Facile Functionalization of Electrospun Poly(ethylene-co-vinyl ... - MDPI

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Facile Functionalization of Electrospun Poly(ethylene-co-vinyl alcohol) Nanofibers via the Benzoxaborole-Diol Interaction Yohei Kotsuchibashi 1, * and Mitsuhiro Ebara 2,3 1

2 3

*

International Center for Young Scientists (ICYS) and International Center for Materials Nanoarchitectonics (WPI-MANA), National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan Biomaterials Unit, WPI-MANA, NIMS, 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan; [email protected] Graduate School of Industrial Science and Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika, Tokyo 125-8585, Japan Correspondence: [email protected]; Tel.: +81-29-851-3354 (ext. 8918); Fax: +81-29-860-4762

Academic Editor: Hatsuo Ishida Received: 1 December 2015; Accepted: 29 January 2016; Published: 5 February 2016

Abstract: A facile functionalization method of poly(ethylene-co-vinyl alcohol) (EVOH) nanofiber meshes was demonstrated by utilizing the benzoxaborole-diol interaction between EVOH and benzoxaborole-based copolymers (BOP). EVOH and BOP were firstly mixed to prepare the quasi-gel-state solution with enough viscosity for electro-spinning. The fiber morphology was controlled via changing the mixing ratio of EVOH and BOP. The prepared EVOH/BOP nanofiber mesh showed good stability in aqueous solution. Over 97% of the nanofibers remained after the immersion test for 24 h in acid or alkali aqueous solutions without changing their morphology. Temperature and pH-responsive moieties were copolymerized with BOP, and cationic dye was easily immobilized into the nanofiber mesh via an electrostatic interaction. Therefore, the proposed functionalization technique is possible to perform on multi-functionalized molecule-incorporated nanofibers that enable the fibers to show the environmental stimuli-responsive property for the further applications of the EVOH materials. Keywords: benzoxaborole; boroxole; poly(ethylene-co-vinyl alcohol); nanofiber; electrospinning; gel; smart polymer; stimuli response; pH response

1. Introduction Poly(ethylene-co-vinyl alcohol) (EVOH) has been one of the best known flexible thermoplastic materials and used in a wide range of fields, such as food packaging, funnel tanks, and medical applications for the high gas-barrier and biocompatibility [1–3]. The fabrication of EVOH materials has been customized dependent on the choice of applications, such as films [4], (nano)particles [5], porous materials [6] and (nano)fibers [7,8]. Moreover, these EVOH materials have been modified with molecules/polymers to enhance their functionalities. The modification, however, is limited due to the multi-steps of the reaction processes. Therefore, a simple modification system for EVOH materials has been focused on recently. Zhou et al. prepared a phosphorylcholine-modified EVOH in two reaction steps to prevent nonspecific protein adsorption [9]. Zhu et al. modified chelating groups on EVOH nanofibers via a three-step treatment, the modified nanofibers were used for a selective protein separation system [10]. Plasma treatment achieves a one-step modification for EVOH materials. By the treatment, however, the EVOH Polymers 2016, 8, 41; doi:10.3390/polym8020041

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Polymers 2016, 8, 41 Polymers 2016, 8, 41

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EVOH composition was changed via the newly-generated groups, such as acid. carboxylic acid. The composition was changed via the newly-generated groups, such as carboxylic The conversion conversion leads of to the a change of the physicochemical properties the original [11]. As also leads toalso a change physicochemical properties of the originalof EVOH [11]. AsEVOH other one-step other one-step modifications for EVOH materials, there is a blending method with hydrophobic modifications for EVOH materials, there is a blending method with hydrophobic polymers via the polymers via the hydrophobic-hydrophobic interaction [12]. properties The mechanical properties of EVOH hydrophobic-hydrophobic interaction [12]. The mechanical of EVOH nanofibers were nanofibers were by mixingsuch withaspolymers, such as(PP) polypropylene (PP)acid) [13], (PLA) poly(lactic strengthened by strengthened mixing with polymers, polypropylene [13], poly(lactic [14], acid) (PLA) [14], cellulose [15] and Nylon 6/12 [16]. However, it is difficult to obtain stable EVOH cellulose [15] and Nylon 6/12 [16]. However, it is difficult to obtain stable EVOH blend materials blenda materials with a hydrophilic polymer in aqueous solution because of their weak with hydrophilic polymer in aqueous solution because of their weak interactions. Stableinteractions. EVOH and Stable EVOH and hydrophilic polymer blends in aqueous solution were reported using hydrophilic polymer blends in aqueous solution were reported using multi-step treatments,multi-step such as a treatments, suchofashydrophilic a polymerization of hydrophilic polymers from the EVOH surface [17,18] and a polymerization polymers from the EVOH surface [17,18] and a cross-linking method cross-linking method blend process [19]. Moreover,ofaethylene, copolymerization of ethylene, vinyl after the blend processafter [19].the Moreover, a copolymerization vinyl acetate (precursor of acetate (precursor of vinyl alcohol) and hydrophilic monomers is expected to obtain a modified vinyl alcohol) and hydrophilic monomers is expected to obtain a modified EVOH with hydrophilic EVOHHowever, with hydrophilic units. However, typical (meth)acrylate and (meth)acrylamide monomers units. typical (meth)acrylate and (meth)acrylamide monomers have different reaction ratios have ethylene different and reaction ratios withdue ethylene and vinyl acetate to their structure, which leads to with vinyl acetate to their structure, whichdue leads to low yields and a gradient low yields and a gradient composition Therefore, remainsa asimple challenge to propose simple composition [20]. Therefore, it remains[20]. a challenge to itpropose EVOH functiona system. EVOH function system.we Tofocused achieve on thisa system, focusedchemistry on a dynamic covalent chemistry [21,22]. To achieve this system, dynamicwe covalent [21,22]. Recently, we have reported a hydrogel gel system using reversible covalent bonding between between Recently, we have reported a hydrogel gel system using reversible covalent bonding benzoxaborole-based temperature-responsive and glyco-based copolymers [23,24]. The benzoxaborole-based temperature-responsivecopolymers copolymers and glyco-based copolymers [23,24]. benzoxaborole unit unit can reversibly combine with with the cis-diol in the copolymers. The The benzoxaborole can reversibly combine the cis-diol in glyco-based the glyco-based copolymers. mixed hydrogels displayed temperature-responsive, pH-responsive and glucose-responsive The mixed hydrogels displayed temperature-responsive, pH-responsive and glucose-responsive properties. Moreover, possesses aa properties. Moreover, aa photo-acid photo-acid generator generator of of 2-nitrobenzaldehyde 2-nitrobenzaldehyde (2-NBA) (2-NBA) that that possesses proton-release by UV irradiation [25] was encapsulated into the hydrogel. The UV irradiation caused proton-release by UV irradiation [25] was encapsulated into the hydrogel. The UV irradiation caused the disintegration of the hydrogel structure in the exposed region, resulting in the local pH decrease. the disintegration of the hydrogel structure in the exposed region, resulting in the local pH decrease. The benzoxaborole benzoxaborole units units have have also also been been applied applied in in biomedical biomedical fields, fields, such such as as the the selective selective binding binding The with the Thomsen–Friedenreich (TF)-antigen disaccharide, delivery of a protein toxin in the cytosol, with the Thomsen–Friedenreich (TF)-antigen disaccharide, delivery of a protein toxin in the cytosol, neutralization of (HIV) and antitrypanosomal agent [26–30]. neutralization ofhuman humanimmunodeficiency immunodeficiencyvirus virus (HIV) and antitrypanosomal agent [26–30]. In this was mixed withwith the water-soluble benzoxaborole-based copolymers (BOPs) In thisstudy, study,EVOH EVOH was mixed the water-soluble benzoxaborole-based copolymers in organic solvent and was found to show a gelation via a benzoxaborole-diol interaction. The (BOPs) in organic solvent and was found to show a gelation via a benzoxaborole-diol interaction. functionalized EVOH nanofiber meshes with BOP were the The functionalized EVOH nanofiber meshes with BOP wereprepared preparedby byelectro-spinning electro-spinning using using the mixed viscous solution (Figure 1). The nanofiber meshes had a stable cross-linked structure in mixed viscous solution (Figure 1). The nanofiber meshes had a stable cross-linked structure in aqueous aqueous solution due to the reversible covalent bonding. Using this simple method, the EVOH solution due to the reversible covalent bonding. Using this simple method, the EVOH nanofibers were nanofibers were successfully functionalized with stimuli-responsive To the best of our successfully functionalized with stimuli-responsive copolymers. To thecopolymers. best of our knowledge, this is knowledge, this is the first report on functionalized EVOH materials with BOPs. The stability and the first report on functionalized EVOH materials with BOPs. The stability and functionality of the functionality nanofiber of the EVOH/BOP nanofiber meshesinwere investigated in various solution conditions. EVOH/BOP meshes were investigated various solution conditions.

