Spin-Casting Polymer Brush Films for Stimuli-Responsive and Anti

Feb 24, 2016 - Jagoba Iturri , Alberto Moreno-Cencerrado , José L. Toca-Herrera. Colloids and Surfaces B: Biointerfaces 2017 158, 270-277 ...
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Spin-Casting Polymer Brush Films for Stimuli-Responsive and AntiFouling Surfaces Binbin Xu,† Chun Feng,*,† Jianhua Hu,*,‡ Ping Shi,§ Guangxin Gu,‡ Lei Wang,‡ and Xiaoyu Huang*,† †

Key Laboratory of Synthetic and Self-Assembly Chemistry for Organic Functional Molecules, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032, People’s Republic of China ‡ State Key Laboratory of Molecular Engineering of Polymers, Department of Macromolecular Science, Fudan University, 220 Handan Road, Shanghai 200433, People’s Republic of China § State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, People’s Republic of China S Supporting Information *

ABSTRACT: Surfaces modified with amphiphilic polymers can dynamically alter their physicochemical properties in response to changes of their environmental conditions; meanwhile, amphiphilic polymer coatings with molecular hydrophilic and hydrophobic patches, which can mitigate biofouling effectively, are being actively explored as advanced coatings for antifouling materials. Herein, a series of welldefined amphiphilic asymmetric polymer brushes containing hetero side chains, hydrophobic polystyrene (PS) and hydrophilic poly(ethylene glycol) (PEG), was employed to prepare uniform thin films by spin-casting. The properties of these films were investigated by water contact angle, X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and quartz crystal microbalance (QCM). AFM showed smooth surfaces for all films with the roughness less than 2 nm. The changes in water contact angle and C/O ratio (XPS) evidenced the enrichment of PEG or PS chains at film surface after exposed to selective solvents, indicative of stimuli- responsiveness. The adsorption of proteins on PEG functionalized surface was quantified by QCM and the results verified that amphiphilic polymer brush films bearing PEG chains could lower or eliminate protein-material interactions and resist to protein adsorption. Cell adhesion experiments were performed by using HaCaT cells and it was found that polymer brush films possess good antifouling ability. KEYWORDS: polymer brush, thin film, stimuli-responsive, antifouling, spin-casting

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INTRODUCTION In the past many years, polymeric films have been long used to modify the surface properties of a wide range of materials.1−13 These polymeric films have novel and potential applications, especially nontoxic antifouling surfaces.3,12,14−17 Most of surface modifications by polymeric films were obtained via the strategy of surface initiation. Most of surface-initiated polymerizations, which must be performed in inert conditions, are rapidly terminated by oxygen so that the molecular weight and molecular weight distribution of the obtained polymers are difficult to control and the thickness of films is uneven.18−20 Moreover, the initiating sites on two-dimensional surface can not be uniformly distributed and two-dimensional surface is difficult to be covered uniformly, therefore, surface initiated polymerizations accomplishing this over large surfaces would be costly and technically difficult.20 Obviously, these issues are difficult to resolve by the widely used approach based on surface-initiated polymerization. Thus, the use of polymeric films constructed by surface initiation strategy may be impractical. © XXXX American Chemical Society

Polymer brush is a special type of polymers in which multiple polymer chains are attached to a linear polymer, i.e., every repeated unit on the linear polymer backbone possesses an attached polymeric side chain.21−25 Because of high grafting density and conformational flexibility of side chains, such polymers may be attractive for the preparation of antifouling and stimuli-responsive surfaces.21 Polymer brush films can be prepared by spin-casting so that the preparation process is not only easy and convenient, but the films are reproducible.26 In comparison with the surface-initiated polymerization, spincasting can greatly simplify some common laboratory procedures, which implied that polymer brush films can be a better alternative to the polymeric films obtained via surface initiation strategy. On the other hand, there has been considerable effort focusing on antifouling materials.14−17,27−30 Amphiphilic Received: December 30, 2015 Accepted: February 24, 2016

