- Email: [email protected]
polyethersulfone composite membranes for versatile applications
Materials Science and Engineering C 59 (2016) 556–564
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
A facile approach toward multi-functional polyurethane/ polyethersulfone composite membranes for versatile applications Rui Wang a, Tao Xiang a, Wei-Feng Zhao a,⁎, Chang-Sheng Zhao a,b,⁎ a b
College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu 610065, People's Republic of China National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, People's Republic of China
a r t i c l e
i n f o
Article history: Received 28 May 2015 Received in revised form 23 September 2015 Accepted 20 October 2015 Available online 21 October 2015 Keywords: Polyurethane Polyethersulfone Adsorption capacity Blood compatibility Antibacterial property
a b s t r a c t The complex synthesis through multistep reactions and tedious purifications based on different monomers or macromolecules limits the practical applications of functional polymers. Herein, a facile approach toward a series of functional polyurethanes (PUs) is designed for versatile biological applications within fewer step reactions under mild conditions. The tertiary amino groups in the PU are converted into zwitterions or quaternary ammonium salt via simple one-step synthesis, and then used to prepare PU/polyethersulfone composite membranes. The composite membrane with tertiary amine groups exhibits significant adsorption capability to anionic dye Congo red (CR) and toxin bilirubin. The membrane bearing zwitterionic PU displays excellent blood compatibility; while which with quaternary ammonium salts has antibacterial property. Furthermore, carboxybetainefunctional composite membrane is exploited to bear Ag nanoparticles to endow with dual functions of antibacterial and antifouling properties. This work demonstrates the potential of PUs as readily available, multifunctional, and easy-to-use materials for biological applications. © 2015 Elsevier B.V. All rights reserved.
1. Introduction With the development of polymeric materials, desired properties could be achieved flexibly by utilizing either different common monomers or some specific monomers with elaborate modifications [1–3]. Besides, the macromolecules could be further modified for extensive applications using the functional groups such as –COOH, –OH and –NH2 on the macromolecular chains [4–6]. For example, the macromolecules or monomers that contained quaternary ammonium salt or Ag nanoparticles [7] could endow the materials with broad-spectrum antimicrobial property; while antifouling property was generally achieved using the monomers or macromolecules with zwitterions, hydroxyl groups and/or PEG chains, such as Sulfobetaine methacrylate (SBMA) [8], Hydroxyethyl methacrylate (HEMA) [9–11] and Poly(ethylene glycol) methacrylate (PEGMA) [12,13], which could also improve biocompatibility of materials [14]. On the other hand, for the polymers like polyesters and polyurethanes, most of the active functional groups like –OH, –NH2 or –COOH in monomers are usually sacrificed in the polymerization process, thus the possibility of further modifications is less than the polymers prepared by vinyl monomers [15,16]. Despite that the types of monomers and macromolecules have been increasingly diversified through the modification, a considerable amounts of synthetic processes generally required multistep synthesis ⁎ Corresponding authors. E-mail addresses: [email protected], [email protected] (W.-F. Zhao), [email protected], [email protected] (C.-S. Zhao).
http://dx.doi.org/10.1016/j.msec.2015.10.058 0928-4931/© 2015 Elsevier B.V. All rights reserved.
with many reactants or catalysts [17,18], restricting the practical applications due to the lack of efficiency and universality. Regarding this concern, developing different functions based on the same monomers or macromolecules through fewer step reactions under mild conditions is an indispensable strategy for both the productions and the applications. The aim of this study is to develop a facile strategy to combine various functions in polyurethane (PU) via straightforward reactions. 4,4′-Diphenylmethane diisocyanate (MDI) was used as the hard segment to enhance the miscibility of PU with other polymeric materials; while N-methyldiethanolamine (MDEA, containing tertiary amino group) was used as the soft segment. Additionally, citric acid (CA) was used as blocking agent to endow with anticoagulation property. Different from the modification of the macromolecules that contained –OH, –NH2 or –COOH, the various functionalities in this study are based on the tertiary amino groups, which could be further converted into zwitterions (sulfobetaine and carboxybetaine) [19,20] or quaternary ammonium salt [21]. The process was performed through one-step reaction without any catalysts or additional reactants. The synthesized PUs were exploited to prepare composite membranes with polyethersulfone (PES), and the adsorption capability of the composite membrane that contained tertiary amino groups was evaluated firstly by using dye Congo red (CR) and toxin bilirubin. The blood compatibility and antifouling property of the zwitterionic composite membranes were then evaluated in terms of clotting times, protein adsorption and platelet adhesion; while the activities of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were performed to demonstrate the
R. Wang et al. / Materials Science and Engineering C 59 (2016) 556–564
antibacterial property of the membrane bearing quaternary ammonium salts. Furthermore, it was found that the carboxybetaine could load silver, which has been seldom reported [22]; thus the carboxybetaine membrane was used to bear Ag nanoparticles to endow with dual functions of antibacterial and antifouling properties. 2. Experimental 2.1. Materials 4,4′-Diphenylmethane diisocyanate (MDI, 98%, Aladdin), Nmethyldiethanolamine (MDEA, 98%, Aladdin), 1,3-propanesulfonate (98%, Aladdin), 3-bromopropionic acid (98%, Aladdin), and iodomethane (98%, Xiya) were used without further purification. N,Ndimethylformamide (DMF, 99%, Kelong), 1-methyl-2-pyrrolidinone (NMP, 99%, Kelong) and dimethyl sulfoxide (DMSO, 99%, Kelong) were distilled under vacuum. Deionized water (DI water) was used throughout the study. Congo red (CR) and methylene blue (MB) were obtained from Kelong Inc. Bilirubin (98%) was purchased from Aladdin Industrial Inc. The LIVE/DEAD BacLight Bacterial Viability Kit L-7012 was purchased from Thermo Fisher Scientific Inc. 2.2. Synthesis and characterization of polyurethanes 2.2.1. Synthesis of polyurethane with tertiary amine (PU) All the polyurethanes were modified from original polyurethane that contained tertiary amine groups (PU) as shown in Scheme 1 and the synthetic process of PU is as follows: MDI (6.92 g) and MDEA (3.08 g) were dissolved in DMAc with the monomer concentration of 20 wt.%, and the molar ratio of the MDI to NMDA was 16:15. The polymerization was carried out in nitrogen gas at 75 °C for 2 h, and then citric acid was introduced in the reaction system and carried out at 80 °C for another 4 h. After the reaction, the polyurethane was precipitated with deionized (DI) water and washed with ethanol for several times. The product was dried in a vacuum oven at 40 °C. 2.2.2. Synthesis of polyurethane with sulfobetaine (SPU) 1,3-Propanesulfonate (1.98 g) was added dropwise into the solution of the as-prepared PU (2.00 g) in DMSO, and the reaction was carried
557
out at 25 °C for 24 h. Then the product was precipitated using ethanol and dried in a vacuum oven at 40 °C. 2.2.3. Synthesis of polyurethane with carboxybetaine (CPU) 3-Bromopropionic acid (2.49 g) and the PU (2.00 g) were dissolved in DMSO, and reaction was carried at 60 °C for 24 h. After the reaction, the product was precipitated using ethanol and dried in a vacuum oven at 40 °C. 2.2.4. Synthesis of polyurethane with quaternary ammonium (QPU) CH3I (12 mL) was added into the solution of the PU (2.00 g) in NMP and reacted at 60 °C for 12 h. After the reaction, the product was precipitated using ethanol and dried in a vacuum oven at 40 °C. 2.2.5. Characterization of the polyurethanes 1 H NMR (400 MHz) spectra were recorded on a BrukerAVII-400 MHz spectrometer (Bruker Co., Germany), using tetramethylsilane (TMS) as the internal standard in DMSO-d6 at room temperature. Gel permeation chromatography (GPC) measurement was performed by using a PL220 GPC analyzer (Britain) to measure molecular weight. N,N-dimethyl formamide (DMF) was chosen as the eluent and polystyrene (PS) as the reference. 2.3. Preparation of membranes PES was dissolved in DMSO to keep the concentration at 16 wt.%. Then the PU was added to the solution and the concentration was controlled at 4 wt.%. The solution was prepared into membranes by spin coating coupled with a phase inversion technique as described in our earlier reports [23,24]. The membranes modified with PU, SPU, CPU and QPU were termed PU/PES, SPU/PES, CPU/PES and QPU/PES, respectively. To obtain membrane bearing Ag nanoparticles, the CPU/PES membrane was firstly immersed into 0.1 M NaOH for 10 min and rinsed 3 times in DI water. Then the membrane was immersed into 0.05 mM AgNO3 solution with oscillation for 24 h in the dark, followed by rinsing with DI water for 3 times to remove the excess AgNO3. Then the membrane was dipped in 0.05 mM NaBH4 for 2 h to prepare Ag loaded membrane which was termed CPU-Ag/PES.
Scheme 1. Synthesis of a series of polyurethanes.
558
R. Wang et al. / Materials Science and Engineering C 59 (2016) 556–564
The morphologies of the membranes were observed by using a JSM7500F scanning microscope (JEOL, Japan). Water contact angles were measured with a contact angle goniometer (OCA20, Dataphysics, Germany). 2.4. Ultrafiltration experiments Ultrafiltration experiments were carried out on the apparatus as described in our previous study [25]. A dead-end ultrafiltration (UF) cell with an effective membrane area of 3.90 cm2 was used. The membrane was pre-compacted with deionized water for 30 min at a pressure of 0.1 MPa to get steady before the ultrafiltration experiments. The water flux was determined as follows: Flux ¼
V SPt
ð1Þ
where V (mL) is the volume of the permeated solution; S (m2) is the effective membrane area; P (mm Hg) is the pressure applied to the membrane and t (h) is the time for collecting permeated solution. 2.5. Adsorption to Congo red (CR), methylene blue (MB) and bilirubin The PU/PES membranes (5 pieces) were dipped in 10 mL of 200 mmol/L CR or MB aqueous solution at 25 °C with the stirring speed of 100 rpm. The adsorption amounts of the CR and MB were calculated using the absorbance at the wavelength of 497 and 630 nm, respectively. For bilirubin adsorption, 0.15 g of bilirubin was firstly dissolved in 1 mL of 0.1 M NaOH, and then diluted to 100 mL using phosphatic buffer solution (PBS, pH 7.4) away from light to prepare adsorbate solution. Then, the PU/PES membranes (20 pieces) were dipped in 10 mL bilirubin solution at 25 °C with the stirring speed of 100 rpm. The adsorption amount of the bilirubin was calculated by the absorbance at the wavelength of 438 nm, and all the operations were under dark condition.