Figure 1. 1. Schematic with benzoxaborole-based benzoxaborole-based Figure Schematic representation representation of of modified modified EVOH EVOH nanofiber nanofiber with copolymers (BOPs). copolymers (BOPs).

Polymers 2016, 8, 41

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2. Experimental Section 2.1. Material Five-methacrylamido-1,2-benzoxaborole (MAAmBO) was synthesized and purified according to the protocol given [23,24]. One-pyrenemethyl methacrylate (PyMA) was purified by recrystallization from ethanol. Two-(2-methoxyethoxy)ethyl methacrylate (MEO2 MA), oligo(ethylene glycol) methacrylate (OEGMA, Mn = 475 g/mol) and acrylic acid (Ac) were purchased from Sigma–Aldrich (St. Louis, MO, USA) and purified by passing through a basic alumina column. EVOH (EVAL E105A, 44 mol % ethylene) was kindly supplied from KURARAY (Okayama, Japan). All other chemicals and solvents were used as received. Distilled water used in this study was purified with a Millipore Milli-Q system. 2.2. Preparation of Benzoxaborole-Based Copolymers Reversible addition-fragmentation chain transfer (RAFT) polymerization was employed to synthesize copolymers with a narrow molecular weight distribution. The polymerization methods of BOPs are described in the Supplementary Materials. Briefly, MEO2 MA (540 mg, 2.87 mmol), OEGMA (908 mg, 1.91 mmol), MAAmBO (116 mg, 0.53 mmol), 4-cyanopentanoic acid dithiobenzoate (CTP) (7.42 mg, 2.66 ˆ 10´ 2 mmol) and 4,4’-azobis-4-cyanovaleric acid (ACVA) (2.98 mg, 1.06 ˆ 10´ 2 mmol) ([MEO2 MA]0 /[OEGMA]0 /[MAAmBO]0 /[CTP]0 /[ACVA]0 = 108/72/20/1/0.4) were dissolved in 4 mL of methanol. After degassing with nitrogen gas for 30 min, the mixture was allowed to polymerize for 24 h at 60 ˝ C. The resulting P(MEO2 MA-co-OEGMA-co-MAAmBO) was purified by dialysis against ethanol and acetone and was dried under reduced pressure. The preparation conditions of other BOPs are shown in the Supplementary Materials. 2.3. Preparation of EVOH/BOP Nanofiber Meshes BOPs were dissolved in 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) at room temperature. Electro-spinning was performed using a Nanon-01A (MECC, Fukuoka, Japan). Spinning parameters were kept constant at a 25-kV applied voltage, a 1.0-mL/h solution flow rate, a 15-cm working distance and a 25-gauge pointed needle [8]. The fibers were electrospun onto a sheet of aluminum foil on a stationary plate collector. The fibers were directly extracted from the foil. 2.4. Stability Tests Nanofiber meshes of EVOH and BOPs (P(MEO2 MA-co-MAAmBO-co-PyMA) or P(MEO2 MA-co-PyMA)) were prepared for stability tests in various solution conditions (pH 2/37 ˝ C, pH 2/4 ˝ C, pH 12/37 ˝ C and pH 12/4 ˝ C). The PyMA units were used to detect the amount of released BOPs from the nanofibers. The BOP and EVOH were dissolved in HFIP at 2 and 7 wt % concentrations, respectively. Each 2.5-mL polymer solution was mixed and was kept at least 1 h to reach the equilibrium before electro-spinning. Nanofiber meshes were cut (2.70–3.79 mg) as a square sample and were added to pH 2 (0.01 N HCl) or pH 12 (0.01 N NaCl) solution to be the same concentration (0.3 wt %, e.g., 3 mg of fiber sample was added to 1 mL solution). After 24 h, all nanofiber samples were washed with Milli-Q and were dried under atmospheric pressure. The morphology and weight of the nanofiber meshes were measured. Each nanofiber sample was measured at 3 different places (N = 3). 2.5. Characterizations 1H

