Accepted Manuscript Title: Nano-structural comparison of 2-methacryloyloxyethyl phosphorylcholine- and ethylene glycol-based surface modification for preventing protein and cell adhesion Authors: Tomoyuki Azuma, Ryuichi Ohmori, Yuji Teramura, Takahiro Ishizaki, Madoka Takai PII: DOI: Reference:

S0927-7765(17)30554-4 http://dx.doi.org/10.1016/j.colsurfb.2017.08.039 COLSUB 8795

To appear in:

Colloids and Surfaces B: Biointerfaces

Received date: Revised date: Accepted date:

11-5-2017 15-8-2017 22-8-2017

Please cite this article as: Tomoyuki Azuma, Ryuichi Ohmori, Yuji Teramura, Takahiro Ishizaki, Madoka Takai, Nano-structural comparison of 2-methacryloyloxyethyl phosphorylcholine- and ethylene glycol-based surface modification for preventing protein and cell adhesion, Colloids and Surfaces B: Biointerfaceshttp://dx.doi.org/10.1016/j.colsurfb.2017.08.039 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Research Article

Nano-structural comparison of 2-methacryloyloxyethyl phosphorylcholine- and ethylene glycol-based surface modification for preventing protein and cell adhesion

Tomoyuki Azuma†, Ryuichi Ohmori‡, Yuji Teramura*,†,§, Takahiro Ishizaki‡, Madoka Takai*,†

†

Department of Bioengineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-

8656, Japan ‡

Department of Materials Science and Engineering, College of Engineering, Shibaura Institute

of Technology, 3-7-5 Toyosu, Koto-ku, 135-8548 Tokyo, Japan §

Department of Immunology, Genetics and Pathology (IGP), Rudbeck Laboratory C5:3,

Uppsala University, SE-751 85 Uppsala, Sweden

* Corresponding authors: Madoka TAKAI; Tel: +81-3-5841-7125, Fax: +81-3-5841-0621, E-mail: [email protected] Yuji TERAMURA; Tel: +81-3-5841-1174, Fax: +81-3-5841-0621, E-mail: [email protected]

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This research article contains 3187 words as the contents and 6 figures and 1 table.

Graphical abstract

Highlights



The brush structure of MPC and EG units prevented protein and cell adhesion regardless of their density.



Multiple polymer layers formed by MPC-based polymer coating showed similar antibiofouling property with brush structure. 2



Single layered EG-based polymer coating was not stable enough for complete prevention of protein and cell adhesion.



Monomer monolayer of MPC could suppress protein adsorption only under the low concentration.



The volume excluded by the polymer structure was not only important in EG units-based surface, but also in MPC-based surface.

[ABSTRACT]

Polymer brush, owing to its precisely controllable nanostructure, has great potential for surface modification in the biomedical field. In this study, we evaluated the bio-inertness of polymer brush, monomer monolayers, and polymer-coated surfaces based on their structures, to identify the most effective bio-inert modification. We focused on two well-known bio-inert materials, 2-methacryloyloxyethyl phosphorylcholine (MPC) and ethylene glycol (EG). The amount of adsorbed proteins on the surface was found to be dependent on the monomer unit density in the case of MPC, whereas this correlation was not observed in the case of EG. Cell adhesion was suppressed on the brush structure of both MPC and EG units, regardless of their density. The brush structure of MPC and EG units showed better anti-protein- and anti-celladhesion than monolayers and polymer-coated surfaces. Thus, the steric repulsion was not only important in EG units-based surface, but also in MPC-based surface. In addition, multiple polymer layers formed by MPC-based polymer coating also displayed similar properties.

