Controlling the Hydration Structure with a Small Amount of Fluorine To

Article Cite This: Biomacromolecules 2019, 20, 2265−2275

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Controlling the Hydration Structure with a Small Amount of Fluorine To Produce Blood Compatible Fluorinated Poly(2methoxyethyl acrylate) Ryohei Koguchi,†,‡ Katja Jankova,†,§ Noriko Tanabe,∥ Yosuke Amino,∥ Yuki Hayasaka,∥ Daisuke Kobayashi,∥ Tatsuya Miyajima,∥ Kyoko Yamamoto,‡ and Masaru Tanaka*,†

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†

Soft Materials Chemistry, Institute for Materials Chemistry and Engineering, Kyushu University, Build. CE41, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan ‡ AGC Incorporation New Product R&D Center, 1150 Hazawa-cho, Kanagawa-ku, Yokohama, Kanagawa 221-8755, Japan § Department of Energy Conversion and Storage, Technical University of Denmark, Elektrovej, Build. 375, 2800 Kongens Lyngby, Denmark ∥ AGC Incorporation Innovative Technology Research Center, 1150 Hazawa-cho, Kanagawa-ku, Yokohama, Kanagawa 221-8755, Japan S Supporting Information *

ABSTRACT: Poly(2-methoxyethyl acrylate) (PMEA) shows excellent blood compatibility because of the existence of intermediate water. Various modifications of PMEA by changing its main or side chain’s chemical structure allowed tuning of the water content and the blood compatibility of numerous novel polymers. Here, we exploit a possibility of manipulating the surface hydration structure of PMEA by incorporation of small amounts of hydrophobic fluorine groups in MEA polymers using atom-transfer radical polymerization and the (macro) initiator concept. Two kinds of fluorinated MEA polymers with similar molecular weights and the same 5.5 mol % of fluorine content were synthesized using the bromoester of 2,2,3,3,4,4,5,5,6,6,7,7,8,8-pentadecafluoro-1octanol (F15) and poly(2,2,2-trifluoroethyl methacrylate) (PTFEMA) as (macro) initiators, appearing liquid and solid at room temperature, respectively. The fibrinogen adsorption of the two varieties of fluorinated MEA polymers was different, which could not be explained only by the bulk hydration structure. Both polymers show a nanostructured morphology in the hydrated state with different sizes of the features. The measured elastic modulus of the domains appearing in atomic force microscopy and the intermediate water content shed light on the distinct mechanism of blood compatibility. Contact angle measurements reveal the surface hydration dynamicswhile in the hydrated state, F15-b-PMEA reorients easily to the surface exposing its PMEA part to the water, the small solid PTFEMA block with high glass-transition temperature suppresses the movement of PTFEMA-b-PMEA and its reconstruction on the surface. These findings illustrate that in order to make a better blood compatible polymer, the chains containing sufficient intermediate water need to be mobile and efficiently oriented to the water surface. appears, and then it melts just below that of FW (0 °C) or often appears as a shoulder of the FW melting. Each state of water contributes differently to the properties of polymers when hydrated. The best example is the considerable lower glass-transition temperature (Tg) of biomacromolecules when hydrated. Together with the IW content (IWC), the decreased in the hydrated state Tg may be accepted as biocompatibility indices.

1. INTRODUCTION Water molecules play an important role in the interface between polymers and biological systems. In hydrated polymers,1−4 water is found to exist in three states: free water (FW), intermediate water (IW), and nonfreezing water (NFW). As in the cited order the interactions with the polymer increase, and the freezing-bound water was called IW because of having an intermediate association with the polymer. This type of water is expressed as cold crystallization (CC) in the differential scanning calorimetry (DSC) cooling scan (−40 °C) below the freezing of FW (−25 °C) and above NFW, which actually was not found yet to freeze at all even until −100 °C. IW can also appear in the heating scanfirst CC © 2019 American Chemical Society

Received: February 9, 2019 Revised: April 25, 2019 Published: May 1, 2019 2265

DOI: 10.1021/acs.biomac.9b00201 Biomacromolecules 2019, 20, 2265−2275

Article

Biomacromolecules

side chains in the hydrated state, which can happen by at least the above-mentioned different scenario. As a continuation of the authors’ efforts to deeply and systematically study the mechanism of blood compatibility using the IW concept, here we start exploiting another strategy for changing the IWC in MEA polymersincluding small amounts of heteroatoms in MEA polymers and analogues, herein the electronegative and hydrophobic fluorine. Even nature is including mostly C, H, O, N, S, and P, some F has been found in it. Very recently, the fluorine metabolism was engineered, and the fluoropolymer production in living cells was realized.14 Small amounts of fluorine have been previously introduced in polystyrene (PS)15 by atom-transfer radical polymerization (ATRP) from various hydroxyl-containing fluorine compounds (or oligomers)15,16 using the macroinitiator concept.17 The fluorine-containing PSs with similar MWs show different surface characteristics depending on the position of the fluorine chainterminal fluorine-containing groups were more effective than internal because of better mobility. Furthermore, by using fluorinated initiators for the free-radical polymerization of various vinyl monomers, many telechelic polymers have been synthesized bearing fluorine-end groups from the initiator.18,19 Additionally, a number of fluorine monomers have been accepted as nontoxic, and the chemical resistance and great lubricity of their polymers were used from the medicinal industry to produce biocompatible materials such as tubing, catheters, and surgical devises. Finally, some fluorine-containing block copolymers with PMEA have already been synthesized, called bioacceptable, and their biocompatibility was attributed to the hydrophobicity and inertness of fluorinated polymers, as well as to the presence of the blood compatible PMEA or analogues, without any further investigations.20,21 In other examples as in blends of amphiphilic polymers possessing PEG and semifluorinated chains with another polymer, surface analysis reveals that the fluorinated segments showed surface segregation. In the case of amphiphilic polymers, the longer semifluorinated F10H10 chains aggregate at the surface more efficiently than F8H6.22 The surface properties were changed upon immersion in water as the more hydrophilic PEG groups migrated to the surface after adsorption of water molecules.23 The presence of both these groups on the surface resulted in better foaling-release performance. However, when other hydrophilic monomers, for example, EG24 and HEMA,25 were copolymerized with small amounts of 2-perfluorooctylethyl methacrylate (FMA), the surface structures were found to change, and the amount of adsorbed protein was found to exhibit a minimum value. This was also the result from the atomic force microscopy (AFM) investigations of the copolymer mPEG-b-polyacrylic acid-bPMMA-b-PMFA, when the mPEG part was exposed to water.26 It was also reported that the receding contact angle (CA) decreases by copolymerization of small amounts of FMA with MMA and methoxy PEG methacrylate. The authors assume that fluorine having a low cohesive force segregates at the air interface so that when it comes into contact with water, the fluorine part is easily switched to the hydrophilic site.27 As a first attempt to study the IW and the blood compatibility of PMEA containing small amounts of fluorine, we have utilized here the possibility of using a fluorinecontaining initiator for the ATRP of MEA.28 A short chain of a monomer bearing fluorine was also inserted in a block copolymer structure with PMEA.15 We compare here the