NMR spectra of copolymers were taken with a JNM-GSX300 spectrometer operating at 300 MHz (JEOL, Tokyo, Japan) to confirm successful synthesis and to determine the chemical composition of the synthesized copolymers. Molecular weight and polydispersity of the synthesized copolymers were determined by gel permeation chromatography (GPC) at 40 ˝ C (DMF, including


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10 mM LiBr, 1 mL/min) with a TOSOH TSK-GEL a-2500 and a-4000 and (Tosoh, Tokyo, Japan) connected to an RI-2031 refractive index detector (JASCO International Co. Ltd., Tokyo, Japan). Transmittance of a copolymer solution at 500 nm was continuously recorded at a heating rate of 1.0 ˝ C/min by a UV–Vis spectrometer V-550 (JASCO International Co. Ltd., Tokyo, Japan) to measure the lower critical solution temperature (LCST). Synthesized copolymers were dissolved in aqueous solution at the given concentration. LCSTs of copolymers were determined at 50% transmittance. Fluorescence spectra were recorded using a fluorescence spectrometer F-2500 (Hitachi High-Technologies Corporation, Tokyo, Japan). The morphologies of nanofiber meshes were observed using a NEO-Scope JCM-5000 SEM (JEOL, Tokyo, Japan). 3. Results and Discussion 3.1. Preparation of Benzoxaborole-Based Copolymers The BOPs were polymerized by the reversible addition-fragmentation chain transfer (RAFT) polymerization. Boronic acids and their esters can reversibility interact with the cis-diol group. The interaction between phenylboronic acid (PBA) and glucose has been utilized as a trigger for an insulin release system that was incorporated into newly-designed polymeric materials [31–33]. Recently, Hall and co-workers reported that the benzoxaborole molecule showed a higher affinity than boronic acid toward saccharides in phosphate-buffered saline (PBS) [34]. The high affinity comes from the relatively low pKa of the benzoxaborole (pKa 7–8) as compared to that of boronic acid (pKa 8–9) [35,36]. In this study, benzoxaborole-based monomers were copolymerized with functional monomers for the functionalization of EVOH nanofiber meshes (Figure 2). The compositions of the BOPs are summarized in Table 1. The copolymers, P(MEO2 MA-co-MAAmBO), P(MEO2 MA-co-OEGMA-co-MAAmBO) and P(MEO2 MA-co-MAAmBO-co-Ac) were prepared. The monomers of MEO2 MA and OEGMA were selected in order to control the temperature-responsive properties, i.e., lower critical solution temperature (LCST). The LCSTs of P(MEO2 MA) and P(OEGMA) homopolymers were 28 and 90 ˝ C, respectively [37]. The Ac monomer with carboxylic acid functionality was used to donate the pH-responsive property to the BOPs. Moreover, the anionic charge can interact with cationic molecules via electrostatic interactions. The compositions (mol %) of the BOPs were measured using 1 H NMR and are shown at the right side of monomer unit (P(MEO2 MA91.0 -co-MAAmBO9.0 ), P(MEO2 MA56.5 -co-OEGMA38.2 -co-MAAmBO5.3 ) and P(MEO2 MA86.7 -co-MAAmBO4.8 -co-Ac8.5 ). Molecular weight (Mn ) and Mw /Mn of the BOPs were calculated from GPC measurement. However, broad GPC peaks were observed in all BOPs (40 ˝ C, DMF including 10 mM LiBr, 1 mL/min). This might be an adsorption between MAAmBO units and the filler of the GPC column. Therefore, the benzoxaborole units were protected with 1,4-butanediol to prevent the adsorption [38], and the molecular weights were measured using the same GPC conditions. The molecular weights were smaller than those of their targeted monomer units (200 units) (P(MEO2 MA91.0 -co-MAAmBO9.0 ): Mn = 3000 g/mol, Mw /Mn = 1.26; P(MEO2 MA56.5 -co-OEGMA38.2 -co-MAAmBO5.3 ): Mn = 6000 g/mol, Mw /Mn = 1.80; and P(MEO2 MA86.7 -co-MAAmBO4.8 -co-Ac8.5 ): Mn = 2300 g/mol, Mw /Mn = 1.32). The estimated low molecular weights might come from the unprotected benzoxaborole units and the solubility change that occurred due to the protecting groups. The stimuli-responsive properties of BOPs were measured using the transmittance change as a function of temperature (Figures S1 and S2). The LCST of the P(MEO2 MA91.0 -co-MAAmBO9.0 ) was around 18.3 ˝ C at pH 2 (0.01N HClaq. ) (Figure S1A). The hydrophobic MAAmBO units led to a decrease in the LCST. On the other hand, at pH 12 (0.01 N NaOHaq. ), the transmittance change of P(MEO2 MA91.0 -co-MAAmBO9.0 ) disappeared due to the anionic charged MAAmBO units, resulting in the electrostatic repulsion. The LCST of P(MEO2 MA56.5 -co-OEGMA38.2 -co-MAAmBO5.3 ) was observed at 63.5 ˝ C at pH 2 due to the hydrophilic OEGMA units, and no transmittance change was observed at pH 12 (Figure S1B). These results suggested that the LCST of the BOPs could be easily controlled by the copolymerized


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monomer compositions. P(MEO2 MA86.7 -co-MAAmBO4.8 -co-Ac8.5 ) had the carboxylic acid groups as the pH-responsive units. At pH 2, the LCST was 17.6 ˝ C. On the other hand, at pH 12, no transmittance Polymers 8, 41 5 of 11S1C). change was2016, observed due to the electrostatic repulsion of both MAAmBO and Ac units (Figure

co

co

co

8

P(MEO2MA-co-MAAmBO)

P(MEO2MA-co-OEGMA-co-MAAmBO)

co

co

co

Poly(ethylene-co-vinyl alcohol) (EVOH)

P(MEO2MA-co-MAAmBO-co-Ac)

co

co

co

P(MEO2MA-co-PyMA)

P(MEO2MA-co-MAAmBO-co-PyMA)

Figure 2. Chemical structure of BOPsofandBOPs EVOH. two-(2-methoxyethoxy)ethyl methacrylate; Figure 2. Chemical structure andMEO EVOH. 2MA, two-(2-methoxyethoxy)ethyl 2 MA, MEO methacrylate; MAAmBO, five-methacrylamido-1,2-benzoxaborole; OEGMA, oligo(ethylene glycol) Ac, MAAmBO, five-methacrylamido-1,2-benzoxaborole; OEGMA, oligo(ethylene glycol) methacrylate; methacrylate; Ac,one-pyrenemethyl acrylic acid; PyMA,methacrylate. one-pyrenemethyl methacrylate. acrylic acid; PyMA, Table 1. Characterization of BOPs. LCST, lower critical solution temperature.