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KEYWORDS: MPC polymer / PEG / Polymer brush / Polymer coating / Monolayer / Cell adhesion

[INTRODUCTION]

Adsorption of biomolecules on material surfaces depends on surface properties such as wettability, surface charge, and microstructure 1-3. Surface modification methods to prevent biofouling, such as polymer coating and grafting, have been investigated for biomedical applications. Polymer brush, owing to its high chain density and well-organized nanostructure, could be a useful surface modification method to generate surfaces with effective anti-biofouling properties 4-7. Therefore, polymer brush-based surface modification is expected to be applied for advanced biomedical devices. Polymer brush is synthesized by the surface-initiated polymerization of functional monomer units from substrates. Poly(N-isopropylacrylamide) (PIPAAm) brushes have been used for selecting appropriate surfaces for the fabrication of cell sheets 8. Poly(2-hydroxyethyl methacrylate) (PHEMA) brushes with different graft densities, which show different sizeexclusion properties, have been used for the protein separation 9. Before the practical use of polymer brush-based surface modification is possible, some technical issues need to be solved. One such issue is the immobilization of the initiator to the substrate. The graft density of the polymer brush is highly dependent on the density of the initiator. Currently, because of the lack of initiators, the use of polymer brush in biomedical applications is limited. A successful approach to overcome this challenge involves photo-induced surface-initiated radical polymerization, and this technology has been used for surface modification of artificial joints

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10,11

. However, the graft density cannot be precisely controlled in this surface modification; its

application in devices that are in contact with blood is therefore risky. In contrast, polymer coating and casting methods are simple and have been used in the biomedical field. For example, a ventricular assist device, EVAHEART, has been successfully coated with the 2methacryloyloxyethyl phosphorylcholine (MPC) polymer 12,13. However, the recipients of this implant need lifelong treatment with anti-coagulants such as warfarin or aspirin, although MPCcoated pumps showed excellent anti-coagulant properties. Being on an anti-coagulant therapy is inevitable, partly because of the risk of thrombus formation on MPC polymer-coated surfaces 12. Although coating with the MPC polymer repels plasma proteins, the adsorption of even small amounts of serum proteins could potentially trigger blood coagulation and complement activation. Therefore, surface modification should render the surface completely bio-inert to suppress protein adsorption or cell adhesion, and the polymer brush approach might be useful to achieve this. In the polymer brush approach, high graft density has been reported, and this might be an effective parameter for suppressing protein adsorption 6,7. Therefore, the polymer chain density is considered an important parameter for anti-protein- and anti-cell-adhesive properties. In this study, our objective was to identify the most effective design for bio-inert modification by comparing two biocompatible polymers and three different approaches. Here, we used three types of MPC- and ethylene glycol (EG)-based surfaces, polymer brush, monolayer, and polymer-coated surface (Figure 1); they were selected as they have been reported to be excellent bio-inert surfaces 6,7,14-18. The structure, particularly chain density, of all the fabricated surfaces was evaluated to investigate the relationship between the structure and antiprotein- and anti-cell-adhesive property.

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[EXPERIMENTAL SECTION]

Materials MPC was purchased from NOF Co. (Tokyo, Japan). Poly(EG) methyl ether methacrylate (Mn~300) (mOEGMA), copper (I) bromide (CuBr), 2,2’-bipyridyl (bpy), ethyl-2bromoisobutyrate, dodecyltrichlorosilane, methanol-d4, bovine serum albumin (BSA), Albumin– fluorescein isothiocyanate conjugate (FITC-BSA) and Pluronic (F-127) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Diisopropylamine and n-butyl methacrylate (BMA) were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). (3-mercaptopropyl) trimethoxysilane was purchased from Johnson Matthey Japan G.K. (Tochigi, Japan). MPC, mOEGMA, and BMA were used as purchased. Hexane, ethanol, methanol, tetrahydrofuran (THF), acetone, and toluene were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). These solvents were of extra-pure grade and used without further purification. Dulbecco’s phosphate buffered saline (PBS, without calcium chloride and magnesium chloride), Dulbecco's modified Eagle's medium (DMEM), and fetal bovine serum (FBS) were purchased from Invitrogen Co. (Carlsbad, CA, USA). L929 (mouse fibroblast) cells were purchased from RIKEN Cell Bank (Ibaraki, Japan). Synthesis of (1-(2-butyroiloxyethylphosphorylcholine)propylsulfanyl)trimethoxysilane (MPC-S-Si)

