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Photoinduced Surface Zwitterionization for Antifouling of Porous Polymer Substrates Sheng-Han Chen, Kyoko Fukazawa, Yuuki Inoue, and Kazuhiko Ishihara Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01089 • Publication Date (Web): 24 Jun 2018 Downloaded from http://pubs.acs.org on June 26, 2018

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Figure 1. The synthetic route for the PMA and MPC polymers. 352x244mm (72 x 72 DPI)

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Figure 2. Photoreaction mechanism in the polymer system. 352x244mm (72 x 72 DPI)


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Figure 3. FT-TR spectra of porous PE substrate and those modified with the MPC polymers. 352x244mm (72 x 72 DPI)


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Figure 4. XPS charts of the porous PE substrate and those modified with the MPC polymers. 352x244mm (72 x 72 DPI)


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Figure 5. SEM images of porous PE substrate and those modified with the MPC polymers. 352x244mm (72 x 72 DPI)


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Figure 6. Fluorescence microscope images of the porous PE substrate surface and those modified with the fluorescence dye-conjugated MPC polymers. 352x244mm (72 x 72 DPI)


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Figure 7. Confocal microscope images of cross-sectional porous PE substrate and those modified with the fluorescence dye-conjugated MPC polymers. 352x244mm (72 x 72 DPI)


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Wetting by water on the surface of porous PE substrate and those modified with the MPC polymers. 352x244mm (72 x 72 DPI)


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Figure 9. The relative amount of fibrinogen adsorbed on the porous PE substrate and that modified with the MPC polymers. 352x244mm (72 x 72 DPI)


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Synthetic results of MPC polymers 352x244mm (72 x 72 DPI)


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Photoinduced Surface Zwitterionization for Antifouling of Porous Polymer Substrates

Sheng-Han Chen, Kyoko Fukazawa, Yuuki Inoue, and Kazuhiko Ishihara*

Department of Materials Engineering, School of Engineering, The University of Tokyo

7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan

*To whom all correspondence should be addressed


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ABSTRACT: Surface functionalization of polymeric porous substrates is one of the most important requirements to enhance their applications in the biomedical field. In this study, we achieved photoinduced surface modification using a highly efficient reaction of hydrophilic polymers bearing phosphorylcholine groups. Polymers composed of 2-methacryloyloxyethyl phosphorylcholine (MPC) units and 2-(N-ethylanilino)ethyl methacrylate units were synthesized with attention to the polymer architectures. The surface modification of the porous polyethylene (PE) substrates was carried out by the coating of the MPC polymers with a photochemical radical generator, followed by photoirradiation for a few minutes. Surface analysis by attenuated total reflectance Fourier transform IR spectroscopy and X-ray photoelectron spectroscopy indicated that the MPC polymer layer was generated on the PE surface. Cross-sectional confocal microscopy images showed that the MPC polymers were coated on the polymer surface, even inside the porous structure of the PE substrate. After modification, the porous PE substrates showed a significant increase in hydrophilicity and the water-penetration rate through the pores. Furthermore, the amount of protein adsorbed on the PE substrate was reduced significantly by the surface modification. These functionalities were dependent on the MPC polymer architectures. Thus, we concluded the photoreactive polymer system developed furnished the porous substrates with antifouling properties.

KEYWORDS:

photoinduced

surface

reaction;

porous

polymer

substrate;

2-methacryloyloxyethyl phosphorylcholine polymer; hydrophilicity; antifouling property


