Surface Characterization and Platelet Adhesion on Thin Hydrogel

Dec 6, 2017 - Poly(vinyl ether), with short oxyethylene side chains which possess a simple and relatively polar structure, should be a unique candidat...
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Surface Characterization and Platelet Adhesion on Thin Hydrogel Films of Poly(vinyl ether) Nozomi Itagaki,† Yukari Oda,*,† Toyoaki Hirata,† Hung Kim Nguyen,† Daisuke Kawaguchi,‡ Hisao Matsuno,*,†,§ and Keiji Tanaka*,†,§ †

Department of Applied Chemistry, ‡Education Center for Global Leaders in Molecular Systems for Devices, and §International Institute for Carbon-Neutral Energy Research (WPI-I2CNER), Kyushu University, Fukuoka 819-0395, Japan S Supporting Information *

ABSTRACT: Poly(vinyl ether), with short oxyethylene side chains which possess a simple and relatively polar structure, should be a unique candidate for a bioinert material thanks to its solubility in water. On the basis of living cationic copolymerization and subsequent ultraviolet light irradiation, thin films of poly(2-methoxyethyl vinyl ether) with different cross-linking densities were prepared on solid substrates. The films were thickened in water, and the extent was dependent on the cross-linking density. Although the surface chemistry and aggregation states were almost identical to one another, the stiffness, or the softness, of the outermost region in the film was strongly dependent on the cross-linking density. That is, the interface between polymer and water became thicker, or more diffused, with decreasing cross-linking density. The blood compatibility based on the platelet adhesion on to the hydrogel films was better for a more diffused interface.

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after fixing other conditions must be collected initially and a huge amount of data acquired under various conditions should eventually be analyzed using methods such as artificial intelligence. This motivates us to study only the effect of surface chain dynamics and thereby stiffness on the adsorption of biorelated substances on polymer films without changing other factors such as surface chemistry and morphology. Poly(vinyl ether) (PVE), with short oxyethylene side chains which possess a simple and relatively polar structure,22 should be a unique candidate for a bioinert material thanks to its solubility in water. However, because PVE generally has a glasstransition temperature which is lower than room temperature, its thin film geometry is not stable even under ambient conditions and is easily broken because of the dewetting. The film of PVE also disappears in water because of its solubility, as mentioned above. A plausible way to fix PVE chains onto a solid surface is to use a diblock copolymer.23 We have recently synthesized a diblock copolymer composed of a rubbery poly(2-methoxyethyl VE) (PMOVE) with a glassy poly(cyclohexyl VE) (PCVE) and prepared thin films, in which the PMOVE component formed the segregation layer at the water interface.24 In this case, although the PMOVE chains are in the water phase, they are able to be near to the solid surface because of the chemical junction with the water-insoluble PCVE blocks. The films then exhibit a successful suppression of platelet adhesion and subsequent activation on them.25

INTRODUCTION Construction of bioinert interfaces, having little or no effect on the human organism, has been urgently required for the development of functional biomedical devices such as artificial blood vessels, dialyzers, and artificial heart−lung apparatus.1−5 There are many factors associated with their bioinert properties, including hydrophilicity,6−8 morphology,9 and hydration structure, at the water interfaces.10−12 Thus far, the correlation between these interfacial features and bioinertness has been widely examined, and the results have led to the successful development of a variety of synthetic polymers exhibiting excellent bioinertness.6,9,10,13−15 Considering from the aspect of polymers, the physical properties at the water interface are of pivotal importance in addition to the above for the bioinertness. For instance, the excluded volume effect, which is derived from the steric repulsion of hydrated chains in water, plays a key role in the expression of bioinertness.11,12,16 If that is the case, controlling the chain length is directly related to the extent of the excluded volume effect as can actually be seen for poly(ethylene oxide) or poly(ethylene glycol).6,16 The local dynamics of chains at the water interface is also crucial in determining the blood compatibility.12,17,18 Faster dynamics in the interfacial region more effectively suppresses the attachment of proteins and platelets on it.17,18 Mechanical stiffness, or softness, which is another way of describing chain dynamics, also affects the protein adsorption behaviors.19−21 However, despite a growing body of literature on the development of bioinert polymers, little is known about the optimal strategy for the design of these materials. To overcome this problem, a lot of data on one factor © XXXX American Chemical Society

