Controlling the Integration of Polyvinylpyrrolidone ... - ACS Publications

Research Article www.acsami.org

Controlling the Integration of Polyvinylpyrrolidone onto Substrate by Quartz Crystal Microbalance with Dissipation To Achieve Excellent Protein Resistance and Detoxification Jian Zheng,†,∥ Lin Wang,*,‡,∥ Xiangze Zeng,‡,§ Xiaoyan Zheng,‡,§ Yan Zhang,† Sa Liu,† Xuetao Shi,∥ Yingjun Wang,† Xuhui Huang,*,‡,§ and Li Ren*,† †

School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641, China Department of Chemistry and §Hong Kong Branch of Chinese National Engineering Research Center for Tissue Restoration & Reconstruction, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong China ∥ National Engineering Research Center for Tissue Restoration & Reconstruction, South China University of Technology, Guangzhou 510006, China ‡

S Supporting Information *

ABSTRACT: Blood purification systems, in which the adsorbent removes exogenous and endogenous toxins from the blood, are widely used in clinical practice. To improve the protein resistance of and detoxification by the adsorbent, researchers can modify the adsorbent with functional molecules, such as polyvinylpyrrolidone (PVP). However, achieving precise control of the functional molecular density, which is crucial to the activity of the adsorbent, remains a significant challenge. In the present study, we prepared a model system for blood purification adsorbents in which we controlled the integration density of PVP molecules of different molecular weights on an Au substrate by quartz crystal microbalance with dissipation (QCM-D). We characterized the samples with atomic force microscopy, X-ray photoelectron spectroscopy, and QCM-D and found that the molecular density and the chain length of the PVP molecules played important roles in determining the properties of the sample. At the optimal condition, the modified sample demonstrated strong resistance to plasma proteins, decreasing the adsorption of human serum albumin (HSA) and fibrinogen (Fg) by 92.5% and 79.2%, respectively. In addition, the modified sample exhibited excellent detoxification, and the adsorption of bilirubin increased 2.6-fold. Interestingly, subsequent atomistic molecular dynamics simulations indicated that the favorable interactions between PVP and bilirubin were dominated by hydrophobic interactions. An in vitro platelet adhesion assay showed that the adhesion of platelets on the sample decreased and that the platelets were maintained in an inactivated state. The CCK-8 assay indicated that the modified sample exhibited negligible cytotoxicity to L929 cells. These results demonstrated that our method holds great potential for the modification of adsorbents in blood purification systems. KEYWORDS: protein resistance, detoxification, surface modification, bilirubin, quartz crystal microbalance with dissipation

■

INTRODUCTION Blood purification systems, which can remove exogenous and endogenous toxins from the blood of patients, have been widely used in the treatment of liver failure,1 kidney failure,2 and adult respiratory distress syndrome.3 However, the bottleneck in blood purification is often the adsorbent. Many common adsorbents, such as cellulose,4 poly(ether sulfone),5 and polystyrene− divinylbenzene,6 exhibit poor blood compatibility during the detoxification process. These materials can adsorb plasma © 2016 American Chemical Society

proteins/platelets in the blood and cause thrombus, which could lead to serious consequences for the patients.7−13 Surface modification of the adsorbent is an effective method to resolve this problem. Many functional molecules (e.g., poly(ethylene glycol) (PEG),14 zwitterions,15 poly(vinyl alcohol),16 Received: April 12, 2016 Accepted: July 1, 2016 Published: July 1, 2016 18684

DOI: 10.1021/acsami.6b04348 ACS Appl. Mater. Interfaces 2016, 8, 18684−18692

Research Article

ACS Applied Materials & Interfaces and polyvinylpyrrolidone (PVP)17−19) have been used for modification. Among these molecules, researchers have screened the hydrophilic and nonionic PVP molecule. By reversible addition− fragmentation chain transfer (RAFT) polymerization, PVP molecules of controlled chain lengths can be synthesized,20−22 and these PVP molecules can be integrated onto the adsorbent by functional groups at their molecular terminal, such as thiolterminated, amino-terminated,23 or acid-terminated24 PVP, to improve the blood compatibility of the adsorbent.25,26 Despite these promising properties, difficulty in controlling the integration of PVP molecules onto the adsorbent presents a challenge for achieving desired polymer densities, which is critical for optimal protein resistance and detoxification by the adsorbent.27,28 In particular, an insufficient number of functional molecules could lead to poor protein resistance, while excessive functional molecules would block the pores of the adsorbents and impair detoxification.29,30 To control molecular density on the surface, we adopted the quartz crystal microbalance with dissipation (QCM-D) technique. QCM-D has high sensitivity to detecting adsorption, conformation, and interactions between molecules and a surface in real time and thus serves as a powerful approach to regulating the integration of molecules onto material surfaces.31,32 Moreover, the mass of the molecules adsorbed onto the surface can be obtained in situ.33−35 In the present study, we successfully designed a model PVP surface that is resistant to plasma proteins but strongly adsorbs toxins in the blood and has promising properties for application in blood purification. We utilized QCM-D to precisely control the surface density of PVP molecules on an Au substrate. Au substrates have been widely used as model surfaces33−36 due to their simple structure and nonspecific adsorption of plasma proteins.37,38 We synthesized PVP molecules with different and narrowly distributed chain lengths by RAFT and introduced a thiol group at the end of the molecules (abbreviated as HS-PVP). Then, we utilized QCM-D to monitor the integration density of PVP molecules on the surface and used atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) to characterize the sample. We also used QCM-D to characterize the protein resistance of the sample against human serum albumin (HSA) and fibrinogen (Fg) and the detoxification of the common toxin bilirubin, which is difficult to remove from patients’ blood.39,40 Then, we employed molecule dynamics simulations to illustrate the interaction between the PVP molecules and bilirubin. The platelet adhesion of the sample was tested with platelet-rich plasma from rabbit blood, and the cytotoxicity of the sample was characterized with L929 cells by the CCK-8 assay.

