Thermoresponsive Self-Assembled Î²-Cyclodextrin-Modified Surface

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Thermoresponsive Self-Assembled #-CyclodextrinModified Surface for Blood Purification sa liu, Chunting Zhong, Junjian Chen, Jiezhao Zhan, Jingcai He, Yuchen Zhu, Ying-Jun Wang, Lin Wang, and Li Ren ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00156 • Publication Date (Web): 13 Apr 2017 Downloaded from http://pubs.acs.org on April 18, 2017

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ACS Biomaterials Science & Engineering

Thermoresponsive Self-Assembled β-Cyclodextrin-Modified Surface for Blood Purification Sa Liu1, Chunting Zhong 2, Junjian Chen1, Jiezhao Zhan2, Jingcai He2, Yuchen Zhu1, Yingjun Wang1, Lin Wang2*, Li Ren1* 1. School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641, China. 2. National Engineering Research Center for Tissue Restoration and Reconstruction, South China University of Technology, Guangzhou 510006, China. * [email protected] * [email protected]

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ABSTRACT For patients with liver failure, bilirubin (BR) is one of the endogenous toxins in their blood. Although blood purification can remove the bilirubin from the body in clinic, the detoxification system needs to be improved, and the cost needs to be decreased. In the present study, we developed a recyclable model surface that can strongly remove bilirubin. We first prepared adamantane (Ad) on a model gold surface by self-assembly. Then, we integrated the βcyclodextrin dimer (CDD) onto the surface with host-guest interactions between one of the CD cavities in the CDD and Ad. We characterized the surface with XPS, static contact angle measurements and AFM. In addition, we employed QCM-D to characterize the preparation process as well as the detoxification of the surface. We demonstrated that this modified surface could strongly adsorb bilirubin through host-guest interactions between the CD cavities in the CDD and bilirubin, and the detoxification was improved by 1.7 times (compared to the surface only with Ad). Interestingly, after characterization with QCM-D, this surface could be recycled due to the thermoresponsive property of the host-guest interaction between the CDD and Ad. After adsorbing the toxin and increasing the temperature to 45 °C, the CDD with bilirubin could be removed from the surface. Then, the refreshed surface with CDD could be prepared again at room temperature. This cycle could be repeated at least 3 times. Additionally, during each cycle, the modified surface exhibited good detoxification to bilirubin. This modified surface also showed strong resistance to plasma proteins, decreasing the adsorption of human serum albumin (HSA) and fibrinogen (Fg). An in vitro platelet adhesion assay showed that the adhesion of the platelets on the modified surface decreased and that the platelets were in an inactivated state. The hemolysis assay showed that this surface exhibited no hemolysis activity in the samples to red blood cells (RBCs). The CCK-8 assay showed that this surface had negligible cytotoxicity to


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L929 cells. This work has taken the advantages of the host-guest self-assembly between β-CD and BR/Ad for special recognizing adsorption, as well as the thermoresponse of β-CD-Ad inclusion for recyclable application, and these results demonstrate that this technology has great potential for removing bilirubin and decreasing clinic costs. KEYWORDS: Bilirubin; β-Cyclodextrin; Thermoresponsive; Host-guest; Self-assembly


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1. INTRODUCTION Blood purification is an important technology for removing the toxins in the blood of patients with liver failure,1 adult respiratory distress syndrome2 and other diseases in clinic.3 As reported, there is an increasing number of patients that require blood purification in the world.4-6 As the most common small molecular toxin for patients with liver failure, bilirubin is a pathogenic toxin that is metabolized from porphyrin chemicals.7 The metabolism disorders of bilirubin

may cause

yellow

discoloration

of

the

skin,

called

hyperbilirubinemia.8

Hyperbilirubinemia causes hemolytic or hepatocellular jaundice and selectively affects the central nervous system, which results in cerebral palsy, mental retardation, hearing loss, epilepsy, and even death.9 To remove bilirubin from a patient’s blood, researchers have employed many kinds of adsorbents, such as mesoporous silica,10 activated carbon,7, 11-13 resins1416

or carbon nanotubes,12-14,

17

for application in hemoperfusion. Meanwhile, many modified

methods have been used to improve the adsorbing properties of removing bilirubin. For example, Timin, A. S. et al. modified the hybrid-silica materials with octyl, phenyl and urea-propyl functional groups or the guanidine-containing polymers to increase the bilirubin adsorption capacity.18-19