Table 1. Characterization of BOPs. LCST, lower critical solution temperature. Copolymer

MEO2MAAmBO MA MAAmBO MEO2 MA OEGMA OEGMA Ac

P(MEO2MA91.0-

P(MEO2 MA91.0 co-MAAmBO9.0)91.0 co-MAAmBO9.0 ) 2MA56.5P(MEOP(MEO 2 MA56.5 38.2- 56.5 co-OEGMA co-OEGMA 38.2 co-MAAmBO 5.3) co-MAAmBO 5.3 ) P(MEOP(MEO 2 MA86.72-MA86.7co-MAAmBO 86.7 4.8 4.8co-MAAmBO co-Ac8.5 ) co-Ac8.5) P(MEO2 MA93.9 P(MEO2-MA93.9-93.9 co-MAAmBO 5.2 5.2co-MAAmBO co-PyMA 0.9 ) P(MEO2 MA 0.9) co-PyMA 99.0 99.0 co-PyMA 1.0 ) 2MA99.0P(MEO

Mn b

In Copolymer (mol%) a

In Copolymer (mol%) a

Copolymer 91.0

9.0

9.0

–

Mw/Mn b

b (g/mol) M w /M(–) (g/mol) n (–)

Ac PyMA PyMA

–

–

–

b

Mn

–

–

LCST (°C) c ˝

LCST ( C) c

pH 2 dpH 2 dpH 12 e pH 12 e 3000

3,000

1.26

1.26

18.3

no LCST f

18.3

no LCST f

56.5

5.3

5.338.2

38.2 –

––

–

6000 6,000

1.80 1.80

f 63.5 63.5no LCST no LCST f

86.7

4.8

4.8 –

8.5 –

8.5–

–

2300 2,300

1.32 1.32

g g LCST 17.6 17.6no LCST no

2600

1.59

5.3

93.9

–

5.3 –

99.0

–

–

0.9

–

–

–

1.0

0.9

2,600 12,500

1.59 1.31

15.2

15.2

no LCST f

no LCST f

21.3 (pH 7.4 PBS)

–

– – 1.0 12,500 1.31 21.3 (pH 7.4 PBS) a The 1.0) co-PyMA copolymer compositions were calculated by 1 H NMR; b determined by gel permeation chromatography

a The b determined (GPC) using 10 mM LiBr DMF; c LCSTs determined the temperature withby 50% transmittance; copolymer compositions werewere calculated by 1Hat NMR; gelofpermeation d 0.01 N-HCl e 0.01 N-NaOH was used; was used. No transmittance change was observed by fwith 90 and c LCSTs aq. aq. were determined at the temperature chromatography (GPC) using 10 mM LiBr DMF; g 70˝ C. d e 50% of transmittance; 0.01 N-HClaq. was used; 0.01 N-NaOHaq. was used. No transmittance change was observed by f 90 and g 70 °C.

3.2. Preparation of EVOH/BOP Nanofiber Meshes by Electro-Spinning 3.2. Preparation of EVOH/BOP Nanofiber Meshes by Electro-Spinning

BOPs can interact with OH groups in the EVOH. Kikuchi et al. reported a hydrogel that was interact with OH groups in the and EVOH. Kikuchi et al. reported a hydrogel was [39]. composedBOPs of acan boronic acid-based copolymer a water-soluble poly(vinyl alcohol)that (PVA) composed of a boronic acid-based copolymer and a water-soluble poly(vinyl alcohol) (PVA) [39]. We We have prepared hydro(nano)gels consisting of glyco-based copolymers and temperature-responsive have prepared hydro(nano)gels consisting of glyco-based copolymers and temperature-responsive P(N-isopropylacrylamide (NIPAAm)-co-MAAmBO)s [23,24]. The water-soluble glyco-based P(N-isopropylacrylamide (NIPAAm)-co-MAAmBO)s [23,24]. The water-soluble glyco-based copolymers were mixed with P(NIPAAm-co-MAAmBO) in aqueous solution. However, EVOH