■

cycles and sealed with a PTFE seal. Then, the system was placed in an oil bath at 70 °C and was terminated by liquid nitrogen at the desired time. The polymer was precipitated by ether and was dried in a vacuum box at room temperature until a constant weight was achieved. The PVP molecules were characterized using gel permeation chromatography (GPC, Viscotek GPC Max VE 2001, Malvern), and the results are shown in Figure S1. According to calculations performed using Omni SEC software, the PVP molecules under different preparation conditions had molecular weights of 1.36 × 103, 2.14 × 103, and 3.74 × 103, which were designated PVP1, PVP2, and PVP3, respectively. One gram of PVP1, PVP2, or PVP3 and 10 mL of DMF were added to a 25 mL round-bottom flask. The solution was degassed by nitrogen bubbling for 30 min and then was mixed with 72.1 mg of 2-aminoethanethiol, 197.1 mg of 1-(3-(dimethylamino)propyl)-3ethylcarbodiimide hydrochloride, and 108.4 mg of N-hydroxysuccinimide. The reaction preceded at room temperature in a nitrogen atmosphere in the dark for 24 h. Then, the mixture was dialyzed against water for 1 day in the dark and was subsequently lyophilized and marked as HS-PVP1, HS-PVP2, or HS-PVP3. We used the quartz crystal microbalance with the dissipation (QCM-D) technique with a Q-Sense E4 system (Q-Sense AB, Sweden) to control the integration of HS-PVP chains onto the Au substrate in real time to prepare Au-PVP. The HS-PVP molecules of different molecular weights were dissolved in ethanol at a concentration of 5 mg/mL. Before the preparation, the Au substrate was immobilized in the QCM chamber, and ethanol was injected to obtain a baseline. Then, the HS-PVP solution was injected to prepare the samples. After the desired time interval, ethanol was injected into the QCM chamber again to remove the nonadsorbed molecules. The Au-PVP samples were abbreviated as Au-PVP1-nmin, Au-PVP2-nmin, and Au-PVP3-nmin, with PVP1, PVP2, and PVP3 representing HS-PVP with molecular weights of 1.36 × 103, 2.14 × 103, and 3.74 × 103, respectively, and nmin representing the duration of the HS-PVP injection. All the processes were performed in continuous flow mode (30 μL/min) at a stable temperature (25 ± 0.1 °C). Characterization. The samples were analyzed using a Kratos AXis Ultra (DLD) (England) operated using an Al K (1486.4 eV) monochromatic X-ray source at a pressure of 2 × 10−9 Torr and a scan area of 0.7 mm × 0.3 mm. Analyses consisted of a survey scan performed at a pass energy of 140 eV to identify all of the species present, followed by high resolutions scans (55 eV) of the species of interest. The generated data were analyzed using XPSPEAK 41 software. The topography of the sample was characterized using an MFP-3D-S atomic force microscope (Asylum Research, America). The AFM images were collected under dry conditions at room temperature. Samples were analyzed over a 2.0 × 2.0 μm region at a resolution of 512 × 512 pixels. The root-meansquare roughness (RMS) was determined from a height retrace image of each sample. The static contact angle of the sample was characterized with a contact angle goniometer (OCA15, DADAPHYSICS, England) at 25 °C with distilled water. One microliter of distilled water was pumped onto the surface of the sample through a stainless steel needle at a rate of 1.0 μL/s. Protein Resistance and Detoxification Assay. The protein resistance and the detoxification of the samples were measured by QCM-D. Briefly, human serum albumin (HSA) and fibrinogen (Fg) were dissolved in PBS buffer (pH = 7.4) at a concentration of 1 mg/mL. The toxin bilirubin was dissolved in 0.01 M NaOH solution at a concentration of 2.5 mg/mL and then diluted 50 times with PBS buffer (pH = 7.4). During the assay, the samples were immobilized in the QCM chamber, and PBS buffer was injected to obtain a baseline. Then, the protein or bilirubin solution was injected into the chamber. After balancing, the PBS buffer was injected into the QCM chamber again to remove the nonadsorbed molecules. All of the processes were performed in a continuous flow mode (50 μL/min) at a stable temperature (25 ± 0.1 °C). Molecular Dynamics Simulation. In addition to the QCM-D assay, we also used molecular dynamics simulations to illustrate the interactions between HS-PVP and bilirubin. All simulations were performed using the Gromacs 4.5.4 package41 with a general Amber force field.42 The partial charge was derived by RESP fitting to a

EXPERIMENTAL SECTION

Materials. N-Vinylpyrrolidone (NVP, 98%, Sigma-Aldrich, St. Louis, MO) was purified by distillation under reduced pressure to remove the inhibitors. 2,2-Azobis(isobutyronitrile) (AIBN, Acros, Geel, Belgium) was recrystallized in methanol three times. 1-Succinimidyl-4-cyano-4[N-methyl-N-(4-pyridyl)carbamothioylthio]pentanoate (97%, SigmaAldrich, St. Louis, MO), bilirubin (BR, Sigma-Aldrich, St. Louis, MO), human serum albumin (HSA, Sigma-Aldrich, St. Louis, MO), and fibrinogen (Fg, Sigma-Aldrich, St. Louis, MO) were purchased and used directly. Sample Preparation. We used reversible addition−fragmentation chain transfer (RAFT) polymerization to synthesize PVP with different molecular weights. Briefly, the monomer (NVP), AIBN, and chain transfer agent (CTA) were dissolved in 4 mL of CH3CN in a roundbottom flask, and the concentrations of the reagents are shown in Table S1. The solution was degassed by three freeze−evacuate−thaw 18685


Research Article

ACS Applied Materials & Interfaces

Figure 1. Scheme of the preparation for HS-PVP.

