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Synthesis and Postpolymerization Modification of Thermoresponsive Coatings Based on Pentaerythritol Monomethacrylate: Surface Analysis, Wettability, and Protein Adsorption Yurij Stetsyshyn,*,† Joanna Raczkowska,*,‡ Andrzej Budkowski,‡ Andrij Kostruba,§,▽ Khrystyna Harhay,† Halyna Ohar,† Kamil Awsiuk,‡ Andrzej Bernasik,∥,#,⊥ Nazar Ripak,† and Joanna Zemła‡ †

“Lvivska Polytechnika” National University, S. Bandery 12, 79013 Lviv, Ukraine Smoluchowski Institute of Physics, Jagiellonian University, Łojasiewicza 11, 30-348 Kraków, Poland § Lviv Academy of Commerce, Samtshuk 9, Dragomanov 19, 79011 Lviv, Ukraine ▽ Lviv Institute for Physical Optics, Dragomanov 19, 79011 Lviv, Ukraine ∥ AGH University of Science and Technology, Faculty of Physics and Applied Computer Science, al. A. Mickiewicza 30, 30-059 Kraków, Poland # Faculty of Physics and Applied Computer Science and ⊥Academic Centre for Materials and Nanotechnology, AGH University of Science and Technology, Mickiewicza 30, 30-059 Kraków, Poland ‡

S Supporting Information *

ABSTRACT: Properties of novel temperature-responsive hydroxyl-containing poly(pentaerythritol monomethacrylate) (PPM) coatings, polymerized from oligoperoxide grafted to glass surface premodified with (3-aminopropyl)triethoxysilane, are presented. Molecular composition, chemical state, thickness, and wettability are examined with time of flight-secondary ion mass spectrometry (ToF-SIMS), X-ray photoelectron spectroscopy (XPS), ellipsometry, and contact angle measurements, respectively. Temperatureinduced changes in hydrophobicity of grafted PPM brushes are revealed by water contact angle and ellipsometric measurements. Partial postpolymerization modification of hydroxyl groups (maximum a few percent), performed with acetyl chloride or pyromellitic acid chloride, is demonstrated to preserve thermal response of coatings. Adsorption of bovine serum albumin to PPM brushes, observed with fluorescence microscopy, is higher than on glass in contrast to similar hydroxyl-containing layers reported as nonfouling. Enhanced and temperature-controlled protein adsorption is obtained after postpolymerization modification with pyromellitic acid chloride.

1. INTRODUCTION In the last decades, thermoresponsive polymers have drawn growing interest, mainly due to their potential applications in medicine and biotechnology, e.g., for fabrication “smart” nanocontainers, nanopatterns, biomedical devices, and intelligent surfaces.1−6 Considerable part of performed research, broadly examined in two recent reviews7,8 has been devoted to thermoresponsive polymer brushes grafted to solid surfaces. Such brushes are required for production of selective membranes, sensors, and separation systems, for targeted drug delivery and nanoparticle translocation as well as for fabrication of mechanical actuators and transducers.1,2,6,8 Among diversities of thermoresponsive polymers, special attention should be paid to the polymers with hydroxyl groups,9−11,15−20 as they offer two advantages. First, the polymers with hydroxyl fragments have low toxicity as a rule.12−14 For example, poly(2,3-dihydroxypropyl methacrylate) and poly(2-hydroxyethyl methacrylate) have been used to prepare biocompatible amphiphilic networks and soft contact lenses.15,16 Second, these polymers can be partly modified, © 2015 American Chemical Society

altering lower critical solution temperature (LCST) and inducing novel functionalities. For instance, esterification of polyglycidol with acetate groups leads to LCST that varies between 0 and 100 °C.17 Moreover, free hydroxyl groups are used to attach biomolecules. In our previous papers,21,22 we have reported the method of fabrication for thermoresponsive coatings of PNIPAM or POEGMA246 brushes grafted “from the surface” of oligoperoxides. Moreover, we have synthesized and investigated temperature-responsive polymeric brushes of poly(N-methacryloylleucine).23 This strategy is extended in this work, where the brushes “from the surface” of the oligoperoxide were fabricated by grafted polymerization of pentaerythritol monomethacrylate (PM). The poly(pentaerythritol monomethacrylate)−PPM brushes exhibit temperature sensitivity that has not been reported previously for this polymer. Received: February 18, 2015 Published: August 7, 2015 9675

DOI: 10.1021/acs.langmuir.5b02285 Langmuir 2015, 31, 9675−9683

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Langmuir Postpolymerization modification of grafted polymer brushes, transforming old and introducing new functional moieties, has been widely recognized as a route to fabricate the coatings for novel applications.24−29 In this work, postpolymerization reaction of the grafted PPM brushes not only preserves thermal response of synthesized coatings but also enhances protein adsorption or enables its temperature control. Molecular composition, chemical state, thickness, and wettability of resulting coatings were analyzed using time of flight-secondary ion mass spectrometry (ToF-SIMS), X-ray photoelectron spectroscopy (XPS), ellipsometry, and contact angle measurements, respectively. Protein adsorption to the coatings was observed with fluorescence microscopy. As a result, transition of surface properties as well as the effect of the postpolymerization modifications of PPM brushes on the temperature-responsive wettability and protein adsorption were demonstrated.

