Design of Antibacterial Poly(ether sulfone) Membranes via Covalently

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Design of Antibacterial Poly(ether sulfone) Membranes via Covalently Attaching Hydrogel Thin Layers Loaded with Ag Nanoparticles Min He, Qian Wang, Rui Wang, Yi Xie, Weifeng Zhao,* and Changsheng Zhao* College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu 610065, P. R. China S Supporting Information *

ABSTRACT: To inhibit bacteria attachment and the subsequent formation of biofilms on poly(ether sulfone) (PES) membranes, poly(sulfobetaine methacrylate)/poly(sodium acrylate) antibacterial hydrogel thin layers were covalently attached onto the membranes, followed by loading with Ag nanoparticles. In our strategy, double bonds were firstly introduced onto the PES membrane surfaces to provide anchoring sites, and then the hydrogel layers were synthesized on the membrane surfaces via UV light-initiated crosslinking copolymerization. Then, Ag ions were adsorbed into the hydrogel layers and reduced to Ag nanoparticles by sodium borohydride. The amounts of the adsorbed Ag ions were controlled by the mole ratios of carboxylate groups in the hydrogel layers. After attaching the hydrogel layers, a typical 3D porous structure was observed by scanning electron microscopy, and the surface chemical composition variations were characterized by attenuated total reflection-Fourier transform infrared spectroscopy. The live/dead staining, inhibition zone, and the optical degree of co-culture solution demonstrated that the designed surfaces could not only effectively resist bacteria attachment but also kill the surrounding bacteria Escherichia coli and Staphylococcus aureus. It was noteworthy that the strong antibacterial ability could be maintained for more than 5 weeks. Additionally, the excellent hemocompatibility of the modified membranes was confirmed by undetectable plasma protein adsorption, suppressed platelet adhesion, prolonged clotting time, low hemolysis ratio, and suppressed blood-related complement activation. Cell culture tests indicated that the membranes showed no cytotoxicity, but strong anti-cell adhesion properties. The proposed method to fabricate antibacterial hydrogel thin layers has great potential to be widely used to inhibit the formation of biofilms on various biomedical devices. KEYWORDS: hydrogel thin layer, Ag nanoparticles, antifouling, poly(ether sulfone), antibacteria preventing the initial bacteria attachment;7 and (iii) bacteriadetaching surfaces for detaching the attached bacteria by an external force.8 Each surface has its inherent advantages and disadvantages, which limit the actual applications. For example, bactericidal surfaces will be contaminated by the remaining dead bacteria. Bacteria-resistant surfaces cannot reach complete prevention of bacteria attachment;9,10 then, the residual bacteria would inevitably attach onto the surfaces and colonize, finally leading to the formation of biofilms. Therefore, these kinds of antibacterial surfaces with only one antibacterial mechanism cannot effectively inhibit the formation of biofilms. An ideal antibacterial surface requires the following functions: firstly, preventing initial bacteria attachment; subsequently, killing the attached bacteria; finally, detaching the dead bacteria. To achieve this goal, several groups have tried to prepare

1. INTRODUCTION Poly(ether sulfone) (PES) membranes have been widely used in water treatment and blood purification because of their outstanding oxidative, thermal, and hydrolytic stability, as well as their good mechanical and membrane-forming properties.1,2 However, surface bacterial infection is one of the major drawbacks of the membrane, and may result in unfavorable bioresponses at the membrane interface, thereby reducing the ultrafiltration performance.3 Therefore, much effort has been devoted to PES membrane modification to confer it with the desired long-term antibacterial and antifouling properties. Zhang et al. blended Ag nanoparticle-loaded halloysite with PES to prepare an antibacterial PES membrane.4 Zhao et al. incorporated a Ag nanoparticles/halloysite nanotubes−reduced graphene oxide nanocomposite into PES to prepare a longlasting antibacterial PES membrane.5 Antibacterial surfaces can be classified into three categories based on their operating mechanisms: (i) bactericidal surfaces for killing attached bacteria;6 (ii) bacteria-resistant surfaces for © 2017 American Chemical Society

Received: March 5, 2017 Accepted: April 25, 2017 Published: April 25, 2017 15962

DOI: 10.1021/acsami.7b03176 ACS Appl. Mater. Interfaces 2017, 9, 15962−15974

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ACS Applied Materials & Interfaces

surfaces via in situ crosslinking polymerization of 2-hydroxyethyl methacrylate (HEMA), and then double bonds were introduced via the reaction between the hydroxyl groups and acryloyl chloride to provide anchoring sites for the hydrogel layers. The hydrogel thin layers were formed and covalently attached onto the PES membranes by a UV light-initiated copolymerization. Ag ions were adsorbed into the hydrogel layers through the carboxylate groups, and were reduced to Ag nanoparticles by sodium borohydride. The loading amounts of Ag nanoparticles were controlled by the mole ratios of AANa in the hydrogel layers. The surface morphology, chemical compositions, thickness control, and stability of the hydrogel layer were investigated. Studies on the live/dead staining, inhibition zone, and optical degree of co-culture solution were conducted to evaluate the antibacterial property. Then, the blood compatibility of the modified PES membranes was also evaluated by investigating the protein adsorption behavior, platelet adhesion, clotting times, hemolysis ratios, and bloodrelated complement activation. Finally, the cell compatibility was also investigated.

