Temperature-controlled orientation of proteins on temperature

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Temperature-controlled orientation of proteins on temperatureresponsive grafted polymer brushes. Poly(butyl methacrylate) versus poly(butyl acrylate): Morphology, wetting and protein adsorption. Kamil Awsiuk, Yurij Stetsyshyn, Joanna Raczkowska, Ostap Lishchynskyi, Pawe# D#bczy#ski, Andrij Kostruba, Halyna Ohar, Yana Shymborska, Svyatoslav Nastyshyn, and Andrzej Budkowski Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.9b00030 • Publication Date (Web): 24 Apr 2019 Downloaded from http://pubs.acs.org on April 25, 2019

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Temperature-controlled orientation of proteins on temperature-responsive grafted polymer brushes. Poly(butyl methacrylate) versus poly(butyl acrylate): Morphology, wetting and protein adsorption.

Kamil Awsiuk1*, Yurij Stetsyshyn2*, Joanna Raczkowska1, Ostap Lishchynskyi2, Paweł Dąbczyński1, Andrij Kostruba3, Halyna Ohar2, Yana Shymborska2, Svyatoslav Nastyshyn1, Andrzej Budkowski1 1Smoluchowski

Institute of Physics, Jagiellonian University, Łojasiewicza 11, 30-348 Kraków, Poland

2Lviv

3Stepan

Polytechnic National University, St. George’s Square 2, 79013 Lviv, Ukraine

Gzhytskyi National University of Veterinary Medicine and Biotechnologies, Pekarska 50, 79000 Lviv, Ukraine

*Corresponding

authors [email protected]

E-mail:

Yurij

Stetsyshyn

[email protected],

1 Environment ACS Paragon Plus

Kamil

Awsiuk

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KEYWORDS: stimuli-responsive polymer coatings, grafted polymer brushes, glass transition temperature, orientation of the proteins, PCA, ToF-SIMS. ABSTRACT The poly(n-butyl methacrylate) – (PBMA) or poly(n-butyl acrylate) – (PBA) grafted brush coatings attached to glass were successfully prepared using ATR polymerization “from the surface.” The thicknesses and composition of the PBMA and PBA coatings were examined using ellipsometry and ToF-SIMS, respectively. For PBMA the glass transition temperature constitutes a range close to the physiological, this being in contrast to PBA where the glass transition temperature is around -55˚C. AFM studies at different temperatures suggest a strong morphological transformation for PBMA coatings, in contrast to PBA where such essential changes in the surface morphology are absent. Besides, for PBMA coatings, protein adsorption depicts a strong temperature dependence. The combination of BSA and anti-IgG structure analysis with the PCA of ToF-SIMS spectra revealed a different orientation of proteins adsorbed to PBMA coatings at different temperatures. In addition, the biological activity of anti-IgG molecules adsorbed in different temperatures was evaluated through tracing the specific binding with goat IgG.


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1. Introduction n-Butyl methacrylate and n-butyl acrylate are two extremely widespread synthetic monomers which have found applications for the fabrication of copolymers for adhesives and coatings. Poly(n-butyl acrylate) – (PBA) has glass transition temperature (Tg) of around -55˚C,1-2 which is an important requirement for soft and elastic polymer networks. In contrast, although n-butyl methacrylate has only one additional methyl group its properties are entirely different, for instance, the Tg of poly(n-butyl methacrylate) – (PBMA) is around 20-25˚C,3-7 that is within the range of physiological temperatures. In recent years, there have been noted numerous examples n the applications of PBA and PBMA surfaces and coatings for biomedical applications. Soft PBA networks were introduced as polymer networks with adaptable mechanical properties and proposed as soft substrates for the passive mechanical stimulation of some types of cells, which could potentially be used in vivo as implant coatings. Besides, it has been shown that they are absolutely non-cytotoxic in in-vitro tests using fibroblast-like cells (L929)1 and primary endothelial cells.8 Moreover, copolymer networks from n-butyl acrylate and oligo(caprolactone) enabling a reversible shape-memory effect at human body temperature have been developed in work by M. Saatchi et al.9 PBMA films decorated by end primary amine groups have been successfully used for cell adhesion and the proliferation of renal epithelial cells.10 In works11-12 the influence of the PBMA chain mobility and temperature-dependent surface softness on bovine serum albumin adsorption have been demonstrated. For protein adsorption a key role is played not only by the wettability and water adsorption of the polymers but also by the polymer chain flexibility.13-15 In works by M. C. Vyner et al.,13-14 it has been shown that two elastomers fabricated from acrylated star-poly(D, L lactide-co-εcaprolactone) with a different bulk modulus and polymer chain stiffness are different in their 3 Environment ACS Paragon Plus