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MPC-S-Si was synthesized as described in a previous study 19. Briefly, MPC (10 mmol), (3Mercaptopropyl)trimethoxysilane (10 mmol), and diisopropylamine (0.4 mmol) were dissolved in methanol (20 mL). The reaction mixture was bubbled with Ar for 15 min. After 20 h of reaction at 25 °C, methanol was evaporated from the resultant solution (EYELA N-1110, Tokyo Rikakikai Co., Ltd., Tokyo, Japan). The residual solution was washed with hexane and then THF, followed by evaporation again. The obtained product was dissolved in water, freeze-dried, and collected (yield: 1.53 mg [31 %]). The obtained product was evaluated by 1H-NMR (JNMGX 270, JEOL, Tokyo, Japan). 1H-NMR (CDCl3, 400 MHz, δ ppm): 3.45 (9H, -CH3 of choline group), 0.70 (2H, Si-CH2-), 3.55 (8.3H, -OCH3 of trimethoxysilane group). Synthesis of (1-(2-butyroiloxy(oligoethylene glycol)methyl ether)propylsulfanyl)trimethoxysilane (mOEGMA-S-Si) mOEGMA-S-Si was synthesized for fabricating the mOEGMA monolayer surface. mOEGMA (10 mmol), (3-Mercaptopropyl)trimethoxysilane (10 mmol), and diisopropylamine (0.4 mmol) were dissolved in methanol (20 mL), and the solution was bubbled with Ar for 15 min. After allowing the reaction to happen for 20 h at 25 °C, methanol was evaporated. The residual mixture was then washed with hexane and THF, evaporated again, and then dried in vacuo (yield: 122 mg [2.5%]). The obtained product was evaluated by 1H-NMR. 1H-NMR (CDCl3, 400 MHz, δ ppm): 1.25 (3H, -CH3 of methacrylic group), 3.35 (3H, -CH3 of methyl ether group), 0.70 (2H, Si-CH2-), 3.55 (8.1H, -OCH3 of trimethoxysilane group). Fabrication of polymer brush surfaces (11-(2-Bromo-2-methyl)propionyloxy)undecyltrichlorosilane (BrC10TCS) was synthesized as described in a previous study and used as the initiator for polymer brush fabrication 20. Si wafers with 10-nm-thick SiO2 (Furuuchi Chemical Co., Ltd., Tokyo, Japan), SiO2-coated quartz crystal

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microbalance with energy dissipation (QCM-D) sensor chips (Q-Sense, Gothenburg, Sweden), and a slide glass (1 cm × 1 cm, Matsunami glass Ind., Ltd., Osaka, Japan) were used as the substrates. These substrates were sonicated in hexane, ethanol, and acetone for 3 min and then subjected to O2 plasma treatment for 5 min (High voltage, 600 mTorr, PDC-001; Harrick Plasma, Ithaca, NY, USA) for cleaning. The cleaned substrates were kept immersed in 2 mM BrC10TCS solution in toluene at 25 °C overnight. They were then rinsed thoroughly with toluene and dried in vacuo overnight. Polymer brush surfaces of poly(MPC) and poly(mOEGMA) were fabricated on the BrC10TCS-treated substrates by surface-initiated atom transfer radical polymerization (SI-ATRP), according to the protocol reported in a previous study by our group 21. Briefly, CuBr, 2,2’-bipyridyl (bpy), and MPC or mOEGMA monomer units were dissolved in degassed methanol. The concentrations of CuBr, bpy, and MPC monomer were 0.01 M, 0.02 M, and 0.5 M, respectively. In the case of mOEGMA polymerization, the concentrations of CuBr, bpy, and mOEGMA monomer were 0.03 M, 0.06 M, and 1.5 M, respectively. After the reaction mixture was bubbled with Ar for 10 min at 25 °C, ethyl-2bromoisobutyrate (the sacrificial initiator; 0.01 M for MPC and 0.03 M for mOEGMA) and the BrC10TCS-treated substrates were immersed in the reaction solution. After 20 h at 25 °C, the reaction was stopped by adding O2 into the solution. The obtained substrates were sonicated in methanol for 3 min and dried in vacuo overnight. The resultant solution in the fluid phase was collected to examine the reaction ratio by 1H-NMR with methanol-d4 (Reaction ratio > 99%). Fabrication of MPC and mOEGMA monolayer surfaces Si wafers with 10-nm-thick SiO2, QCM sensor chips, and slide glasses were cleaned, as described in the previous section. The substrates were immersed in MPC-S-Si or mOEGMA-S-