antibacterial surfaces by combining two or three of the abovementioned antibacterial mechanisms into one system.11,12 There are three types of antibacterial surfaces based on the combination of two antibacterial mechanisms: resisting−killing, resisting−detaching, and killing−detaching.13 For biomedical applications, the resisting−killing type is more effective to inhibit biofilm formation,14 and is usually designed by introducing nonfouling materials onto substrates, and then incorporating biocide agents in three models: (i) tethered onto nonfouling hydrophilic polymers,15,16 (ii) alternately deposited with antiadhesive layers,17 and (iii) stored in nonfouling matrixes.18 However, for models (i) and (ii), insufficient biocide agent loading and instability of the functional layer are the main drawbacks. For model (iii), the key points are how to immobilize the biocide loaded nonfouling matrix onto substrates and how to control the thickness of the nonfouling matrix. Covalently attaching a nonfouling hydrogel thin layer onto a substrate, followed by loading a biocide agent is a promising method, and it presents several advantages: the robust bonds formed between the substrate surfaces and the hydrogel layers guarantee the long-term stability of the hydrogel layers, and the hydrogel thickness can be effectively controlled;19 the hydration capacity of the hydrogel layer is robust to resist bacteria adhesion; the storage function of the hydrogel layer can facilitate loading of sufficient biocide agent. Many researchers have attached a Ag nanoparticle-loaded hydrogel layer onto substrates to prepare antibacterial surfaces. For instance, Yang et al. immobilized a Ag nanoparticle-loaded PNIPAAm functional hydrogel thin layer onto glass slides to achieve nonfouling, antibacterial surfaces.20 Marion Fischer et al. attached Ag nanoparticle-loaded multilayer hydrogel coatings onto a polyurethane catheter to improve its hemocompatibility and antimicrobial activity.21 However, no report about applying a Ag nanoparticle-loaded antifouling hydrogel layer to modify a PES hemodialysis membrane has been reported. Recently, many techniques have been used to attach functional hydrogel layers onto substrates.22,23 Zhu et al. attached a zwitterionic hydrogel nonfouling thin film onto a PES membrane surface via five step chemical reactions.24 Chollet et al. applied ene-functionalized polymers to form hydrogel thin films and simultaneously covalently attached them onto thiol modified Si wafers via ene−thiol click chemistry after four step reactions.25,26 In our previous studies, a robust and straightforward method was developed to covalently attach functional hydrogel layers onto substrates by three steps:27 introducing hydroxyl groups on the substrate surface, grafting double bonds as anchoring points for the hydrogel layer, and forming and simultaneously attaching a hydrogel thin layer by surface crosslinking polymerization. This technique is also versatile, and can be applied to modify various materials, such as polymer materials, inorganic materials, and metal materials, and confer them with multifunctions. For instance, Beinn V. O. Muir et al. treated a titanium surface with 3-(trimethoxysilyl)propyl methacrylate to graft double bonds as anchoring points for a hydrogel layer, and then the hydrogel layer was formed and attached via surface crosslinking radical polymerization.28 With this method, super-anticoagulant PES membranes were prepared.29 In this study, sulfobetaine methacrylate (SBMA) (for its excellent antifouling property) and sodium acrylate (AANa) (to adsorb Ag ions) were selected as the functional monomers to form hydrogel layers onto PES membrane surfaces. In our strategy, hydroxyl groups were introduced onto the membrane

2. MATERIALS AND EXPERIMENTS 2.1. Materials. Commercial PES (Ultrason E6020P) was purchased from BASF. AANa (98%, CAS no. 7446-81-3), HEMA (98%, CAS no. 868-77-9), 2-ketoglutaric acid (99%, CAS no. 328-507), and N,N′-methylenebisacrylamide (MBA) (99%, CAS no. 110-269) were purchased from Aladdin Chemistry Co. Ltd. HEMA was purified through an aluminum oxide column to remove the inhibitors prior to use. SBMA was synthesized according to our previous reported procedures.30 N-methyl-2-pyrrolidinone (NMP) (AR, CAS no. 872-50-4), silver nitrate (CAS no. 7761-88-8), sodium borohydride (CAS no. 16940-66-2), and azo-bis-isobutryonitrile (AIBN) (AR, CAS no. 78-67-1) were purchased from Chengdu Kelong Inc. (Chengdu, China), and used as received. Thiethylamine (98%, CAS no. 121-44-8) and acryloyl chloride (99%, CAS no. 814-68-6) were purchased from Best-reagent Ins. Bovine serum albumin (BSA) and bovine serum fibrinogen (BFG) were obtained from Sigma Chemical Co. The Micro BCA Protein Assay Reagent kit was purchased from PIERCE Inc. Activated partial thromboplastin time (APTT) reagent, thrombin time (TT) reagent, Owren’s Veronal Buffer, and Factor XII-deficient plasma were purchased from Siemens Co. Ltd. All other chemical reagents were obtained from Chengdu Kelong Inc. (Chengdu, China), and were used without further purification. Dulbecco’s modified Eagle medium, fetal bovine serum, 0.05% Trypsin EDTA, CCK-8 agent, MTT agent, and phosphate-buffered saline (PBS, pH 7.4) were purchased from Gibco. Deionized (DI) water was used throughout the experiments. 2.2. Membrane Preparation. The treatment procedures of the membrane were taken from our previous works with a minor modification.27,29 Firstly, hydroxyl groups were introduced onto the PES membrane surface via in situ crosslinking polymerization of HEMA in PES solution, followed by spin-coating coupled with a phase inversion technique. The in situ crosslinking polymerization technique has been widely used to introduce functional groups onto PES membranes, and the mechanism forms microgels in situ to avoid eluting of hydrophilic polymers.31−33 A typical procedure was as follows: 9.6 g of PES (16 wt % of the total solution) was dissolved in 40 g of NMP to reach a homogeneous solution, and then a mixture of HEMA (1.8 g, 3 wt % of the total solution), AIBN (1 mol % with respect to HEMA), MBA (1.5 mol % with respect to HEMA), and NMP was added into the PES solution. A crosslinking polymerization was carried out at 70 °C for 5 h under a nitrogen atmosphere and mechanical stirring at 300 rpm. The solution was then exposed to air at room temperature to terminate the polymerization. After degassing the air, the solution was spin-coated on a glass surface, then immersed into DI water. The prepared membrane was washed with DI water several 15963


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ACS Applied Materials & Interfaces times and stored in DI water for 1 week before use, and called PESOH. To obtain the double bond enriched PES membrane, the dried PESOH membrane with an area of 1 × 1 cm2 was immersed into 50 mL of diethyl ether solution with 4 mL of triethylamine, and 6 mL of acryloyl chloride was then added dropwise. The reaction was carried out at 0 °C for 1 h, and then the membrane was moved to a 25 °C water bath for another 23 h, and called PES-DB. To determine the density of the double bonds on the membrane surface, 10 pieces of PES-DB membrane were immersed into 2 mL of 0.01 mol/L NaOH solutions for 48 h, and then the amount of AANa hydrolyzed from the membrane surface was detected by high-performance liquid chromatography (HPLC). Then, a mixture of functional monomers, crosslinking agent, and initiator was coated onto the PES-DB membrane surface, and covered by a quartz glass sheet. The surface crosslinking copolymerization was carried out under 365 nm UV light between two glass sheets. The compositions of the hydrogel layers are listed in Table 1. To load the Ag nanoparticles into the hydrogel layers, the prepared

also investigated. The detailed procedures are shown in the Supporting Information. 2.4. Total Ag Content and Release Tests. To calculate the total Ag loading amounts, a 1.0 × 1.0 cm2 sample was digested with HNO3 (80%, 5.0 mL) for 1 h, and then the digestate was diluted to a final volume of 50 mL. Determination of Ag was carried out by inductively coupled plasma-mass spectrometry (ICP-MS). Three replicates of each sample were analyzed. To test the Ag release, a 1.0 × 1.0 cm2 sample was immersed into 2.0 mL DI water. The solution was then taken out and diluted to 20 mL with 5% HNO3 at given intervals. The solution was analyzed by ICP-MS to determine the Ag content. 2.5. Antibacterial Activity Tests. Escherichia coli (Gram negative) and Staphylococcus aureus (Gram positive) bacteria were used as the model bacteria to evaluate the antibacterial properties of the Ag nanoparticle-loaded hydrogel thin layers by the live/dead twocolor fluorescence, inhibition zone, and bactericidal efficiency methods. The detailed procedures are presented in the Supporting Information. 2.6. Blood Compatibility. The blood compatibility of the modified membranes was evaluated by plasma protein adsorption (BSA and BFG), platelet adhesion experiments, and clotting time, contact activation, complement activation, and hemolysis tests. The detailed procedures are shown in the Supporting Information. 2.7. Cell Tests. The cell compatibility of the modified membranes was assessed by a CCK-8 assay with L929 cells. The cytotoxicity of the modified membranes was evaluated by an MTT assay with L929 cells. The detailed procedures are provided in the Supporting Information. 2.8. Ultrafiltration of Pure Water and BSA Solution. The barrier properties of the membranes were evaluated by testing the fluxes of pure water and BSA solution, the protein rejection ratio, and the flux recovery ratio. The detailed procedures are provided in the Supporting Information.