A

DOI: 10.1021/acsami.5b12820 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Scheme 1. Synthesis of (PtBA-g-PS)-co-PPEGMEMA Well-Defined Amphiphilic Asymmetric Polymer Brush via the Graftingfrom Strategy

used in the preparation of stimuli-responsive and antifouling surface. In this study, we used amphiphilic asymmetric polymer brushes 2 containing PEG and PS side chains to develop an optimized PEG/PS functionalized surface coating. Thin films were prepared by spin-casting polymer solution onto a solid surface, and the properties of these films were investigated by water contact angle measurement, X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and quartz crystal microbalance (QCM). Water-contact angle and XPS results indicated the enrichment of PEG or PS chains at the film surface after exposing the film to methanol or cyclohexane solvent vapors, which are selective to PEG or PS side chains, respectively. Furthermore, we used QCM technique to quantitatively evaluate the adsorption of proteins on PEG/PS functionalized QCM sensors. The ability of these polymer brush films to reduce cell adhesion was investigated using HaCat cells. The antifouling characteristics of amphiphilic asymmetric polymer brushes with different compositions were examined in detail. This work demonstrated that stimuliresponsive antifouling surface coatings can be readily prepared using amphiphilic asymmetric polymer brushes via simply and economically viable solution process.

polymer coatings with molecular hydrophilic and hydrophobic patches, which can mitigate biofouling effectively, are being actively explored as advanced coatings for antifouling materials. Surfaces with compositional heterogeneities may discourage thermodynamically favorable interactions between the foulant and the surface, which in turn would limit adsorption events.31,32 Most of these reports were aimed at the studies on polymeric films;32 however, to our best knowledge, few reports could be found concerning on amphiphilic polymer brush films.20 There are two major difficulties in such studies: First, the preparation of such films requires amphiphilic polymer brushes containing the random hydrophilic and hydrophobic hetero side chains; the mediation of the lengths of hetero side chains and the random distribution of hetero side chain are more complicated. Second, it is difficult for such polymer brushes to prepare the smooth films required for property measurement and practical application. In previous work, our group has synthesized a series of welldefined amphiphilic asymmetric polymer brushes 2 containing hetero side chains (Scheme 1), hydrophobic polystyrene (PS), and hydrophilic poly(ethylene glycol) (PEG). Inspired by previous literatures,3,14,15 we can reasonably infer that hydrophilic PEG coatings may form “brushlike” structures to prevent proteins from penetrating the substrate surface and hydrophobic PS side chains with low values of polymer−protein interfacial energy may show good fouling release properties.14 Meanwhile, in analogy to the switchable wettability that has been observed in polymeric films,33−37 amphiphilic asymmetric polymer brushes with mixed hydrophobic and hydrophilic side chains may show switchable wettability when exposed in different solvents. For an amphiphilic polymer brush film with hydrophilic PEG and hydrophobic PS side chains, exposure to methanol (which is selective to PEG) may result in enrichment of PEG chains at the film surface with a decrease in the water contact angle. Whereas, the film exposed to cyclohexane (which is selective for PS) may lead to enrichment of PS chains at the film surface with an increase of the water contact angle. Thus, amphiphilic asymmetric polymer brushes can be conveniently

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RESULTS AND DISCUSSION Preparation of Polymer Brush Thin Film. In our previous work,38 a series of PtBBPMA-co-PPEGMEMA 1 macroinitiators was first prepared by RAFT copolymerization of PEGMEMA macromonomer and tBBPMA trifunctional acrylate monomer (Table S1, these abbreviations are defined in Supporting Information). Using these macroinitiators, (PtBA-gPS)-co-PPEGMEMA 2 asymmetric polymer brushes comprising randomly distributed hydrophilic PEG and hydrophobic PS side chains were constructed via the grafting-from strategy and their structural parameters are summarized in Table 1. The lengths of PS side chains of brushes 2a and 2c are about 16.0, and those of 2b, 2d, and 2e are ranging from 26.0 to 27.6; all PS side chains are obviously longer than PPEGMEMA side chains (9.0). B

DOI: 10.1021/acsami.5b12820 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces Table 1. Structure Parameters of (PtBAx-g-PSn)-coPPEGMEMAy 2 Polymer Brusha

Table 2. Water Contact Angle for Amphiphilic Polymer Brush 2 Film

entry

x:y

Mn,GPCe (g/mol)