protein adsorption. Similar tendencies for differences in fibrinogen adsorption on spin-coated poly(isobutyl methacrylate), poly(butyl methacrylate) and poly(lauryl methacrylate) with different flexibility at room temperature have also been described.15 In some works16-18 the strong impact of substrate elasticity on cell behavior has been shown. Moreover, in the work by J. Raczkowska et al.,19 the highly cell-dependent impact of mechanical properties on cellular behavior has been shown for cells co-cultured from the mixture. In recent years, significant efforts have been concentrated in developing “smart” polymer materials,20-24 especially grafted temperature-responsive polymer brushes25-35 for biomedical applications. Temperature stimulus changes the affinity these materials have towards proteins and cells and therefore they are used in the production of a surface cell culture dish for the attachment/detachment of the tissues.36-37 A new trend in polymer brushes is temperatureresponsive grafted brushes with temperature transitions based on Tg (glass transition temperature).3,38 Tg is expressed as the transition from a glassy to a rubbery state.39 In the work40 where the grafted PBA brushes were studied, it has been revealed that tethered PBA chains have very different surface dynamics from those of the viscous liquid of untethered PBA chains. In general we can conclude that the properties of the grafted polymer brushes differ significantly from untethered polymers and should consequently be studied in detail. In our previous papers3,

25, 32-33, 41-43

the temperature-responsive as well as the pH and

temperature dual-responsive coatings based on Tg or on a low critical solution temperature were reported. A significant part of those studies was devoted to protein adsorption at different temperatures. In this paper this strategy is extended, using an atom transfer radical polymerization (ATRP) initiator, grafted to a glass surface previously functionalized with (3aminopropyl)triethoxysilane (APTES) for the fabrication of the PBMA (Tg= 20-25˚C) or PBA 4 Environment ACS Paragon Plus

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(Tg= -55˚C) grafted brushes. The composition, thickness, wettability and morphology of the synthesized coatings were studied using Time of Flight-Secondary Ion Mass Spectrometry (ToFSIMS), ellipsometry, contact angle measurements and Atomic Force Microscopy (AFM), respectively. The huge impact of the temperature on the morphology of the PBMA coating in contrast to the PBA coating was here shown. Finally, the effect of the temperature on the proteins adsorption onto the obtained coatings using fluorescently labeled bovine serum albumin (BSA) and anti-species specific antibody (anti-IgG) is presented. BSA and anti-IgG adsorption onto PBMA coatings were studied with ToF-SIMS and PCA that revealed a BSA as well as an anti-IgG molecule temperature dependent orientation, one strongly affecting biological activity, and evaluated for anti-IgG molecules by tracing the specific binding with goat IgG. 2. Experimental Section 2.1. Materials. 2-bromoisobutyryl bromide (BIBB), (3-aminopropyl)triethoxysilane (APTES), n-butyl methacrylate (BMA), n-butyl acrylate (BA), CuBr2, 2,2′-dipyridyl (bpy), triethylamine (Et3N), sodium L-ascorbate and solvents were provided by Sigma-Aldrich.

Bovine serum

albumin (BSA), rabbit anti-goat IgG (anti-IgG) and goat anti-mouse IgG, all labeled with Alexa Fluor 488, as well as unlabeled rabbit anti-goat IgG (anti-IgG) was purchased from Invitrogen (USA). Unlabeled BSA used to blocking buffer was purchased form ACROS Organics. 2.2. Synthesis of coatings. Functionalization of glass surfaces with ATRP initiator.44 In the first stage, glass plates (20x20 mm) were placed in a vacuum oven with a vial containing 10 drops of APTES (Schema 1 (2)). The chamber was then pumped down to 565 nm), a 100 W halogen lamphouse, camera type DP72 and the Cell^F software. All the fluorescence images were recorded for the dried samples.


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In all statistical analyses of the fluorescence data a one-way ANOVA followed by Bonferroni's post-hoc comparisons tests were performed

ToF-SIMS combined with PCA. High resolution mass spectra were acquired from randomly chosen, non-overlapping spots (200 µm×200 µm area each) using the ToF.SIMS 5 (ION-ToF GmbH) instrument. To examine the surface chemistry after each fabrication step of the PBMA or PBA coatings as well as protein adsorption Bi3 clusters were used as the primary ions. To ensure static mode conditions the ion dose density was kept below 1012 ion/cm2. Additionally, a pulsed low energy electron flood gun was used for charge compensation. All mass spectra were recorded for positive ions with the minimal mass resolution being m/Δm>7500 (at C4H5, m/z = 53, peak). To analyse the BSA and anti-IgG orientation a principal component analysis (PCA) of the data set consisting of positive ion signals, collected from PBMA and PBA with adsorbed BSA as well as for PBMA with adsorbed anti-IgG, was conducted using the PLS Toolbox (Eigenvector Research, Manson, WA) for MAT-LAB (Math-Works, Inc., Natick, MA). Additionally, the glass slides with BSA adsorbed at different temperatures were analysed as a reference. The data set consisted of peaks characteristic only for amino acids.48 Prior to PCA, the data was preprocessed - the peak intensities were normalized to the sum of their intensities and then meancentered.