Scheme 1. Synthesis of Pentaerythritol Monomethacrylate

temperature, and then the solution was neutralized by addition of 1g of KOH, resulting in the formation of two separated layers. The supernatant water phase was concentrated to 150 mL, and then the resulting precipitate was filtered, washed with ethanol, and subsequently redissolved in water and reprecipitated in ethanol. The obtained product was purified by chromatography. Dried in vacuum (100−200 Pa) at 40 °C for 3 h. The yield was 18 g (80%). Chemical structure of the synthesized monomer was confirmed by IR (Figure S1a, Supporting Information), 1H NMR spectroscopies, and elemental analysis. The presence of methacrylic fragments was confirmed by a characteristic esteric (C−O−C), carbonyl (CO), and (CC) absorption bands of vibrations at 1250 and 1150, 1712, and 1650 cm−1, respectively. Additionally, the measured intensity of the band at 3320 cm−1, corresponding to OH groups, is significantly decreased in the spectrum of the pentaerythritol monomethacrylate, as compared to the spectrum of pentaerythritol. NMR analysis, performed to confirm the chemical structure of polymerized PPM yields 1H NMR (300 MHz, DMSO-d6), ppm: 1.92 (3H, CH3); 3.28 (3H, 3OH); 3.80 (2H, CH2); 4.42 (6H, 3CH2); 5.57 (1H, CH2); 6.10 (1H, CH2). Elemental analysis was performed on standard equipment for microanalysis. Calculated (C9H16O5): C, 52.94%; H, 7.84%. Found: C, 53.21%; H, 7.93%. 2.2. Preparation of Coatings. 2.2.1. Modification of Glass Surfaces with Oligoperoxides. The procedure for the preparation of PPM coatings is presented in Scheme 2. First, glass plates (20 × 20 mm) (1) were dipped into a 0.2% (w/w) methanolic solution of APTES for 24 h. After the incubation, loosely attached silane molecules were removed with methanol in a Soxhlet apparatus. Then the APTES-functionalized plates (2) were dipped into a 1% solution of oligoperoxide in dry dioxane for a certain period (grafting time of 24 h). Similarly, loosely attached oligoperoxides were removed with dioxane in a Soxhlet apparatus during 1 h. As a result, oligoperoxides grafted to aminated surfaces were obtained (3).21,22,34 2.2.2. Polymerization of PPM Brushes. Glass plates with grafted oligoperoxides (step 3 in Scheme 2) were placed in a container with a 0.1 M aqueous solution of the monomer PM and heated under argon atmosphere at 90 °C for a certain period (polymerization time from 20 to 48 h), resulting in the oligoperoxide-graf t-PPM coatings (4). Then the plates coated with polymerized PPM were washed with water in a Soxhlet apparatus for 4 h and dried. 2.2.3. Postpolymerization Modification Reactions. 2.2.3.1. Modification of PPM Brushes with Acetyl Chloride (PPM-AE). Glass plates with grafted PPM coatings were placed into a 1% solution of acetyl chloride in dry dioxane cooled previously to 5 °C for a certain period (modification time of 5 h). Then the plates were rinsed with water. As a result, partially acetylated PPM brushes were obtained. 2.2.3.2. Modification of PPM Brushes with Pyromellitic Acid Chloride (PPM-PE). Glass plates with a grafted PPM coating were placed into a 1% solution of pyromellitic acid chloride in dry dioxane cooled previously to 5 °C for a certain period (modification time of 5 h). In the next step, plates were rinsed by water. As a result, PPM brushes partially modified by pyromellitic ester were obtained. 2.3. Characterization of Coatings. 2.3.1. Water Contact Angle Measurements (CA). Static contact angle experiments were performed by the sessile drop technique using a Kruss EasyDrop (DSA15) instrument with a Peltier temperature-controlled chamber. The measurements with distilled water (pH ≈ 7) were carried out at temperatures ranging from 4 to 30 °C to study the thermal response of the oligoperoxide-graft-PPM coatings. Contact angles were determined as the average of 10 measurements at different spots.