Mw/ Mne

nStf

NStg

Mn,NMRh (g/mol)

sample

2ab 2bb 2cc 2dc 2ed

17.4/7.4 17.4/7.4 10.7/11.4 10.7/11.4 5.6/15.5

28 000 41 700 16 800 35 000 14 400

1.19 1.19 1.21 1.26 1.23

16.2 26.1 15.9 26.0 27.6

281.9 454.1 170.1 278.2 154.6

38 300 56 200 26 700 37 900 25 700

2a 2b 2c 2d 2e

a Polymerization temperature: 80 °C. [St]:[Br group]:[CuBr]: [PMDETA] = 400:1:1:1. bInitiated by PtBBPMA-co-PPEGMEMA 1a. cInitiated by PtBBPMA-co- PPEGMEMA 1b. dInitiated by PtBBPMA-co-PPEGMEMA 1c. eMeasured by GPC at 35 °C in THF. fThe number of St repeated unit per PS side chain obtained from 1H NMR. gThe total number of St repeated unit obtained from 1 H NMR. hObtained from 1H NMR.

a

Polymer brush thin films, which were found to be unstable on silicon and tended to dewet,39 were prepared by spin-casting dilute solutions of (PtBA-g-PS)-co- PPEGMEMA 2 asymmetric polymer brushes onto ITO glass substrate. In order to obtain smooth and uniform films, it is necessary to choose a suitable solvent with good solubility for both PS and PEG side chains, plus a suitable speed of spin-casting and a suitable polymer concentration.20,26 In the current work, chloroform, a common organic solvent, was used as the solvent and the spin-casting procedure was 4500 rpm for 10 s and then 3000 rpm for additional 30 s with a polymer concentration of 7.5 mg/mL. Note that QCM-D sensor film was prepared using a procedure of 2500 rpm for 30 s with a polymer concentration of 15 mg/ mL for protein adsorption experiment. Smooth films (thickness: 200−400 nm) were achieved on substrate for surface property measurements and AFM image of the film prepared from (PtBA-g-PS)-co- PPEGMEMA 2c polymer brush (see Figure S1A) indicated that the roughness of surface was just about 2 nm. There was no significant change for film uniformity after the film was exposed to methanol or cyclohexane vapor in a sealed annealing chamber (see Figure S1B, C). AFM analysis indicated that the roughness of film increased slightly after methanol and cyclohexane vapor treatments, but still both below 2 nm. Stimuli-Responsiveness of Polymer Brush Thin Film. Since (PtBA-g-PS)-co-PPEGMEMA 2 amphiphilic polymer brushes possess hetero PEG and PS side chains, it can be inferred that thin films prepared from (PtBA-g-PS)-coPPEGMEMA 2 polymer brushes may be responsive to diverse solvents with different hydrophobicity/hydrophilicity.20,37 To examine the solvent- dependent stimuli-responsiveness of polymer brush thin film, we used mixed polymer brushes 2 composed of hydrophilic PEG side chains with a fixed chain length (9 repeated units) and hydrophobic PS side chains with variable chain lengths (16−27 repeated units) for the studies on self-assembly induced by two common organic solvents, hydrophobic cyclohexane and hydrophilic methanol. Polymer brush 2 thin films prepared by spin-casting on clean ITO-coated glass were first characterized with contact angle measurements. The contact angle of water on the surface of polymer brush 2 film exhibited a transition from 83.5 to 95.0° as shown in Table 2 and Figure 1. It should be noted that the contact angle was dependent on the length of PS side chain and the content of PPEGMEMA segment. For the films with the same number of PEGMEMA repeated unit, the contact angle increased with the rising of the length of PS side chain, such as

contact angle (deg) 88.7 95.0 83.5 87.7 85.8

± ± ± ± ±

0.8 0.7 0.6 0.7 0.4

methanol treateda (deg) 87.3 93.6 80.8 84.1 84.0

± ± ± ± ±

0.3 0.5 0.7 0.4 0.6

cyclohexane treateda (deg) 91.7 97.1 90.2 93.4 91.2

± ± ± ± ±

0.9 0.9 0.9 0.4 0.6

Treated with methanol or cyclohexane for 8 h.

Figure 1. Micrographs of water droplets on (PtBA-g-PS)-coPPEGMEMA 2 polymer brush films, (A) 2a, (B) 2b, (C) 2c, (D) 2d, and (E) 2e.