3. Results and discussion The PBMA or PBA grafted brush coatings attached to glass were prepared using ATR polymerization “from the surface” in a three-step process, for the glass surface previously



functionalized by APTES and then ATRP molecules. The thicknesses and composition of the PBMA and PBA coatings were evaluated using ellipsometry and ToF-SIMS respectively and are presented in Section 3.1. Next, the temperature-sensitive wettability and morphology of the PBMA and PBA coatings, fabricated with different polymerization times (result in different coating thicknesses) were compared and are described in Section 3.2. Finally, the impact of the temperature on proteins adsorption onto the obtained coatings, examined using fluorescently labeled BSA and anti-IgG, is presented in Section 3.3. In addition, ToF-SIMS and PCA studies of BSA and anti-IgG adsorbed onto PBMA coatings revealed the orientation of those adsorbed molecules at different temperatures. Moreover, biological activity as well as different orientation of adsorbed anti-IgG molecules were evaluated by specific binding between rabbit anti-goat IgG and goat IgG (highly cross-adsorbed against rabbit IgG).

3.1. Composition of polymer grafted brush coatings (ellipsometry and ToF-SIMS). The thicknesses of grafted coatings as well as APTES layer were checked using ellipsometry. The thickness of APTES, measured for the conditions used for glass functionalization, was equal to 0.5-1.5 nm (and accord with the subject literature).45, 49 In turn, the thickness of the ATRP coating that was used as a substrate for the PBMA and PBA polymerization was ~1 nm. In this work a coating with different thicknesses of PBMA and PBA was used. For PBMA coatings prepared with polymerization times of 5 and 12 hours the average thickness was 43±4 and 82±3 nm, respectively. In turn, for the PBA coatings prepared with the same polymerization times the average thickness was slightly lower and was equal to 38±3 and 75±8 nm, respectively. To examine the fabrication process of the PBA and PBMA grafted brush coatings positive ion mass spectra were collected. The ToF-SIMS spectra depict the series of alkyl and alkanoyloxy


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fragments characteristic for both polymers. The normalized intensities of ToF-SIMS peaks are presented in Figure 1. Moreover, peaks identified previously3,50 as characteristic for PBA (C3H3O2) as well as for PBMA (C4H5O and C4H7O2) were observed. The presented results confirmed the effectiveness of the proposed fabrication process of both (PBMA and PBA) polymer brushes. 3.2. Impact of the temperature on wetting and morphology: PBMA versus PBA (CA and AFM). According to the subject literature4-6 and our previous study3 the temperature of transition (from glass to rubber state) Tg for PBMA is around 13-25 °C. In contrast to PBMA the glass transition temperature for PBA is around -55˚C,1-2 which is much lower than room temperature. As has been shown in our previous work,3 wetting contact angles measurements cannot be used to determine precisely Tg. In order to examine the temperature-sensitive properties of PBMA and PBA grafted brush coatings fabricated with a different thickness and surface morphologies of the coatings (as a result of different polymerization times), AFM measurements were performed between 5 and 35 °C. The representative AFM images for the PBMA coatings with a thickness of 43 and 82 nm as well as for the PBA coatings with a thickness equal to 38 and 75 nm and their comparable cross-sections at different temperatures are presented in Figures 2 and 3, respectively. The root-mean-square (RMS) roughness values estimated from the AFM topography images of the PBMA and PBA grafted brush coatings with a different thickness at different temperatures (Figure 4 a) and b)), suggest a morphological transformation for the PBMA coatings from being highly rough and structured to being relatively smooth at an elevated temperature (Figure 2 and 3). Moreover, the observed changes are weaker for a thicker PMBA coating (82 nm). Based on



RMS roughness values Tg were calculated for PBMA grafted brush coatings with a different thickness (see Figure 4). For the PBMA coatings with a thickness of around 43 nm, the Tg equals approximately 16 °C. A similar value was revealed for PBMA coating with a thickness close to 82 nm, with the Tg ~ 18 °C being around 2 deg lower than the Tg of the bulk PBMA. In contrast, for PBA grafted brush coatings with thicknesses equal to 38 nm as well as 75 nm (Figure 2, 3 and 4 red lines) no essential changes in the surface morphology at different temperatures were observed. In addition, the morphology of the PBA coatings with different thicknesses is similar to PBMA coatings at temperatures above Tg. The absence of the morphological changes at elevated temperatures for PBA grafted brush coatings may be correlated with a glass transition temperature equal to approximately -55˚C. The contact angles were measured for PBMA grafted brush coatings with a thickness equal to 43 nm (Figure 5a; squares, blue lines) and 82 nm (Figure 5b; squares, blue lines) as well as for PBA with a thickness of 38 nm (Figure 5a; circles, red lines) and 75 nm (Figure 5b; circles, red lines) at different temperatures (between 5 and 35 ºC). The curves obtained have a complicated character and are difficult to interpret. In general, coatings with a higher thickness are more hydrophobic. For PBMA coatings, the elevation of temperature leads to increased hydrophobicity with a well expressed maximum at 15-20 ºC, followed by the decrease of the wetting contact angle with any subsequent increase in temperature. For PBMA coatings, a strongly expressed decrease in the water contact angle for temperatures higher than 20 ºC is observed only for the first temperature examination of the samples and almost disappears over the course of subsequent examination cycles as the values of the wetting contact angles at 20 ºC as well as at 35 ºC are comparable (Figure 5, stars, blue lines).3 This effect can be related to a horizontal reordering of the grafted coatings. 3