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INTRODUCTION

Surface functionalization of conventional polymer substrates increases their applications.1-3 There are many surface treatment methodologies for obtaining suitable functionalities on polymer substrates. The most convenient methodology is the combination of a functional polymer at the surface. However, knowledge of how to immobilize the functional polymer on the surface while avoiding damage to the bulk properties of the base polymer is required. Generally, plasma treatment and corona treatment are used to generate reaction points the functional polymer and polymer substrate.4,5 However, these high-energy treatments sometimes induce the degradation of the base polymer substrate and change the mechanical properties of the substrate. A much more convenient surface functionalization technique is the photoinduced chemical reaction of a functional polymer at a surface.6-8 In this process, less energy is generated by photoirradiation, and the setup of this continuous process is easier compared with those of plasma and corona treatments. Usually, photoreactive groups such as phenylazide (PAz) groups and benzophenone (BP) groups are introduced into the polymer chain, and these groups can generate radicals by photoirradiation.7,8 The radicals can abstract hydrogen atom from the polymer substrate when the photoreactive polymer is in close contact with the polymer substrate, and, thus, stable chemical bonding between the photoreactive polymer and substrate is achieved. Other photoreaction for binding the polymer on the substrate is photoinduced crosslinking between the polymer chains.9-11 In this case, the polymers do not react with the polymer substrate directly. A highly efficient photoreaction is the most important factor to achieve stable immobilization on the substrate, but this process is not dependent on the nature of the substrate. We consider that the photoinduced radical formation and transportation along the polymer chains can be used to obtain a highly efficient reaction.

Thus,

we

investigated

a

photoinduced

crosslinking

system

using

a

bromine-substituted compound and N-alkyl-substituted aniline group. The photoinduced cross-linking system of the polymers bearing N-alkyl-substituted aniline groups based on the polymer reactions can provide an effective modification process and tough polymer modification layer on the substrate. Comparison with the other photoreactive system, BP groups and PAz groups bearing polymers, the surface modification layer may be much stable


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by the cross-linking between polymer chains even when they could not contact with the substrate. As the BP groups and PAz groups absorb photoenergy at the most outer surfaces of the coated polymer layer, the reaction part at the real contacting interface with the substrate hardly had enough photoenergy for reacting. This is very weak point for making thick polymer modification layer at the surface. In the present system, photoenergy easily convert to the radicals of the low molecular weight compounds and they withdraw hydrogen atom of the N-alkyl-substituted aniline groups in the polymer side chains. It provides cross-linking points with other polymer chains. Therefore, significant reducing effects of the photoenergy may be ignored and it is applicable for modification of porous substrates. The reaction mechanism of this polymer system is as follows. First, the bromine-substituted compound is decomposed by photoirradiation, and radicals are generated. The radicals attack the p-position of the N-alkyl-substituted

aniline

compound.

Then,

the

second

decomposition

of

the

p-bromine-substituted N-alkyl-substituted aniline occurs by photoirradiation. During this reaction step, new chemical bonds between the N-alkyl-substituted anilines are induced. By using this photoreaction process with a polymer system, a new photoreactive polymer, formed by cross-linking by photoirradiation, may be obtained. Based on this reaction mechanism, we synthesized photoreactive polymers for surface modification of a substrate. Obtaining antifouling polymer surfaces is essential in biomedical research12-14. Protein adsorption and cell adhesion to the substrates used in diagnosis, protein separation, and cell culture should be controlled. In addition, protein adsorption and cell adhesion cause severe problems when medical devices are applied for treatment of patients. Therefore, many investigations concerning the preparation of antifouling surfaces have been carried out from viewpoint of surface modification. Among these investigations, the most promising method is the zwitterionization of the surface15-20. A zwitterionic surface provides super-hydrophilic and electrically neutral characteristics. These are favorable to prevent protein adsorption and conformational change if proteins come into contact with the surface. This results in the inhibition of cell adhesion and bacterial attachment. In our previous reports, we synthesized photoreactive polymers bearing phosphorylcholine groups in the side chain and found that the polymer could be immobilized on a polymer substrate and prevent protein adsorption and cell adhesion. However, one weak point of the photoinduced surface modification methodology