The term blood compatibility is related to materials which do not undergo some side effects caused by contact with blood and the adsorbed protein layer, such as platelet denaturation and activation, plasma coagulation, leucocyte interaction leading to inflammation and cell depletion, thrombosis, hemolysis, cancer, toxic or allergic reactions, and so forth. More information of this complex property can be found in the review about the state of the art of blood compatible materials.5 The scientific literature abounds with examples dealing with the synthesis of many (co)polymers claimed of being biocompatible surfaces. However, the reasons and mechanisms for this are seldom discussed. Poly(2-methoxyethyl acrylate) (PMEA) is a simple acrylic polymer with one unit ethylene oxide in the side chain and excellent blood compatibility because of the existence of IW in the hydrated state.1 The first artificial lung oxygenator was therefore coated with a thin film of PMEA, and the blood compatibility was evaluated using an in vitro test.6 Polymers containing IW are defined blood compatible, evaluated by having low platelet adhesion and protein adsorption. The properties of many famous biocompatible polymers [polyethylene glycol (PEG), poly(2-methacryloyloxyethyl phosphorylcholine), polyvinylpyrrolidone (PVP), etc.] were explained with the concept of IW too. There are different ways to change and tune the IWC of materials. By synthesizing multiple homopolymer analogues of MEA polymers7,8 containing more than one ethylene oxide unit in the side chain or different substituentsmethyl, ethyl, and butylat the side chain end, it was noticed that their IWC increases with the number of ethylene oxide groups in the side chain and decreases when comparing the acrylate with the methacrylate polymers. Thus, by choosing acrylate or methacrylate polymers with various nEO or different substituents, the water content in the hydrated polymers could be tuned. The methacrylate analogue of PMEA, poly(2methoxyethyl methacrylate), shows much lower IWC than PMEA and was according to this found less blood compatible polymer.9 Another number of PMEA analogues have been investigated for the existence of IW, and one of these poly(ωmethoxyalkyl acrylate)s, poly(methoxypropyl acrylate), was found to possess even more IW and thus better blood compatibility than PMEA.3 Its properties were utilized to coat a heart of a rat and to realize a long-time electrocardiographic monitoring of the hearts dynamically moving.10 Copolymerization of MEA with 2-hydroxyethyl acrylate (HEMA) was also attempted, basically because in the hydrated poly-HEMA (PHEMA) there was less sign of IW.2,11,12 By evaluation of the water structure of random,2 block,11 and graft12 copolymers of HEMA and MEA, IW due to the presence of PMEA and decent compatibility was found, when the amount of PHEMA was less than 40%.2,11 However, when PHEMA was grafted with PHEMA with molecular weights (MWs) 5400 and 2600, respectively, IW appeared.13 It was the contribution of Prof. Tsuruta to suggest that NFW participates in a stable molecular network system together with the hydroxyl group of HEMA, which can be destroyed using small amounts of key comonomers. Copolymerization with aminocontaining monomers resulted in loosening of the H-bonding network to form a soft biological surface where IW predominates and in increase of the blood compatibility.13 All these investigations show that the amount of IW can be manipulated by tuning the chemical structure of the polymers and the molecular mobility of the polymer backbone or of the 2266