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For PBA coatings a slow and almost linear increase in the wetting contact angles is revealed with increasing temperature. We can assume that this phenomenon strongly depends on an increase in the mobility of the polymer segments at temperatures above Tg.5, 51-52 In this case, increasing temperature causes larger scale molecular motions until the translational mobility of whole polymer molecules eventually begins. When the temperature is high enough, local chain interactions are no longer of a sufficiently high energy (level) to prevent molecular flow.5, 51-52 Grafted PBA brush-coatings with a thickness equal to 75 nm are more hydrophobic than PBA with a thickness of 38 nm. The initial and subsequent cycles of the temperature examinations for grafted PBA brush coatings have similar curves. In subject works5,

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the behaviour of the amorphous polymer as a function of the tensile

modulus versus temperature is described. Five regions of elastic behaviour were determined: glassy, “leathery,” rubbery “plateau,” rubbery flow and liquid flow. Basing ourselves on the subject literature5, 51-52 and our experimental data (AFM and water contact angles measurements) we propose a hypothetical scheme for the temperature-sensitivity of the PBMA as well as PBA grafted brush-coatings at temperatures close to the physiological (10-36 °C). At TTg the properties of the PBMA grafted brush-coatings are essentially different (Scheme 2b). Thus in this temperature region, short-range diffusion motions of the polymer segments occur much faster but the long-range cooperative motions of chains are strongly limited due to strong local interactions between neighboring chains. The transition from glassy to rubber state is manifested in sharp transformations of the RMS (Figure 4) and the water contact angles (Figure 5).



The studied temperature interval for PBA grafted brush-coatings is far from Tg and in the rubber state (T>>Tg) (according to the subject literature5 this is typically more than 45 °C from Tg), therefore we shall state that PBA coatings at 10 °C as well as at 36 °C are in the region of a rubbery flow or the state of liquid flow (Scheme 2c and d). Although the polymer chains in our system are grafted, their segments are able to intensively move relative to each other, which are reflected by changes in the water contact angles at an increased temperature.

3.3. Protein adsorption 3.3.1. Impact of the temperature on protein adsorption. Being motivated by the presented results, ones showing that despite the marked similarity in chemical structures of PBA and PBMA, their physicochemical properties do differ significantly we decided to examine adsorption of proteins for both coatings. PBMA and PBA coatings with average thicknesses of 43±4 nm and 38±3 nm respectively were chosen for these studies as more rough and nanostructured surfaces than thicker coatings. As wettability53-55 and roughness56-58 are two factors strongly affecting this process, we expect to see completely different PBMA fouling properties when compared to PBA. Moreover, also the polymer chain flexibility, so crucial for the adsorption of proteins,13-15 is different for PBA and PBMA at some temperatures, something that should even enhance the difference in their biocompatibility. To verify this hypothesis BSA labelled with Alexa Fluor was used as a model protein to evaluate protein adsorption to both coatings using fluorescence microscopy, ToF-SIMS measurements and PCA. The results of the adsorption experiments recorded using fluorescence microscopy and analyzed semi-quantitatively with an integral geometry approach59 are presented in Figure 6.


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For BSA adsorption to a PBA coating, the fluorescence intensities determined from the fluorescence micrographs (the red bars in Figure 6) and proportional to the amount of adsorbed proteins are almost constant for all analyzed temperatures and slightly weaker than the control glass sample (the dashed line in Figure 6). In contrast, for PBMA coatings, protein adsorption depicts a strong temperature dependence and increases almost twice, for temperature raised from 10 °C (below the transition) to 35 °C (above the transition). The BSA surface density on glass samples, estimated form XPS measurements60 (not reported here) equals to 0.47±0.03mg/m2.

3.3.2. Study on BSA orientation adsorbed to PBMA brushes in different temperatures To analyze the differences in the orientation of BSA molecules adsorbed to PBMA and PBA coatings further studies with ToF-SIMS and PCA were performed. The PCA was performed using a data set containing peaks originating only from amino acids, with the results of the analysis for PBMA coatings being presented in Figure 7. As seen in Figure 7a, PC1 predictably shows that the greatest variation (86.39 %) within the data set is between BSA adsorbed onto PBMA coatings and glass used as reference. But importantly, PC2 captures the difference (6.42%) originating from the BSA adsorbed to PBMA brushes at different temperatures. Samples with BSA adsorbed at 10 °C (below the transition) are loaded negatively in PC2, whereas, samples incubated in temperatures above the transition are loaded positively in PC2. Moreover, data points from reference samples are distributed randomly around the zero value of PC2 independently of temperature itself. Based on the loadings plot for PC2 (Figure 7 b)) it can be seen that the negative scores for PC2 are related with fragments of histidine, arginine, tyrosine, serine and phenylalanine. In turn, the positive scores on PC2 are due to the