2. EXPERIMENTAL SECTION 2.1. Materials. Pyridine and other organic solvents were purified as reported by Weissberger et al.30 Poly(ethylene glycol) (PEG-9) was supplied by Merck Chem. Co. 3-Aminopropyltriethoxysilane (APTES) was supplied by Sigma-Aldrich. Bovine serum albumin (BSA) labeled with fluorescein isothiocyanate was purchased from Biokom (Poland). tert-Butyl hydroperoxide was obtained31 and purified by vacuum rectification. The fraction boiling in the temperature range of 45−47 °C (at 1.6 kPa) was collected. It had a refractive index nd20 = 1.40020 ± 0.00002, in accord with previous reports (nd20 = 1.401031). Pyromellitic acid chloride: In a 500 mL round-bottomed flask equipped with a thermometer and a reflux condenser, connected with water scrubber, 43.6 g (0.2 mol) of pyromellitic dianhydride and 91.6 g (0.44 mol) of PCl5 were mixed and boiled in a oil bath until the mixture became homogeneous. Afterward, it was continuously mixed for 15−16 h at 130−135 °C. The reflux condenser was then replaced by a Liebig condenser, and approximately 60−63 g of POCl3 was distilled off during 8 h. Then the temperature of the mixture was increased to 180−185 °C. The crude product was then recrystallized from gasoline, yielding 51.2 g (78.1%) of a colorless crystalline product with a melting point of 67 °C (in accord with the literature value 68 °C32) and the acid number AN = 1373 mg KOH/g (calculated value is 1368 mg KOH/g). Oligoperoxide with residual acid chloride groups: 4.6 g (0.014 mol) of pyromellitic acid chloride was dissolved in 15 mL of anhydrous dichlorethane and placed in a three-necked flask equipped with stirrer, and 1.26 g (0.014 mol) of tert-butyl hydroperoxide was added. The mixture was cooled to 5 °C, and then 1.1 g (0.014 mol) of pyridine, dissolved in 10 mL of anhydrous dichlorethane, was added dropwise at 5 °C. Then the suspension was mixed for 1 h. Subsequently, 5.6 g (0.014 mol) of PEG-9 was added, and again a solution of 2.2 g (0.028 mol) of pyridine in 10 mL of anhydrous dichlorethane was admixed dropwise. The mixture was then stirred for another 3 h, and the temperature was raised gradually up to room temperature. A precipitate of pyridinium chloride was filtered out. The solvent was distilled out, and the pellet was dried in vacuum (100−200 Pa) at 40 °C for 3 h, yielding 8.2 g (81%) of oligoperoxide. The pellet had a yellowish resin-like appearance. Its characteristics are summarized as follows: content of active oxygen, 1.9% (calcd 2.2%); content of active chlorine, 5.4% (calcd 4.9%); AN, 163.1 mg KOH/g (calculated value is 155.3 mg KOH/g); infrared spectra showed characteristic bands of ν (CO) in Ar−C(O)Cl, ν (CO) in ester group at 1760 and 1752 cm−1; doublet at 1390, 1365 cm−1, referring to d(C(CH3)3) and a band for a tert-butoxy group at 848 cm−1. Pentaerythritol monomethacrylate was synthesized similarly to the method described in ref 33, and its synthesis is sketched in Scheme 1. Pentaerythritol (15 g, 0.11 mol) was dissolved in 270 mL of 2.5 wt % KOH and stirred at 5 °C, while methacryloyl chloride (11.5 g, 0.11 mol) and 60 mL of 1,2-dichloroethane were added dropwise within 2 h. The mixture was stirred for 1 h at 5 °C and for 10 h at room 9676

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Scheme 2. Functionalization of a Glass Surface (1) with Amino-Terminated APTES Film (2), Followed by Subsequent Grafting of the Oligoperoxide (3) and Polymerization of PM, Initiated by Peroxide Groups of the Oligoperoxide, Resulting in PPM Brushes (4)

2.3.2. Ellipsometry. Measurements were carried out with a serial null-ellipsometer LEF-3 M equipped with the “polarizer−compensator−specimen-analyzer” arrangement, enabling angular positions of polarization elements to be determined within 0.01° precision. A He− Ne single-mode laser (light wavelength λ = 632.8 nm) was used as a light source. Polarization parameters of light reflected from a sample (angles Ψ and Δ) were determined using the two-zone technique (in the third and fourth measuring zone) for angle of incidence ϕ varied between 58° and 63° (with a 1° step). This ϕ-range, corresponding to the region of the pseudo-Brewster angle (where Δ ≈ π/2 or 3π/2), ensures maximal sensitivity. The iterative procedure using mono- and two-layer models was used to fit the (Ψ, Δ) data recorded at optimal experimental conditions35 and yield average thickness d and refractive index n for APTES and oligoperoxide-graft-PPM coatings, respectively. The molecular mass M (g/mol) of the grafted polymer brushes was calculated from ellipsometry data and kinetic parameters of polymerization using the following equation:

M=

calibration was performed with H, H2, CH, C2H2, and C4H5 peaks. The multiple ToF-SIMS spectra acquired to examine postpolymerization modification of PPM coatings were examined with principal component analysis (PCA) to enhance detection of subtle differences in the surface chemistry of the samples. 2.3.4. XPS. To verify the chemical state of analyzed samples, XPS measurements were carried out using a PHI 5000 VersaProbe II spectrometer (ULVAC-PHI, Chigasaki, Japan) with a microfocused (100 μm, 25 W) monochromatic Al Kα beam with a photoelectron takeoff angle of 45°. All XPS peaks were charge referenced to the neutral (C−C) carbon C 1s peak at 284.8 eV. The spectrum background was subtracted using the Shirley method. 2.3.5. Protein Adsorption. Bovine serum albumin (BSA) labeled with fluorescein isothiocyanate (adsorbing blue, λabs = 490 nm, and emitting green fluorescence light, λemit = 525 nm) was used as a model protein to examine adsorption to the oligoperoxide-graft-PPM and postpolymerization modified coatings (PPM-AE, PPM-PE) at different temperatures. A BSA solution with constant concentration, equal to 125 μg/mL, was prepared using phosphate buffer saline (PBS) as the neutral buffer (pH = 7.4). To examine protein adsorption, a 50 μL drop of protein solution was placed on the oligoperoxide-graf t-PPM (obtained after 48 h of PM polymerization) and postpolymerization modified substrates for an incubation time of 15 min at 10 and 30 °C. Then each sample was rinsed carefully with the buffer and finally dried in a nitrogen stream. 2.3.6. Optical Fluorescence Microscopy. Protein adsorption to the oligoperoxide-graft-PPM coatings was examined using an Olympus BX51 optical microscope, equipped with a 100 W halogen lamphouse, a U-MNG2 filter (λexit = 470−490 nm, λemit > 510 nm), and a type DP72 camera. All images were recorded for dried samples using the Cell F̂ program. The recording conditions were adjusted carefully to enable subsequent semiquantitative analysis of protein adsorption.

Cp C0(1 − e−kτ )n

where Cp the is concentration of PPM on the surface (from Figure 2) (g/m2), C0 is the initial concentration of peroxide groups of the oligoperoxide initiator on the surface (2.3 × 10−6 mol/m2), k is the first-order constant of initiation at 90 °C (k = 8.1 × 10−6 s−1), τ is the polymerization time (s), and n is the polymerization effectiveness on the surface (n = 0.2).36 2.3.3. ToF-SIMS. To examine the surface chemistry after fabrication of the oligoperoxide-graf t-PPM coatings as well as after postpolymerization modifications (PPM-AE, PPM-PE), time of flight-secondary ion mass spectrometry (ToF-SIMS) measurements were performed using a TOF.SIMS 5 (ION-TOF GmbH) instrument equipped with a 30 keV bismuth liquid metal ion gun. Bi3 clusters were used as primary ions with an ion dose density lower than 1012 ion/cm2 to ensure static mode conditions. A pulsed low energy electron flood gun was used for charge compensation. For each sample, high mass resolution spectra were acquired from a few different and nonoverlapping spots (100 μm × 100 μm area each). For all spectra, the minimal mass resolution (m/ Δm) at the C4H5 (m/z = 53) peak was higher than 5500. Mass

3. RESULTS AND DISCUSSION The PPM brushes were fabricated using polymerization “from the surface” of the oligoperoxide, in a three-step process, outlined in Scheme 2 and described in detail in Experimental Section. 9677

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Langmuir Molecular composition, chemical state, and temperaturesensitive wettability of the oligoperoxide-graf t-PPM coatings are described in section 3.1. The postpolymerization modification of the PPM grafted brushes with acetyl chloride or pyromellitic acid chloride and temperature-sensitive properties of the modified coatings are discussed in section 3.2. In turn, the BSA adsorption on oligoperoxide-graf t-PPM and subsequently modified coatings at different temperatures is discussed in section 3.3. 3.1. Composition and Wettability of Oligoperoxidegraft-PPM Coatings. 3.1.1. Chemical Analysis and Surface Coverage with PPM. Overall chemical analysis, verifying the surface coverage with PPM brushes polymerized from the surface of oligoperoxide, was examined using X-ray photoelecton spectroscopy (XPS) and time of flight-secondary ion mass spectrometry (ToF-SIMS). XPS measurements were performed for glass surfaces premodified with APTES after subsequent grafting of oligoperoxide for 24 h (Figure 1a) as well as for brushes after 48 h of PM polymerization from the surface of the peroxide (Figure 1b). Recorded C 1s XPS spectra are presented in Figure 1.

