Novel anti-biofouling and drug releasing materials for contact lenses

Colloids and Surfaces B: Biointerfaces 189 (2020) 110859

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Novel anti-biofouling and drug releasing materials for contact lenses a

Hiroaki Ogawa , Tadashi Nakaji-Hirabayashi Hiromi Kitanoa,e, Yoshiyuki Saruwatarif

a,b,c,

d

T c

*, Kazuaki Matsumura , Chiaki Yoshikawa ,

a

Department of Applied Chemistry, Graduate School of Science and Engineering, University of Toyama, Toyama 930-8555, Japan Department of Advanced Nano-bioscience, Graduate School of Innovative Life Science, University of Toyama, Toyama 930-0194, Japan International Center for Materials Nanoarchitectonics, National Institute of Material Science, Ibaraki 305-0047, Japan d School of Materials Science, Japan Advanced Institute of Science and Technology, Ishikawa 923-1292, Japan e R and D Head Office, Institute for Polymer-Water Interfaces, Toyama 939-2376, Japan f Business Operation Division, Osaka Organic Chemical Industry Ltd., Osaka 541-0052, Japan b c

A R T I C LE I N FO

A B S T R A C T

Keywords: Allergic conjunctivitis Contact lens Drug release Hot-melt press method Anti-biofouling property Zwitterionic polymer

Contact lens users very often become patients of allergic conjunctivitis, which is caused by protein and bacteria adsorption to the eye, because contact lenses easily adsorb proteins and bacteria. However, even if contact lens users develop eye diseases such as allergic conjunctivitis, most of them continue to use contact lenses to avoid interference to daily life or a decrease in their quality of life. If novel contact lenses able to prevent and additionally cure eye diseases can be manufactured, they could improve the quality of life of contact lens users worldwide. Thus, we aim to develop a novel material for contact lenses to prevent diseases by incorporating a zwitterionic polymer with the ability to suppress protein and bacteria adsorption. In addition, we also aim to effectively introduce and release a drug against allergic conjunctivitis from the contact lens material. Because the poorly water-soluble drug for allergic conjunctivitis (pranoprofen) forms a rigid crystal structure, we developed the novel “hot-melt press method” to construct a contact lens able to effectively release it. In the present study, polymer sheets containing carboxymethyl betaine (a kind of zwitterionic monomer), 2-hydroxyethyl methacrylate, and 1-vinyl-2-pyrrolidone were prepared using three different procedures. The sheets were hydrophilic and showed a strong resistance against protein and bacteria adsorption. The sheets prepared by the hot-melt press method were transparent and seemed to have potential as a material for contact lenses. In addition, the drug introduced into the sheets during preparation was observed to release at a practically appropriate dose. Therefore, it is expected that the sheets could possibly be used as a material for contact lenses which not only protect against the development of eye trouble due to protein and bacterial adsorption, but also heal allergic conjunctivitis.

1. Introduction Allergic conjunctivitis, which has common symptoms including burning, itching, foreign body sensations, photophobia, and tearing [1], is one of the most common eye problems and is increasingly prevalent worldwide [2,3]. An estimated 14–20 % of the US and 15–20 % of the Japanese population are thought to suffer from the disease [4,5]. The disease is a recurrent condition initiated by antigens [6], most of which are proteins such as Cry j1 [7]. Moreover, allergic conjunctivitis is thought to develop through inflammation initially associated with bacteria adsorption [1,6]. Thus, avoiding proteins and bacteria which adsorb to the eye is very important to prevent the disease.

The number of contact lens users worldwide has been increasing and now stands at over 71 million. Contact lenses are worn for both vision correction and cosmetic purposes [8]. However, contact lenses have drawbacks such as drying and the deposition of proteins and bacteria [9]. Because of this, contact lens users are at high risk of easily developing eye diseases such as allergic conjunctivitis. A major problem faced by most contact lens users is that, even if they develop eye disease, they prefer not to stop using their contact lenses, whether for medical or fashion purposes, because of the decreased quality of life [8,10]. Given these facts, the development of novel contact lenses to prevent allergic conjunctivitis is required. Additionally, if these novel contact lenses are able to prevent and also “cure” allergic conjunctivitis,

⁎ Corresponding author at: Department of Applied Chemistry, Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama, 9308555, Japan. E-mail address: [email protected] (T. Nakaji-Hirabayashi).

https://doi.org/10.1016/j.colsurfb.2020.110859 Received 21 October 2019; Received in revised form 23 January 2020; Accepted 10 February 2020 Available online 11 February 2020 0927-7765/ © 2020 Published by Elsevier B.V.

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2-hydroxyethyl methacrylate (HEMA) and 1-vinyl-2-pyrrolidone (VP), known contact lens materials, and carboxymethyl betaine (CMB), a zwitterionic monomer with anti-biofouling properties. In order to demonstrate the validity of our hot-melt press method, we also fabricate two additional polymer sheets using commonly-used crosslinking methods. The surface hydrophilicity of the polymer sheets was examined using water droplet and air bubble contact angle measurements. The mechanical properties, Young’s modulus, and transparency of the polymer sheets were confirmed using tensile tests and light transmittance measurements, respectively. We also evaluated protein adsorption and bacteria adhesion to assess the anti-biofouling properties of the polymer sheets. In addition, we loaded a hydrophobic drug (pranoprofen) into the sheets using the hot-melt press method and confirmed sustained drug release. Our final goal is to create a contact lens with the capacity not only to prevent, but also to cure, allergic conjunctivitis. This work is the first step towards this goal.