positive loadings of ions originating from valine, lysine, threonine, isoleucine and leucine. This implies that regions of BSA rich in those amino acids are exposed out of the PBMA coatings. Our previous studies have revealed that BSA might undergo conformational changes during adsorption process.60 As a result the molecules denature and expose the hydrophobic amino acids typically hidden deeper in the protein structure. To verify whether the observed temperature induced changes in the BSA adsorbed to PBMA coatings and that these are related with BSA denaturation, a relative measure of the hydrophobicity of amino acid side chains was defined as the difference in retention time, ΔtR, relative to glycine peptide61 and was plotted against the loadings on PC 2 (Figure S2 Supporting Information). The graph obtained clearly shows that no correlation could be indicated, one relating the PC2 loadings with the temperature-induced conformation changes of adsorbed BSA molecules. Therefore, the differences in the composition of the outermost region of adsorbed protein layer revealed by PCA suggest changes in the BSA orientation. To evaluate this hypothesis an analysis of the BSA molecule structure was performed. BSA consists of three domains (Albumin 1, Albumin 2, Albumin 3) and the size of the molecule is about 9 x 5.5 x 5.5 nm3.62 Since the sampling depth of ToF-SIMS is lower than the size of the proteins, the orientation of BSA molecules can be examined.63-64 The lengths of each domain are comparable (Albumin 1 - 191 aa, Albumin 2 - 193 aa and Albumin 3 - 198 aa) but their amino acid compositions are different. For this reason, amino acid compositions for all three domains of BSA (4F5S) were calculated from a protein data bank,65 as shown in Table S1. Based on this result it can be seen that Albumin 1 and Albumin 2 when compared to Albumin 3 are rich in histidine (8 and 6 vs. 3) and tyrosine (9 and 7 vs. 4). Moreover, threonine (7 and 7 vs. 17) and valine (6 and 11 vs. 19) are more abundant in Albumin 3 than in Albumin 1 and


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Albumin 2. The structure of a BSA molecule with a marked position of histidine (Figure 7d and S3 a), tyrosine (Figure S3b), threonine (Figure 7c and S3c) and valine (Figure S3d) clearly shows that His and Tyr are more abundant in Albumin 1, whereas Albumin 3 is rich in Val and Thr. Moreover, serine is the most abundant in Albumin 2 (12) as compared to Albumin 1 and Albumin 3 (7 and 8). The combination of the BSA structure analysis with PCA have revealed a different orientation of BSA when adsorbed to PBMA coatings, schematically presented in Figure 9, below (Figure 9a) and above (Figure 9b) the transition Tg. The positions of Val and Thr amino acids, more abundant in Albumin 3 (coloured in green in Figure 9), are marked in yellow (Figure 9). BSA adsorbed to PBMA coatings below Tg adopts such an orientation that Albumin 1 and Albumin 2 (rich in His, Tyr and Ser) are exposed out of the surface, whereas, for temperatures above transition Albumin 3 is exposed. Differences in BSA molecule orientation might also explain the differences in the amount of adsorbed proteins revealed by fluorescence microscopy. Rezwan et al. have reported62 that albumin adsorbed side-on with exposed Albumin 3 (at pH 7) interacts with other molecules forming a dimer. In our studies, some of BSA molecules adsorbed to PBMA brushes at a temperature above the transition might also form dimers and as a result observed is an increase in the fluorescence signal. A similar analysis of ToF-SIMS data was performed for BSA adsorbed to PBA coatings. The obtained scores plot (Figure S4 in Supporting Information) did not reveal any temperatureinduced differences in protein adsorption. Again PC1 reveals that the greatest variation (94.24%) within the data set is between BSA adsorbed onto PBA coatings and glass used as a reference. In turn to PBMA coating, PC 2 captures only 2.01% of the variances and is spanned by reference



samples. These results accord with the data obtained from fluorescence microscopy where no differences in the amount of proteins adsorbed to PBA coatings were observed.

3.3.3. Study on anti-IgG orientation at different temperatures and the impact of the orientation on biological activity. Since the study on BSA adsorption revealed a temperature-dependent orientation of protein molecules adsorbed at different temperatures we evaluated this effect for anti-IgG, proteins commonly used in immunosensors or enzyme-linked immunosorbent assay. Orientation of the antibodies using ToF-SIMS analysis or combination of the ToF-SIMS and fluorescence imaging was intensively studied in works presented by the Pigram66-67, the Grainger68 and the Budkowski69 groups. First, the amount of anti-IgG adsorbed at different temperatures to PBMA coatings with thickness equal to 43±4 nm was evaluated using fluorescence microscopy and the integral geometry approach. In a way similar to BSA, anti-IgG adsorption displays a strong temperature dependence and one that increases with temperature (Figure 8a, red columns). Next, the PCA of the ToF-SIMS data obtained for anti-IgG immobilized at different temperatures onto PBMA coatings were performed. The PC1 (Figure 8c) clearly separates the data points corresponding to anti-IgG adsorbed onto the surface PBMA below and above Tg. The positive PC1 values correspond to the anti-IgG adsorbed at 10 and 20 °C, whereas the anti-IgG adsorbed at 26 and 35 °C exhibit negative values on the PC1. The corresponding loadings on PC1 (Figure 8d, see also the loading plot in Figure S5) indicate that the positive PC1 scores are related with threonine and tryptophan, that contribute decisively to the mass signals that loads PC1 positively. In turn, the negative PC1 scores correspond mainly to the fragments originating