2a (88.7°) to 2b (95.0°) and 2c (83.5°) to 2d (87.7°). For the films with similar length of PS side chain, the contact angle decreased with the ascending of the content of PPEGMEMA segment, for example 2a (88.7°) to 2c (83.5°) and 2b (95.0°) to 2e (85.8°). Next, polymer brush 2 thin films were treated with a selective solvent (methanol or cyclohexane) for 8 h and dried in vacuo overnight. The treatment by selective solvent may preferentially swell either PEG or PS side chains, resulting in a measurable change in the surface composition and water contact angle. Cyclohexane is a good solvent for PS but a poor solvent for PEG, whereas methanol is a good solvent for PEG but a poor solvent for PS, which may result in enrichment of PS or PEG segment at the surface of film after treating with cyclohexane or methanol vapor. In the current case, polymer brush 2 films were treated with cyclohexane or methanol vapor for 8 h followed by drying in vacuo overnight to remove residual solvent. Water contact angle of the film was measured again after solvent treatment to check the change of hydrophobicity/ hydrophilicity of polymer brush 2 film surface. Methanoltreated film surfaces displayed a decrease in contact angle, and in contrary, cyclohexane-treated surfaces demonstrated an increase in contact angle compared to the as-cast films (Table 2). As shown in Figure 2, the orginal contact angle of

Figure 2. Micrographs of water droplets on solvent-treated polymer brush films, (A) 2c as cast, (B) 2c treated with cyclohexane, and (C) 2c treated with methanol. C

DOI: 10.1021/acsami.5b12820 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces as-cast film 2c was 83.5°; it increased by 6.7° with cyclohexane vapor treatment, but decreased by 2.7° after methanol vapor treatment. The results of water contact angle measurements seemed to imply the changing of surface composition (i.e., enrichment of PEG or PS segment) after solvent treatment. This inference was verified by XPS data as listed in Table 3. Because sampling depths were within 10 nm, XPS measure-

vapor but decreased to 1.80 after methanol vapor treatment, which showed that polymer brush 2 thin films could response to the treatment with different solvents accompanied by the enrichment of different segments. From the aforementioned results, either water contact angle or XPS measurements, it can be concluded that solvent treatment can indeed change the interface composition of amphiphilic polymer brush 2 thin film, resulting in a higher PS content after cyclohexane treatment or a higher PEG content after methanol treatment, that is to say that polymer brush 2 thin film is stimuli-responsive. Resistance of Polymer Brush Thin Film to Protein Adsorption. Because amphiphilic asymmetric polymer brush 2 thin films contained PEG segment, we can infer that this kind of film can lower or eliminate protein−material interactions according to previous reports.14,15 A good estimation of this ability is to monitor dynamic protein adsorption on the film surface, as this adsorption will evaluate the interaction level of proteins with the material. Herein, quartz crystal microbalance with dissipation monitoring (QCM-D) was employed to measure the adsorption of proteins on different films. Different proteins may exhibit different adsorption with the same surface in which isoelectric point (PI) of proteins played a certain role.40,41 Therefore, different proteins with different PI values (for BSA, PI = 4.8; cytochrome c, PI = 10.7) were used as model proteins and the assessment of protein adsorption from BSA or cytochrome c solution was a stringent test, which directly probed whether substrates were resistant to fouling by different proteins.30,32 The antifouling ability of polymer brush 2 thin film was first investigated by using PBS (phosphate buffered saline, pH 7.4) solution containing BSA. As seen in Figure 4A, QCM-D time traces of all five polymer brush 2 thin films showed a sharp negative shift in the fifth harmonic frequency of the crystal when the polymer- coated SiO2 sensor was exposed to a solution of BSA. The decrease in the harmonic frequency of sensor indicated a mass increase at the film surface, and the decrease of harmonic frequency of sensor was physically correlated with the increased mass of protein vibrating with the sensor.31 We can clearly notice that protein adsorbed readily to the bare sensor (−32.38 Hz) without polymer brush 2 film and all polymer coatings showed less protein adsorption for exhibiting a good antifouling ability. For the amphiphilic polymer brush 2 thin films, protein adsorption was also dependent on the length of PS side chain and the content of PPEGMEMA segment. The protein adsorption increased with the rising of the length of PS side chain when polymer brush 2 films possessed same number of PEGMEMA repeated unit, for example, 2a (−26.12 Hz) to 2b (−27.23 Hz) and 2c (−17.36 Hz) to 2d (−20.40 Hz). For the films with similar length of PS side chain, the protein adsorption decreased with the ascending of the content of PPEGMEMA segment, such as 2a (−26.12 Hz) to 2c (−17.36 Hz) and 2b (−27.23 Hz) to 2e (−16.15 Hz). Therefore, polymer brush film 2e with the highest PEG content displayed the lowest adsorbed protein density. Note that for polymer brush films 2c, 2d, and 2e, the harmonic frequency increased slightly with the extending of time after a maximum load of BSA, meanwhile, the harmonic frequencies of polymer brush films 2a and 2b monotonously decreased after the introduction of BSA protein, which revealed that polymer brush films 2c, 2d, and 2e had weaker interaction with BSA protein and showed better protein-resistance.

