Safe and Effective Removal of Urea by Urease-Immobilized, Carboxyl

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Article Cite This: ACS Omega 2019, 4, 2853−2862

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Safe and Effective Removal of Urea by Urease-Immobilized, Carboxyl-Functionalized PES Beads with Good Reusability and Storage Stability Jue Zhang,† Zhoujun Wang,† Chao He,*,† Xiaoling Liu,† Weifeng Zhao,† Shudong Sun,† and Changsheng Zhao*,†,‡ †

ACS Omega 2019.4:2853-2862. Downloaded from pubs.acs.org by 178.159.97.58 on 02/07/19. For personal use only.

College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu 610065, China ‡ National Engineering Research Center for Tissue Restoration & Reconstruction, South China University of Technology, Guangzhou 510006, China S Supporting Information *

ABSTRACT: Urea is one of the uremic toxins threating people’s health. Herein, we prepared a kind of ureaseimmobilized beads to remove urea, which exhibited good hemocompatibility and high removal efficiency. Urease was covalently grafted onto carboxyl-group-functionalized polyethersulfone beads, which were previously prepared by in situ crosslinking polymerization and phase inversion methods. The ureaseimmobilized beads had sufficient blood compatibility with low protein adsorption amount, undiminished clotting times, low hemolysis ratio, and suppressed complement activation and contact activation. Furthermore, the urease-immobilized beads showed good urea removal ability with a urea removal amount of 75.1 mg/g after incubating in 80 mg/dL urea solution for 480 min. In addition, the prepared urease-immobilized beads could maintain urea removal activity after being used for five cycles and showed satisfied activity even when stored for 15 days in phosphate buffer saline solution. Thus, the urease-immobilized beads may meet the potential application demand for safe and effective blood detoxification.

1. INTRODUCTION Chronic kidney disease (CKD) is a syndrome in which the renal structure and function become disordered, resulting in the inability to effectively remove toxins that might lead to end-stage renal disease (ESRD), thus endangering people’s health.1 In 2015, more than 124 000 new cases of ESRD were reported, according to the US Renal Data System 2017 Annual Data Report.2 Even though patients can receive renal transplantation, there are some restrictions such as donor shortage, postoperative immune rejection, and high treatment expenditure. Besides hemodialysis, hemoperfusion is another effective treatment for CKD,3,4 which is a blood purification technology in which patient’s blood is introduced into a device with solid absorbents to remove uremic toxins. Urea is a major uremic toxin and can further form some other lethal substrates, such as guanidines or carbamylation products.5,6 Hence, the removal of urea is essential. There are some absorbents that can be used to remove urea, such as activated carbons,7 zeolites,8 ion-exchange resins,9 and so on. However, the poor blood compatibility and low adsorption ability limit their applications.10,11 Although some research works report that a Cu(II)/chitosan complex or Cu(II)/ synthetic polymer complex can be used for urea removal owing © 2019 American Chemical Society

to the coordination affinity between metal ions in the complex and the carbonyl oxygen in the urea division,12 metal ion shedding is still a big problem, which will lead to poor blood compatibility. Actually, it is difficult to find a suitable adsorbent for direct urea absorption, since urea has high hydrophilic property.13 Urease is a kind of hydrolase containing one bi-nickel center per active site, which can be used as a catalyst to decompose urea into ammonia and carbonic acid.14,15 Even though the enzymatic method is an effective way to remove urea, there are still some drawbacks, such as nonreusability and low storage stability of native urease.11 To solve these problems, much attention has been paid to immobilize native urease onto suitable solid matrixes. Biopolymers such as chitosan,16,17 alginate,18,19 and cellulose20 are used as supports to immobilize urease, since they have many functional groups that can be easily modified. Synthetic polymers such as polyacrylonitrile21 and polypropylene22 have also been used. Moreover, some studies report that inorganic materials such as magnetic Received: November 26, 2018 Accepted: January 30, 2019 Published: February 7, 2019 2853