after PM polymerization for 48 h (Figure 1b) reveals a very intense C−C signal accompanied by two weak peaks corresponding to the C−O and CO bonds. Comparison of the shapes of both spectra unambiguously indicates coverage of the initial oligoperoxide layer with the PPM brush. This result is confirmed by ToF-SIMS measurements performed for the analogous pair of samples. The ToF-SIMS spectrum recorded for the oligoperoxide layer depicts a series of peaks characteristic for peroxide molecules,22 which are no longer visible in the ToF-SIMS spectrum recorded for PPM brushes after 48 h of polymerization (see Figure S2, Supporting Information). ToF-SIMS analysis of oligoperoxide-graf t-polymer coatings was reported in our previous works.21,22 Thicknesses of grafted layers were examined using ellipsometry. Average thicknesses of grafted APTES and oligoperoxide initiator, measured by ellipsometry, were reported to be equal to 0.5 and 1.5 nm, respectively.21,22,34 The calculated concentration of peroxide groups of the oligoperoxide on the surface is (2.3 ± 0.2) × 10−6 mol/m2. The average thickness of PPM brushes increased with polymerization time to 38 nm, whereas the refractive index did not show any significant changes (see Figure S3, Supporting Information). The PPM concentration in grafted nanolayers was calculated assuming the bulk density of the PPM equal to 1.255 g/cm3 (calculated using ACD/ChemSketch 14.01). The calculated concentration of the PPM increases from 0 to 48 mg/m2 for coatings prepared with increasing polymerization time (Figure 2).

Figure 2. Concentration of grafted PPM coating vs polymerization time. The solid line is a guide for the eye.

The calculation of the grafting density of PPM chains on the peroxided glass surface was performed using the following equation:

Figure 1. Representative C 1s XPS spectra of the APTES-modified glass surface after subsequent steps of oligoperoxide grafting for 24 h (a) and PM polymerization for 48 h (b). Blue, violet, and green lines correspond to the signals characteristic for C−C, C−O, and CO bonds, respectively.

hρNA M where σ is the grafting density (chains/nm2), h is the dry layer thickness measured by ellipsometry (nm), ρ is the PPM bulk density, NA is Avogadro’s constant, and M (g/mol) is the molecular mass of the PPM brushes grafted onto the surface. Calculated molecular masses obtained for polymerization times of 30 and 40 h are equal to 93 000 and 145 000, respectively. Grafting densities for these polymerization times are equal to 0.17 and 0.2 chains/nm2, respectively. These values are close to the values previously described in similar works (0.1−0.4 chains/nm2).21,23,36 σ=

For both analyzed samples, the measured XPS spectra consist of three peaks corresponding to C−C, C−O, and CO bonds (284.8 eV, 286.4, and 288.4 eV, respectively). However, the relative intensities of signals recorded for each sample differ significantly. For the oligoperoxide layer (Figure 1a), the intensities of signals characteristic for C−C and C−O bonds are comparable and approximately three times higher than the intensity of the CO signal. In turn, the spectrum recorded 9678

