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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, Ying-Jun Wang, Xuhui Huang, and Li Ren ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b04348 • Publication Date (Web): 01 Jul 2016 Downloaded from http://pubs.acs.org on July 3, 2016

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ACS Applied Materials & Interfaces

Controlling the Integration of Polyvinylpyrrolidone onto Substrate by Quartz Crystal Microbalance with Dissipation to Achieve Excellent Protein Resistance and Detoxification Jian Zheng1,3, Lin Wang2,3*, Xiangze Zeng2,4, Xiaoyan Zheng2,4, Yan Zhang1, Sa Liu1, Xuetao Shi3, Yingjun Wang1, Xuhui Huang2,4*, Li Ren1* 1. School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641, China. 2. Department of Chemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China. 3. National Engineering Research Center for Tissue Restoration & Reconstruction, South China University of Technology, Guangzhou 510006, China. 4. 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. * [email protected] * [email protected] * [email protected]

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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 2.6fold. 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.


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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 failure1, kidney failure2 and adult respiratory distress syndrome3. However, the bottleneck in blood purification is often the adsorbent. Many common adsorbents, such as cellulose4, polyethersulfone5 and polystyrenedivinylbenzene6, exhibit poor blood compatibility during the detoxification process. These materials can adsorb plasma proteins/platelets in the blood and cause thrombus, which could lead to serious consequences for the patients7-13. Surface modification of the adsorbent is an effective method to resolve this problem. Many functional molecules (e.g., polyethylene glycol (PEG)14, zwitterions15, poly(vinyl alcohol)16 and polyvinylpyrrolidone (PVP)17-19) have been used for modification. Among these molecules, researchers have screened the hydrophilic and nonionic PVP molecule. By reversible additionfragmentation chain transfer (RAFT) polymerization, PVP molecules of controlled chain lengths can be synthesized20-22, and these PVP molecules can be integrated onto the adsorbent by functional groups at their molecular terminal, such as thiol-terminated, amino-terminated23 or acid-terminated24 PVP, to improve the blood compatibility of the adsorbent25-26. Despite these promising properties, difficulty in controlling the integration of PVP molecules onto the adsorbent present a challenge for achieving desired polymer densities, which is critical for optimal protein resistance and detoxification by the adsorbent27-28. 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 detoxification29-30. 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


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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 surfaces3132

. Moreover, the mass of the molecules adsorbed onto the surface can be obtained in situ33-35. 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 proteins37-38. 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’ blood39-40. 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.

EXPERIMENTAL SECTION 1. Materials. N-vinylpyrrolidone (NVP, 98 %, Sigma-Aldrich, St. Louis, MO) was purified by distillation under reduced pressure to remove the inhibitors. 2,2-Azobisisobutyronitrile (AIBN, Acros, Geel, Belgium) was recrystallized in methanol three times. 1-Succinimidyl-4-cyano-4-[N-


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methyl-N-(4-pyridyl)carbamothioylthio]pentanoate (97 %, Sigma-Aldrich，St. Louis, MO, USA), bilirubin (BR, Sigma-Aldrich, St. Louis, MO, USA), human serum albumin (HSA, Sigma-Aldrich, St. Louis, MO, USA) and fibrinogen (Fg, Sigma-Aldrich, St. Louis, MO, USA) were purchased and used directly. 2. 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 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, 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-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 108.4 mg of N-hydroxysuccinimide. The reaction preceded at room temperature in nitrogen atmosphere in the dark for 24 h. Then, the mixture was dialyzed against


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water for 1 d in the dark and was subsequently lyophilized and marked as HS-PVP1, HS-PVP2 or HS-PVP3. We used the quartz crystal microbalance with 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, 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). 3. 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 mean square roughness (RMS) was determined from a height retrace image of


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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. 4. 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 non-adsorbed molecules. All of the processes were performed in a continuous flow mode (50 µL/min) at a stable temperature (25 ± 0.1 °C). 5. 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 package 41 with a general Amber force field 42

. The partial charge was derived by RESP fitting to a HF/6-31G* electrostatic potential 43. The

initial structure of HS-PVP and bilirubin were optimized at a HF/6-31G* level using an Gaussian package 44 and then placed in a cubic box of 6 × 6 × 6 nm3. The simulation box was then solvated by TIP3P water molecules

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. 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-


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rescale thermostat 46 in the NPT ensemble. The pressure was coupled by the Parrinello-Rahman method at 1 bar

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. A LINCS algorithm was used to restrain all the bonds

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, and the PME

method was applied to calculate the long-range electrostatic potential49. The cutoffs of the shortrange electrostatic potential and van der Waals potential were set to 1.2 nm and 1.1 nm, respectively. 6. 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. 7. 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 non-adherent 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).


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8. 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

Controlling the Integration of Polyvinylpyrrolidone ... - ACS Publications

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