1.36 × 103, 2.14 × 103, and 3.74 × 103, respectively. The H NMR results for the NVP, PVP2, and HS-PVP2 molecules shown in Figure S2 demonstrated that we successfully synthesized the compounds with thiol. We then used QCM-D to characterize the integration of different HS-PVP molecules onto the model Au substrate, and the results are shown in Figure 2. A decrease in Δf reflects the

HF/6-31G* electrostatic potential.43 The initial structures of HS-PVP and bilirubin were optimized at a HF/6-31G* level using an Gaussian package44 and then placed in a cubic box of 6 × 6 × 6 nm3. The simulation box was then solvated by TIP3P water molecules.45 Two Na ions were added to neutralize the system. The whole system was energy minimized by the steepest descent method followed by 500 ps position restrained equilibration in the NPT ensemble. Five 100 ns independent trajectories were performed with different initial velocities while the temperature was maintained at 300 K by a V-rescale thermostat46 in the NPT ensemble. The pressure was coupled by the Parrinello−Rahman method at 1 bar.47 A LINCS algorithm was used to restrain all the bonds,48 and the PME method was applied to calculate the long-range electrostatic potential.49 The cutoffs of the short-range electrostatic potential and van der Waals potential were set to 1.2 and 1.1 nm, respectively. Cytotoxicity Assay. L929 cells were obtained from ATCC (ATCCCCL-1) and were cultured in RMPI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (Excell) at 37 °C and in a 5% CO2 incubator. Before cell seeding, the materials were immersed in the medium for 24 h at 37 °C, and the extraction liquid was passed through a 0.22 μm filter membrane. The cells were dissociated using 0.25% trypsin/EDTA and seeded in a 96-well plate at a density of 2000 cells per well with the culture medium. After 24 h, the medium was replaced with fresh culture medium for the control group and with the extraction liquid for the experimental group. After being cultured for another 24 h, cell cytotoxicity was characterized using a CCK-8 kit (Dojindo) in accordance with the manufacturer’s instructions. Platelet Adhesion Assay. Platelet-rich plasma (PRP) was prepared by centrifuging whole rabbit blood at a rate of 1000 rpm for 15 min. Then, 1 mL of PRP was overlaid onto each sample and incubated in a water bath at 37 °C. After 1 h, the samples were gently rinsed with PBS buffer to remove the nonadherent platelets. The samples were fixed in a 2.5% glutaraldehyde solution at room temperature for 4 h, dehydrated in a gradient ethanol/distilled water mixture (50%, 60%, 70%, 80%, 90%, and 100% (v/v)) for 15 min each, and freeze-dried. Then, the samples were covered with a layer of gold and observed with a scanning electron microscopy (SEM, LEO1530VP, Zeiss, Germany). Statistical Analysis. The contact angle and cytotoxicity assay data are the mean values of three independent measurements and are shown as the mean ± standard deviation. Experimental results were analyzed using analysis of variance (ANOVA) to determine significant differences among the groups. Statistical significance was defined as p < 0.05.

1

Figure 2. QCM-D assay for the integration of different HS-PVP molecules on Au substrate.

growth of the sample mass, which was caused by the integration of the HS-PVP molecules. The results in Figure 2 illustrated that after the injection of the HS-PVP solution the mass of the sample reached a balance in 25 min. After 150 min of injection and washing with ethanol, the final Δf of Au-PVP1-150min, Au-PVP2-150min, and Au-PVP3-150min were −19.67, −21.56, and −24.42 Hz, respectively. Calculated using Q-Tool software, the results for the HS-PVP molecular density on Au-PVP1150-min, Au-PVP2-150-min and Au-PVP3-150min are shown in Table 1 and were 2.029, 2.408, and 3.108 μg/cm2, respectively. Furthermore, the PVP molecular ratios on Au-PVP1-150min, Au-PVP2-150min, and Au-PVP3-150min were 1:0.75:0.56, and the NVP repetitive units on Au-PVP1-150min, Au-PVP2150min, and Au-PVP3-150min demonstrated a 1:1.37:1.95 ratio. The AFM results in Figure 3a−d show that the pristine Au substrate possessed a plain and neat surface (Figure 3a). After the integration of HS-PVP molecules, Figure 3b−d shows that the morphologies of the substrates clearly changed to a rounded feature, and the PVP molecules distributed homogeneously on the surfaces. The root-mean-square roughness (RMS) increased with the growth of the PVP chain lengths and were 2.583, 3.091, and 3.595 nm, corresponding to Au-PVP1-150min, Au-PVP2150min, and Au-PVP3-150min, respectively. The XPS results of Au-PVP2-150min in Figure 3e show that the C 1s signal could be

■

RESULTS AND DISCUSSION We first used reversible addition−fragmentation chain transfer (RAFT) polymerization to synthesize PVP molecules with different chain lengths, and the process is shown in Figure 1. The GPC (gel permeation chromatography) results in Figure S1 indicated that PVP1, PVP2, and PVP3 had molecular weights of 18686


Research Article


Table 1. PVP Molecules Density and the Mass Decrease Percentage of HSA/Fg on the Indicated Samples Were Calculated Using Q-Tool Software HS-PVP1

5min 8min 30min 150min

HS-PVP2

HS-PVP3

density (μg/cm2)

decrease of HSA (%)

decrease of Fg (%)

density (μg/cm2)

decrease of HSA (%)

decrease of Fg (%)

density (μg/cm2)

decrease of HSA (%)

decrease of Fg (%)

0.806 1.207 1.708 2.029

46.3 64.0 76.1 84.6

18.9 36.4 58.1 66.2

1.075 1.421 2.036 2.408

49.1 71.3 85.2 92.5

29.1 52.7 71.0 79.2

1.779 2.239 2.727 3.108

47.3 65.6 81.0 87.3

23.3 40.5 60.4 70.9

Figure 3. AFM images of (a) Au, (b) Au-PVP1-150min, (c) Au-PVP2150min, (d) Au-PVP3-150min and the XPS results of (e) C 1s highresolution spectrum and (f) N 1s high-resolution spectrum of Au-PVP2150min.