the global occurrence of eye diseases associated with the use of contact lenses might decrease drastically. A contact lens for suppressing protein adsorption has previously been developed [11,12] and commercialized by the company involved. However, commercialized contact lens materials have to wash regularly for removing adsorbed protein because the surface of these materials cannot completely suppress the protein adsorption. The development of contact lens materials enable to ultimately suppress the protein adsorption is very important to prevent the eye disease for contact lens user. Until now, we demonstrated that materials containing zwitterionic polymer, which have the same number of cations and anions (mostly in their side chains), and surfaces covered with zwitterionic polymer can be ultimately suppress the protein adsorption and cell adhesion [13–16]. Therefore, it is considered that contact lens materials incorporated zwitterionic polymer can be suppressed the adsorption of proteins and bacteria. Previous studies reported that the zwitterionic polymers have anti-biofouling properties such as the suppression of protein adsorption and cell adhesion [11]. As the key material, poly (methacryloyloxyethyl phosphorylcholine) and polyethylene oxide are used for the suppression of protein adsorption and is coated on the surface of contact lens using a silane coupling reaction. However, surface coatings can easily detach from the surface of contact lenses due to the scraping associated with blinking and during the insertion and removal of the contact lenses. Therefore, improvement to the stability and durability of these suppression properties is required. In addition, considering that it is difficult for contact lens users to stop wearing them even after the onset of an allergic reaction, contact lenses that can treat diseases while being worn are in strong demand. There are some previous reports on the use of contact lenses for drug release. The drugs are typically incorporated into the contact lens materials by charge interactions [17] or physical adsorption [18,19]. For these methods, the drugs need to be soluble in water, while most ophthalmic drugs are hydrophobic and therefore difficult to dissolve in water. In order to overcome these limitations of contact lenses, we designed a novel contact lens material for ophthalmic drug release. Scheme 1 shows the design concept: We achieved (1) excellent antibiofouling properties on the surface of the contact lens by incorporating a zwitterionic polymer into the material, and (2) the sustained drug release of poorly water-soluble drugs (pranoprofen) from the material. In order to load the drugs into the material, we developed a new technique, namely the hot-melt press method which combines hot-melt extrusion and the hot-press molding method. Hot-melt extrusion is used to dissolve drugs (especially highly crystalline drugs) into polymers [20,21], while the hot-press molding method is used in various fields, especially contact lens development [22–24]. Both these methods are often applied in manufacturing, however, they have never been combined. Therefore, our developed material and technique could be valuable in terms of their industrial applications. Herein, we prepare the contact lens material (hydrogel sheets) using

2. Materials and methods 2.1. Materials The 1-carboxy-N,N-dimethyl-N-(2-methacryloyloxyethyl)methanaminium hydroxide inner salt (CMB); commercial name, GLBT, 97 %) was donated by Osaka Organic Chemistry Industry Co., Ltd. (Osaka, Japan). The HEMA (95 %), copper(I) bromide, 2-2′-bipyridyl, 4-cyano4-(thiobenzoylthio)pentanoic acid (CTA-1), N,N,N’,N’-tetramethyl ethylenediamine (TEMED), potassium peroxodisulfate (KPS), glutaraldehyde, and ammonium acetate were purchased from Wako Pure Chemicals (Osaka, Japan). The 2-(5H-Chromeno[2,3-b]pyridin-7-yl) propanoic acid (pranoprofen) was obtained from Sigma-Aldrich (St. Louis, MO, USA). The VP (97 %) and sodium L-ascorbate were from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). The copper sulfate and 2-(2-bromoisobutyryloxy)ethyl methacrylate (BIEM) were from Sigma-Aldrich. The propargyl methacrylate (PGMA, 98 %) was purchased from Hydrus Chemical Inc. (Tokyo, Japan). The polyethylene glycol dimethacrylate (PEGDMA, EG units ≈ 8) was a gift from ShinNakamura Chemical Co., Ltd. (Wakayama, Japan). The ethyl-2-bromoisobutyrate (EBiB) was from Nacalai Tesque (Kyoto, Japan). All other reagents and solvents were commercially available. Ultrapure water (18 MΩ cm, MilliporeSystem) was used for the preparation of samples. 2.2. HEMA-VP-base polymer sheets incorporating CMB or PCMB Three different methods were adopted for the preparation of sheets: (Method-A) the glutaraldehyde cross-linkage of a copolymer containing HEMA and VP, grafted with CMB polymer (PCMB); (Method-B) the copolymerization of HEMA, VP, a PCMB-macromonomer, and PEGDMA using free radical polymerization; and (Method-C) the Scheme 1. Design concept of novel anti-biofouling and drug releasing materials for contact lenses. These materials are based on 2hydroxyethyl methacrylate and 1-vinyl-2-pyrrolidone which are the same components as used in soft contact lenses. By incorporating carboxymethyl betaine, the novel material can drastically suppress the nonspecific adsorption of proteins and bacteria. Additionally, by using the hot-melt press method, which is a novel method of encapsulating poorly water-soluble drugs that form crystal structures, the antibiofouling hydrogel can effectively store and release the drug.

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and 100 °C for 30 min on a hot-press machine (AH-2003, As One Corporation, Osaka, Japan). The constructed sheets were washed by incubating them in water for 1 week at 37 °C and sterilized in the same manner as the sheets described above.

copolymerization of HEMA, VP, CMB or a PCMB-macromonomer, and PEGDMA using the hot-melt press method. Various tests were conducted on the sheets prepared using these methods. 2.2.1. Preparation of polymer sheets cross-linked by glutaraldehyde (Method-A) Polymer sheets were prepared in three steps (Scheme S1 in Supporting information (SI)). First, a macroinitiator, P(HEMA-VPBIEM) with an initiating group for atom transfer radical polymerization (ATRP), was prepared by RAFT polymerization of HEMA, VP, and BIEM, as described in the SI. Then, PCMB was prepared from the initiating group of BIEM residues in P(HEMA-VP-BIEM) by the ATRP of CMB, as described in the SI (P(HEMA-VP-BIEM-g-PCMB)). Nine kinds of polymers, which differed from PCMB in their graft number per main polymer chain and their degree of polymerization (DP) of CMB (Table S1), were prepared using this method. The reaction efficiency of the monomers was examined using proton nuclear magnetic resonance (1H NMR) measurements, as described in the SI. After preparing the (P(HEMA-VP-BIEM-g-PCMB)), polymer sheets were constructed using glutaraldehyde cross-linkages between the hydroxyl groups on the HEMA residues. The synthesized polymer, P (HEMA-VP-BIEM-g-PCMB) (20 mg), was dissolved in 800 μL of methanol. The solution was mixed with 200 μL of 25 % glutaraldehyde solution and the mixed solution was poured into a 26 mm × 38 mm × 0.5 mm glass chamber (silicon sheets (thickness, 0.5 mm) were sandwiched by the glass plates) to form films (Scheme S1). After forming the sheets by cross-linking, the films were dried overnight at 25 °C and then at 80 °C for 6 h. To remove unreacted glutaraldehyde, the films were incubated in water at 37 °C for 1 week and sterilized in an autoclave at 120 °C for 15 min.

