Synthesis and Thrombogenicity Evaluation of Poly (3

Kazuhiro Sato†, Shingo Kobayashi‡ , Asuka Sekishita†, Miyuki Wakui†, and Masaru Tanaka‡. † Department of Biochemical Engineering, Graduate...
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Synthesis and Thrombogenicity Evaluation of Poly(3methoxypropionic acid vinyl ester): A Candidate for BloodCompatible Polymers Kazuhiro Sato,† Shingo Kobayashi,*,‡ Asuka Sekishita,† Miyuki Wakui,† and Masaru Tanaka*,‡ †

Department of Biochemical Engineering, Graduate School of Science and Engineering, Yamagata University, 4-3-16 Jonan, Yonezawa, Yamagata 992-8510, Japan ‡ Institute for Materials Chemistry and Engineering, Kyushu University, CE41 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan Biomacromolecules 2017.18:1609-1616. Downloaded from pubs.acs.org by UNIV OF WINNIPEG on 11/23/18. For personal use only.

S Supporting Information *

ABSTRACT: A poly(vinyl acetate) derivative, poly(3-methoxypropionic acid vinyl ester) (PMePVE), was synthesized to develop a new candidate for blood compatible polymers. The monomer MePVE was synthesized by a simple twostep reaction, and then the MePVE was polymerized via free radical polymerization to obtain PMePVE. Human platelet adhesion tests were performed to evaluate the thrombogenicity, and the platelet adhesion was suppressed on the PMePVE-coated substrate. To determine the expression of the nonthrombogenicity of the PMePVE, the plasma protein adsorption and a conformationally altered state of fibrinogen were analyzed by a microBCA assay and enzyme-linked immunosorbent assay. The adsorption and denaturation of the plasma proteins were inhibited on the PMePVE; thus, PMePVE exhibited blood compatibility. A distinctive hydration water structure in the nonthrombogenic polymer, intermediate water (IW), was observed in the hydrated PMePVE by differential scanning calorimetry analysis. The nonthrombogenicity of PMePVE is considered to be brought about by the presence of IW.

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INTRODUCTION Blood compatible polymers have been widely studied in the biomedical field for the development of blood-contacting medical devices such as artificial organs and drug delivery carriers.1−4 When a polymer material comes in contact with blood, various biological responses are evoked on the polymer surface. Among the responses, thrombus formation is the significant problem. Although blood clotting is a vital part of hemostasis to stop bleeding and fibrin clots act as a scaffold for repairing the injured blood vessel, the blood coagulation and the clot formation on the blood-contacting polymer surface lead to thrombosis. In fact, all blood-contacting medical devices still have the drawback of thrombus formation.5 Therefore, nonthrombogenicity is the most important characteristic and the minimum requirement for blood compatible polymeric materials. Because platelet adherence onto polymer surfaces, the main step toward thrombus formation, is mediated by the denatured adsorbed protein, inhibiting the adsorption or denaturation of the plasma proteins is one of the simplest methods for suppressing the thrombogenicity. Consequently, polymers with excellent nonthrombogenicity should be developed with a focus on the protein adhesion/denaturation behavior at the surface of the polymeric materials. In general, poly(ethylene glycol) (PEG) and zwitterionic polymers are polymers that are well-known to exhibit nonthrombogenicity.6−8 Nagasaki et al. reported that material surfaces coated with diblock copolymers of PEG-b-poly(lactide) (PLA) suppressed protein adsorption because PEG © 2017 American Chemical Society

has a low interfacial free energy and a high chain mobility in aqueous medium. These properties of PEG produce a large exclusion volume, and thus, PEG-b-PLA exhibits a high resistance toward protein adsorption.9,10 Ishihara et al. reported that poly(2-methacryloyloxyethyl phosphorylcholine0.3-co-nbutyl methacrylate0.7) (PMPC) exhibited excellent nonthrombogenicity when a PMPC-coated surface came in contact with human whole blood.11 A cell membrane-like surface was formed on the PMPC surface due to the adsorption of phospholipids from the blood, thus suppressing platelet adhesion on the PMPC surface. In addition, Zhang et al. reported that polymer brushes composed of poly(sulfobetaine methacrylate) (PolySBMA) or poly(carboxybetaine methacrylate) (PCBMA) showed a high resistance toward fibrinogen or plasma protein adsorption; these polymer brushes exhibited low platelet adhesion.12 However, generally, these zwitterionic polymers were water-soluble. Therefore, their homopolymers could not be used as the coating material without covalently fixing them on the material surfaces or using them as waterinsoluble copolymers that were modified with hydrophobic monomers. Poly(vinyl acetate) (PVAc) is a commonly used polymer, similar to polyacrylates, that can serve as an adhesive, a binder, and a biomedical material. For example, it has been used as a Received: February 12, 2017 Revised: April 7, 2017 Published: April 10, 2017 1609