2.8. Platelet adhesion experiments To study platelet adhesion, the membrane with an area of 1 × 1 cm2 was dipped in normal saline at 37 °C for 1 h. After removing the normal saline, 200 μL of fresh PRP was dropped in each well of the culture plate and then incubated at 37 °C for 2 h. Then the PRP was decanted off and the membrane was rinsed three times with normal saline. The adhered platelets on the membrane surface were fixed using 2.5 wt.% glutaraldehyde in normal saline at 4 °C for 24 h. Finally, the sample was washed with normal saline, and dehydrated with a series of normal saline/ ethanol mixtures with increasing ethanol concentration (25, 50, 75 and 100 wt.%), and then dried at room temperature. The platelet adhesion was observed using scanning electron microscopy. 2.9. Antibacterial tests Firstly, bacterial inhibition zone method was used to study the antibacterial property. The sterilized membrane with an area of 1 × 1 cm2 was placed on E. coli or S. aureus bacteria agar plate at an inoculum concentration of 107 colony forming units per mL (cfu mL− 1) and then incubated at 37 °C for 12 h. The presence of the inhibition zone was recorded by a digital camera. Then, the antibacterial activity to bacteria suspension was investigated. The membranes (two pieces, 1 × 1 cm2 for each) were immersed in 2 mL of E. coli or S. aureus suspension at 106 cfu mL−1 and incubated in a shaking incubator at 37 °C for 2, 4, 6, and 8 h, respectively. Then the optical degree of the bacterial suspension was determined at the wavelength of 500 nm. Finally, live/dead two-color fluorescence method was used. The membrane that co-cultured with E. coli or S. aureus suspension at 106 cfu mL−1 for 12 h was washed with normal saline. Then the membrane was stained with the LIVE/DEAD BacLight Bacterial Viability Kit, and observed under a fluorescent microscopy. 3. Results and discussion
2.6. Protein adsorption experiments 3.1. Synthesis of polyurethanes Firstly, the membrane with an area of 1 × 1 cm2 was dipped in PBS (pH 7.4) containing bovine serum albumin (BSA) or bovine fibrinogen (BFG) with a concentration of 1 mg/mL, and incubated at 37 °C for 1 h, followed by slight rinsing with PBS and double distilled water. Then the membrane was immersed into a washing solution (2% sodium dodecyl sulfate at 37 °C) and shaken for 2 h to remove the adsorbed protein. The protein concentration in the washing solution was determined by using the Micro BCA™ Protein Assay Reagent Kit (PIERCE), and the adsorbed protein amount was calculated. 2.7. Blood clotting time experiments The activated partial thromboplastin time (APTT) test was performed as follows. Fresh human blood was centrifuged at 1000 rpm for 15 min to obtain platelet-rich plasma (PRP) or at 4000 rpm for 15 min to obtain platelet-poor plasma (PPP). The membrane with an area of 1 × 1 cm2 was firstly dipped in 0.2 mL PBS (pH 7.4) for 1 h. After removing the PBS, 0.1 mL of fresh PPP was introduced and incubated at 37 °C for 30 min. Then 50 μL of the incubated PPP was added into a test-tube, followed by the addition of 50 μL of APTT agent (Dade Actin Activated Cephaloplastin Reagent, Siemens; incubated 10 min before use), and incubated at 37 °C. Thereafter, 50 μL of 0.025 M CaCl2 solution was added and then the APTT was measured. For prothrombin time (PT) test, 50 μL of the incubated PPP was mixed well with 100 μL of TT agent (Thromborel®S, Siemens; incubated 10 min before use) at 37 °C and the thrombin time (TT) was measured. For PT test, 50 μL of the incubated PPP was mixed with 100 μL of PT agent (Thromborel®S, Siemens; incubated 10 min before use) at 37 °C and then the PT was measured.
1 H NMR is an effective way to analyze the molecular structure of polyurethanes. The 1H NMR for the PU with tertiary amine groups is shown in Fig. 1(A): δ = 2.24 ppm (a, N–CH3), δ = 2.67 ppm (b, N–CH2–), δ = 4.15 ppm (c, O = C–O–CH2–), δ = 8.54 ppm (d, O = C–NH–), δ = 7.35 and 7.09 ppm (e and f, benzene ring), δ = 3.77 ppm (g, –CH2–Ar), δ = 9.55 ppm (h, –COOH). As shown in Fig. 1(B), for the SPU, the chemical shifts of hydrogen in a, b and c increased to 2.35 ppm (N–CH3), 2.76 ppm (N–CH2–), 4.17 ppm (O = C–O–CH2–), respectively, which was caused by the N+ after that the tertiary amine was converted into sulfobetaine. In addition, new peaks at 3.0–3.5 ppm for the SPU were attributed to the chemical shift of the hydrogen in propanesultone. Fig. 1(C) and (D) shows the 1H NMR spectra for the CPU and QPU. As shown in the figure, the chemical shifts in a, b and c for CPU and QPU increased similarly due to the existence of N+. For the CPU, the chemical shifts in a, b and c increased to 2.43 ppm (N–CH3), 2.86 ppm (N–CH2–) and 4.22 ppm (O = C–O–CH2–), respectively; while for the QPU, the chemical shifts in a, b and c increased to 2.46 ppm (N–CH3), 2.90 ppm (N–CH2–), and 4.25 ppm (O = C–O–CH2–), respectively. On the other hand, for the CPU and QPU, the peaks of bromopropionic acid and iodomethane groups were overlapped with original peaks. The results indicated that sulfobetaine, carboxybetaine and quaternary ammonium salt were grafted onto the polyurethane. For the PU without further modification, the number average molecular weight (Mn) was 40,284 g/mol; the weight average molecular weight (Mw) was 73,512 g/mol; and the molecular weight distribution was 1.823, which presented relatively narrow molecular weight distribution. In addition, the further modifications did not break the molecular bonds.
R. Wang et al. / Materials Science and Engineering C 59 (2016) 556–564
559
Fig. 1. 1H NMR spectra for PU, SPU, CPU and QPU.
3.2. Morphologies of membranes Scanning electron microscopy (SEM) was performed to observe the morphologies of the membranes. As shown in Fig. 2, for the pristine PES membrane, the skin layer was not obvious; while the finger-like structure could be clearly observed. The skin layer was different from those observed in our previous report [26], since the solvent (DMSO as the solvent) used in this study was different from those (DMAc and NMP) used in the earlier studies, demonstrating that different solvents would affect the structure of the membranes. However, for the composite membranes, the figure-like structures disappeared instead of spongy structures. Besides, it could be observed that the pore sizes in cross sections were different for the composite membranes. The SPU/PES had macroporous structure, while the other membranes had relatively denser structure, demonstrating that the PUs with different groups would affect the morphologies of the membranes, which will affect the membrane properties as discussed in the following sections.
The distribution of the Ag nanoparticles on the surface of the CPUAg/PES membrane is shown in Fig. 2. The result indicated that the carboxybetaine polyurethane could load silver, and the silver nanoparticles were distributed homogenously on the membrane surface. 3.3. Water contact angles Water contact angles (WCAs) reflect the surface hydrophilicity of membranes, which might further affect the surface properties such as biocompatibility and antifouling property. As shown in Fig. 3, compared with the WCA (64.5°) for PES, the WCA for the composite membrane PU/PES was as high as 105.0° due to the existence of large amounts of hydrophobic groups such as benzene rings and ester groups. However, the WCAs for the SPU/PES, CPU/PES and QPU/PES membranes decreased to about 60°, demonstrating that the grafting of sulfobetaine, carboxybetaine and quaternary ammonium salt onto the polyurethane could change the hydrophilicity of polyurethanes and improve the surface wettability of the composite membranes. On the other hand,
560
R. Wang et al. / Materials Science and Engineering C 59 (2016) 556–564
Fig. 2. SEM images of membranes and distribution of Ag nanoparticles on the surface of CPU-Ag/PES measured by EDS.
the decrease in the WCAs for the SPU/PES, CPU/PES and QPU/PES was not significant compared to the pristine PES membrane. The PUs also had great effect on the membrane permeability. Different from those of the PES membranes prepared using DMAc or NMP as the solvents [27], the water flux for the pristine PES membrane prepared using DMSO was as high as 208.12 mL/m2·h·mm Hg, indicating that the solvents also affected the membrane permeability. On the other hand, the water fluxes for the PU/PES, SPU/PES, CPU/PES and QPU/PES were 218.43, 383.68, 442.45 and 508.33 mL/m2·h·mm Hg, respectively, demonstrating that the PUs could increase the permeability, especially for the SPU, CPU and QPU composite membranes which contained hydrophilic groups.
adsorption capacity to the anionic dye of Congo red (CR) compared to the cationic dye of methylene blue (MB), which is attributed to the abundant tertiary amine groups in each repetitive structure unit of PU. Together with the simple synthetic process of PU and the preparation of composite membranes, the PU/PES had potential in the area of water treatment.
3.4. Adsorption capacity Dyes including cationic dyes and anionic dyes are common toxins in industrial waste water, harming the aquatic organism and human health. Therefore, PU that contained tertiary amine groups is designed for improving the removal ability of dyes for the polymeric membranes. Fig. 4(a) showed that the PU/PES membrane had a significantly selective
Fig. 3. Water contact angles for the PES, PU/PES, SPU/PES, CPU/PES, and QPU/PES membranes, respectively.