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using conventional photoreactive polymers is the lower reaction efficiency in the area from photoirradiation. Recently, some porous substrates have drawn significant attention in biomedical field. They can be applied, for example, as separation membranes, cell attachment supports, and sensing substrates, and porous substrates with antifouling properties are critical for these applications. In this study, we examined the surface modification of a porous polyethylene (PE) substrate using the new photoreactive polymer system, which is composed of a soluble phosphorylcholine-based

polymer

with

N-alkyl-substituted

aniline

groups

and

tribromine-substitute compounds as a photoinitiator. The polymers were synthesized by the copolymerization

of

2-methacryloyloxyethyl

phosphorylcholine

(MPC)

and

2-(N-ethylanilino)ethyl methacrylate. Random-type and block-type copolymers were compared to help us understand the effects of the polymer architecture on the photoreactivity and the functionality after modification. Surface analysis was carried out to confirm the modification and stability of the polymer layer not only at the surface but also inside the pores of the substrate. The water penetration and protein adsorption resistance were examined to understand the function of the polymer immobilized on the surface of the substrate.

MATERIALS AND METHODS

Materials MPC was purchased from NOF Co., Ltd. (Tokyo, Japan) and was synthesized by the previously reported procedure.21 2, 2’-Azobisisobutyronitrile (AIBN) was purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Ethylene bis(2-bromoisobutyrate) (EBBiB), tris(2-pyridylmethyl)amine (TPMA), and copper(II) bromide (CuBr2) were purchased from Sigma-Aldrich (St. Louis, MO, USA). L-Ascorbic acid was obtained from Wako Pure Chemicals Industries, Ltd. (Osaka, Japan). 2,2,2-Tribromoethanol (TBE) was obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). 2-Methacryloxyethyl thiocarbamoyl rhodamine B was purchased from Polysciences, Inc. (Warrington, PA, USA). All organic solvents and reagents were extra-pure grade and were used as received without further purification. Phosphate-buffered saline (PBS) was purchased from Gibco (Waltham, MA,


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USA). Deionized water (DI) was purified using the ultra-pure MilliQ system and had a minimum resistivity of 18.0 MΩ·m. The 2-mm thick porous PE substrate had an average pore diameter of 17 µm and a porosity of 30%; it was provided by Nitto Denko Corp., Tokyo, Japan.

Monomer Synthesis 2-(N-Ethylanilino)ethyl methacrylate (PMA) was synthesized by the reaction between methacryloyl chloride (MC) and 2-(N-ethylanilino)ethanol (2-NE). Briefly, MC (55 mmol, 5.75 g) was dissolved in 20 mL of dichloromethane and then added into a dropping funnel. Then, 2-NE (50 mmol, 8.26 g) and triethylamine (TEA) (55 mmol, 5.57 g) were dissolved in 80 mL of dichloromethane and mixed in a 300-mL three-necked round-bottomed flask. The MC solution was slowly dropped into the flask equipped with a magnetic stirrer and reacted at -2 °C. After reaction for 24 h, the resulting mixture of trimethylamine hydrochloride was ﬁltered and then evaporated by rotary evaporator. Extraction in a 200-mL HCl (10 mM) aqueous solution was used to purify the PMA. A light-yellow liquid was separated from HCl aqueous solution, which was collected and dehydrated for 3 h by the addition of anhydrous magnesium sulfate. Finally, PMA was obtained by the evaporation of the residual solvent under reduced pressure. The chemical structure of PMA was confirmed using 1H-NMR spectroscopy (JNM-GX400, JEOL Tokyo, Japan). 1H-NMR results (CDCl3, ppm): 1.08–1.12 (t, 3H, N-CH2CH3); 1.95 (s, 3H, α-CH3); 3.32−3.38 (q, 2H, -CH2CH2); 3.51–3.55 (t, 2H, N-CH2CH3); 4.21–4.25 (t, 2H, -CH2CH2); 5.49 (s, 1H, =CH2); 6.02 (s, 1H, =CH2); 6.58–6.68 (t, d, 1H, 2H, phenyl); 7.12–7.18 (m, 2H, phenyl).

Polymer Synthesis and Characterization The random copolymers, poly(MPC-random-PMA) (PMrP), were synthesized by conventional free radical polymerization.