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Si solution (10 mM; in methanol solution bubbled with Ar for 15 min) overnight. The treated substrates were then sonicated with methanol for 3 min and dried in vacuo overnight. Fabrication of MPC- and PEG-coated surfaces To fabricate MPC-coated surfaces, poly(MPC-co-BMA) (PMB30) was used. PMB30 polymerization was performed as described in a previous report 22. Pluronic (F-127) was used without modification as a PEG-coated surface. Si wafers with 10-nm-thick SiO2, QCM sensor chips, and slide glasses were cleaned as described in the previous sections. The substrates were treated with dodecyltrichlorosilane solution (10 mM in toluene) overnight to fabricate CH3 surfaces (CH3-SAM). The treated substrates were incubated in PMB30 solution (1 wt% in ethanol) at 25 °C overnight. They were also incubated in the Pluronic solution (1 mg/mL in H2O) at 25 °C overnight. After washing thoroughly, the coated substrates were used for further experiments. X-ray reflectivity (XRR) measurement. The thickness and density of the fabricated surfaces were evaluated by XRR measurements (SmartLab (9 kW), Rigaku Co., Japan) in air at 25 °C, as described in a previous study 23. A CuK source was used for X-ray radiation. X-ray was focused on the substrates using a collimating mirror with an incident angle of approximately 0°. The detector was rotated by 2 (0° < 2 < 10°), while the substrates were rotated by  during the measurements. The Global Fit (Rigaku Co.) software was used to fit the obtained data. In the case of poly(MPC) or poly(mOEGMA), fitting was performed with a three-layer model (SiO2, BrC10TCS and poly(MPC) or poly(mOEGMA)). In the case of MPC or mOEGMA monolayer, fitting was performed with a two-layer model (SiO2 and MPC-S-Si or mOEGMA-S-Si). In the case of MPC- or PEG-coated

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surface, fitting was performed with a three-layer model (SiO2, dodecyltrichlorosilane, and PMB30 or Pluronic). Protein adsorption on fabricated surfaces The interaction of proteins with the fabricated polymers on the surface was evaluated using QCM-D (QCM-D E4, Q-Sense, Gothenburg, Sweden). The measurement was performed in flow chambers at 25 °C. The AT-cut quartz crystal sensors with SiO2 coating (fundamental resonance frequency = 4.95 MHz) were used, and three types of surface modification with MPC and PEG (polymer brush, monolayer, and coating) were performed on these surfaces, as described in a previous section. The treated sensor chips were prepared in advance, except for the ones coated with Pluronic solution. The surfaces were exposed to 1 mg/mL BSA solution in PBS for 30 min, and PBS was then injected into the flow chamber to wash away the unbound BSA. For Pluronic coating, 1 mg/mL Pluronic solution in PBS was allowed to flow through the chamber for 30 min, after which it was rinsed with PBS. Finally, BSA solution was added. The change in resonance frequency (f) was calculated to evaluate the amount of adsorbed BSA using the Sauerbrey equation (m = −17.7fn/n) 14. Here, m indicates mass change and fn indicates frequency change at the overtone of n (n = 7 in this study). Cell experiments L929 cells were cultured in DMEM supplemented with 10% FBS, 50 U/mL penicillin, and 50 g/mL streptomycin. The cells were cultured at 37 °C in 5% CO2 and 95% air. Slide glasses treated with MPC and PEG as described in the previous sections were used for this experiment. These glasses were placed on tissue culture polystyrene dishes (TCPSs). After the L929 cells were collected by trypsinization, they were seeded into the TCPS (2.0 × 104/mL of the

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culture medium) and cultured for 1 day. The cells were observed through an optical microscope 24 h after seeding (IX73, Olympus Co., Tokyo, Japan).