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Scheme 1. (A) Preparation of the Polymerized Solution via in Situ Cross-Linking; (B) Procedure for Preparing the PES−PAA Beads by Electrostatic Spraying; and (C) Preparation of PES−PAA−U Beads by Conjugating Urease onto the PES−PAA Beads

beads23 and TiO2 beads24 have also been used to immobilize the enzyme. The urease-immobilized materials have been used in various fields including food and beverages, biosensors, wastewater treatment, and dialysate regeneration.20 However, there is barely research that focuses on choosing suitable materials as supports for urease immobilization for application in blood detoxification through hemoperfusion. Furthermore, most of the studies on urease immobilization mainly focus on retaining or improving the stability of urease but ignore blood compatibility.25−28 As a kind of blood-contacting material, sufficient blood compatibility is necessary. Polyethersulfone (PES) has been widely applied in various fields such as water purification and hemodialysis due to its good mechanical property, thermal stability, chemical resistance, and blood compatibility.29,30 Thus, it may be a suitable support material for urease immobilization and can be further applied to remove urea from blood. Urease can be immobilized onto matrixes by several methods, such as adsorption, entrapment, and covalent grafting.31−33 Adsorption is a process of accumulating urease on solid supports depending on the intermolecular interactions; although it is used as a typical method, there exists a problem that urease might leach from the solid supports. Entrapment is a process of trapping urease through polymerizing monomers or low-molecular-weight polymers around urease; however, the chemical environment of polymerization solution might denature urease, the pore size is hard to control, and the leaching of urease is also a problem. Compared with the above two methods, covalent grafting is a

promising method that may avoid the elution of urease with retained activity and stability.34 In this study, we aimed to prepare a material that can remove urea by the enzymatic method for the purpose of blood detoxification. To deal with the drawbacks of nonreusability and low storage stability of native urease and endow the material with good blood compatibility, we prepared urease-immobilized, PES-based beads. PES was selected as the initial support with blood compatibility; after in situ crosslinking polymerization of acrylic acid (AA) and N-vinyl-2pyrrolidone (VP) monomers in PES solution, the phase inversion method was utilized to prepare carboxyl-groupfunctionalized PES beads and the carbodiimide reaction was then used for urease immobilization through covalent grafting. Attenuated total reflection-Fourier transform infrared (ATRFTIR) spectroscopy, thermogravimetric analysis (TGA), and scanning electron microscopy (SEM) were used to confirm the successful preparation of urease-immobilized, PES-based beads. Protein adsorption, clotting time, hemolysis, complement activation, and contact activation tests were performed to evaluate the hemocompatibility. Then, urea removal ability, bead reusability, and storage stability were systemically studied.

2. RESULTS AND DISCUSSION 2.1. Characterization of the Beads. In this study, ureaseimmobilized beads were prepared for the removal of urea. To achieve this purpose, the preparation procedure was divided into three steps, which are as follows. First, the functionalized 2854

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Figure 1. (A) Optical image of PES−PAA−U beads. FTIR spectra (B), TGA curves (C), and DTG (D) curves of the native PES beads, PES−PAA beads, and PES−PAA−U beads.

(Figure S1). Thermogravimetric analysis (TGA) and derivative thermogravimetry (DTG) were used to evaluate the thermal stability of the beads. As shown in Figure 1C, there was slight weight loss before 400 °C for the PES−PAA−U beads, which indicated that the prepared beads were thermally stable for common use. The thermal decomposition temperature of PES−PAA beads was lower than that of native PES beads. This phenomenon might be associated with the thermal degradation property of PAA moieties, which had lower thermal stability than that of the PES material.40,41 As shown in Figure 1D, the PES−PAA−U and PES−PAA beads reached the maximum degradation rate at 560 and 575 °C, respectively. The lower degradation temperature for PES−PAA−U beads might be caused by the introduction of urease, since urease showed lower thermal stability and began to degrade when heated to 200 °C; the corresponding results of the TGA curve and DTG curve for native urease are shown in Figure S2 in the Supporting Information. The results of the SEM for the prepared beads are presented in Figure 2. The average size of the beads was about 600 μm (the scar bar was 200 μm) according to the SEM image. Also, the cross-sectional morphology of native PES beads contained a skin layer and an inner region with a fingerlike structure, which were the typical structures and commonly observed in previous studies.42,43 The same structures were also observed after in situ cross-linking (for PES−PAA beads) and urease immobilization (for PES−PAA−U beads). Additionally, many micropores were further formed in PES−PAA beads and PES− PAA−U beads, which might be due to the hydrophilic property of PAA and polyvinylpyrrolidone (PVP), since some short chains of PAA and PVP might be eluted during preparation.36 The viscosity of polymerized solution after cross-linking increased compared to that of native PES solution, which could be attributed to the entanglement of polymer chains. Moreover, the increased viscosity may decrease the driving force and the relative diffusion rate between the solvent and nonsolvent during phase separation, thus leading to a longer time for bead formation and the