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from proline, isoleucine and leucine. Distribution of these two types of amino acids in an antibody (see e.g. Figure S6 for Thr and Pro) suggest its different orientation. To evaluate the anti-IgG orientation more precisely, the loadings on the PC1 were compared with the relative prevalence, (RP), of amino acid ion fragments originating from the F(ab’)2 (negative RP values) versus the Fc region (positive PR values). The RP is defined64 by the results of the PCA performed70 for the ToF-SIMS data of a model antibody molecule and its reference regions. Figure 8d presents the RP parameter values70 plotted against the PC1 loadings determined in this work (Figure S5) for identical ion fragments (red solid squares) and/or amino acids (black open circles). The analysis clearly showed that the outer regions of the anti-IgG adsorbed to PBMA coatings below Tg (positive PC1 values) are dominated by the F(ab’)2 fragments of the molecule (negative RP), while the exposed regions of the anti-IgG immobilized onto PBMA coatings above Tg (negative PC1 loadings) correspond to the Fc fragments. The analysis revealed a dominant head-on (Figure 9 d) orientation for the anti-IgG adsorbed above Tg compared to a dominant end-on orientation (Figure 9 c) for those molecules adsorbed below the temperature of transition. The crucial issue, relevant for any potential biomedical applications of polymer brush-based coatings, especially as biosensors, is their biological activity. In order to examine whether the adsorption process driven at different temperatures affects protein activity, the highly specific antigen-antibody immunoreaction between rabbit anti-goat IgG and goat IgG was analyzed by means of fluorescence (Figure 8a, blue bars). First, PBMA coatings with rabbit anti-goat IgG (Canti-IgG =100 μg/mL) pre-adsorbed at different temperatures (10, 20, 26 and 35 °C) were incubated in a blocking buffer of BSA, which is commonly used to block any non-specific interactions (CBSA =10 mg/mL). Then, the samples were incubated with a 25 μg/mL solution of



goat IgG (labelled with Alexa Fluor 488) dissolved in a blocking buffer at room temperature. In such a procedure fluorescently labelled IgG molecules may bind only specifically with preadsorbed anti-IgG, therefore the intensity of the observed fluorescence corresponds to the biological activity of the protein adsorbed to the PBMA coating. Since antigen binding sites are located at F(ab’)2 fragments, any differences in fluorescence intensity may be correlated with the orientation of the adsorbed anti-IgG, altered for an adsorption process driven at different temperatures. By implementing the integral geometry approach to the fluorescence micrographs, the relative binding activity of the immobilized IgG was calculated as the ratio of fluorescence intensity recorded for IgG after binding with pre-adsorbed anti-IgG (Figure 8a, blue bars) to the intensity of fluorescence recorded for anti-IgG (Figure 8a, red bars). As shown in Figure 8 b, effectiveness of IgG binding to pre-adsorbed anti-IgG is higher for anti-IgG adsorbed at a temperature below Tg as compared to the antibody adsorbed above transition temperature. This accords with the conclusions from the PCA of ToF-SIMS data (Figure 8d), since in an end-on orientation the antigen binding sites are exposed to solution and more antigens can be bounded. The presented results clearly indicate that the anti-IgG adsorbed onto PBMA brushes preserves its biological activity and confirms its temperature dependent orientation.

3.3.4. Discussion on the orientation mechanisms of the proteins adsorbed to PBMA brushes at different temperatures. The main factors which determine the orientation of the proteins adsorbed to the surface are its free energy minimum resulting from attractive Coulomb and van-der-Waals interactions, hydrogen bonds, and the change of entropy due to the counter ions or solvent molecules release.71 Moreover in last years, there is growing attention to interactions between protein


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molecules and surface topographies due to the combination of the attractive and repulsive forces that are governed by local changes in surface properties, including chemistry, which may lead to spatial changes in the amount, surface coverage, conformation and orientation of adsorbed proteins.72-74 This interesting concept is presented in the works by M. Berglin ea al.,15, 75 where the strong influence of polymer flexibility on the conformation of some proteins and immune complement activation were shown. Using our experimental results and analyzed literature sources62-65, 70 we present in Figure 9 the orientation of the BSA and anti-IgG molecules adsorbed to PBMA grafted brush coatings at a temperature below (a, c) and above (b, d) the transition (Tg). One of the major factors determining protein adsorption is protein-substrate electrostatic interaction. Therefore, we have analyzed if this factor may be responsible for the temperaturedependent orientation of BSA and anti-IgG adsorbed onto a PBMA coating. A protein molecule above its isoelectric point (IEP) is negatively charged. However, charge distribution within the molecule is not homogeneous and different domains have a different net charge, due to the different amino acid composition. In addition, even at pH values close to IEP, different parts of the molecule could be charged, although the total net charge of the protein is close to zero. For a BSA (IEP about 4.7-5) at pH 7 Rezwan at al. present that the net charge of Albumin 1, 2 and 3 is equal to -7.8, -9 and -1.3 (Figure S7a), respectively.62 On the other hand, the isoelectric point of F(ab’)2 and Fc fragments of anti-IgG (IEP~7) is equal to 8.5 and 6.1, respectively70. As a results at pH 7.4 the F(ab’)2 and Fc fragments are positively and negatively charged, respectively, and the whole anti-IgG molecule has a dipole moment (Figure S7b) pointing from a Fc to a F(ab’)2 fragment.64, 70