[RESULTS AND DISCUSSION]

Fabrication and evaluation of MPC- and EG-based surfaces Here, three types of MPC- and EG-based surfaces, polymer brush type, monolayer, and polymer-coated surface, were fabricated to detect the parameter most important for practical applications. The microstructure of each fabricated surface was evaluated using XRR measurements. Raw XRR charts and the theoretically fitted curve are shown in Figure 2, and the calculated parameters are summarized in Table 1. The thickness of the poly(MPC) brush and poly(mOEGMA) brush was 6.8  0.5 nm and 7.7  0.3 nm, respectively. The chain density of the poly(MPC) brush and the poly(mOEGMA) brush was 0.38  0.03 chains/nm2 (1.41  0.08 g/cm3) and 0.32  0.02 chains/nm2 (1.05  0.06 g/cm3), respectively, indicating that both the surfaces could have the polymer brush structure, as the chain density was more than 0.1 chains/nm2 24,25. The thickness of the MPC monolayer and the mOEGMA monolayer was 1.2  0.1 nm and 1.2  0.1 nm, respectively, and the chain density of the MPC monolayer and the mOEGMA monolayer was 1.76 0.01 chains/nm2 (1.10  0.01 g/cm3) and 1.92  0.09 chains/nm2 (1.32  0.06 g/cm3), respectively. In contrast, other studies demonstrated that the self-assembled monolayer (SAM) of alkanethiols and thiolated MPC, which was closely packed, had around 4.5 and 3.7 chains/nm2, respectively16,26. Thus, the prepared MPC and mOEGMA monolayers were less closely packed than the SAM surface; however, the chain density was high enough to cover the surface with the MPC and the mOEGMA monomers. When Pluronic- and

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PMB30-coated surfaces were analyzed with XRR, the Pluronic-coated surface could not be fitted with the theoretical curve, although the PMB30-coated surfaces could be well fitted, and were found to have a thickness of 4.4 0.4 nm and a chain density of 0.04  0.01 chains/nm2 (0.32  0.06 g/cm3). As PMB30 is not soluble in water, the coating was performed with ethanol solution; as a result, the coated PMB30 formed a thick layer made of multiple polymer layers. In contrast, the Pluronic solution formed a single polymer layer with poly(propyrene oxide) (PPO) units on the surface, resulting in a very thin layer that made analysis more difficult. In addition, the wettability was analyzed by measuring the contact angle (Table 1). The static contact angles of air in water for poly(MPC), MPC monolayer, and PMB30 were 168  2°, 157  4°, and 151  4°, respectively, while that for poly(mOEGMA), mOEGMA monolayer, and Pluronic solution were 140  7°, 132  4°, and 142  5°, respectively, indicating that MPC- and EG-based surfaces were hydrophilic and fully covered with their component polymers, MPC and EG, respectively.

Measurement of proteins adsorbed on MPC- and EG-based surfaces The adsorption of BSA onto MPC- and EG-based surfaces was quantitatively analyzed with QCM-D (Figure 3a, b). The amount of adsorbed BSA in the different surfaces is summarized in Figure 3c. In the case of the Pluronic-coated surface, the adsorption of Pluronic solution onto CH3-SAM surface was also monitored, while the other surfaces were pretreated