21-23

The desired amounts of MPC and PMA with

AIBN as an initiator were dissolved in 20 mL of ethanol at room temperature. The total monomer concentration and AIBN concentration were adjusted to 0.50 mol/L and 2.5 mmol/L, respectively. After the solution had been placed into a glass polymerization tube, the oxygen in the glass tube was removed by bubbling with argon gas for 10 min. Polymerization was performed in a sealed glass tube at 60 °C for 3.0 h. Finally, the reacted solution was poured into


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a mixed solvent of diethyl ether/chloroform (80/20, v/v) to precipitate the polymer. The collected polymer was dried in a vacuum desiccator overnight. The ABA-type block copolymers, poly(PMA)-block-poly(MPC)-block-poly(PMA) (PMbP), were synthesized via an activator regenerated by electron transfer atom transfer radical polymerization (ARGET-ATRP). First, the poly(MPC) prepolymer as the B segment in PMbP was synthesized, as follows. The MPC, TPMA, Cu(II)Br2, and ascorbic acid were dissolved in degassed methanol in a glass tube. The MPC concentration was 0.50 mol/L, and the molar ratio of MPC/EBBiB/Cu(II)Br2/TPMA/ascorbic acid was 140/1.0/0.01/0.2/1.0. The mixture was bubbled with argon, and EBBiB (bifunctional initiator), prepared in methanol, was injected into the mixture. The polymerization was carried out with stirring at 20 °C in a sealed tube. After 20 h, PMA, which was the second monomer, was added once the conversion of MPC to poly(MPC) had reached 90%. The PMA was dissolved in 1, 4-dioxane, and the concentration was adjusted to 0.50 mol/L. The solution was injected into the poly(MPC) solution with a continuous flow of argon. The second polymerization step was carried out at 40 °C for 48 h. The reaction solution was precipitated in diethyl ether/chloroform (80/20, v/v) after the solution had cooled to room temperature, thus precipitating the final polymer. The collected polymer was further purified by dialysis (Spectra/Por 7 dialysis membranes, MWCO: 1 kDa) in water for 72 h. PMbP was lyophilized in a freeze dryer (EYELA FDU-1100, Tokyo Rikakikai Co., Ltd., Japan) at -45 °C for 72 h. The synthetic routes for the preparation of PMrP and PMbP are shown in Figure 1. 2-Methacryloxyethyl thiocarbamoyl rhodamine B was selected as a fluorescent monomer to synthesize the fluorescent-dye-labeled random and block-type MPC polymers, denoted FPMrP and FPMbP, respectively. The synthetic procedures were the same as those of PMrP and PMbP. The chemical structure and the molar ratio of these polymers were determined by 1

H-NMR spectroscopy. PMrP was dissolved in 70 vol% methanol aqueous solution containing 10 mM LiBr, and

PMbP was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) containing 10 mM CF3COONa. These polymer solutions were analyzed by gel permeation chromatography (GPC, JASCO Co., Ltd., Tokyo, Japan) to evaluate the weight-averaged (Mw) and number-averaged (Mn) molecular weights of the synthesized polymers. The elution time was calibrated with


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poly(ethylene oxide) in the case of PMrP and poly(methyl methacrylate) standards in the case of PMbP. The UV absorption spectra of the 0.050 mg/mL polymer ethanol solutions were obtained with a UV/Vis spectrophotometer (V-560; JASCO, Tokyo, Japan) from 220 to 400 nm. The average size of the MPC polymers in the solution was determined by dynamic light scattering (DLS) (Zetasizer Nano, Malvern Instruments Ltd., Worcestershire, UK). Fifty microliters of the polymer solution was loaded into a quartz cuvette for measurement. All polymers were dissolved in ethanol and the polymer concentration was 0.50 wt%.

Photoinduced Surface Modification of the Porous PE Substrate First, the porous PE substrates were treated by a simple dip-coating process with the MPC polymer solutions. The substrates of 1.0 × 1.0 × 0.20 cm3 in area were immersed into 0.50 wt% MPC polymer solution in ethanol with the addition of 0.25 wt% of 2,2,2-tribromoethanol (TBE) as a photoinitiator and ultrasonicated for 5 min. After the substrates had been dried in an ethanol vapor atmosphere for 30 min, the substrates were directly photoirradiated using a UV lamp (λ = 254 nm, intensity = 10 mW/cm2) for 6 min under gentle rotation. After photoirradiation, the substrates were immersed and shaken for 24 h in ethanol to remove the unreacted MPC polymer. All samples were dried and stored in a vacuum desiccator in the dark.

