the ethylene glycol ether carbons, the intensity of which increases upon extending the side chain length by one additional ethylene glycol unit from poly(L-EG2-Glu) to poly(L-EG3-Glu). Evidence for the ability of the polypeptide brushes to adopt secondary structures, exclusively comes from FTIR and CD spectroscopies. Figure 3A compares the 1800−1500 cm−1 region of the FTIR spectra of poly(L-EG2-Glu), poly(D-EG2Glu), and poly(rac-EG2-Glu) brushes. Full FTIR spectra of poly(L-EG2-Glu) and poly(D-EG3-Glu) brushes are included in the Supporting Information (Figure S2). In addition to providing additional support for the chemical structure of the brushes, FTIR spectroscopy also provides insight in the conformation of the surface-grown polypeptide chains. The appearance of the amide A absorbance (backbone N−H

Figure 2. XPS survey spectra and C1s high-resolution scans of (A) an APS-modified silicon substrate (APS layer thickness = 3.2 nm), (B) a poly(L-EG2-Glu) brush modified substrate (polypeptide film thickness = 78 nm), and (C) a poly(L-EG3-Glu) brush (modified silicon substrate (polypeptide film thickness = 24 nm) (all film thicknesses are ellipsometric film thicknesses).

stretching), ethylene (C−H stretching), CO ester, amide I (backbone carbonyl stretching), and amide II (mainly C−N stretching) indicates the successful grafting of the polypeptide brushes. The FTIR spectra of the poly(L-EG2-Glu) and poly(D-EG2-Glu) brush films reveal amide I and amide II bands at 1653 and 1548 cm−1, respectively, which are indicative of an α-helical secondary structure.32 The shift of the amide I band from 1653 cm−1 for the poly(L-EG2-Glu) and poly(DEG2-Glu) films to 1660 cm−1 for the poly(rac-EG2-Glu) brush suggests the existence of random coil conformation.33 The D


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Figure 3. (A) FTIR and (B) CD spectra of poly(L-EG2-Glu) (thickness = 78 nm), poly(D-EG2-Glu) (thickness = 75 nm), and poly(rac-EG2-Glu) (thickness = 43 nm) brush films (all film thicknesses are ellipsometric film thicknesses).

FTIR spectra thus provide direct evidence for the α-helical secondary structure of the poly(L-EG2-Glu) and poly(D-EG2Glu) brushes and also show that SI-ROP of the racemic NCA monomer leads to polypeptide brush films that do not possess an ordered secondary structure. CD spectroscopy provides further insight into the secondary structure of the surface-grafted polypeptides (Figure 3B). The CD spectrum of the poly(rac-EG2-Glu) brush does not show any characteristic signals, which is in agreement with the absence of an ordered secondary structure of these surface grafted polypeptide chains. Analysis of the poly(L-EG2-Glu) and poly(D-EG2-Glu) brush films, in contrast, reveals CD spectra that are characteristic for α-helical polypeptides. The opposite signal intensities for the poly(L-EG2-Glu) and poly(DEG2-Glu) brushes reflect the difference in handedness of the Land D-based helices. Figures 4 and 5 present CD spectra of poly(L-EG2-Glu) and poly(L-EG3-Glu) brushes that were recorded at temperatures varying from 10 to 70 °C. While Figure 5. CD spectra of a 24 nm thick poly(L-EG3-Glu) brush film recorded at different temperatures.

the intensity of the spectra slightly decreases with increasing temperature (i.e., become less negative) they retain their characteristic α-helical signature (minima at 208 and 222 nm) over the entire temperature range, illustrating the “shape persistence” of the polypeptide brushes. The temperature responsiveness of the poly(EG2-Glu) and poly(EG3-Glu) brushes was studied with QCM-D and compared with that of a PPEG2MEMA brush. Figure 6 shows the results of QCM-D experiments with poly(L-EG2Glu), poly(L-EG3-Glu), PPEG2MEMA, and poly(rac-EG2-Glu) brushes exposed to water at a range of temperatures between 10 and 40 °C. The frequency shift Δf for all samples increases with increasing temperature, which is due to the dehydration of the brushes. The dissipation shift ΔD decreases with increasing temperature, indicating the increasing rigidity of these polymer films as they dehydrate. From the inflection point of the frequency shift curves, a collapse temperature (Tc) was estimated, which is indicated in each of the plots presented in Figure 6. For the poly(L-EG2-Glu) brush, the collapse

Figure 4. CD spectra of a 78 nm thick poly(L-EG2-Glu) brush film recorded at different temperatures. E


