Antithrombogenic Properties of Amphiphilic Block Copolymer

Article pubs.acs.org/journal/abseba

Antithrombogenic Properties of Amphiphilic Block Copolymer Coatings: Evaluation of Hemocompatibility Using Whole Blood Kazutoshi Haraguchi,*,†,‡ Toru Takehisa,†,§ Toshihide Mizuno,∥ and Kazuomi Kubota†,§ †

Material Chemistry Laboratory, Kawamura Institute of Chemical Research, Sakura, Chiba 285-0078 Japan Department of Applied Molecular Chemistry, College of Industrial Technology, Nihon University, Narashino, Chiba 275-8575 Japan § Central Research Laboratories, DIC Co., Sakura, Chiba 285-8668, Japan ∥ Department of Artificial Organs, Research Institute, National Cerebral and Cardiovascular Center, Suita, Osaka, 565-8565, Japan ‡

ABSTRACT: Antithrombogenicity is one of the most critical properties required for materials used in biomedical devices, particularly in devices that contact blood. The antithrombogenicity of surfaces coated with amphiphilic block copolymers composed of hydrophobic poly(2-methoxyethyl acrylate) (M) and hydrophilic poly(N,N-dimethylacrylamide) (D) segments was investigated using plasma protein and whole blood with regard to protein adsorption, thrombus formation, platelet activation, and clotting kinetics. Three types of block copolymers and a random copolymer were synthesized using one-pot reversible addition−fragmentation chain-transfer (RAFT) polymerization under conditions of high yield and high molecular weight. Triblock and 4-arm block copolymers with MDM and (MD)4 architecture, respectively, showed good adhesion to both organic and inorganic substrates, including polyvinyl chloride (PVC) tubes, and the resulting coated surfaces showed superior protein repellency and hemocompatibility compared to the diblock or random copolymer coatings and noncoated control. In a Chandler-loop method with whole blood, PVC tubes coated with MDM and (MD)4 showed improved thromboresistance and adsorption resistance to blood-derived proteins. This high hemocompatibility was also confirmed with human whole blood by thrombelastography (suppression of blood-clotting behavior in both intrinsic and extrinsic coagulation pathways) and platelet function analyses (significant reductions in the aggregation activity of platelets under two types of stimulation). The antithrombogenicity has been discussed based on the structural analyses of the MDM-coated surface. The results of this study will enable the development of more effective biomedical and analytical devices with excellent antithrombogenic characteristics by using a simple and environmentally friendly approach. KEYWORDS: antithrombogenicity, hemocompatibility, amphiphilic block copolymer, coating, thromboresistance, thrombelastography, platelet function analysis

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and their block copolymers,35−37 are nonfouling and prevent both nonspecific protein adsorption and cell adhesion. However, in general, the simultaneous achievement of biocompatible properties and good attachment to the substrate is difficult with simple coating of polymer solutions. Therefore, in many cases, intricate treatments such as graft polymerization20,21,23,26,29,31 and specific assemblies22,30,34 have been adopted. Among the properties required for development of a biomaterial surface that contacts blood, antithrombogenicity is the most important one for providing long-term blood contacting ability and implant tolerance.38,39 Therefore, it is critical to determine the interactions between blood and the coating material, such as protein adsorption, platelet activation,

INTRODUCTION

The development of biocompatible materials with proteinresistant surfaces exhibiting nonfouling, low cellular adhesion, and antithrombogenic characteristics is important for both fundamental and applied research in the biological and biomedical sciences.1−4 Such surfaces are required in medical implants,5−7 biosensors,8,9 drug delivery carriers,10,11 and biomedical and analytical devices,12,13 in addition to various industrial applications. 14−18 Among the several surface modification procedures currently employed, polymer coating is the most economical and versatile method as it can be applied to various types of organic and inorganic substrates with a wide variety of sizes and shapes and is also performed under sterile conditions. For example, hydrophilic surfaces with bound water, chain flexibility, and neutral or balanced charge groups, such as polyethylene glycol (PEG),19−23 poly(2methoxyethyl acrylate),24−26 zwitterionic (e.g., phosphorylcholine,27−31 carboxybetaine,32,33 and sulfobetaine33,34) polymers, © 2015 American Chemical Society

Received: September 17, 2014 Accepted: May 12, 2015 Published: May 12, 2015 352

DOI: 10.1021/acsbiomaterials.5b00079 ACS Biomater. Sci. Eng. 2015, 1, 352−362

Article

ACS Biomaterials Science & Engineering

Scheme 1. Reversible Addition−Fragmentation Radical-Transfer Polymerization (RAFT) Agents for the Triblock Copolymer (CTA-1), Diblock Copolymer (CTA-2), and 4-Arm Block Copolymer (CTA-3)

and thrombus formation. Protein adsorption is the first step in the inflammatory response, which leads to platelet adhesion, thrombosis, and microbial infections in response to implantation of medical devices and other materials in vivo.40−42 Various types of biochemical and rheological tests have been adopted for blood analysis and coagulopathy in clinical practice43−45 and for the evaluation of potential biomaterials.46−48 In clinical practice, thrombelastographic monitoring systems (e.g., ROTEM)43,49−51 and platelet function analyzers (e.g., Multiplate)52−54 are used for reliable and rapid perioperative coagulation tests that enable differential diagnosis and for distinguishing surgical bleeding from significant coagulopathy. Thus far, we have developed new types of nanocomposite hydrogels55,56 and soft nanocomposites57 that can be used in vivo as mechanically durable hydrogels and as thermoresponsive substrates for a cell harvesting system.58−60 In a previous study,61 by extending previous findings for nanocomposite materials, we developed new amphiphilic block copolymers composed of hydrophobic poly(2-methoxyethyl acrylate) (PMEA; M) and hydrophilic poly(N,N-dimethylacrylamide) (PDMAA; D) segments by using a reversible addition−fragmentation chain-transfer (RAFT) polymerization method. We found that two types of amphiphilic block copolymers, triblock (MDM) and 4-arm block ((MD)4) copolymers with specific compositions (D/M = [750−1500]/ 250) and high molecular weight (Mw = 1.5−2 × 105 g/mol), showed good adhesion to various substrates, including polystyrene, polypropylene (PP), Ti, and glass, and that the surface coating showed high protein repellency and a low water contact angle regardless of the substrate to which it was applied. Furthermore, we showed that a polystyrene dish coated with the MDM block copolymer served as a good substrate for suspension cell culture, as it exhibited low cell adhesion and enabled good cell growth. Thus, amphiphilic MDM and (MD)4 block copolymers are promising coating materials with superior biomedical characteristics. In the present study, we investigated the hemocompatibility of the MDM and (MD)4 block copolymer coatings with respect to adsorption of plasma proteins, in vitro thrombus formation of whole blood, activation and adhesion of platelets, and clotting kinetics of whole blood by using a Chandler-loop method, thrombelastographic monitoring system (ROTEM), and multiple platelet function analyzer (Multiplate). All results indicated high hemocompatibility of the MDM and (MD)4 block copolymers.

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(V-601) (Wako Co., Japan) was used as an initiator. Methyl isobutyl ketone (MIBK) (99.5%; Wako Pure Chemicals) was used as a solvent after purification by passage through activated alumina. Diisopropyl ether (99.5%; Wako Pure Chemicals; Japan, 99.5%), a poor solvent, was used without purification. RAFT Agents. The chemical structures of the three RAFT agents used in the present study, 2-(1-carboxy-1-methylethylsulfanylthiocarbonylsulfanyl)-2-methylpropionic acid (CTA-1), 2-dodecylsulfanylthiocarbonylsulfanyl-2-methylpropionic acid (CTA-2), and pentaerythritol tetrakis(3-(S-(1-methoxycarbonylethyl)trithiocarbonyl)propionate) (CTA-3), are shown in Scheme 1. CTA-1, CTA-2,

and CTA-3 were used for synthesis of the triblock, diblock, and 4-arm block copolymers, respectively. All RAFT agents, CTA1−3, were prepared according to the procedures reported in our previous study.61 The three RAFT agents obtained were stored in the dark at low temperature (4 °C). The nuclear magnetic resonance (NMR) data were as follows: (CTA-1, 13C NMR data in CD3OD): δ 25.6 (CH3), 57.2 (C(CH3)2), 176.1 (CO2H), 220.3 (CS); (CTA-2, 1H NMR data in CDCl3): δ 0.88 (t, J = 8 Hz, 3H, CH2CH3), 1.25−1.30 (m, 16H, (CH2)8), 1.30−1.40 (m, 2H, SCH2CH2CH2), 1.68 (quintet, J = 8 Hz, 2H, SCH2CH2), 1.73 (s, 6H, C(CH3)2), 3.28 (t, J = 8 Hz, 2H, SCH2); (CTA-3, 1H NMR data in CDCl3): δ 1.60 (d, J = 8 Hz, 12H, CHCH3COOCH3), 2.80 (t, J = 8 Hz, 8H, CH2CO), 3.61 (t, J = 8 Hz, 8H, CH2S), 3.74 (s, 12H, COOCH3), 4.16 (s, 8H, CH2O), 4,81 (q, J = 8 Hz, 4H, SCHCH3).

Sample Nomenclature. The block copolymers consisting of hydrophobic M and hydrophilic D segments, i.e., the diblock copolymer (PMEA-b-PDMAA), triblock copolymer (PMEA-bPDMAA-b-PMEA), and 4-arm block copolymer (PMEA-bPDMAA)4, are designated as MD, MDM, and (MD)4, respectively. In addition, the compositions of the block copolymers are denoted using the molar ratio of each monomer relative to the corresponding CTA (RAFT agent), e.g., M(250)-D(1500), M(250)-D(1500)M(250), and (M(250)-D(750))4. The random copolymer is similarly represented as M(1500)-co-D(500). In the present study, the block copolymers were synthesized using the one-pot procedure, which is described in the next section. Thus, the resulting block copolymer was composed of pure M segments and a D segment that was slightly copolymerized with MEA. For example, because the conversion of MEA in the first polymerization step was 90 mol %, the residual MEA monomer (10 mol %) was incorporated in the second polymerization (D). Therefore, the actual composition of the block copolymer prepared by the one-pot procedure is represented as M(230)[D(1500)-co-M(40)]-M(230) and (M(230)-[D(750)-co-M(20)])4. Syntheses of Polymers. Preparation of the Triblock (MDM) Copolymer by One-Pot Synthesis. A solution of MEA (2.92 g, 22.4 mmol), CTA-1 (12.7 mg, 0.045 mmol), V-601 (0.9 mg, 0.004 mmol), and MIBK (10 mL) was purged with nitrogen for 90 min, and the reaction flask was placed in an oil bath (70 °C). After 8 h, a DMAA solution (6.66 g, 67.2 mmol DMAA in 10 mL MIBK) was added to the heated MEA solution. Heating was continued for another 24 h, and the polymerization was quenched by cooling the solution and exposing it to air. The polymer solution was precipitated into diisopropyl ether. The precipitate (powder) was collected, washed three times with diisopropyl ether, and dried in vacuo at 40 °C. The final conversions of

MATERIALS AND METHODS

Materials. Two monomers, N,N-dimethylacrylamide (DMAA; Kohjin Co.; Japan) and 2-methoxyethyl acrylate (MEA; Toagosei Co.; Japan), were purified by passage through a column of activated alumina to remove the inhibitor and were then stored in the dark at low temperature (4 °C) until use. Dimethyl 2,2′-azobis(isobutyrate) 353


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ACS Biomaterials Science & Engineering MEA and DMAA as determined by 1H NMR were 99.9% and 98.1%, respectively. Preparation of the 4-Arm ((MD)4) Block Polymer by One-Pot Synthesis. The 4-arm block copolymer was synthesized with CTA-3 as the chain-transfer agent and V-601 as the initiator in a manner similar to that described for the triblock copolymer. The final conversions of MEA and DMAA as determined by 1H NMR were 99.9 and 98.5%, respectively. Preparation of the Diblock (MD) Copolymer by One-Pot Synthesis. The diblock copolymer was synthesized using CTA-2 as the chain-transfer agent and V-601 as the initiator. MD was collected using the same procedure described for the triblock copolymer. The final conversions of MEA and DMAA as determined by 1H NMR were 99.0 and 96.5%, respectively. Preparation of the Random (M-co-D) Copolymer. The random copolymer was synthesized using a solution of MEA (2.92 g, 22.4 mmol), DMAA (6.66 g, 67.2 mmol), CTA-1 (12.7 mg, 0.045 mmol), V-601 (0.9 mg, 0.004 mmol), and MIBK (20 mL). M-co-D was collected following the same procedure used for the triblock copolymer. The conversions of MEA and DMAA as determined by 1 H NMR were 99.2 and 98.8%, respectively. Coatings. Surface Coating. A uniform copolymer solution was prepared by dissolving the copolymer (1 g) in a mixed solvent (99 g of ethanol and 1 g of water) to achieve a polymer concentration (Cp) of 1 wt %. Here, 1 wt % H2O in ethanol was added to improve the stability of the copolymer solution. The copolymer solution was coated on the surface of a PVC tube representative of those used in biomedical devices. The copolymer solution (10 mL) was poured into a PVC tube (Senko medical Instrument Mfg. Co., Ltd.; inner (outer) diameter of 3/32 (3/8) inches, 30 cm length), which was then secured with silicone caps at both ends. The PVC tube containing the solution was slowly rotated for 1 min to sufficiently wet the inner surface. After 1 min, the solution was drained from the tube, and the tube was dried in an oven at 50 °C for 30 min. The tube was then washed by immersion in warm water (50 °C) for 10 min and finally dried in an oven at 50 °C for 3 h. A polycarbonate (PC) connector (Senko medical Instrument Mfg. Co., Ltd.) was also coated in a manner similar to that described above. The MDM block copolymer solution was coated on the surface of various substrate plates (PVC, PC, and PP) by dipping the plates in the copolymer solution (1 wt %), followed by drying at 50 °C for 30 min. After coating, the substrate (plate) was washed by immersion in an excess amount of sterile water at 50 °C for 10 min followed by drying at 50 °C for 1 h. Extraction from the Coated Surface. After the PVC tube was coated with the copolymer, the inner surface of the PVC tube was extracted by filling the tube with hot pure water (21 mL, 50 °C) and incubating it for 10 min at 50 °C. After the extract water was drained, the tube was dried in an oven at 50 °C for 30 min. This procedure was repeated three times. The extract water was subjected to ultraviolet (UV) spectrometry (Nihon Denshi; UV-300) to evaluate the amount of copolymer that detached from the coated surface during each extraction process. After extraction, the PVC tube was filled with ethanol (21 mL) for 4 h at room temperature (∼25 °C) to completely detach the polymer coating. The ethanol containing the detached copolymer was then subjected to UV measurement to evaluate the amount of coated copolymer in the PVC tube. The thickness of the coating was estimated based on the amount of detached copolymer present in the ethanol. Hemocompatibility Testing. Thrombus formation in PVC tubes was evaluated using fresh bovine whole blood by the modified Chandler-loop method, which simulates blood circulation in a medical device such as an extracorporeal bypass circuit. The PVC tube and PC connector (both copolymer-coated and noncoated) were sterilized prior to use by ethylene oxide gas (EOG). The sterilization by EOG was conducted by maintaining the sample in an atmosphere containing EOG (3 wt %) at 30 °C for 2 h, followed by gas release for 72 h in fresh air. Blood samples were used immediately after collection from a calf, without addition of any anticoagulant; 5 mL of fresh blood was drawn into 3/8 in. PVC tubes coated with the four types of copolymers. The sample PVC tubes were looped with a polycarbonate

(PC) connector (coated with the same copolymer as that used in each PVC tube) (Figure 1a). Then, the loop tube was rotated at 60

Figure 1. (a) Round PVC tube with both ends connected by a PC connector. The inside wall surfaces of both the PVC tube and the connector surface were coated with the same copolymer. (b) Chandler-loop method (top view; 37 °C, 60 rpm) using PVC tubes and bovine whole blood maintained at 37 °C.

rpm in a water bath at 37 °C for 15 min (Figure 1b). The PVC tube was separated from the system, the blood was drained, and the tube was rinsed with saline. When large blood coagulates formed, saline was also used to drain the blood. The bloodderived protein adsorption was measured using a commercially available microbicinchoninic acid (BCA) assay (Wako; Japan), and the thrombus formation on the surface was visualized by scanning electron microscopy (SEM).

Measurements. 1H NMR Spectroscopy. 1H NMR measurements were conducted using a JEOL JNM-LA300 spectrometer operating at 300 MHz with CDCl3 as a solvent. High-Performance Liquid Chromatography (HPLC). The MDM block copolymer detached from the coated surface of the PVC tube during the extraction process was measured with HPLC (Alliance System, Waters 2695 Separations Module, Japan Waters Ltd.) performed using a dual λ absorbance detector and Inertsil ODS-3 column with a 20% acetonitrile aqueous solution at 40 °C. Molecular Weight. The weight-average and number-average molecular weights (Mw and Mn, respectively) and the Mw/Mn ratio of the copolymers were measured using size exclusion chromatography (SEC) on a Tosoh HLC-8220GPC system equipped with a refractive index detector, a set of two TSK-gel α-M columns (length of 30 cm each), and N,N-dimethylformamide with 0.1 mM LiBr at 40 °C with a flow rate of 1.0 mL/min. Calibration was performed with PMMA standards (Shodex Standard M-75). SEM and Surface Contact Angle. The surface of the copolymercoated PVC tube (inner wall surface) was observed using SEM (SEM007; Nihon Denshi Co.) before and after the whole blood circulation and plasma protein adsorption tests. The surface contact angles for water (θw) on the surfaces of the various substrates (PVC, PC, and PP plates) coated with the MDM block copolymer were measured using a surface-contact-angle measuring instrument (WPI3000: Kyowa Kagaku) by depositing a drop of water (8 μL) on the surfaces of pristine and coated substrates (n = 9). Atomic Force Microscopy (AFM). The surface topography of noncoated and coated PVC plate samples was characterized using dynamic force microscope mode atomic force microscopy (DFMAFM). The DFM-AFM analysis was performed using BRUKER Nano Scope(R) IIIa AFM equipment with a Si-DF20 cantilever (purchased from SII nanotechnologies Inc.). Typically, the scan rate was 0.5 Hz and the measured area was 10 × 10 and 2 × 2 μm2. Five replicate measurements were performed to determine the roughness value, expressed as root-mean-square roughness (Ra). Protein Adsorption. The adsorption of plasma protein was examined on the inner surfaces of PVC tubes coated with the four types of block copolymers and the random copolymer. The adsorption 354


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ACS Biomaterials Science & Engineering assay was performed by drawing 1 mL of bovine plasma (Sigma) into the coated PVC tube (1.5 cm length) with caps at both ends and storing the tube at 37 °C for 1 h. After three light rinses with 3 mL of saline, the plasma proteins adsorbed on the coated surface were measured using a micro-BCA assay kit (Thermo SCIENTIFIC, USA) with UV spectrophotometer. Here, the adsorption time, 1 h, was selected to ensure sufficient adsorption.62−64 As a control, the protein adsorption on the surface of a noncoated PVC tube was also measured in a similar manner. The plasma protein adsorption was also measured for different types of substrate plates (PVC, PC, and PP) coated with the MDM triblock copolymer as described above for the PVC tube. The adsorption of blood proteins on the surface of the copolymercoated PVC tube in ECC tests was also measured using a micro-BCA assay kit. Clot Formation Test. Functional analysis for anticoagulation on the surface of the copolymer coating was performed using a thromboelastometry monitoring system (ROTEM; TEM International GmbH; Munich, Germany)44,47 and whole human blood from a healthy volunteer (male adult). We used surface-modified test cells (consisting of cups and pins) for measurement in the ROTEM system. This modification involved coating of the surfaces of a cup and pin with the copolymer solution, followed by drying in a manner similar to that described above for PVC tube coating. Thromboelastometry was performed according to routine procedures for 60 min using human whole fresh blood with 3.2% sodium citrate and two types of liquid reagents (EXTEM for activating extrinsic pathway and INTEM for activating intrinsic pathway; TEM International GmbH). The clot formation on the block copolymer surface was evaluated using specific parameters, such as clotting time (CT) and maximum clotting firmness (MCF), captured from the thromboelastgram. Platelet Aggregation Test. Platelet aggregation and platelet adhesion to the surface of copolymer coatings were measured by the multiple electrode aggregometry method using the Multiplate (Roche Diagnostics International Ltd.; Switzerland) for assessment of the platelet reaction.65,66 To evaluate the effect of copolymer coating on platelet aggregation and adhesion, we coated all of the surfaces of the assay test cells, including the blood cuvettes, poles, and a magnetic stirring bar, with copolymers as described above for PVC tube coating. Human whole blood with hirudin was poured in the cells, and subsequently, two types of analyses for platelet activation stimulated by adenosine diphosphate (ADP test) and collagen-induced aggregation (COL test) were performed. The ADP test analyzes the agglutinability of platelets through activation of the ADP receptor, which indicates the secondary aggregation ability of platelets. The COL test evaluated primary aggregation by assessment of collageninduced activation. Platelet function was analyzed following routine procedures for 15 min. Two replicate measurements (n = 4) were performed simultaneously. The impedance value during the measurement has been expressed in arbitrary aggregation units for the 2 determinations. To express the aggregation response over the measured time interval, we calculated the area under the aggregation curve (AUC) from the mean value of two curves. The AUC value was used as a quantitative parameter for the aggregation activity of platelets against the coated surface.

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Scheme 2. Amphiphilic Block Copolymers Consisting of Hydrophobic M (white) and Hydrophilic D (black) Segments: (a) Triblock MDM Copolymer, (b) 4-Arm (MD)4 Block Copolymer, and (c) Diblock MD Copolymer

The characteristics of the block and random copolymers synthesized in the present study, such as their conversion (1H NMR), molecular weight (Mw), and Mw/Mn ratio (SEC), are summarized in Table 1. The block architecture consisting of pure M segments and slightly copolymerized D segments was confirmed based on the 13C NMR and differential scanning calorimetry (DSC) measurements in addition to 1H NMR, as shown in our previous study. The block copolymers obtained herein showed fairly high dispersity (Mw/Mn ≈ 2.2) compared with those previously generated using RAFT polymerization. This increased dispersity mainly resulted from differences in the synthetic conditions applied in the present study, such as high monomer concentration (4.5 M), low molar ratio of RAFT agent (1/1540) in the reaction solution, and high monomer conversion rate (98−100%), resulting in high yield (∼100%) and high molecular weight (1.5−2.0 × 105 g/mol). Although synthesis of a block copolymer with a narrow molecular weight distribution is generally desirable, in this case, the use of the one-pot synthesis method, high molecular weight, and high conversion were necessary to achieve the goals of high substrate adhesion, high protein resistance, and antithrombogenicity, and to enable economical mass production of the coating materials. The block and random copolymers listed in Table 1 showed different solubilities in water; transparent and translucent aqueous dispersions were obtained for M-co-D and MD, respectively, and opaque (white) dispersions were obtained for MDM and (MD)4, with an average particle size of 470 ± 10 nm. Although the latter aqueous dispersions were fairly unstable, they became more stable and the average particle size decreased to 340 ± 10 nm with the addition of a small amount of surfactant (e.g., sodium dodecylbenzenesulfonate [SDBS], 0.3 wt %). In contrast, all block and random copolymers were soluble in organic solvents such as ethanol, methanol, and tetrahydrofuran. In the present study, we used ethanol as a solvent because it can be readily applied to coat most substrates commonly used in biomedical devices. In practice, we used an ethanol solution consisting of 99 wt % ethanol and 1 wt % H2O because the MDM and (MD)4 solutions showed increased stability with the addition of 1 wt % H2O. Thus, uniform and transparent ethanol solutions were obtained for all block and random copolymers tested. PVC Tube Coatings. PVC tubes are used in many biomedical devices and clinical operations, such as extracorporeal blood circuits (artificial hearts, artificial lungs, and hemodialyzers), percutaneous cardio-pulmonary support, and extracorporeal membrane oxygenation. The block and random copolymers listed in Table 1 were coated on the inner surface of PVC tubes by using an ethanol solution (Cp = 1 wt %, H2O = 1 wt %) followed by sterilization with the EOG method, as described in the Materials and Methods section. Here, the EOG sterilization did not affect the properties and structure of the

RESULTS AND DISCUSSION

Synthesis and Characterization of Block Copolymers. In the present study, we evaluated two main types of block copolymers, triblock MDM and 4-arm block (MD)4 copolymers (Scheme 2a, b), consisting of M (hydrophobic)-D (hydrophilic)-M (hydrophobic) sequences, in comparison with diblock M-D (Scheme 2c) and random M-co-D copolymers. In a previous study,61 we demonstrated that the characteristics of low cell adhesion and ultralow adsorption of specific proteins [immunoglobulin G (IgG) and bovine serum albumin (BSA)] could be achieved using MDM and (MD)4 with an appropriate composition (M/D ratio) and high molecular weight (block length). 355


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Table 1. Characteristics of Amphiphilic Block Copolymers, Triblock MDM, 4-Arm Block (MD)4, Diblock MD, and the Random M-co-D Copolymer Prepared by Reversible Addition−Fragmentation Chain-Transfer Polymerization of MEA and DMAA with Trithiocarbonate RAFT Agents conversionb (%) block copolymer

compositiona

CTA

MEA

DMAA

Mwc (× 103 g/mol)

Mw/Mnc

MDM (MD)4 MD M-co-D

M(230)-[D(1500)-co-M(40)]-M(230) (M(230)-[D(750)-co-M(20)])4 M(230)-[D(1500)-co-M(20)] D(1500)-co-M(500)

CTA-1 CTA-3 CTA-2 CTA-1

99.9 99.9 99.0 99.2

98.1 98.5 96.5 98.8

140 202 173 141

2.15 2.39 2.27 2.42

a

Numbers in parentheses represent the molar ratio of MEA or DMAA relative to CTA. bDetermined by 1H NMR spectroscopy. cDetermined by SEC.

Figure 2. Surface of a PVC tube coated with MDM. (a) Optical microscopy, (b) SEM.

Table 2. Effects of Coating with MDM, (MD)4, MD, and Mco-D on the Water Contact Angle (θw) on PVC Plates and Adsorption of Plasma Proteins and Blood-Derived Proteins on the Inner Surfaces of PVC Tubes sample

composition

MDM

M(230)[D(1500)-coM(40)]-M(230) (M(230)[D(750)-coM(20)]4 (M(230)[D(1500)-coM(20)]) D(1500)-coM(500) noncoated substrate

(MD)4 MD M-co-D

θwa (deg)

plasma adsorptionb (μg/cm2)

whole blood protein adsorptionc (μg/ cm2)

25

6.56

7.7

27

7.02

10.2

53

10.33

12.4

63

18.98

32.0

75

19.51

30.1

Figure 3. Appearance of PVC tubes coated with different copolymers after rotation tests using a modified Chandler loop with fresh bovine whole blood. The coating copolymers were (b) MDM, (c) (MD)4, (d) MDM, (e) MD, and (f) M-co-D. Here, two MDM-coated tube samples, (b, d), were used to verify the effectiveness. A noncoated PVC tube is shown in a.

In a previous study,61 we found that the MDM and (MD)4 copolymers showed good adhesion to various types of substrates. To confirm the strong adhesion of MDM to the inner surface of a PVC tube, hot-water (50 °C) extraction for 10 min was conducted three times. In the first extraction, slight detachment of MDM (0.0095 mg/cm2) was observed, whereas detached polymer was not detected in the second and third extractions. Considering this result and the total amount of MDM coated on the PVC tube (0.23 mg/cm2), the amount of polymer detached in the first and second extractions with water was estimated to be approximately 4 and 0 wt %, respectively, of the total MDM coating. In contrast, approximately 38 and 3 wt % and 65 and 4 wt %, respectively, of the MD and M-co-D copolymers was detached from the coated surface of the PVC tubes following the first and second washing steps, respectively. To determine the water contact angle (θw) on the PVCcoated surface, the four types of copolymers were coated on PVC plates in the same manner used for PVC tubes. The noncoated PVC plate had a θw of 75°, and the changes observed following coating are shown in Table 2. In the case of MD and M-co-D, the contact angle on the coated surface was moderately and highly similar, respectively, to that observed on the noncoated surface, which indicates that

a Water contact angle on a PVC plate. bAdsorption of plasma protein on the inner surface of a PVC tube. cAdsorption of blood-derived proteins on the inner surface of a PVC tube.

coated surface. The resulting coated surfaces were transparent and uniform in all cases. The PVC tube surface observed by optical microscopy and SEM is shown in Figure 2a, b, respectively. The coating thickness of the MDM block copolymer on the inner surface of PVC tubes was calculated based on the total amount of MDM desorbed from a PVC tube by extraction with ethanol for 4 h. From the amount of MDM (0.23 mg/cm2) and the assumption of copolymer density (= 1.2 g/cm3), the coating thickness of MDM was determined to be approximately 1.9 μm. The relatively higher thickness of the MDM coating on PVC tubes compared with that on PS dishes observed in our previous study (0.13 μm) can likely be attributed to the different coating methods used; dip-coating was used in the present study, whereas spin-coating was used in our previous study. 356


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copolymers sufficiently adhered to the PVC plate even after washing. In fact, barely any detached polymer was observed in the freeze-dried washing water. Furthermore, MDM was sufficiently coated on both the PC and PP plates (θw = 26° on the PC plate and 25° on the PP plate). Thus, the MDM and (MD)4 block copolymers listed in Table 1 were well-coated on the inner surface of PVC tubes and on the surfaces of the PVC, PC, and PP plates. Protein Adsorption. Nonspecific protein adsorption on the surface of PVC tubes coated with the block and random copolymers was examined using plasma proteins prior to performing hemocompatibility tests with whole blood. As shown in Table 2, the PVC tube surfaces coated with MDM and (MD)4 showed higher resistance to plasma protein adsorption than noncoated PVC tube surfaces did. M-co-Dcoated surfaces showed almost the same level of adsorption as the noncoated surface, whereas MD-coated surfaces showed an intermediate level of adsorption. These results for plasma protein adsorption were consistent with the results reported in our previous study for specific adsorption of IgG and BSA on a PS dish surface coated with the same block and random copolymers, although the improvement in protein resistance by coating [adsorption (coated surface)/adsorption (noncoated surface)] was quite different: 0.002, 0.15, and 0.336 for IgG, BSA, and plasma protein, respectively. This difference probably resulted from intrinsic differences among protein types. In general, plasma proteins are adsorbed more readily than IgG because plasma proteins consist of three different proteins, that is, albumin, globulin, and fibrinogen, including adhesive proteins. The adsorption of BSA was intermediate to that of IgG and plasma proteins. Hemocompatibility Testing. Hemocompatibility is one of the most critical properties required for a material destined for use in a biomedical device, particularly for devices that contact blood, such as medical devices for cardiovascular surgery (e.g., vascular grafts, oxygenators, artificial hearts, heart valves, hemodialyzers, stents, and catheters). 5,6,67−69 Here, the hemocompatibility of PVC tube surfaces coated with MDM and (MD)4 was evaluated by using the Chandler-loop method, which enables simulation of extracorporeal circulation (ECC), and was compared to that on the MD- and M-co-D- coated and control (noncoated) surfaces. In the Chandler-loop test, we adopted 15 min as the rotation time because the fresh whole blood samples were used immediately after collection from a calf, without addition of any anticoagulants such as heparin70 or acid citrate dextrose.71 In general, such nontreated flowing whole blood tends to coagulate in a short time (15−25 min with rotation)69 because of thrombus formation, and a short rotation time is thus normally used with Chandler-loop and fresh blood systems.72 In the present study, occlusion occurred and flow ceased within 10 min in the case of the noncoated PVC tube. Therefore, 15 min was defined as an adequate time to evaluate the effect of surface coating on hemocompatibility. This Chandler loop-nonanticoagulated whole blood system was the most sensitive method available to evaluate the hemocompatibility of a coated surface. For the noncoated PVC tube, a large amount of thrombi appeared during the rotation test to the extent that the blood was no longer liquid and could not circulate (Figure 3a) by the end of the experiment. Thus, owing to the difficulty in removing coagulated blood from the noncoated PVC tube, an excessive saline rinse was used to remove the coagulant. Conversely, the blood was readily removed from the

Figure 4. SEM observations of the blood-derived proteins adsorbed on the inner surface of PVC tubes after a Chandler-loop test with whole blood for 15 min at 37 °C. The coating copolymers were (a) none, (b) MDM, (c) (MD)4, (d) MD, and (e) M-co-D.

Figure 5. (a) ROTEM thrombelastometry (Pentapharm GmbH), (b) thrombelastogram, and (c) pin and cup.

the MD and M-co-D coatings were removed to a large extent during the washing process. The poor adhesion of MD and Mco-D mainly resulted from their molecular architecture, as their overall composition was identical to that of MDM. In contrast, the contact angles on the surfaces coated with MDM and (MD)4 were approximately 20−30°, indicating that these 357


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Figure 6. Evaluation of coating materials by using ROTEM. (a) Intrinsic measurement, (b) extrinsic measurement. (a-1, b-1) Surface coated with MDM triblock copolymer. (a-2, b-2) Noncoated surface. Scale bar is 10 min.

The total amount of blood-derived proteins that adhered to the surfaces of the PVC tubes during the rotation test was determined using a micro-BCA assay kit. The amount of protein adsorption varied substantially depending on the coating polymer used, as shown in Table 2. The adsorption values were consistent with the corresponding SEM observations, i.e., MDM < (MD)4 < MD < M-co-D ≈ noncoated. The effect of polymer architecture on antithrombogenicity was nearly identical for adsorption of plasma proteins and bloodderived proteins. Thus, it was concluded that MDM and (MD)4 block copolymers exhibit high adsorption repellency, not only for plasma proteins but also for whole blood (blood-derived proteins), and high hemocompatibility in the Chandler-loop− fresh whole blood system. Clot Formation Test. The effect of the MDM block copolymer coating on whole blood coagulation was investigated using thromboelastometry (ROTEM), in which interactions occurring at the blood−surface interface are evaluated. Hemostasis involves complicated feedback reactions between blood cells and plasma-derived factors.40−42,73 A blood clot represents the formation of a three-dimensional network of massive thrombin, which is formed by strong interactions between plasma-derived factors and cellular components. Thromboelastometry (ROTEM) is commonly used to measure blood-clotting behavior for clinical analyses of hemostasis in whole blood and of hypercoagulability and coagulopathy.74,75 Two types of activators, i.e., EXTEM and INTEM reagents, which include extrinsic and intrinsic coagulation activating

copolymer-coated PVC tubes. The visual appearance of the PVC tube after light rinsing with saline is shown in Figures 3b-f. PVC tubes coated with M-co-D showed a fair amount of clots attached to the surface (Figure 3f), whereas PVC tubes coated with MD showed minimal attachment of coagulated blood (Figure 3e). In contrast, no clot was visually detectable in the PVC tubes coated with MDM and (MD)4 (Figures 3b−d). These results indicate that PVC tubes whose inner surfaces were modified by coating with MDM and (MD)4 showed high hemocompatibility for fresh bovine whole blood. The amount of blood-derived proteins adsorbed on the surface of the PVC tube after the rotation tests was evaluated by SEM (Figure 4). The protein on the surface was fixed by cross-linking with glutaraldehyde, followed by washing with saline and drying at room temperature. In the case of the noncoated PVC tube (Figure 4a), a large amount of deposited material was observed, which may indicate the aggregation of blood-derived proteins attached to the whole surface. By contrast, the surface of the MDM-coated PVC tube was smooth and showed little attachment of blood-derived proteins (Figure 4b), which indicates that the MDM coating produced a good antithrombogenic surface. The PVC surface coated with (MD)4 showed a similarly low level of protein adhesion, although some adhesive proteins were observed in a few areas (Figure 4c). In contrast, the surfaces coated with MD and M-co-D showed substantial adsorption of blood-derived proteins on all surfaces, as shown in Figure 4d, e. 358


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Figure 7. (a) Graph shows the electrical impedance curve between two pair of electrodes with activated platelets in the ADP test for different coating conditions: (1) MDM, (2) M-co-D, and (3) noncoated (control). (b) Mean AUC values (n = 4) of the ADP test and COL test: MDM-coated surfaces show significant reduction in platelet adhesion stimulated by ADP and COL reagents, as compared with that seen in noncoated cells.

factors, respectively, were applied. The change in torsion stress caused by clot formation was monitored through a torsion wire connected to a pin, which was set in the center of a rotating cup filled with human whole blood (Figure 5a). Once plasma proteins adsorbed on the surface, a series of biochemical events causing platelet adhesion and activation was initiated, resulting in the generation of a fibrin clot. Through continuous assessment of blood clot firmness after addition of an activator, either EXTEM or INTEM, blood coagulation was evaluated with regard to CT, MCF, and clot stability (A10 and A20, the amplitudes at 10 and 20 min, respectively) (Figure 5b). The blood contacting surfaces (Figure 5c) were coated with MDM, MD, or M-co-D, in the same manner as PVC tube coating. In this study, the results obtained in the thromboelastometry assay for the MDM-coated surfaces differed greatly from those for the noncoated vessel, as shown in Figure 6. The noncoated surfaces showed standard (normal) thrombelastographic tracking (Figure 6a-2, b-2). In contrast, although CT in both the INTEM and EXTEM assays was maintained in the normal range (slightly (10−15%) increased), clot firmness parameters, such as MCF, A10, and A20, remarkably decreased when MDM coating was adopted (Figure 6a-1, b-1). In particular, A10 and A20 were very small even when compared with the decreased MCF. These results indicate that primary process of clot formation first occurred on the coated surface; however, the secondary process of clot maturation was interrupted, and the immature clots were rapidly peeled off. These remarkable changes in the thrombelastogram were observed in both INTEM and EXTEM assays, which indicates that the MDM coating shows effective antithrombogenicity regardless of intrinsic and extrinsic coagulation pathways. However, in the case of MD and M-co-D coatings, very few changes were observed in the thrombelastogram, which indicates that these

Figure 8. AFM measurements of PVC plate surfaces. (a) Noncoated surface. (b) MDM-coated surface. Area: (1) 10 × 10 μm2, (2, 3) 2 × 2 μm2.

Figure 9. Schematic representation of the surface structure of the MDM coating consisting of hydrophobic M and hydrophilic D segments. The thickness was decreased for simplicity.

polymer coatings did not improve the hemocompatibility of the surface. Platelet Function Analyses. Multiple electrode aggregometry (Multiplate) was used to evaluate the aggregation activity of activated fresh platelets when the blood test cells (including all surfaces of cuvettes and electrodes) were coated with an antithrombogenic polymer, MDM, or M-co-D. Typical plots of the ADP tests for MDM-coated and noncoated surfaces are shown in Figure 7a-1, a-3, respectively. For MDM-coated 359


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The MDM coating adheres to the substrate because M segments are preferentially attached thereon. Then, MDM may form layered and/or subdivided structures because of the M-D-M architecture, and at the outermost surface, D segments may exist mainly because PDMAA has higher affinity to solvents (e.g., ethanol and water) than PMEA. Additionally, the M-D-M sequences may generate fine, uneven morphology at the outermost surface. Thus, in MDM-coated surfaces, high protein repellency and strong adhesion to the substrate in addition to a low water contact were simultaneously achieved, suggesting high hemocompatibility. (MD)4-coated surfaces also have these characteristics because the (MD)4 block copolymers consist of two MDM units.

cells, the rate of aggregation (slope of the trace plotted in the graph) was remarkably lower than that in noncoated (control) surfaces. The results clearly show that, on an MDM-coated surface, it was difficult for activated platelets with ADP to aggregate and adhere. In contrast, the M-co-D coating exhibited aggregation activity intermediate to that observed for MDMcoated and noncoated chips (Figure 7a-2). The AUC values in two types of platelet aggregation tests using the ADPtest and COLtest are shown together in Figure 7b. In MDM-coated cells, the AUC value significantly decreased in both the ADP test and COL test. In contrast, M-co-D-coated cells did not show changes in the COL test, although the AUC value in the ADP test was relatively diminished. These results demonstrated that the MDM-coated surface prevented collagen absorption more effectively than the M-co-D-coated surface. Thus, from platelet function analysis, it was concluded that the MDM polymer coatings were superior in preventing platelet adhesion and aggregation onto the surface that they coated. Mechanism of the Antithrombogenicity of the MDM Coating. It was confirmed that MDM and MD4 block copolymers exhibit high hemocompatibility in biomedical tests widely used in clinical practice, e.g., the Chandler-loop (simulation of extracorporeal circulation), ROTEM (thromboelastometry), and Multiplate (platelet function analysis) tests. The high antithrombogenicity of MDM-coated surfaces is primarily attributed to the molecular architecture and chemical composition of MDM. Here, it should be noted that the chemical structure of MDM was completely different from those of well-known antithrombogenic coating polymers such as 2-methacryloyloxyethyl phosphorylcholine (MPC)-based polymers, which have biomimetic zwitterionic structures consisting of anionic phosphorylcholine and cationic ammonium groups.27,28 In contrast, MDMs are neutral (nonionic) with an amphiphilic triblock structure designed to satisfy the requirements of high resistance to protein adsorption and good attachment to the substrate, based on the combined effects of the components of the polymer blocks. Specifically, the use of hydrophobic and flexible M chains at both ends promoted attachment to the substrate via a hydrophobic effect, which was enhanced by the low glass-transition temperature (Tg = −34 °C), and both the M and D segments contributed to high resistance to protein adsorption because of their hydrophilic chain and hydrophobic but intermediately water−absorbed chain,24 respectively. The quite high molecular weight of M segment (Dp = 230) may play an important role in promoting the adhesiveness. Thus, MDM block copolymers coated on various substrates showed good attachment to the substrate and simultaneously showed high resistance to protein adsorption. To clarify the surface structure, we conducted AFM measurements for PVC plates that were either not coated or were coated with MDM. The results are shown in Figure 8a for noncoated surfaces and Figure 8b for MDM-coated surfaces. The original PVC plate, which was fairly rough, became smoother after coating with MDM. The average surface roughness, Ra (nm), decreased from 5.50 nm (noncoated: Figure 8a-1) to 3.01 nm (MDM-coated: Figure 8b-1). Additionally, 3D imaging (Figure 8b-3) clearly showed that the MDM-coated surface had fine uneven morphology. Taking into account all of its aspects, e.g., the low water contact angle, low protein adsorption, high adhesivity to the substrate, and uniform but fine morphology at the outermost surface, the MDM-coated surface is schematically represented in Figure 9.

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CONCLUSIONS Great concerns have been raised regarding the coating materials used in biomedical devices, both for fundamental research and for clinical practice, as such materials have the potential to induce high thrombogenicity on coated surfaces. In the present study, we investigated the hemocompatibility of amphiphilic block copolymers consisting of hydrophobic M and hydrophilic D segments, namely, triblock and 4-arm block copolymers with an MDM and (MD) 4 architecture, respectively. The hemocompatibility of surfaces coated with MDM and (MD)4 was investigated using a Chandler-loop (simulation of extracorporeal circulation), ROTEM (thrombelastography), and Multiplate (platelet activation and clot formation), all of which are used in clinical practice. MDM and (MD) 4 sufficiently adhered to all substrates of test cells, including PVC tubes, which are utilized in many cardiovascular devices. The resulting MDM- and (MD)4-coated surfaces on PVC tubes showed high plasma protein repellency and good hemocompatibility (reduced thrombus formation and decreased adsorptions of blood-derived proteins), whereas the other block and random copolymer coatings and noncoated (control) surfaces showed blood coagulation and attachment of blood-derived proteins. The superior hemocompatibility of MDM was also confirmed using human whole blood by thrombelastometry measurements, in which the blood-clotting behavior was largely suppressed in both intrinsic and extrinsic coagulation pathways, and platelet function analyses, in which the aggregation activity of platelets was significantly reduced in two types of stimulation. The mechanism of antithrombogenicity has been discussed with respect to surface structure. Thus, MDM could potentially be used as coating materials on the inside or outside of materials, including tubing, catheters, and valves, that are employed in a variety of biomedical devices that contact blood.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +81-47-4742567. Fax: +81-47-474-2579. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Mr. H. Nakaguma for the help with the synthesis of block copolymers, and Dr. T. Takada for the fruitful discussion. This work was supported by the Ministry of Education, Science, Sports, and Culture, Japan (Grant-in-Aid 23350117 and 15H03870). 360


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Antithrombogenic Properties of Amphiphilic Block Copolymer

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