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was run through a column of basic Al oxide, concentrated, and precipitated in hexane. After decantation, the polymer was washed with water under stirring, collected as a transparent liquid, and dried. The yield was determined gravimetrically (57%). 1 H NMR (CDCl3): δ (ppm) 1.1−1.3 (m, 6H, −C(CH3)2), 1.4−2.1 (m, 72H, −CH2), 2.1−2.6 (m, 36H, −CH), 3.3−3.5 (m, 108H, −O− CH3), 3.5−3.7 (m, 36H, −O−CH2), 3.7−3.8 (m, 2H, −O−CH2CF2), 4.0−4.4 (m, 36H, −O−CH2). 19 F NMR (CDCl3 + 1,3-(CF3)2C6H4): δ (ppm) −62.1 (1,3(CF3)2C6H4), −78.4 (−8CF3), −117.0 (−2CF2), −119.4 (−3CF2, −4CF2), −120.4 (−5CF2), −120.9 (−6CF2), −123.7 (−7CF2). 2.1.3.2. PTFEMA4-b-PMEA27 (2). Block copolymer was synthesized in a pressure glass vessel, placed in a glovebox in the beginning for loading all the components. The vessel was equipped with a magnetic stirrer and sealed with a metal lid. After that, 2.58 mL (20 mmol) of MEA, 2.60 mL of toluene, 14 mg (0.1 mmol) of Cu(I)Br, 88 mg (0.1 mmol) of the PTFEMA4−Br macroinitiator, and 21 μL (0.1 mmol) of PMDETA were added. The glass vessel was sealed with the metal lid, and the reaction was performed at 80 °C for 18 h. The reaction mixture was diluted by adding toluene (∼20 mL), and Al2O3 was then inserted to remove the catalyst residue. The mixture was stirred at room temperature for 5 min, Al2O3 was filtered off, and the filtrate was precipitated in hexane. 1 H NMR (CDCl3): δ (ppm) 0.8−1.4 (m, 21H, −C(CH3)), 1.4− 2.1 (m, 62H, −CH2), 2.2−2.4 (m, 27H, −CH), 3.2−3.5 (m, 81H, −O−CH3), 3.5−3.7 (m, 54H, −O−CH2), 4.0−4.3 (m, 56H, −O− CH2), 4.3−4.5 (m, 8H, −O−CH2-CF3). 19 F NMR (CDCl3 + 1,3-(CF3)2C6H4): δ (ppm) −61.2 (1,3(CF3)2C6H4), −72.4 (−CF3). 2.1.4. Synthesis and Characterization of the Homopolymers. 2.1.4.1. Synthesis of PMEA169 (3). PMEA was synthesized by freeradical polymerization using azobis(isobutyronitrile) as the initiator in 1,4-dioxane at 75 °C for 6 h.9 The polymer was purified by precipitation in THF/hexane mixture, collected, and dried under reduced pressure at 60 °C for 1 day. 1 H NMR (CDCl3): δ (ppm) 1.3−2.1 (m, 2H, −CH2), 2.1−2.6 (m, 1H, −CH), 3.3−3.5 (m, 3H, −O−CH3), 3.5−3.7 (m, 2H, −O− CH2), 4.0−4.4 (m, 2H, −O−CH2). 2.1.4.2. Synthesis of PTFEMA71 (4). TFEMA [21.4 mL (150 mmol)] and 30.0 mL of toluene were charged to a pressure glass vessel, as described above. Cu(I)Br [215 mg (1.5 mmol)], 223 μL (1.5 mmol) of EBIB, and 260 μL (1.5 mmol) of PMDETA were added, and the reaction was run at 80 °C for 13 h. PTFEMA (16.5 g, 66% yield) was obtained as a white solid by precipitation of the toluene solution in ethanol. 1 H NMR (CDCl3): δ (ppm) 0.7−1.2 (m, 213H, −C(CH3)), 1.2− 1.4 (m, 9H, −C(CH3)), 1.8−2.3 (m, 142H, −CH2), 4.0−4.2 (m, 2H, −O−CH2), 4.0−4.7 (m, 142H, −O−CH2-CF3). 19 F NMR (CDCl3 + 1,3-(CF3)2C6H4): δ (ppm) −62.1 (1,3(CF3)2C6H4), −72.4 (−CF3). 2.1.5. Analyses. 2.1.5.1. Characterization of the Chemical Structures and Investigation of the Thermal Properties of Polymers. The fluorinated (macro) initiators and the fluorinated block polymers were characterized by 1H NMR, using a JEOL 500 MHz JNM-ECX spectrometer, and 19F NMR, employing Bruker AVANCE 300 MHz at room temperature in DMSO-d6 or CDCl3 as solvents. Chemical shifts for 1H NMR are reported in δ ppm downfield from tetramethylsilane, whereas for 19F NMR, the assignments are given downfield from 1,3-(CF3)2C6H4. MWs were determined by sizeexclusion chromatography (SEC) employing an integrated SEC unit of Tosoh HLC-8220 chromatograph equipped with three TSK-gel columns connected in series (super HZ4000, super HZ3000, Super HZ2500, and Super HZ2000) and a refractive index (RI) detector. Measurements were performed in THF at 30 °C with a 0.35 mL/min flow. MWs were calculated using PS narrow MW standards in the range of 5 × 102 to 5.48 × 106 g/mol. DSC measurements were performed using Q1000 from TA Instruments in the temperature range of −80 to 100 °C at a heating rate of 5 °C min−1 under nitrogen. Tg was determined automatically

water structure, platelet adhesion, and protein adsorption of these fluorinated MEA polymers (F-PMEAs) with virgin surfaces of polyethylene terephthalate (PET) and polypropylene (PP). PMEA is further synthesized by free-radical polymerization and included in the investigations for comparison.

2. EXPERIMENTAL SECTION 2.1. Experimental Part. 2.1.1. Materials. 2,2,3,3,4,4,5,5,6,6,7,7,8,8-Pentadecafluoro-1-octanol (F15-OH), dimethylaminopyridine (DMAP), bromoisobutyrylbromide (BIBB), and ethyl 2-bromoisobutyrate (EBIB) (all from Sigma-Aldrich) were used as received without further purification. The MEA monomer (SigmaAldrich) was passed through a ready-to-use, disposable prepacked inhibitor remover column. 2,2,2-Trifluoroethyl methacrylate (TFEMA) and N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDETA) were purchased from TCI Chemicals and deoxygenated by three pump−thaw cycles. TFEMA was passed through a column of activated neutral Al2O3 before use to remove the inhibitor. All other solvents or chemicals were used as received unless otherwise stated. Phosphate-buffered saline (PBS) was purchased from FUJIFILM Wako Pure Chemical Corporation. 2.1.2. Synthesis and Characterization of Fluorinated (Macro) Initiators. 2.1.2.1. Fluorinated Initiator 2-Bromo-2-methylbutyrylic Acid-(2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-pentadecafluorooctyl)ester (F15Br). The fluorinated initiator 2-bromo-2-methylbutyrylic acid(2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-pentadecafluorooctyl)ester (F15-Br) was obtained by esterification15−17 of F15-OH with BIBB: 3 g (7.49 mmol) of F15-OH and 60 mL of dry tetrahydrofuran (THF) were suspended in a three-neck round-bottom flask equipped with a magnetic stirrer and an addition funnel. The mixture was cooled down with ice water, and 1.3 mL (9.44 mmol) of triethyl amine, 100 mg of DMAP recrystallized from toluene, and 1.2 mL (9.44 mmol) of BIBB were added slowly with continuous stirring. The temperature was allowed to come to room temperature, and the reaction was performed overnight. The suspension was filtered, and THF was removed by a rotary evaporator. The brown-colored liquid formed was diluted with diethyl ether, and the ether layer was washed extensively with saturated NaHCO3 solution, 1 M HCl, and finally distilled water. The ether layer was dried over Na2SO4, and the solvent was removed on a rotary evaporator to leave a yellowish liquid. Yield: 97%. 1 H NMR (DMSO-d6): δ (ppm) 1.9 (s, 6H, 2 −C(CH3)2), 4.99 (t, 2H, −O−CH2). 19 F NMR (CDCl3 + 1,3-(CF3)2C6H4): δ (ppm) −62.1 (1,3(CF3)2C6H4), −80.0 (−8CF3), −117.8 (−2CF2), −122.2 (−3CF2, −4CF2), −121.0 (−5CF2), −121.5 (−6CF2), −124.3 (−7CF2). 2.1.2.2. Fluorinated Macroinitiator PTFEMA4−Br. TFEMA [8.6 mL (60 mmol)] and 9.6 mL of toluene were charged to a pressure glass vessel, equipped with a magnetic stirrer and sealed with a metal lid. The glass vessel was placed in a glovebox. Cu(I)Br [430 mg (3.0 mmol)], 447 μL (3.0 mmol) of EBIB, and 627 μL (3.0 mmol) of PMDETA were added, and the reaction was run at 80 °C for 20 min. After precipitation in hexane, PTFEMA4−Br (1.70 g, 16% yield) was obtained as light green solids. 1 H NMR (CDCl3): δ (ppm) 0.7−1.4 (m, 21H, −C(CH3)), 1.4− 1.8 (m, 3H, −CBr(CH3)), 1.8−2.3 (m, 8H, −CH2), 4.0−4.7 (m, 10H, −O−CH2). 19 F NMR (CDCl3 + 1,3-(CF3)2C6H4): δ (ppm) −62.1 (1,3(CF3)2C6H4), −72.4 (−CF3). 2.1.3. Synthesis and Characterization of Fluorinated (Block Co)polymers. 2.1.3.1. F15-b-PMEA36 (1). In a typical experiment,28 0.141 g (0.256 mmol) of F15-Br was dissolved in 5.06 g (0.0389 mol) of MEA in a Schlenk tube, 37 mg (0.256 mmol) of CuBr and 0.108 μL (0.512 mmol) of PMDETA were added, and a magnetic stirrer bar was placed into the tube. The system was deoxygenated by three freeze−pump−thaw cycles using dry nitrogen, and polymerization was initiated by immersing the tube in a preheated oil bath at 90 °C for 3.5 h. The viscous polymer was diluted with THF; the solution 2267