2.3. Characterization of the surface of the polymer sheets by contact angles Water drop and air bubble contact angle measurements were measured using a contact angle meter, the DMs-401 (Kyowa Interface Science Co., Ltd., Saitama, Japan). The detailed procedure is described in the SI. 2.4. Mechanical strength of the polymer sheets The mechanical strength of the polymer sheets was examined using a tensile test (TENSILON RTG-1210, A&D Co., Ltd., Tokyo, Japan), as described in the SI. The mechanical strength of the wet polymer sheets was evaluated by measuring the stress and strain. 2.5. Light transmittance of the polymer sheets The light transmittance of the sheets was examined using a UV–vis spectrophotometer (UV-2600, Shimadzu Corporation, Kyoto, Japan), as described in the SI. 2.6. Observation of the surfaces of the polymer sheets The surfaces of the polymer sheets were observed with a field emission scanning electron microscope (FE-SEM, JSM-6700 F, JEOL Ltd., Tokyo, Japan), as described in the SI. 2.7. Investigation of reaction efficiency of monomers

2.2.2. Preparation of polymer sheets using KPS and TEMED (Method-B) Polymer sheets were prepared in four steps (Scheme S2). Detailed procedures are described in the SI. Briefly, PCMB synthesized by ATRP has an azide group at the terminal end of the polymer. When mixed with sodium azide, the PCMB terminal group changes from a Br group to an azide group. Finally, methacrylate groups were introduced to the terminals of PCMB using a click reaction between the azide group of the PCMB and the alkyne group of PGMA (PCMB-MA). The purity of the PCMB-MA macromonomer was analyzed using 1H NMR measurements. Using PCMB-MA, PCMB-graft chains were introduced to the HEMAVP base polymer sheet. An aqueous solution of PCMB-MA (100 mg/mL) was added to a monomer mixture (HEMA : VP : PEGDMA = 239 : 60 : 20) while the ratio of CMB in the total amount of substance was set to 2 mol%, 4 mol%, and 6 mol%. KPS was added to this solution at 1 mg/mL and, after mixing, 1 μL/mL of TEMED was added. This mixture was poured into the 26 mm × 38 mm × 0.5 mm glass chamber. After leaving them overnight, the gel sheets obtained were incubated in water for 1 week at 37 °C in order to remove byproducts and then sterilized in an autoclave at 120 °C for 15 min (Scheme S2).

To investigate the reaction efficiency of HEMA, VP, and CMB and to discuss the location of CMB residues in the polymer chains, three monomers were polymerized by free radical polymerization in methanol using AIBN as an initiator. The detailed procedure and analysis method are described in the SI. 2.8. Evaluation of protein adsorption to polymer sheets The amount of protein (bovine serum albumin, BSA) adsorbed to the sheets was evaluated by the bicinchoninic acid (BCA) method using a microBCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA), as described in the SI. 2.9. Evaluation of bacteria adsorption to polymer sheets To investigate the bacterial adsorption to the polymer sheets incorporating CMB in the main chain or PCMB as a graft chain, the adsorption of the DH5α strain of Escherichia coli, which is well known as the EK1 family of E. coli, to polymer sheets was observed by FE-SEM. The detailed procedure is described in the SI.

2.2.3. Preparation of polymer sheets using the hot-melt press method (Method-C) HEMA, VP, PEGDMA, and 2,2′-azobisisobutyronitrile (AIBN) were dissolved in water or alcohol solutions of CMB or PCMB-MA with or without the drug (pranoprofen). The molar ratio of HEMA : VP : PEGDMA : AIBN was 239 : 60 : 20 : 0.5. The ratio of CMB or PCMB-MA in the total amount of substances (HEMA, VP, and PEGDMA) was set to 0.5 %, 1.0 %, and 2.0 %. As shown in Scheme 2, to construct the polymer sheets, silicon sheet with an inner window of W25 mm × D20 mm × H1.0 mm (silicon frame) were prepared. Each 200 μL sample of the solution containing monomers and the initiator, with or without the drug, was placed into the inner window of a silicon frame which was attached to a silicon sheet, and another silicon sheet was used to cover it. The sheets were interposed between iron plates. After that, the polymer sheets were prepared using the hot-melt press method at 5 MPa

2.10. Drug releasing test The release of the drug for treating allergic conjunctivitis, pranoprofen, which is a poorly water-soluble drug, was investigated using polymer sheets prepared using the hot-melt press method. The drug was added to the alcohol that was used as a solvent in the hot-melt press method at concentrations of 0, 1, 10, 30, and 50 mg/mL. After processing, 0, 27, 270, 810, and 1350 μg of the drug were present in the sheets. The sheets were immersed in 10 mL PBS at 37 °C and stirred. The solution (100–500 μL) was collected 1, 2, 3, 5, 12, and 24 h after incubation and mixed with a water-alcohol mixture (7:3), containing 0.03 M ammonium acetate, to completely dissolve the pranoprofen. UV 3

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Scheme 2. Illustration of the set-up of the hot-melt press method. The sample window of the silicon sheet was W25 × D20 × H1.0 mm. A monomer solution containing an initiator, with or without the drug, was placed in the sample window. The polymer sheet was then prepared at 5 MPa and 100 °C for 30 min.

number of PCMB grafts compared to the main chain of the HEMA-VP polymer. From a financial standpoint, it is difficult to manufacture contact lenses by RAFT polymerization and ATRP. Additionally, the mechanical properties of polymer sheets cross-linked with glutaraldehyde are insufficient for practical use. Glutaraldehyde cannot be used as a regent for medical devices because of its strong toxicity in the human body. Next, we attempted to prepare the sheet using Method-B, which is a single-step method using free radical polymerization and PEGDMA as a cross-linker. For this purpose, we synthesized the PCMBmacromonomer. Although the polymer sheets prepared using Method-B showed good suppression of protein adsorption, the mechanical properties of the sheets were still low and presented a fatal flaw in a lack of transparency. In the end, we developed a third, novel method for constructing polymer sheets, the hot-melt press method (Method-C). This novel method was inspired by three ideas: 1) the preparation of “clear” gel sheets composed of poly(vinyl alcohol) using the hot press method previously studied by Matsumura et al. [26]; 2) the hot-melt extrusion method widely used in the development of drug delivery system materials [20,21,27]; and 3) the hot-press molding method generally used for manufacturing contact lenses [25]. In Method-C, polymer sheets incorporating CMB displayed anti-biofouling properties due to the chemical properties of their zwitterionic moieties and the exclusion volume effects of their CMB moieties. However, the polymer sheets with PCMB-graft chains became slightly cloudy even though we used the hot press method, which may be a serious obstacle to practical use. We additionally evaluated the characterization of the polymer sheets that incorporated CMB in the main chain. Essentially, the CMB composition and the influence of incorporating CMB on the capabilities of the material were investigated using polymer sheets prepared using Method-A. In contrast, the polymer sheets prepared using Methods-B and -C were carefully investigated for practical use. Finally, the stiffness (mechanical strength), optical transparency, anti-biofouling properties against proteins and bacteria, and poorly