Jiang,

X.

et

al.

found

that

amino-grafted

PES/PGMA

(polyethersulfone/poly(glycidyl methacrylate)) particles have shown great potential for the removal of bilirubin.9 Kavoshchian, M., et al. have examined thehuman serum albumin (HSA) immobilized poly(2-hydroxyethylmethacrylate) as an alternative sorbent in hemoperfusion columns that achieved a maximum bilirubin removal of 25.4 mg/g at 37.5 °C.20 These adsorbents exhibited good hemocompatibility and have been applied in clinic. However, there is a challenge for improving the removal rate of bilirubin and decreasing patient costs.8, 21


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Recently, researchers screened the interactions between bilirubin and cyclodextrin (CD).2223

Cyclodextrin is an abbreviation of a series of annular oligosaccharides that are connected by α-

1,4- glucosidic bonds.24-25 The most widely used cyclodextrins have six, seven or eight glucopyranose molecules, which are named α-, β- or γ-cyclodextrin, respectively. Due to their specific structures with cavities and abundant hydroxyl groups, the cyclodextrin molecules exhibit external hydrophilicity and internal hydrophobicity.26 This structure contributes to a particular property of cyclodextrin that enables self-assembly with hydrophobic molecules that are similar in size to the cavity through host-guest interactions.27 Driven by electrostatic forces/hydrogen-bonds, these host-guest interactions form inclusion complexes.28 Researchers have found host-guest interactions between β-CD and bilirubin. In addition, there have been several kinds of adsorbents with β-CD for bilirubin removal, such as β-CD-chitosan,29 β-CD-PEI (polyethyleneimine),30 and β-CD-co-EGDMA (ethylene glycol dimethylacrylate)31. In addition to interacting with bilirubin, β-CD exhibits another classical host-guest interaction with adamantane (Ad).32 In the last few decades, this interaction has been applied in several fields, including hydrogel crosslinking,33 drug loading,34 biological detection35 and water purification.36-37 Interestingly, this host-guest interaction has thermoresponsive property38-39, which means self-assembly occurs below the corresponding temperature (T-point) and would disappear above the T-point. The T-point is often located between 20 °C and 70 °C, depending on the distance between the inclusion complex and polymer backbone.40-42 In the present study, to provide a method for improving the removal rate of bilirubin and decreasing patients’ costs, we prepared a recyclable model surface that could remove bilirubin based on the host-guest interactions between β-CD and bilirubin/Ad in this study. We employed gold as our model surface because it has a simple structure and shows nonspecific adsorption


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toward plasma proteins43. First, we modified the Ad molecules with the sulfhydryl polyethylene glycol (abbreviated as HS-PEG-Ad), and integrated this HS-PEG-Ad onto the Au surface using self-assembly. And this model should be easily modified on the adsorbents with C=C bonds, e.g., poly(styrene-divinylbenzene)microsphere for clinical application.44-45 Then, we synthesized the β-cyclodextrin dimer (CDD). We used one β-CD cavity to integrate the CDD onto the Au surface with HS-PEG-Ad, and this interaction exhibited thermoresponsive property, making the surface recyclable. After that, we used the other β-CD cavity of the CDD to interact with bilirubin. We employed a quartz crystal microbalance with dissipation (QCM-D) to characterize the whole process of the assembly, as well as the bilirubin removal, recyclable utilization and protein resistance of the surface. As a gravimetrical device, QCM-D can measure adsorbed mass through decreases in resonant frequency and was initially used to analyze the protein assembly and conformation changes.46 The famous Sauerbrey equation elaborates the directly proportional relation between ∆F, the frequency change of the quartz crystal, and the adsorbed mass: a decrease in ∆F means an increase in the mass.47 In addition, we characterized the surface by Xray photoelectron spectroscopy (XPS), atomic force microscopy (AFM) and contact angle goniometer (CA) measurements. We used platelet-rich plasma and red blood cells suspension to evaluate the hemocompatibility of the surface, and used a CCK-8 assay to research the cytotoxicity toward L929 cells. 2. MATERIALS AND METHODS 2.1. Materials. The gold substrate was purchased from Foxconn Nanotechnology Research Center (Beijing, China), which was made by depositing gold particles on a silicon wafer (10*10 mm-2).