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before QCM-D was performed. The change in the frequency of Pluronic adsorption was 39.7  5.1 Hz, which was calculated to correspond to a chain density of approximately 0.33 0.04 chains/nm2. The amount of adsorbed BSA on MPC-based surfaces was −4  5 ng/cm2 on poly(MPC), 10  2 ng/cm2 on MPC monolayer, and 20  6 ng/cm2 on PMB30-coated surface. The value for poly(MPC) was negative, likely due to the slight shift of the baseline, suggesting that poly(MPC) brush could almost completely repel BSA. The amount of adsorbed BSA on EGbased surfaces was 10  2 ng/cm2 on poly(mOEGMA), 290 17 ng/cm2 on mOEGMA monolayer, and −67  53 ng/cm2 on Pluronic-coated surface. The value for the Pluronic-coated surface was also negative. From these QCM-D results, it was unclear whether the Pluronic solution completely inhibited BSA adsorption or some of the adsorbed Pluronic was replaced or detached during the interaction between BSA molecules. To clarify this issue, we further analyzed the Pluronic-coated surfaces with another approach. We directly measured the FITCBSA adsorption through image analysis and compared it with the QCM-D results (Figure 3d). We observed some binding of FITC-BSA to the Pluronic-coated surface, which was significantly higher than the background level and lower than that of positive control. However, no apparent BSA adsorption was observed in the QCM-D results. Further investigation was done by using QCM-D. Briefly, anti-albumin antibody was induced after albumin was exposed on Pluroniccoated surface. The representative QCM chart was shown in the Figure S1. The mass change at the anti-albumin exposure was 18.6 ± 7.6 ng/cm2 (1.05 ± 0.43 Hz). These results strongly suggested that replacement of Pluronic with BSA did indeed occur during the interaction. Therefore, the amount of adsorbed BSA was dependent on the density of MPC units, regardless of the structure (polymer brush, monolayer, or coated-membrane). In contrast, no correlation was

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observed between the amount of adsorbed BSA and the density or chain density of EG-based surfaces.

Cell adhesion test on MPC- and EG-based surfaces L929 cells were cultured on MPC- and EG-based surfaces for 1 day (Figure 4a, b). For MPC surfaces, L929 adhesion was not observed on poly(MPC) brush and PMB30-coated surfaces, whereas L929 cells adhered to the MPC monolayer. Thus, although three types of MPC-based surfaces prevented BSA adsorption, L929 adhesion was observed on the MPC monolayer. To further investigate this result, two different concentrations of BSA (1 and 10 mg/mL) were used for the BSA adsorption test (Figure 5a, b). We found no change in the amount of adsorbed BSA on poly(MPC) for these two concentrations. However, the amount of adsorbed BSA significantly increased on the MPC monolayer when the fed concentration was increased. The amount of adsorbed BSA on PMB30-coated surfaces has been shown to be not dependent on the protein concentration 15. While MPC-based polymers such as poly(MPC) and PMB30 prevented protein adsorption and cell adhesion stably, the MPC monolayer with its low molecular weight could not effectively repel protein adsorption, particularly under higher concentrations; cell adhesion was also not prevented by the monolayer. The volume excluded by the MPC monolayer likely could not efficiently prevent protein adsorption. For EG-based surfaces, L929 adhesion was not observed on the poly(mOEGMA) surfaces; L929 adhesion was partly observed on the Pluronic-coated surface, as well as on the mOEGMA monolayer. These results were consistent with those of the BSA adsorption test. We confirmed that the brush

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structure of MPC and EG units had excellent anti-protein-adhesion and anti-cell-adhesion properties. Surfaces coated with EG units (Pluronic) were less effective at repelling cell and protein adhesion than those coated with MPC-based polymers (PMB30), indicating that the adsorbed polymers were not stable enough during the interaction with proteins, leading to them being eventually replaced by the protein. Therefore, adsorbing multiple layers of polymers would be a reasonable structure. Thus, the polymer structure should be carefully considered when designing biomaterials.

[CONCLUSION]

The amount of protein adsorbed was dependent on the monomer unit density in the case of MPC, whereas this correlation was not observed in the case of EG. Cell adhesion was suppressed by the brush structure of both MPC and EG units, regardless of the density. The brush structure of MPC and EG units showed excellent anti-protein- and anti-cell-adhesive properties, compared to the monolayers and polymer-coated surfaces. Thus, the volume excluded by the polymer structure was not only important in EG units-based surface, but also in MPCbased surface. In addition, multiple polymer layers formed by the MPC-based polymer coating also showed similar properties. 15

Funding Sources: This study was supported by a Grant-in-Aid for Scientific Research (A) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (24241042). Disclosure of competing interests: The authors declare no conflicts of interest.