Fig. 4. (a) The color changes of the membranes and solutions after contacting with CR and MB for 50 h; (b) Bilirubin adsorption amounts for the pristine PES and PU/PES membranes.
R. Wang et al. / Materials Science and Engineering C 59 (2016) 556–564
561
Furthermore, as the adsorption capacity to anion was proven, the PU/PES membrane was used for the removal of bilirubin, a metabolite that damages nervous systems [28,29]. As shown in Fig. 4(b), the PU/PES had higher bilirubin adsorption amount compared to pristine PES membrane due to the tertiary amine groups. Since PES has been widely used as blood-contacting materials [30], the PU/PES membrane has great potential application as an adsorbent used for blood purification. 3.5. Blood compatibility Blood-contacting materials require good blood compatibility. On the contacting surface, protein adsorption is believed to be an important role in material-associated clotting. Serum albumin is the most abundant protein in blood, and is always used to evaluate protein adsorption and anti-biological fouling property; while fibrinogen adsorption on the material surface is the key event to cause final blood coagulation [31]. Fig. 5 showed that the BSA and BFG adsorption amounts for the SPU/PES and CPU/PES decreased sharply compared to the pristine PES, suggesting that the zwitterionic membranes had good blood compatibility and antifouling property, as reported by many studies [8,32,33]. However, the PU/PES had high adsorption amounts to BSA and BFG due to the hydrophobicity as revealed by water contact angle; the QPU/PES also exhibited high protein adsorption amounts due to the positive charge of the quaternary ammonium salt groups. Blood clotting times are very important to evaluate the blood compatibility of materials. APTT is an indicator of the efficacy of the intrinsic and common plasma coagulation pathway; while PT is measuring the extrinsic pathway. TT was used to observe the clot formation time taken for the thrombin conversion of fibrinogen into fibrin. The shorter clotting time indicated the faster conversion of fibrinogen into insoluble fibrin protein, which may lead to thrombus [34,35]. As shown in Fig. 6, for all the composite membranes, the clotting times were prolonged compared to the pristine PES membrane, which was attributed to the existence of the citric acid groups in the PUs [36]. The results indicated that the PUs could improve the anticoagulant property of membranes. Activation of platelets is considered as a key factor in thrombus formation [37]. Thus, the number and morphology of the adhering platelets on the material surfaces are widely used to evaluate the blood compatibility. As shown in Fig. 7, many activated platelets with pseudopodia were adhered on the pristine PES membrane surface, even some platelets were ruptured. However, the platelet adhesion was significantly suppressed on the SPU/PES and CPU/PES surfaces, and the morphology of the platelets was not deformed, demonstrating the beneficial effect of zwitterions on the blood compatibility of SPU/ PES and CPU/PES membranes. Despite that the anticoagulant properties of the composite membranes had been improved by the PUs, the zwitterionic membranes had the least influence on blood ingredients such as protein adsorption
Fig. 5. BSA and BFG adsorbed amounts on pristine PES and composite membranes.
Fig. 6. APTT, PT and TT values of the PPP, pristine PES and composite membranes.
and platelet adhesion, which exhibited excellent blood compatibility and had potential applications in blood-contacting materials. 3.6. Antibacterial property The antibacterial properties of two membranes bearing quaternary ammonium salts and Ag nanoparticles were evaluated via bacterial inhibition zone, optical degree of co-cultured solution, and live/dead two-color fluorescence method, respectively. The bacterial inhibition zone is an effective way to reveal the bacteriostatic efficacy of materials which could release free antimicrobial substance [38,39]. As shown in Fig. 8, the QPU/PES had no bacterial inhibition zone since the antimicrobial macromolecules were immobilized on the membrane surface. However, for the CPU-Ag/PES, the sizes of inhibition zone were 0.6 mm toward E. coli and 1.5 mm toward S. aureus, respectively, which indicated that the CPU-Ag/PES bearing Ag nanoparticles could release Ag+ and had inhibition capacity toward both Gram-negative and Gram-positive bacteria, especially for the S. aureus. To further confirm the antibacterial property of the membranes, optical degree of the bacterial-membrane co-cultured solutions at different times was measured [40]. As shown in Fig. 9, both the E. coli and S. aureus growth rates were fast for the control sample, pristine PES and PU/PES membranes; for the QPU/PES, the E. coli growth was not inhibited significantly, while the S. aureus growth became slower, revealing the higher antibacterial activity toward S. aureus. Remarkably,
Fig. 7. SEM images of the platelets adhering on the pristine PES and composite membranes.
562
R. Wang et al. / Materials Science and Engineering C 59 (2016) 556–564
Fig. 8. The inhibition zone images for E. coli (Gram negative) and S. aureus (Gram positive).
the antibacterial activity of the CPU-Ag/PES was obviously toward both of E. coli and S. aureus. On the other hand, with the increase of times, the antibacterial properties decreased for both of the QPU/PES and CPU-Ag/ PES membranes. The bactericidal efficacy of the membranes was also assayed in terms of the live/dead two-color fluorescence method [41]. After contacting with the solutions of either E. coli or S. aureus for 12 h, the viable bacteria appeared green, while the dead bacteria appeared red under the fluorescence microscope. As shown in Fig. 10, a large amount of viable E. coli aggregated into clusters on the surfaces of pristine PES and PU/PES membranes, and few dead bacteria adhered on the surfaces,
which demonstrated that the pristine PES and PU/PES were susceptible to bacterial fouling. However, despite the low antifouling property of the QPU/PES surface which resulted in the bacterial adhesion, most of the E. coli on the surface were dead, exhibiting bactericidal activity. For the CPU-Ag/PES membrane, there were few viable or dead E. coli adhered on the surface owing to the antifouling property of the carboxybetaine groups and the bactericidal activity of the Ag nanoparticles. The co-culture of S. aureus and membranes showed similar results and the number of S. aureus on the surface was fewer due to the lower growth rate and adhesion compared to E. coli. In summary, the QPU/PES could kill adhered bacteria efficiently, and then get the antibacterial property; while the CPU/PES was proven to load Ag effectively, and then the CPU-Ag/PES showed the dual functions of higher antibacterial and antifouling properties. In addition, it was proven that the CPU/PES had good antifouling property against different biological pollutants due to low protein adsorption, platelet adhesion and bacterial adhesion. 4. Conclusion We provided a facile approach toward the functionalization of PU via simple reactions, and the PU/PES composite membranes for versatile biological applications. The composite membranes with tertiary amine had significant adsorption capability to toxin bilirubin, exhibiting adsorption capacities in the areas of blood purification. After converting into zwitterionic PUs including sulfobetaine and carboxybetaine, the composite membranes showed prolonged clotting times, decreased protein adsorption and suppressed platelet adhesion, indicating excellent blood compatibility and potential application as blood-contacting materials. Besides, the composite membranes containing quaternary ammonium salt exhibited bactericidal property without antifouling property. After loading Ag nanoparticles, the carboxybetaine membranes showed dual functions of bactericidal and antifouling properties. This work demonstrates the potential of PUs prepared by the facile approach as readily available, multi-functional, and easy-to-use materials to prepare composite membranes for versatile biological applications. Acknowledgments
Fig. 9. The optical degrees for E. coli (Gram negative) and S. aureus (Gram positive); the absorbance represents the bacterial amount after exposure to functionalized membranes at different times.
This work was financially sponsored by the National Natural Science Foundation of China (No. 51225303 and 51433007), and the Sichuan Province Youth Science and Technology Innovation Team (No. 2015TD0001). We should also thank our laboratory members for their generous help, and gratefully acknowledge the help of Ms. Hui. Wang, of the Analytical and Testing Center at Sichuan University, for the SEM.
R. Wang et al. / Materials Science and Engineering C 59 (2016) 556–564
563
Fig. 10. Fluorescence microscopy images for PES, PU/PES, QPU/PES and CPU/PES-Ag surfaces after exposure to E. coli (Gram negative) and S. aureus (Gram positive), respectively.
References [1] E.R. Gillies, J.M.J. Frechet, Designing macromolecules for therapeutic applications: polyester dendrimer-poly(ethylene oxide) "bow-tie" hybrids with tunable molecular weight and architecture, J. Am. Chem. Soc. 124 (2002) 14137–14146. [2] L. Tian, L. Yam, N. Zhou, H. Tat, K.E. Uhrich, Amphiphilic scorpion-like macromolecules: design, synthesis, and characterization, Macromolecules 37 (2004) 538–543. [3] Q. Zhang, P. Wilson, A. Anastasaki, R. McHale, D.M. Haddleton, Synthesis and aggregation of double hydrophilic diblock glycopolymers via aqueous SET-LRP, ACS Macro Lett. 3 (2014) 491–495. [4] C. Deng, H.Y. Tian, P.B. Zhang, J. Sun, X.S. Chen, X.B. Jing, Synthesis and characterization of RGD peptide grafted poly(ethylene glycol)-b-poly(L-lactide)-b-poly(L-glutamic acid) triblock copolymer, Biomacromolecules 7 (2006) 590–596. [5] L. Zou, W. Zhu, Y.M. Chen, F. Xi, Modification of side chain terminals of PEGylated molecular bottle brushes—a toolbar of molecular nanoobjects, Polymer 54 (2013) 481–484. [6] R. Narain, S.P. Armes, Synthesis and aqueous solution properties of novel sugar methacrylate-based homopolymers and block copolymers, Biomacromolecules 4 (2003) 1746–1758. [7] Z.J. Fan, B. Liu, J.Q. Wang, S.Y. Zhang, Q.Q. Lin, P.W. Gong, L.M. Ma, S.R. Yang, A novel wound dressing based on Ag/graphene polymer hydrogel: effectively kill bacteria and accelerate wound healing, Adv. Funct. Mater. 24 (2014) 3933–3943. [8] W.W. Yue, H.J. Li, T. Xiang, H. Qin, S.D. Sun, C.S. Zhao, Grafting of zwitterion from polysulfone membrane via surface-initiated ATRP with enhanced antifouling property and biocompatibility, J. Membr. Sci. 446 (2013) 79–91. [9] H. Qin, C. Sun, C. He, D. Wang, C. Cheng, S. Nie, S. Sun, C. Zhao, High efficient protocol for the modification of polyethersulfone membranes with anticoagulant and antifouling properties via in situ cross-linked copolymerization, J. Membr. Sci. 468 (2014) 172–183. [10] D. Paripovic, H. Hall-Bozic, H.A. Klok, Osteoconductive surfaces generated from peptide functionalized poly(2-hydroxyethyl methacrylate-co-2-(methacryloyloxy)ethyl phosphate) brushes, J. Mater. Chem. 22 (2012) 19570–19578. [11] S.A.A.N. Nasreen, S. Sundarrajan, S.A.S. Nizar, R. Balamurugan, S. Ramakrishna, In situ polymerization of PVDF–HEMA polymers: electrospun membranes with improved flux and antifouling properties for water filtration, Polym. J. 46 (2014) 167–174. [12] D. Pranantyo, L.Q. Xu, K.-G. Neoh, E.-T. Kang, W. Yang, S. Lay-Ming Teo, Photoinduced anchoring and micropatterning of macroinitiators on polyurethane surfaces for graft polymerization of antifouling brush coatings, J. Mater. Chem. B 2 (2014) 398. [13] S. Chen, L. Li, C. Zhao, J. Zheng, Surface hydration: principles and applications toward low-fouling/nonfouling biomaterials, Polymer 51 (2010) 5283–5293.