Table 3. C/O Ratio of Amphiphilic Bottlebrush Polymer Filma

a

sample

as cast

methanol-treatedb

cyclohexane-treatedb

2a 2b 2c 2d 2e

5.35 9.00 1.98 4.80 2.78

4.60 7.83 1.80 4.25 2.41

6.40 9.82 3.62 5.37 5.08

Obtained from XPS measurement. bTreated for 8 h.

ment can certainly provide direct evidence of composition change near the top film surface. In the present work, all XPS measurements were carried out at an incidence angle of 45°. It can be seen from Table 3 that C/O ratio of polymer brush 2 film surface decreased after treating with hydrophilic methanol, indicative of swelling of PEG segment; or increased after treating with hydrophobic cyclohexane, indicating enrichment of PS segment. It was found that C/O ratio was also dependent on the length of PS side chain and the content of PPEGMEMA segment, consistent with the results obtained from contact angle measurements. For the polymer brush 2 films with the same number of PEGMEMA repeated unit, C/O ratio increased with the lifting of the length of PS side chain, for example, 2a (5.35) to 2b (9.00) and 2c (1.98) to 2d (4.80). For the films with similar length of PS side chain, C/O ratio decreased with the rising of the content of PPEGMEMA segment, such as 2a (5.35) to 2c (1.98) and 2b (9.00) to 2e (2.78). As shown in Figure 3, C/O ratio on the surface of ascast polymer brush 2c thin film before solvent treatment was 1.98. This ratio increased to 3.62 after treating with cyclohexane

Figure 3. Survey XPS data for (PtBA-g-PS)-co-PPEGMEMA 2c polymer brush films before and after solvent treatment. D

DOI: 10.1021/acsami.5b12820 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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cytochrome c adsorption. It is clear that polymer brush 2 thin film presented worse resistance to cytochrome c in comparison with BSA. There are a large number of carbonyls in the backbone of polymer brushes, which could serve as hydrogen-bonding acceptors, whereas various hydrogen-bonding donors are located in solvent-exposed surfaces of BSA and cytochrome c. We speculated that the difference in the composition, size, shape, and structure for BSA and cytochrome c might lead to different microenvironments for hydrogenbonding donors in BSA and cytochrome c. Thus, the interaction between proteins of BSA and cytochrome c with polymer films via hydrogen-bonding might be different, which resulted in the different resistance for BSA and cytochrome c.42 To further demonstrate the change on film surface vs. solvent treatment, we also used selective solvent (methanol or cyclohexane) to treat polymer brush 2e thin film on SiO2coated QCM-D sensor. Solvent treatment can result in a measurable change on the surface composition so as to affect the interaction of protein on surface. For example, the enrichment of PEG chains at the surface after treatment with methanol vapor may result in the decrease of the protein adsorption and the enrichment of PS chains at the surface after treatment with cyclohexane vapor may lead to the increase of the protein adsorption. This hypothesis was certified by QCMD measurements as shown in Figure 5. Methanol-treated

Figure 4. Representative time traces of frequency shifts in 5th harmonic of QCM-D crystal sensor coated with (PtBA-g-PS)-coPPEGMEMA 2 polymer brush films in (A) BSA (PI = 4.8) and (B) cytochrome c (PI = 10.7) protein solutions.