The PCA of the ToF-SIMS data indicates that the BSA adsorbed to PBMA coatings above Tg adopts such an orientation so that negatively charged Albumin 1 (net charge -7.8) and Albumin 2 (net charge -9) are close to the surface, whereas for anti-IgG positively charged a F(ab’)2 fragment attaches to the surface (Figure S7c). In turn, below Tg both molecules changed their orientation and as a result strongly negatively charged Albumin 1 and Albumin 2 are exposed out of the surface, whereas a negatively charged Fc fragment of anti-IgG is directed to the PBMA coatings (Figure S7d). This finding clearly presents that the protein-substrate electrostatic interaction is not a major factor responsible for the temperature-dependent protein orientation on PBMA coatings. In turn, analysis of the Lifshitz–van der Waals apolar and the Lewis acid–base polar components surface energy76 of the PBMA at 10 and 35 ºC, equal to 33.9 and 4.7 mJ/m2 at 10 °C, respectively, and 36.7 and 5.1 mJ/m2 at 35 °C, points to their small increase. Therefore we conclude that the slight modification of surface energy, as well as its components is probably not the dominant factor determining proteins orientation onto PBMA coatings. Based on the above mentioned information we can assume that surface topography impact as in works by M. Lord et al.,72 and P. Roach et al.,73 and polymer elasticity (flexibility) as in the work by M. Berglin et al.,15 on the orientation of those proteins adsorbed to PBMA coating at different temperatures. In general, elasticity of the spin-coated polymer layers as well as grafted polymer brushes at temperatures below the glass transition temperature is in the range 1-3 GPa, whereas in rubber state Young modulus equals to 1-100 MPa.77-78 For PBMA, at temperatures below glass transition the elastic modulus is 1 GPa.79-80 At elevated temperatures, a gradual decrease in the elastic modulus values from 1 GPa to 30−50 MPa for the rubbery state is observed.80-81


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4. Summary and Conclusions The PBMA and PBA grafted brush coatings attached to glass were prepared using ATR polymerization “from the surface” in a three-step process, and this for a glass surface previously functionalized by APTES and then ATRP molecules. To gain information about the composition, morphological transformation and thicknesses of the PBMA and PBA grafted brush coatings multi-technique (ToF-SIMS, AFM, contact angle and ellipsometry) characterizations were performed. According to the subject literature4-6 and our previous study3 the temperature of transition (from the glassy to the rubbery state) Tg for PBMA equals approximately 13-25 °C. In contrast to PBMA the glass transition temperature for PBA is around -55 °C,1-2 that is much lower than room temperature. The AFM data from the PBMA grafted brush coatings with a different thickness collected at different temperatures suggest the transformation of the surface morphology from the very rough and structured to the relatively smooth at an increased temperature. In turn, for PBA grafted brush coatings no substantial morphological changes at different temperatures were observed. The water contact angles measured for PBMA coatings confirmed our previous observation3 that the increase in the temperature leads to a rise in the hydrophobicity with a well expressed maximum at 15-20 ºC, followed by the decrease in the wetting contact angles with a subsequent increase in temperature. Moreover, a strongly expressed decrease in the water contact angle for temperatures higher than 20 ºC is manifest only during the first temperature examination of the samples. In turn, for PBA coatings a slow and almost linear increase in the wetting contact angles with increasing temperature was shown. We can assume that this phenomenon depends strongly on the increase in the mobility of the polymer segments at temperatures above Tg.5, 51-52 Using subject literature sources5,

51-52

and our experimental data, a hypothetical scheme of



PBMA temperature-sensitivity as well as PBA grafted brush-coatings at temperatures close to the physiological (10-36 °C) was proposed. Fluorescently labelled BSA was used to study the protein adsorption onto PBMA as well as PBA grafted brush coatings. For PBMA coatings, protein adsorption depicts a strong temperature dependence and increases almost twofold, for a temperature rise from 10 °C (below the transition) to 35 °C (above the transition). In contrast, for the PBA coating, protein adsorption almost displays no temperature dependence. Since fluorescence microscopy has revealed the strongest temperature dependence for BSA adsorption to be onto the PBMA coatings, further studies with ToF-SIMS and PCA were performed for these types of brushes. BSA consist of three domains (Albumin 1, Albumin 2, Albumin 3), therefore it is a very convenient object to study the protein orientation. The combination of BSA structure analysis with PCA and loadings plot for PC2 revealed a different orientation of BSA adsorbed to PBMA coatings at different temperatures. BSA adsorbed to PBMA coatings below Tg adopts such an orientation that Albumin 1 and Albumin 2 are exposed out of the surface, whereas for temperatures above transition Albumin 3 is exposed. Differences in BSA molecule orientation might also explain differences in the amount of adsorbed proteins revealed by fluorescence microscopy.62 Similarly to BSA, anti-IgG adsorption depicts a strong temperature dependence and increases with temperature. The PCA of the ToF-SIMS data obtained for anti-IgG immobilized at different temperatures onto PBMA coatings clearly separates the anti-IgG adsorbed onto the surface PBMA below and above Tg. The analysis revealed a dominant head-on orientation for anti-IgG adsorbed above Tg compared to dominant end-on orientation for molecules adsorbed below the temperature of transition.