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Biomacromolecules Scheme 1. Synthetic Routes to Fluorinated (Macro) Initiators and Block Copolymers

by the instrument from the second heating trace and is reported as the midpoint of the thermal transition. 2.1.5.2. Quantification of the Interactions between Water and Polymers. From the interactions of water with the particular polymer, we classify three kinds of water in hydrated macromolecules: NFW, IW, and FW, whose contents are given as NFWC, IWC, and FWC, respectively. The equilibrium water content (EWC) of the polymers is estimated by DSC as the water content at which a peak for the melting of ice around 0 °C and a small shoulder or peak for the melting of IW below this temperature appear and is given by the following eq 1

EWC (wt %) = ((W1 − W0)/W1) × 100

XPS was carried out on an ESCA-5500 (Physical Electronics, Inc.) equipped with a monochromated Al Kα X-ray source. XPS measurements were performed at photoelectron take-off angles (measured with respect to the plane of the sample) of 15 and 45° to acquire the outer and inner compositions of the polymer-coated substrates, respectively. The information depth provided by the angle of 15° is shallower than 45°. Charge compensation was achieved with a low-energy electron flood gun. 2.1.5.4. Protein Adsorption and Deformation by the μBCA Assay and ELISA.1,3,7,8 The μ-bicinchoninic acid (BCA) assay was performed in order to measure the amount of adsorbed proteins on the polymer surfaces. In addition, the enzyme-linked immunosorbent assay (ELISA) was executed to determine the amount of the exposed γ chains in fibrinogen. The polymer surfaces were prepared for both the μBCA and the ELISA assays by the following manner: 15 μL of the particular fluorinated polymer solution (0.2 wt/vol %) in THF or of PMEA in methanol was added to the polypropylene 96-well plate. The plate was slowly air-dried over 3 days at room temperature. Two types of polymer-casted 96-well plates were prepared. The one is the priming substrate, which was immersed in 25 μL of PBS for 1 h at 37 °C, and the other is the nonpriming substrate, which was not subjected to PBS prior to the μBCA and ELISA assays. In the case of μBCA, 25 μL of 2 mg/mL fibrinogen (Sigma) in PBS and 50 μL of 1 mg/mL fibrinogen in PBS were added to each well and incubated for 10 min at 37 °C. After that, the wells were washed five times with 200 μL of PBS. The adsorbed proteins were extracted with a solution of 5% sodium dodecyl sulfate and 0.1 N NaOH by incubation for 10 min at 37 °C. The extracted proteins were assessed by the μBCA assay (Thermo Scientific, Rockford, IL) according to the manufacturer’s instructions. The protein amount was calculated using the albumin standard curve. When ELISA was applied, the fibrinogen in PBS was added in the same volumes but in different concentrations40 and 20 μg/mL and incubated as above. After that, the samples were incubated with Blocking-One (Nacalai Tesque) for 30 min at 37 °C to prevent nonspecific reactions. Further incubation was carried out with antifibrinogen γ′ antibody (Ab) (Millipore) for 2 h at 37 °C and then with peroxidase-conjugated anti-mouse immunoglobulin G (IgG) Ab for 1 h at 37 °C. Finally, the samples were incubated with 2,2′azinobis(3-ethylbenzothiazoline-6-sulfonic acid)ammonium salt in a buffer for the latter from Roche Diagnostics. The absorbance was measured at a wavelength of 405 nm using a plate reader (Bio-Rad). The reported values are the average of five measured points on a plate excluding the lowest and highest value. 2.1.5.5. Preparation of Polymer Substrates and Human Platelet Adhesion Test.1,3,7,8 The polymer substrates used in the human platelet adhesion tests were prepared as follows: the polymer-coated substrates were cut into 8 × 8 mm squares. The substrates were then fixed on a scanning electron microscopy (SEM) specimen stage using a double-sided tape. Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were obtained from human blood by two stages of centrifugation: first at 1500 rpm for 5 min to obtain PRP and then the same blood was centrifuged at 4000 rpm for 10 min to yield PPP. The

(1)

where W0 and W1 are the weight of the dry and hydrated samples, respectively. The amounts of different types of water in the hydrated polymers are given by the following equations

EWC (wt %) = NFW (wt %) + IW (wt %) + FW (wt %)

(2)

IW (wt %) = ΔHcc/334 (J g −1)

(3)