measurement at 271 nm was performed using a spectrophotometer to quantify the amount of the drug released into the PBS. As a negative control, polymer sheets without the drug were immersed for 24 h in 0.1 % pranoprofen solution (solvent: PBS). After that, the amount of drug released from the sheets was also measured following the method shown above. A calibration curve was made from 0 to 50 μg/mL of pranoprofen. 2.11. Statistical analysis The results of contact angle, Young’s module and assays for protein adsorption were statistically evaluated using the JMP software (JMP Pro 13, SAS Institute Inc., Tokyo, Japan). One-way analysis of variance (ANOVA) was carried out to determine the significant differences. In addition, Tukey’s honestly significant difference (HSD) test was carried out for multiple comparisons of all data at a significance level of p < 0.05. 3. Results and discussion 3.1. Strategy for constructing novel soft contact lens material We prepared the sheet material composed of HEMA and VP, with and without CMB, using three different methods. HEMA and VP are the main components in soft contact lenses. The composition ratio of HEMA and VP in all three types of polymer sheets containing CMB was equivalent to their composition in commercialized soft contact lenses [8,25]. Our strategy for creating a novel, soft contact lens material is detailed below. First, P(HEMA-VP-g-PCMB) synthesized with RAFT polymerization and ATRP was cross-linked with glutaraldehyde (Method-A). This is because This method allows us to clearly examine the relationships between the capabilities of the material for use in contact lenses and the composition ratio of CMB, the length of the PCMB-graft chains, and the 4

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polymer sheets could be obtained. However, the preparation of sheets using RAFT polymerization and ATRP for the base polymer and glutaraldehyde for cross-linking is problematic. For the viable application of this material to contact lenses, easy and safe preparation methods are essential. Therefore, we prepared sheets cross-linked with PEGDMA during the polymerization of KPS and TEMED (Method-B) and evaluated the characteristics.

water-soluble drug release properties of the sheet materials prepared using Method-C were evaluated. 3.2. Evaluation of CMB content using polymer sheets cross-linked by glutaraldehyde (Method-A) 3.2.1. Characterization of polymer sheets incorporating PCMB grafts First, three types pre-polymer composed of HEMA, VP and BIEM was prepared. The composition of pre-polymers was determined by 1H NMR measurement (P(HEMA235-VP60-BIEM5), P(HEMA238-VP60BIEM2), and P(HEMA239-VP60-BIEM1), Table S1). And then CMB was polymerized to the side chain of the pre-polymer by ATRP. The average degree of polymerization of CMB was calculated with molecular weight (Mn) obtained by GPC measurement. Clear sheets could be prepared by glutaraldehyde cross-linking a copolymer of HEMA-VP grafted with PCMB (Fig. S1). The wettability of the sheets with PCMB-graft chains was evaluated by both water drop and air bubble contact angles (Figs. S2A and S2B). The water contact angles did not drastically change, probably because the PCMB-graft chains penetrated the main chain, including the HEMA and VP residues, in a dry state and, therefore, the composition at the surface might not noticeably change. The air bubble contact angle of the sheets with PCMB-graft chains was larger than that of the sheets without PCMB-graft chains by over 10 degrees, which shows that the sheets with PCMB-graft chains were more hydrophilic. It is inferred that PCMB-graft chains occur outside of the main chains, including the HEMA and VP residues, in water. The air bubble contact angle increased with CMB content, because CMB is hydrophilic [28,29]. In general, surfaces have environmental responsiveness if the sum of the water and air bubble contact angles is above 180 degrees. The sum of the contact angles for almost all the sheets with PCMB-graft chains drastically exceeded 180 degrees (Fig. S2C), so the sheets with PCMBgraft chains were environmentally responsive. Therefore, it is inferred that PCMB-graft chains were in the main chains, including the HEMA and VP residues, in dry conditions while they were outside of the main chains in water.

3.3.1. Preparation of polymer sheets To construct the polymer sheets in one step, the PCMB-MA macromonomer (DP of CMB = 53) was synthesized while being incorporated into a polymer sheet. To investigate the chemical composition of polymer chain in the sheet, the polymer sheets without washing by water were evaluated with 13C solid-state NMR measurement (data not shown). The peak derived from vinyl group of monomers completely disappeared. This indicated that all monomers were consumed by polymerization, namely, the chemical composition of the sheet (polymers) is equivalent to the feeding ratio of monomers. Sheets were prepared with various CMB contents (Fig. S4). Heterogeneous sheets were obtained when the CMB content was over 8%, whereas homogeneous sheets were obtained with CMB contents less than 6%. Therefore, only the sheets prepared with less than 6% CMB were used in the various tests described hereafter. However, all the polymer sheets obtained using this method were cloudy, which is highly problematic for their application as contact lens material. We discuss this further in section 3.3.3. Tensile test of the mechanical strength of polymer sheets, below. 3.3.2. Anti-biofouling properties of polymer sheets The adsorption of proteins to the sheets with PCMB-graft chains was investigated using BSA (Fig. S5). Less protein adsorbed to the sheets with PCMB-graft chains than to those without them. Furthermore, the protein adsorption of the sheets decreased with an increase in CMB content, because CMB suppresses protein adsorption [28,29,32,35]. This agrees with the results obtained from the polymer sheets prepared using Method-A. 3.3.3. Tensile test of the mechanical strength of polymer sheets The strength of the sheets in wet conditions was measured with a tensile test (Fig. S6). There was a tendency for both strength and strain to decrease with increasing CMB content. It is possible that polymerization efficiency decreased due to the increase in PCMB-graft chains. Additionally, all the sheets produced by this method were very weak. Due to 1) the very weak mechanical properties and 2) the poor transparency of the polymer sheets obtained by Method-B, we concluded that the HEMA-VP main chains and PCMB-graft chains caused a phase separation. In the PCMB, especially, domains over 100 nm might be formed in the polymer sheet. As shown in Fig. S6, stress-strain curves of the sheets incorporating PCMB-graft chains displayed several decreasing stress points before the rupture point. This indicates that domains caused by the different strengths were formed in the polymer sheets. It has been well established that, when micro-domains over 100 nm are contained in a polymer material, the material becomes cloudy [26]. Consequently, although the polymer sheets prepared by Method-B displayed anti-biofouling properties, it is difficult to apply this method to the production of contact lens material.

