A facile approach toward multifunctional polyethersulfone membranes via in situ cross-linked copolymerization.

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A facile approach towards multifunctional polyethersulfone membranes via in situ cross-linked copolymerization a

a

a

a

ab

Chuangchao Sun , Haifeng Ji , Hui Qin , Shengqiang Nie , Weifeng Zhao

& Changsheng

a

Zhao a

College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu 610065, People’s Republic of China b

Fiber and Polymer Technology, School of Chemical Science and Engineering, Royal Institute of Technology (KTH), Teknikringen 56-58 SE-100 44, Stockholm, Sweden Accepted author version posted online: 13 Jul 2015.

Click for updates To cite this article: Chuangchao Sun, Haifeng Ji, Hui Qin, Shengqiang Nie, Weifeng Zhao & Changsheng Zhao (2015): A facile approach towards multifunctional polyethersulfone membranes via in situ cross-linked copolymerization, Journal of Biomaterials Science, Polymer Edition, DOI: 10.1080/09205063.2015.1071929 To link to this article: http://dx.doi.org/10.1080/09205063.2015.1071929

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Publisher: Taylor & Francis Journal: Journal of Biomaterials Science, Polymer Edition DOI: http://dx.doi.org/10.1080/09205063.2015.1071929

A facile approach towards multifunctional polyethersulfone membranes via in situ cross-linked copolymerization Chuangchao Suna, Haifeng Ji, Hui Qin, Shengqiang Nie, Weifeng Zhao a,b* and

Downloaded by [New York University] at 02:01 24 July 2015

Changsheng Zhaoa** a

College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu 610065, People's Republic of China. b Fiber and Polymer Technology, School of Chemical Science and Engineering, Royal Institute of Technology (KTH), Teknikringen 56-58 SE-100 44, Stockholm, Sweden. *Corresponding author. E-mail: [email protected] (*), [email protected] (**) Tel.: +86-28-85400453; Fax: +86-28-85405402. Abstract In this study, multifunctional polyethersulfone (PES) membranes are prepared via in situ cross-linked copolymerization coupled with a liquid-liquid phase separation technique. Acrylic acid (AA) and N-vinylpyrrolidone (VP) are copolymerized in PES solution, and the solution is then directly used to prepare PES membranes. The infrared and X-ray photoelectron spectroscopy (XPS) testing, scanning electron microscopy (SEM) and water contact angles (WCA) measurements confirm the successful modification of pristine PES membrane. Protein adsorption, platelet adhesion, plasma recalcification time (PRT), and activated partial thromboplastin time (APTT) assays convince that the modified PES membranes have a better biocompatibility than pristine 1

PES membrane. In addition, the modified membranes showed good protein antifouling property and significant adsorption property of cationic dye. The loading of Ag nanoparticles into the modified membranes endows the composite membranes with antibacterial activity. Key words: In situ cross-linked copolymerization; Polyethersulfone membrane;


Multifunctional; Facile

2

1. Introduction Over the last few decades, artificial membranes have been widely used in the fields of hemodialysis, water treatment, tissue engineering and regenerative medicine [1-5]. However, several fatal problems are revealed during practically using of these artificial membranes, such as thrombus generation, membrane fouling, biomaterials induced bacterial infection and so on [6-8]. Therefore, the current focus and tendency on


membrane modification are to confer them with satisfied blood compatibility, antifouling and antibacterial properties. Multifunctional membranes confer two or more properties have attracted more and more researchers` attention. Xia et al. [9] prepared novel 3D multifunctional nanolayers on biomedical membrane surfaces via layer-by-layer (LBL) self-assembly of nanogels and heparin-like polymers, and the resultant membranes showed excellent blood compatibility, cell proliferation and antibacterial properties. Jiang et al. [10] prepared modified PP membranes through a simple dip-coating process in dopamine aqueous solution and further modified by poly(N-vinyl pyrrolidone) (PVP). The modified membranes showed enhanced antifouling property and remarkable antimicrobial activity after iodine complication with the PVP layer. In order to confer the artificial membranes with two or more properties, a lot of monomers as additives with special functional groups have been developed. Among these functional groups, carboxyl group (-COOH) has been extensively used to modify the blood compatibility of artificial membranes due to its good hydrophilicity [11, 12]. In addition, the ionizable carboxyl acid groups blended in membranes can be used for 3

loading of Ag nanoparticles, which indicates that the modified membranes are able to be conferred with antibacterial activity [13]. Meanwhile, acrylic acid (AA), a common monomer with high activity in polymerization and contains carboxyl group, is also used in cationic dye adsorption [14]. With these mentioned advantages, AA has become an ideal candidate for membrane substrate modification with multifunction. However, pure PAA has a poor miscibility with membrane matrix like polyethersulfone (PES).


Another monomer, N-Vinylpyrrolidone (VP), possessing good miscibility with membrane matrix and good blood compatibility becomes an excellent choice. Meanwhile, the addition of VP also confers the membranes with a new property of antifouling. Xu et al. [15] prepared a series of PVDF/PVDF-g-PVP blend porous membranes, and the modified PVDF membranes showed excellent antifouling property. Ran et al. [16] fabricated modified PES membranes by blending with a synthesized amphiphilic triblock co-polymer, poly(vinyl pyrrolidone)-b-poly(methyl methacrylate)-b-poly-(vinyl pyrrolidone) (PVP-b-PMMA-b-PVP). The resultant membranes showed improved blood compatibility, good cytocompatibility, ultrafiltration and protein anti-fouling properties. To integrate the additives with membrane matrix, various methods have been developed, such as grafting [17], coating [18] and physical blending [19, 20]. Among these methods, physical blending has usually been recognized as a convenient and versatile method to improve the properties of membrane surfaces. However, physical blending usually changes the mechanical property of the membrane matrix. The grafting method usually needs complicated physical or chemical treatment, which may 4

not be suitable for large-scale modification of the artificial membranes and practical use. Though, the coating method can meet the needs of ideal modification, the modified membranes are not stable during long time use. Therefore, a facile approach towards a large-scaled production is desired to fabricate multifunctional membranes with the stability to some content. In recent studies, in situ cross-linked copolymerization has gained much attention in


the industrial fields because of its easy operations and high efficiency. In situ cross-linked copolymerization has been applied to designing functional materials in biomaterials, fuel cells and water treatment [21-25]. Our group has also developed a series of modified PES membranes via in situ cross-linked copolymerization. Xiang et al. [26] demonstrated that in situ cross-linked copolymerization was a simple method because of its easy preparation and post-processing. Thus it can be facile to be applied to commercial process. Meanwhile, there were many cross-linked sites on the chain of additives via in situ cross-linked copolymerization. Therefore the additives could be tightly entangled with PES matrix, and the elution of additives would be prevented. Herein, multifunctional PES membranes are prepared via in situ cross-linked copolymerization coupled with a liquid-liquid phase separation by directly adding functional monomers along with the initiator and cross-linker into the matrix solution. The ATR-FTIR (Attenuated Total internal-Reflectance Fourier Transform Infrared spectroscopy), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and water contact angles (WCA) of the modified membranes are studied in detail. The blood compatibility, antifouling property, cationic dye and heavy metal ions 5

adsorptions are systematically investigated. Furthermore, the modified membranes can be used for loading of Ag nanoparticles, which endows the membranes with antibacterial activity. The facile approach towards multifunctional polyethersulfone membranes via in situ cross-linked copolymerization is shown in Scheme 1. Scheme 1 2. Experimental


2.1. Materials Polyethersulfone (PES, Ultrason E6020P) was purchased from BASF, Germany. N,N-dimethylacetamide (DMAc), was purchased from Chengdu Kelong Chemical Reagent Company, and distilled before used as a solvent. N-vinylpyrrolidone (VP) (99.0 %, Alfa Aesar) was distilled under reduced pressure to remove the inhibitor before use. Acrylic acid (AA) (C3H4O2, AR, CAS No. 79-10-7) was purchased from Aladdin Chemistry Co. Ltd. and purified through a neutral aluminum oxide column. Azo-bis-isobutryonitrile (AIBN) (C8H12N4, AR, CAS No. 78-67-1) was purchased from Chengdu Kelong Inc. (Chengdu, China), and used as initiator. N, N’-Methylenebisacryl amide (MBA) (C7H10N2O2, ≥99%, CAS No. 110-26-9) was purchased from Aladdin Chemistry Co. Ltd. and used as a cross-linking agent. Bovine serum albumin (BSA) and bovine serum brinogen (BFG) were obtained from Sigma Chemical Co. Micro BCATM Protein Assay Reagent kit was purchased from PIERCE Inc. Activated partial thromboplastin time (APTT) reagent, calcium chloride reagent for APTT test, and Owren’s Veronal Buffer were purchased from Siemens Co. Ltd. All the other chemicals (AR grade) were obtained from Aladdin reagent Co. Ltd. (China) and used without further purification before use. Deionized (DI) water was obtained 6

from a pure water production system and used throughout the experiments. 2.2. Preparation and characterization of modified PES membranes 2.2.1 Preparation PES casting solution was prepared via in situ cross-linked copolymerization of VP and AA in PES solutions. PES was dissolved in DMAc at 70 °C, then a mixture composed of VP, AA, MBA and AIBN was added into the PES solution (the feed


compositions are shown in Table 1). After bubbling nitrogen for 30 min, cross-linked copolymerization was carried out at 70 °C for 24 h under a nitrogen atmosphere with mechanical stirring (200 rpm); the solution was then exposed in air at room temperature to terminate the polymerization. Except for M-0 and M-4-0, other solutions were turbid. Moreover, with increasing the total contents, the solution became more turbid. PES membranes were prepared via spin coating and liquid–liquid phase inversion technique which was similar to our previous study , and the membrane thickness was controlled at about 70 μm. The names of each sample for different compositions are shown in Table 1. Table 1 2.2.2. Characterization The chemical compositions of the prepared membrane surfaces were characterized by ATR-FTIR (Nicolet 560) between 4000 cm-1 and 1000 cm-1 and XPS (XSAM800, Kratos Analytical, UK). The samples were dried in an oven at least 10 h before ATR-FTIR and XPS tests. The surfaces and cross-section structures of the modified membranes were observed 7

by a scanning electron microscopy (SEM) (JSM-7500F, JEOL, Japan). The membranes were quenched and fractured in liquid nitrogen, and then were attached to the sample supports and coated with gold layers before characterization. 2.3 Hydrophilic/hydrophobic property of modified membrane Water contact angle (WCA) can be used to evaluate the hydrophilicity/hydrophobicity of the membrane surfaces by using a contact angle


goniometer (OCA20, Dataphysics, Germany) equipped with a video capture. A piece of membrane (1 × 3 cm2, dried in a vacuum oven at 60 °C overnight) was attached on a glass slide and mounted on the goniometer. One drop of water (3 μL) was dropped on the airside surface of the membrane at room temperature, and the static WCA was measured after 30 s. Each sample was measured for five times to obtain a reliable value at least. 2.4. Blood compatibility 2.4.1. Plasma collection Healthy human fresh blood (man, 27 years old) was collected using vacuum tubes (5 mL, Jiangsu Kangjian Inc., China), containing sodium citrate as an anticoagulant (anticoagulant to blood ratio, 1:9 (v/v)) [27]. In order to avoid the differences caused by different experimental objectives between clinical test and material modification research, the plasma was obtained from one donor. The blood was centrifuged at 1000 rpm for 10 min to obtain platelet-rich plasma (PRP) or at 4000 rpm for 15 min to obtain platelet-poor plasma (PPP). To evaluate the blood compatibility of the modified membranes, protein adsorption, 8

platelet adhesion, plasma recalcification time and activated partial thromboplastin time experiments were carried out. 2.4.2. Protein adsorption Protein adsorption as the first factor should be considered when blood contact with biomaterial surfaces [28]. BSA or BFG solution (1 mg/mL in PBS, pH = 7.4) was used to investigated the protein adsorption behavior of PES membranes [29]. The detailed


operations were as follows: The membrane (with an area of 1 × 1 cm2) was first immersed in PBS for 24 h, and then incubated in BSA or BFG solution at 37 °C for 1 h. After protein adsorption, the membrane was slightly rinsed with PBS and deionized water for three times, followed by immersing in sodium dodecyl sulfate (SDS) aqueous solution (2 wt. %) with a shake speed of 200 rpm at 37 °C for 1 h. Nearly 90 % of adsorbed protein could be washed into SDS solution. The amount of the protein eluted in the SDS solution was determined by Micro BCATM protein assay reagent kits (PIERCE) and ELIASA (DNM-9602, Beijing Perlong new technology Inc., China) at 568 nm. The adsorbed amounts of BSA and BFG were then calculated. At least three measurements were averaged to reach a reliable value. 2.4.3. Platelet adhesion Platelet adhesion assay can eliminate the interference of other components in blood, such as erythrocyte and leukocyte [30], and the detailed operations are according to the method which has been reported previously [31]. The membranes (1 × 1 cm2 for each) were immersed in physiological saline using a 24-well culture plate and equilibrated at 37 °C for 1 h. Then, the physiological saline was removed and 300 μL fresh PRP was 9

introduced in each well and incubated at 37 °C for 2 h. Afterwards, the PRP was decanted off, and the membranes were slightly rinsed with physiological saline for three times. Finally, the membranes were treated with 2.5 wt. % glutaraldehyde in physiological saline at 4 °C for 24 h. The prepared samples were washed with physiological saline, and dehydrated by a series of graded alcohol/physiological saline solutions (30%, 50%, 70%, 80%, 90%, 95% and 100 wt. %, respectively; and 15 min


for each solution). The platelets adhered on the membrane surfaces were observed using SEM (JSM-7500F, JEOL, Japan). The number of the adherent platelets on the membrane surfaces was calculated from five SEM pictures at 1000× magnification from different places on the same sample. 2.4.4. Plasma recalcification time (PRT) Plasma recalcification time (PRT) is an indicator of intrinsic coagulation cascade activation [32]. Thus this test was used to evaluate the anticoagulant property of the membranes followed our previous report [33]. In brief, the membranes (1 × 1 cm2) were immersed in PBS solution (pH = 7.4) and equilibrated at 37 °C in a 24-well culture plate for 15 min. Then PBS was removed and 100 μL of fresh PPP was introduced and incubated statically at 37 °C. Subsequently, 100 μL of 25 mM CaCl2 aqueous solution was added to the PPP, and the plasma solution was monitored for clotting by manually dipping a stainless-steel hook coated with silicone into the solution to detect fibrin threads. Clotting time was recorded as the first fibrin strand formed on the hook. All the plasma recalcification tests were performed for three times. 2.4.5. Activated partial thromboplastin time (APTT) 10

The APTT test can evaluate the intrinsic pathway of coagulation cascade when biomaterials contact with blood [34, 35], thus the APTT tests were performed by a semi-automatic blood coagulation analyzer CA-50 (Sysmex Corporation, Kobe, Japan) in this study. The specific test operations are described as follows: First of all, membranes were cut into pieces of 0.5 × 0.5 cm2, and 4 pieces of the membranes were placed in a 96-well


plate. And the membranes were immersed in 200 μL of physiological saline at 4 °C for 24 h, and then kept at 37 °C for 1 h. After that, the physiological saline was removed followed by adding 100 μL of PPP. After incubating at 37 °C for 30 min, 50 μL of the incubated PPP was added into a test cup, followed by the addition of 50 μL of APTT agent (incubated 10 min before use). After incubating at 37 °C for 3 min, 50 μL of 0.025 M CaCl2 solution was added to measure APTT. At least three measurements were averaged independently to get a reliable value, and the results were analyzed by a statistical method. 2.5. Protein antifouling property It is indispensible to evaluate protein antifouling property of blood-compatible membranes when used in practice, such as hemodialysis [36]. Ultrafiltration of BSA solution through the membranes can be used to assess the protein antifouling property of blood-compatible membranes. The apparatus was described in our previous studies [37] and a dead-end ultrafiltration cell with an effective membrane area of 3.9 cm2 was used. The membrane was pre-compacted by PBS (pH = 7.4) under the pressure of 0.07 11

MPa for 20 min to get a steady flux. Afterwards, the pressure was reduced to 0.06 MPa, and the PBS flux was measured. The measurement lasted for 30 min, and the data recorded every 5 min was similar indicating that the PBS flux was steady. After 30 min of filtration, the feed solution was changed to 1.0 mg/mL BSA solution. For BSA solution ultrafiltration experiment, the same method was used, and the data were recorded after the flux was steady. Finally the membrane was washed with PBS for 1 h,


and the PBS flux was measured again. The fluxes of PBS and BSA solutions through the membrane were calculated by using the following equation: F(mL/(h×m2×mmHg))=

௏

(1)

ௌ௧௉

where V (mL) is the volume of the permeated solution; t (h) is the time of the permeated solution collecting; S (m2) is the effective membrane area; and P (mmHg) is the pressure applied to the membrane. Flux recovery ratio (FRR) was defined and calculated by using the following expression to assess the fouling-resistance ability of the membranes: FRR (%) = F 2 × 100 F1

(2)

where F1 and F2 (mL/(h×m2×mmHg)) are the PBS fluxes before and after the BSA solution ultrafiltration, respectively. 2.6. Adsorption experiments To investigate cationic dye adsorption to the modified membranes, methylene blue (MB) was used as a model. In order to discuss the effect of different compositions/amounts of additives in the membranes on the adsorption, 5 pieces (1 × 1 cm2, about 0.0089 g of dry weight) of the membranes were applied in 10 mL of 0.1 12

mmol/L MB solution with a stirring speed of 100 rpm at 30 ± 1 °C for 24 h, and the pH value of the solution was controlled at 7. The experiments were repeated for three times. The residual concentrations were determined with an UV-vis spectrophotometer 756PC at the wavelength of 631 nm. 2.7. Antibacterial activity tests According to our previous study [9], ionizable carboxyl acid groups on the


cross-linked chitosan nanogels could load Ag nanoparticles by in situ binding of Ag ions within the nanogel networks and reducing them with vitamin C (VC). Therefore, the modified membranes were used for loading Ag nanoparticles to gain antibacterial property. The membranes were immersed in 0.1 M NaOH solution for 2 h. Then the membranes were washed with RO water several times until the pH of washed water reached neutral. After immersing in 20 mL of 0.1 M AgNO3 solution with continuous oscillation at 25 °C in dark for 12 h, the membranes were took out and washed with DI water to remove the excess AgNO3. Subsequently, the membranes were immersed in 0.1 M NaBH4 solution with continuous oscillation at 25 °C in dark for another 12 h; after that, the membranes were stored in RO water for a day. Escherichia coli (E. coli, gram negative) and Staphylococcus aureus (S. aureus, gram positive) bacteria were to evaluate the antibacterial ability and bactericidal efficacy for the membranes. The detailed operations were the same as our previous study [9]. 2.8. Statistics for biological assays: All the values of biological assays in bar diagrams are presented as averages ± standard deviations (SD). The statistical significance was assessed by Student’s t-test, 13

with the level of significance set at P < 0.05. 3. Results and Discussions 3.1. Preparation and characterization of modified PES membrane. ATR-FTIR and XPS were firstly used to characterize the membrane surfaces; and the results are shown in Figure 1 and table 3. As for the ATR-FTIR, the peak at 1650 cm-1 was attributed to the C=O stretching vibrations of VP, and the peak at 1720 cm-1 was


attributed to the C=O stretching vibrations of AA. As shown in Figure 1 (a), the vibrations of these peaks became stronger with the increase of total monomer amounts. The content of the copolymer on the membrane surface increased except for M-4-4 membrane. Due to the high viscosity of M-4-4 solution (the viscosity of all the membranes was shown in Figure S1), its phase separation was taken much longer than other solutions. We can observe a big leap between the viscosities of M-3-3 and M-4-4. Thus, the hydrophilic polymer of P(VP-AA) in the solution may be more difficult to be immigrated from the inner to the surface of the membrane during the membrane formation for M-4-4. Thus the content of the additive of the M-4-4 membrane was less on the membrane surface than that for the M-3-3 membrane. As shown in Figure 1 (b), as the content of VP decreased and AA increased, the intensity of the peak at 1650 cm-1 became weaker and that at 1720 cm-1 became stronger except for the M-0-4 membranes. This may be due to the better compatibility of VP than AA with PES matrix. The copolymerization might provide the M-1-3 membranes with more AA than the M-0-4 membranes. Figure 1 In order to make the contents of additives on the membrane surfaces more clear, we 14

have calculated the intensity ratio of the C=O peak to that of the PES sulfone peak 1500 cm-1. The results are shown in Table 2. Table 2 The A1604-1766 represents the area of the C=O peak, and the A1454-1517 represents the area of the PES sulfone peak at 1500 cm-1. The R represents the ratio of A1604-1766 to A1454-1517. We can observe that with the increase of the total monomer amounts, the R


increased obviously except for M-4-4, which indicated more P(VP-AA) on the membrane surfaces. Among the M-4-0, M-3-1, M-2-2, M-1-3 and M-0-4 membranes, the M-2-2 membrane owned the largest R about 0.9226 due to the good miscibility of PVP and PES, and M-0-4 owned the least R about 0.3304 due to the poor miscibility of PAA and PES. As for the XPS test, the element contents (atom%) of membranes were calculated out as shown in Table 3, and the XPS spectra are shown in Figure S2. As shown in Figure S2, for the M-0 and M-0-4 membranes, the XPS C 1s core-level spectrum was curve-fitted into three peaks with the binding energies (BEs) at 284.1, 284.9 and 286.0 eV, which were attributed to the C-C/C-H, C-S and C-O, respectively. For the M-2-2, M-4-4 and M-4-0 membranes, the XPS C 1s core-level spectrum was curve-fitted into four peaks with the binding energies (BEs) at 284.1, 284.9, 286.0 and 287.0 eV. The new peak at 287.0 eV was attributed to the C-N. We can clearly observe that with the increase of the total monomer amounts, the C-O and C-N peaks became more intensive as for the M-0, M-2-2 and M-4-4 membranes, which indicated there were more P(VP-AA) contents on the membrane surfaces. Compared to M-0 membrane, a new 15

peak of C-N was observed for the M-4-0 membrane; while the C-O peak became more intensive. All the results indicated the successful modification of the pristine PES membrane. Table 3 Figure 2 shows the SEM images of the cross-section morphologies for M-0, M-1-1, M-2-2, M-3-3 and M-4-4 membranes. The cross-section views for the membranes


changed with increasing the amounts of P(VP-AA). For the M-1-1 membrane, the finger-like holes were on the top of the membrane, followed by an orderly porous structure. The structures became more and more irregular with the increase of the P(VP-AA) amounts. For the M-4-4, no finger-like structure was observed but a porous structure. Due to the hydrophilicity of P(VP-AA), the phase separation rate became lower and lower with the increase of the P(VP-AA) amounts, and the viscosity of the solutions might also affect the structure of the membranes. The SEM images of the cross-section views for M-0-4, M-1-3, M-2-2, M-3-1 and M-4-0 membranes are shown in Figure S3. Figure 2 In general, the hydrophilic/hydrophobic of the membrane surface could be directly assessed by water contact angle . As shown in Figure 3 (a), WCA decreased with the increase of the P(VP-AA) contents in the membranes according to the results shown in Figure 1. However, the WCAs of the modified membranes were not obviously decreased compared to the pristine PES membrane as shown in Figure 3 (b). Other factors might also affect WCA like the surface morphologies of the composite membranes [38]. As the M-1-1 and M-2-2 membranes showed, the hydrophilicity of 16

the membrane surface surely can affect the WCA, thus the WCA of M-2-2 should be lower than M-1-1. However, the membrane surface of M-2-2 was rougher than M-1-1, and the results of WCA should be considered by the integrated factors. The surface morphologies of all the membranes are shown in Figure S4. Figure 3 3.2. Blood-compatibility


3.2.1. Protein adsorption The adsorption of contact-phase proteins may cause the activation of the intrinsic cascade, and then promote thrombus formation [39]. Thus protein adsorption is indispensible to evaluate blood compatibility of the membrane, and the results are shown in Figure 4. As shown in Figure 4 (a), the amount of the binding proteins decreased with increasing the amount of the P(VP-AA). The more P(VP-AA) added to the membrane, the more hydrophilic groups were immigrated to the membrane surface. Therefore, the increase of the hydrophilicity resulted in the decrease of protein adsorption. Other factors like the porosity and the surface area of the pores may have great effect on the adsorbed amounts of proteins. According to the results of BSA and BFG adsorption, the binding amount for the M-4-4 was larger than that for the M-3-3, since the M-4-4 was more porous than M-3-3 (according to Figure S4), which may cause the larger binding amount of BSA and BFG. As shown in Figure 4 (b), the binding amounts of proteins for M-4-0 and M-0-4 membranes were more than those of M-3-1, M-2-2 and M-1-3. It indicated that AA and VP might have a synergistic effect on the protein adsorption. The good compatibility of 17

VP with PES made the membranes contain more additives. Thus, the membrane may contain more AA according to Table 2. AA was negatively charged, and repulsed the negatively charged proteins. Moreover, VP can also repulse proteins by the formation of hydrogen bond. Thus, the synergistic effect of AA and VP can decrease the binding amounts of proteins. The effect of hydrophilicity on the protein adsorption may be different for BSA and BFG. As for BFG, the maximum change of the binding amounts


between M-0 and M-3-3 was about 7 µg/cm2, less than that of BSA. Thus, in the case of the BFG binding, the change of the amounts for M-1-1, M-2-2, and M-4-4 (or for M-3-1, M-2-2, M-1-3 and M-0-4) is not significant. It was believed that the more hydrophilic the membrane was, the less protein it absorbed [40-42]. Figure 4 3.2.2. Platelet adhesion Platelet adhesion is activated by the plasma protein layer adsorbed on the material surface or stimulation induced by material surface when the blood contacts with biomaterials. Then the activated platelets can activate many kinds of coagulation factors, resulting in thrombus and coagulation on the material surface [43, 44]. In vitro platelet adhesion test can be carried out to evaluate the amount and morphology of platelets adhering on material surfaces. Figure 5 shows the micrographs and numbers of the platelets adhering onto the surfaces of the M-0, M-1-1, M-2-2, M-3-3 and M-4-4 membranes. Many pseudopodia were observed on the surface of M-0, and the pseudopodia became fewer with increasing the total amount of P(VP-AA). There was almost no pseudopodium for M-3-3 and M-4-4 membranes. Moreover, the platelets adhering on the modified 18

membranes were round, and the morphologies of platelets were became spherical with increasing the total amounts of P(VP-AA). It is well known that the surface absorbing fibrinogen activate platelets, thus the binding amounts of BFG may be relative to the Platelet adhesion. The results of BFG adsorption were consistent with platelet adhesion except for M-4-4. According to the results of BFG adsorption, the binding amount for the M-4-4 was more than that of M-3-3. The porosity of the membranes may also have


an effect on the protein binding amounts [45]. The M-4-4 was more porous than M-3-3 (according to Figure S4), which may cause the larger BFG binding amount. Since platelet adhesion occurred on the membrane surfaces, the porosity of the membranes was less important to platelet adhesion. The pore sizes of the membrane were in micron-scale according to Figure S4, and the diameters of the platelet were also in micron-scale. Thus, it may be more difficult to adsorb platelets into the pores than BFG (in nano-scale). The surface hydrophilicities of M-3-3 and M-4-4 (according to Table 2) were almost in the same level. Thus the results of platelet adhesion were consistent with the surface hydrophilicity for M-3-3 and M-4-4. Pseudopodia and platelet aggregation are two important parameters to evaluate the activation of platelet. As shown in the figure, the pseudopodia are more obvious for the M-0 membrane compared to the M-2-2 and M-1-1 membranes. For the M-1-1 and M-2-2 membranes, we only showed the platelet aggregation zone. The distribution of platelets for M-0 was homogenous on the membrane surface, while those for the M-1-1 and M-2-2 membranes were discrete. Actually, the number of the adsorbed platelets on M-0 is larger than that on the M-2-2. It inferred that the modified membranes could prevent platelet adhesion when 19

contacted with blood compared to pristine PES membrane. Figure 5 3.2.3. Plasma recalcification time (PRT) Plasma recalcification time (PRT) is to evaluate the anticoagulant property of the membranes, indicating intrinsic coagulation cascade activation [32]. Figure 6 shows the PRTs for M-0, M-1-1, M-2-2, M-3-3 and M-4-4 membranes, and the control sample is


plasma. The PRT was prolonged with increasing the total monomer ratio. The results for M-4-0, M-3-1, M-1-3 and M-0-4 membranes are shown in Figure S5, and it indicated that the anticoagulant property was improved after the modification. Figure 6 3.2.4. Activated partial thromboplastin time (APTT) APTT test is widely used in evaluating coagulation abnormalities in the intrinsic pathway, which can detect functional deficiencies in factor II, V, X or fibrinogen [46, 47]. APTT assay is thus performed in order to determine whether the blood factors would be activated or not when blood contacts with materials. As shown in Figure 7(a), the APTT for the M-0 membrane was about 46 s. The APTT was prolonged with the increase of P(VP -AA) ratios, and reached a maximum value for the M-4-4 membrane. The APTTs of various VP/AA ratios are shown in Figure 7(b). Longer APTT was observed with increasing the content of AA. It demonstrated that AA was more effective in APTT prolongation than VP. The reason might be that -COOH could chelate Ca2+ [27], resulting in the increased APTT. Figure 7 The blood coagulation can be initiated by intrinsic and extrinsic pathways. The 20

formation of blood clots after the blood contacting with biomaterials belongs to intrinsic pathway. For the parameters to evaluate anticoagulant properties, APTT test is widely used in evaluating coagulation abnormalities in the intrinsic pathway. Only the results of APTT cannot reflect surface induced blood coagulation. Therefore, we also use protein adsorption, and platelet adhesion to evaluate the anticoagulant properties of the membranes. Protein adsorption is to evaluate the adsorption of contact-phase


proteins, which may cause the activation of the intrinsic cascade, and then promote thrombus formation; platelet adhesion is to evaluate the activated platelets when biomaterials contact with platelets, which can activate many kinds of coagulation factors, resulting in thrombus and coagulation on the material surface. In conclusion, it is an effective system to evaluate the blood compatibility of the membranes by combination of APTT, protein adsorption and platelet adhesion. Other methods such as platelet activation, and thrombin–antithrombin III (TAT) generation could also be employed to characterize the blood compatibility of polymeric materials [48]. 3.3. Protein antifouling property The antifouling property of the membranes was improved with increasing the hydrophilicity of membranes. In the present study, hydrophilic P(VP-AA) was used to modify PES membrane, and ultrafiltration experiments were carried out to study the antifouling property. The fluxes of PBS solution or BSA solution for the modified membranes are shown in Figure S6. The fluxes of all the membranes could be influenced by many factors, such as pore sizes and their distribution [49, 50]. 21

The recovery ratios were calculated using formula (2), and the recovery ratios of all the membranes are shown in Figure 8. When applying BSA solution through the membranes, the protein molecules would deposit/absorb onto the membrane surfaces and/or in the membrane pore surfaces. The flux thus became lower when the flux of PBS was tested again. The more the antifouling property the membrane had, the higher recovery ratio reached [31]. As is known, PVP has been widely used as an additive


during membrane preparation via phase separation method to improve the antifouling property of PES ultrafiltration membranes [51], and AA can hardly influence on protein antifouling property [31]. Thus, the protein antifouling property mainly was depended on the contents of PVP in the membrane. The results of the recovery ratios were consistent with the contents of PVP in the membrane according to the former characterizations. As shown in Figure 8 (a), the PBS recovery ratios became higher with the increase of the P(VP-AA) amounts. The recovery ratio decreased with decreasing the VP contents as shown in Figure (b). The recovery ratio of M-4-0 was the highest, indicating the best antifouling property among all the modified membranes. Figure 8 3.4. Adsorption experiments The MB adsorbed amounts per unit mass of the membranes are shown in Figure 9. The adsorbed amounts increased with the increase of the total mass of the polymer in the membranes. In addition, pure PVP adsorbed MB by forming association complexes, and the adsorption was weaker compared to the charge attraction[52]. The adsorption of MB by PAA relies on its strong charge attraction, but the nature of poor misibility with PES led to a low content. Fortunately, the synergistic effects of VP and AA [53] 22

made the adsorbed amounts improved obviously. The introduction of VP could stabilize PAA in PES membranes via in situ cross-linked copolymerization. Therefore the adsorption amount of M-4-4 was as high as 34.9 mg/g; while the adsorption amounts of M-0, M-2-2, M-4-0 and M-0-4 were 12.7, 17.3, 18.0 and 13.1 mg/g, respectively. Figure 9


3.5. Antibacterial activity tests In order to confirm the antibacterial activity of the modified membranes in aqueous solution, the optical degree of the bacterial-membranes co-cultured solution was detected. a As shown in Figure 10 (a) and (b), the bacteria grown obviously on the control sample and the pristine PES membrane after 24 h. Meanwhile, the optical degree for the loading-Ag nanoparticles membranes, especially for M-4-4, showed considerable reduction for both S. aureus and E. coli. The results indicated that the loading of Ag nanoparticles onto the modified membranes conferred the composite membrane with antibacterial property, especially for S. aureus. E.coli and S.aureus. are gram negative and gram positive bacterial, respectively. The difference between the results of E.coli and S.aureus. may be caused by the intrinsic activity of E.coli and S.aureus. The high activity observed in M-4-4 may be due to the large loaded amount of Ag nanoparticles. Thus, we added an EDS test of the membrane surfaces to ensure the loading Ag nanoparticles. After loading Ag nanoparticles, the membranes were dried in an oven at least 10 h before EDS test (JSM-7500F, JEOL, Japan). The results have been shown in Figure S7. The M-4-4 has the largest Ag content of about 3.89 wt. % of the membrane, while the M-2-2 and M-0-4 have only about 0.69 wt. % and 0.59 wt. % 23

respectively. Thus, the antibacterial property of M-4-4 was the most obvious. The results indicated that the more Ag nanoparticles the membrane surface loaded, the more the membrane antibacterial was. Figure 10 4. Conclusions P(VP-AA) was successfully synthesized with various VP/AA ratios in PES solution


via in situ cross-linked copolymerization in this study. The modified PES membranes exhibited more hydrophilic, enhanced anticoagulant and improved antifouling properties than pristine PES membrane. With the increase of the P(VP-AA) content, the blood compatibility of modified PES was improved. In addition, the film-forming ability of the M-4-4 membrane was the lowest in all of modified membranes, and the surface content of P(VP-AA) for M-4-4 was lower than that for M-3-3. Thus P(VP-AA) content for M-4-4 may be the maximum in the in situ cross-linked copolymerization. The results of blood compatibility tests with various VP/AA ratios suggested that AA played a more important role in the improvement of blood compatibility than VP. The ultrafiltration experiment indicated the antifouling property of the modified membranes was improved, and the recovery ratio of M-4-0 was the highest. The results of MB adsorptions of the modified membranes indicated that the modified membranes, especially for the M-4-4 membrane, could be used to adsorb cationic dyes because of the highest adsorbed amounts. After loading of Ag nanoparticles for the modified membranes, they were conferred with antibacterial activity according to the results of optical degree of the bacterial-membranes co-cultured solution. In a word, the modified PES membranes showed multifunction in the present study. Combing with the facile in 24

situ cross-linked copolymerization, the modified membranes have great potential in many application fields. 5. Acknowledgements This work was financially sponsored by the National Natural Science Foundation of China (No. 51225303 and 51433007), and the Sichuan Province Youngth 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.

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31

Appendices (Nomenclature) F

water flux of membranes (mL/(h×m2×mmHg))

V

volume of the permeated solution (mL)

T

time (h)

S

effective membrane area (m2)

P

pressure applied to the membrane (mmHg)


FRR

flux recovery ratio (%)

F1

PBS flux before BSA solution ultrafiltration (mL/(h×m2×mmHg))

F2

PBS flux after BSA solution ultrafiltration (mL/(h×m2×mmHg))

32

Caption of table Table 1 Feed compositions for in situ cross-linked copolymerization. Table 2 The intensity ratio of the C=O peak and PES sulfone peak from ATR-FTIR spectrum


Table 3 Elemental surface compositions of the membranes determined from XPS

33

Captions of figures Scheme 1 Facile approach towards multifunctional polyethersulfone membranes via in situ cross-linked copolymerization Figure 1 (a) ATR-FTIR spectra of M-0, M-1-1, M-2-2, M-3-3 and M-4-4 membranes; (b) ATR-FTIR spectraof M-4-0, M-3-1, M-2-2, M-1-3 and M-0-4 membranes. Figure 2 SEM images of the cross-section morphologies for the membranes.


Magnification: 500 × (a) and 3000 × (b). Figure 3 Static water contact angles for the membranes. (Values are expressed as mean ± SD, n=5) Figure 4 BSA and BFG adsorption for the membranes. Values are expressed as mean ± SD, n=3, *P, #P < 0.05 compared to the value of M-0. Figure 5 The scanning electron micrographs of the adherent platelets on the membrane surfaces with the magnification of 5000 ×, and the number of the adhering platelets onto the membranes from platelet-rich plasma estimated by SEM images. Figure 6 Plasma recalcification times (PRT) for the M-0, M-1-1, M-2-2, M-3-3, M-4-4 membranes and control sample. Data are presented as mean ± SD, n=3, *P < 0.05 compared to the value of the control sample. Figure 7 APTTs for the membranes. Values are expressed as mean ± SD, n=3, *P < 0.05 compared to the value of M-0. Figure 8 PBS flux recovery ratios for the membranes through the ultrafiltration experiments. (n=3) Figure 9 The adsorbed amounts per unit mass for M-0, M-2-2, M-4-4, M-4-0 and 34

M-0-4 membranes after applying in 10 mL of 0.1 mmol/L MB solution. The adsorption experiment were carried out at 30 ± 1°C with the stirring speed of 100 rpm for 24 h, pH=7. Figure 10 The optical degree for E. coli (gram negative) and S. aureus (gram positive), the absorbance represented the bacterial amount after exposure to functionalized


membranes for 24 h

35

Table 1 Feed compositions for in situ cross-linked copolymerization.


Membranes PES

VP

AA

AIBN

MBA

DMAc

(wt. %)

(wt. %)

(wt. %)

(wt. %)a

(mol. %)b

(wt. %)

M-0

16.0

-

-

-

-

84.0

M-1-1

16.0

1.0

1.0

1.0

2.0

82.0

M-2-2

16.0

2.0

2.0

1.0

2.0

80.0

M-3-3

16.0

3.0

3.0

1.0

2.0

78.0

M-4-4

16.0

4.0

4.0

1.0

2.0

76.0

M-4-0

16.0

4.0

-

1.0

2.0

80.0

M-3-1

16.0

3.0

1.0

1.0

2.0

80.0

M-1-3

16.0

1.0

3.0

1.0

2.0

80.0

M-0-4

16.0

-

4.0

1.0

2.0

80.0

a

AIBN is 1.0 wt. % of the total monomers. b MBA is 2.0 mol. % of the total monomers.

36

Table 2 The intensity ratio of the C=O peak and PES sulfone peak from ATR-FTIR


spectrum Membranes

A1604-1766

A1454-1517

R

M-0

0

219.854

0

M-1-1

153.952

215.200

0.7154

M-2-2

184.355

199.830

0.9226

M-3-3

248.615

153.060

1.6243

M-4-4

218.500

152.404

1.4337

M-4-0

182.023

207.396

0.8777

M-3-1

211.408

235.289

0.8985

M-1-3

122.278

142.390

0.8588

M-0-4

53.394

161.588

0.3304

37


Table 3 Elemental surface compositions of the membranes determined from XPS. Element (atom%) Membranes O N C S M-0 2.428 73.64 1.763 22.17 M-2-2 2.38 74.98 2.272 20.37 M-4-4 1.579 69.43 3.071 25.92 M-4-0 2.425 72.77 2.358 22.44 M-0-4 2.351 73.95 2.058 21.64

38

39


40


41


42


43


44


45


46


47


48


49


In situ re-endothelialization via multifunctional nanoscaffolds.

Thermosensitive ionic microgels via surfactant-free emulsion copolymerization and in situ quaternization cross-linking.

A facile access to substituted benzo[a]fluorenes from o-alkynylbenzaldehydes via in situ formed acetals.

Versatile antifouling polyethersulfone filtration membranes modified via surface grafting of zwitterionic polymers from a reactive amphiphilic copolymer additive.

Electrocyclization.

A facile approach toward protein-resistant biointerfaces based on photodefinable poly-p-xylylene coating.

Toward multifunctional "clickable" diamond nanoparticles.

An in situ approach for facile fabrication of robust and scalable SERS substrates.

Copolymerization of carbon dioxide and butadiene via a lactone intermediate.

Fabrication and cytocompatibility of in situ crosslinked carbon nanomaterial films.

Preparation and characterization of in-situ crosslinked pectin-gelatin hydrogels.

Facile synthesis of 1H-imidazo[1,2-b]pyrazoles via a sequential one-pot synthetic approach.

Polyethersulfone Hybrid Hollow Fiber Membranes with Enhanced Mechanical and Anti-Fouling Properties.

Preparation and characterizations of EGDE crosslinked chitosan electrospun membranes.

Facile and highly efficient approach for the fabrication of multifunctional silk nanofibers containing hydroxyapatite and silver nanoparticles.

Polymeric molecular sieve membranes via in situ cross-linking of non-porous polymer membrane templates.

A Novel Fabrication Approach for Multifunctional Graphene-based Thin Film Nano-composite Membranes with Enhanced Desalination and Antibacterial Characteristics.

Adsorption of silver ions on polypyrrole embedded electrospun nanofibrous polyethersulfone membranes.

Ultrasound irradiation combined with hydraulic cleaning on fouled polyethersulfone and polyvinylidene fluoride membranes.

Dynamical structure of the short multifunctional peptide BP100 in membranes.

Toward a Personalized Approach in Prebiotics Research.

nanocrystal reinforced capsules: a fast and facile approach toward assembly of liquid-core capsules with high mechanical stability.

From metal-organic framework to carbon: toward controlled hierarchical pore structures via a double-template approach.

Facile production of stable silicon nanoparticles: laser chemistry coupled to in situ stabilization via room temperature hydrosilylation.