Figure 4. (a) Controlling the integration of HS-PVP2 molecule on Au substrate with QCM-D. (b) Contact angle of the indicated samples with different HS-PVP2 molecular densities (n = 3).

adsorbed and unstable HS-PVP molecules were removed from Au-PVP-30min with washing by ethanol. The AFM results in Figure S4 show that there were rounded features on Au-PVP25min and Au-PVP2-30min, too. And compared to Au-PVP2150min, both of these surfaces exhibited smaller RMS of 1.308 and 2.489 nm as the lower density of PVP molecules, which corresponded to the QCM-D results in Figure 4a. In addition, the AFM results show that PVP molecules on Au-PVP2-5min and Au-PVP2-30min showed good homogeneity. It illustrated that during the preparation the PVP molecules could be integrated homogeneously onto the surface regardless of the density of PVP molecules. Figure S5 indicates that the integration of HS-PVP1 and HS-PVP3 can also be regulated by QCM-D. Table 1 show that the HS-PVP1 densities on Au-PVP1-5min, Au-PVP1-8min, Au-PVP1-30min, and Au-PVP1-150min were 0.806, 1.207, 1.708, and 2.029 μg/cm2, respectively, and the HS-PVP3 densities on Au-PVP3-5min, Au-PVP3-8min, Au-PVP3-30min, and Au-PVP3150min were 1.779, 2.239, 2.727, and 3.108 μg/cm2, respectively. We also found that the larger surface density of HS-PVP2 resulted in a more hydrophilic surface. We utilized contact angle analysis to measure surface hydrophobicity, and the results of the studied samples are shown in Figure 4b, which shows that as HS-PVP2 molecular density increased, the hydrophilicity of the surface obviously increased. The contact angle of the pristine Au surface was 68.6°, and the contact angles of Au-PVP2-5min, Au-PVP2-8min, Au-PVP2-30min, and Au-PVP2-150min were

deconvoluted into peaks for N−CO, C−N/C−S, and C−C/ C−H, which correspond to the binding energies 285.0, 286.0, and 287.8 eV, respectively.50−52 The XPS results in Figure 3f also show an evident N 1s peak from the PVP molecules on Au-PVP2-150min. In contrast, the XPS spectrum of the pristine Au substrate in Figure S3 contained a narrow C 1s peak and no evident N 1s peak. These AFM and XPS results suggested that the PVP molecules were integrated onto the Au substrate. Furthermore, we demonstrated that we could achieve precise control of surface density using QCM-D when integrating various HS-PVP molecules (e.g., HS-PVP2, HS-PVP1, and HSPVP3) of different chain lengths onto the substrate. As shown in Figure 4a, we could regulate the density of HS-PVP2 on the substrate by terminating the reaction at the desired time (5, 8, 30, or 150 min) to obtain Au-PVP2-5min, Au-PVP2-8min, Au-PVP2-30min, and Au-PVP2-150min, respectively. After washing with ethanol, the Δf values of these samples were −9.71, −12.82, −18.34, and −21.69 Hz, respectively. From calculations using Q-Tool software, the HS-PVP2 density on Au-PVP2-5min, Au-PVP2-8min, Au-PVP2-30min, and Au-PVP2-150min was determined to be 1.075, 1.421, 2.036, and 2.408 μg/cm2, respectively (shown in Table 1). Moreover, as shown in Figure 2, although the mass of the samples reached a balance after 25 min, the final PVP molecular densities of Au-PVP-30min and AuPVP-150min differed. This result illustrates that more physically 18687


Research Article

ACS Applied Materials & Interfaces 51.1°, 42.5°, 35.1°, and 28.7°, respectively. This change likely resulted from the presence of a PVP polymer brush on the surface, which can form hydrogen bonds with water molecules and generate a hydration layer on the surface to increase hydrophilicity.52 We further demonstrated that our designed surfaces are highly resistant to plasma proteins, including human serum albumin (HSA) and fibrinogen (Fg). The results in Figure 5 show the

Figure 6. QCM-D assay for protein resistance of Au, Au-PVP2-5min, Au-PVP2-8min, Au-PVP2-30min, and Au-PVP2-150min: (a) against human serum albumin (HSA) and (b) against fibrinogen (Fg).

explained by the hypothesis that a hydration layer is formed on the hydrophilic surface through hydrogen bonds between water and PVP, which become a “barrier” and reduce protein adsorption onto the material surface.54−59 Interestingly, although our surfaces did not adsorb plasma proteins, they demonstrated strong adsorption of toxins in blood. We characterized the interaction between Au-PVP2-150min and the toxin bilirubin by QCM-D (see Figure 7) and found that

Figure 5. QCM-D assay for the protein resistance of Au, Au-PVP1150min, Au-PVP2-150min, and Au-PVP3-150min: (a) against human serum albumin (HSA) and (b) against fibrinogen (Fg).

protein resistance of Au, Au-PVP1-150min, Au-PVP2-150min, and Au-PVP3-150min to HSA and Fg. The results indicate that after modification the protein resistance of the samples increased significantly. Table 1 show the results of calculations using Q-Tool software and comparisons to Au, which indicate that the mass of adsorbed HSA decreased by 84.6%, 92.5%, and 87.3%, and the mass of Fg decreased by 66.2%, 79.2%, and 70.9% on Au-PVP1-150min, Au-PVP2-150min, and Au-PVP3-150min, respectively. These results correspond to those of other studies, in which PVP molecules decreased the adsorption of plasma protein.53 Interestingly, the results also indicate that although Au-PVP2-150min did not possess the highest PVP molecular ratio or the most NVP repetitive units (as shown in Figure 2), this surface demonstrated better protein resistance than AuPVP1-150min or Au-PVP3-150min. Figure 6 shows the protein resistance of samples with different HS-PVP2 integration densities. With higher HS-PVP2 densities, the protein resistance of the sample clearly increased. The results in Table 1 show that the adsorbed mass of HSA decreased compared to Au by 49.2%, 71.2%, 85.2%, and 92.5%, and the mass of Fg decreased by 29.1%, 52.7%, 71.0%, and 79.2% on Au-PVP2-5min, Au-PVP2-8min, Au-PVP2-30min, and Au-PVP2-150 min, respectively. The results shown in Figure S6 also illustrate a similar trend in protein resistance for Au-PVP1-nmin and Au-PVP3-nmin, in which sample protein resistance was improving with HS-PVP molecular density increased. The improved protein resistance could be

Figure 7. QCM-D assay for bilirubin adsorption to Au and Au-PVP2150min.