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Figure 6. Temperature dependence of the third overtone of the frequency shift (Δf) and of the dissipation shift (ΔD) for (A): a poly(L-EG2-Glu) brush (thickness = 78 nm), (B) a poly(L-EG3-Glu) brush (thickness = 24 nm), (C) a PPEG2MEMA brush (thickness = 78 nm), and (D) a poly(racEG2-Glu) brush (thickness = 43 nm) as measured by QCM-D in water (all film thicknesses are ellipsometric film thicknesses).

temperature was estimated to ∼25 °C, which is slightly lower compared to the solution LCST of 32 °C that has been reported for this polymer.24 Poly(L-EG3-Glu) in solution has been reported to display a solution LCST of 57 °C.24 This is outside the range of temperatures accessible with the QCM-D setup that was used, but the evolution of Δf in Figure 6B is in agreement with a collapse temperature >40° for the poly(LEG3-Glu) brush. The QCM-D results obtained on a 78 nm thick PPEG2MEMA brush are very similar to those measured for a poly(L-EG2-Glu) brush of comparable thickness, revealing a Tc of ∼25 °C, which is slightly lower than the bulk LCST of 26 °C34 but in good agreement with the value of 22.6 °C reported earlier by Laloyaux et al. on a 100 nm thick PPEG 2 MEMA brush. 3 5 The poly(L-EG 2 -Glu) and PPEG2MEMA brushes have identical side chains but differ in the polymer main chain chemistry. The poly(EG2-Glu) brush has a polypeptide backbone, which potentially can undergo hydrogen-bonding interactions with water. The α-helical secondary structure of the surface-tethered poly(L-EG2-Glu) brush chains, however, minimizes the interactions with water since the hydrogen bonds are used to stabilize the α-helix. As a consequence, hydration/dehydration mainly involves the EG2 side chain as it is the case for the PPEG2MEMA brushes. The similarity in the collapse temperature of poly(L-EG2-Glu) and PPEG2MEMA underlines that the thermoresponsiveness of these polymer brush films is primarily due to the hydration/ dehydration properties of the EG2 side chains. The poly(racEG2-Glu) brush shows a much higher collapse temperature as

compared to the poly(L-EG2-Glu) and PPEG2MEMA brushes (>40 °C). The surface-tethered poly(rac-EG2-Glu) chains, however, do not possess a regular secondary structure, and as a consequence, the main chain amide bonds (in addition to the EG2 side chains) can also undergo hydrogen bonding with water, leading to the observed increase in the collapse temperature. The dehydration of the polypeptide brushes can also be monitored by measuring the water contact angles of these films at different temperatures. As an example, Table S1 in the Supporting Information summarizes water contact angles recorded for poly(L-EG2-Glu) and poly(L-EG3-Glu) brushes at 20 and 60 °C. The increase in water contact angles upon increasing the temperature from 20 to 60 °C is in agreement with the dehydration that can be inferred from the QCM-D experiment. The thermoresponsive behavior of the different polypeptide brushes as well as the PPEG2MEMA reference sample was further investigated by measuring the film thickness of the different samples in water at different temperatures. Film thicknesses were determined using both AFM (at 10 and 40 °C) as well as by in situ multiangle ellipsometry (at 25 and 50 °C). As discussed in the Experimental Section, while the thicknesses of strongly swollen brushes measured by AFM and ellipsometry have limitations in probing the smoothly decaying segment density distribution at the periphery,36 nevertheless they provide useful insight into structural changes. The results of these experiments are summarized in Tables 1 and 2 as well as in Supporting Information Tables S2−S4. We note that film F


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CONCLUSIONS In this article, we have described the use of vapor deposition surface-initiated N-carboxyanhydride ring-opening polymerization to prepare thermoresponsive polypeptide brushes that are composed of a polyglutamate backbone and substituted with oligo(ethylene glycol) side chains. Poly(L-EG2-Glu), poly(D-EG2-Glu), and poly(L-EG3-Glu) brushes produced in this way are densely packed arrays of surface-tethered helical peptides, which retain their secondary structure over the entire investigated temperature range (10−70 °C). QCM-D measurements revealed that poly(L-EG2-Glu) brushes significantly dehydrate upon heating from 10 to 40 °C (Tc ∼ 25 °C), reminiscent of the behavior of various thermoresponsive vinyl polymers that collapse and dehydrate upon heating above their lower critical solution temperature. In contrast to conventional thermoresponsive polymers, however, poly(L-EG x -Glu) brushes are shape persistent, i.e., they retain their ordered helical secondary structure upon heating, and the observed changes in (surface) properties are due to collapse of the oligo(ethylene glycol) side chains. The results presented herein illustrate the potential of surface-initiated N-carboxyanhydride polymerization to generate densely grafted polymer brush films that possess ordered secondary structures and tunable surface properties. The combination of the putative (piezo)electric properties of the helical peptide backbone with the PEG-like side chains may offer prospects to explore these polypeptide brushes as platforms for e.g. bioelectronic interfaces.