solution was prepared via in situ cross-linking polymerization with the PES matrix,35 as shown in Scheme 1A. AA was selected as a functional monomer to introduce carboxyl groups; PES (previously dissolved in solution) was used due to its good blood compatibility and good mechanical property.36,37 VP was further introduced to increase the blending compatibility of poly(acrylic acid) (PAA) and PES after in situ cross-linking.38 Second, an electrostatic spraying machine was utilized to prepare the beads (PES−PAA) with carboxyl groups through phase inversion. As shown in Scheme 1B, the polymerized functional solution was transferred into an injector and then dropped into a coagulating bath (deionized (DI) water containing sodium dodecyl sulfate (SDS)) with a voltage of 10 kV; thus, the beads with carboxyl groups (PES− PAA) were prepared. Finally, urease was immobilized onto PES−PAA beads via covalent grafting to prepare PES−PAA− U beads. Briefly, the amino groups of urease reacted with the carboxyl groups of PES−PAA beads by the carbodiimide reaction, as shown in Scheme 1C; 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC) and Nhydroxysuccinimide (NHS) were used as coupling agents to activate the carboxyl groups via producing an intermediate.39 The optical image of the obtained PES−PAA−U beads is shown in Figure 1A, showing a good spherical shape. The results of ATR-FTIR are shown in Figure 1B, which were used to characterize the functional groups of the prepared beads. According to the results, a new peak at 1720 cm−1 was observed for the PES−PAA beads when compared with native PES beads, which was due to the stretching vibration of carbonyl groups. After the immobilization of urease, new peaks at 3300 and 1650 cm−1 were obviously observed for PES− PAA−U beads, which belonged to the characteristic absorption peaks of secondary amide, owing to the vibration of N−N and CO, respectively. Thus, the results of the FTIR spectra indicated that urease was immobilized onto PES−PAA beads successfully. Also, the amount of urease grafting was 12 mg/g, whereas the grafting efficiency was 36%; the detailed experimental process is shown in the Supporting Information 2855

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PES−PAA beads. This might be caused by the electrostatic repulsion between urease (negatively charged in the solution with a isoelectric point lower than 746) and negatively charged BSA or BFG.47,48 2.2.2. Platelet Adhesion. Platelet adhesion and platelet aggregation play an important role in the process of coagulation.37 Platelet adhesion on material surfaces might lead to thrombin formation; thus, it was tested in this study. According to the results shown in Figure 3B, platelet adhesion and aggregation could be observed on the surface of native PES beads, which were highlighted in yellow. Compared to that on native PES beads, almost no platelet aggregation occurred on the surface of PES−PAA−U beads, which indicated that the urease-immobilized beads had good antiplatelet adhesion property. 2.2.3. Clotting Times. To apply the beads for urea removal from blood, good hemocompatibility is necessary, especially with sufficient anticoagulant property when contacting with blood.49 Clotting times were tested to confirm the anticoagulant property of the beads, as shown in Figure 3C. In vitro antithrombogenicity of the beads was determined by APTT and TT tests, whereas the role of the exogenous coagulation system was confirmed by the PT test.50,51 The values of APTT, TT, and PT for the control sample platelet-poor plasma (PPP) were 44, 21.5, and 14.1 s, respectively, whereas these were 44.4, 20.9, and 13.8 s, respectively, for PES−PAA−U beads, which showed no obvious difference; similar results were also observed for the native PES beads. Thus, the results indicated that the prepared beads showed no coagulant trend with undiminished clotting times, which was coincident with our previous work.52 However, a little prolonged APTT could be observed for PES−PAA beads. This phenomenon might be due to the carboxyl groups of the PES−PAA beads that performed anticoagulation function by binding Ca2+ to inhibit the activation of Factor IX, Factor X, and prothrombin, which

Figure 2. SEM images of the native PES beads, PES−PAA beads, and PES−PAA−U beads.