Surface Characterization of the Porous PE Substrate The functional groups of the PMrP- and PMbP-modified surfaces were characterized by Fourier transform (FT)-IR spectroscopy combined with attenuated total reflection (ATR) equipment (FT/IR-6300, JASCO, Tokyo, Japan) at a resolution of 4.0 cm−1. The surface compositions of the porous PE substrate and prepared substrates were determined via X-ray photoelectron spectroscopy (XPS, AXIS-HSi165, Shimadzu/Kratos, Kyoto, Japan) equipped with a 15 kV Mg Kα radiation source at the anode. The take-off angle of the photoelectron was maintained at 90°. All binding energies (BE) are referenced to the C1s peak at 285.0 eV. The surface morphologies of the porous PE substrates was observed using a scanning electron microscope (SEM, SM-200, Topcon Corp., Tokyo, Japan) at an acceleration voltage of 20 kV and a working distance of 10 cm.


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Confocal laser scanning microscopy (CLSM, FV1000-D IX81, Olympus, Tokyo, Japan) was used to observe the surface and cross-section of the PE substrates modified with FPMPMA and FPMbP. This high-resolution microscope is equipped with 405, 473, 559, and 635 nm lasers and a high-resolution photomultiplier (PMT) detector. The fluorescent polymers show peak excitation at 548 nm and peak emission at 570 nm. The depth to which the photoreactive polymers had penetrated and become fixed in the pores was determined using thin samples cut out in the cross-section. The surface water-in-air and air-in-water static contact angles were measured using a contact angle goniometer (CA-W; Kyowa Interface Science Co., Ltd., Saitama, Japan). The contact angles were measured continuously for 30 s. All contact angles were directly obtained from the photographic images.

Determination of the Amount of Protein Adsorbed on the Porous Substrate The amount of protein adsorption on the prepared PE substrates was determined by MicroBCA protein assay (Thermo Scientific Inc., Waltham, MA, USA). Fibrinogen was chosen for the evaluation of the protein adsorption and the discussion of the antifouling property. Porous PE substrates cut to 1.0 × 1.0 × 0.20 cm3 were placed in individual wells of a 24-well plate and then pre-wetted with water overnight to allow surface equilibration. The pre-wetted samples were transferred to another 24-well plate, and 1.0 mL of the fibrinogen solution at a concentration of 0.30 mg/mL in PBS was added into each well and incubated at 37 °C for 2.0 h. The non-adsorbed protein was removed via rinsing with PBS several times. The adsorbed protein was detached and collected in a 0.10 wt% sodium n-dodecyl sulfate (SDS) solution. Then, 150 µL of the SDS solution was gently mixed with 150 µL of bicinchoninic acid (BCA) in a 96-well plate. The absorbance was measured at 562 nm using a microplate reader (Wallac 1420 ARVOsx; PerkinElmer Inc., Waltham, MA, USA) after reaction at 37 °C for 2.0 h. The amount of protein adsorbed on the substrates was calculated using a calibration curve. Each measurement was repeated four times for each sample (n = 4).