3.2.2. Protein adsorption and cell adhesion to the polymer sheets The amount of BSA, a well-known model for proteins [30,31], adsorbed to the polymer sheets with or without PCMB-graft chains was investigated using BCA measurement (Fig. S3). Less protein was adsorbed to the polymer sheets with PCMB-graft chains than to those without them. Comparing polymer sheets with the same number of graft chains per main chain, there was a tendency towards decreased protein adsorption with increasing CMB content. This is because CMB has the ability to suppress protein adsorption [28,29,32,33]. Interestingly, polymer sheets with longer PCMB graft chains suppressed protein adsorption more effectively than those with shorter graft chains, even if the number of graft chains per HEMA-VP polymer chain increased. Furthermore, a substantial amount of protein adsorbed to the sheets with 10 and 15 % CMB content and two graft chains per main chain. The anti-biofouling properties of the sheets was obtained by the exquisite balance between the chemical properties of the zwitterionic moieties and the exclusion volume effect associated with the chain length and chain number of the graft chains, as we described previously [14,34]. We concluded that the exclusion volume effect of the polymer chain, or in other words its physical and mechanical properties, is also a very important factor in the suppression of protein adsorption.

3.4. Polymer sheets constructed using the hot-melt press method (Method-C) 3.4.1. Preparation of polymer sheets Polymer sheets were prepared with various CMB contents. The chemical composition of polymer chain in the sheet prepared by Method-C was evaluated with 13C solid-state NMR measurement. When the polymer sheet without washing with water was measured, the peak of vinyl group of monomers completely disappeared, indicating that all monomers were consumed by polymerization. Therefore, it was

3.3. Polymer sheets cross-linked by PEGDMA using KPS and TEMED (Method-B) Using the polymer sheets prepared by Method-A, the anti-biofouling properties associated with PCMB incorporation in HEMA-VP-based 5

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assumed that the chemical composition of polymers in the sheet prepared with Hot-melt press method is equivalent to the feeding ratio of monomers. When the CMB content was above 2%, the turbidity of the sheets became conspicuous. The maximum CMB content of the sheets was therefore 2%. In addition, water and alcohol were evaluated as solvents in preparing the sheets. The polymer sheets displayed turbidity and were heterogeneous when water was used as the solvent, probably because phase separation occurred. In contrast, homogeneous sheets could be prepared when alcohol was used as the solvent (Fig. S7). This is probably because the reaction solutions in which water was used as the solvent were highly viscous, resulting in the easy separation of CMB and HEMA-VP in the solution. Therefore, sheets were prepared by introducing a PCMB macromonomer or a CMB monomer in alcohol. Polymer sheets with PCMB-graft chains were homogeneous, but slightly turbid. In contrast, sheets with CMB residues in the main chains were homogeneous and clear (Fig. S8).

Fig. 2. Visible region light transmittance of polymer sheets with various CMB contents. The polymer sheets without CMB (a, solid line), with CMB in the main chain at a concentration of 0.5 % (b, dotted line), 1.0 % (c, dashed line), and 2.0 % (d, long dashed-dotted line), and with CMB as graft chains at a concentration of 0.5 % (e, short dashed-dotted line), 1.0 % (f, long dashed line), and 2.0 % (g, long dashed-double dotted line) were prepared using Method-C.

3.4.2. Wettability of polymer sheets containing CMB in the main chain and PCMB-graft chains The wettability of sheets was measured by water drop and air bubble contact angle methods. The water contact angle decreased with increasing CMB content, while the bubble contact angle increased (Fig. 1). This was because the wettability of the polymer sheets was increased by the introduction of CMB residues, which provided hydrophilicity [29].

PCMB-graft chains can be ascribed to light scattering by the PCMB domains. The formation of the PCMB domain is discussed in depth in the next section. The light transmittance of the sheets with CMB in the main chain was similar to that of most commercialized contact lenses (transmittance = 92 ∼ 97 %) [36,37].

3.4.3. Measurement of light transmittance of the polymer sheets The transmittance of polymer sheets without CMB was over 92.3 % in the visible region in water (Fig. 2). The transmittance of sheets with CMB residues in the main chain was also over 91.5 % in the visible region, whereas that of the sheets with PCMB-graft chains was lower than that of either of the other sheet types. In addition, the transmittance of the sheets with PCMB-graft chains decreased with increasing CMB content. The decrease in the transmittance of the sheets with

3.4.4. Observation of polymer sheet surfaces by FE-SEM First, the roughness of the polymer sheet was observed by FE-SEM. As shown in Fig. S9, the roughness of the polymer sheets containing CMBs in the main chain was significantly small in comparison of that of control polymer sheet. It was probably because the mobility of HEPA and VP in the polymer chains are affected by the CMB content. In contrast, the surface of the polymer sheets with PCMBs as the graft chain increased roughness. This suggests that the roughness was ascribed to the phase separation between PCMB grafts and HEMA-VP main chains. Next, the polymer sheets with and without CMB were investigated to evaluate the phase separation between HEMA-VP and CMB (Fig. S9). White dots were observed both on sheets with CMB in the main chain and on sheets without CMB (Figs. S9c and S9f). Therefore, it was thought that these white dots were not related to phase separation. In contrast, a scaly pattern was observed on the sheets with PCMB-graft chains (Fig. S9i, black arrows). Therefore, it is suggested that PCMB caused phase separation in the sheets with PCMB-graft chains. This implies that the light transmittance of sheets with PCMB-graft chains is lower than that of the other sheet types. The polymer sheets with CMB in the main chain, prepared using Method-C, were clear and demonstrated high light transmittance, whereas the polymer sheets prepared using Method-B were opaque. This is probably due to the macro-domains formed in the polymer sheets prepared with Method-B. In contrast, the polymer sheets prepared with Method-C hardly formed any macro-domains. In other words, phase separation might not occur in the polymer sheets prepared using Method-C. Generally, it is well known that even if the domains formed in association with a phase separation are very small, the light transmittance of a polymer material decreases when the difference in refractive index of the monomers is over 0.02 [38]. The refractive indices of HEMA, VP, and CMB are 1.452 [39], 1.513 [39], and 1.491 [40], respectively. If the polymer sheets prepared using Method-C formed micro domains due to microphase separation, the light transmittance of the sheets would decrease because of the large differences in the refractive indices of the monomers. Therefore, we assumed that hardly any domains formed in the polymer sheets prepared using Method-C.