formation of micropores.44 According to the results of Table S1 and Figure S3, the surface area and porous volume of PES− PAA−U beads were 12.227 m2/g and 5.8020 × 10−3 cm3/g, respectively, whereas these were 16.203 m2/g and 7.3061 × 10−3, respectively, for PES−PAA beads. The results indicated that the surface area and porous volume decreased after urease immobilization. 2.2. Blood Compatibility. 2.2.1. Protein Adsorption. When materials contact with blood, plasma proteins might be absorbed on the material surface. Hence, protein adsorption was measured in this study. Bovine serum albumin (BSA) and bovine serum fibrinogen (BFG) were selected as representative proteins, since they are of great importance to thrombus formation.45 As shown in Figure 3A, the adsorption amounts of BSA and BFG for urease-immobilized beads (PES−PAA− U) were decreased compared to those of native PES beads and

Figure 3. (A) Protein adsorption of the PES beads, PES−PAA beads, and PES−PAA−U beads. (B) Platelet adhesion of the PES beads and PES− PAA−U beads. (C) Activated partial thromboplastintime (APTT), thrombin time (TT), and prothrombin time (PT) of the beads. (D) Hemolysis rates of the beads. 2856

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Figure 4. C3a (A), C5a (B), platelet factor 4 (PF4) (C), and thrombin−antithrombin (TAT) (D) concentrations of the beads after incubated with whole blood for 2 h.

Figure 5. (A) Urea decomposing diagram under urease catalysis. (B) Residual urea quantities after incubating PES−PAA−U beads with urea solutions of different concentrations (40, 60, and 80 mg/dL) along time. (C) Amounts of urea removal for PES beads, PES−PAA beads, and PES− PAA−U beads after incubating for 480 min.

blood.57,58 The generated concentrations of human complement fragment 3a (C3a) and human complement fragment 5a (C5a) were evaluated to trace the hemocompatibility of the beads. As exhibited in Figure 4A,B, there was no significant difference in the generated concentrations of C3a and C5a between the control sample and the PES−PAA−U beads. The results indicated that the urease-immobilized beads would not activate the inflammation responses when in contact with blood. Contact activation is regarded as a pathophysiological surface defense mechanism for foreign materials.59 Platelet activation can lead to platelet aggregation and coagulation cascade activation. The platelet activation level can be evaluated by the generated concentration of platelet factor 4 (PF4). When foreign materials contacted with blood, thrombin

played an important role in the blood coagulation cascade via activating clotting factors from Factor IX to IXa, Factor X to Xa, and prothrombin to thrombin.53−55 2.2.4. Hemolysis Analysis. Hemolysis is also a crucial factor that could affect the blood compatibility of materials.48 In this study, hemolysis was tested by incubating red blood cells (RBCs) with the beads. As shown in Figure 3D, the hemolysis ratios of the beads were all far below 5% (as the ASTM standard56) and no hemolysis phenomenon (red color) was observed from the inset figure; thus, it could be concluded that the prepared beads had good red blood cell compatibility. 2.2.5. Complement Activation and Contact Activation. Complement activation is a process in which the complement changes from an inactive form to an active form, and it is considered as a trigger in the host defense mechanism of 2857

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Figure 6. (A) Relative activity of PES−PAA−U beads for urea removal with different cycles. (B) Relative activity of PES−PAA−U beads stored in dry state, DI water, physiological saline solution, and phosphate buffer saline (PBS) buffer solution at 4 °C for urea removal after storing for different days.

given.1 Moreover, HEMA-incorporated poly(ethylene glycol) dimethacrylate microbeads were also reported to immobilize urease; the average removal amount was 3.525 mg/g, which was far below than that for PES−PAA−U beads.63 Thus, it could be concluded that the urease-immobilized beads owned good urea removal ability with satisfactory efficiency. 2.4. Reusability and Storage Stability. Reusability of the urease-immobilized beads (PES−PAA−U) was determined, and the results are presented in Figure 6A. As can be seen, the relative activity for urea removal was almost maintained without an obvious decrease after being used for five cycles. Thus, the urease-immobilized beads showed good reusability with persistent urea decomposition property during repeated operations, whereas free urease cannot be cyclically reused.1 The storage stability was also tested, and the results are shown in Figure 6B. For the PES−PAA−U beads stored in PBS buffer solution, the relative activities for urea removal were 94.6 and 89.2% after storing for 7 days and 15 days, respectively, which were higher than those of the PES−PAA− U beads stored in dry state, DI water, and physiological saline solution. This might be due to the slight change of the pH in PBS buffer solution, which could influence the activity of urease.14,46 Additionally, the PES−PAA−U beads stored in dry-state conditions showed lower relative activity for the later few days, with a relative activity of about 50% after storing for 15 days. These phenomena gave an inspiration that the PES−PAA−U beads should be stored in PBS buffer solution during transportation. Moreover, for free urease, the relative activity decreased to 6.3% after storing in PBS for 7 days according to Table S2. The urease-immobilized beads showed higher stability than that of free urease, which might be caused by the reduced conformational change of immobilized urease, and the conformation can influence urease inactivation.64 The results indicated that the urease-immobilized beads had good storage stability.