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oligoperoxide-graf t-PPM coatings fabricated with a polymerization time of 20 and 24 h (data not presented here) demonstrate only a small thermal response of the water contact angle (approximately 8°). Similarly, there is no thermal response of the pure oligoperoxide layer (stars in Figure 3). One of the most important properties of the temperaturesensitive systems is the applicability in temperature-controlled multiple cycles. The water contact angle (Figure S4) recorded during multiple cycles of heating and cooling for coatings fabricated with a polymerization time of 30 h demonstrates good replicability. To study the swelling process and temperature-controlled conformational changes of the PPM coating, ellipsometric examination was performed. The sample fabricated with a polymerization time of 30 h, investigated previously in the dry state (see results in Figure 2 and Figure S3), was fixed in a cuvette. Then the cuvette was filled with distilled water and was kept for 0.5 h at fixed temperature. The thickness of the grafted PPM coating decreases from 30 nm at 5 °C (swollen state) to 23 nm at 18 °C (collapsed state) (Figure S6, Supporting Information). This is accompanied by an increase in refractive index from 1.482 to 1.493 (Figure S6). The water content in the PPM coating, determined using the Maxwell−Garnett effective medium approximation for bulk PPM refractive index of 1.501, equals 11.1% and 4.4% at 5 and 18 °C, respectively, suggesting temperature transition of the PPM coating. PPM brushes, like the majority of temperature-sensitive brushes, transit from a hydrated state with loose coils (at lower temperatures) to a hydrophobic state with collapsed chains (at higher temperatures).41 The gradual change in contact angle values for a wide range of temperatures, observed for the PPM coatings, can be most probably related to the gradual release of water molecules bonded to the surface by large amounts of hydroxyl groups. 3.2. Postpolymerization Modification of the PPM Grafted Brushes. Postpolymerization modification of the grafted polymer brushes allows introduction of specific functional groups or transformation of their reactive functional moieties, enabling fabrication coatings with novel potential applications.24,25 For example, postpolymerization modifications of poly(2-hydroxyethyl methacrylate) brushes by alkanoyl chlorides26 or perfluoroalkyl acid chloride27 and fluorinated agents bearing various chemical head-groups, including acyl chlorides, anhydrides, and organosilanes,28 were reported. Poly(glycidyl methacrylate) brushes postmodified with propylamine and bovine serum albumin (BSA) have been described.24 In turn, brushes of poly-N-[(2,2-dimethyl-1,3-dioxolane)methyl]acrylamide, displaying LCST behavior, were altered chemically by hydrolyzing the dioxolane groups, resulting in modified wetting behavior.29 The authors reported the dependence of wettability on the degree of chemical modification of the parent polymer. In the present work we demonstrate the possibility of partial postpolymerization modification of hydroxyl groups in grafted PPM brushes using acetyl chloride or pyromellitic acid chloride. As a result of postpolymerization modification of PPM (4), we obtained acetyl ester (AE, OC(O)CH3) (5) or pyromellitic ester (PE, OC(O)C6H2(COOH)3) (6) groups covalently bonded to PPM brushes (Scheme 3). Modification of the PPM brushes by pyromellitic acid chloride can result in formation of different fractions including intermolecular crosslinking, explained by the multifunctionality of the pyromellitic

To investigate the morphology of PPM coatings obtained after different polymerization times (partially and completely modified surface), AFM measurements were performed at room temperature. Recorded phase images (see Figure S4, Supporting Information) demonstrate high homogeneity of the PPM coatings fabricated for times of polymerization longer than 30 h. With increasing polymerization time, the surface of the PPM coating evolved from initially relatively rough (Figure S4a) to more smooth (Figure S4b,c), confirming fabrication of a completely modified surface. These characteristics were quantified with root-mean squares (RMS) roughness decreasing from 3.4 to 1.3 nm. Using the contact angle data corresponding to 5 °C (see Figure 3) and the Cassie relation, we determined the effective

Figure 3. Temperature dependencies of water contact angles determined for the oligoperoxide-graf t-PPM coatings fabricated with different polymerization times (marked by different symbols as indicated in the legend).

fractional surface coverage of oligoperoxide surface with PPM. Water contact angles were determined for two reference surface types of the Cassie “‘mosaic’” surface. The values of 67 ± 3° and 15.3 ± 2.8° were obtained for oligoperoxide and “pure” PPM coating, respectively. Maximal effective fractional surface coverage with PPM reaches approximately 96%. 3.1.2. Temperature-Sensitive Wettability of PPM Coatings. PPM systems have been described only in a very limited number of literature reports. The temperature-response of wettability, widely described for PNIPAM, POEGMA, or other polymers and coatings,7,8,21−23,37 has never been reported previously for PPM. Temperature sensitivity of similar systems was observed for poly(2-hydroxyethyl methacrylate)s or poly(2-hydroxypropyl acrylate)s,9−11 where the effect of molecular weight on both water solubility and LCST behavior was reported.9,38,39 Poly(glycerol monomethacrylate) is completely soluble,16 but copolymers including glycerol monomethacrylate and hydroxypropyl methacrylate demonstrate LCST between 7 and 20 °C.40 To examine the temperature-sensitive wettability of grafted PPM coatings, contact angles of sessile water droplets were measured between 4 and 30 °C following coatings fabrication with different PM polymerization times (varied from 0 to 48 h). Distinct, close to 30°, gradual changes in the water contact angle with a transition temperature of 14 °C are visible for oligoperoxide-graf t-PPM coatings fabricated with polymerization times longer than 28 h (Figure 3). In turn, the 9679

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are presented in Figure 5 for the original PPM coating (blue) as well as for the PPM brush modified with acetyl ester (PPM-AE, green) or pyromellitic ester (PPM-PE, violet) groups.