Fig. 1. (A) Water drop contact angle and (B) air bubble contact angle on the surface of polymer sheets with various CMB contents prepared by Method-C. 6

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Fig. 4. The amount of protein adsorption on sheets prepared using Method-C. Dotted line shows the detection limit (8.88 μg/mg) of microBCA measurement.

Fig. 3. Young’s modules of polymer sheets with and without CMB in the main chain and with PCMB as a graft chain.

using the hot-melt press method. From these results, it is understood that both the sheets with CMB residues in the main chain and those to which PCMB is introduced as a graft chain have environmental responsiveness, whereas sheets without CMB do not.

3.4.5. Mechanical strength of polymer sheets The mechanical strength of the polymer sheets prepared using Method-C was over 500 times higher than that of the polymer sheets prepared using Method-B (Fig. S2). This is because the polymer sheets can form high-density polymer networks during construction using the hot-melt press method. The stress and strain of the polymer sheets with CMB in the main chain decreased with increasing CMB concentration (Fig. S10A). As shown in Fig. 3, polymer sheets containing 0.5 % and 1.0 % CMB produced almost the same Young’s module, whereas the values of the Young’s module of the polymer sheet containing 2.0 % CMB were drastically lower. These results were attributed to a decrease of hydrogen bonding between the hydroxyl groups of the HEMA and an increased swelling ratio of the polymer sheet associated with the inclusion of CMB. In contrast, the mechanical strength of the polymer sheets containing PCMB-graft chains gave more distinguishing results (Fig. S10B). Although the stress and strain decreased with the addition of PCMBgraft chains (concentration of CMB: 0.5 %), the stress and strain tended to increase with increasing CMB concentration in the graft chain. Especially, the stress and strain of the polymer sheets with PCMB-grafts at a concentration of 2% were higher than those of sheets without CMB. This is probably because the PCMB-graft chains penetrated the HEMAVP networks or, in other words, a network structure similar to a semiinterpenetrating polymer network was formed. Additionally, the mechanical properties of the sheets obtained using Method-C were almost the same as those of commercialized soft contact lenses (0.5–3.5 MPa) [38,39].

3.4.7. Interaction of proteins and bacteria with polymer sheets Protein adsorption (using BSA) was measured for the sheets prepared by the hot-melt press method. The protein adsorptions of the sheets with CMB in the main chain and PCMB-graft chains were lower than that of the sheets without CMB (which suppresses protein adsorption) (Fig. 4). Many researchers have pointed out that the structure of water on the polymer surface is related to anti-biofouling properties [35,41,42]. We propose that the anti-biofouling properties of zwitterionic polymers are attributable to the mildness of the polymers’ reaction to the hydrogen bonded network structure of vicinal water [43–46], resulting in a reduction in the non-specific adsorption of proteins and affording biocompatibility [47]. As shown in the Fig. 4, the amount of protein adsorption did not change with CMB content. This is because the amount of protein adsorption on the sheets with CMB was close to the detection limit of the microBCA method (Fig. 4, dashed line). Protein adsorption on the sheets with CMB residues in the main chain was suppressed, partly because the CMB domains extending into the liquid phase had exclusive volume effects, similar to a PCMB-graft chain. This suggestion in based on the results of the time evolution of the air bubble contact angle and the reaction efficiency of monomers (section 3.4.6.). As preliminary test, we also examined adsorptions of human lysozyme and human serum albumin, that are contained in tears was investigated (n = 1). As shown in Table S2, all the polymer sheets containing CMBs suppressed the adsorption of human serum albumin. In contrast, the adsorption of lysozyme was significantly suppressed to the surface of the polymer sheets with CMBs in main chain, whereas slightly adsorbed to the surface with CMB as graft chain. These results indicated that incorporation of CMBs to the HEMA-VP polymer sheets can effectively suppress protein adsorptions. Additionally, the bacterial adsorption to polymer sheets with CMB in the main chain or PCMB-graft chains was investigated using the E. coli. As the controls, the adsorptions of E. coli to the surface of glass, the polymer sheet without CMB, and a commercialized soft contact lens were also investigated to allow comparisons between E. coli adsorption numbers (Fig. 5). The adsorption of E. coli on the surface of polymer sheets with CMB was significantly lower than that on the surface of glass or the polymer sheets without CMB. Interestingly, the adsorption of E. coli was most suppressed on the polymer sheets with 1% CMB either in the main chain or as the graft chain, though it was expected that E. coli adsorption would decrease with increasing CMB concentration. On the low-magnification SEM images (Fig. S13), the polymer sheets with 1%

3.4.6. Environmental response of surface of polymer sheets containing CMB in the main chain and PCMB-graft chains The change in wettability of the surfaces over time was evaluated by air bubble contact angle (Fig. S11). The contact angles of the sheets with CMB in the main chain and those with PCMB-graft chains tended to increase as time elapsed after immersing them in water and plateaued after 10 min of immersion. In contrast, the contact angles of the sheets without CMB did not change during 60 min of immersion in water. This indicates that the PCMB or CMB residue was exposed on the outside of the sheets when they were immersed in water. Therefore, it can be inferred that sheets with CMB in the main chain have domains composed of a few CMB residues. To demonstrate the domain formation of CMBs in the main chain of the HEMA-VP, the reaction efficiency of monomers of HEMA, VP, and CMB was investigated by 1H NMR (Fig. S12). Almost equal amounts of HEMA, VP, and CMB were included before the polymerization reaction, but the ratio of remaining monomer residues (HEMA : VP : CMB) was 1.71 : 3.99 : 1.00 after the reaction. Therefore, it can be inferred that CMB reacts quickly and that domain structures are formed by extending the few CMB residues in the system when the sheets were prepared 7