might be activated and would be inactivated immediately through reacting with antithrombin III to form a thrombin− antithrombin (TAT) complex.60,61 Hence, the generated concentrations of PF4 and TAT were tested to confirm the hemocompatibility of the prepared beads. As shown in Figure 4C,D, the generated concentrations of PF4 and TAT after incubating whole blood with the beads exhibited no difference compared to that of the control sample, indicating the prepared beads with suppressed contact activation. The results also suggested that platelet adhesion and aggregation could occur on the surface of native PES beads according to the SEM image (Figure 3B); however, there was almost no activation. 2.3. Urea Removal Ability of the Beads. As shown in Figure 5A, urea could be decomposed into ammonia and carbonic acid under urease catalysis. Batch urea removal experiments were carried out to evaluate the urea removal ability of the urease-immobilized beads (PES−PAA−U beads). As shown in Figure 5B, after incubating for 240 min, the residual quantities of urea were all below 50% for the selected urea concentrations. Also, when the incubation time was prolonged to 480 min, the residual quantity for the solution with urea concentration of 40 mg/dL was 2.7%, whereas the residual quantities for the solutions with urea concentrations of 60 and 80 mg/dL were 5.3 and 14.4%, respectively. As shown in Figure 5C, the amounts of urea removal were 44.1, 63.1, and 75.1 mg/g for the PES−PAA−U beads after incubating for 480 min in urea solutions of concentrations 40, 60, and 80 mg/dL, respectively; however, there was almost no urea removal for PES beads and PES−PAA beads. The results indicated that the immobilized urease maintained its activity. The amounts of urea removal at different urea concentrations along time were also calculated, and the results are shown in Figure S4. The amount of urea removal was higher for 80 mg/ dL urea solution than those for 60 and 40 mg/dL for the same incubation time. This might be caused by the phenomenon that the urease-immobilized beads could contact with urea more effectively at a higher concentration. A patient of chronic kidney disease (CKD) carries 40−85 mg/dL blood urea nitrogen (BUN), which is equivalent to 856−1819 mg/L urea. An adult carries about 5 L of blood, which means the total amount of urea is about 4.28−9.09 g.62 Therefore, about 57− 121 g of PES−PAA−U beads could remove almost 60% urea within 480 min to reach the normal level of BUN. According to previously reported studies, silk fibroin was used to immobilize urease to remove urea, the urea removal ratio of this prepared material was 20% at 50 mg/dL urea solution for incubation time of 240 min, and blood compatibility was not

3. EXPERIMENTAL SECTION 3.1. Materials. Commercial polyethersulfone (PES, Ultrason E6020P) was purchased from BASF. Acrylic acid (AA, containing MEHQ, 180 ppm), N-vinyl-2-pyrrolidone (VP, containing NaOH 100 ppm), urea (99%), urease from jack bean (≥45 units/mg dry weight), N,N-methylenebisacrylamide (MBA), N-hydroxysuccinimide (NHS), and 1-[3(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC) were purchased from Aladdin Chemistry Co. Ltd. (Shanghai, China). N,N-dimethylacetamide (DMAC, 99%), sodium dodecyl sulfate (SDS), and 2,2′-azobis(2-methylpro2858