Table 1. Thickness of Different Polymer Brushes Measured by AFM in Air or in Water thickness (nm) sample

in air (RT)

in water (10 °C)

in water (40 °C)

poly(L-EG2-Glu) PPEG2MEMA

59 ± 8.1 62 ± 2.3

93 ± 9.6 125 ± 1.7

128 ± 4.6 82 ± 0.6

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thicknesses measured in air are nominally referred to as “dry” film thicknesses, in reference to the fact that hydrophilic brushes in air absorb some moisture from the ambient.36 The film thicknesses determined by AFM both at 10 and 40 °C in water are measurably larger than the dry film thicknesses (Table 1). While the absolute thickness measured when brush chains are highly solvated may not be an accurate measure of the true extent of stretching of those chains, particularly in the case of the poly(L-EG2-Glu) brush, where 10 °C is far below the Tc estimated by the QCM-D experiments (vide supra) (25 °C), the behavior is qualitatively different from that of the PPEG2MEMA brush, where a decrease in thickness with increasing temperature is observed. This decrease in thickness is in agreement with the nonordered secondary structure of the polymer backbone and reflects the dehydration induced collapse of these chains at the LCST (or, more accurately, the volume phase transition temperature). In contrast, while it appears that the thickness of the poly(L-EG2-Glu) brush did not decrease upon increasing the temperature from 10 to 40 °C, we again note that this may be more due to the sensitivity of the method than reflecting the absolute height of the brush. At this point it is also important to reiterate that secondary structural information on the surface-grafted polypeptide chains and the primary evidence for the shape-persistence of the poly(L-EG2-Glu), poly(D-EG2-Glu), and poly(L-EG3-Glu) brushes comes from FTIR and CD spectroscopy. The ellipsometric dry film thicknesses reported in Table 2, which represent the average of four replicate samples, are in good agreement with the AFM data reported in Table 1. Note, however, that, as seen from the dry film thickness, Tables 1 and 2 report data from different sets of samples. The small standard deviation for each brush indicates both the consistency of multiple measurements across a given sample and the reproducibility of brush growth on multiple surfaces. The results from the ellipsometric measurements indicate that the brushes swell by a factor of ∼2 when solvated by water (at 25 °C). (Ellipsometry measurements at temperatures below room temperature were complicated by the problem of condensation on the fluid cell.) In contrast to the AFM thickness measurements, the ellipsometric film thicknesses determined at 25 and 50 °C do not reveal any significant differences. This is likely due to the fact that the lowest possible temperature in these experiments was 25 °C, which is already within the collapse regime, whereas the AFM measurements could be performed as low as at 10 °C, far below the collapse temperature and well in the swollen regime.

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ASSOCIATED CONTENT

* Supporting Information S

IR spectra, AFM images, ellipsometry data, and water contact angles. 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 harm-anton.klok@epfl.ch (H.-A.K.). *E-mail [email protected] (Z.L.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the Sino-Swiss Science and Technology Cooperation (project EG41-092011) as well as the Chinese Academy of Sciences (Visiting Professorship for Senior International Scientists to H.-A.K.). B.A. and S.M.K. gratefully acknowledge support from the National Science Foundation (Award No. 1133320). A portion of this research was conducted at the Center for Nanophase Materials Sciences, which is a DOE Office of Science User Facility.

Table 2. Average Dry and Swollen Film Thicknesses Measured by Ellipsometry

a

sample

dry thickness (nm)

swollen thickness at 25 °C (nm)

na

swelling ratiob

swollen thickness at 50 °C (nm)

na

PPEG2MEMA poly(D-EG2-Glu) poly(L-EG2-Glu)

53 ± 1 65 ± 9 57 ± 5

117 ± 16 133 ± 11 130 ± 10

1.40 ± 0.005 1.42 ± 0.010 1.41 ± 0.006

2.22 ± 0.34 2.03 ± 0.45 2.27 ± 0.38

114 ± 11 128 ± 6 128 ± 4

1.40 ± 0.007 1.41 ± 0.007 1.41 ± 0.005

Refractive index. bRatio of swollen to dry thickness (at 25 °C). G


Article

Macromolecules

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REFERENCES

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