Received: September 30, 2017 Revised: November 13, 2017

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DOI: 10.1021/acs.langmuir.7b03427 Langmuir XXXX, XXX, XXX−XXX

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Scheme 1. Synthesis of MrV by Living Cationic Polymerization in Toluene, Spin-Coating Thin Films onto Substrates, and Photo-Cross-Linking of MrV in a Thin-Film State

stopcock. The polymerization was initiated by the addition of an Et1.5AlCl1.5 toluene solution into a mixture of monomers, 1,4-dioxane, and MOEA in toluene at 273 K via a dried syringe. The initial concentrations of monomers ([MOVE]0 and [VEM]0) were set at three cases: 0.64 M and 0.16 M, 0.48 M and 0.32 M, or 0.32 M and 0.48 M, respectively. The initial concentrations of other reagents were fixed as follows: [MOEA]0 = 4.0 mM, [Et1.5AlCl1.5]0 = 20 mM, and [1,4-dioxane] = 1.2 M. After 24 h, the polymerization was terminated with prechilled methanol containing a small amount of aqueous ammonia solution (0.1 vol %). The quenched reaction mixture was diluted with dichloromethane and then washed at least 10 times with water to remove the residual initiator. The products were vacuumdried for more than 6 h at 273 K. Then, they were dissolved into methanol and purified using a dialysis membrane with a molecular weight (MW) cutoff of 3.5k (Spectrum Laboratories Inc., Rancho Dominguez, CA, USA) against methanol to remove the unreacted VEM. The purified MrVs were stored at 245 K in the dark. The MW distribution of the MrVs was measured by gel permeation chromatography (GPC) equipped with three polystyrene (PS) gel columns (TSKgel MultiporeHXL-M, Tosoh Corp., Tokyo, Japan) connected to a PU-4180 pump and an RI-4030 refractive detector (JASCO Corp., Tokyo, Japan). Tetrahydrofuran was used as an eluent, and the flow rate was set to 1.0 mL·min−1 at 313 K. The numberaverage MW (Mn) and polydispersity index (Mw/Mn), where Mw is the weight-average MW, were calculated from GPC curves with respect to PS standards. The chemical structure of the polymers was examined using 400 MHz 1H nuclear magnetic resonance spectroscopy (1H NMR; JNM-ECP400, JEOL Ltd., Tokyo, Japan) in CDCl3 with tetramethylsilane (TMS). Preparation of Cross-Linked Thin Films. Cross-linked MrV films were prepared according to Scheme 1. First, thin films of MrVs were prepared on silicon wafers with a native oxide layer and quartz prisms, or on silicon substrates modified with chloro(decyl)dimethylsilane (only for the platelet adhesion test), by a spin-coating method from toluene solutions. Then, the films were irradiated by UV−visible light with a wavelength ranging from 250 to 500 nm using an R300-3J Xe light (Eagle Engineering Ltd., Tokyo, Japan) for 6 min at room temperature or approximately 298 K to promote the crosslinking reaction. After that, the films were dried in a vacuum oven for 12 h at 318 K. The films were soaked in water, which was purified using a Milli-Q system (Merck KGaA, Darmstadt, Germany), at room temperature for 6 h to remove un-cross-linked chains and dried again under vacuum at 318 K for 24 h. The progress of the cross-linking reaction was confirmed by Fourier transform infrared (FT-IR) spectroscopy using an FT/IR-620 spectrometer (JASCO) at a resolution of 2 cm−1 based on the disappearance of methacrylate double bonds. For the measurements, thicker samples were prepared on CaF2 substrates by a solvent-casting method. Hereafter, the crosslinked MrV films obtained are referred to as c-MrV. Characterization of c-MrV Films. The surface morphology and thickness of the c-MrV films in air and water were examined by AFM (Cypher ES, Asylum Research, an Oxford Instruments Company, Santa Barbara, CA, USA) at room temperature. A cantilever with a nominal spring constant of 0.12 N·m−1, which has resonance frequencies of 122 and 26 kHz in air and water, respectively, was used for the observations. Also, the films were partly cut by a blade so that the thickness could be determined from the height of the crosssection. The thickness in water was characterized after soaking the film