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clotting time, hemolysis analysis, complement activation, and contact activation tests. All of the beads were pretreated by immersing in physiological saline overnight. Whole blood was acquired from donors aged about 24 years. Platelet-rich plasma (PRP), platelet-poor plasma (PPP), and red blood cells (RBC) were obtained from the whole blood by centrifugation. Details of the test procedures are given in the Supporting Information. All of the tests were complied with the laws and relevant national guidelines, and were upheld by West China Hospital, Sichuan University. 3.6. Urea Removal. The urea removal ability of the beads was investigated by the QuantiChrom Urea Assay Kit (DIUR500), which can produce a colored complex with urea. The intensity of the color is proportional to the urea concentration.11 Briefly, different concentrations of urea solutions (40, 60, and 80 mg/dL) were first prepared and then certain amounts of PES−PAA−U beads (about 0.28 g) were introduced into 30 mL of the above solutions with oscillation at 37 °C. The concentration of urea solution was tested at set intervals via the QuantiChrom Urea Assay Kit (DIUR-500); thus, the amount of removed urea could be further calculated. The same operating procedure was also carried out to evaluate the urea removal ability of PES and PES−PAA beads. 3.7. Reusability and Storage Stability of the UreaseImmobilized Beads. The reusability of the PES−PAA−U beads was tested, and the procedure is described as follows. First, 0.28 g of the PES−PAA−U beads were added into 30 mL of 80 mg/dL urea solution and oscillated at 37 °C for 2 h. Then, the PES−PAA−U beads were isolated from the urea solution and washed with deionized water several times. Afterward, the treated beads were introduced into another fresh 30 mL of 80 mg/dL urea solution and oscillated at 37 °C for 2 h again. The same operation was repeated five times, as described above, and the concentration of each treated urea solution was obtained via the QuantiChrom Urea Assay Kit (DIUR-500). The relative activity of each cycle was calculated by comparing to the urea removal ability in the first operation, which was defined with a relative activity of 100%. To study the storage stability, PES−PAA−U beads were previously stored in dry state (surface dried by a filter paper previously and then stored directly), DI water, physiological saline solution, or phosphate buffer saline (PBS) solution at 4 °C for different times (1 day, 7 days, and 15 days). After that, the treated beads were added into 30 mL of 80 mg/dL urea solution and oscillated at 37 °C for 480 min; then, the urea solution was transferred into a 96-well plate and the absorbance was tested by the QuantiChrom Urea Assay Kit (DIUR-500). The relative activity of urea removal for the 1 day stored PES−PAA−U beads was set to 100%, and then, the relative activity of the PES−PAA−U beads stored for 7 days and 15 days could be calculated. The storage stability of free urease was also tested, which was the same as the above procedure, and the detailed description is given in the Supporting Information.

pionitrile) (AIBN) were obtained from Chengdu Kelong Chemical Reagent Company (Chengdu, China). 4-morpholineethanesulfonic acid (MES) was purchased from Best-reagent Co. Ltd., China. Bovine serum albumin (BSA) and bovine serum fibrinogen (BFG) were purchased from Sigma Chemical Co. Ltd. Activated partial time (APTT) reagent, thrombin time (TT) reagent, and prothrombin time (PT) reagent were purchased from Siemens Co. Ltd. The Micro BCA Protein Assay Reagent kit was purchased from Pierce Inc. The QuantiChrom Urea Assay Kit (DIUR-500) was purchased from Beijing Qbioscience Co. Ltd. (Beijing, China). Deionized (DI) water was homemade and employed throughout the study. 3.2. Bead Preparation. The beads were prepared by the phase inversion method using a polymer solution. First, the polymer solution was prepared via in situ cross-linking polymerization. Briefly, 6 g of PES was dissolved in 44 g of DMAC with magnetic stirring until a homogeneous solution is obtained. A mixture of monomers (1 g of AA and 0.15 g of VP), an initiator (0.01 g of AIBN), and a cross-linker (0.1 g of MBA) was introduced into the above-mentioned PES solution. The reaction was proceeded at 70 °C for 24 h under a nitrogen atmosphere with a constant mechanical stirring rate of 400 rpm. After that, the obtained polymerized solution was pumped into a coagulating bath (deionized water containing SDS) using an electrostatic spraying machine with an extruding speed rate of 3 cm/min. Finally, the beads were washed thoroughly using deionized water for one week and the deionized water was changed frequently (every 3−4 h) to make sure that the water-soluble SDS was completely removed. The prepared beads were named as PES−PAA. The native PES beads were also prepared using the same method with the native PES solution. 3.3. Immobilization of Urease onto PES−PAA Beads. To immobilize urease onto PES−PAA beads, a covalent grafting method was used, and the procedure is presented as follows. First, 0.96 g of EDC and 0.2 g of NHS were added into 100 mL of MES aqueous solution (0.1 M). Then, 3 g of PES− PAA beads was immersed into the solution for 1 h to activate the carboxyl groups of PES−PAA beads. After that, 0.1 g of jack bean urease was further added into the mixture with continuous stirring at room temperature. Then, the reaction was stopped after 12 h by separating the beads from the solution. The urease-immobilized beads (named as PES− PAA−U) were washed several times with deionized water and then stored at 4 °C before use. 3.4. Characterization of the Urease-Immobilized Beads. An attenuated total reflection infrared (ATR-FTIR) spectroscope (Nicolet 560) was used to confirm the surface functional groups of the prepared beads, with wavenumber ranging from 500 to 4000 cm−1. To investigate the thermal property, a Q-500 thermogravimetric analyzer (TG209F1, Netzsch, Germany) was used, with heating from 30 to 800 °C under a nitrogen atmosphere at the heating speed of 10 °C/ min. The cross-sectional morphologies of the beads were observed via a scanning electron microscope (SEM, JSM7500F, JEOL, Japan) with a voltage of 5 kV, and the magnifications were 300× and 1000×. The nitrogen adsorption isotherm were tested by a dynamic specific surface analyzer (MicrotracBEL, Bel Japan, Inc.) to determine the surface area and porous volume of the prepared beads. 3.5. Blood Compatibility. The hemocompatibility of the beads was confirmed by protein adsorption, platelet adhesion,