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is a water-insoluble copolymer, which means a good solvent for both copolymers has to be selectedPolymers for the electro-spinning method. HFIP was selected as a good solvent. 6The EVOH 2016, 8, 41 of 11 solutionPolymers (35 2016, mg,8, 500 µL HFIP) was mixed with P(MEO2 MA91.0 -co-MAAmBO9.0 ) 6atof 11 different 41 copolymersand werethe mixed with P(NIPAAm-co-MAAmBO) in aqueous solution. However, a a low concentrations, mixed solutions were used in electro-spinning (FigureEVOH 3). isAt water-insoluble copolymer, which means a good solvent for both copolymers has to be selected fora copolymers were mixed with P(NIPAAm-co-MAAmBO) in500 aqueous solution. However, EVOH is concentration of P(MEO MA -co-MAAmBO ) (5 mg, µL HFIP), nanofibers and aggregated 2 91.0 9.0 the electro-spinning method.which HFIP means was selected a goodfor solvent. The EVOH has solution mg, 500 water-insoluble copolymer, a goodassolvent both copolymers to be (35 selected for particles were observed (Figure 3A). It has been observed that electro-spinning with a low μL was mixed method. with P(MEO 2MA 91.0selected -co-MAAmBO 9.0) atsolvent. different concentrations, and(35 themg, mixed theHFIP) electro-spinning HFIP was as a good The EVOH solution 500 viscoussolutions solution can result inP(MEO aggregated particles [8]. When the concentration was increased used in electro-spinning (Figure 3). At concentration of P(MEO 91.0-coμL HFIP) were was mixed with 2MA91.0-co-MAAmBO 9.0)aatlow different concentrations, and2MA the mixed using P(MEO -co-MAAmBO ) (10(Figure mg, 500 HFIP), fine formation was MAAmBO 9.0) (5 mg, 500 HFIP),9.0 nanofibers and3). aggregated particles were nanofiber observed (Figure 3A). 91.0 solutions2 MA were used in μL electro-spinning At µL a low concentration of P(MEO 2MA 91.0 -coIt has been9.0observed that with a aggregated low viscousparticles solution can result aggregated observed (Figure The interaction between and OH units ledinto the increase of MAAmBO )3B). (5 mg, 500 μLelectro-spinning HFIP), nanofibers andMAAmBO were observed (Figure 3A). particles [8]. When the wassuperficial increased P(MEO 91.0-co-MAAmBO 9.0 (10 mg, 500 It has been observed that increasing electro-spinning with ausing low viscous2MA solution canAt result in)concentrations aggregated the viscosity, resulting in concentration the molecular weight. high of μL HFIP),[8]. fine nanofiber formation was 3B). The interaction between MAAmBO When the concentration was observed increased (Figure using P(MEO 2MA 91.0-co-MAAmBO 9.0) (10 mg, 500 P(MEO2particles MA91.0 -co-MAAmBO 9.0 ) (25 mg, 500 µL HFIP), gel formation was observed, which could and OH units led to the increase of the viscosity, resulting in the increasing superficial molecular μL HFIP), fine nanofiber formation was observed (Figure 3B). The interaction between MAAmBO not be weight. from electro-spinning (Figure 3C).MA Fine nanofibers9.0were also obtained from other BOPs. highled concentrations of P(MEO 91.0-co-MAAmBO (25 increasing mg, 500 μLsuperficial HFIP), gel formation and OHAtunits to the increase of the 2viscosity, resulting in )the molecular Figure 4A,B shows the nanofiber structures of EVOH/P(MEO MA -co-OEGMA 2 ) (2556.5 5.3 ) was observed, could not be from2MA electro-spinning (Figure 3C). FineμLnanofibers were also weight. At highwhich concentrations of P(MEO 91.0-co-MAAmBO 9.0 mg, 500 HFIP),38.2 gel-co-MAAmBO formation (25 mg,obtained 500observed, µL from HFIP) and EVOH/P(MEO -co-MAAmBO -co-Ac ) (10 mg, 500 µL HFIP), other BOPs. Figure 4A,B shows the structures ofFine EVOH/P(MEO 2MA 56.5also -cowas which could not be from electro-spinning (Figure 3C). nanofibers were 2 MA 86.7nanofiber 4.8 8.5 OEGMA 38.2 -co-MAAmBO 5.3) Figure (25 mg, 500 μL HFIP) and for EVOH/P(MEO 86.7-co-MAAmBO 4.8-coobtained from other BOPs. 4A,B shows the need nanofiber structures of2MA EVOH/P(MEO 2MA56.5 -co- for the respectively. High MAAmBO content led to the a small amount of copolymers Ac 8.5) (10 38.2 mg, 500 These μL HFIP), led tostructures the need forwere a smallaffected amount OEGMA -co-MAAmBO 5.3)respectively. (25 mg, 500High μL MAAmBO HFIP) and content EVOH/P(MEO 2MA 86.7-co-MAAmBO 4.8-conanofiber structure. results suggested that the nanofiber by the of for μL theHFIP), nanofiber structure.High TheseMAAmBO results suggested the nanofiber were Accopolymers 8.5) (10 mg, 500 respectively. content that led to the need for structures a small amount MAAmBO contents, polymer concentrations and their mixture ratios. For example, imperfect nanofiber affected by thefor MAAmBO contents, polymer concentrations and their mixture ratios. For example, of copolymers the nanofiber structure. These results suggested that the nanofiber structures were formation (with particle aggregation) was observed in the mixture of P(MEO MA -co-OEGMA 2 69.7 30.3 ) imperfect nanofiber formation (with particle aggregation) was observed in the mixture of affected by the MAAmBO contents, polymer concentrations and their mixture ratios. For example, (25 mg)P(MEO (Mn =2MA 25,400 g/mol, M30.3w)/M = 1.28 [39]) (Figure 4C).MThe MA -co-OEGMA 69.7-co-OEGMA (25n(with mg) (M n = 25,400 g/mol, w /MnP(MEO =observed 1.28 2[39]) (Figure 4C). The imperfect nanofiber formation particle aggregation) was in69.7 the mixture of30.3 ) has P(MEO -co-OEGMA )) has MAAmBO unit and cannot interact with EVOH. The lack of no MAAmBO unit and cannot30.3 interact with The lack ofwincrease in[39]) viscosity led theThe imperfect P(MEO22MA MA69.7 69.7 -co-OEGMA 30.3 (25 no mg) (MEVOH. n = 25,400 g/mol, M /M n = 1.28 (Figure 4C). increase in viscosity led the30.3 imperfect P(MEO 2MA 69.7-co-OEGMA ) has no nanofiber MAAmBOformation. unit and cannot interact with EVOH. The lack of nanofiber formation. increase in viscosity led the imperfect nanofiber formation. (A) P(MEO2MA91.0-co-MAAmBO9.0) 5 mg (A) EVOH 35 mg P(MEO2MA91.0-co-MAAmBO9.0) 5 mg EVOH 35 mg

(C) (B) P(MEO2MA91.0-co-MAAmBO9.0) 10 mg P(MEO2MA91.0-co-MAAmBO9.0) 25 mg (C) (B) EVOH 35 mg EVOH 35 mg P(MEO2MA91.0-co-MAAmBO9.0) 10 mg P(MEO2MA91.0-co-MAAmBO9.0) 25 mg EVOH 35 mg EVOH 35 mg

Figure 3. SEM image of electro-spinning samples of EVOH (35 mg) and (A) P(MEO2MA91.0-co-