[14] S. Abraham, S. Brahim, K. Ishihara, A. Guiseppi-Elie, Molecularly engineered p(HEMA)-based hydrogels for implant biochip biocompatibility, Biomaterials 26 (2005) 4767–4778. [15] L. García-Fernández, A. Specht, A. del Campo, A polyurethane-based positive photoresist, Macromol. Rapid Commun. 35 (2014) 1801–1807. [16] L. Ma, H. Qin, C. Cheng, Y. Xia, C. He, C. Nie, L. Wang, C. Zhao, Mussel-inspired selfcoating at macro-interface with improved biocompatibility and bioactivity via dopamine grafted heparin-like polymers and heparin, J. Mater. Chem. B 2 (2014) 363–375. [17] A. Narumi, K. Fuchise, R. Kakuchi, A. Toda, T. Satoh, S. Kawaguchi, K. Sugiyama, A. Hirao, T. Kakuchi, A versatile method for adjusting thermoresponsivity: synthesis and ‘click’ reaction of an azido end-functionalized poly(N-isopropylacrylamide), Macromol. Rapid Commun. 29 (2008) 1126–1133. [18] K. Fuchise, R. Kakuchi, S.T. Lin, R. Sakai, S.I. Sato, T. Satoh, W.C. Chen, T. Kakuchi, Control of thermoresponsive property of urea end-functionalized poly(Nisopropylacrylamide) based on the hydrogen bond-assisted self-assembly in water, J. Polym. Sci. Pol. Chem. 47 (2009) 6259–6268. [19] L. Tauhardt, D. Pretzel, K. Kempe, M. Gottschaldt, D. Pohlers, U.S. Schubert, Zwitterionic poly(2-oxazoline)s as promising candidates for blood contacting applications, Polym. Chem. 5 (2014) 5751–5764. [20] Q. Zhang, X.D. Tang, T.S. Wang, F.Q. Yu, W.J. Guo, M.S. Pei, Thermo-sensitive zwitterionic block copolymers via ATRP, RSC Adv. 4 (2014) 24240–24247. [21] H.S. Lee, M.Q. Yee, Y.Y. Eckmann, N.J. Hickok, D.M. Eckmann, R.J. Composto, Reversible swelling of chitosan and quaternary ammonium modified chitosan brush layers: effects of pH and counter anion size and functionality, J. Mater. Chem. 22 (2012) 19605–19616. [22] R. Hu, G.Z. Li, Y.J. Jiang, Y. Zhang, J.J. Zou, L. Wang, X.W. Zhang, Silver-zwitterion organic–inorganic nanocomposite with antimicrobial and antiadhesive capabilities, Langmuir 29 (2013) 3773–3779. [23] C. Cheng, S. Li, W.F. Zhao, Q. Wei, S.Q. Nie, S.D. Sun, C.S. Zhao, The hydrodynamic permeability and surface property of polyethersulfone ultrafiltration membranes with mussel-inspired polydopamine coatings, J. Membr. Sci. 417 (2012) 228–236. [24] L. Ma, B.H. Su, C. Cheng, Z.H. Yin, H. Qin, J.M. Zhao, S.D. Sun, C.S. Zhao, Toward highly blood compatible hemodialysis membranes via blending with heparin-mimicking polyurethane: study in vitro and in vivo, J. Membr. Sci. 470 (2014) 90–101. [25] L.L. Li, Z.H. Yin, F.L. Li, T. Xiang, Y. Chen, C.S. Zhao, Preparation and characterization of poly(acrylonitrile-acrylic acid-N-vinyl pyrrolidinone) terpolymer blended polyethersulfone membranes, J. Membr. Sci. 349 (2010) 56–64. [26] F. Ran, J. Li, Y. Lu, L.R. Wang, S.Q. Nie, H.M. Song, L. Zhao, S.D. Sun, C.S. Zhao, A simple method to prepare modified polyethersulfone membrane with improved hydrophilic surface by one-pot: the effect of hydrophobic segment length and molecular weight of copolymers, Mater. Sci. Eng. C 37 (2014) 68–75.
564
R. Wang et al. / Materials Science and Engineering C 59 (2016) 556–564
[27] T. Xiang, H. Fu, W.W. Yue, S.D. Sun, C.S. Zhao, Preparation and characterization of poly(acrylonitrile-co-maleic anhydride) copolymer modified polyethersulfone membranes, Sep. Sci. Technol. 48 (2013) 1627–1635. [28] A. Denizli, M. Kocakulak, E. Piskin, Bilirubin removal from human plasma in a packed-bed column system with dye-affinity microbeads, J. Chromatogr. B 707 (1998) 25–31. [29] K. Ishihara, E. Hayashi, A. Watanabe, N. Nakabayashi, T. Akisawa, S. Koshikawa, S. Kimura, Y. Yamamoto, Adsorption of bilirubin by direct hemoperfusion using macroporous polymer beads, Kobunshi Ronbunshu 48 (1991) 283–288. [30] C.S. Zhao, J.M. Xue, F. Ran, S.D. Sun, Modification of polyethersulfone membranes — a review of methods, Prog. Mater. Sci. 58 (2013) 76–150. [31] F. Ran, X.Q. Niu, H.M. Song, C. Cheng, W.F. Zhao, S.Q. Nie, L.R. Wang, A.M. Yang, S.D. Sun, C.S. Zhao, Toward a highly hemocompatible membrane for blood purification via a physical blend of miscible comb-like amphiphilic copolymers, Biomater. Sci. 2 (2014) 538–547. [32] F.L. Hu, K.M. Chen, H. Xu, H.C. Gu, Functional short-chain zwitterion coated silica nanoparticles with antifouling property in protein solutions, Colloids Surf. B 126 (2015) 251–256. [33] B.R. Li, M.Y. Shen, H.H. Yu, Y.K. Li, Rapid construction of an effective antifouling layer on a Au surface via electrodeposition, Chem. Commun. 50 (2014) 6793–6796. [34] Y. Xia, C. Cheng, R. Wang, H. Qin, Y. Zhang, L. Ma, H. Tan, Z. Gu, C. Zhao, Surfaceengineered nanogel assemblies with integrated blood compatibility, cell proliferation and antibacterial property: towards multifunctional biomedical membranes, Polym. Chem. 5 (2014) 5906–5919.
[35] S.Q. Nie, M. Tang, C. Cheng, Z.H. Yin, L.R. Wang, S.D. Sun, C.S. Zhao, Biologically inspired membrane design with a heparin-like interface: prolonged blood coagulation, inhibited complement activation, and bio-artificial liver related cell proliferation, Biomater. Sci. 2 (2014) 98–109. [36] L. Li, C. Cheng, T. Xiang, M. Tang, W. Zhao, S. Sun, C. Zhao, Modification of polyethersulfone hemodialysis membrane by blending citric acid grafted polyurethane and its anticoagulant activity, J. Membr. Sci. 405-406 (2012) 261–274. [37] H. Mirzadeh, M. Dadsetan, N. Sharifi-Sanjani, Platelet adhesion on laser-induced acrylic acid-grafted polyethylene terephthalate, J. Appl. Polym. Sci. 86 (2002) 3191–3196. [38] A. Roguska, A. Belcarz, M. Pisarek, G. Ginalska, M. Lewandowska, TiO2 nanotube composite layers as delivery system for ZnO and Ag nanoparticles — an unexpected overdose effect decreasing their antibacterial efficacy, Mater. Sci. Eng. C 51 (2015) 158–166. [39] R. Sridhar, S. Sundarrajan, A. Vanangamudi, G. Singh, T. Matsuura, S. Ramakrishna, Green processing mediated novel polyelectrolyte nanofibers and their antimicrobial evaluation, Macromol. Mater. Eng. 299 (2014) 283–289. [40] S.H. Yu, F.L. Mi, S.S. Shyu, C.H. Tsai, C.K. Peng, J.Y. Lai, Miscibility, mechanical characteristic and platelet adhesion of 6-O-carboxymethylchitosan/polyurethane semi-IPN membranes, J. Membr. Sci. 276 (2006) 68–80. [41] W.J. Yang, D. Pranantyo, K.G. Neoh, E.T. Kang, S.L.M. Teo, D. Rittschof, Layer-by-layer click deposition of functional polymer coatings for combating marine biofouling, Biomacromolecules 13 (2012) 2769–2780.
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
A facile approach toward multi-functional polyurethane/ polyethersulfone composite membranes for versatile applications Rui Wang a, Tao Xiang a, Wei-Feng Zhao a,⁎, Chang-Sheng Zhao a,b,⁎ a b
College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu 610065, People's Republic of China National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, People's Republic of China
a r t i c l e
i n f o
Article history: Received 28 May 2015 Received in revised form 23 September 2015 Accepted 20 October 2015 Available online 21 October 2015 Keywords: Polyurethane Polyethersulfone Adsorption capacity Blood compatibility Antibacterial property
a b s t r a c t The complex synthesis through multistep reactions and tedious purifications based on different monomers or macromolecules limits the practical applications of functional polymers. Herein, a facile approach toward a series of functional polyurethanes (PUs) is designed for versatile biological applications within fewer step reactions under mild conditions. The tertiary amino groups in the PU are converted into zwitterions or quaternary ammonium salt via simple one-step synthesis, and then used to prepare PU/polyethersulfone composite membranes. The composite membrane with tertiary amine groups exhibits significant adsorption capability to anionic dye Congo red (CR) and toxin bilirubin. The membrane bearing zwitterionic PU displays excellent blood compatibility; while which with quaternary ammonium salts has antibacterial property. Furthermore, carboxybetainefunctional composite membrane is exploited to bear Ag nanoparticles to endow with dual functions of antibacterial and antifouling properties. This work demonstrates the potential of PUs as readily available, multifunctional, and easy-to-use materials for biological applications. © 2015 Elsevier B.V. All rights reserved.
1. Introduction With the development of polymeric materials, desired properties could be achieved flexibly by utilizing either different common monomers or some specific monomers with elaborate modifications [1–3]. Besides, the macromolecules could be further modified for extensive applications using the functional groups such as –COOH, –OH and –NH2 on the macromolecular chains [4–6]. For example, the macromolecules or monomers that contained quaternary ammonium salt or Ag nanoparticles [7] could endow the materials with broad-spectrum antimicrobial property; while antifouling property was generally achieved using the monomers or macromolecules with zwitterions, hydroxyl groups and/or PEG chains, such as Sulfobetaine methacrylate (SBMA) [8], Hydroxyethyl methacrylate (HEMA) [9–11] and Poly(ethylene glycol) methacrylate (PEGMA) [12,13], which could also improve biocompatibility of materials [14]. On the other hand, for the polymers like polyesters and polyurethanes, most of the active functional groups like –OH, –NH2 or –COOH in monomers are usually sacrificed in the polymerization process, thus the possibility of further modifications is less than the polymers prepared by vinyl monomers [15,16]. Despite that the types of monomers and macromolecules have been increasingly diversified through the modification, a considerable amounts of synthetic processes generally required multistep synthesis ⁎ Corresponding authors. E-mail addresses: [email protected], [email protected] (W.-F. Zhao), [email protected], [email protected] (C.-S. Zhao).
http://dx.doi.org/10.1016/j.msec.2015.10.058 0928-4931/© 2015 Elsevier B.V. All rights reserved.
with many reactants or catalysts [17,18], restricting the practical applications due to the lack of efficiency and universality. Regarding this concern, developing different functions based on the same monomers or macromolecules through fewer step reactions under mild conditions is an indispensable strategy for both the productions and the applications. The aim of this study is to develop a facile strategy to combine various functions in polyurethane (PU) via straightforward reactions. 4,4′-Diphenylmethane diisocyanate (MDI) was used as the hard segment to enhance the miscibility of PU with other polymeric materials; while N-methyldiethanolamine (MDEA, containing tertiary amino group) was used as the soft segment. Additionally, citric acid (CA) was used as blocking agent to endow with anticoagulation property. Different from the modification of the macromolecules that contained –OH, –NH2 or –COOH, the various functionalities in this study are based on the tertiary amino groups, which could be further converted into zwitterions (sulfobetaine and carboxybetaine) [19,20] or quaternary ammonium salt [21]. The process was performed through one-step reaction without any catalysts or additional reactants. The synthesized PUs were exploited to prepare composite membranes with polyethersulfone (PES), and the adsorption capability of the composite membrane that contained tertiary amino groups was evaluated firstly by using dye Congo red (CR) and toxin bilirubin. The blood compatibility and antifouling property of the zwitterionic composite membranes were then evaluated in terms of clotting times, protein adsorption and platelet adhesion; while the activities of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were performed to demonstrate the
R. Wang et al. / Materials Science and Engineering C 59 (2016) 556–564
antibacterial property of the membrane bearing quaternary ammonium salts. Furthermore, it was found that the carboxybetaine could load silver, which has been seldom reported [22]; thus the carboxybetaine membrane was used to bear Ag nanoparticles to endow with dual functions of antibacterial and antifouling properties. 2. Experimental 2.1. Materials 4,4′-Diphenylmethane diisocyanate (MDI, 98%, Aladdin), Nmethyldiethanolamine (MDEA, 98%, Aladdin), 1,3-propanesulfonate (98%, Aladdin), 3-bromopropionic acid (98%, Aladdin), and iodomethane (98%, Xiya) were used without further purification. N,Ndimethylformamide (DMF, 99%, Kelong), 1-methyl-2-pyrrolidinone (NMP, 99%, Kelong) and dimethyl sulfoxide (DMSO, 99%, Kelong) were distilled under vacuum. Deionized water (DI water) was used throughout the study. Congo red (CR) and methylene blue (MB) were obtained from Kelong Inc. Bilirubin (98%) was purchased from Aladdin Industrial Inc. The LIVE/DEAD BacLight Bacterial Viability Kit L-7012 was purchased from Thermo Fisher Scientific Inc. 2.2. Synthesis and characterization of polyurethanes 2.2.1. Synthesis of polyurethane with tertiary amine (PU) All the polyurethanes were modified from original polyurethane that contained tertiary amine groups (PU) as shown in Scheme 1 and the synthetic process of PU is as follows: MDI (6.92 g) and MDEA (3.08 g) were dissolved in DMAc with the monomer concentration of 20 wt.%, and the molar ratio of the MDI to NMDA was 16:15. The polymerization was carried out in nitrogen gas at 75 °C for 2 h, and then citric acid was introduced in the reaction system and carried out at 80 °C for another 4 h. After the reaction, the polyurethane was precipitated with deionized (DI) water and washed with ethanol for several times. The product was dried in a vacuum oven at 40 °C. 2.2.2. Synthesis of polyurethane with sulfobetaine (SPU) 1,3-Propanesulfonate (1.98 g) was added dropwise into the solution of the as-prepared PU (2.00 g) in DMSO, and the reaction was carried
557
out at 25 °C for 24 h. Then the product was precipitated using ethanol and dried in a vacuum oven at 40 °C. 2.2.3. Synthesis of polyurethane with carboxybetaine (CPU) 3-Bromopropionic acid (2.49 g) and the PU (2.00 g) were dissolved in DMSO, and reaction was carried at 60 °C for 24 h. After the reaction, the product was precipitated using ethanol and dried in a vacuum oven at 40 °C. 2.2.4. Synthesis of polyurethane with quaternary ammonium (QPU) CH3I (12 mL) was added into the solution of the PU (2.00 g) in NMP and reacted at 60 °C for 12 h. After the reaction, the product was precipitated using ethanol and dried in a vacuum oven at 40 °C. 2.2.5. Characterization of the polyurethanes 1 H NMR (400 MHz) spectra were recorded on a BrukerAVII-400 MHz spectrometer (Bruker Co., Germany), using tetramethylsilane (TMS) as the internal standard in DMSO-d6 at room temperature. Gel permeation chromatography (GPC) measurement was performed by using a PL220 GPC analyzer (Britain) to measure molecular weight. N,N-dimethyl formamide (DMF) was chosen as the eluent and polystyrene (PS) as the reference. 2.3. Preparation of membranes PES was dissolved in DMSO to keep the concentration at 16 wt.%. Then the PU was added to the solution and the concentration was controlled at 4 wt.%. The solution was prepared into membranes by spin coating coupled with a phase inversion technique as described in our earlier reports [23,24]. The membranes modified with PU, SPU, CPU and QPU were termed PU/PES, SPU/PES, CPU/PES and QPU/PES, respectively. To obtain membrane bearing Ag nanoparticles, the CPU/PES membrane was firstly immersed into 0.1 M NaOH for 10 min and rinsed 3 times in DI water. Then the membrane was immersed into 0.05 mM AgNO3 solution with oscillation for 24 h in the dark, followed by rinsing with DI water for 3 times to remove the excess AgNO3. Then the membrane was dipped in 0.05 mM NaBH4 for 2 h to prepare Ag loaded membrane which was termed CPU-Ag/PES.
Scheme 1. Synthesis of a series of polyurethanes.
558
R. Wang et al. / Materials Science and Engineering C 59 (2016) 556–564
The morphologies of the membranes were observed by using a JSM7500F scanning microscope (JEOL, Japan). Water contact angles were measured with a contact angle goniometer (OCA20, Dataphysics, Germany). 2.4. Ultrafiltration experiments Ultrafiltration experiments were carried out on the apparatus as described in our previous study [25]. A dead-end ultrafiltration (UF) cell with an effective membrane area of 3.90 cm2 was used. The membrane was pre-compacted with deionized water for 30 min at a pressure of 0.1 MPa to get steady before the ultrafiltration experiments. The water flux was determined as follows: Flux ¼
V SPt
ð1Þ
where V (mL) is the volume of the permeated solution; S (m2) is the effective membrane area; P (mm Hg) is the pressure applied to the membrane and t (h) is the time for collecting permeated solution. 2.5. Adsorption to Congo red (CR), methylene blue (MB) and bilirubin The PU/PES membranes (5 pieces) were dipped in 10 mL of 200 mmol/L CR or MB aqueous solution at 25 °C with the stirring speed of 100 rpm. The adsorption amounts of the CR and MB were calculated using the absorbance at the wavelength of 497 and 630 nm, respectively. For bilirubin adsorption, 0.15 g of bilirubin was firstly dissolved in 1 mL of 0.1 M NaOH, and then diluted to 100 mL using phosphatic buffer solution (PBS, pH 7.4) away from light to prepare adsorbate solution. Then, the PU/PES membranes (20 pieces) were dipped in 10 mL bilirubin solution at 25 °C with the stirring speed of 100 rpm. The adsorption amount of the bilirubin was calculated by the absorbance at the wavelength of 438 nm, and all the operations were under dark condition.
2.8. Platelet adhesion experiments To study platelet adhesion, the membrane with an area of 1 × 1 cm2 was dipped in normal saline at 37 °C for 1 h. After removing the normal saline, 200 μL of fresh PRP was dropped in each well of the culture plate and then incubated at 37 °C for 2 h. Then the PRP was decanted off and the membrane was rinsed three times with normal saline. The adhered platelets on the membrane surface were fixed using 2.5 wt.% glutaraldehyde in normal saline at 4 °C for 24 h. Finally, the sample was washed with normal saline, and dehydrated with a series of normal saline/ ethanol mixtures with increasing ethanol concentration (25, 50, 75 and 100 wt.%), and then dried at room temperature. The platelet adhesion was observed using scanning electron microscopy. 2.9. Antibacterial tests Firstly, bacterial inhibition zone method was used to study the antibacterial property. The sterilized membrane with an area of 1 × 1 cm2 was placed on E. coli or S. aureus bacteria agar plate at an inoculum concentration of 107 colony forming units per mL (cfu mL− 1) and then incubated at 37 °C for 12 h. The presence of the inhibition zone was recorded by a digital camera. Then, the antibacterial activity to bacteria suspension was investigated. The membranes (two pieces, 1 × 1 cm2 for each) were immersed in 2 mL of E. coli or S. aureus suspension at 106 cfu mL−1 and incubated in a shaking incubator at 37 °C for 2, 4, 6, and 8 h, respectively. Then the optical degree of the bacterial suspension was determined at the wavelength of 500 nm. Finally, live/dead two-color fluorescence method was used. The membrane that co-cultured with E. coli or S. aureus suspension at 106 cfu mL−1 for 12 h was washed with normal saline. Then the membrane was stained with the LIVE/DEAD BacLight Bacterial Viability Kit, and observed under a fluorescent microscopy. 3. Results and discussion
2.6. Protein adsorption experiments 3.1. Synthesis of polyurethanes Firstly, the membrane with an area of 1 × 1 cm2 was dipped in PBS (pH 7.4) containing bovine serum albumin (BSA) or bovine fibrinogen (BFG) with a concentration of 1 mg/mL, and incubated at 37 °C for 1 h, followed by slight rinsing with PBS and double distilled water. Then the membrane was immersed into a washing solution (2% sodium dodecyl sulfate at 37 °C) and shaken for 2 h to remove the adsorbed protein. The protein concentration in the washing solution was determined by using the Micro BCA™ Protein Assay Reagent Kit (PIERCE), and the adsorbed protein amount was calculated. 2.7. Blood clotting time experiments The activated partial thromboplastin time (APTT) test was performed as follows. Fresh human blood was centrifuged at 1000 rpm for 15 min to obtain platelet-rich plasma (PRP) or at 4000 rpm for 15 min to obtain platelet-poor plasma (PPP). The membrane with an area of 1 × 1 cm2 was firstly dipped in 0.2 mL PBS (pH 7.4) for 1 h. After removing the PBS, 0.1 mL of fresh PPP was introduced and incubated at 37 °C for 30 min. Then 50 μL of the incubated PPP was added into a test-tube, followed by the addition of 50 μL of APTT agent (Dade Actin Activated Cephaloplastin Reagent, Siemens; incubated 10 min before use), and incubated at 37 °C. Thereafter, 50 μL of 0.025 M CaCl2 solution was added and then the APTT was measured. For prothrombin time (PT) test, 50 μL of the incubated PPP was mixed well with 100 μL of TT agent (Thromborel®S, Siemens; incubated 10 min before use) at 37 °C and the thrombin time (TT) was measured. For PT test, 50 μL of the incubated PPP was mixed with 100 μL of PT agent (Thromborel®S, Siemens; incubated 10 min before use) at 37 °C and then the PT was measured.
1 H NMR is an effective way to analyze the molecular structure of polyurethanes. The 1H NMR for the PU with tertiary amine groups is shown in Fig. 1(A): δ = 2.24 ppm (a, N–CH3), δ = 2.67 ppm (b, N–CH2–), δ = 4.15 ppm (c, O = C–O–CH2–), δ = 8.54 ppm (d, O = C–NH–), δ = 7.35 and 7.09 ppm (e and f, benzene ring), δ = 3.77 ppm (g, –CH2–Ar), δ = 9.55 ppm (h, –COOH). As shown in Fig. 1(B), for the SPU, the chemical shifts of hydrogen in a, b and c increased to 2.35 ppm (N–CH3), 2.76 ppm (N–CH2–), 4.17 ppm (O = C–O–CH2–), respectively, which was caused by the N+ after that the tertiary amine was converted into sulfobetaine. In addition, new peaks at 3.0–3.5 ppm for the SPU were attributed to the chemical shift of the hydrogen in propanesultone. Fig. 1(C) and (D) shows the 1H NMR spectra for the CPU and QPU. As shown in the figure, the chemical shifts in a, b and c for CPU and QPU increased similarly due to the existence of N+. For the CPU, the chemical shifts in a, b and c increased to 2.43 ppm (N–CH3), 2.86 ppm (N–CH2–) and 4.22 ppm (O = C–O–CH2–), respectively; while for the QPU, the chemical shifts in a, b and c increased to 2.46 ppm (N–CH3), 2.90 ppm (N–CH2–), and 4.25 ppm (O = C–O–CH2–), respectively. On the other hand, for the CPU and QPU, the peaks of bromopropionic acid and iodomethane groups were overlapped with original peaks. The results indicated that sulfobetaine, carboxybetaine and quaternary ammonium salt were grafted onto the polyurethane. For the PU without further modification, the number average molecular weight (Mn) was 40,284 g/mol; the weight average molecular weight (Mw) was 73,512 g/mol; and the molecular weight distribution was 1.823, which presented relatively narrow molecular weight distribution. In addition, the further modifications did not break the molecular bonds.
R. Wang et al. / Materials Science and Engineering C 59 (2016) 556–564
559
Fig. 1. 1H NMR spectra for PU, SPU, CPU and QPU.
3.2. Morphologies of membranes Scanning electron microscopy (SEM) was performed to observe the morphologies of the membranes. As shown in Fig. 2, for the pristine PES membrane, the skin layer was not obvious; while the finger-like structure could be clearly observed. The skin layer was different from those observed in our previous report [26], since the solvent (DMSO as the solvent) used in this study was different from those (DMAc and NMP) used in the earlier studies, demonstrating that different solvents would affect the structure of the membranes. However, for the composite membranes, the figure-like structures disappeared instead of spongy structures. Besides, it could be observed that the pore sizes in cross sections were different for the composite membranes. The SPU/PES had macroporous structure, while the other membranes had relatively denser structure, demonstrating that the PUs with different groups would affect the morphologies of the membranes, which will affect the membrane properties as discussed in the following sections.
The distribution of the Ag nanoparticles on the surface of the CPUAg/PES membrane is shown in Fig. 2. The result indicated that the carboxybetaine polyurethane could load silver, and the silver nanoparticles were distributed homogenously on the membrane surface. 3.3. Water contact angles Water contact angles (WCAs) reflect the surface hydrophilicity of membranes, which might further affect the surface properties such as biocompatibility and antifouling property. As shown in Fig. 3, compared with the WCA (64.5°) for PES, the WCA for the composite membrane PU/PES was as high as 105.0° due to the existence of large amounts of hydrophobic groups such as benzene rings and ester groups. However, the WCAs for the SPU/PES, CPU/PES and QPU/PES membranes decreased to about 60°, demonstrating that the grafting of sulfobetaine, carboxybetaine and quaternary ammonium salt onto the polyurethane could change the hydrophilicity of polyurethanes and improve the surface wettability of the composite membranes. On the other hand,
560
R. Wang et al. / Materials Science and Engineering C 59 (2016) 556–564
Fig. 2. SEM images of membranes and distribution of Ag nanoparticles on the surface of CPU-Ag/PES measured by EDS.
the decrease in the WCAs for the SPU/PES, CPU/PES and QPU/PES was not significant compared to the pristine PES membrane. The PUs also had great effect on the membrane permeability. Different from those of the PES membranes prepared using DMAc or NMP as the solvents [27], the water flux for the pristine PES membrane prepared using DMSO was as high as 208.12 mL/m2·h·mm Hg, indicating that the solvents also affected the membrane permeability. On the other hand, the water fluxes for the PU/PES, SPU/PES, CPU/PES and QPU/PES were 218.43, 383.68, 442.45 and 508.33 mL/m2·h·mm Hg, respectively, demonstrating that the PUs could increase the permeability, especially for the SPU, CPU and QPU composite membranes which contained hydrophilic groups.
adsorption capacity to the anionic dye of Congo red (CR) compared to the cationic dye of methylene blue (MB), which is attributed to the abundant tertiary amine groups in each repetitive structure unit of PU. Together with the simple synthetic process of PU and the preparation of composite membranes, the PU/PES had potential in the area of water treatment.
3.4. Adsorption capacity Dyes including cationic dyes and anionic dyes are common toxins in industrial waste water, harming the aquatic organism and human health. Therefore, PU that contained tertiary amine groups is designed for improving the removal ability of dyes for the polymeric membranes. Fig. 4(a) showed that the PU/PES membrane had a significantly selective
Fig. 3. Water contact angles for the PES, PU/PES, SPU/PES, CPU/PES, and QPU/PES membranes, respectively.
Fig. 4. (a) The color changes of the membranes and solutions after contacting with CR and MB for 50 h; (b) Bilirubin adsorption amounts for the pristine PES and PU/PES membranes.
R. Wang et al. / Materials Science and Engineering C 59 (2016) 556–564
561
Furthermore, as the adsorption capacity to anion was proven, the PU/PES membrane was used for the removal of bilirubin, a metabolite that damages nervous systems [28,29]. As shown in Fig. 4(b), the PU/PES had higher bilirubin adsorption amount compared to pristine PES membrane due to the tertiary amine groups. Since PES has been widely used as blood-contacting materials [30], the PU/PES membrane has great potential application as an adsorbent used for blood purification. 3.5. Blood compatibility Blood-contacting materials require good blood compatibility. On the contacting surface, protein adsorption is believed to be an important role in material-associated clotting. Serum albumin is the most abundant protein in blood, and is always used to evaluate protein adsorption and anti-biological fouling property; while fibrinogen adsorption on the material surface is the key event to cause final blood coagulation [31]. Fig. 5 showed that the BSA and BFG adsorption amounts for the SPU/PES and CPU/PES decreased sharply compared to the pristine PES, suggesting that the zwitterionic membranes had good blood compatibility and antifouling property, as reported by many studies [8,32,33]. However, the PU/PES had high adsorption amounts to BSA and BFG due to the hydrophobicity as revealed by water contact angle; the QPU/PES also exhibited high protein adsorption amounts due to the positive charge of the quaternary ammonium salt groups. Blood clotting times are very important to evaluate the blood compatibility of materials. APTT is an indicator of the efficacy of the intrinsic and common plasma coagulation pathway; while PT is measuring the extrinsic pathway. TT was used to observe the clot formation time taken for the thrombin conversion of fibrinogen into fibrin. The shorter clotting time indicated the faster conversion of fibrinogen into insoluble fibrin protein, which may lead to thrombus [34,35]. As shown in Fig. 6, for all the composite membranes, the clotting times were prolonged compared to the pristine PES membrane, which was attributed to the existence of the citric acid groups in the PUs [36]. The results indicated that the PUs could improve the anticoagulant property of membranes. Activation of platelets is considered as a key factor in thrombus formation [37]. Thus, the number and morphology of the adhering platelets on the material surfaces are widely used to evaluate the blood compatibility. As shown in Fig. 7, many activated platelets with pseudopodia were adhered on the pristine PES membrane surface, even some platelets were ruptured. However, the platelet adhesion was significantly suppressed on the SPU/PES and CPU/PES surfaces, and the morphology of the platelets was not deformed, demonstrating the beneficial effect of zwitterions on the blood compatibility of SPU/ PES and CPU/PES membranes. Despite that the anticoagulant properties of the composite membranes had been improved by the PUs, the zwitterionic membranes had the least influence on blood ingredients such as protein adsorption
Fig. 5. BSA and BFG adsorbed amounts on pristine PES and composite membranes.
Fig. 6. APTT, PT and TT values of the PPP, pristine PES and composite membranes.
and platelet adhesion, which exhibited excellent blood compatibility and had potential applications in blood-contacting materials. 3.6. Antibacterial property The antibacterial properties of two membranes bearing quaternary ammonium salts and Ag nanoparticles were evaluated via bacterial inhibition zone, optical degree of co-cultured solution, and live/dead two-color fluorescence method, respectively. The bacterial inhibition zone is an effective way to reveal the bacteriostatic efficacy of materials which could release free antimicrobial substance [38,39]. As shown in Fig. 8, the QPU/PES had no bacterial inhibition zone since the antimicrobial macromolecules were immobilized on the membrane surface. However, for the CPU-Ag/PES, the sizes of inhibition zone were 0.6 mm toward E. coli and 1.5 mm toward S. aureus, respectively, which indicated that the CPU-Ag/PES bearing Ag nanoparticles could release Ag+ and had inhibition capacity toward both Gram-negative and Gram-positive bacteria, especially for the S. aureus. To further confirm the antibacterial property of the membranes, optical degree of the bacterial-membrane co-cultured solutions at different times was measured [40]. As shown in Fig. 9, both the E. coli and S. aureus growth rates were fast for the control sample, pristine PES and PU/PES membranes; for the QPU/PES, the E. coli growth was not inhibited significantly, while the S. aureus growth became slower, revealing the higher antibacterial activity toward S. aureus. Remarkably,
Fig. 7. SEM images of the platelets adhering on the pristine PES and composite membranes.
562
R. Wang et al. / Materials Science and Engineering C 59 (2016) 556–564
Fig. 8. The inhibition zone images for E. coli (Gram negative) and S. aureus (Gram positive).
the antibacterial activity of the CPU-Ag/PES was obviously toward both of E. coli and S. aureus. On the other hand, with the increase of times, the antibacterial properties decreased for both of the QPU/PES and CPU-Ag/ PES membranes. The bactericidal efficacy of the membranes was also assayed in terms of the live/dead two-color fluorescence method [41]. After contacting with the solutions of either E. coli or S. aureus for 12 h, the viable bacteria appeared green, while the dead bacteria appeared red under the fluorescence microscope. As shown in Fig. 10, a large amount of viable E. coli aggregated into clusters on the surfaces of pristine PES and PU/PES membranes, and few dead bacteria adhered on the surfaces,
which demonstrated that the pristine PES and PU/PES were susceptible to bacterial fouling. However, despite the low antifouling property of the QPU/PES surface which resulted in the bacterial adhesion, most of the E. coli on the surface were dead, exhibiting bactericidal activity. For the CPU-Ag/PES membrane, there were few viable or dead E. coli adhered on the surface owing to the antifouling property of the carboxybetaine groups and the bactericidal activity of the Ag nanoparticles. The co-culture of S. aureus and membranes showed similar results and the number of S. aureus on the surface was fewer due to the lower growth rate and adhesion compared to E. coli. In summary, the QPU/PES could kill adhered bacteria efficiently, and then get the antibacterial property; while the CPU/PES was proven to load Ag effectively, and then the CPU-Ag/PES showed the dual functions of higher antibacterial and antifouling properties. In addition, it was proven that the CPU/PES had good antifouling property against different biological pollutants due to low protein adsorption, platelet adhesion and bacterial adhesion. 4. Conclusion We provided a facile approach toward the functionalization of PU via simple reactions, and the PU/PES composite membranes for versatile biological applications. The composite membranes with tertiary amine had significant adsorption capability to toxin bilirubin, exhibiting adsorption capacities in the areas of blood purification. After converting into zwitterionic PUs including sulfobetaine and carboxybetaine, the composite membranes showed prolonged clotting times, decreased protein adsorption and suppressed platelet adhesion, indicating excellent blood compatibility and potential application as blood-contacting materials. Besides, the composite membranes containing quaternary ammonium salt exhibited bactericidal property without antifouling property. After loading Ag nanoparticles, the carboxybetaine membranes showed dual functions of bactericidal and antifouling properties. This work demonstrates the potential of PUs prepared by the facile approach as readily available, multi-functional, and easy-to-use materials to prepare composite membranes for versatile biological applications. Acknowledgments
Fig. 9. The optical degrees for E. coli (Gram negative) and S. aureus (Gram positive); the absorbance represents the bacterial amount after exposure to functionalized membranes at different times.
This work was financially sponsored by the National Natural Science Foundation of China (No. 51225303 and 51433007), and the Sichuan Province Youth Science and Technology Innovation Team (No. 2015TD0001). We should also thank our laboratory members for their generous help, and gratefully acknowledge the help of Ms. Hui. Wang, of the Analytical and Testing Center at Sichuan University, for the SEM.
R. Wang et al. / Materials Science and Engineering C 59 (2016) 556–564
563
Fig. 10. Fluorescence microscopy images for PES, PU/PES, QPU/PES and CPU/PES-Ag surfaces after exposure to E. coli (Gram negative) and S. aureus (Gram positive), respectively.
References [1] E.R. Gillies, J.M.J. Frechet, Designing macromolecules for therapeutic applications: polyester dendrimer-poly(ethylene oxide) "bow-tie" hybrids with tunable molecular weight and architecture, J. Am. Chem. Soc. 124 (2002) 14137–14146. [2] L. Tian, L. Yam, N. Zhou, H. Tat, K.E. Uhrich, Amphiphilic scorpion-like macromolecules: design, synthesis, and characterization, Macromolecules 37 (2004) 538–543. [3] Q. Zhang, P. Wilson, A. Anastasaki, R. McHale, D.M. Haddleton, Synthesis and aggregation of double hydrophilic diblock glycopolymers via aqueous SET-LRP, ACS Macro Lett. 3 (2014) 491–495. [4] C. Deng, H.Y. Tian, P.B. Zhang, J. Sun, X.S. Chen, X.B. Jing, Synthesis and characterization of RGD peptide grafted poly(ethylene glycol)-b-poly(L-lactide)-b-poly(L-glutamic acid) triblock copolymer, Biomacromolecules 7 (2006) 590–596. [5] L. Zou, W. Zhu, Y.M. Chen, F. Xi, Modification of side chain terminals of PEGylated molecular bottle brushes—a toolbar of molecular nanoobjects, Polymer 54 (2013) 481–484. [6] R. Narain, S.P. Armes, Synthesis and aqueous solution properties of novel sugar methacrylate-based homopolymers and block copolymers, Biomacromolecules 4 (2003) 1746–1758. [7] Z.J. Fan, B. Liu, J.Q. Wang, S.Y. Zhang, Q.Q. Lin, P.W. Gong, L.M. Ma, S.R. Yang, A novel wound dressing based on Ag/graphene polymer hydrogel: effectively kill bacteria and accelerate wound healing, Adv. Funct. Mater. 24 (2014) 3933–3943. [8] W.W. Yue, H.J. Li, T. Xiang, H. Qin, S.D. Sun, C.S. Zhao, Grafting of zwitterion from polysulfone membrane via surface-initiated ATRP with enhanced antifouling property and biocompatibility, J. Membr. Sci. 446 (2013) 79–91. [9] H. Qin, C. Sun, C. He, D. Wang, C. Cheng, S. Nie, S. Sun, C. Zhao, High efficient protocol for the modification of polyethersulfone membranes with anticoagulant and antifouling properties via in situ cross-linked copolymerization, J. Membr. Sci. 468 (2014) 172–183. [10] D. Paripovic, H. Hall-Bozic, H.A. Klok, Osteoconductive surfaces generated from peptide functionalized poly(2-hydroxyethyl methacrylate-co-2-(methacryloyloxy)ethyl phosphate) brushes, J. Mater. Chem. 22 (2012) 19570–19578. [11] S.A.A.N. Nasreen, S. Sundarrajan, S.A.S. Nizar, R. Balamurugan, S. Ramakrishna, In situ polymerization of PVDF–HEMA polymers: electrospun membranes with improved flux and antifouling properties for water filtration, Polym. J. 46 (2014) 167–174. [12] D. Pranantyo, L.Q. Xu, K.-G. Neoh, E.-T. Kang, W. Yang, S. Lay-Ming Teo, Photoinduced anchoring and micropatterning of macroinitiators on polyurethane surfaces for graft polymerization of antifouling brush coatings, J. Mater. Chem. B 2 (2014) 398. [13] S. Chen, L. Li, C. Zhao, J. Zheng, Surface hydration: principles and applications toward low-fouling/nonfouling biomaterials, Polymer 51 (2010) 5283–5293.
[14] S. Abraham, S. Brahim, K. Ishihara, A. Guiseppi-Elie, Molecularly engineered p(HEMA)-based hydrogels for implant biochip biocompatibility, Biomaterials 26 (2005) 4767–4778. [15] L. García-Fernández, A. Specht, A. del Campo, A polyurethane-based positive photoresist, Macromol. Rapid Commun. 35 (2014) 1801–1807. [16] L. Ma, H. Qin, C. Cheng, Y. Xia, C. He, C. Nie, L. Wang, C. Zhao, Mussel-inspired selfcoating at macro-interface with improved biocompatibility and bioactivity via dopamine grafted heparin-like polymers and heparin, J. Mater. Chem. B 2 (2014) 363–375. [17] A. Narumi, K. Fuchise, R. Kakuchi, A. Toda, T. Satoh, S. Kawaguchi, K. Sugiyama, A. Hirao, T. Kakuchi, A versatile method for adjusting thermoresponsivity: synthesis and ‘click’ reaction of an azido end-functionalized poly(N-isopropylacrylamide), Macromol. Rapid Commun. 29 (2008) 1126–1133. [18] K. Fuchise, R. Kakuchi, S.T. Lin, R. Sakai, S.I. Sato, T. Satoh, W.C. Chen, T. Kakuchi, Control of thermoresponsive property of urea end-functionalized poly(Nisopropylacrylamide) based on the hydrogen bond-assisted self-assembly in water, J. Polym. Sci. Pol. Chem. 47 (2009) 6259–6268. [19] L. Tauhardt, D. Pretzel, K. Kempe, M. Gottschaldt, D. Pohlers, U.S. Schubert, Zwitterionic poly(2-oxazoline)s as promising candidates for blood contacting applications, Polym. Chem. 5 (2014) 5751–5764. [20] Q. Zhang, X.D. Tang, T.S. Wang, F.Q. Yu, W.J. Guo, M.S. Pei, Thermo-sensitive zwitterionic block copolymers via ATRP, RSC Adv. 4 (2014) 24240–24247. [21] H.S. Lee, M.Q. Yee, Y.Y. Eckmann, N.J. Hickok, D.M. Eckmann, R.J. Composto, Reversible swelling of chitosan and quaternary ammonium modified chitosan brush layers: effects of pH and counter anion size and functionality, J. Mater. Chem. 22 (2012) 19605–19616. [22] R. Hu, G.Z. Li, Y.J. Jiang, Y. Zhang, J.J. Zou, L. Wang, X.W. Zhang, Silver-zwitterion organic–inorganic nanocomposite with antimicrobial and antiadhesive capabilities, Langmuir 29 (2013) 3773–3779. [23] C. Cheng, S. Li, W.F. Zhao, Q. Wei, S.Q. Nie, S.D. Sun, C.S. Zhao, The hydrodynamic permeability and surface property of polyethersulfone ultrafiltration membranes with mussel-inspired polydopamine coatings, J. Membr. Sci. 417 (2012) 228–236. [24] L. Ma, B.H. Su, C. Cheng, Z.H. Yin, H. Qin, J.M. Zhao, S.D. Sun, C.S. Zhao, Toward highly blood compatible hemodialysis membranes via blending with heparin-mimicking polyurethane: study in vitro and in vivo, J. Membr. Sci. 470 (2014) 90–101. [25] L.L. Li, Z.H. Yin, F.L. Li, T. Xiang, Y. Chen, C.S. Zhao, Preparation and characterization of poly(acrylonitrile-acrylic acid-N-vinyl pyrrolidinone) terpolymer blended polyethersulfone membranes, J. Membr. Sci. 349 (2010) 56–64. [26] F. Ran, J. Li, Y. Lu, L.R. Wang, S.Q. Nie, H.M. Song, L. Zhao, S.D. Sun, C.S. Zhao, A simple method to prepare modified polyethersulfone membrane with improved hydrophilic surface by one-pot: the effect of hydrophobic segment length and molecular weight of copolymers, Mater. Sci. Eng. C 37 (2014) 68–75.
564
R. Wang et al. / Materials Science and Engineering C 59 (2016) 556–564
[27] T. Xiang, H. Fu, W.W. Yue, S.D. Sun, C.S. Zhao, Preparation and characterization of poly(acrylonitrile-co-maleic anhydride) copolymer modified polyethersulfone membranes, Sep. Sci. Technol. 48 (2013) 1627–1635. [28] A. Denizli, M. Kocakulak, E. Piskin, Bilirubin removal from human plasma in a packed-bed column system with dye-affinity microbeads, J. Chromatogr. B 707 (1998) 25–31. [29] K. Ishihara, E. Hayashi, A. Watanabe, N. Nakabayashi, T. Akisawa, S. Koshikawa, S. Kimura, Y. Yamamoto, Adsorption of bilirubin by direct hemoperfusion using macroporous polymer beads, Kobunshi Ronbunshu 48 (1991) 283–288. [30] C.S. Zhao, J.M. Xue, F. Ran, S.D. Sun, Modification of polyethersulfone membranes — a review of methods, Prog. Mater. Sci. 58 (2013) 76–150. [31] F. Ran, X.Q. Niu, H.M. Song, C. Cheng, W.F. Zhao, S.Q. Nie, L.R. Wang, A.M. Yang, S.D. Sun, C.S. Zhao, Toward a highly hemocompatible membrane for blood purification via a physical blend of miscible comb-like amphiphilic copolymers, Biomater. Sci. 2 (2014) 538–547. [32] F.L. Hu, K.M. Chen, H. Xu, H.C. Gu, Functional short-chain zwitterion coated silica nanoparticles with antifouling property in protein solutions, Colloids Surf. B 126 (2015) 251–256. [33] B.R. Li, M.Y. Shen, H.H. Yu, Y.K. Li, Rapid construction of an effective antifouling layer on a Au surface via electrodeposition, Chem. Commun. 50 (2014) 6793–6796. [34] Y. Xia, C. Cheng, R. Wang, H. Qin, Y. Zhang, L. Ma, H. Tan, Z. Gu, C. Zhao, Surfaceengineered nanogel assemblies with integrated blood compatibility, cell proliferation and antibacterial property: towards multifunctional biomedical membranes, Polym. Chem. 5 (2014) 5906–5919.
[35] S.Q. Nie, M. Tang, C. Cheng, Z.H. Yin, L.R. Wang, S.D. Sun, C.S. Zhao, Biologically inspired membrane design with a heparin-like interface: prolonged blood coagulation, inhibited complement activation, and bio-artificial liver related cell proliferation, Biomater. Sci. 2 (2014) 98–109. [36] L. Li, C. Cheng, T. Xiang, M. Tang, W. Zhao, S. Sun, C. Zhao, Modification of polyethersulfone hemodialysis membrane by blending citric acid grafted polyurethane and its anticoagulant activity, J. Membr. Sci. 405-406 (2012) 261–274. [37] H. Mirzadeh, M. Dadsetan, N. Sharifi-Sanjani, Platelet adhesion on laser-induced acrylic acid-grafted polyethylene terephthalate, J. Appl. Polym. Sci. 86 (2002) 3191–3196. [38] A. Roguska, A. Belcarz, M. Pisarek, G. Ginalska, M. Lewandowska, TiO2 nanotube composite layers as delivery system for ZnO and Ag nanoparticles — an unexpected overdose effect decreasing their antibacterial efficacy, Mater. Sci. Eng. C 51 (2015) 158–166. [39] R. Sridhar, S. Sundarrajan, A. Vanangamudi, G. Singh, T. Matsuura, S. Ramakrishna, Green processing mediated novel polyelectrolyte nanofibers and their antimicrobial evaluation, Macromol. Mater. Eng. 299 (2014) 283–289. [40] S.H. Yu, F.L. Mi, S.S. Shyu, C.H. Tsai, C.K. Peng, J.Y. Lai, Miscibility, mechanical characteristic and platelet adhesion of 6-O-carboxymethylchitosan/polyurethane semi-IPN membranes, J. Membr. Sci. 276 (2006) 68–80. [41] W.J. Yang, D. Pranantyo, K.G. Neoh, E.T. Kang, S.L.M. Teo, D. Rittschof, Layer-by-layer click deposition of functional polymer coatings for combating marine biofouling, Biomacromolecules 13 (2012) 2769–2780.