4. CONCLUSIONS In this study, urease was successfully immobilized onto carboxyl-functionalized PES beads by covalent grafting; the urease-immobilized beads exhibited good thermal stability. The cross-sectional morphology of native PES beads was consistent, which had a skin layer and an inner region with a fingerlike structure. Blood compatibility tests indicated that the 2859

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thrombin−antithrombin; CKD, chronic kidney disease; BUN, blood urea nitrogen; ESRD, end-stage renal disease

urease-immobilized beads had low protein adsorption amount, undiminished clotting times, low hemolysis ratio, and suppressed complement activation and contact activation; thus, the urease-immobilized beads had good blood compatibility. In addition, the urease-immobilized beads exhibited good urea removal ability with satisfactory efficiency. Furthermore, the urease-immobilized beads could maintain bioactivity for urea removal during recycling. The storage stability was also good with maintained relative activity after storing in PBS buffer solution for several days. In summary, we designed a kind of PES-based bead with good hemocompatibility, which had the potential to be applied in the field of urea removal from blood.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b03287.

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REFERENCES

Experiment details of grafting efficiency, blood compatibility, nitrogen adsorption isotherm, storage stability for purchased urease, TGA and DTG curves for native urease, and the urea removal amounts along time (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel: +86-28-85400453. Fax: +8628-85405402 (C.H.). *E-mail: [email protected] (C.S.Z.). ORCID

Weifeng Zhao: 0000-0003-2689-0251 Changsheng Zhao: 0000-0002-4619-3499 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially sponsored by the National Natural Science Foundation of China (Nos. 51503125, 51673125, 51773127, 51803131, 51803134, and 51873115), the State Key Research Development Programme of China (2016YFC1103000 and 2016YFC1103001), the State Key Laboratory of Polymer Materials Engineering (Nos. sklpme2017-3-07 and sklpme2015-1-03), and the Consulting project of Chinese Academy of Engineering (2017-XZ-08).

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ABBREVIATIONS PES, polyethersulfone; AA, acrylic acid; VP, N-vinyl-2pyrrolidone; MBA, N,N-methylenebisacrylamide; NHS, Nhydroxysuccinimide; EDC, 1-[3-(dimethylamino)propyl]-3ethylcarbodiimide hydrochloride; DMAC, N,N-dimethylacetamide; SDS, sodium dodecyl sulfate; AIBN, 2,2′-azobis(2methylpropionitrile); MES, 4-morpholineethanesulfonic acid; BSA, bovine serum albumin; BFG, bovine serum fibrinogen; APTT, activated partial time; TT, thrombin time; PT, prothrombin time; ATR-FTIR, attenuated total reflectionFourier transform infrared; TGA, thermogravimetric analysis; DTG, derivative thermogravimetry; SEM, scanning electron microscopy; PRP, platelet-rich plasma; PPP, platelet-poor plasma; RBC, red blood cells; AMTS, American Material Test Society; C3a, human complement fragment 3a; C5a, human complement fragment 5a; PF4, platelet factor 4; TAT, 2860

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