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Sulfhydryl/thiol and NHS heterofunctionalized polyethylene glycol (HS-PEG-NHS, MW 2,000) were purchased from NANOCS (New York, USA). β-Cyclodextrin (β-CD) was purchased from Sinopharm Chemical Reagent Company (Shanghai, China). 2-Iodoxybenzoic acid (IBX) was purchased from Jinmao Chemical Ltd. (Shanghai, China). 1,12-Diaminododecane (DAD, 98%) was purchased from J&K Chemical Ltd. (Beijing, China). Sodium borohydride was from Tianjin Yongda Chemical Reagent Co., Ltd. (Tianjin, China). The cellulose ester dialysis bags (1,000 Da) were purchased from Shanghai Haling Biological Technology Co., Ltd. (Shanghai, China). Sodium chloride, bilirubin (95%), dichloromethane (AR), ethylenediaminetetraacetic acid (EDTA, AR) and isoamyl acetate (AR) were purchased from Aldrich Industrial Corporation (California, USA). Human serum albumin (HSA, ≥ 96%) and fibrinogen (Fg, 35-65%) from human plasma were purchased from Sigma (Missouri, USA). The anticoagulant rabbit blood was purchased from Guangzhou Hongquan Biotechnology Co., Ltd. (Guangzhou, China). Paraformaldehyde-glutaraldehyde immobile liquid was purchased from Beijing Leagene Biotech. Co., Ltd. (Beijing, China). The L929 cells were obtained from ATCC (Maryland, USA). The cell count kit-8 (CCK-8) was purchased from Shanghai Beyotime Institute (Shanghai, China). RMPI 1640 medium, calcium- and magnesium-free phosphate buffered saline (PBS, pH 7.4, 1X), fetal bovine serum and 0.25 % trypsin/EDTA were purchased from Gibco BRL Co. Ltd. (California, USA). The 0.22 µm filter membrane was purchased from Merck Millipore Ltd. (Massachusetts, USA). All other materials and solvents were of analytical reagent grade. 2.2. Synthesis of the sulfhydryl and adamantane heterofunctionalized polyethylene glycol (HS-PEG-Ad). The sulfhydryl and adamantane heterofunctionalized polyethylene glycol (HS-PEG-Ad) was synthesized according to a previous report48 with a few modifications. Briefly, we mixed Ad-


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NH3Cl (4.69 mg, 0.025 mmol, 5.0 equiv.), HS-PEG-NHS (10 mg, 0.005 mmol, 1.0 equiv.) and Et3N (2.58 mg, 0.0255 mmol, 5.1 equiv.) into 1 mL of dichloromethane sequentially. After stirring at 25 °C for 2 h, the solvent was subsequently removed in a vacuo drying oven. Then, we dissolved the residue in water and centrifuged the system at 4,000 rpm for 10 min to remove the unreacted Ad-NH3Cl. The solution was lyophilized to yield HS-PEG-Ad. The composition of HS-PEG-Ad was determined using 1H NMR and 13C NMR (AVANCE Digital 400 MHz, Bruker, Karlsruhe, Germany) spectral measurements at 400 MHz. 2.3. Synthesis of the β-cyclodextrin dimer (CDD). First, we synthesized the β-cyclodextrin monoacetal (CDMA) as follows. Briefly, we dissolved β-CD (1.135 g, 0.001 mol) and IBX (364 mg, 0.001 mol) in 12 mL of DMSO in a 50 mL stand-up round bottom flask and stirred the system at 25 °C for 24 h. We obtained the residue by adding 180 mL of acetone and centrifuging the heterogeneous system at 10,000 rpm for 10 min. The residue was dried at 30 °C for 12 h in the vacuo drying oven and dissolved in water. The CDMA was obtained after freeze drying. Then, we used the CDMA to synthesize the β-cyclodextrin dimer (CDD) according to previous reports49-50 with some modifications. Briefly, we dissolved the CDMA (500 mg, 0.44 mmol, 2.1 equiv.) and 1,12-diaminododecane (42 mg, 0.21 mmol, 1 equiv.) in 50 µL of glacial acetic acid and 20 mL of DMSO in a 100 mL stand-up round bottom flask. The solution was stirred at 60 °C for 7 h. After, we added 237 mg of NaBH4 into the solution and stirred it at 30 °C for another 24 h. Then, we added 200 mL of acetone in the solution and centrifuged the system at 10,000 rpm for 10 min. The deposit was dried at 30 °C for 12 h in a vacuo drying oven. We dissolved the residue in water, and the solution was transferred into a dialysis bag