Scheme 3. Postpolymerization Modification of PPM (4) Brushes (after 48 h of PM polymerization) with Acetyl Chloride or Pyromellitic Acid Chloride, Resulting in Acetyl Ester (AE, 5) or Pyromellitic Ester (PE, 6) Groups in PPM Brushes, Respectively

acid chloride. However, most probably the monoester with free carboxylic groups is formed (Scheme 3). To examine postpolymerization modification of PPM brushes, surface techniques such as ToF-SIMS, XPS, and CA measurements were used. For original PPM coatings, as well as for both modified surfaces, PPM-AE and PPM-PE, ToF-SIMS spectra were recorded. The resulting spectra, not presented here, were analyzed using PCA22,42 to enhance detection of subtle differences in surface chemistry. PCA defined two directions of uncorrelated major variations in the ToF-SIMS data set, the so-called principal components (PC1 and PC2), capturing 92.25% and 5.08% of the total variance in the data set. The calculated plot of the scores on PC1 and PC2 is presented in Figure 4a. It clearly separates ToF-SIMS results into three groups of data points, corresponding to the samples PPM, PPM-AE, and PPM-PE, indicating their differences in surface chemistry. The negative values of PC1 scores (Figure 4a) are related mostly to the presence of alkyl chains with O2 (Figure 4b), that mark surfaces (rich in oxygen) modified with acetyl ester (PPM-AE). In turn, the positive values of PC2 scores (Figure 4c) correspond to CHO+ and CH 3O + ions, separating the results obtained for pyromellitic ester (PPM-PE)-modified surfaces. The separation of the PCA scores is well pronounced for all postmodified surfaces, suggesting an effective modification process for all analyzed samples. Postpolymerization modification of PPM coatings was analyzed additionally using XPS. The resulting XPS spectra

Figure 5. XPS C 1s spectra of PPM coating (blue) and PPM brush modified with acetyl ester (PPM-AE, green) and pyromellitic ester (PPM-PE, violet) groups.

The comparison of recorded spectra (Figure 5) depicts no visible difference between the original PPM brush and the coatings modified with acetyl ester (Figure 5, green line) groups. However, for the second type of modification, using pyromellitic ester groups (Figure 5, violet line), the shape of the spectra is changed; the additional peak, corresponding to the CO bond characteristic for both the carboxyl and carbonyl group present in the analyzed sample, appears at 288.4 eV. This observation is confirmed by quantitative analysis of relative intensities of the C−C, C−O, and CO signals. The intensity of the CO signal increases by approximately 50% for the sample modified with pyromellitic ester groups, as compared to the original PPM coating. To summarize, the measurements performed with surfacesensitive ToF-SIMS and XPS techniques, providing information about the chemical composition of the sample, confirm the postpolymerization modification of the original PPM coatings.

Figure 4. PCA analysis of ToF-SIMS spectra. PC1 vs PC2 score plots (a) and loadings on PC1 (b) and PC2 (c). Data points marked with different symbols in part a correspond to the spots of different samples (PPM, PPM-AE, and PPM-PE). 9680

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in our previous works, for PNIPAM- or POEGMA-based thermo- and pH-responsive coatings.21,22 In the present work we show the influence of partial modification of the PPM brushes (only a few percent of all hydroxyl groups) on adsorption of bovine serum albumin (BSA), labeled with fluorescein isothiocyanate, at different temperatures. Albumin-adsorbing surfaces are commonly known as antithrombogenic and biocompatible.48,49 Protein adsorption to modified coating fabricated for 48 h of the polymerization was studied for two temperatures, T = 10 °C and 30 °C, i.e., below and above the LCST transition temperature, respectively. The results of adsorption experiments are presented in Figure 7. Surprisingly, we observe high adsorption of BSA on

This effect is clearly visible for all samples analyzed with ToFSIMS, whereas recorded XPS spectra depict noticeable changes only for the sample modified with pyromellitic ester groups. As the sampling depth of XPS (6.2 nm43) is considerably larger than for ToF-SIMS (1−1.5 nm44), these results suggest the modification of only the top layer of the coatings in the case of acetyl ester groups (cf., distinct negative PC1 from alkyl chains with O2). In contrast, the modification of the hydroxyl groups in the deeper layers of the coating is postulated for the pyromellitic ester group. Moreover, all samples were modified only very weakly: no more than a few percent from all hydroxyl groups. The temperature dependence of water contact angles determined for the oligoperoxide-graf t-PPM and postmodified coatings is shown in Figure 6. Presented results confirm

Figure 7. Calculated fluorescence intensities of BSA adsorbed to glass, PPM, and postpolymerization-modified coatings PPM-AE and PPMPE.