FW (wt %) = (ΔHm/334 (J g −1)) − IW

(4)

where ΔHcc and ΔHm are enthalpy changes during CC and the melting of ice, respectively. The weight of the samples was 3−7 mg. The hydrated samples were prepared by immersing in ultrapure water for 7 days before measurements. 2.1.5.3. Preparation and Characterization of the Polymer Surfaces. Film samples were prepared as follows and used for CAs, AFM, and X-ray photoelectron spectroscopy (XPS) measurements, as well as for the human blood platelet adhesion test. The polymer samples were dissolved in THF or methanol to give a 0.2 wt/vol % solution, filtered, and spin-coated onto round-shaped PET surfaces, 14 mm in diameter, previously washed with methanol. The substrates were coated twice with the particular solution using a Mikasa spin coater MS-A100 at rates of 500 rpm for 5 s, 2000 rpm for 10 s, slope for 5 s, 4000 rpm for 5 s, and slope for 4 s, and then the substrates were dried at room temperature. CAs of water toward the airside of the polymer films with an accuracy of ±2° were measured at 25 °C by the sessile drop method. The captive bubble method is a special arrangement for measuring the CA between a liquid and a solid using drop shape analysis. Air bubble (2 μL) is injected to the hydrated surface immersed in water.3 The reported values for both measurements are the average of three measurements made at different positions of a film. AFM observations were conducted using a Cypher (Oxford Instruments), and a cantilever HQ-75-Au was used (spring constant k = 2.5 N/m, resonance frequency f = 75 kHz in air, tip radius < 10 nm; Oxford Instruments). AFM topographies were imaged in PBS using ac mode. The images were obtained 10, 30, and 60 min after the immersion in PBS. The arithmetical mean height (Sa) and the elastic modulus of convex and flat portions were determined before and after immersion for 60 min in PBS. 2268


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Biomacromolecules cells in PRP and PPP were measured using a hemocytometer, and a plasma solution containing 4 × 107 cells cm−2 of platelets was prepared by mixing the PRP and PPP solutions in the calculated ratio. This solution (200 μL) was transferred onto the polymer-coated substrates, either with priming or not. The first type of substrates was immersed in PBS for 1 h at 37 °C before the platelet adhesion test. The polymer-coated substrates were then washed twice with PBS. The adhered platelets were fixed by immersion in 1% glutaraldehyde in PBS for 120 min at 37 °C. Finally, the substrates were washed four times in the following order: PBS, diluted PBS (1:1, PBS/water), and pure water twice. SEM was used to quantify the amount of platelets adhered onto the substrates. For statistical purpose, the platelet adhesion test was performed three times with human blood from different donors. The reported values are the average of five measured points on three different films, excluding the maximum and the minimum value. 2.1.5.6. Statistical Analysis. All of the data are expressed as the means ± SD. The significance of the differences between two samples was determined using an unpaired Student’s t-test using Microsoft Excel 2013. Differences with P values less than 0.05 were considered to be statistically significant.

The NMR spectra of all these polymers are shown in Figures S7−S9. Table 1. Composition, Glass-Transition Temperatures, and the MW Characteristics of the Synthesized F-PMEAs, PMEA169, and PTFEMA71 fluorine contenta polymers

Tg (°C) Mn theob (Da)

Mnc (Da)

−49

5200

−31

−37

4200

−35

−50

74

71

10 600 44 100 22 000 29 200

mol %

wt %

dry

hydrated

1

3.6

5.5

−43

2

12.9

5.5

3

0.0

0.0

4

100.0

33.9

11 900

Mw/Mnc 1.30 1.32 2.81 1.59

The fluorine content was calculated using the theoretical Mn value. The mol and wt % were calculated from the mol or wt ratio of the fluorine atoms in the polymer, respectively. bMn theo was calculated from the block copolymer composition identified by 1H NMR. c Estimated by SEC employing the PS calibration curve. a

3. RESULTS AND DISCUSSION Fluorinated polymers of the blood compatible PMEA were prepared using either a fluoroalkyl initiator or a TFEMA macroinitiator. The synthetic route to the F-PMEAs is outlined in Scheme 1. ATRP and the macroinitiator concept have been employed. 3.1. Synthesis and Characterization of the F-PMEAs. Fluoroalkyl chains are known to crystallize by packing when having 8 or higher number of carbons.29 In order to maintain the high mobility in water, the fluorinated alcohol F15-OH consisting of C7 chains was selected for the synthesis of the fluorinated initiator, F15-Br, used further for the ATRP of MEA. In addition, it is also known that the bioaccumulation problem is low in polymers containing about C7 or lower fluoroalkyl chains.30 The esterification reaction of F15-OH was performed with BIBB.25−27 Evidence for the quantitative conversion of the hydroxyl to the bromoester group could be found in the 1H NMR spectra (in dry DMSO-d6).17,31 As shown in Figures S1 and S2, the signal of OH at 6.11 ppm disappeared, and a new triplet at 4.99 for F15-Br and the 2methylene protons in CH2−O appeared. The synthesized macroinitiator was utilized to design a PMEA having 15 fluorine atoms at the chain end (F15-b-PMEA36) and shown in the NMR spectra in Figure S3. Another macroinitiator was also synthesized by the ATRP of TFEMA. Conversions were estimated from the 1H NMR analysis of the reaction mixture using the ratio of the peak for −O−CH2− in the fluorinated methacrylate at δ 4.35 ppm (for the polymer) to that at δ 4.53 ppm (for the monomer). The crude 1H NMR spectra for the homopolymerization are shown in Figures S4. From the first-order kinetic plot presented in Figure S5, the reactivity apparent rate constant, kapp p , was found to be 1.1 × 10 −4 s−1. This value is in reasonably good agreement with the one of 1.6 × 10 −4 s−1, previously published in a similar reaction with the same monomer.20 The NMR spectra of purified macroinitiator are shown in Figure S6. Employing the PTFEMA4−Br macroinitiator for the ATRP of MEA yielded fluorinated block copolymers of MEA. We present here one PTFEMA4-b-PMEA27 which has the same fluorine content as F15-b-PMEA36. Their characteristics together with a higher MW PTFEMA used for comparison are given in Table 1, and the structures are shown in Figure 1.

Figure 1. Structures of the evaluated polymers.