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(1,000 Da) to be dialyzed against water for 5 d. Finally, the CDD was obtained after freezedrying. We characterized the absorbance spectra of the CDMA and CDD using a VECTOR-22 FTIR spectrometer (Vector 33, Bruker, Germany) with the KBr pellet method and determined the molecular structures using 1H NMR (AVANCE Digital 400 MHz, Bruker, Karlsruhe, Germany) spectral measurements. The mass of the CDD was characterized using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS, autoflex-III smartbeam, Bruker, Karlsruhe, Germany). 2.4. Preparation of the substrates. The gold substrate was cleaned with acetone and ethyl alcohol for 10 min, respectively. Then, it was treated with 2 mL of a freshly prepared “piranha” solution (3:7 volume ratio of 30% H2O2 and 98% H2SO4) at 60 °C for 15 min. Next, the substrate was washed again with ultrapure water and ethyl alcohol and dried with flowing nitrogen (hereafter abbreviated as Au). The Au was immersed in an aqueous solution of HS-PEG-Ad (2.5 mg/mL) at 37 °C for 24 h. Then, the Au was washed with ultrapure water and ethyl alcohol and dried under flowing nitrogen. This modified substrate was abbreviated as Au-Ad. To prepare the CDD on the substrate, we immersed the Au-Ad into a CDD aqueous solution (0.5 mg/mL) at 37 °C for 24 h. Then, the substrate was washed with ultrapure water and ethyl alcohol and dried with flowing nitrogen. This substrate was abbreviated as Au-Ad-CDD. The process is shown in Scheme 1.


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Scheme 1. The preparation of the functionalized surface using host-guest interactions. 2.5. Characterization of the substrates. The atomic composition of the substrate was characterized using a Kratos Axis Ultra (DLD) (Thermo ESCALAB 250Xi, Massachusetts, USA) X-ray photoelectron spectrometer operated using an Al Kα (1486.6 eV) monochromatic X-ray source with a power of 150 W and a 500 µm beam spot. The static contact angle of the substrate was characterized using a contact angle goniometer (OCA15, Dataphysics, Germany) at room temperature (n=3). The surface morphology was characterized using atomic force microscope (MFP-3D-S, New Jersey, USA). 2.6. The QCM-D assay. We employed a quartz crystal microbalance with dissipation (QCM-D, E4, Q-Sense, Goteborg, Sweden) to characterize the molecular self-assembly and bilirubin removal process. The bilirubin solution (0.2 mg/mL)31 was prepared by dissolving 0.01 g of bilirubin and 0.01 mg


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of EDTA in 0.5 mL of 0.1 M NaOH solution and diluted with phosphate buffered saline (PBS, pH 7.4, 1X) to 50 mL. The EDTA was used to maintain the solution stability. As the process mentioned above, we first introduced the HS-PEG-Ad (2.5 mg/mL) and CDD (0.5 mg/mL) solution on the Au sensor in the QCM-D chamber. Then, we introduced the bilirubin solution onto the substrate to characterize the adsorption. Based on blood purification applications, we chose the lowest point from the curve during the transflux of the bilirubin solution to evaluate the adsorption effects. Each step was followed by PBS washing until the curve kept horizontal. We also used QCM-D to characterize the thermo-responsive and recyclable properties of the surface. Briefly, after the adsorption of the BR mentioned above on the Au sensor, we increased the temperature from 37 °C to 45 °C to remove the CDD with bilirubin. Then, we decreased the temperature to 37 °C and introduced the CDD as well as the bilirubin solution again. This process was repeated 3 times. To illustrate the effect of temperature on ∆F, we characterized the blank control (Au) with the injection of PBS under the condition of changing temperature mentioned above. Finally, we employed QCM-D to characterize the protein resistance of the sensors developed above. We injected HSA solution (10 mg/mL in PBS) and the Fg solution (0.1 mg/mL in PBS) into the chamber at 37 °C. After balancing, the PBS buffer was injected into the QCM-D chamber again to remove the non-adsorbed proteins. All of the characterizations during QCM-D used AT-cut, 4.95 MHz quartz crystals (purchased from Biolin scientific Co. Ltd., Shanghai, China) in standard liquid cells at a flow rate of 70 µL/min.8 2.7. Platelet adhesion.