Figure 3. SEM image of electro-spinning samples of EVOH (35 mg) and ) (5image mg) of and (B) P(MEO2MA 91.0-co-MAAmBO ) (10 mL 1,1,1,3,3,3MAAmBO Figure 3. 9.0 SEM electro-spinning samples of EVOH 9.0(35 mg)mg) and in (A) 1P(MEO 2MA91.0-co(A) P(MEO MA ) (5 mg) and (B) P(MEO(35 -co-MAAmBO ) (10 mg) 2 91.0 -co-MAAmBO 9.0 2 MA 91.0and 9.0-coMA91.0 hexafluoroisopropanol (HFIP). (C) Gel formation of EVOH mg) P(MEO MAAmBO9.0) (5 mg) and (B) P(MEO2MA91.0-co-MAAmBO9.0) (10 mg) in 1 mL 21,1,1,3,3,3in 1 mL 1,1,1,3,3,3-hexafluoroisopropanol (HFIP). (C) Gel formation of EVOH (35 mg) and MAAmBO 9.0) (25 mg) in 1 mL HFIP. hexafluoroisopropanol (HFIP). (C) Gel formation of EVOH (35 mg) and P(MEO2MA91.0-coP(MEO2MAAmBO MA91.0 -co-MAAmBO (25 mg) in 1 mL HFIP. 9.0) (25 mg) in 1 9.0 mL) HFIP. (A) P(MEO2MA56.5-co-OEGMA38.2-co(A) MAAmBO 5.3): 25 mg P(MEO35 EVOH: mg 2MA 56.5-co-OEGMA38.2-coMAAmBO5.3): 25 mg EVOH: 35 mg

(B) P(MEO2MA86.7-co-MAAmBO4.8-co(B)8.5):10 mg Ac P(MEO35 EVOH: mg 2MA 86.7-co-MAAmBO4.8-coAc8.5):10 mg EVOH: 35 mg

(C) P(MEO2MA69.7-co-OEGMA30.3): 25 mg (C) EVOH: 35 mg P(MEO2MA69.7-co-OEGMA30.3): 25 mg EVOH: 35 mg

Figure 4. SEM images of electro-spinning samples: (A) P(MEO2MA56.5-co-OEGMA38.2-co-MAAmBO5.3) 86.7-co-MAAmBO -co-Ac 8.5) (10 mg) and EVOH (35 mg) (25 mg)4.and EVOH (35ofmg), (B) P(MEO2MA Figure SEM images electro-spinning samples: (A) P(MEO4.82MA 56.5-co-OEGMA 38.2-co-MAAmBO 5.3) Figure 4. SEM images2MA of electro-spinning samples: (A) P(MEO MA -co-OEGMA 2 56.5 38.2 -co-MAAmBO 5.3 ) 69.7 -co-OEGMA 30.3 ) (25 mg) and EVOH (35 mg) in 1 mL HFIP. and (C) P(MEO (35 mg) (25 mg) and EVOH (35 mg), (B) P(MEO2MA86.7-co-MAAmBO4.8-co-Ac8.5) (10 mg) and EVOH

(25 mg) and and(C) EVOH (35 mg); (B) P(MEO) MA -co-MAAmBO ) (10 mg) and EVOH (35 mg) 86.7 and 4.8 -co-Ac 2MA69.7-co-OEGMA30.32 (25 mg) EVOH (35 mg) in 1 mL8.5 HFIP. P(MEO and (C) P(MEO2 MA69.7 -co-OEGMA30.3 ) (25 mg) and EVOH (35 mg) in 1 mL HFIP.