As can be seen from Table 1, the synthesized polymer 1 and block copolymer 2 have low fluorine contents of 5.5 wt %. This content was estimated from the MW found by 1H NMR. Although the interactions of the synthesized F-PMEAs with the SEC columns should be weak, the MW measured by SEC and calculated with PS standards are much bigger than that calculated from the 1H NMR data. The inaccurate match of SEC and 1H NMR data indicates that different standards and/ or solvents are necessary for the PMEA polymers that contain fluorine. SEC was also carried out in dimethylformamide (DMF), and the numbers for the MWs were calculated using PMMA and PEG standards. The theoretical values for 1, 2, and 4 calculated from 1H NMR are relatively consistent with the results, measured in DMF using PEG as standards. However, the Mw/Mn ratio remains almost the same for all polymers 1−4 by either method used. Also, the SEC overlay presented in Figure S10 shows almost complete shift of the SEC trace of the macroinitiator to polymer 2 as a block copolymer is formed. SEC data of the polymers 1, 3, and 4 are shown in Figure S11. Because the RI of fluorine is low and close to that of THF, the fluorine homopolymer 4 showed a weak intensity peak. The almost 15° lower Tg of PMEA in the hydrated state is already known and is connected to the water−polymer interactions, which will be discussed later in the paper. Likewise, a decrease of Tg in the hydrated fluorinated block 2269


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Table 3. Contact Anglesa of the F-PMEAs, PMEA169, and PTFEMA71 Used in the Investigation

copolymer is observed too, however, to a lower extent than in PMEA. In polymer 1, the fluorine site (F15) plasticizes PMEA, and the Tg in the dry state decreases. In the hydrated state, further decrease was detected. For the block copolymer 2, the fluorinated block (PTFEMA) suppresses the hydration of PMEA, and therefore, the Tg of this hydrated polymer was relatively higher than that of PMEA169, 3. Block copolymer 2 shows only one Tg (that of the PMEA block), which is not surprising either as the small amount of the fluorine block is difficult to detect, thus making also the observation of potential phase separation problematic. 3.2. Hydration Behavior of F-PMEAs. DSC was used to evaluate the hydration behavior of the F-PMEAs, and the results are shown in Table 2. It is important to notice that F-

sessile drop (WCA) polymers 1 2 3 4 PET

EWC (wt %)

NFW (wt %)

IWheatinga (wt %)

IWcoolingb (wt %)

IWtotal (wt %)

FW (wt %)

1 2 3 4

10.03 7.91 9.78 1.60

3.92 2.20 3.32 1.60

1.80 2.45 3.30 0.00

2.83 0.00 0.00 0.00

4.63 2.45 3.30 0.00

1.48 3.27 3.17 0.00

76.7 94.7 68.5 96.9 81.1

± ± ± ± ±

captive air bubble (ACA)

30 s 4.3 3.3 4.2 1.3 1.1

40.7 89.0 42.5 96.0 77.3

± ± ± ± ±

16 h 2.1 3.5 1.2 1.3 1.0

129.3 124.1 130.6 88.5 109.5

± ± ± ± ±

1.1 2.5 0.8 2.6 4.8

a

WCA (sessile drop method) and ACA (captive bubble method). The data represent the means ± SD (n = 3).

third kind applies to the most hydrophobic sample PTFEMA71, 4, showing the lowest ACA at the air−water interface even when hydrated. The present investigations show that small amounts of fluorine play an important role not only to the bulk but also to the surface structure of the synthesized F-PMEAs. Therefore, in order to evaluate the hydration dynamics of the surface layer, the time dependence of the WCA after the water droplet was put on each of the polymer-coated substrates was measured as shown in Figure 2.

Table 2. Water Content of the F-PMEAs, PMEA169, and PTFEMA71 Used in the Investigation polymers

1s

a Crystallizes at a temperature different from the crystallization of FW in the heating process. bCrystallizes at a temperature different from the crystallization of FW in the cooling process.

PMEAs 1 and 2 show IW, most probably imparting it from the adjacent PMEA block. This is one requirement for a material to show blood compatibility but not the only one. From the results of the DSC analysis in the heating scan, PMEA169 3 and F-PMEAs show CC of water around −40 °C and its melting below 0 °C. Focusing on the amount of hydration water, polymer 1 has higher NFWC and lower FWC than its nonfluorinated analogue 3 and block copolymer 2, and conversely 2 has higher FWC and lower NFWC than its nonfluorinated analogues 3 and 1. This phenomenon is probably due to the mobility of the fluorine; the high mobility fluorine prevents the formation of FW and makes possible larger NFWC and IWC with stronger binding forces to be formed. In this case, because of the effect to the amount and mobility of the introduced fluorine, a part of IW of 1 was observed around −40 °C in the cooling process, and as a result, not only the amount of NFWC but also the amount of IWC increases. The combined enthalpy changes (the sum of both parts of the crystallized IW in the cooling and heating cycles) gives the total IWC of this polymer (Figure S12 and Table 2), greater than that of PMEA. 3.3. Contact Angle Measurements of Dry and Hydrated MEA Polymer Substrates. The water in air CAs (WCA) of the F-PMEAs (sessile drop method) and the air bubble in water CAs (ACA) of the polymers (captive bubble method) are listed in Table 3. The results for the WCA show change of the sessile drop with time to a different extent. Clearly, three kinds of samples are observed. The first kind are the hydrophilic samples 1 and 3 with higher ACA at the air− water interface, where additionally, the WCA decreases in the beginning, and at 30 s, it levels up. As these samples are highly mobile, the interface between water and the polymer changes fast. The second kind are the hydrophobic samples. Including MEA in a block copolymer structure 2 with a short block of PTFEMA lowers the ACA and increases the WCA of 2. The

Figure 2. Time dependence of WCA onto the surface of 1−4 and PET. The data represent the means ± SD (n = 3).