Figure 6. Temperature dependence of water contact angles determined for the oligoperoxide-graft-PPM and postmodified coatings (marked by different symbols and colors, as indicated in the legend).

the PPM coatings although previous reports50−54 described similar hydroxyl-contained coatings [e.g., polyhydroxypropyl methacrylate, poly(2-hydroxyethyl methacrylate), and poly(glycerol monomethacrylate] as nonfouling surfaces. Direct examination of recorded fluorescence intensities reveals a noticeable increase in protein adsorption for all examined coatings, as compared to the control glass sample. However, this effect is most pronounced for the PPM coating modified by pyromellitic ester. Moreover, protein adsorption depicts different thermosensitive behaviors for different coatings. The native PPM brush and acetylated PPM coating adsorbs BSA in the same manner at different temperatures. In contrast, adsorption of BSA to PPM coating modified by pyromellitic ester increases significantly as compared to the control glass sample. Moreover, in this case the effect of temperature on protein adsorption is mostly pronounced; fluorescence intensity increases by more than 60% for temperatures above the transition temperature. Enhanced temperature-sensitive BSA adsorption is most probably due to insertion of characteristic groups. It is known that surfaces modified with polystyrene55 or substances with free carboxyl groups56 have very good adsorption effectiveness, especially for BSA. On the other hand, the surfaces modified by ethers often have low-fouling properties.45,57,58

temperature-sensitive wetting for all samples. However, modification conducted by acetyl chloride, leading to a partial conversion of hydroxyl groups into acetyl esters, results in significantly more hydrophobic coating with a less pronounced change in the value of the water contact angle (nearly 17°). The transition temperature remain unchanged, as compared to unmodified PPM coating (LCST = 14 °C). In turn, for partial postpolymerization modification using pyromellitic acid chloride, the transition temperature of the PPM-PE coating increases slightly up to 17 °C. Additionally, it leads to noticeably diminished hydrophilicity of the coating accompanied by a change in the water contact angle value close to 30°. The results of CA measurements suggest that partial postpolymerization modification of the grafted PPM brushes (a few percent from all hydroxyl groups) probably has a stronger impact on wettability of the coatings than on the LCST transition temperature of PPM brushes. 3.3. Adsorption of BSA on the Surface at Different Temperatures. Protein adsorption onto solid surfaces is a crucial factor determining the properties of novel materials as biocompatible or not biocompatible. Moreover, information about the nonspecific adsorption of proteins to the surface has essential theoretical significance.45,46 Prospective biocompatible materials with switchable wettability allow controlled nonspecific protein adsorption and cell detachment.21,22,47,48 Similar systems for controlled protein adsorption were reported

4. CONCLUSIONS Properties of novel temperature-responsive hydroxyl-containing PPM coatings attached to glass, prepared using polymerization from oligoperoxide initiator grafted to the glass surface premodified with APTES, are presented. The surface properties 9681

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of the coatings after subsequent fabrication steps was confirmed by XPS, static ToF-SIMS, and ellipsometry. Measurements of water contact angle and ellipsometric investigations revealed temperature-induced changes in hydrophobicity as well as in refractive index and thickness of PPM coating. Thermal response of wettability was manifested by gradual change in water contact angle values in a wide range of temperatures, with a transition temperature of about Tc = 14 °C. The change in surface properties is realized by the transition from relatively hydrated loose coils to relatively hydrophobic collapsed chains. The possibility of partial postpolymerization modification of hydroxyl groups in grafted PPM brushes using acetyl chloride and pyromellitic acid chloride was demonstrated. As a result of postpolymerization modification, acetyl ester (OC(O)CH3) or pyromellitic ester (OC(O)C6H2(COOH)3) groups covalently bonded to PPM brushes were obtained. Modified PPM brushes were examined using surface-sensitive techniques such as ToFSIMS, XPS, and CA measurements. Obtained results suggest that partial postpolymerization modification of the grafted PPM brushes (a few percent of all hydroxyl groups) probably has a stronger impact on the changes in wettability of the coating than on the transition temperature of the PPM brushes. Surprisingly, adsorption of bovine serum albumin to PPM brushes, observed with fluorescence microscopy, was higher than on glass in contrast to similar hydroxyl-containing layers reported as nonfouling.50−54 Moreover, the influence of postpolymerization modification on protein adsorption was revealed. Enhanced and temperature-controlled protein adsorption was obtained after postpolymerization modification with pyromellitic acid chloride.

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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.langmuir.5b02285. Additional information as noted in the text (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The research reported in this work was partly funded by financial support in the framework of the Polish Innovation Economy Operational Program (contract no. POIG.02.01.0012-023/08) and M. Smoluchowski Krakow Scientific Consortium in the framework of the KNOW (grant FOCUS).

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