RESULTS AND DISCUSSION


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Characterization of the MPC Copolymers A photoreaction system composed of the tribromine-compound (TBE) and polymer bearing N-alkyl-substituted aniline groups in the side chain (PMrP and PMbP) was developed, as shown in Figure 2. Photoirradiation induces the decomposition of the tribromine-compounds and radicals are formed. The radicals react at the p-position of the N-alkyl-substituted aniline group. Further photoreaction was carried out to remove further bromine atoms attached to the polymer. During this photoreaction, new chemical bonds between the radicals are formed and cross-linking of the polymer chains occurs; finally, the polymers are immobilized on the surface of the substrate. To achieve this photochemical system, first, PMA was synthesized by the reaction of MC and the hydroxyl group in 2-NE. Then, the random-type copolymers (PMrP) and block-type copolymers (PMbP) composed of MPC and PMA were synthesized via conventional free radical polymerization and ARGET-ATRP, respectively. The results of polymer synthesis are summarized in Table 1. The monomer unit compositions in the polymer were calculated from the integration of characteristic peaks in the 1H-NMR spectrum. For the PMrP polymers, the monomer unit composition in polymers was approximately comparable to that in the feed. The composition of PMbP was also precisely controlled by ARGET-ATRP. The molecular weight (Mw) of the PMrP polymers was greater than 1.1 × 105, and the PMbP polymers had Mw's of 1.0 × 105 and 7.9 × 104. The polydispersity (PDI) of the PMbP polymers was less than 1.5, which results from the characteristics of ARGET-ATRP. In previous studies, we used an MPC copolymer with photoreactive monomer moieties for the surface modification of the polymer substrate. A photoreactive monomer composition of around 30 units mol% yields a stable coating layer of the MPC copolymer. Thus, in this study, we adjusted the concentration of PMA units from 10 to 30 mol%. In addition, the effects of the polymer architecture on the efficiency of polymer immobilization on the porous PE substrate were evaluated by comparing the PMrP and PMbP polymers.

Surface Characterization of the Modified Porous PE Substrates Photoinduced surface modification was carried out by dip-coating the substrates with the MPC copolymer solution containing TBE, followed by UV irradiation. There are two


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viewpoints regarding the surface modification of porous substrates. First, the preservation of original physical structure is of crucial importance for any kind of surface modification treatment. Secondly, the modified layer should not only be fixed by physical attachment on the surface but also chemically immobilized on the substrate. To identify the MPC polymers immobilized on the porous surface of the PE substrate through photoirradiation, MPC polymers with fluorescence dye units were applied. Figure 3 shows the surface morphologies of the porous PE substrates before and after 6-min photoirradiation with PMrP80 and PMbP80. The porous structure of the original PE substrate remained, even after photochemical reaction with PMrP80. There was also no significant difference in the case of the PMbP80-modified substrate. The functional groups of PMrP- and PMbP-modified porous PE substrates were characterized qualitatively by ATR-FTIR spectra. In Figure 4, representative IR spectra are shown. On the unmodified porous PE substrate, IR absorption peaks at 1500, 2800, and 2900 cm-1 were observed. After modification with PMrP80 and PMbP80, additional IR absorption peaks at 1720 cm-1, attributed to the carbonyl groups, and the characteristic peaks at 967, 1073, and 1240 cm-1, attributed to the phosphorylcholine group in the MPC units, were observed. These results suggested the MPC polymer layer had been successfully constructed on the porous PE substrates after UV irradiation for 6 min. The stability and durability of the MPC polymer layer on the surface are of importance to understand the photochemical reaction. To examine the coating stability, we used an ultrasonication treatment on the PMrP90 and PMbP90-modified substrates, and the surface was subsequently analyzed by ATR-FTIR spectroscopy. The ultrasonication treatment in water for 15 min did not induce a significant change in the IR absorptions of the substrate modified with MPC after photoirradiation. Thus, the stability of the modified MPC polymer layer was confirmed. XPS analysis of the porous PE substrate was carried out before and after surface modification. Figure 5 shows representative XPS charts of the unmodified porous PE substrate and that modified with PMrP80 and PMbP80 for carbon (C1s), oxygen (O1s), nitrogen (N1s), and phosphorus (P2p). In the case of the unmodified porous PE substrate, an XPS signal was observed at 285 eV, and this was assigned to the C-C and C-H bonds in C1s region. A small XPS signal in the O1s region was observed. However, this is due to contamination of the