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Anticoagulant rabbit blood (10 mL) was added into centrifugal tubes and centrifuged (1,000 rpm, 10 min) to obtain platelet-rich plasma. The samples of Au, Au-Ad and Au-Ad-CDD were placed in 24-well plates. Each sample was immersed in 0.4 mL of platelet-rich plasma and was cultured at 37 °C for 2 h. After that, the samples were washed with PBS three times, and the adsorbed platelets on the surface were immobilized with 1 mL of paraformaldehydeglutaraldehyde immobilizing liquid for 4 h. Then, the samples were washed with deionized water three times and treated with 30%, 50%, 60%, 70%, 80%, 90% and 100% ethyl alcohol/water (v/v) solutions (each for 15 min in sequence) as well as isoamyl acetate for 30 min to dewater. The morphology of the platelets on the surfaces was characterized with high-resolution fieldemission scanning electron microscopy (Merlin, Jena, Germany). 2.8. Hemolysis assay. The samples, Au-Ad, Au-Ad-CDD and Au-Ad-HSA, Au-Ad-CDD-HSA (Au-Ad, Au-Ad-CDD immersed in 10 mg/mL HSA solution for 2 h) were put into the 24-well plates for the hemolysis assay.51 Briefly, 2 mL of rabbit blood was added to physiological saline (0.9% sodium chloride) and then centrifuged at 2,000 rpm for 5 min to isolate red blood cells (RBCs) from the serum. After the RBCs were further washed three times with physiological saline, the purified blood was diluted to 10 mL of saline to obtain a RBC suspension. Then, the samples were immersed in the RBC suspension (0.2 mL) complexed with physiological saline (0.8 mL). The Au immersed in the RBC suspension (0.2 mL) with physiological saline (0.8 mL) and RBC suspension (0.2 mL) with ultrapure water (0.8 mL) was used as a negative and positive control, respectively. All the samples were incubated in 37 °C for 2 h. Finally, the supernatants (100 µL) of each sample were transferred to 96-well plates after being centrifuged at 2,000 rpm for 5 min. The absorbance values of the supernatants were determined using an ELISA plate reader (Thermo3001,


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California, USA) under a 490 nm wavelength. The hemolysis ratios of the RBCs were calculated using the following equation: ((sample absorbance – negative control absorbance) / (positive control absorbance – negative control absorbance)) × 100%. After that, the samples were washed with physiological saline three times. Then, the adsorbed RBCs were immobilized and dehydrated as mentioned in 2.7. The morphology of the RBCs on the surfaces was characterized with high-resolution field-emission scanning electron microscopy (Merlin, Jena, Germany). 2.9. Cytotoxicity assay. The Au, Au-Ad and Au-Ad-CDD samples were put into the 24-well plates and soaked in RMPI 1640 medium complementary with 10% fetal bovine serum at room temperature. After 24 h, the leach liquor was passed through a 0.22 µm filter membrane and then stored in a 4 °C refrigerator. Meanwhile, L929 cells were cultured in the medium in a 5 % CO2 incubator at 37 °C. When the cell density reached the standard density, the cells were dissociated using a 0.25% trypsin/EDTA solution. These cells were seeded in a 48-well plate (2,000 cells per well) with the culture medium and cultured for 24 h. After removing the medium, the cells were washed using PBS once and 500 µL of the prepared extracted fluid was added to each disk. An empty well was used as a positive control. After 24 h of incubation, the viability of the cells was characterized using a CCK-8 kit. Briefly, the samples were washed three times by PBS. Then, 400 µL of the complete medium containing 40 µL of the CCK-8 solution was added to each disk. After 3 h, the CCK-8 solution was transferred to 96-well plates (100 µL in each disk). The optical density (OD) value of the medium was measured using an ELISA plate reader (Thermo3001, California, USA) under a 450 nm wavelength.


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2.10 Statistic. The results from the contact angle measurements and cytotoxicity assay are shown as the mean±standard deviation of more than three independent measurements. The experimentally significant differences among the groups were evaluated using analysis of variance (ANOVA). The statistical significance was defined as p

Thermoresponsive Self-Assembled Î²-Cyclodextrin-Modified Surface

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