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3.3. Stability Test of EVOH/BOP Nanofiber Meshes Multi-step organic reactions are needed to modify molecules in EVOH materials. By simple mixing, the EVOH nanofiber mesh was easily modified with the BOPs. As the copolymers, P(MEO2 MA93.9 -co-MAAmBO5.2 -co-PyMA0.9 ) and P(MEO2 MA99.0 -co-PyMA1.0 ) were selected (Table 1 Polymers 2016, 8, 41 7 of 11 and Figure 2). The fluorescent molecule of PyMA can trace the morphology change of a polymer, 3.3.the Stability Test of EVOH/BOP chain and fluorescent intensityNanofiber can alsoMeshes be an indicator for cell biology [40]. To prevent the LCST change via the hydrophobic PyMA units, the PyMA contents in the copolymers were adjusted less Multi-step organic reactions are needed to modify molecules in EVOH materials. By simple ˝ C (Table 1 and Figure S2) in than 1 mol %. In fact, the LCST of P(MEO MA -co-PyMA ) was 21.3 mixing, the EVOH nanofiber mesh was easily modified with the BOPs. As the copolymers, 2 99.0 1.0 P(MEO 2MA93.9was -co-MAAmBO 5.2-co-PyMA 0.9) and P(MEO 2MA99.0 -co-PyMA1.0) were selected 1 [40]). pH 7.4 PBS, which similar to that of the P(MEO homopolymer (LCST: 23 ˝(Table C in PBS 2 MA) and Figure 2). The fluorescent molecule of PyMA can trace the morphology change of a polymer, The stability of the nanofiber meshes of EVOH/BOP was measured at various solution conditions. chain and the fluorescent intensity can also be an indicator for cell biology [40]. To prevent the LCST EVOH/P(MEO2 MA93.9 -co-MAAmBO5.2 -co-PyMA0.9 ) nanofiber meshes were immersed for 24 h in pH change via the hydrophobic PyMA units, the PyMA contents in the copolymers were adjusted less 2 (HClaq.than ) and 12 (NaOH ) atLCST different solution temperatures (4 and 37 ˝ C). Figure 5A shows the SEM aq.the 1 mol %. In fact, of P(MEO 2MA99.0-co-PyMA1.0) was 21.3 °C (Table 1 and Figure S2) in images pH of EVOH/P(MEO meshes. immersion 7.4 PBS, which was similar to that of the P(MEO 2MA) homopolymer (LCST: 23 °C inAfter PBS [40]). 2 MA 93.9 -co-MAAmBO 5.2 -co-PyMA 0.9 ) nanofiber Thethere stability theoutward nanofibermorphology meshes of EVOH/BOP was measured at various solution conditions. treatment, wasofno change on the nanofiber structure (Figure S3A,B). At the EVOH/P(MEO 2MA93.9-co-MAAmBO 5.2-co-PyMA0.9) nanofiber meshes in were immersed for 24 h in pHnanofiber 2 same immersion conditions, particle aggregation was observed the modified EVOH (HClaq.) and 12 (NaOHaq.) at different solution temperatures (4 and 37 °C). Figure 5A shows the SEM with P(MEO2 MA99.0 -co-PyMA1.0 ) (Figure 5B and Figure S3C–F). The weight loss of the nanofiber images of EVOH/P(MEO2MA93.9-co-MAAmBO5.2-co-PyMA0.9) nanofiber meshes. After immersion meshes after immersion is shown in Figure 5C. In the P(MEO2 MA93.9 -co-MAAmBO5.2 -co-PyMA0.9 ) treatment, there was no outward morphology change on the nanofiber structure (Figure S3A,B). At at pH 12, weight losses were small at both 4 and was 37 ˝observed C; over 97% copolymers thethe same immersion conditions, particle aggregation in theof modified EVOH remained nanofiber in the nanofiber structure. The99.0high stability is a result of the interaction between and MAAmBO with P(MEO2MA -co-PyMA 1.0) (Figures 5B and S3C–F). The weight loss ofEVOH the nanofiber meshes units. ˝ after immersion is shown in Figure 5C. In the P(MEO 2 MA 93.9 -co-MAAmBO 5.2 -co-PyMA 0.9 ) at pHcopolymer 12, Interestingly, at pH 2, the weight losses were also small at both 4 and 37 C (over 98% of the weight losses were small at both 4 and 37 °C; over 97% of copolymers remained in the nanofiber remained in the nanofiber). The small weight loss might be due to three reasons: (1) polymeric structure. The high stability is a result of the interaction between EVOH and MAAmBO units. entanglement; (2) hydrophobic-hydrophobic interaction; and (3) boroxole and cis-diol interaction Interestingly, at pH 2, the weight losses were also small at both 4 and 37 °C (over 98% of copolymer at acidicremained condition [35,36]. On The the small otherweight hand,loss themight weight losses of P(MEO -co-PyMA1.0 ) 2 MA in the nanofiber). be due to three reasons: (1) 99.0 polymeric ˝ that cannot interact with EVOH via the covalent bonding were 86.6 and 86.7% at 4 and entanglement; (2) hydrophobic-hydrophobic interaction; and (3) boroxole and cis-diol interaction at 37 C, respectively. Moreover, afterOn thethe immersion supernatant liquid was also measured acidic condition [35,36]. other hand,test, the the weight losses of P(MEO 2MA 99.0-co-PyMA 1.0) that using cannot detector interact with via the bonding wereof86.6 86.7% 2atMA 4 and °C, respectively. a fluorescent to EVOH estimate thecovalent released amount theand P(MEO -co-PyMA 99.037 1.0 ) from the Moreover, immersion test, the supernatant liquid was alsovs. measured using a fluorescent nanofiber meshes.after Thethe standard straight line (fluorescent intensity concentration) was made from detector to estimate the released amount of the P(MEO2MA99.0-co-PyMA1.0) from the nanofiber meshes. P(MEO2 MA99.0 -co-PyMA1.0 ) (R2 = 0.991). The calculated release of P(MEO2 MA99.0 -co-PyMA1.0 ) was The standard straight line (fluorescent intensity vs. concentration) was made from P(MEO2MA99.0-co˝ C. There was a difference in the calculated release between the weight loss measurements 5% at 4 PyMA 1.0) (R2 = 0.991). The calculated release of P(MEO2MA99.0-co-PyMA1.0) was 5% at 4 °C. There was and the aconcentration determined by fluorescent intensity. The supernatantand liquid might have a very difference in the calculated release between the weight loss measurements the concentration determined by fluorescent supernatant liquid might have aas very small amount of of free small amount of EVOH, whichintensity. led to a The different external environment compared to that led to a different external environment as compared tostandard that of free P(MEO2line. MA99.0-coP(MEO2EVOH, MA99.0which -co-PyMA ), resulting in the discrepancy with the straight 1.0 PyMA1.0), resulting in the discrepancy with the standard straight line. (A) P(MEO2MA93.9-coMAAmBO5.2-co-PyMA0.9) 5 mg EVOH 35 mg in 1 mL HFIP

105

100

co

37 oC

4 oC

37 oC

85

4 oC

90

37 oC

(B) P(MEO2MA99.0-coPyMA1.0) 5 mg EVOH 35 mg in 1 mL HFIP

95

4 oC

co

Weight (W/W0, %)

co

(C)

80

pH 2

pH 12

P(MEO2MA93.9-coMAAmBO5.2-co-PyMA0.9) + EVOH

pH 12 P(MEO2MA99.0-coPyMA1.0) + EVOH

Figure 5. SEM images of electro-spinning samples: (A) P(MEO2MA93.9-co-MAAmBO5.2-co-PyMA0.9) (5

Figure 5. SEM of mg); electro-spinning (A) P(MEO MA93.9(35 -co-MAAmBO 5.2 -co-PyMA 0.9 ) 2MA99.0-co-PyMA1.0) and2 EVOH mg) in 1 mL HFIP. (C) mg) andimages EVOH (35 and (B) P(MEOsamples: (5 mg) and EVOH (35 mg); and (B) P(MEO MA -co-PyMA ) and EVOH (35 mg) in 1 mL HFIP. Weight loss ((weight after immersion test, W)/(weight test, W0) × 100 %) at various 2 99.0 before immersion 1.0 immersion conditionsafter for 24immersion h. (C) Weight loss ((weight test, W)/(weight before immersion test, W 0 ) ˆ 100 %) at various immersion conditions for 24 h.