However, the synthesis of a diblock copolymer with a welldefined block length and thereby composition by living cationic polymerization is currently difficult. Also, when the copolymer film is thin, the structure at the outermost region might be dependent on the thickness26 as well as the kind of the substrate used.27 This means that the right condition for the preparation of copolymer films must be chosen to reach the best performance of the bioinertness. To overcome these difficulties, we here propose an alternative method of preparing the PMOVE surface based on a cross-linking reaction.28 2-(Vinyloxy)ethyl methacrylate (VEM)29 having a photo-cross-linkable part was copolymerized with MOVE with different monomer feed compositions,30,31 yielding a PMOVE gel with different cross-linking densities after ultraviolet (UV) irradiation. The methacryloyl group of VEM is intact under cationic polymerization conditions but can be radically polymerized in the presence of heat or light.29,32 Thin hydrogel films of PMOVE so-prepared in water can be characterized by atomic force microscopy (AFM), contact angle measurements, and sum frequency generation (SFG) vibrational spectroscopy. The platelet adhesion behavior on the films is then examined. Combining all data, we clearly demonstrate that the surface stiffness, or softness, is one of the crucial factors in expressing a highly blood-compatible polymer surface.

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EXPERIMENTAL SECTION

Materials. MOVE was kindly supplied by Maruzen Petrochemical Co. Ltd. (Tokyo, Japan). To remove impurities, it was washed with a 10 vol % aqueous sodium hydroxide solution, followed by deionized water, and dried over potassium hydroxide pellets overnight. It was then distilled twice over calcium hydride and metallic sodium before use. VEM was synthesized by the reaction of 2-chloroethyl vinyl ether (94 mL, 0.92 mol, Tokyo Chemical Industry Co. Ltd., Tokyo, Japan) with sodium methacrylate (50 g, 0.46 mol, Sigma-Aldrich, St. Louis, MO, USA) in dimethyl sulfoxide (230 mL) in the presence of tetrabutylammonium iodide as a phase-transfer catalyst and 4-tertbutylcatechol as an inhibitor at 353 K for 4 h, as described elsewhere.29,30 The product was distilled twice over calcium hydride and stored in the dark. Ethylaluminum sesquichloride (Et1.5AlCl1.5, 1.0 M solution in toluene, Nippon Aluminum Alkyls Ltd., Osaka, Japan) was used without further purification. Toluene was washed with a 10 vol % aqueous sulfuric acid solution, a 10 vol % aqueous sodium hydroxide solution, and deionized water and dried over potassium hydroxide pellets. It was then distilled over calcium hydride and metallic sodium just before use. 1,4-Dioxane was distilled over calcium hydride and then lithium aluminum hydride. 1-(Methoxyethoxy)ethyl acetate (MOEA) was prepared by the addition reaction of MOVE (30 mL, 0.26 mol) to acetic acid (12.5 mL, 0.22 mol, Wako Pure Chemical Industries Ltd., Osaka, Japan) and purified by distilling twice over calcium hydride under reduced pressure according to the literature.33 Synthesis of Poly(MOVE-r-VEM). Scheme 1 shows a schematic illustration of our synthetic route for a series of poly(MOVE-r-VEM), referred to as MrV. They were synthesized by base-assisting living cationic polymerization34 of MOVE and VEM at 273 K under a dry nitrogen atmosphere in a glass tube equipped with a three-way B

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Langmuir in water for 3 h to reach an equilibrium swollen state. The thickness in a swollen state was also examined by optical reflectivity (OR).35,36 The mechanical properties of the c-MrV films in the vicinity of the water interface were examined based on the force (F)−distance (d) curves acquired by AFM with a contact mode at room temperature. In this case, silicon nitride cantilever tips with a radius (r) of curvature of 8 nm and a spring constant of 0.12 N·m−1 were used. Force curves were collected at a scan rate of 60 nm·s−1, and a total of 40 force curves were taken at four randomly selected points on each hydrogel film. Static contact angle measurements were carried out with a Drop Master 500 (Kyowa Interface Science Co. Ltd., Saitama, Japan) at room temperature in water using air bubbles of 2 μL as a probe. The aggregation states at the c-MrV/water interface were examined by SFG spectroscopy. SFG spectra were collected with visible light with a wavelength of 532 nm and tunable infrared (IR) beams traveling through the quartz prism and overlapping at the water interfaces on the polymer films, as previously reported.37 The measurements were taken at room temperature with polarization combinations of ssp and ppp (SF output, visible input, and IR input) after 3 h of water immersion. Platelet Adhesion Test. Platelet adhesion tests were conducted on the c-MrV films and a poly(ethylene terephthalate) (PET) film as a reference. Prior to the test, the films were immersed in a phosphatebuffered saline (PBS) solution for 3 h. Platelet-rich plasma (PRP) and platelet-poor plasma were obtained by the centrifugation of commercially available whole human blood as described elsewhere.38 PRP suspension was poured onto the films, and they were incubated for 1 h at 310 K. After the films were washed two times with PBS, they were immersed into 1 wt % glutaraldehyde of PBS for 2 h at 310 K to fix adhered platelets. Then, the films were washed with PBS, PBS/ water mixture (1:1; v/v), and water and dried in air and then under vacuum at room temperature. The number and morphology of adhered platelets on the films were examined by scanning electron microscopy (SEM, SS-500, Shimadzu Corp., Kyoto, Japan) at an acceleration voltage of 15 kV. Before SEM observation, the films were sputter-coated using a JFC-1600 Pt coater (JEOL).

