Controlling the hydration structure with small amount of fluorine to

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Controlling the hydration structure with small amount of fluorine to produce blood compatible fluorinated poly(2-methoxyethyl acrylate) Ryohei Koguchi, Katja Jankova, Noriko Tanabe, Yosuke Amino, Yuki Hayasaka, Daisuke Kabayashi, Tatsuya Miyajima, Kyoko Yamamoto, and Masaru Tanaka Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.9b00201 • Publication Date (Web): 01 May 2019 Downloaded from http://pubs.acs.org on May 1, 2019

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Controlling the hydration structure with small amount of fluorine to produce blood compatible fluorinated poly(2-methoxyethyl acrylate) Ryohei Koguchi,1,2 Katja Jankova,1,3 Noriko Tanabe,4 Yosuke Amino,4 Yuki Hayasaka,4 Daisuke Kobayashi,4 Tatsuya Miyajima, 4 Kyoko Yamamoto,2 Masaru Tanaka1,**

1 Soft

Materials Chemistry, Institute for Materials Chemistry and Engineering, Kyushu

University, Build. CE41, 744 Motooka Nishi-ku, Fukuoka, 819-0395, Japan

2 AGC

Inc. New Product R&D Center, 1150 Hazawa-cho, Kanagawa-ku, Yokohama,

Kanagawa 221-8755, Japan

3

Department of Energy Conversion and Storage, Technical University of Denmark,

Elektrovej, Build. 375, 2800 Kongens Lyngby, Denmark

4 AGC

Inc. Innovative Technology Research Center, 1150 Hazawa-cho, Kanagawa-ku,

Yokohama, Kanagawa 221-8755, Japan

ACS Paragon Plus Environment

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KEYWORDS Atom transfer radical polymerization (ATRP), intermediate water, blood compatibility, poly(2-methoxyethyl acrylate), fluorinated polymers, block copolymers


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Abstract: Poly(2-methoxyethyl acrylate) (PMEA) shows excellent blood compatibility due to 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 to manipulate 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 kind 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-1-octanol (F15) and the 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, that couldn’t be explained only by the bulk hydration structure. Both polymers show nanostructured morphology in hydrated state with different sizes of the features. The measured elastic modulus of the domains appearing in atomic force microscopy (AFM) and the intermediate water content shine light on the distinct mechanism of blood compatibility. Contact angle measurements


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reveal the surface hydration dynamics - while in hydrated state the 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.


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Introduction Water molecules play an important role on the interface between polymers and biological systems. In hydrated polymers1-4 water is found to exist in 3 states: free water (FW), intermediate water (IW) and non-freezing water (NFW). As in the cited order the interactions with the polymer increase, 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 DSC cooling scan (-40 oC) below the freezing of FW (25 oC), and above NFW, which actually was not found yet to freeze at all even until -100 oC.

IW can appear also in the heating scan – first CC appears, then it melts just below

that of FW (0 oC), 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.


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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, like platelet denaturation and activation, plasma coagulation, leucocyte interaction leading to inflammation and cell depletion, thrombosis, hemolysis, cancer, toxic or allergic reactions, etc. 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 bio compatible 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 due to 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 bio compatible polymers


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(polyethylene glycol (PEG), poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC), polyvinylpyrrolidone (PVP) etc.) were explained with the concept of IW too.

There are different ways to change and tune the IWC of materials. Synthesizing multiple homopolymer analogs of MEA polymers7-8 containing more than one ethylene oxide unit in the side chain, or different substituents – methyl-, ethyl-, 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 analog of PMEA, the poly(2-methoxyethyl methacrylate) (PMEMA) shows much lower IWC than PMEA, and was according to this found less blood compatible.9 Another number of PMEA analogs have been investigated for the existence of IW, and one of those poly(ω-methoxyalkyl acrylate)s, the poly(methoxypropyl acrylate) was found to possess even more IW, and thus better blood compatibility than PMEA.3 Its


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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 PHEMA there was less sign of IW. 2,11-12 By evaluation of the water structure of random2, block11 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 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 amino containing monomers resulted in loosening of the H-bonding network to form soft biological surface where IW predominates and in increasing 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


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backbone or of the side chains in the hydrated state, which can happen by at least the mentioned above 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 analogs, herein the electronegative and hydrophobic fluorine. Even nature is implementing 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 PS15 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 due to better mobility. Furthermore, by using fluorinated initiators for the free-radical polymerization of various vinyl monomers, many telechelic polymers have been


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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 was 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 bio-acceptable 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 analogs, 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 reveal 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 of these groups on the surface resulted in better foaling-release performance.


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However, when other hydrophilic monomers, e.g. 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 to exhibit a minimum value. This was also the result from the AFM investigations of the copolymer mPEG-bpolyacrylicacid-b-PMMA-b-PMFA, when the mPEG part was exposed to water.26 It was also reported that the receding contact angle decreases by copolymerization of small amounts of FMA with MMA and methoxy polyethylene glycol 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 fluorine containing initiator for the ATRP of MEA.28 A short chain of a monomer bearing fluorine was inserted in a block copolymer structure with PMEA15 too. We compare here the water structure, platelet adhesion and protein adsorption of these fluorinated MEA polymers (F-


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PMEAs) with virgin surfaces of PET and PP. PMEA is further synthesized by free radical polymerization and included in the investigations for comparison.