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Fig. 5. SEM images of bacteria adsorption on the surface of polymer sheets with various contents of CMB in the main chain or as the graft chain. The size of the E. coli used as the model is generally known: short axis = 1 μm and long axis = 2 – 3 μm. Black arrows represent E. coli.

its mechanical properties and surface roughness on the suppression of E. coli adsorption [48,49]. The hydrophilicity and swelling ratio significantly increase with the incorporation of CMB in the polymer sheet, resulting in decreased mechanical strength and increased surface roughness. Therefore, the polymer sheet with 1% CMB might have the best stiffness and roughness for the suppression of E. coli adsorption. 　Cell adhesion was also investigated on the polymer sheets containing CMBs. Herein, we used NIH3T3, one of common fibroblast cells, as a model cell (Fig. S14). The cell adhesions were significantly suppressed on the surface of the polymer sheet with CMBs in the main chain. In addition, the surface of polymer sheet containing CMBs shows high wettability (Fig. 1), indicating that these surfaces are covered with thick water layer in comparison of polymer sheet surface with HEMA and VP. Taken with the results of protein adsorption and bacteria adhesion, it is expected that a contact lens prepared by the polymer material containing CMBs is bio-inactive not to affect human corneal epithelial cells and cornea.

Fig. 6. The amount of drug (pranoprofen) released from sheets prepared using Method-C in water. ⬤, 0 μg/0.12 cm3 (the amount of drug per sheet); ○, 27 μg/0.12 cm3; ⬜, 270 μg/0.12 cm3; ⬛, 810 μg/0.12 cm3; ◇, 1350 μg/0.12 cm3; ◆, sheet immersed in 0.1 % drug solution/0.12 cm3.

3.4.8. Drug releasing test Finally, the amount of drug released from the sheets was investigated (Fig. 6). It is known that the pranoprofen crystallizes and dissolve in dimethylformamide and alcohols, whereas hardly dissolve in water (solubility: < 0.05 %) [50]. However, the monodispersed pranoprofen can dissolve in water. Thus, we expected that the incorporated pranoprofen in the polymer sheets by the hot-melt press method could be released into the solution (tears) as the monodispersed pranoprofen. The melting and degradable temperature of pranoprofen is 186–190 °C and 200–205 °C, respectively [50,51]. In addition, pranoprofen dissolves in alcohol/water mixture around 95−100 °C. Therefore, we expected that pranoprofen should be stable during our hot-melt press. In order to confirm this, we heated pranoprofen in a mix solvent of alcohol

CMB can be seen to totally suppress E. coli adsorption. Moreover, the bacteria’s adsorption to the surface of a commercialized soft contact lens (ACUVUE®, Jonson & Jonson Vision Care Companies, New Brunswick, NJ, US) was compared with their adsorptions to the polymer sheets with and without CMB. The commercialized soft contact lens could not completely suppress bacteria adsorption, although the number of adsorbed bacteria was less than that of the polymer sheet without CMB (the HEMA-VP control polymer sheet). These results suggest that E. coli adsorption to polymer sheets can be suppressed by the incorporation of CMB, which has anti-biofouling properties. The reason the polymer sheet with 1% CMB could suppress E. coli adsorption so effectively, might be explained by the influence of 8

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Acknowledgments

and water at 100 °C for 30 min, the same condition as the hot-melt press. 1H NMR confirmed that the structure of pranoprofen hardly changed before and after the heating. This result indicates that the hot melt press method would not affect the effective components of pranoprofens. The drug was released over time, and the amount of drug released increased with the amount of drug introduced. Generally, it is suggested that two drops of 0.1 w/v% pranoprofen are used four times per day. The dotted and dashed lines in Fig. 6 show the amount of drug recommended per dose (a) and per day (b). A sheet containing 1350 μg of pranoprofen released more than the recommended daily amount of the drug. Therefore, it would be possible to release the optimal amount of the drug to the eye by controlling the amount of drug introduced. In contrast, the amount of drug released from the sheets immersed in drug solution was less than the recommended amount per dose (Fig. 6, ♦). This is because it is difficult to efficiently load drugs in water into sheets, since the entropy of the system determines that the amount of drug in the water is higher than that within the sheets.