[ACKNOWLEDGEMENTS]

The authors would like to acknowledge the Research Hub for Advanced Nano Characterization, where a part of the study was performed. [ASSOCIATED CONTENT]

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[23] K. Nagahashi, Y. Teramura, M. Takai, Stable surface coating of silicone elastomer with phosphorylcholine and organosilane copolymer with cross-linking for repelling proteins, Colloids. Surf., B 134 (2015) 384-391. [24] T. Wu, K. Efimenko, J. Genzer, Combinatorial study of the mushroom-to-brush crossover in surface anchored polyacrylamide, J. Am. Chem. Soc. 124 (2002) 9394-9395. [25] T. Azuma, Y. Teramura, M. Takai, Cellular response to non-contacting nanoscale sublayer: cells sense several nanometer mechanical property, ACS Appl. Mater. Interfaces 8 (2016) 10710-10716. [26] J. C. Love, L. A. Estroff, J. K. Kriebel, R. G. Nuzzo, G. M. Whitesides, Self-assembled monolayers of thiolates on metals as a form of nanotechnology, Chem. Rev. 105 (2005) 11031170.

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Figure 1. The chemical structure of MPC monolayer, mOEGMA monolayer, poly(MPC), poly(mOEGMA), PMB30 and Pluronic (F-127).

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Figure 2. The XRR charts of (a) MPC-based surfaces (poly(MPC), MPC monolayer and PMB30) and (b) EG-based surfaces (poly(mOEGMA), mOEGMA monolayer and Pluronic (F-127)).

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Figure 3. (a) The QCM charts of pluronic (F-127) coating and BSA adsorption. (b) The QCM charts of BSA adsorption on poly(MPC), MPC monolayer, PMB30, poly(mOEGMA) and mOEGMA monolayer. (c) The mass change at BSA exposure on poly(MPC), MPC monolayer, PMB30, poly(mOEGMA), mOEGMA monolayer and pluronic. (d) The

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fluorescence intensity of adsorbed FITC-labelled BSA on Pluronic-coated surface and CH3SAM.

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Figure 4. (a) L929 adhesion on TCPS, PMB30, MPC monolayer, and poly(MPC). (b) L929 adhesion on CH3-SAM, Pluronic (F-127), mOEGMA monolayer, and poly(mOEGMA). (Scale bar = 200 m).

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Figure 5. (a) Amount of adsorbed BSA on poly(MPC) and MPC monolayers when the concentration of BSA was changed. (b) QCM charts for BSA adsorption on MPC monolayer and poly(MPC) at BSA concentration of 10 mg/mL.

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Table 1. The thickness, density, roughness, and static contact angles of air in water of poly(MPC), MPC monolayer, PMB30, poly(mOEGMA), mOEGMA monolayer, and Pluronic (F-127).

Monomeric

Morphology

unit of

Thickness

Chain

Density

Roughness

SCA of air

(nm)

density

(g/cm3)

(nm)

in water

(chain/nm2)

water-

(deg)

soluble domain Brush

6.8  0.5

0.38  0.03

1.41  0.08

0.6

168  2

Monolayer

1.2  0.1

1.76  0.01

1.10  0.01

0.1

157  4

PMB30

Coating

4.4  0.4

0.04  0.01

0.32  0.06

0.5

151  4

Poly(mOEGMA)

Brush

7.7  0.3

0.32  0.02

1.05  0.06

1.2

140  7

Monolayer

1.2  0.1

1.92  0.09

1.32  0.06

0.5

132  4

Coating

ND

ND

ND

ND

142  5

Poly(MPC) MPC monolayer

mOEGMA

MPC

EG

monolayer Pluronic (F-127)

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