the presence of PVP molecules on the surface could improve bilirubin removal. After reaching a balance and being washed with PBS buffer, the Δf values for Au and Au-PVP2-150min were −2.63 and −6.84 Hz, respectively. This observation indicated that the final mass of bilirubin on Au-PVP2-150min was 2.6-fold of that on a pristine substrate. We theorized that the strong adsorption of our surfaces to the blood toxin bilirubin is mainly due to hydrophobic interactions. To reveal the molecular interactions between bilirubin and the PVP surface, we performed atomistic molecular dynamics (MD) simulations (see Platelet Adhesion Assay section for details). We found a large contact surface area between HS-PVP and bilirubin, 18688


Research Article


bilirubin, surfaces consisting of HS-PVP demonstrate great promise for removing toxins such as bilirubin from blood samples. These results above show that compared to the conditional PVP blood purification systems, the PVP integration density could be controlled precisely in our system. Meanwhile, compared to the existing materials used for blood purification, such as PEGMA60,61 or zwitterionic polymers,62 the PVP system we developed not only resisted protein adsorption but also increased the removal of bilirubin. To examine the applicability of our surfaces for blood purification, we demonstrated that the HS-PVP surfaces are bloodcompatible and displayed negligible cytotoxicity. The results of the platelet adhesion assay shown in Figure 10 indicate that after

Figure 8. Distribution of the contact area between HS-PVP and bilirubin.

supporting the experimental observation of strong adsorption between these two molecules (see Figure 8). We further demonstrated that the interactions between the molecules are dominated by hydrophobic interactions, as 64.9% of the intermolecular contact pairs were hydrophobic−hydrophobic and less than 1% were of a hydrophilic−hydrophilic type. To further support this conclusion, we also found that no salt bridge interactions and negligible intermolecular hydrogen bonds (see Figure S7). In addition, we clearly identified two different binding modes based on the relative orientation of the first and fourth pyrrole-like rings of bilirubin (see Figure 9a). As shown in

Figure 10. SEM image of the rabbit blood platelet on different samples: (a) 1500× image of Au, (b) 5000× image of Au, (c) 1500× image of Au-PVP2-150min, and (d) 5000× image of Au-PVP2-150min.

being cultured with platelet-rich plasma for 2 h, large numbers of platelets were present on the pristine Au, and these platelets displayed evident pseudopodia. This result suggests that the platelets were activated to the stage of blood coagulation.63−65 Meanwhile, few platelets were found on Au-PVP2-150min, and most of them maintained their original round shape, reflecting the inactivated state of the platelets, which would not contribute to functional blood coagulation.63,66,67 The platelet adhesion assay with Au-PVP2-5min, Au-PVP2-8min, and Au-PVP2-30min in Figure S8 also indicated that platelet resistance could be improved with increased PVP molecular density on the surface. The CCK-8 results shown in Figure 11 indicate that compared to pure culture medium or that of the pristine substrate, the

Figure 9. (a) Distribution of the wing angles between the first and fourth pyrrole-like rings in bilirubin when interacting with HS-PVP molecule. (b) The two combination modes between bilirubin and HS-PVP molecule.

Figure 9b, in mode I, the bilirubin molecule is extended and parallel to the backbone of HS-PVP, whereas it is folded and holds the HS-PVP molecule with a hand-like shape in mode II. Because of these strong interactions between HS-PVP and

Figure 11. CCK-8 assay for the cytotoxicity of the indicated samples to L929 cells (n = 3). 18689


Research Article

ACS Applied Materials & Interfaces extracted fluid of Au-PVP2-150min did not exhibit evident cytotoxicity to L929 cells.