From the data in Figure 2 and Table 3, it is seen that polymers 1, 2, and 4 act as more hydrophobic polymers than PMEA immediately after the droplet of water is put, which is understandable as the fluorine block having low surface energy is segregated at the air−polymer interface. However, it takes time to stabilize the shape of 2. The reason for this is seen in the low mobility of the PTFEMA partTg is above room temperature and the water content is low. On the contrary, the high mobility of the fluorine part in 1 makes the polymer appear hydrophobic at the beginning, but the picture changes fast, and this liquid fluorinated MEA polymer has even lower WCA as compared to its nonfluorinated analogue PMEA. 3.4. Protein Adsorption, Denaturation Behavior, and Platelet Adhesion of Substrates, Coated with the FPMEAs, PMEA169, and PTFEMA71. The amount of adsorbed fibrinogen, the exposure of gamma chain of the fibrinogen, and the adhesion of platelets from human blood on the surface of PMEA and F-PMEAs are presented in Figure 3. The samples of F-PMEAs have lower platelet adhesion as compared to the substrates they are coated onPP or PET and to the 2270


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The correlation between the IWC and the biological response is shown in Figure 4. As the IWC increases, the

Figure 3. Amount of fibrinogen adsorption (a), the exposure of gamma chain of fibrinogen (b), and human platelet adhesion (c) onto the surface of 1−4 and PET using surfaces without or with priming. The data represent the means ±SD (n = 3) of fibrinogen adsorption and denaturation and ±SD (n = 11) of the platelet adhesion. *: P < 0.05, **: P < 0.01 vs PP or PET substrate without priming.

Figure 4. Correlation between the amount of IW and biological response of 1−4, and PET: fibrinogen adsorption (a), the exposure of gamma chain of fibrinogen (b), and human platelet adhesion (c). The data represent the means ± SD (n = 3) of fibrinogen adsorption and denaturation and ± SD (n = 11) of the platelet adhesion.

substrates coated with the F-PMEAs turn more blood compatible. In particular, this tendency became more prominent by priming, and the relationship between the IWC and the biological response after priming agrees well with the previous work.7 However, the platelet adhesion is not sufficiently decreased in case of the block copolymer 2. This phenomenon seems to be attributed to the low mobility of the block copolymer, already discussed before, and investigated further by AFM measurements. 3.5. AFM Measurements of Substrates, Coated with the F-PMEAs, PMEA169, and PTFEMA71. AFM measurements were performed to evaluate the interface morphology and the elastic modulus of the samples in dry and hydrated conditions. In the dry state, all samples are flat and the arithmetical mean height (Sa) is around 5 nm. After priming,

PTFEMA71 4. Some of the samples have even lower adhesion than PMEA169 3. Priming helps the hydrophobic block copolymer samples to reorient their PMEA block to the water−polymer interface, thus decreasing the platelet adhesion and protein adsorption. Figure S13 shows the dependence of the amount of adsorbed fibrinogen from the priming time. Increasing the priming time of the F-PMEAs and PMEA samples up to 30 min decreases the protein adsorption. Longer priming times do not affect the amount of protein adsorption. Similar result was obtained in previous investigations by using a film of a PMEA−PMMA blend.32 The SEM photos visually show the adhered platelets on the F-PMEAs. They are presented in Figure S14. 2271


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Figure 5. AFM topographic images of 2, PTFEMA4-b-PMEA27/PBS interfaces, in dry and hydrated state (10, 30, and 60 min after dropping the PBS).

only the PTFEMA71 sample 4 kept its flat structure, whereas the other samples were gradually forming periodic structures because of the microphase separation into polymer-rich and water-rich domains.33 1, F15-b-PMEA36, and 3, PMEA169, exhibit a similar tendency, and Sa changes to about 10 nm (Figures S15 and S16). As there is no big difference between priming for 10 and 30 min, it is accepted that the morphology changes saturate within 10 minutes. Particularly large phase separation demonstrates the block copolymer 2, PTFEMA4-bPMEA27. To investigate the reason for the appearance of these structures, the time dependence of the surface structure after the PBS droplet was put is evaluated (Figure 5). With an increase in the hydration of the surface with PBS, it can be seen that the protrusions of 2, PTFEMA4-b-PMEA27, gradually increase at the polymer−PBS interface. This suggests that the microphase separation due to surface hydration is progressively developing. The results for Sa and the elastic modulus of convex and flat portions before and after immersion for 60 min in PBS are presented in Table 4.

for 16 h using vacuum of 20 Pa during freezing. To compare the amount of the elements on the surfaces with and without priming, XPS analysis was conducted on the samples without freeze drying as a control too. To acquire the outer and inner compositions of the polymer-coated substrates, two different degrees of the take-off angle for photoelectron were used15 and 45°in the dry and hydrated states. The XPS spectra of block copolymer 2, PTFEMA4-bPMEA27, are presented in Figure 6. The calculated F1s/O1s area

Table 4. Elastic Modulus in PBS onto the F-PMEAs, PMEA169, and PTFEMA71-Coated Substrates Sa (nm) air

elastic modulus (MPa)

in PBS

air

in PBS

polymers

-

-

flat portions

convex portions

flat portions

1 2 3 4

4.6 2.4 4.5 4.0

7.1 52.9 6.9 6.1

1000∼ 100∼ 1000∼ 1000∼

100−1000 1−10 100−1000 1000∼

100−1000 1000∼ 100−1000 1000∼

Figure 6. XPS wide spectra of the substrate surface coated with 2, PTFEMA4-b-PMEA27, at degree of the take-off angle for photoelectron (a) 45° at the dried state, (b) 45° at the hydrated state, (c) 15° at the dried state, and (d) 15° at the hydrated state.

By immersing 2, PTFEMA4-b-PMEA27-coated substrate, in PBS, the surface is continuously hydratedphase separation occurs to form a nanoscale structure. The elastic modulus greatly differs between the convex and the flat portions. From the results of the other elastic modulus, listed in Table 4, we assume that the soft protrusions are from the PMEA block, where the hard flat portion is that of the phase-separated PTFEMA block. The reason why the amount of adhered platelets did not decrease even after priming is considered that the PTFEMA block with high Tg suppresses the reconstruction of the PMEA block on the polymer−PBS interface, and as a result, the PTFEMA block part functioned as a scaffold for the blood cells. 3.6. XPS Measurements of Substrates, Coated with the F-PMEAs, PMEA169, and PTFEMA71. To confirm the AFM results, surface analysis in the hydrated state was carried out by freeze drying of spin-coated substrate samples. For this reason, the samples were immersed in water for 1 h before decompression at room temperature and vacuumed at −30 °C

ratio of this sample was only 0.13, whereas more fluorine was detected on the surface despite being without or with freeze drying. These ratios were 0.73 and 0.52 at 45° of take-off angle for photoelectron. Moreover, this phenomenon is more remarkable when the detection depth is shallow at 15° of detection angle. This result indicates that more fluorine is aggregated closer to the surface. On the other hand, less fluorine was detected by freeze drying at both detection angles. These XPS results indicated that the PTFEMA block was segregated at the top surface because of its high hydrophobicity and was easily exchanged with the PMEA block by priming the substrate with water. The wide spectra and atomic ratio for other samples are summarized in Figure S17 and Table S1, respectively. At the surface coated by poly(perfluoroalkyl acrylate)s, low values of F1s/C1s and high values of O1s/C1s are probably derived from the reorientation of fluoroalkyl groups and the 2272