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surface. After the porous PE substrate had been modified with the MPC polymers, XPS signals at 288 eV in the C1s region, 403 eV in the N1s region, and 134 eV in the P2p region were observed. These signals were attributed to the MPC units in the polymers used for the surface modification. Furthermore, an XPS signal appeared at 399 eV in the N1s region. This arises from the tertiary amine group in the PMA unit. Therefore, this XPS analysis revealed that the surfaces of the porous PE substrates were covered with PMrP or PMbP. Moreover, the modifications were stable even when the substrates were washed with a large quantity of ethanol, which is a good solvent for MPC polymers, for 24 h. Thus, the photoreaction of the MPC polymers occurred under the reported conditions, and a stable polymer layer was generated. We tried to visualize the modified MPC polymer layer on the surface and in the pores of the PE substrate using fluorescence-labeled MPC polymers. Figure 6 shows representative fluorescence microscopic images of the surface of the porous PE substrates and those modified with FPMrP80 and FPMbP80. Although there was no fluorescence on the unmodified PE substrates, the substrates modified with FPMrP80 and FPMbP80 are obviously marked with red fluorescence. From this result, we determined that the MPC polymer layer had been constructed on the surface of the porous PE substrate after photoirradiation. These results completely correspond to the results of IR and XPS spectroscopies, as shown in Figures 4 and 5, respectively. The cross-sectional views of the FPMrP80 and FPMbP80-modified porous PE substrates were obtained. The results are shown in Figure 7. These images clearly show fluorescence in the pores of the PE substrates. The dip-coating of the MPC polymer solution in ethanol occurred first, distributing the polymer into the porous substrates. Then, the polymers were immobilized by photochemical reaction inside the pores. The strong fluorescence intensity was recorded in the region at a depth of approximately 450 nm from the surface for all FPMrP-modified substrates with various monomer unit compositions. This result indicates that a large amount of the polymer was immobilized in this region. The composition of PMA units in the PMrPs had no influence on the photoreaction. In contrast, FPMbP-modified substrates had the strong fluorescence intensity in much deeper region of approximately 570 nm. Thus, some effects of the polymer architecture on the penetration or


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photochemical reactions of the MPC polymer were considered. As indicated in Table 1, the molecular weight and molecular weight distribution of the PMrPs were larger compared with those of the PMbPs. General, the viscosity of the polymer solution is dependent on the molecular weight of the polymer. In addition, highly viscous solutions penetrate small-diameter pores less than more free-flowing solutions. This is one reason for this difference. Another possible effect is the reactivity of the MPC polymer in the photochemical system. The PMA units in the polymer are important for immobilizing the polymer by cross-linking and reaction with the substrate. Therefore, the density of the PMA units after the coating process is a significant factor in determining the reactivity. By comparison with the random sequence polymer (PMrP), we concluded that PMbP may preferentially form a higher density of the PMA units because of the formation of molecular aggregates in solution. During the evaporation process, the structure of the molecular aggregates is maintained and fixed at the surface. The photoinduced radical formation of TBE occurs in the PMA domain, and the reaction progresses. Thus, the immobilization efficiency of the PMbP system is higher than that of PMrP system, and deeper modification in the pores can be achieved. Thus, we obtained UV spectra corresponding to the PMA units in the MPC polymers in ethanol solution. The maximum UV absorption at 256 nm appeared in each polymer solution. The absorption intensities in the spectra of the PMrP polymers were greater than those of the PMbP polymers, even when they had the same PMA composition. The UV absorption intensity is sensitive to the surrounding polarity. Generally, under low polarity conditions, the intensity of the UV absorbance is decreased. From these considerations, it is concluded that the PMA units in PMrP face the MPC units and polar ethanol, on the other hand, the poly(PMA) segments in PMbP form aggregate by themselves.