DOI: 10.1021/acs.biomac.7b00221 Biomacromolecules 2017, 18, 1609−1616

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Biomacromolecules

acrylate) (PMA, Mn = 29 kg/mol, Mw/Mn = 2.5) was prepared in the same manner as PMEA. Poly(n-butyl methacrylate70-co-2methacryloyloxyethyl phosphorylcholine30) (PMPC, Mw = 600 kg/ mol) was kindly donated from NOF Corporation. Poly(n-butyl acrylate) (PBuA, Mn = 73 kg/mol, Mw/Mn = 2.2) was purchased from Scientific Polymer Product, Inc. and was purified by precipitation in methanol. Human whole blood was purchased from Tennessee Blood Services Corporation. Synthesis of 3-Methoxypropionic acid vinyl ester (MePVE). An aqueous solution of 20% NaOH (40.1 g, 1060 mmol) was slowly added to methyl 3-methoxypropionate (118.2 g, 1000 mmol) in a three-neck round-bottom flask cooled with a water-ice bath, and then the mixture was stirred for 31 h at reflux temperature. The reaction mixture was cooled down to room temperature, and hydrochloric acid (100 mL) was added to acidify the reaction mixture. The reaction mixture was extracted by dichloromethane three times. The organic layer was dried over anhydrous Na2SO4. After the solution was concentrated, the resulting crude product was purified by fractional vacuum distillation, which resulted in 3-methoxypropionic acid (75.9 g, 729 mmol, 73% yield, bp = 85−90 °C at 5 mmHg) as a colorless liquid (see Supporting Information Figure S1). 1 H NMR (500 MHz, CDCl3): δ = 2.6 (t, J = 11.3 Hz, 2H, −CH2− CH2−O−CH3), 3.3 (s, 3H, O−CH3), 3.6 (t, J = 6.3 Hz, 2H, −CH2− O−CH3), and 11.7 (br, 1H, −COO−OH). MePVE was synthesized via transesterification according to a literature procedure reported by Alloua et al.20 3-Methoxypropionic acid (36.8 g, 350 mmol), KOH (1.78 g, 31.7 mmol), and palladium(II) acetate (223 mg, 1.0 mmol) were added to vinyl acetate (268 g, 3100 mmol) in a three-neck round-bottom flask, and then the mixture was stirred for 20 h at 60 °C. After the reaction completed, 5% NaOH aqueous solution was added to the reaction mixture, and the reaction mixture was extracted by dichloromethane three times. The organic layer was dried over anhydrous K2CO3. The organic layer was roughly purified by passing through a column of silica gel and neutral aluminum oxide. After the solution was concentrated, the resulting crude product was purified by fractional vacuum distillation over CaH2, which resulted in MePVE (20.7 g, 159 mmol, 51% yield, bp = 70−75 °C at 5.6 mmHg) as a colorless liquid (1H NMR spectrum showed in Supporting Information Figure S2). 1 H NMR (500 MHz, CDCl3) δ = 2.6 (t, J = 6.3 Hz, 2H, −CH2− CH2−O−CH3), 3.3 (s, 3H, O−CH3), 3.7 (t, J = 6.5 Hz, 2H, −CH2− O−CH3), 4.6 (dd, J = 6.0, 1.5 Hz, 1H, CH2CH−), 4.9 (dd, J = 14, 1.5 Hz, 1H, CH2CH−), and 7.3 (dd, J = 14, 6.0 Hz, 1H, CH2 CH−). Synthesis of Poly(3-methoxypropionic acid vinyl ester) (PMePVE). PMePVE was synthesized according to a literature procedure reported by Hatada et al.21 A monomer solution of MePVE and AIBN initiator were added to benzene or ethyl acetate in a three-neck round-bottom flask. The solution was stirred at 75 °C for 20 h under N2 gas flow. The polymer was purified by precipitation three times using THF/hexane as the good/bad solvent pair system and then purified by stirring in distilled water for 24 h to remove water-soluble impurities. After purification, the polymer was collected and dried under reduced pressure at 60 °C for 23 h (1H NMR is presented in Supporting Information Figure S3). 1 H NMR (500 MHz, CDCl3) δ = 1.5−2.0 (br, polymer backbone), 2.5 (br, 2H, −CH2−CH2−O−CH3), 3.3 (s, 3H, −CH2−CH2−O− CH3), 3.5 (br, 2H, −CH2−CH2−O−CH3), and 4.8−5.0 (br, polymer backbone). Polymer Characterization. The chemical structures of these polymers were characterized by 1H NMR spectroscopy (JEOL ECX 500 MHz). The number-average molecular weights (Mn) and molecular weight distributions (Mw/Mn) of the PMEA analogues were determined by conducting gel permeation chromatography using a Tosoh HPLC HLC-8220 system equipped with a refractive index and ultraviolet detector at 40 °C. The column set, four consecutive columns (Tosho TSK-GELs (bead size, exclusion-limited molecular weight)), for GPC was as follows: G4000HXL (5 μm, 4 × 105), G3000HXL (5 μm, 6 × 104), G2000HXL (5 μm, 1 × 104), 30 cm each, and a guard column (TSK-guard column HXL-L, 4 cm). The