Membrane-Based Blood Purification System for Bioartificial Liver Support. Artif. Organs 1999, 23, 319−330. (2) Ronco, C.; Honore, P. Renal Support in Critically Ill Patients with Acute Kidney Injury. N. Engl. J. Med. 2008, 359, 1961−1962. (3) Abraham, E.; Matthay, M. A.; Dinarello, C. A.; Vincent, J.-L.; Cohen, J.; Opal, S. M.; Glauser, M.; Parsons, P.; Fisher, C. J., Jr.; Repine, J. E. Consensus Conference Definitions for Sepsis, Septic Shock, Acute Lung Injury, and Acute Respiratory Distress Syndrome: Time for a Reevaluation. Crit. Care Med. 2000, 28, 232−235. (4) Yan, L.; Ishihara, K. Graft Copolymerization of 2-methacryloyloxyethyl Phosphorylcholine to Cellulose in Homogeneous Media using Atom Transfer Radical Polymerization for Providing New Hemocompatible Coating Materials. J. Polym. Sci., Part A: Polym. Chem. 2008, 46, 3306−3313. (5) Ran, F.; Nie, S.; Zhao, W.; Li, J.; Su, B.; Sun, S.; Zhao, C. Biocompatibility of Modified Polyethersulfone Membranes by Blending an Amphiphilic Triblock co-Polymer of Poly (vinyl pyrrolidone)−bPoly (methyl methacrylate)−b-Poly (vinyl pyrrolidone). Acta Biomater. 2011, 7, 3370−3381. (6) Weber, V.; Linsberger, I.; Hauner, M.; Leistner, A.; Leistner, A.; Falkenhagen, D. Neutral Styrene Divinylbenzene Copolymers for Adsorption of Toxins in Liver Failure. Biomacromolecules 2008, 9, 1322−1328. (7) Lee, B. S.; Lee, J. K.; Kim, W.-J.; Jung, Y. H.; Sim, S. J.; Lee, J.; Choi, I. S. Surface-Initiated, Atom Transfer Radical Polymerization of Oligo (ethylene glycol) Methyl ether Methacrylate and Subsequent Click Chemistry for Bioconjugation. Biomacromolecules 2007, 8, 744−749. (8) Courtney, J.; Lamba, N.; Sundaram, S.; Forbes, C. Biomaterials for Blood-Contacting Applications. Biomaterials 1994, 15, 737−744. (9) Chen, H.; Yuan, L.; Song, W.; Wu, Z.; Li, D. Biocompatible Polymer Materials: Role of Protein−surface Interactions. Prog. Polym. Sci. 2008, 33, 1059−1087. (10) Castner, D. G.; Ratner, B. D. Biomedical Surface Science: Foundations to Frontiers. Surf. Sci. 2002, 500, 28−60. (11) Brisbois, E. J.; Hitesh, H.; Meyerhoff, M. E. In Advanced Polymers in Medicine; Puoci, F., Ed.; Springer International Publishing: Switzerland, 2015; Chapter 16, pp 481−511. (12) Horbett, T. A. Principles Underlying the Role of Adsorbed Plasma Proteins in Blood Interactions with Foreign Materials. Cardiovasc. Pathol. 1993, 2, 137−148. (13) Schopka, S.; Schmid, T.; Schmid, C.; Lehle, K. Current Strategies in Cardiovascular Biomaterial Functionalization. Materials 2010, 3, 638−655. (14) Ahmad, A.; Abdulkarim, A.; Ooi, B.; Ismail, S. Recent Development in Additives Modifications of Polyethersulfone Membrane for Flux Enhancement. Chem. Eng. J. 2013, 223, 246−267. (15) Liu, P.-S.; Chen, Q.; Liu, X.; Yuan, B.; Wu, S.-S.; Shen, J.; Lin, S.-C. Grafting of Zwitterion from Cellulose Membranes via ATRP for Improving Blood Compatibility. Biomacromolecules 2009, 10, 2809− 2816. (16) Eichhorn, T.; Ivanov, A. E.; Dainiak, M. B.; Leistner, A.; Linsberger, I.; Jungvid, H.; Mikhalovsky, S. V.; Weber, V. Macroporous Composite Cryogels with Embedded Polystyrene Divinylbenzene Microparticles for the Adsorption of Toxic Metabolites from Blood. J. Chem. 2013, 2013, 348412. (17) Wetzels, G. M.; Koole, L. H. Photoimmobilisation of Poly (Nvinylpyrrolidinone) as a Means to Improve Haemocompatibility of Polyurethane Biomaterials. Biomaterials 1999, 20, 1879−1887. (18) Wan, L. S.; Xu, Z. K.; Huang, X. J.; Wang, Z. G.; Ye, P. Hemocompatibility of Poly (acrylonitrile-co-N-vinyl-2-pyrrolidone): Swelling Behavior and Water States. Macromol. Biosci. 2005, 5, 229− 236. (19) Wan, L.-S.; Xu, Z.-K.; Huang, X. J.; Wang, Z. G.; Wang, J. L. Copolymerization of Acrylonitrile with N-vinyl-2-pyrrolidone to Improve the Hemocompatibility of Polyacrylonitrile. Polymer 2005, 46, 7715−7723. (20) Jana, S.; Parthiban, A. Cyclic-Amine-Based Dithiocarbamate Chain Transfer Agents for the RAFT Polymerization of Less Activated Monomers. Macromol. Chem. Phys. 2011, 212, 790−798.

■

CONCLUSIONS In the present study, we were able to control the integration density of PVP molecules of different chain lengths onto a substrate by quartz crystal microbalance with dissipation (QCM-D). After being modified with PVP molecules at an optimal condition (Au-PVP2-150min), the sample demonstrated decreased adsorption of HSA and Fg by 92.5% and 79.2%, respectively. In addition, bilirubin removal by the surface increased 2.6-fold, and the results of all-atom MD simulations indicated that the strong adsorption of bilirubin to the PVP molecule is mainly due to hydrophobic interactions. This modified surface was able to decrease the adhesion of platelets and keep platelets in an inactivated status. Meanwhile, the material demonstrated negligible cytotoxicity to L929 cells. This model surface modification method provides a means of achieving precise control of the surface density of the PVP and demonstrates significant potential as an adsorbent for blood purification systems in the clinic.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b04348. Synthesis and gel permeation chromatography of the PVP molecules, 1H NMR spectra of the molecules, XPS results for Au, AFM results of Au-PVP2-5min and Au-PVP230min, the results of controlling the integration of HS-PVP1 and HS-PVP3 onto the substrates, protein resistance of Au-PVP1-nmin and Au-PVP3-nmin, hydrophobic solvent accessible surface area of HS-PVP and bilirubin in the adsorption process, intermolecular hydrogen bonds between bilirubin and HS-PVP, and SEM image of rabbit blood platelets on Au-PVP2-5min, Au-PVP28min, and Au-PVP2-30min (PDF)

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (L.W.). *E-mail: [email protected] (X.H.). *E-mail: [email protected] (L.R.). Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors thank the National Basic Research Program of China (2012CB619100), the National Natural Science Foundation of China (Grants 51232002, 51273072, and 51302088), the Hong Kong Scholars Program (XJ2015016), the Innovation and Technology Commission (ITC-CNERC14SC01), the Natural Science Foundation of Guangdong (2012A080800015), the Guangdong Scientific and Technological Project (2014B090907004), and Guangdong Natural Science Funds for Distinguished Young Scholar (2016A030306018). We thank Miss Carmen K. M. Tse for the helpful discussions.

■

REFERENCES

(1) Stange, J.; Mitzner, S.; Risler, T.; Erley, C.; Lauchart, W.; Goehl, H.; Klammt, S.; Peszynski, P.; Freytag, J.; Hickstein, H. Molecular Adsorbent Recycling System (MARS): Clinical Results of a New 18690


Research Article

ACS Applied Materials & Interfaces (21) Guinaudeau, A.; Mazières, S.; Wilson, D. J.; Destarac, M. Aqueous RAFT/MADIX Polymerisation of N-vinyl pyrrolidone at Ambient Temperature. Polym. Chem. 2012, 3, 81−84. (22) Moad, G.; Rizzardo, E.; Thang, S. H. Toward Living Radical Polymerization. Acc. Chem. Res. 2008, 41, 1133−1142. (23) Tang, Z.; Li, D.; Wang, X.; Gong, H.; Luan, Y.; Liu, Z.; Brash, J. L.; Chen, H. A t-PA/Nanoparticle Conjugate with Fully Retained Enzymatic Activity and Prolonged Circulation Time. J. Mater. Chem. B 2015, 3, 977−982. (24) McDowall, L.; Chen, G.; Stenzel, M. H. Synthesis of Seven-Arm Poly (vinyl pyrrolidone) Star Polymers with Lysozyme Core Prepared by MADIX/RAFT Polymerization. Macromol. Rapid Commun. 2008, 29, 1666−1671. (25) Liu, X.; Xu, Y.; Wu, Z.; Chen, H. Poly (N - vinylpyrrolidone)Modified Surfaces for Biomedical Applications. Macromol. Biosci. 2013, 13, 147−154. (26) Higuchi, A.; Shirano, K.; Harashima, M.; Yoon, B. O.; Hara, M.; Hattori, M.; Imamura, K. Chemically Modified Polysulfone Hollow Fibers with Vinylpyrrolidone having Improved Blood Compatibility. Biomaterials 2002, 23, 2659−2666. (27) Feng, W.; Brash, J. L.; Zhu, S. Non-biofouling Materials Prepared by Atom Transfer Radical Polymerization Grafting of 2-methacryloloxyethyl Phosphorylcholine: Separate Effects of Graft Density and Chain Length on Protein Repulsion. Biomaterials 2006, 27, 847−855. (28) Feng, W.; Zhu, S.; Ishihara, K.; Brash, J. L. Adsorption of Fibrinogen and Lysozyme on Silicon Grafted with Poly (2methacryloyloxyethyl Phosphorylcholine) via Surface-initiated Atom Transfer Radical Polymerization. Langmuir 2005, 21, 5980−5987. (29) Edmondson, S.; Osborne, V. L.; Huck, W. T. Polymer Brushes via Surface-initiated Polymerizations. Chem. Soc. Rev. 2004, 33, 14−22. (30) Davankov, V.; Pavlova, L.; Tsyurupa, M.; Brady, J.; Balsamo, M.; Yousha, E. Polymeric Adsorbent for Removing Toxic Proteins from Blood of Patients with Kidney Failure. J. Chromatogr., Biomed. Appl. 2000, 739, 73−80. (31) Munro, J. C.; Frank, C. W. Polyacrylamide Adsorption from Aqueous Solutions on Gold and Silver Surfaces Monitored by the Quartz Crystal Microbalance. Macromolecules 2004, 37, 925−938. (32) Höök, F.; Vörös, J.; Rodahl, M.; Kurrat, R.; Böni, P.; Ramsden, J.; Textor, M.; Spencer, N.; Tengvall, P.; Gold, J. A Comparative Study of Protein Adsorption on Titanium Oxide Surfaces using in Situ Ellipsometry, Optical Waveguide Lightmode Spectroscopy, and Quartz Crystal Microbalance/Dissipation. Colloids Surf., B 2002, 24, 155−170. (33) Wu, J.; Mao, Z.; Gao, C. Controlling the Migration Behaviors of Vascular Smooth Muscle Cells by Methoxy Poly (ethylene glycol) Brushes of Different Molecular Weight and Density. Biomaterials 2012, 33, 810−820. (34) Liu, G.; Cheng, H.; Yan, L.; Zhang, G. Study of the Kinetics of the Pancake-to-brush Transition of Poly (N-isopropylacrylamide) Chains. J. Phys. Chem. B 2005, 109, 22603−22607. (35) Slavin, S.; Soeriyadi, A. H.; Voorhaar, L.; Whittaker, M. R.; Becer, C. R.; Boyer, C.; Davis, T. P.; Haddleton, D. M. Adsorption Behaviour of Sulfur Containing Polymers to Gold Surfaces using QCM-D. Soft Matter 2012, 8, 118−128. (36) Slavin, S.; Haddleton, D. M. An Investigation into Thiol−ene Surface Chemistry of Poly (ethylene glycol) Acrylates, Methacrylates and CCTP Polymers via Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D). Soft Matter 2012, 8, 10388−10393. (37) Senaratne, W.; Andruzzi, L.; Ober, C. K. Self-assembled Monolayers and Polymer Brushes in Biotechnology: Current Applications and Future Perspectives. Biomacromolecules 2005, 6, 2427−2448. (38) Kanagaraja, S.; Lundström, I.; Nygren, H.; Tengvall, P. Platelet Binding and Protein Adsorption to Titanium and Gold after Short Time Exposure to Heparinized Plasma and Whole Blood. Biomaterials 1996, 17, 2225−2232. (39) Wu, G.; Brown, G. Adsorption of Bilirubin by Amine-containing Polyacrylamide Resins. React. Polym. 1991, 14, 49−61.