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Finally, the biological activity of anti-IgG molecules adsorbed at different temperatures was evaluated by tracing the specific binding between rabbit anti-goat IgG and goat IgG. More IgG was bonded to pre-adsorbed anti-IgG: for the antibody adsorbed at a temperature below the temperature of transition when compared to the anti-IgG adsorbed above Tg. Control of the orientation of immobilized proteins plays a significant role in obtaining the good steric accessibilities of active binding sites and in increasing stability. Such a study could help not only to increase the production of preparative procedures but also to widen knowledge about cell-fate decisions.82

Conflict of interest There are no conflicts to declare.

5. Acknowledgements This work was financially supported by the Ministry of Science and Higher Education in Poland (7150/E-338/M/2017) and by the National Science Centre of Poland under Grants No. UMO-2016/21/D/ST5/01633. Equipment was purchased thanks to the financial support of the European Regional Development Fund (POIG.02.01.00-12-023/08), the European Regional Development Fund Operational Program on Infrastructure and Environment (POIS 13.01.00-00062/08). 6. Supporting Information. Schematic representation of model immunoreaction on surface coated with PBMA after rabbit anti-IgG antibody adsorption in different temperatures, blocking with BSA and specific binding of goat IgG (Figure S1); Hydrophobicity of amino acid side chains defined as the difference in retention time, ΔtR, relative to glycine peptide plotted against



the loadings on PC 2 (Figure S2); BSA structure with marked as grey spheres amino acids: (a) histidine, (b) tyrosine, (c) threonine and (d) valine. The three domains of BSA molecule are distinguished by the color: red - Albumin 1, blue – Albumin 2 and green – Albumin 3 (Figure S3); PCA scores plot of BSA adsorbed to PBA and glass substrates in 10 °C, 20 °C, 26 °C and 35 °C and corresponding loadings plot for PC2 with peaks named by origin amino acids (Figure S4); PCA Loadings plot for PC1 (corresponding scores polot shown in Figure 8c) of anti IgG adsorbed to PBMA in different temperature with peaks named by origin amino acids (Figure S5); IgG structure (1IGT) with marked as grey spheres amino acids: (a) threonine and (b) proline. The Fab and Fc regions are marked as red and blue, respectively (Figure S6); The three domains of BSA with calculated net charge at pH 7. The structure of anti-IgG with the dipole moment (depicted with yellow arrow) associated with the different net charge of Fc and F(ab’)2 fragments. The orientation of proteins molecules (c) above and (d) below Tg with negatively and positively charged regions schematically marked (Figure S7); Amino acid compositions for different domains for BSA (Table S1).


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Scheme 1. Functionalization of glass surface (1) with amino-terminated APTES film (2), subsequent grafting of ATRP initiator (3) and polymerization of BMA or BA, initiated by an ATRP initiator, resulting in PBMA (4) or PBA (5) brushes. Figure 1. Normalized intensities of ToF-SIMS positive ions originating from PBA and PBMA. Peaks characteristic for PBA and PBMA were marked with a red and black asterisk, respectively. Figure 2. AFM images and cross-sections of the PBMA (blue line) and PBA (red line) grafted brush coatings with a thickness of 43 nm and 38 nm respectively at different temperatures. Figure 3. AFM images and cross-sections of the PBMA (blue line) and PBA (red line) grafted brush coatings with a thickness of 82 nm and 75 nm respectively at different temperatures. Figure 4. The RMS roughness obtained from the AFM images of the PBMA grafted brush coatings with a thickness of 43 (a) and 82 (b) nm (squares, blue lines) and of PBA with a thickness of 38 (a) and 75 (b) nm (circles, red lines) at different temperatures. Figure 5. The wetting contact angles of the PBMA grafted brush coatings with a thickness of 43 (a) and 82 (b) nm (squares, turquoise lines – for the first temperature cycle, and stars, blue lines – for the second and subsequent temperature cycles) and PBA with thickness 38 (a) and 75 (b) nm (circles, red lines) at different temperatures Scheme 2. Hypothetical conformations of PBMA (a, b) and PBA (c, d) grafted brushes at TTg (b) and T>>Tg (c, d). Figure 6. Fluorescence intensities of proteins adsorbed to PBMA (blue bars) and PBA (red bars) coatings as wells as to glass reference samples (dashed line). (*Significantly different from each other, pTg (b) and T>>Tg (c, d) 249x165mm (96 x 96 DPI)

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Figure 6. Fluorescence intensities of proteins adsorbed to PBMA (blue bars) and PBA (red bars) coatings as wells as to glass reference samples (dashed line). (*Significantly different from each other, p

Temperature-controlled orientation of proteins on temperature

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