carrier for drugs and cells, and scaffold for tissue engineering.13−16 In addition, PVAc has a number of features, such as thermal stability and melt processability.17 Accordingly, there are many opportunities for the further development of PVAc and its derivatives for applications in the biomedical field as coating materials, similar to the case of polyacrylates. However, in spite of the versatility of PVAc, the homopolymer itself does not possess nonthrombogenicity. Hence, this polymer has to be modified to improve its nonthrombogenicity for application as a blood compatible polymer. We have reported that poly(2-methoxyethyl acrylate) (PMEA) shows excellent blood compatibility, even though this polymer has a simple chemical structure.18 PMEA is approved by the Food and Drug Administration (FDA) for medical use. Moreover, PMEA has the largest market share worldwide as an antithrombogenic coating agent for artificial oxygenators. However, the performance against long-term use is not yet sufficient and further improvement to maintain nonthrombogenicity over a long-term is demanded. Therefore, considering these facts, we focused on the chemical structure of PMEA to improve the nonthrombogenicity of PVAc. The chemical structure of PMEA is composed of an acrylate backbone and monomethoxy terminated ethylene glycol side chain. If the placement of the ester unit in the PMEA framework is inverted, the polymer becomes a poly(vinyl acetate) derivative, poly(3-methoxypropionic acid vinyl ester) (PMePVE, Figure 1). We predicted that this polymer,

Figure 1. Chemical structures of poly(2-methoxyethyl acrylate) (PMEA) and poly(3-methoxypropionic acid vinyl ester) (PMePVE).

composed of a vinyl ester backbone and methoxy-terminated hydroxy acid side chain, could have great potential as a coating material with excellent nonthrombogenicity. In this work, we synthesized PMePVE as a new candidate exhibiting excellent nonthrombogenicity. The nonthrombogenicity characteristic of the synthesized polymer was evaluated by performing human platelet adhesion tests. In addition, the adsorption and conformational change of plasma proteins on PMePVE were estimated by using a microBCA assay and enzyme-linked immunosorbent assay (ELISA) to evaluate the nonthrombogenicity of PMePVE. Furthermore, the water structures within hydrated PMePVE were analyzed by differential scanning calorimetry (DSC) measurements.

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EXPERIMENTAL SECTION

Materials. All reagents were purchased from commercial sources and were used as received unless otherwise specified. 3-Methoxypropionic acid methyl ester and vinyl acetate were purchased from Tokyo Chemical Industry. Palladium(II) acetate and bovine serum fibrinogen (FNG) were purchased from Sigma-Aldrich. Potassium hydroxide (KOH), sodium hydroxide (NaOH), sodium carbonate (anhydrous) (Na2CO3), and potassium carbonate (anhydrous) (K2CO3) were purchased from Wako Pure Chemical Industries. Benzene, ethyl acetate, n-hexane, tetrahydrofuran (THF), and 2,2′azobis(isobutyronitrile) (AIBN) were purchased from Kanto Chemical Co., Inc. PMEA (Mn = 22 kg/mol, Mw/Mn = 2.8) was synthesized according to the previously reported procedure.19 Poly(methyl 1610

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Biomacromolecules Scheme 1. Synthesis of PMePVE

Table 1. PMePVE Prepared by Free Radical Polymerization with Various Conditions

a

entry

solvent

M/I [-]

[M]0 [mol/L]

time [h]

conv. [-]a

Mn [kg/mol]b

Mw/Mn [-]b

1 2 3 4

benzene benzene ethyl acetate ethyl acetate

1:0.03 1:0.02 1:0.02 1:0.02

1.65 3.45 2.50 5.00

20 20 18 23

64 71 83 81

7.4 10.8 9.9 14.0

1.6 2.1 1.9 2.6

Determined by 1H NMR spectroscopy. bDetermined by SEC using polystyrene standards in THF at 40 °C.

system was operated at a flow rate of 1.0 mL/min using THF as the eluent. Polystyrene standards were employed for calibration. Preparation of Polymer-Coated Substrates. The polymercoated substrates were prepared by using spin coating method. PMePVE, PMEA, and PMPC were dissolved in methanol, and PVAc and PBuA were dissolved in THF; all polymer solutions were prepared at 0.2 wt/vol %. The polymer solution was spin coated twice onto PET substrates (ϕ = 14 mm, thickness =125 μm) using a Mikasa Spin Coater MS-A100 at the consecutive rates of 500 rpm for 5 s, 2000 rpm for 10 s, a ramp up for 5 s, 4000 rpm for 5 s, and a ramp down for 4 s and then dried. The water contact angle was measured on the polymer-coated substrates using the sessile drop method by placing a 2 μL water droplet. Human Platelet Adhesion Test. The human platelet adhesion tests were performed according to a previously reported procedure.19 Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were obtained from human whole blood by centrifugation, and then a plasma solution containing 4 × 107 cells/cm2 of platelets was prepared by mixing the PRP and PPP solutions. The plasma solution (200 μL) was placed on each polymer substrate, and the substrate was incubated for 1 h at 37 °C. After 1 h, each polymer substrate was rinsed with PBS, and then the platelets adhered on polymer substrates were fixed by immersion in 1% glutaraldehyde in PBS for 120 min at 37 °C. Finally, each polymer substrate was rinsed with PBS and Milli-Q-water. The platelet adhesion number was evaluated by using scanning electron microscopy (SEM). Quantification of Protein Adsorption on Polymer Surface. The amount of adsorbed proteins was evaluated by conducting a microBCA assay according to a previously reported method.19 All polymer solutions were casted to 96-well polypropylene (PP) plates. The plates were slowly air-dried over 3 days at room temperature. PPP was added to each well and incubated for 60 min at 37 °C. After incubation, each well was rinsed with PBS. The adsorbed proteins were extracted by incubation with a solution of 5% sodium dodecyl sulfate (SDS) and 0.1 N NaOH for 60 min at 37 °C. The extracted proteins were assessed by conducting a microBCA assay (Thermo Scientific, Rockford, IL). Evaluation of Conformationally Altered Fibrinogen after Enzyme-Linked Immunosorbent Assay (ELISA). The conformationally altered fibrinogen on the polymer surface was evaluated by using a previously reported procedure.19 PPP was added to each well and incubated for 60 min at 37 °C. Conformationally altered fibrinogen was evaluated by using antifibrinogen γ′ antibody (Ab) (Millipore) as a primary Ab and peroxidase-conjugated antimouse IgG Ab as a secondary Ab. The secondary Ab was detected with a 2, 2′azinobis(3-ethylbenzothiazoline-6-sulfonic acid ammonium salt) (ABTS) substrate (Roche Diagnostics). The absorbance was measured at a wavelength of 405 nm by using a plate reader (BIO-RAD).