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Figure 7. Conceptual images of F-PMEAs in dry and hydrated conditions that suppress fibrinogen: (a) solid F-PMEA 2 (PTFEMA4-b-PMEA27) and (b) liquid F-PMEA 1 (F15-b-PMEA36).

exposure of carbonyl groups to the water interface.29 As shown in Table S1, polymer 1, F15-b-PMEA36, shows low values of F1s/C1s and high values of O1s/C1s; therefore, the exposure of carbonyl groups to the water interface is assumed. Furthermore, in this case, because the mobility of the fluorine being oriented is also different, it is considered that the above effect can be further accelerated. Block copolymer 2, PTFEMA4-b-PMEA27, has high Tg part, so it is possible to keep the wet state orientation during freeze drying and in the XPS equipment. These results confirm the AFM results and indicate that more fluorine is aggregated closer to the surface. However, for polymer 1, less changes were observed without and with freeze drying than for block copolymer 2. It is considered that the reason for this is that the PMEA block aggregated closer to the surface during immersing in water was exchanged again to the fluorine block during the freeze drying or the XPS measurement vacuum process because of the low Tg of the fluorine block and its high mobility. 3.7. Relationship between the Bulk or the Surface Hydration Structure and the Biological Response. From the above results, the relationship between the bulk hydration structure or the hydration dynamics of the surface and the biological response is interpreted as follows. 3.7.1. Bulk Structure. Introducing small fluorine molecules from, for example, fluorinated initiators into PMEA changed the interaction of 1, F15-b-PMEA36, with water. This was substantiated by the crystallization of water observed even during the cooling process. On the contrary, by inserting short TFEMA block into block copolymer structures with MEA, no crystallization of water or an increase in the water content was observed during the cooling process. 3.7.2. Surface structure. The WCA of fluorinated block copolymer 2 where solid fluorinated block is introduced continuously decreases with time, whereas the WCA of 1, F15b-PMEA36, is already after 30 s lower than that of pure PMEA169, 3. The reason for this is seen in the fast interface reorientation of the flexible and with weak cohesion polymer 1 to the water−polymer interface. The introduction of fluorine in polymer 1 helps to promote the surface hydrophilization. The reason why this polymer 1 has a smaller amount of adsorbed proteins or platelets than 3 is that the surface was made hydrophilic before the protein adsorption could happen by hydrophobic interactions (Figure 7). This reveals that if FPMEA is spontaneously and efficiently oriented to the water interface, it will be a better protein-repelling material than pure PMEA. Small amounts of fluorine introduced herein into PMEAs by using a fluorinated macroinitiator contributed to the surface hydration dynamics. Figure 7 presents the suggested differences of F-PMEAs in the dry and hydrated states that suppress the adsorption of fibrinogen and platelets. These results show that by considering not only the amount but also the mobility of the introduced fluorine in F-PMEAs, the macromolecular flexibility and the water content are

increasing, efficiently reorienting the hydration layer at the water−polymer interface, thus achieving a material with better blood compatibility.

4. CONCLUSIONS We have synthesized MEA polymers containing small amounts of fluorine by ATRP employing fluorinated macroinitiators, having 15 fluorine atoms either in a bromoester structure or in a short block of the fluoromonomer TFEMA, appearing liquid and solid at room temperature, respectively. Inserting fluorine changes the hydration behavior of the prepared F-PMEAs as evidenced by DSC measurements in the dry and hydrated states. Small amounts of fluorinated molecules were found to play an important role not only in the bulk but also in the surface structure. The high mobility of F15-b-PMEA36, its greater IWC, and the dynamic change of the surface structure detected by CA measurements are responsive for the lower fibrinogen adsorption as compared to not fluorinated PMEA and for the low platelet adhesion of this polymer. Block copolymer 2, PTFEMA4-b-PMEA27, with the same amount of fluorine atoms as in 1, F15-b-PMEA36, shows low mobility because of its high Tg and lower water content. Accordingly, its platelet adhesion is not sufficiently changed. AFM measurements of both coatings in the hydrated state display phase separation to nanoscale structures in water-poor (polymerrich) and water-rich domains with different sizes. Acquired data for the elastic modulus suggest that the flat part of PTFEMA4-b-PMEA27 consists of the phase-separated fluorinated block functioning as a scaffold for the blood cells, whereas both convex and flat portions of F15-b-PMEA36 were made of soft contacts of PMEA. Performed XPS analyses show the abundance of fluorine close to the surface and exposure of more oxygen species after hydration (freeze drying). The possibility of minimizing biofouling with F-PMEA was realized by the rapid switching of the fluorine block to the PMEA block, which has higher IWC because of the small amount of high mobility fluorine at the water interface. These results suggest opportunity for further improvement of the biocompatibility by the introduction of a more flexible fluorine as, for example, perfluoropolyether in a small amount in a block copolymer structure with MEA.34 Controlling appropriately the hydration structure of polymers by introducing small amounts of fluorine high blood compatibility can also be imparted into other materials. Such investigations are under way.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.9b00201. 2273


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Experimental details, detailed characterization data, DSC profiles, SEM and additional AFM images, and XPS and NMR spectra for the discussed polymers (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Ryohei Koguchi: 0000-0003-0017-4783 Masaru Tanaka: 0000-0002-1115-2080 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Project PROGRESS 100 from Kyushu University is acknowledged for financial support. This work was partially supported by Dynamic Alliance for Open Innovation Bridging Human, Environment and Materials. The authors are grateful to Dr. Shingo Kobayashi for fruitful discussions about DSC in hydrated conditions and other characterization methods and Dr. Daiki Murakami for surface analysis with AFM.

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REFERENCES

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