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3.4. Controlled Adsorption and Desorption of Cationic Dye on Anionic EVOH/Benzoxaborole Nanofiber Meshes 3.4. Controlled Adsorption and Desorption of Cationic Dye on Anionic EVOH/Benzoxaborole Nanofiber Meshes The nanofiber of EVOH/P(MEO2 MA86.7 -co-MAAmBO4.8 -co-Ac8.5 ) was utilized for its pH-responsive property in a molecule catch and release system. The pH-responsive units The nanofiber of EVOH/P(MEO 2MA86.7-co-MAAmBO4.8-co-Ac8.5) was utilized for its pHof the P(MEO MA -co-MAAmBO -co-Ac occur due to the carboxylic acid located in the the 2 86.7in a molecule 4.8 8.5 ) release responsive property catch and system. The pH-responsive units of ˝ Ac unit. At pH 2, the LCST was 17.6 C. On the other hand, at pH 12, no transmittance P(MEO 2MA86.7-co-MAAmBO4.8-co-Ac8.5) occur due to the carboxylic acid located in the Ac unit. At pH change was observed duethe toother the hand, electrostatic repulsion of both change MAAmBO and Ac due units 2, the LCST was 17.6 °C. On at pH 12, no transmittance was observed to (Table 1). Therefore, theof surface properties nanofiber mesh will be the electrostatic repulsion both MAAmBO andofAcfunctionalized units (Table 1).EVOH Therefore, the surface properties altered at different pHnanofiber via the mesh mixedwill P(MEO -co-MAAmBO -co-Ac The 2anionic 2 MA86.7 4.8the 8.5 ). P(MEO of functionalized EVOH be altered at different pH via mixed MA86.7EVOH/P(MEO MA -co-MAAmBO -co-Ac ) nanofiber mesh was immersed in cationic methylene 86.7 8.5 co-MAAmBO4.82-co-Ac 8.5). The anionic 4.8 EVOH/P(MEO 2MA86.7-co-MAAmBO4.8-co-Ac8.5) nanofiber mesh blue immersed solution atin pHcationic 7.4 PBSmethylene (Figure 6).blue The anionic nanofiber mesh6). was expected adsorb was solutioncharged at pH 7.4 PBS (Figure The anionictocharged the methylene strongly via an electrostatic interaction as compared to that of only EVOH nanofiber mesh blue was expected to adsorb the methylene blue strongly via an electrostatic interaction nanofiber mesh. In fact, the EVOH/P(MEO MA -co-MAAmBO -co-Ac ) nanofiber mesh strongly 2 mesh. 86.7 In fact, the EVOH/P(MEO 4.8 8.5 2MA86.7-co-MAAmBO4.8as compared to that of only EVOH nanofiber adsorbed the blue dye more than that of the EVOH nanofiber mesh. The nanofiber co-Ac8.5) nanofiber mesh strongly adsorbed the blue dye more than that of thedyed EVOH nanofibermeshes mesh. were immersed in pH 2 solution to release the methylene blue via the pH-responsive property of The dyed nanofiber meshes were immersed in pH 2 solution to release the methylene blue via the the Ac units inproperty the P(MEO -co-MAAmBO ). After 15 min, the blue color15 clearly 2 MA 86.7 4.8 -co-Ac 8.5-co-MAAmBO pH-responsive of the Ac units in the P(MEO 2MA86.7 4.8-co-Ac 8.5). After min, turned light. In other words, the functionalized EVOH nanofiber mesh achieved a controlled molecule the blue color clearly turned light. In other words, the functionalized EVOH nanofiber mesh achieved desorption depending on the solution pH.onThe were forwere the aadsorption controlled and molecule adsorption and desorption depending thenanofibers solution pH. The reusable nanofibers controlledfor adsorption/desorption of methylene blueof(Figure S4). These results suggested the reusable the controlled adsorption/desorption methylene blue (Figure S4). Thesethat results pH-responsive properties of BOPs were imparted into the electro-spun EVOH nanofiber meshes. This suggested that the pH-responsive properties of BOPs were imparted into the electro-spun EVOH simple functionalization system functionalization will be a useful tool in expanding application field of EVOH nanofiber meshes. This simple system will be a the useful tool in expanding the (nano)materials. application field of EVOH (nano)materials.

Figure dye tests of the EVOH 2MA86.7 -co-MAAmBO 4.8Figure 6. pH-responsive 6. pH-responsive dye nanofiber tests and of EVOH/P(MEO the EVOH nanofiber and 8.5) nanofiber using methylene blue solution. Blue and red colors in the illustration are cationic co-Ac EVOH/P(MEO MA -co-MAAmBO -co-Ac ) nanofiber using methylene blue solution. 2 86.7 4.8 8.5 methylene blue andin anionic Ac units, are respectively. Blue and red colors the illustration cationic methylene blue and anionic Ac units, respectively.

4. 4. Conclusions Conclusions In In conclusion, conclusion, we we proposed proposed aa facile facile functionalization functionalization method method of of EVOH EVOH nanofiber nanofiber meshes meshes using using the BOPs via their reversible benzoxaborole-diol interaction. The mixture solution of EVOH the BOPs via their reversible benzoxaborole-diol interaction. The mixture solution of EVOH and and BOPs BOPs was was prepared prepared in in HFIP HFIP for for electro-spinning; electro-spinning; it it was was found found that that the the benzoxaborole-diol benzoxaborole-diol interaction interaction occurred which supported supported the occurred in in the the organic organic solvent. solvent. The The interaction interaction resulted resulted in in increased increased viscosity, viscosity, which the fabrication of a fine nanofiber structure. The structure of the nanofiber mesh was strongly affected fabrication of a fine nanofiber structure. The structure of the nanofiber mesh was strongly affected by by the benzoxaborolecontent, content,polymer polymerconcentration concentration and mixture ratios. Particles and bulk gels the benzoxaborole and thethe mixture ratios. Particles and bulk gels were were also formed depending the preparation conditions. The modified EVOH nanofiber meshes also formed depending on the on preparation conditions. The modified EVOH nanofiber meshes showed showed a high stability in acid or alkali aqueous solutions. After a 24-h immersion test, over 97% of EVOH and BOP remained within the nanofiber mesh. According to the SEM images, there was no morphology change in the nanofiber structure after the immersion test. Moreover, the EVOH


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a high stability in acid or alkali aqueous solutions. After a 24-h immersion test, over 97% of EVOH and BOP remained within the nanofiber mesh. According to the SEM images, there was no morphology change in the nanofiber structure after the immersion test. Moreover, the EVOH nanofiber mesh was also functionalized with BOP having pH-responsive COOH groups. The anionic charged nanofiber mesh was strongly dyed by methylene blue due to the electrostatic interaction as compared to that of the EVOH nanofiber mesh in pH 7.4. The dye was rapidly released from the fiber mesh in acidic condition. This simple and effective functionalization system for EVOH (nano)materials could lead to an expansion of their applications into new fields. Supplementary Materials: Supplementary materials can be found at www.mdpi.com/2073-4360/8/2/41/s1. Acknowledgments: This study was partially supported by research funds from the International Center for Young Scientists (ICYS), National Institute for Materials Science (NIMS), Japan. Author Contributions: Yohei Kotsuchibashi conceived of and conducted the experiments. This paper was written by Yohei Kotsuchibashi and Mitsuhiro Ebara. Conflicts of Interest: The authors declare no conflict of interest.

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