Experimental section Experimental Part

1. Materials

2,2,3,3,4,4,5,5,6,6,7,7,8,8-pentadecafluoro-1-octanol dimethylaminopyridine

(DMAP),

bromoisobutyrylbromide

(F15-OH), (BIBB),

ethyl

2-

bromoisobutyrate (EBIB) (all from Sigma-Aldrich) were used as received without further purification. The MEA monomer (Sigma-Aldrich) was passed through a ready to use, disposable pre-packed inhibitor remover column. 2,2,2-Trifluoroethyl methacrylate (TFEMA) and N,N,N',N'',N''-pentamethyldiethylenetriamine (PMDETA) were purchased from TCI Chemicals, and deoxygentated by three pump-thaw cycles. TFEMA was passed through a column of activated neutral Al2O3 before use to remove the inhibitor. All other


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solvents or chemicals were used as received unless otherwise stated. Phosphatebuffered saline (PBS) was purchased from FUJIFILM Wako Pure Chemical Corporation.

2. Synthesis and characterization of fluorinated (macro) initiators:

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 the F15-OH with BIBB: 3 g (7.49 mmol) of F15-OH and 60 mL of dry 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 DMAP recrystallized from toluene and 1.2 mL (9.44 mmol) 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, 1M HCl and finally with distilled water. The ether layer was


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dried over Na2SO4 and the solvent was removed on a rotary evaporator to leave a yellowish liquid. Yield: 97%.

1H-NMR

19F-NMR

(DMSO-d6), δ (ppm): 1.9 (s, 6H, 2 -C(CH3)2), 4.99 (t, 2H, -O-CH2).

(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).

Fluorinated macroinitiator PTFEMA4-Br: 8.6 mL (60 mmol) TFEMA, 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 glove box. 430 mg (3.0 mmol) of Cu(I)Br, 447 μL (3.0 mmol) of EBIB and 627 μL (3.0 mmol) of PMDETA were added and the reaction was run at 800C for 20 minutes. After precipitation in hexane PTFEMA4Br (1.70 g, 16% yield) was obtained as light green solids.

1H-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).

19F-NMR

(CDCl3+1,3-(CF3)2C6H4) δ (ppm): -62.1 (1,3-(CF3)2C6H4), -72.4 (-CF3)


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3. Synthesis and characterization of fluorinated (block co)polymers:

F15-b-PMEA36 (1). In a typical experiment28 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, 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 the polymerization was initiated by immersing the tube in a preheated oil bath at 90 C for 3.5 hours. The viscous polymer was diluted with THF; the solution 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%).

1H-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).

19F-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).


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PTFEMA4-b-PMEA27 (2). Block copolymer was synthesized in a pressure glass vessel, placed in a glove box in the beginning for loading all the components. The vessel was equipped with a magnetic stirrer and sealed with 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 21μL (0.1mmol) of PMDETA were added. The glass vessel was sealed with the metal lid, and the reaction was performed at 80 oC for 18 hours. 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., the Al2O3 was filtered off, and the filtrate was precipitated in hexane.

1H-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).

19F-NMR

(CDCl3+1,3-(CF3)2C6H4) δ (ppm): -61.2 (1,3-(CF3)2C6H4), -72.4 (-CF3),

4. Synthesis and characterization of the homopolymers:


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Synthesis of PMEA169 (3). PMEA was synthesized by free radical polymerization using azobis(isobutyronitrile) as the initiator in 1,4-dioxane at 75 oC for 6 hours.9 The polymer was purified by precipitation in THF/hexane mixture, collected and dried under reduced pressure at 60 oC for one day. 1H-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).

Synthesis of PTFEMA71 (4). TFEMA, 21.4 mL (150mmol), and 30.0 mL of toluene were charged to a pressure glass vessel, 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 oC for 13 hours. PTFEMA (16.5 g, 66% yield) was obtained as a white solid by precipitation of the toluene solution in ethanol.

1H-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).

19F-NMR

(CDCl3+1,3-(CF3)2C6H4) δ (ppm): -62.1 (1,3-(CF3)2C6H4), -72.4 (-CF3)


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5. Analyses.

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 TMS, whereas for 19FNMR the assignments are given downfield from 1,3-(CF3)2C6H4. Molecular weights were determined by Size-Exclusion 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 oC with a 0.35 mL/min flow. Molecular weights were calculated using PS narrow molecular weight standards in the range of 5 · 102 – 5.48 · 106 g/mol.


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Differential Scanning Calorimetry (DSC) measurements were performed using Q1000 from TA Instruments in a temperature range of -80 to 100 oC at a heating rate of 5 oC min-1 under nitrogen. Tg was determined automatically by the instrument from the second heating trace and is reported as the midpoint of the thermal transition.

5-2) Quantification of the interactions between water and polymers. From the interactions of water with the particular polymer, we classify 3 kinds of water in hydrated macromolecules: NFW, IW and FW, their content is 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 oC and a small shoulder or peak for melting of IW below this temperature appears, and is given by the following Equation (1):

EWC (wt%) = ((W1-W0) / W1)×100 (1)


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where W0 and W1 are the weight of the dry and hydrated sample, 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(Jg-1) (3)

FW (wt%) = (ΔHm / 334(Jg-1)) - IW (4)

ΔHcc and ΔHm are enthalpy changes during cold crystallization and the melting of ice, respectively. The weight of the samples was 3 to 7 mg. The hydrated samples were prepared by immersing in ultrapure water for 7 days before measurements.

5-3) Preparation and characterization of the polymer surfaces.


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Film samples were prepared as follows and used for contact angles (CAs), atomic force microscopy (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 MSA100 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 towards the airside of the polymer films with an accuracy of  2 were measured at 25 oC by the sessile drop method. The captive bubble method is a special arrangement for measuring the contact angle between a liquid and a solid using drop shape analysis. 2μL of air bubble 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


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HQ-75-Au was used (spring constant k = 2.5 N/m, resonance frequency f =75 kHz in air, tip radius

Controlling the hydration structure with small amount of fluorine to

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