Quantification of Amount of Hydration Water. The amount of hydration water was analyzed by using a DSC (X-DSC7000, Seiko Instruments Inc.) with a previously reported method.19 Briefly, approximately 4 mg of the hydrated polymer sample was prepared, and the water content was adjusted to the respective content. The hydrated sample was placed in an aluminum pan, and the pan was sealed by an auto sealer (Seiko Instruments Inc.). The hydrated samples were cooled to −100 °C at a rate of 5.0 °C/min, held at −100 °C for 5 min, and then heated to 50 °C at the same rate under a nitrogen purge flow. The equilibrium water content (EWC) of the polymers is given by eq 1:

EWC (wt%) = (W1 − W0)/W1 × 100

(1)

where W0 and W1 are the weights of the dry sample and hydrated samples, respectively. The amounts of the different types of water in the polymers are given by the following (eq 2):

EWC (wt%) = Wnf + Wfb + Wf

(2)

where Wfb = ΔHcc/334 (J/g). Wf = (ΔHm/334 (J/g)) − Wfb where Wnf, Wfb, and Wf are amounts of nonfreezing water, intermediate water, and free water, respectively; ΔHcc and ΔHm are the enthalpy changes during cold crystallization and ice melting, respectively.

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RESULTS AND DISCUSSION Synthesis of PMePVE: PMePVE was synthesized as shown in Scheme 1. The monomer of MePVE was synthesized by a straightforward two-step reaction. First, 3-methoxypropionic acid was synthesized via hydrolysis of 3-methoxypropionic acid methyl ester in basic conditions. Second, 3-methoxypropionic acid vinyl ester was synthesized via transesterification between vinyl acetate and 3-methoxypropionic acid using Pd(II) acetate. These synthesized compounds were readily purified by extraction from the reaction mixture followed by fractional vacuum distillation. PMePVE was synthesized via free radical polymerization with AIBN as the initiator. The results of the free radical polymerization are summarized in Table 1. Radical polymerization is one of the most common and useful polymerization techniques for making polymers, and a large number of commodity polymers are industrially22 produced by the radical polymerization process. PVAc and its derivatives (e.g., poly(ethylene-co-VAc)) are also produced by the radical polymerization process; however, high levels of chain transfer reactions (hydrogen abstraction) between the growing radicals and 1611

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Biomacromolecules −CH3 in the VAc terminal groups inevitably occur, and these make the production of high molecular weight polymers rather difficult.23 In fact, very low molecular weight PVAc was obtained using our previously reported reaction conditions for the preparation of poly((meth)acrylate)s.19 The radical polymerization of VAc is strongly influenced by the reaction conditions such as the reaction solvent and initiator concentration. Thus, we examined the radical polymerization conditions in benzene or ethyl acetate for preparing the highest molecular weight PMePVE and PVAc possible. The synthesized PMePVE was characterized by NMR spectroscopy and GPC analysis. The chemical structure of the resulting product was analyzed by 1H NMR measurements, and we confirmed that PMePVE was obtained, as shown in Figure S3. From the results of GPC analysis shown in Table 1, the molecular weight of PMePVE was higher when polymerization was performed in ethyl acetate than in benzene. Notably, the molecular weight of the synthesized polymer increased with the increasing the monomer concentration in the reaction solvent. These results indicate that ethyl acetate with a high monomer concentration is a suitable condition for the polymerization of MePVE, and thus, we used PMePVE with the highest molecular weight obtained under the experimental conditions (entry 4). As a reference, PVAc (Mn = 11 kg/mol, Mw/Mn = 1.9) was obtained under the same conditions as entry 4, and we used PVAc as a control sample for the following evaluations. Hydrophilicity Evaluation of PMePVE. First, each polymer substrate was prepared by a spin-coating method. We have reported that the film thickness on PET substrate is always within 70−80 nm under the applied condition and the surface is sufficiently covered by polymer to exhibit a sufficient nonthrombogenicity.24 The hydrophilicity of the polymercoated surface was evaluated by static contact angle measurements in air or in water. The results of the static contact angle measurements are shown in Table 2. The results for PET,

that of the PMEA-coated substrate. Additionally, for the captive air bubble contact angle in water measured after immersion in water for 24 h, the PMePVE-coated surface exhibited a higher value than the PMEA surface. These static contact angle measurement results on PMePVE indicated the presence of a highly hydrophilic surface, similar to PMEA, and the hydrophilicity of PMePVE was comparable to that of the PMEA. These two polymers were composed of the same chemical composition. The PMePVE surface could potentially exhibit good blood compatibility. Regarding the comparison between PMA and PVAc, PVAc exhibited higher hydrophilicity than PMA, which was the same as the relationship between PMePVE and PMEA. Tanaka et al. reported an investigation of the local conformation of poly(methyl methacrylate) (PMMA) chains at the nitrogen gas/polymer and water/ polymer interface by using infrared-visible sum-frequency generation (SFG) spectroscopy.25 The shape of the CO peak in the SFG spectrum at the PMMA interface was dramatically changed after contact with water molecules; the peak shape became broader, and the peak was shifted from 1734 to 1714 cm−1. Tanaka et al. attributed this result to the formation of hydrogen bonds between the carbonyl groups present at the interface and water molecules. In the present study, the CO group of PMePVE moved away from the hydrophobic polymer backbone in comparison with the case of PMEA, and the molecular mobility of the CO group on the side chain may have been higher than that on the PMEA. Consequently, the CO group in PMePVE could be easily oriented toward the water phase under a water-contacting environment in comparison with that of PMEA; thus, the hydrophilicity of PMePVE was slightly higher than that of PMEA, although the two polymers have the same chemical composition. Thrombogenicity Evaluation of PMePVE. Platelet adhesion onto the polymer surface is the main step during thrombus formation, and it is the key step of the biological response evoked when the polymer surface contacts with blood.26,27 Therefore, the platelet adhesion test was performed on PMePVE to evaluate the thrombogenicity. The result of the platelet adhesion test for each polymer-coated substrate is shown in Figure 2. Bare PET and PBuA-coated PET substrates were used as the negative controls, and the PMPC-coated substrate was used as

Table 2. Contact Angles on the Polymer Surfacea captive bubble [deg] polymer PET PMPC PBuA PMA PVAc PMEA PMePVE

sessile drop (30 s) [deg] 71.4 110.1 85.5 75.6 70.1 40.0 38.3

(±1.4) (±1.2) (±1.4) (±1.8) (±1.0) (±1.1) (±0.8)

30 s 125.6 151.4 123.6 122.2 125.6 130.3 130.1

(±0.9) (±1.0) (±2.8) (±0.5) (±2.7) (±2.0) (±2.6)

24 h 121.6 153.8 128.8 127.5 129.2 128.9 129.4

(±0.7) (±2.1) (±1.1) (±1.2) (±1.5) (±1.2) (±1.4)

a

Water in air (sessile drop) and an air bubble in water (captive bubble). The data represent the means ± SD (n = 5).

PMPC, and PBuA are depicted as the control and the reference samples, respectively. PMA and PVAc were chosen as typical commodity polyacrylate and poly(vinyl ester) polymers, and their results are also included because these two polymers possess the same chemical composition and are structural isomers, the same as the relationship between PMEA and PMePVE. As shown in Table 2, the contact angle values of the samples were different from that of PET, which indicated that the PET substrates were surely coated with each polymer sample. The water droplet contact angle in air on the PMePVE-coated substrate was the lowest, and the value was slightly lower than

Figure 2. Number of adhered platelets on the polymer substrates. PET, PBuA (negative control), and PMPC (positive control) are included. The data represent the mean ± SD (n = 10). Stars indicate P < 0.01 versus PVAc. 1612

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Figure 3. Analysis of the interactions between plasma proteins and polymers surfaces. (a) Amount of adsorbed plasma proteins from PPP after 1 h. The data represent the mean ± SD (n = 3). Stars indicate P < 0.01 versus PVAc. (b) Amount of conformationally altered fibrinogen in the adsorbed proteins detected with an antifibrinogen γ chain antibody after 1 h. The data represent the mean ± SD (n = 5). Stars indicate P < 0.01 versus PVAc.

that on PMA. Notably, the structural difference between the two groups is only due to the presence of the methoxy terminal group (or −CH2−O−CH3). From this protein adsorption result, the introduction of the methoxy group at the side chain terminal was deemed as an effective way to provide protein adsorption-resistance properties to polymeric materials. It is well-known that adsorbed fibrinogen on a polymer surface has a dominant role for the promotion of platelet adhesion. Therefore, we determined the degree of exposure of the platelet adhesion sites at the carboxyl terminals of the fibrinogen γ chains by ELISA (Figure 3b). As a result, the evaluation results for the degree of exposure of the platelet adhesion sites completely coincided with the results of the platelet adhesion tests (Figure 2). A considerable difference was found between the number of adsorbed proteins between PMA and PVAc; however, between the two, there was no significant deference in the number of exposed platelet adhesion sites. Similar to PMA and PVAc, no significant difference was found between PMEA and PMePVE. Furthermore, the platelet adhesion sites in the fibrinogen γ chains were hardly detected on PMePVE, similar to PMEA. Latour et al. reported that the relationship between the number of adhered platelets, the loss of α helix content and the platelet adhesion were caused by the conformational change in the fibrinogen.28 The present protein adsorption/conformational change evaluation results imply that the nonthrombogenicity of PMePVE is expressed by not only suppressing the adsorption of plasma proteins onto the polymer surface, but also by suppressing the conformational change in fibrinogen that exposes the platelet adhesion sites. Analysis of Water Structures in Hydrated PMePVE. Platelet adhesion and protein adsorption are strongly influenced by the physical properties of the polymer such as its hydrophilicity, surface roughness, molecular mobility, and water structure.33−37 However, especially, the prevention mechanism of protein adsorption on polymer surfaces is still unclear. We have been hypothesizing that the presence of water molecules at the water/polymer interface significantly influences the prevention of platelet adhesion, and the presence of moderately bounding water molecules, intermediate water (IW), is important for suppressing protein adsorption onto polymer surfaces. The water structures in hydrated polymers are categorized into three types (they are named in order of the decreasing strength of interactions with the polymer): “nonfreezing water (NFW)”, “freezing bound water (intermediate water) (IW)”, and “free water (FW)”. Among the three types of

the positive control. The control samples (PET, PBuA, and PMPC) worked as expected, which indicated a high degree of reliability, as revealed by the results of the platelet adhesion test. The platelet adhesion was inhibited on the PMePVE-coated substrate, and the number of adhered platelet was suppressed to the same level as that of PMEA. In contrast, PVAc- and PMA-coated substrates did not inhibit platelet adhesion. Furthermore, the adhered platelets on PMePVE exhibited small numbers of pseudopodia, which indicated low activation of the platelets in comparison with those of the PET, PBuA, PMA, and PVAc substrates, as shown in Figure S4. These results indicate that PMePVE has excellent nonthrombogenicity, similar to the PMEA. The nonthrombogenicity was expressed only on the PMePVE and PMEA substrates and not the PVAc and PMA substrates; the resultant discrepancy between the two groups was likely caused by the specific chemical structure of PMePVE and PMEA, the methoxy groups (or −CH2−O−CH3) on the terminal side chains. Quantification of the protein adsorption and evaluation of the conformationally altered fibrinogen on the polymer surface. The platelet adhesion is related to the number of adsorbed plasma proteins on the polymer surface.28−30 The plasma proteins, such as albumin and fibrinogen, maintain their native state and original conformation in blood, and thus, the platelet adhesion/aggregation does not arise in the blood vessel. For example, Tsai et al. reported that fibrinogen is the major plasma protein that mediates platelet adhesion on polymer surfaces because the platelet adhesion was suppressed on poly(styrene) preadsorbed with afibrinogenemia plasma.31 In addition, Seo et al. reported that the dodecapeptide sequence present at the Cterminus of the fibrinogen γ chain induces platelet adhesion on the polymer surface.32 Consequently, the evaluation of the adsorbed/denatured plasma proteins, especially fibrinogen, is important for elucidating why materials exhibit blood compatibility/incompatibility. In the present study, the protein adsorption/denaturation behavior on each polymer substrate was evaluated by conducting a microBCA assay and ELISA, and the obtained results are shown in Figure 3. The number of adsorbed proteins on each polymer was evaluated with polymer cast-coated PP wells and platelet poor plasma, and the results are shown in Figure 3a relative to those of the bare PP. The adsorption amount on PMePVE was reduced to the same extent as that of PMEA, and there was no significant difference between the two polymers, although the amount of adsorbed proteins on PVAc was much higher than 1613

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Article

Biomacromolecules

contents did not indicate the cold crystallization of water, as shown in Figure S7. This result suggests that IW was formed in the hydrated PMePVE. Additionally, these results imply that the nonthrombogenicity of PMePVE could be attributed to the presence of IW in the hydrated polymer, similar to the case of PMEA. The amounts of NFW, IW, and FW in the hydrated polymers were quantified by using the equations described in the Experimental Section. The amount of NFW in the hydrated PMePVE, which has an inverted ester unit in the side chain of PMEA, decreased in comparison with that in the PMEA. However, the amount of IW and FW in the hydrated PMePVE was increased in comparison with that of the PMEA, although the EWC in the hydrated PMePVE and PMEA showed quite the same value. Miwa et al. reported a study on the correlation between the amount of intermediate water and the molecular mobility of polymers. Hydrated PMEA, PTHFA, and PHEMA were used as samples, and the mobility of the backbone and side chains in the hydrated state were evaluated by solid-state 2 H NMR and 13C NMR techniques.41,42 The intercept relaxation time, T1 values, of the hydrated polymers were found to be ordered as follows: PMEA > PTHFA > PHEMA at −5 and 37 °C by 2H NMR. Furthermore, Ts values, which indicate the flexibility of the backbone and side chains of the polymer, were determined by using the 13C NMR technique and were ranked as PMEA > PTHFA > PHEMA. From the results, we speculated that the molecular mobility of PMePVE was higher than that of PMEA, and thus, the amount of IW and FW, which possessed higher molecular mobility values in comparison with NFW, increased. The water contact angle of PMePVE showed slightly higher hydrophilicity compared to that of PMEA, as shown in Table 2. The water contact angle reflects the surface free energy, which relates to the molecular mobility of the polymer.43 Blum et al. reported the investigation of the thermal properties and intermolecular interactions of adsorbed PVAc and poly(methyl methacrylate) (PMMA) on silica substrates using temperature-modulated differential scanning calorimetry (TMDSC) characterization and molecular modeling.44,45 The observations suggested that the interactions between PMMA and silica were stronger than those between PVAc and silica because PMMA associates more strongly with silica than does PVAc through additional hydrogen-bonding interactions. Additionally, their simulations showed that the polymer−polymer interactions were stronger in PMMA than PVAc, and the molecular mobility of PVAc was much higher at the air/polymer interface, further away from the polymer/silica interface. Hence, in our case, the molecular mobility of PMePVE could have been higher than that of PMEA, and the molecular interactions of PMePVE, hydrogen-bonding, might also have been weakened in comparison to those of PMEA. Consequently, the amount of IW and FW in hydrated PMePVE increased in comparison with that of PMEA despite possessing the same chemical composition. From the result of the hydration water characterization, the blood compatibility of PMePVE might be attributed to the presence of intermediate water as PMEA where the presence of IW in hydrated PMePVE suppressed the protein adsorption/denaturation at the blood/ polymer interface.

water structures, we have reported that the presence of intermediate water could play a key role in inhibiting the platelet adhesion and the protein adsorption.38−40 For example, no IW was observed in PBuA from the DSC heating profiles for hydrated samples, and the platelet adhesion was hardly suppressed on PBuA. Therefore, to clarify the prevention mechanism of platelet adhesion and protein adsorption on the PMePVE polymer surface, we focused on the water structures in hydrated polymers. The water structures in hydrated PMePVE were analyzed by using a DSC technique. The DSC heating profiles of the dried and hydrated polymer samples are shown in Figure 4. Additionally, the glass transition temper-

Figure 4. DSC heating profiles of each polymer sample for dried and hydrated conditions at 5.0 °C/min. (a) Dried polymers: PMePVE (1), PMEA (2), PMA (3), and PVAc (4). (b) Hydrated polymers at the EWC (see Table 3): PMePVE (5), PMEA (6), PMA (7), and PVAc (8).

ature (Tg) for the dried and the hydrated samples, and the amount of water in each, are summarized in Table 3. The DSC heating profiles and the amount of each type of water absorbed for varying water contents of hydrated PMePVE and PVAc are shown in Figure S5−S8. Table 3. Tg in Dried and Hydrated State and Amount of Each Type of Water in Hydrated PMEA, PMePVE, and PVAc polymer

Tg-da [°C]

Tg-hb [°C]

NFWc [g/g]

IWd [g/g]

FWe [g/g]

EWCf [wt %]

PMEA PMePVE PVAc PMA

−35 −30 40 10.5

−51 −46 16 3.6

0.027 0.018 0.036 0.026

0.040 0.042 0 0

0.028 0.037 0.0041 0.0030

8.7 8.9 3.9 2.8

a

Measured using the dried samples. bMeasured using the hydrated samples. cNonfreezing water. dIntermediate water. eFreezing water. f Equilibrium water content.

The glass transition behavior was observed for dried samples of PMA, PVAc, PMEA, and PMePVE. The absence of crystallization and melting behavior indicated that PMePVE and the other polymers were amorphous. For hydrated samples, however, the cold crystallization of water molecules, which is the characteristic of the presence of IW, was observed at −40 °C in the DSC heating profiles of the hydrated PMEA and PMePVE. This cold crystallization of the water was not observed in the hydrated PVAc and PMA. Furthermore, the DSC heating profiles of the hydrated PVAc with various water

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CONCLUSION An MePVE monomer was synthesized and polymerized via free radical polymerization. The obtained PMePVE was characterized by NMR spectroscopy and SEC measurements. The polymer-coated substrates were prepared using a spin coating 1614

DOI: 10.1021/acs.biomac.7b00221 Biomacromolecules 2017, 18, 1609−1616

Article

Biomacromolecules

(3) Cavadas, M.; Gonzalez-Fernandez, A.; Franco, R. Pathogenmimetic stealth nanocarriers for drug delivery: a future possibility. Nanomedicine 2011, 7 (6), 730−743. (4) Nance, E.; Zhang, C.; Shih, T. Y.; Xu, Q. G.; Schuster, B. S.; Hanes, J. Brain-penetrating nanoparticles improve paclitaxel efficacy in malignant glioma following local administration. ACS Nano 2014, 8 (10), 10655−10664. (5) Ratner, B. D. The catastrophe revisited: Blood compatibility in the 21st century. Biomaterials 2007, 28 (34), 5144−5147. (6) Harris, J. M. E. Poly(Ethylene Glycol) Chemistry, Biotechnical and Biomedical Applications; Springer US: New York, 1992; p 385. (7) Sakata, S.; Inoue, Y.; Ishihara, K. Molecular interaction forces generated during protein adsorption to well-defined polymer brush surfaces. Langmuir 2015, 31 (10), 3108−3114. (8) Ladd, J.; Zhang, Z.; Chen, S.; Hower, J. C.; Jiang, S. Zwitterionic polymers exhibiting high resistance to nonspecific protein adsorption from human serum and plasma. Biomacromolecules 2008, 9 (5), 1357− 1361. (9) Otsuka, H.; Nagasaki, Y.; Kataoka, K. Surface characterization of functionalized polylactide through the coating with heterobifunctional poly(ethylene glycol)/polylactide block copolymers. Biomacromolecules 2000, 1 (1), 39−48. (10) Nagasaki, Y. Construction of a densely poly(ethylene glycol)chain-tethered surface and its performance. Polym. J. 2011, 43 (12), 949−958. (11) Ishihara, K. Bioinspired phospholipid polymer biomaterials for making high performance artificial organs. Sci. Technol. Adv. Mater. 2000, 1 (3), 131−138. (12) Zhang, Z.; Zhang, M.; Chen, S. F.; Horbett, T. A.; Ratner, B. D.; Jiang, S. Y. Blood compatibility of surfaces with superlow protein adsorption. Biomaterials 2008, 29 (32), 4285−4291. (13) Abdal-hay, A.; Hamdy, A. S.; Khalil, K. A.; Lim, J. H. A novel simple one-step air jet spinning approach for deposition of poly(vinyl acetate)/hydroxyapatite composite nanofibers on Ti implants. Mater. Sci. Eng., C 2015, 49, 681−690. (14) Varshosaz, J.; Jannesari, M.; Morshed, M.; Zamani, M. Composite poly(vinyl alcohol)/poly(vinyl acetate) electrospun nanofibrous mats as a novel wound dressing matrix for controlled release of drugs. Int. J. Nanomed. 2011, 6, 993−1003. (15) Zhang, F.; McGinity, J. W. Properties of hot-melt extruded theophylline tablets containing poly(vinyl acetate). Drug Dev. Ind. Pharm. 2000, 26 (9), 931−942. (16) Jeong, H. G.; Kim, Y. E.; Kim, Y. J. Fabrication of poly(vinyl acetate)/polysaccharide biocomposite nanofibrous membranes for tissue engineering. Macromol. Res. 2013, 21 (11), 1233−1240. (17) Vargha, V.; Truter, P. Biodegradable polymers by reactive blending trans-esterification of thermoplastic starch with poly(vinyl acetate) and poly(vinyl acetate-co-butyl acrylate). Eur. Polym. J. 2005, 41 (4), 715−726. (18) Tanaka, M.; Mochizuki, A. Effect of water structure on blood compatibility - Thermal analysis of water in poly(meth)acrylate. J. Biomed. Mater. Res. 2004, 68A (4), 684−695. (19) Sato, K.; Kobayashi, S.; Kusakari, M.; Watahiki, S.; Oikawa, M.; Hoshiba, T.; Tanaka, M. The relationship between water structure and blood compatibility in poly(2-methoxyethyl Acrylate) (PMEA) analogues. Macromol. Biosci. 2015, 15 (9), 1296−1303. (20) Allaoua, I.; Goi, B. E.; Obadia, M. M.; Debuigne, A.; Detrembleur, C.; Drockenmuller, E. (Co)Polymerization of vinyl levulinate by cobalt-mediated radical polymerization and functionalization by ketoxime click chemistry. Polym. Chem. 2014, 5 (8), 2973− 2979. (21) Hatada, K.; Terawaki, Y.; Kitayama, T.; Kamachi, M.; Tamaki, M. Studies on the radical polymerization of vinyl-acetate in benzene, chlorobenzne and ethyl-acetate by 1H NMR spectroscopy. Polym. Bull. 1981, 4 (8), 451−458. (22) Nakamura, Y.; Yamago, S. Termination mechanism in the radical polymerization of methyl methacrylate and styrene determined by the reaction of structurally well-defined polymer end radicals. Macromolecules 2015, 48 (18), 6450−6456.

method, and then the water contact angle was measured on the polymer-coated substrates. The hydrophilicity of PMePVE was higher than that of PMEA despite having a similar chemical composition. The nonthrombogenicity of PMePVE was evaluated after performing human platelet adhesion tests. The platelet adhesion number on PMePVE showed that same level as that of PMEA, which exhibited excellent nonthrombogenicity. To discuss the expressed nonthrombogenicity of PMePVE, microBCA and ELISA tests were performed. The adsorption of the plasma proteins and conformation change in the fibrinogen were suppressed on PMePVE. The water structures in hydrated PMePVE were analyzed by using a DSC technique. The IW and FW in hydrated PMePVE exhibited high values in comparison with those of hydrated PMEA. The nonthrombogenicity of PMePVE was expressed by suppressing the adsorption and conformation changes of the plasma proteins by tuning the hydration water structures at the biointerface, similar to the PMEA. Consequently, PMePVE is expected to be a new addition to the family of nonthrombogenic polymers and a strong candidate as a nonthrombogenic coating material that can be used in bloodcontacting medical devices.

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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.7b00221. NMR spectra of obtained products, SEM images of platelet adhesion tests, DSC analysis results in detail (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Shingo Kobayashi: 0000-0002-8357-8654 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS S.K. gratefully acknowledges financial support from JSPS KAKENHI (No. 15K05512) of Japan Society of the Promotion of Science (JSPS). K.S. acknowledges Grant-in-Aid for JSPS Fellows (No. 15J08130). M.T. acknowledges financial support from the Funding Program for Next-Generation World-Leading Researchers (NEXT Program) of the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan and the Center of Innovation Program from the Japan Science and Technology Agency (JST). This work was partially supported by JST ERATO (No. JPMJER1105).

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DOI: 10.1021/acs.biomac.7b00221 Biomacromolecules 2017, 18, 1609−1616