Moreover, another protein of cytochrome c was also used for examining the antifouling ability of polymer brush 2 thin film. Similarly, QCM-D time traces of all five polymer brush 2 thin films exhibited a distinct negative shift after cytochrome c solution was introduced and the harmonic frequency monotonously decreased after the introduction of cytochrome c. The protein adsorption of polymer brush 2 thin film was also dependent on the length of PS side chain, which increased with the ascending of the length of PS side chain, for example, 2a (−29.90 Hz) to 2b (−33.13 Hz) and 2c (−25.27 Hz) to 2d (−30.59 Hz). The protein adsorption also decreased with the lifting of the content of PPEGMEMA segment, such as 2a (−29.90 Hz) to 2c (−25.27 Hz) and 2b (−33.13 Hz) to 2e (−24.91 Hz). However, polymer brush 2e thin film with the highest PEG content did not show the lowest adsorbed protein density and polymer brush 2c thin film (−25.27 Hz) with lower PEG content and shorter PS side chain exhibited almost same protein adsorption in comparison with polymer brush 2e thin film (−24.91 Hz), which further indicated the influence of the length of PS side chain to the antifouling ability of film. Furthermore, polymer brush 2b thin film with longer PS side chain and the lowest PEG content showed even more protein adsorption (−33.13 Hz) compared to bare SiO2 sensor (−33.16 Hz), which represented the bad resistance to

Figure 5. QCM-D measurements of solvent-treated (PtBA-g-PS)-coPPEGMEMA 2e polymer brush films.

surface exhibited a decrease in cytochrome c absorption (−22.90 Hz) and cyclohexane-treated surface displayed an increase in cytochrome c adsorption (−29.22 Hz) relative to the as-cast film (−24.91 Hz), which also confirmed the chaning of composition near the top film surface. Now, it is clear that polymer brush 2 thin films could resist protein adsorption, either BSA or cytochrome c, which was evidenced by QCM-D measurements; the films exhibited better resistance to BSA compared to cytochrome c. Resistance of Polymer Brush Thin Film to Cell Adhesion. To examine the interaction between polymer brush coating with pollutant in a more relevant manner, cell adhesion experiments were performed to probe the interactions between HaCaT cells and polymer brush 2 thin films.43,44 Samples were prepared identically to ITO substrate to allow for easy visualization of cells. HaCaT cells were seeded and allowed to attach for 6 h on the substrates under standard conditions. E

DOI: 10.1021/acsami.5b12820 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces It was observed that cells were prevented from attaching onto the surface of polymer brush 2 thin film, but preferring to remain rounded in self-associated clusters (Figure 6), i.e.

Figure 7. Quantification of cells appearing on (PtBA-g-PS)-coPPEGMEMA 2 polymer brush films before and after solvent treatment, data are shown as mean ± standard error for five samples.

films prepared from polymer brush 2 were all below 200, generally below 140. This fact also clearly demonstrated that polymer brush 2 thin film could resist cell adhesion of HaCat cells. Moreover, statistical analysis showed that the length of PS side chain and the content of PPEGMEMA segment also had significant effects on the number of single cells presented on surfaces. When the number of PEGMEMA repeated unit in polymer brush 2 thin film was same, the number of adhered HaCaT cells increased with the elevating of the length of PS side chain, such as 2a (55 ± 5) to 2b (136 ± 13) and 2c (36 ± 9) to 2d (38 ± 7). For the films prepared from polymer brush 2a (55 ± 5) and 2c (36 ± 9) with similar length of PS side chain, the number of adhered cells decreased with the ascending of the content of PPEGMEMA segment, which evidenced that PEG played a critical role in determining the resistance ability to cell adhesion of polymer brush 2 thin film.14,15 However, for the films prepared from polymer brush 2b (136 ± 13), 2d (38 ± 7), and 2e (64 ± 2) with similar length of PS side chain, film 2d, not film 2e with the highest content of PPEGMEMA segment, exhibited the best resistance to cell adhesion, which indicated that a suitable content of hydrophilic PPEGMEMA segment (not the highest content) matched the best antifouling ability.31 Solvent-treated films were also used for cell adhesion experiments and the number of adhered HaCaT cells obviously changed after solvent treatment. Less HaCaT cells (19%−58%) were adhered by the films exposed to methanol vapor because of the enrichment of hydrophilic PEG chains; in contrast, more HaCaT cells (118− 227%) were adhered by the films exposed to cyclohexane vapor because of the enrichment of hydrophobic PS chains. This phenomena clearly illustrated that PEG chains could promote the resistance to cell adhesion, whereas PS chains could weaken the resistance to cell adhesion.