Figure 2. 1H NMR spectrum for MrV-17 measured in CDCl3 with TMS as an internal standard. [MOVE]0 and [VEM]0 were 0.64 and 0.16 M, respectively.

was also the case for the other MrVs. The molar ratios of MOVE/VEM for the three MrVs determined by the integrated intensity of each peak were 83/17, 67/33, and 50/50, which were relatively close to the feed ratios of 80/20, 60/40, and 40/ 60, respectively. Hereafter, they are referred to as MrV-17, MrV-33, and MrV-50. Table 1 summarizes the characteristics of the polymers so-obtained. Three MrVs with almost comparable MWs and different VEM contents were obtained. Table 1. Characteristics of Polymers Obtained by Living Cationic Copolymerization

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RESULTS AND DISCUSSION Polymer Characterization. Living cationic copolymerization of MOVE and VEM with various feed ratios was carried out. Figure 1 shows the GPC charts for the MrVs. All three products exhibited a unimodal curve regardless of the feed ratio, meaning that the copolymerization reaction was wellcontrolled. Figure 2 shows the 1H NMR spectrum for the MrV with the lowest feed ratio of VEM as a typical example. All key signals arising from MOVE and VEM repeating units are seen in it without any other signals derived from side reactions. This

polymer

Mn

Mw/Mn

MOVE/VEM (mol/mol)

MrV-17 MrV-33 MrV-50

22.1k 24.3k 23.4k

1.10 1.10 1.09

83/17 67/33 50/50

Figure S1 shows the FT-IR spectra for the cross-linked films after UV light irradiation. On the basis of the areal ratio of absorption peaks for CC stretching vibration at around 1630 cm−1 and CO stretching vibration at around 1720 cm−1, the conversions of CC double bonds to single bonds for MrV17, MrV-33, and MrV-50 were calculated to be 81, 86, and 87%,31 respectively. Postulating that all cleaved CC double bonds were involved in the cross-linking reactions, the maximum cross-linking densities for c-MrV-17, c-MrV-33, and c-MrV-50 were 5.1 × 102, 1.4 × 103, and 2.3 × 103 mol·m−3, respectively. They will be compared with those estimated from Young’s moduli measured by AFM force curves in a later section. Swelling Behavior and Mechanical Properties. The swelling behavior of the c-MrV films was examined. A gel film in which the bottom surface, or interface, is in direct contact with a solid substrate can generally be expanded in a solvent only along the direction normal to the interface.39,40 Hence, the swelling ratio, α, of the film is given by α = dswollen /ddry

(1)

where ddry and dswollen are the film thicknesses in air and water, respectively, and examined by AFM height profiles in conjunction with OR measurements, as shown in Table 2. The values obtained by AFM were in good accordance with those obtained by OR (Supporting Information). The α values calculated for the c-MrV-17, c-MrV-33, and c-MrV-50 films were 1.89 ± 0.07, 1.44 ± 0.03, and 1.27 ± 0.03, respectively. The swelling extent was more striking for a lower VEM

Figure 1. GPC charts for the three products obtained by base-assisting living cationic copolymerization of MOVE and VEM. MW was calibrated with PS standards. Cationic polymerization conditions: [MOVE]0 and [VEM]0 were 0.64 and 0.16 M for MrV-17, 0.48 and 0.32 M for MrV-33, and 0.32 and 0.48 M for MrV-50, respectively. [Et1.5AlCl1.5]0 = 20 mM, [1,4-dioxane] = 1.2 M, in toluene at 273 K for 24 h. EV: elution volume. C

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Langmuir Table 2. Film Thicknesses and Swelling Ratios for c-MrV-17, c-MrV-33, and c-MrV-50 at an Apparent Swollen State film thickness/nm polymer

in air (ddry)

in water (dswollen)

swelling ratio (α)

c-MrV-17 c-MrV-33 c-MrV-50

184 ± 6 181 ± 3 190 ± 3

349 ± 5 261 ± 3 241 ± 4

1.89 ± 0.07 1.44 ± 0.03 1.27 ± 0.03

fraction, meaning that the cross-linking density for the c-MrV films increases with increasing VEM fraction, as expected. Then, the mechanical response of the c-MrV films in water was studied based on the F versus d curve by AFM. Figure 3

Figure 4. Cross-linking density (νe) for c-MrV films as a function of VEM fraction. The solid curve is a fit to the experimental data based on eq 5.

value as a function of VEM fraction for the c-MrV films. The νe values for c-MrV-17, c-MrV-33, and c-MrV-50 were 2.3 × 101, 3.9 × 101, and 1.0 × 102 mol·m−3, respectively. Then, the Mc values were calculated to be 21.8k, 16.9k, and 7.31k in the order of the VEM fraction. Because the physical meaning of νe is essentially the same as Mc, the following discussion is conducted based on νe. Taking into account the probability that the meeting of two cross-linkable VEM units in the film increases with the VEM fraction, the relationship of νe to the VEM fraction can be qualitatively understood. However, the νe value obtained here was much smaller than the maximum value estimated above by 1/20−1/40, depending on the VEM fraction. This implies that only one out of 20−40 VEM units was consumed to form the network structure in the c-MrV film. In other words, the rest of the VEM units contributed to the generation of dangling chains. Thus, it seems most likely that the c-MrV film possesses many dangling chains. According to the classical theory of gelation, the branching coefficient β for a network structure composed of chains having cross-linkable units can be expressed as follows:47

Figure 3. Depth (d) dependence of repulsive force (F) for the c-MrV17, c-MrV-33, and c-MrV-50 films measured by AFM. The thicknesses of all films in the dried state were approximately 185 nm.

shows the data for the films in an equilibrium swollen state. The 0 distance corresponds to the interface between the film and the water phase. F increased with increasing d for all c-MrV films. However, an increment of F against d was clearly dependent on the VEM fraction. At a depth of 5 nm, whereas F was larger than 0.15 nN for c-MrV-50, it was almost 0 for cMrV-17. Thus, it seems most likely that the interface between the polymer and the water phases becomes thicker or more diffused with decreasing VEM fraction. Each measurement was truncated as d reached a distance equal to 10% of the total thickness (for c-MrV-17 and c-MrV-33) or the tip reached the compression limit (for c-MrV-50).41 Because the adhesive force between the film and the tip surfaces was negligible for all cases, the Hertz model was simply applied to the F−d relationship to extract the Young’s modulus (E) as follows:42−44 F = (4/3) ·r1/2·d3/2·E /{1‐ν 2}

β = k 2ω/{1 − k 2(1 − ω)}

where k is the reactivity of cross-linkable units. The parameter ω is the fractional amount of cross-linkable units per chain and thus identical to the VEM fraction. Given that β is proportional to νe, the experimental data shown in Figure 4 might be expressed by eq 5. Because the solid curve based on eq 5 closely matches the experimental data, it is conceivable that νe is controlled by the probability of the formation of branching, or cross-linking, points. Surface Aggregation States. The aggregation states at the outermost region of the c-MrV films were characterized by AFM in conjunction with static contact angle measurements. Figure S2 shows the AFM topographic and phase images of the c-MrV films acquired in air. The root-mean-square surface roughness (Rrms) values were 0.43 ± 0.02, 0.39 ± 0.06, and 0.35 ± 0.02 nm for c-MrV-17, c-MrV-33, and c-MrV-50, respectively. The phase images for all c-MrV films were featureless. Water contact angle for the c-MrV films could not be measured because the water droplets spread and were absorbed immediately into the films regardless of the VEM content. That is, the surface of the c-MrV films was flat, homogeneous, and hydrophilic, independent of the VEM fraction.

(2)

Here, ν and r are the Poisson’s ratio of the film, which is assumed to be 0.5, and the tip radius, respectively. The solid curves in Figure 3 denote the best-fit ones with E values of 139 ± 16, 255 ± 15, and 700 ± 37 kPa for c-MrV-17, c-MrV-33, and c-MrV-50, respectively. The E values so-obtained can be correlated with the crosslinking density (νe) in the film, which is also inversely proportional to the MW between cross-linking points (Mc), as follows:45,46 νe = E /{3RTϕ1/3}

(3)

Mc = ρ /νe

(4)

(5)

Here, R, T, ϕ, and ρ are the gas constant, temperature, volume fraction of the polymer at the equilibrium swollen state, and the mass density of the polymer, respectively. Figure 4 shows the νe D

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polarization combination seem to be quite identical in shape, indicating that the aggregation states of chains at the water interface were almost the same among the c-MrV films. Because peaks derived from antisymmetric C−H stretching of CH2 and OCH2 (CH2,as and OCH2,as) groups at around 2930 cm−1 and 2950 cm−1, respectively, were clearly observed in both of the polarization combinations, it is most likely that the side chains of PMOVE in all films existed at the water interface. On the other hand, the shape of the SFG spectra for the c-MrV films was different from that for a diblock copolymer film containing a PMOVE block, which was segregated at the water interface (shown in the previous paper).25 For instance, whereas the characteristic peaks derived from the symmetric C−H stretching vibration of CH2 and OCH2 (CH2,s and OCH2,s) at around 2865 cm−1 were clearly observed for the diblock copolymer film with the ssp and ppp polarization combinations,25 they were not observed for the c-MrV films. This discrepancy implies that the local conformation of PMOVE chains in the hydrogel films at the water interface is different from that in the block copolymer. The aggregation states of water molecules on the c-MrV films were also examined. Panel (c) of Figure 6 shows the SFG spectra in the O−H region with the ssp polarization combination. Similar broad peaks were observed at around 3160 and 3530 cm−1 for all c-MrV films. The peak position reflects the degree of coordination of water molecules through hydrogen bonding. A higher wavenumber means that the water molecules are in a more disordered state.48−50 The aggregation states of water molecules at the film interface were again independent of the VEM fraction. Thus, on the basis of the AFM observation and contact angle and SFG spectroscopic measurements, it can be claimed that the aggregation structure of both polymer chains and water molecules at the interface is insensitive to the VEM fraction. Platelet Adhesion. As mentioned above, the c-MrV films with different cross-linking densities were successfully prepared. Although they had similar chemistry and thereby aggregation states at the water interface, their stiffness near the interface was strongly dependent on the cross-linking density. We now turn to the blood compatibility, platelet adhesion test, for the c-MrV films so that the relationship between physical properties and function at the polymer interface can be discussed. Panels (a− c) of Figure 7 show the SEM images of platelets adhered onto the films. For comparison, the results for a PET film, on which platelets can easily adsorb, are also shown in Figure 7d. Panels (e) and (f) of Figure 7 represent the number of platelets (NPLT) and their classifications into three types based on the

Figure 5 shows the AFM topographic and phase images for the c-MrV films, which were in contact with water for more

Figure 5. AFM (a−c) topographic and (d−f) phase images for (a,d) the c-MrV-17, (b,e) c-MrV-33, and (c,f) c-MrV-50 films acquired in water. The Rrms values were 1.01 ± 0.07 nm for c-MrV-17, 1.12 ± 0.09 nm for c-MrV-33, and 1.02 ± 0.09 nm for c-MrV-50.

than 3 h. All films were swollen as discussed before, whereas non-cross-linked MrV films completely disappeared from the substrates (data not shown). These results make it clear that the introduction of cross-linking points into PMOVE prevented the films from dissolving in water. The Rrms values for c-MrV17, c-MrV-33, and c-MrV-50 in water were 1.01 ± 0.07, 1.12 ± 0.09, and 1.02 ± 0.09 nm, respectively. Although the surface of the films became rougher in water, the extent is acceptable for further characterization at the interface. The phase images for the c-MrV films were again featureless, meaning that the surface was homogeneous even in water. Static contact angles against an air bubble for the c-MrV-17, c-MrV-33, and c-MrV-50 films were 129.9 ± 1.3°, 130.3 ± 0.9°, and 130.7 ± 1.2° in water, respectively. In this measurement, a larger angle means more hydrophilic. Taking the flatness of the c-MrV film surface into account, it is apparent that the aggregation states of the c-MrV films at the water interface were identical to one another independent of the VEM fraction. Next, the local conformation of polymer chains at the water interface was examined by SFG spectroscopy. Panels (a,b) in Figure 6 show the SFG spectra in the C−H stretching vibration region for the c-MrV films at the water interface with the ssp and ppp polarization combinations. The assignment of peaks was based on the relevant literature.25,37 All spectra at a given

Figure 6. SFG spectra in the C−H stretching vibration region with the (a) ssp and (b) ppp polarization combinations and (c) in the O−H region with the ssp polarization combination for the c-MrV films immersed in water for 3 h. The thicknesses of all films were ca. 185 nm before water immersion. E

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surface and the interface with water was almost independent of the cross-linking density. AFM also found no differences in the surface morphology among the films with different cross-linking densities. On the other hand, the film stiffness, or softness, near the water phase decreased with decreasing cross-linking density. This indicates that the interfacial region of the film with water becomes thicker and softer with decreasing cross-linking density. The number density of platelets adhered on the cMrV films decreased with decreasing cross-linking density. Concurrently, the platelets were also less activated with decreasing cross-linking density. Thus, we come to the conclusion that a softer and more diffused polymer interface with water would be better for the expression of bioinertness under the conditions of the given surface chemistry and morphology.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.7b03427. Experimental details, FT-IR spectra, AFM topographic and phase images, cross-sectional view of the topological images, and OR curves and analytical data for polymers (PDF)

Figure 7. SEM images of platelets adhered on (a) the c-MrV-17, (b) cMrV-33, (c) c-MrV-50 films and (d) a reference, PET, film. The thicknesses of all films were ca. 185 nm before water immersion. Scale bars correspond to 10 μm. (e) Number of platelets (NPLT) on each sample and (f) quantitative data of platelet adhesion behaviors for each c-MrV film. NPLT on each sample is shown with the mean and standard deviation (n = 10) and its breakdown classified into three stages based on the degree of activation for platelets is shown in gray. *P < 0.05 (Student’s t-test).

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (Y.O.). *E-mail: [email protected] (H.M.). *E-mail: [email protected] (K.T.).

degree of activation: the original spherical shapes (type I), the round shapes with a few protruding pseudopods (type II, partially activated), and the flattened shapes with many pseudopods (type III, strikingly activated). The NPLT value was clearly lower for the c-MrV films than that for the PET film and was also strongly dependent on the VEM fraction, or the cross-linking density. These results were well-reproduced in three different experiments (Supporting Information). In addition, the activation of platelets was markedly suppressed with decreasing cross-linking density. When the cross-linking density of the c-MrV film decreases, the swelling of the film becomes more marked, leading to a more diffused polymer interface with water. In that case, the chain dynamics at the water interface becomes faster. This results in more effective suppression of the platelet adhesion and activation.17,18 Thus, it can be concluded that the platelet adhesion is closely related to the cross-linking density, which is directly related to the swelling behavior of the c-MrV film.

ORCID

Yukari Oda: 0000-0002-3322-8365 Daisuke Kawaguchi: 0000-0001-8930-039X Hisao Matsuno: 0000-0002-4096-9006 Keiji Tanaka: 0000-0003-0314-3843 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was partly supported by JSPS KAKENHI, Grantin-Aid for Scientific Research (A) (no. 15H02183) (K.T.), Scientific Research (C) (no. 15K05633) (H.M.), Scientific Research (B) (no. 17H03118) (D.K.), and Young Scientists (B) (no. 16K17917) (Y.O.). We are also grateful for the support from JST SENTANKEISOKU (13A0004) (K.T.), from the Photon and Quantum Basic Research Coordinated Development Program by MEXT (K.T.), and from CSTI Impulsing Paradigm Change through Disruptive Technologies (ImPACT) Program (K.T.).

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CONCLUSIONS A well-defined PMOVE containing photo-cross-linkable VEM units was synthesized by living cationic polymerization. After it was spin-casted onto a substrate, it was irradiated with UV light, resulting in a thin film of cross-linked PMOVE with many dangling chains, named c-MrV. Once the c-MrV film was immersed in water, it thickened because of the water sorption. The swelling ratio of the hydrogel thin film, which was defined as a ratio of the thicknesses in water and air, changed from 1.27 to 1.89, dependent on the VEM fraction, or the cross-linking density. SFG spectroscopy in conjunction with contact angle measurements revealed that the chemistry both at the film

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