The authors are grateful for the funding received through a Grantin-Aid from the Japan Society for the Promotion of Science (15H05353, 17KK0130, 18K19907). We are grateful to Osaka Organic Chemical Industry for the donation of CMB and their continuous support to pursue this work. This work was also supported by the NIMS Joint Research Hub Program. We are indebted to Shin-Nakamura Chemical Co., Ltd., for the gift of polyethylene glycol dimethacrylate. The authors are grateful to Editage for editing English grammar. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfb.2020.110859. References [1] M. Brodsky, Allergic conjunctivitis and contact lenses: experience with olopatadine hydrochloride 0.1% therapy., Acta Ophthalmol, Scand. Suppl. (2000) 56–59, https://doi.org/10.1034/j.1600-0420.2000.078s230056.x. [2] B. Muehleisen, R.L. Gallo, Vitamin D in allergic disease: shedding light on a complex problem, J. Allergy Clin. Immunol. 131 (2013) 324–329, https://doi.org/10. 1016/J.JACI.2012.12.1562. [3] S. Ackerman, L.M. Smith, P.J. Gomes, Ocular itch associated with allergic conjunctivitis: latest evidence and clinical management, Ther. Adv. Chronic Dis. 7 (2016) 52–67, https://doi.org/10.1177/2040622315612745. [4] S.V. Scoper, G.J. Berdy, S.J. Lichtenstein, J.M. Rubin, M. Bloomenstein, R.E. Prouty, C.T. Vogelson, M.R. Edwards, C. Waycaster, T. Pasquine, R.D. Gross, S.M. Robertson, Perception and quality of life associated with the use of olopatadine 0.2% (PatadayTM) in patients with active allergic conjunctivitis, Adv. Ther. 24 (2007) 1221–1232, https://doi.org/10.1007/BF02877768. [5] G. Torkildsen, A. Narvekar, M. Bergmann, Efficacy and safety of olopatadine hydrochloride 0.77% in patients with allergic conjunctivitis using a conjunctival allergen-challenge model, Clin. Ophthalmol. 9 (2015) 1703–1713, https://doi.org/ 10.2147/OPTH.S83263. [6] M.B. Abelson, K. Schaefer, Conjunctivitis of allergic origin: immunologic mechanisms and current approaches to therapy, Surv. Ophthalmol. (38 Suppl) (1993) 115–132, https://doi.org/10.1016/0039-6257(93)90036-7. [7] K. Hirahara, T. Tatsuta, T. Takatori, M. Ohtsuki, H. Kirinaka, J. Kawaguchi, N. Serizawa, Y. Taniguchi, S. Saito, M. Sakaguchi, S. Inouye, A. Shiraishi, Preclinical evaluation of an immunotherapeutic peptide comprising 7 T-cell determinants of Cry j 1 and Cry j 2, the major Japanese cedar pollen allergens, J. Allergy Clin. Immunol. 108 (2001) 94–100, https://doi.org/10.1067/mai.2001. 115481. [8] N.M. Farandos, A.K. Yetisen, M.J. Monteiro, C.R. Lowe, S.H. Yun, Contact Lens sensors in ocular diagnostics, Adv. Healthc. Mater. 4 (2015) 792–810, https://doi. org/10.1002/adhm.201400504. [9] X. Deng, M. Korogiannaki, B. Rastegari, J. Zhang, M. Chen, Q. Fu, H. Sheardown, C.D.M. Filipe, T. Hoare, “Click” Chemistry-Tethered Hyaluronic Acid-Based Contact Lens Coatings Improve Lens Wettability and Lower Protein Adsorption, ACS Appl. Mater. Interfaces 8 (2016) 22064–22073, https://doi.org/10.1021/acsami. 6b07433. [10] S. Baab, E.E. Kinzer, Allergic Conjunctivitis, StatPearls Publishing, 2018. [11] T. Goda, K. Ishihara, Soft contact lens biomaterials from bioinspired phospholipid polymers, Expert Rev. Med. Devices 3 (2006) 167–174, https://doi.org/10.1586/ 17434440.3.2.167. [12] D. Dutta, B. Kamphuis, B. Ozcelik, H. Thissen, R. Pinarbasi, N. Kumar, M.D.P. Willcox, Development of silicone hydrogel antimicrobial contact lenses with Mel4 peptide coating, Optom. Vis. Sci. 95 (2018) 937–946, https://doi.org/10. 1097/OPX.0000000000001282. [13] M. Nishida, T. Nakaji-Hirabayashi, H. Kitano, K. Matsuoka, Y. Saruwatari, Optimization of the composition of zwitterionic copolymers for the easy-construction of bio-inactive surfaces, J. Biomed. Mater. Res. Part B Appl. Biomater. 104 (2016) 2029–2036, https://doi.org/10.1002/jbm.a.35737. [14] M. Nishida, T. Nakaji-hirabayashi, H. Kitano, Biointerfaces Titanium alloy modified with anti-biofouling zwitterionic polymer to facilitate formation of bio-mineral layer, Colloids Surf. B Biointerfaces 152 (2017) 302–310, https://doi.org/10.1016/ j.colsurfb.2017.01.018. [15] L. Li, T. Nakaji-Hirabayashi, H. Kitano, K. Ohno, T. Kishioka, Y. Usui, Gradation of proteins and cells attached to the surface of bio-inert zwitterionic polymer brush, Colloids Surf. B Biointerfaces 144 (2016) 180–187, https://doi.org/10.1016/J. COLSURFB.2016.04.005. [16] L. Li, T. Nakaji-hirabayashi, K. Tokuwa, H. Kitano, K. Ohno, Y. Usui, T. Kishioka, UV-patterning of anti-biofouling zwitterionic copolymer layer with an aromatic anchor group, Macromol. Mater. Eng. 302 (1600374) (2017) 1–10, https://doi.org/ 10.1002/mame.201600374. [29] K. Matsuura, K. Ohno, S. Kagaya, H. Kitano, Carboxybetaine polymer-protected gold nanoparticles: high dispersion stability and resistance against non-specific adsorption of proteins, Macromol. Chem. Phys. 208 (2007) 862–873, https://doi.

4. Conclusion Polymer sheets containing both CMB and HEMA residues could be made using various methods: (Method-A) glutaraldehyde cross-linking, (Method-B) PEGDMA cross-linking using KPS and TEMED, and (Method-C) the hot-melt press method. Every sheet prepared had high wettability and an ability to suppress protein adsorption through the introducing CMB residues. Specifically, the sheets prepared by the hotmelt press method were highly transparent and suppressed protein and bacteria adsorption. Therefore, using these sheets, it would be possible to prevent allergic conjunctivitis. In addition, sheets prepared using the hot-melt press method could release drugs at more than the recommended dose. Thus, a suitable amount of drugs could be released from the sheets to the eye by controlling the amount of the drug loaded into the sheets. Therefore, the sheets show potential for use as contact lenses to cure allergic conjunctivitis by releasing the necessary drugs. In conclusion, these sheets, prepared by the hot-melt press method, are expected to contribute to the improvement of the quality of life of contact lens users by not only preventing, but also curing, allergic conjunctivitis. Credit author statement Hiroaki Ogawa: H. O. carried out almost all experiments and summarized all data. Tadashi Nakaji-Hirabayashi: T. N. -H. generated the idea concerning the novel contact lens material for protecting and curing the eye disease. And the author carried out the preliminary experiment concerning the construction of polymer sheet with anti-biofouling property using zwitterionic monomer. Chiaki Yoshikawa C. Y. carried out the experiment of bacterial adsorption and discussed the antibiofouling property of polymer sheet. Kazuaki Matsumura K. M. supported the construction ofmanufacturing polymer sheet using hot press (Method-C). And he analyzed the phase separation of polymer sheets containing CMB using FE-SEM and XRD. Hiromi Kitano and Yoshiyuki Saruwatari H. K. and Y. S. supported the discussion concerning the relationship between the expression of anti-biofouling property using zwitterionic polymer and the phase separation of polymer sheet Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 9

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