(40) Lavin, A.; Sung, C.; Klibanov, A. M.; Langer, R. Enzymatic Removal of Bilirubin from Blood: a Potential Treatment for Neonatal Jaundice. Science 1985, 230, 543−545. (41) Pronk, S.; Páll, S.; Schulz, R.; Larsson, P.; Bjelkmar, P.; Apostolov, R.; Shirts, M. R.; Smith, J. C.; Kasson, P. M.; van der Spoel, D. GROMACS 4.5: a High-throughput and Highly Parallel Open Source Molecular Simulation Toolkit. Bioinformatics 2013, btt055. (42) Wang, J.; Wolf, R. M.; Caldwell, J. W.; Kollman, P. A.; Case, D. A. Development and Testing of a General Amber Force Field. J. Comput. Chem. 2004, 25, 1157−1174. (43) Bayly, C. I.; Cieplak, P.; Cornell, W.; Kollman, P. A. A Wellbehaved Electrostatic Potential Based Method using Charge Restraints for Deriving Atomic Charges: the RESP Model. J. Phys. Chem. 1993, 97, 10269−10280. (44) Frisch, M.; Trucks, G.; Schlegel, H.; Scuseria, G.; Robb, M.; Cheeseman, J.; Montgomery, Jr., J.; Vreven, T.; Kudin, K.; Burant, J. Gaussian 03, Revision D. 01; Gaussian Inc.: Wallingford, CT, 2004; p 26. (45) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L. Comparison of Simple Potential Functions for Simulating Liquid Water. J. Chem. Phys. 1983, 79, 926−935. (46) Bussi, G.; Donadio, D.; Parrinello, M. Canonical Sampling through Velocity Rescaling. J. Chem. Phys. 2007, 126, 014101. (47) Parrinello, M.; Rahman, A. Polymorphic transitions in single crystals: A New Molecular Dynamics Method. J. Appl. Phys. 1981, 52, 7182−7190. (48) Hess, B.; Bekker, H.; Berendsen, H. J.; Fraaije, J. G. LINCS: a Linear Constraint Solver for Molecular Simulations. J. Comput. Chem. 1997, 18, 1463−1472. (49) Darden, T.; York, D.; Pedersen, L. Particle mesh Ewald: An N· Log (N) Method for Ewald Sums in Large Systems. J. Chem. Phys. 1993, 98, 10089−10092. (50) McArthur, S. L.; McLean, K. M.; St. John, H. A.; Griesser, H. J. XPS and Surface-MALDI-MS Characterisation of Worn HEMA-based Contact Lenses. Biomaterials 2001, 22, 3295−3304. (51) Khan, M.; Armes, S.; Perruchot, C.; Ouamara, H.; Chehimi, M.; Greaves, S.; Watts, J. Surface Characterization of Poly (3, 4ethylenedioxythiophene)-coated Latexes by X-ray Photoelectron Spectroscopy. Langmuir 2000, 16, 4171−4179. (52) Telford, A. M.; James, M.; Meagher, L.; Neto, C. Thermally Cross-linked PVP Films as Antifouling Coatings for Biomedical Applications. ACS Appl. Mater. Interfaces 2010, 2, 2399−2408. (53) Wu, Z.; Chen, H.; Liu, X.; Zhang, Y.; Li, D.; Huang, H. Protein Adsorption on Poly (N-vinylpyrrolidone)-Modified Silicon Surfaces Prepared by Surface-initiated Atom Transfer Radical Polymerization. Langmuir 2009, 25, 2900−2906. (54) Jiang, S.; Cao, Z. Ultralow-fouling, Functionalizable, and Hydrolyzable Zwitterionic Materials and Their Derivatives for Biological Applications. Adv. Mater. 2010, 22, 920−932. (55) Katira, P.; Agarwal, A.; Fischer, T.; Chen, H. Y.; Jiang, X.; Lahann, J.; Hess, H. Quantifying the Performance of Protein-Resisting Surfaces at Ultra-Low Protein Coverages Using Kinesin Motor Proteins as Probes. Adv. Mater. 2007, 19, 3171−3176. (56) Ma, H.; Hyun, J.; Stiller, P.; Chilkoti, A. Non-Fouling” Oligo (ethylene glycol)-Functionalized Polymer Brushes Synthesized by Surface-Initiated Atom Transfer Radical Polymerization. Adv. Mater. 2004, 16, 338−341. (57) Cheng, G.; Li, G.; Xue, H.; Chen, S.; Bryers, J. D.; Jiang, S. Zwitterionic Carboxybetaine Polymer Surfaces and Their Resistance to Long-term Biofilm Formation. Biomaterials 2009, 30, 5234−5240. (58) Raschke, T. M. Water Structure and Interactions with Protein Surfaces. Curr. Opin. Struct. Biol. 2006, 16, 152−159. (59) Herrwerth, S.; Eck, W.; Reinhardt, S.; Grunze, M. Factors that Determine the Protein Resistance of Oligoether Self-assembled Monolayers-internal Hydrophilicity, Terminal Hydrophilicity, and Lateral Packing Density. J. Am. Chem. Soc. 2003, 125, 9359−9366. (60) Chang, Y.; Shih, Y.-J.; Ruaan, R.-C.; Higuchi, A.; Chen, W.-Y.; Lai, J.-Y. Preparation of Poly (vinylidene fluoride) Microfiltration Membrane with Uniform Surface-copolymerized Poly (ethylene glycol) 18691


Research Article

ACS Applied Materials & Interfaces Methacrylate and Improvement of Blood Compatibility. J. Membr. Sci. 2008, 309, 165−174. (61) Salimi, E.; Ghaee, A.; Ismail, A. F.; Othman, M. H. D.; Sean, G. P. Current Approaches in Improving Hemocompatibility of Polymeric Membranes for Biomedical Application. Macromol. Mater. Eng. 2016, 301, 771. (62) Liu, P.-S.; Chen, Q.; Wu, S.-S.; Shen, J.; Lin, S.-C. Surface Modification of Cellulose Membranes with Zwitterionic Polymers for Resistance to Protein Adsorption and Platelet Adhesion. J. Membr. Sci. 2010, 350, 387−394. (63) Goodman, S.; Grasel, T.; Cooper, S.; Albrecht, R. Platelet Shape Change and Cytoskeletal Reorganization on Polyurethaneureas. J. Biomed. Mater. Res. 1989, 23, 105−123. (64) Iwasaki, Y.; Nakabayashi, N.; Ishihara, K. Preservation of Platelet Function on 2-methacryloyloxyethyl Phosphorylcholine-graft Polymer as Compared to Various Water-Soluble Graft Polymers. J. Biomed. Mater. Res. 2001, 57, 72−78. (65) Huang, Q.; Yang, Y.; Hu, R.; Lin, C.; Sun, L.; Vogler, E. A. Reduced Platelet Adhesion and Improved Corrosion Resistance of Superhydrophobic TiO 2-nanotube-coated 316L Stainless Steel. Colloids Surf., B 2015, 125, 134−141. (66) Goodman, S. L. Sheep, Pig, and Human Platelet-material Interactions with Model Cardiovascular Biomaterials. J. Biomed. Mater. Res. 1999, 45, 240−250. (67) Liu, X.; Yuan, L.; Li, D.; Tang, Z.; Wang, Y.; Chen, G.; Chen, H.; Brash, J. L. Blood Compatible Materials: State of the Art. J. Mater. Chem. B 2014, 2, 5718−5738.

18692