Figure 6. Images of HaCaT cells grown on (PtBA-g-PS)-coPPEGMEMA 2 polymer brush films (A, D, G, J, and M) before and after solvent treatment of (B, E, H, K, and N) methanol and (C, F, I, L, and O) cyclohexane.

polymer brush 2 thin film could resist cell adhesion of HaCat cells, which was completely difference from the bare ITO surface where cell morphology showed attachment and cell spreading. Cells were clearly attached in large numbers to the bare ITO surface (Figure 6), on which very few slightly flattened cells were observed in clusters, that is to say bare ITO surfaces could mediate single cell attachment and cell spreading. The spherical clusters of rounded cells have been previously observed on low-fouling surfaces, presumably because the lack of adhesion site on these surfaces resulted in cell−cell adhesion interactions.45 But for an ordinary surface, cells which received stronger adhesion from the surface presented a rounded morphology and remained separate from other cells.45 The number of single and clustered cells was quantified to assess the prevalence of nonspecific protein adsorption on the surfaces as shown in Figure 7.45 The number for bare ITO substrate was 213 ± 11, whereas the numbers for the as-cast

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CONCLUSIONS In the current work, we conveniently prepared uniform polymer brush thin films by spin-casting a solution of welldefined amphiphilic (PtBA-g-PS)-co-PPEGMEMA polymer brush onto a surface. These polymer brush thin films systematically varied in composition and exhibited stimuliresponsiveness. The surface compositions of polymer brush F

DOI: 10.1021/acsami.5b12820 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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films were dynamic and significantly reconstructed due to the flexibility of polymeric side chains20,37 when the films were treated by selective solvent. Solvent treatment with methanol vapor resulted in the enrichment of PEG chains, a more hydrophilic surface, whereas solvent treatment with cyclohexane vapor led to the enrichment of PS chains, a more hydrophobic surface, as demonstrated by water contact angle and XPS measurements. Though the change of contact angle after solvent treatment was not very high, it still implies the potential application of sensing, which is currently being carried out in our laboratory. Polymer brush thin films were also exposed to different protein solutions (BSA and cytochrome c) to characterize the low-fouling properties of the coating by QCM. In contrast to cytochrome c, films exhibited better resistance to BSA because of the different interaction between proteins of BSA and cytochrome c and polymer film via hydrogen-bonding. Analysis of surfaces seeded with HaCaT cells also concluded that polymer brush thin film was a good low-fouling material. This demonstrated that amphiphilic polymer coatings with compositional heterogeneities could mitigate biofouling effectively. The length of hydrophobic PS side chain and the content of hydrophilic PPEGMEMA segment were found to have great effect on either protein adsorption or cell adhesion. In general, films with shorter PS side chains and higher PPEGMEMA content might result in better low-fouling behavior. Furthermore, adsorption of proteins and attachment of cells also changed with solvent treatment for polymer brush thin films and the trend was consistent with the changes of surface composition. This study not only exhibited the excellent antifouling properties of PEG/ PS functionalized surface but provided a new and convenient route to prepare antifouling surfaces.

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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/acsami.5b12820. Experimental details about the synthesis of polymer brush, preparation of polymer brush films, and measurements of the properties of the films (PDF)

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Research Article

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel: +86-21-54925310. Fax: +86-21-64166128. *[email protected]. Tel: +86-21- 55665280. Fax: +86-2165640293. *E-mail: [email protected]. Tel: +86-21-54925606. Fax: +86-21-64166128. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the financial support from National Basic Research Program of China (2015CB931900), International Science & Technology Cooperation Program of China (2014DFE40130), National Natural Science Foundation of China (21474127 and 51373035), and Shanghai Scientific and Technological Innovation Project (13ZR1464800 and 14520720100). G

DOI: 10.1021/acsami.5b12820 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

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DOI: 10.1021/acsami.5b12820 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX