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Biological and Environmental Phenomena at the Interface
Effect of Molecular Weight of Poly(2-methoxyethyl acrylate) on Interfacial Structure and Blood Compatibility Daiki Murakami, Nami Mawatari, Toshiki Sonoda, Aki Kashiwazaki, and Masaru Tanaka Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b02971 • Publication Date (Web): 23 Jan 2019 Downloaded from http://pubs.acs.org on January 30, 2019
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Langmuir
Effect
of
Molecular
Weight
of
Poly(2-
methoxyethyl acrylate) on Interfacial Structure and Blood Compatibility Daiki Murakami1,2*, Nami Mawatari2, Toshiki Sonoda2, Aki Kashiwazaki1, Masaru Tanaka1,2,3* 1
Institute for Materials Chemistry and Engineering, 2 Graduate School of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan. 3
Frontier Center for Organic System Innovations, Yamagata University, 4-3-16 Jonan, Yonezawa, Yamagata, 992-8510, Japan.
KEYWORDS poly(2-methoxyethyl acrylate), molecular weight, interface, atomic force microscopy fibrinogen, blood compatibility
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ABSTRACT
The blood-compatible polymer poly(2-methoxyethyl acrylate) (PMEA) is composed of nanometer-scale interfacial structures because of the phase separation of the polymer and water at the PMEA/phosphate-buffered saline (PBS) interface. We synthesized PMEA with four different molecular weights (19, 30, 44, 183 kg/mol) to investigate the effect of the molecular weight on the interfacial structures and blood compatibility. The amounts of intermediate water and fibrinogen adsorption were not affected by the molecular weight of PMEA. In contrast, the degree of denaturation of adsorbed fibrinogen molecules and platelet adhesion increased as the molecular weight increased. Atomic force microscopy observation revealed that the domain size of the microphase separation structures observed at the PMEA/PBS interfaces drastically (nearly three times in mean area of a domain) changed with the molecular weight. PMEA with lower molecular weight showed a smaller polymer-rich domain size, as expected based on the microphase separation of polymer-rich and water-rich domains. The small domain size suppressed the aggregation and denaturation of adsorbed fibrinogen molecules because only a few fibrinogen molecules were adsorbed on a domain. Increasing the domain size enhanced the denaturation of adsorbed fibrinogen molecules. Controlling the interfacial structures is crucial for ensuring the blood compatibility of polymer interfaces.
Introduction Thrombogenesis on biomaterials is an important limitation reducing the safety of implantation, dialysis, and surgery, among other procedures. An important process in thrombogenesis is adsorption of proteins from the blood onto the material interface.1-4 Particularly, the blood
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protein fibrinogen has been widely examined to reduce the amount of fibrinogen adsorbed on biomaterials.5-10 Furthermore, it has been suggested that denaturation of adsorbed fibrinogen, namely the conformational change and subsequent exposure of platelet adhesion sites triggered by adsorption onto the hydrophobic interface or clustering of adsorbed fibrinogen molecules, is a key process in thrombogenesis.11-15 Thus, both adsorption of fibrinogen and subsequent denaturation should be prevented to develop sufficient antithrombogenesis biomaterials. Poly(2-methoxyethyl acrylate) (PMEA) is a blood-compatible polymer that has been widely utilized in recent years. Tanaka et al. reported that water molecules present in PMEA can be classified as non-freezing water, intermediate water, and free water, in the order of interaction with the polymer chains.16, 17 Particularly, the intermediate water which loosely interacts with the polymer chain, is considered to be a key in blood compatibility. It was demonstrated that polymers containing a higher amount of intermediate water exhibited higher blood compatibility (i.e. lower platelet adhesion). Indeed, fibrinogen adsorption and denaturation were remarkably restrained at the interface of PMEA containing a high amount of intermediate water. However, it remains unclear how PMEA exhibits blood compatibility at polymer interfaces. Recently, we focused on the interfacial phenomena of PMEA and its analogue polymers with and without intermediate water. Atomic force microscopy (AFM) observation revealed that PMEA and analogue polymers with intermediate water showed numerous protrusions of several tens of nanometers and spontaneously formed at polymer/phosphate-buffered saline (PBS) interfaces.5 Polymers without intermediate water such as poly(butyl acrylate) were not associated with such structures. The reversible temperature dependency of the interfacial structures revealed in our recent work indicates that they were formed by microphase separation of polymer-rich and water-rich domains at the interfacial region.18 The predicted mechanism of the interfacial
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structure formation is as follows. PMEA exhibits a lower critical solution temperature in water because the enthalpic gain of hydration increases as temperature decreases. It has been reported that even when the polymer and water phases are separated under bulk conditions with increasing temperature, they are still miscible in higher compositions at the interface.19 Phase separation of polymer-rich domains and water-rich domains in the interfacial region may occur to reduce free energy at the interface as temperature increase over the lower critical solution temperature.20,
21
As the mobility of polymer chains at the interface is restricted because of
partial entanglement to the bulk polymer phase, phase separation occurs on the microscopic scale. Because tiny polymer density in the water-rich domains is not detectable by AFM, polymer-rich domains are observed as nanometer-scale protrusions at the interface. Additionally, in AFM observation of adsorbed fibrinogen, we found that the polymer-rich domains on the PMEA/PBS interface act as scaffold for fibrinogen adsorption. Thus, the interfacial structures should be the dominant factor in preventing fibrinogen adsorption and significantly increasing the blood compatibility of PMEA. In this study, we aimed to control the interfacial structures and investigate how they are responsible for the blood compatibility of PMEA in more detail. PMEA samples with different molecular weights were synthesized and used. We expected that the size of interfacial structures depends on the size of the polymer chain based on the phase separation of polymer-rich and water-rich domains. The topographical changes in PMEA and the relationship with adsorption and denaturation of fibrinogen as well as platelet adhesion were evaluated.
Experimental Polymer synthesis and characterization
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PMEA samples with different molecular weights were synthesized by free radical polymerization as described previously.22 The reaction conditions (ratio of monomer (2methoxyethyl acrylate; MEA) and initiator (2,2′-azobis (isobutyronitrile); AIBN), monomer concentration, and reaction time) were modified to change the molecular weight as shown in Table 1. In entry 4, the initiator AIBN was purified by recrystallization before use to increase the reaction rate. Gel permeation chromatography (HLC-8220 GPC, TOSOH, Tokyo, Japan; Column: TSKgel GMHHR-H. Eluent: tetrahydrofuran) was used to determine the molecular weight. Interpolation by polystyrene standards was employed for calibration. The polymerization conditions, molecular weight (number averaged Mn and weight averaged Mw), and molecular weight distribution (Mw/Mn) of synthesized polymers are shown in Table 1. The synthesized PMEA samples were named based on their Mn values as PMEA19k, PMEA30k, PMEA44k, and PMEA183k. The glass transition temperatures of the polymers were determined by differential scanning calorimetry (DSC) under dry and wet conditions. Wet polymer samples were prepared by immersing the samples in water over 7 days to sufficiently equilibrate the diffusion of water molecules in the polymer. The amount of intermediate water in the wet polymers was evaluated by DSC. The spin-coated films of polymers were prepared from 0.2 wt/vol% methanol solutions (40 µl) twice onto polyethylene terephthalate (PET) substrates (φ = 14 mm, t = 125 µm) using a spin coater MS-A100 (Mikasa, Tokyo, Japan) with the rotational speed 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. Only the solution of PMEA183k was gently heated before spin-coating because it slightly aggregated at room temperature. Prepared films were dried well in a desiccator. The thickness of the spin-coated PMEA film in this condition was known to be around 100 nm by transmission electron
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microscopy.23 Then the contact angle of an air bubble on the PMEA/water interface was measured with a DMo-501 (KYOWA, Tokyo, Japan) on polymer films immersed in water for 24 h in advance.
Table 1. Conditions and results of polymer synthesis.
AFM observation The spin-coated PMEA films were observed in PBS by AFM (Bioscope Resolve, Bruker, Billerica, MA, USA). PBS used in this work doesn’t contain Ca2+ and Mg2+ ions. A cantilever SNL-10 (spring constant k = 0.35 N/m, tip radius < 12 nm; Bruker) was used for observation. Every observation was performed in peak force tapping mode.24, 25 Before imaging in PBS, all samples were observed in air to confirm that all polymer samples were coated on PET with sufficiently low roughness. The AFM image of PMEA183k film in air is shown in Supporting Information (Figure S1) as an example. We observed no influence of gentle heating on the spincoating process. Data analyses were performed using NanoScope Analysis 1.8 (Bruker) and Free softwere Gwyddion 2.49.
MicroBCA and ELISA tests
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The PMEA cast-films were prepared by slowly drying the 0.2 wt/vol% polymer/MeOH solutions (15 µl) on tissue culture-treated polystyrene (TCPS) 96-well plates. The polymer films were immersed in PBS for 1 h (priming process) before use. For the microBCA test, fibrinogen solutions (1 mg/mL in PBS) were added to each well. After incubation for 10 min at 37 °C, the wells were rinsed seven times with PBS to ensure the removal of non-adsorbed fibrinogen. Adsorbed fibrinogen molecules were extracted with 5% sodium dodecyl sulfate and 0.1 M NaOH aqueous solutions. The amount of extracted fibrinogen molecules was determined by measuring the absorbance at 570 nm with a Microplate Reader (iMarkTM, Bio-Rad, Hercules, CA, USA) using a commercial microBCA protein assay kit (Thermo Scientific, Inc., Waltham, MA, USA). For the enzyme-linked immunosorbent assay (ELISA) test, platelet-poor plasma was obtained from human blood purchased from Tennessee Blood Service (Memphis, TN, USA). Incubation and rinsing conducted as described above. After incubation with Blocking-One solution (Nacalai Tesque, Kyoto, Japan) for 30 min at 37 °C to prevent non-specific reactions, the samples were incubated with antibody (mouse anti-fibrinogen γ’, CT, clone 2. G2. H9) for 2 h, and with peroxidase-conjugated goat anti-mouse IgG for 1 h at room temperature. The samples were reacted with 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid ammonium salt) substrate (Roche Diagnostics, Basel, Switzerland) for 10 min to measure the absorbance of colored solutions at a wavelength of 405 nm. The significance of differences among samples was determined by an unpaired t-test using Microsoft Excel. Differences with P