Characterization of the Interior of the Porous PE Substrates Super-hydrophilicity is a surface characteristic of MPC polymers. The time dependence of the water contact angles under dry conditions and the air contact angles in an aqueous medium of the PMrP- and PMbP-modified surfaces were measured to evaluate their surface hydrophilicity. Figure 8 shows the time dependence of the water-contact angle on the porous PE substrates. The water contact angle on the unmodified porous PE substrate was greater than


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120°, and this was maintained during the measurement. Thus, the PE surface has a hydrophobic nature. Additionally, the porous structure filled with air further enhances its properties. In contrast to this, a significant decrease in the water contact angle occurred on the MPC polymer-modified substrates. The water contact angle on PMrP80- and PMbP80-modified surfaces were 90° and 58°, respectively. The values decreased with time, and finally, they became 0° within 12 s. These phenomena were explained as one of the characteristics of porous materials with hydrophilic pore wall24. They affected by the composition of the MPC units in the polymer; that is, for the PMrP90- and PMbP90-modified surfaces, the water droplets entered the pores of the substrates much faster.

Evaluation of Protein Adsorption Resistance The protein adsorption resistance is a measure of the antifouling properties of a material.25-27 To test the materials for use in contact with biological systems, especially blood and soft tissue, fibrinogen can be used as a representative protein correlated closely with blood coagulation cascade and inﬂammatory response. Figure 9 shows the relative amount of fibrinogen adsorbed on the porous PE substrate. It is challenging to determine the total surface area of the porous PE substrate. Thus, the amount of fibrinogen adsorbed per unit area of the PE substrate was determined and presented as a relative value compared to that of the unmodified PE substrate. A significant reduction in the amount of fibrinogen adsorbed on the MPC polymer-modified porous substrates was observed. This depends on the MPC unit composition of the polymer; that is, the order of reduction was PMrP70 > PMrP80 > PMrP90 in the PMrP series. A significant reduction was observed in the PMbP series. It is well known that modification with MPC can reduce the degree of protein adsorption on polymer substrates.28-30 Thus, the observed reduction in the relative amount of fibrinogen adsorbed on the porous PE substrate was due to the construction of the MPC polymer layer, even in the pores.

CONCLUSIONS MPC polymers were immobilized stably on the porous PE substrates by UV irradiation using a photochemical reaction. The PMrP and PMbP polymers had an amphiphilic nature


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based on the hydrophilic MPC units and hydrophobic PMA units. During the dip-coating process, the polymer solution was absorbed and penetrated the porous PE substrate. Photoirradiation induced the photochemical reaction of TBE, forming radicals and inducing reactions with the polymer layer, which resulted in the formation of a stable polymer network-like structure. The PMrP or PMbP polymers successfully entered and were immobilized in the pores of the PE substrates. The polymer layer forms a hydrophilic surface, and water can penetrate the pores of the substrate. Accompanying these changes, the amount of protein adsorbed on the porous substrate was reduced dramatically. These results satisfy our research aim: the coating of the modified polymer on the substrates and into the pores and the maintenance of the excellent antifouling properties of MPC. A significant improvement in the amount of MPC polymer layer modifying the porous structure was obtained by using a simple photoirradiation approach. Our results indicate that the photochemical reaction will be particularly useful for the evolution of biomedical materials.

ACKNOWLEDGMENTS

This research was supported in part by the S-innovation Research Program for the "Development of the biofunctional materials for realization of innovative medicine," Japan Agency for Medical Research and Development (AMED).


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Legends of Figures

Figure 1. The synthetic route for the PMP and MPC polymers. Figure 2. Photoreaction mechanism in the polymer system. Figure 3. FT-TR spectra of porous PE substrate and those modified with the MPC polymers. Figure 4. XPS charts of the porous PE substrate and those modified with the MPC polymers. Figure 5. SEM images of porous PE substrate and those modified with the MPC polymers. Figure 6. Fluorescence microscope images of the porous PE substrate surface and those modified with the fluorescence dye-conjugated MPC polymers. Figure 7. Confocal fluorescence microscope images of cross-sectional porous PE substrate and those modified with the fluorescence dye-conjugated MPC polymers. Figure 8. Wetting by water on the surface of porous PE substrate and those modified with the MPC polymers. Figure 9. The relative amount of fibrinogen adsorbed on the porous PE substrate and that modified with the MPC polymers.


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Schematic illustration of photoinduced surface modification on porous polymer substrates 352x264mm (72 x 72 DPI)


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