Table 1. Compositions of the Hydrogel Precursors code

SBMA (mol)

AANa (mol)

MBA (g)

2-ketoglutaric acid (g)

PES-10-0 PES-9-1 PES-8-2 PES-7-3 PES-6-4 PES-4-6

0.01 0.009 0.008 0.007 0.006 0.004

0 0.001 0.002 0.003 0.004 0.006

0.07 0.07 0.07 0.07 0.07 0.07

0.05 0.05 0.05 0.05 0.05 0.05

membranes were immersed into 0.05 mM AgNO3 solution with oscillation for 24 h in the dark, followed by rinsing with DI water three times to remove the excess AgNO3. Then, the membranes were dipped in 0.05 mM NaBH4 for 2 h to reduce Ag+ to Ag nanoparticles. 2.3. Characterization of Hydrogel Thin-Layer-Attached Membranes. The morphology and chemical composition were characterized by field-emission scanning electron microscopy (FESEM), Attenuated total reflection-Fourier transform infrared (ATRFTIR) spectroscopy, and X-ray diffraction (XRD). The mechanical properties of the modified membranes were also tested by a tensile testing machine (HZ1004B; Dongguan lixian Instrument Scientific Co., Ltd, China). The surface hydrophilicity of the membranes was investigated on the basis of contact angle measurement, using a contact angle goniometer (OCA20; Dataphysics, Germany) equipped with a video capture. The swelling behaviors at various pH values were

3. RESULTS AND DISCUSSION 3.1. Morphology, Chemical Composition, and Physicochemical Properties of the Membranes. The antibacterial hydrogel thin layers were covalently attached onto the PES membrane surfaces by four steps: (i) introducing hydroxyl groups onto the membrane surfaces; (ii) grafting double bonds onto the membrane surfaces via the reaction of the hydroxyl group and acryloyl chloride to provide anchoring sites (according to the results of HPLC, the density of the double bonds on the membrane surface was 3.6 × 10−7 mol/cm2); (iii) the formation and simultaneous attachment of the hydrogel layers onto the membrane surfaces; and (iv) loading Ag

Scheme 1. Chemically Attaching a Hydrogel Thin Layer onto a PES Membrane

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Figure 1. SEM images of pristine and hydrogel thin layers attached to PES membranes.

Figure 2. (A) ATR-FTIR spectra of the membranes; (B) XRD patterns of the membranes; (C) the correlation between the hydrogel precursor concentrations and the hydrogel layer thickness; (D) the residual weights after every cycle of soaking−drying treatment.

After attaching the hydrogel layers, the surface became rougher, and a typical honeycomb-like porous hydrogel structure was observed. As the proportion of AANa increased, the porosity increased due to the charge repulsion of the AANa segments. It is noteworthy that only the freeze-drying samples displayed the porous structure. If the samples were air-dried, only a dense smooth morphology could be observed due to shrinking of the porous structure. Additionally, Ag nanoparticle aggregation could be observed as the AANa proportion increased to 30 mol %. The particle size was analyzed by nano measurement software, and the mean size was around 51.3 nm (Figure S1).

nanoparticles into the hydrogel layers; as shown in Scheme 1. The monomers, initiator, and crosslinking agent used in this system were all water soluble; additionally, the thickness of the formed hydrogel layer was very thin. Therefore, the residual molecules in the final hydrogel layers could be easily washed out. The variations in the surface morphology, chemical composition, and hydrophilicity of the membranes at different steps were characterized by SEM, ATR-FTIR spectroscopy, and water contact angle (WCA) tests. The morphologies of the pristine and modified PES membranes were observed by SEM, as shown in Figure 1. The pristine PES membrane displayed a dense flat morphology. 15965


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Figure 3. Cross-sectional morphologies of the hydrogel layer-attached membranes prepared at various hydrogel precursor concentrations: (A) 0 mol/L; (B) 0.7 mol/L; (C) 1.0 mol/L; (D) 2.0 mol/L; (E) 3.0 mol/L; (F) 4.0 mol/L.

Figure 4. WCAs of the pristine and modified PES membranes. The left images correspond to the WCA values when the water drop immediately contacted with the membrane surface.

The chemical compositions of the membranes at different modification steps were investigated by ATR-FTIR spectroscopy, as shown in Figure 2A. For the PES-OH membrane, the new peaks at 1720 and 3300 cm−1 were assigned to the characteristic peaks of the carbonyl and hydroxyl groups, respectively; which were introduced by the in situ crosslinking polymerization of HEMA. After introducing the double bonds, the hydroxyl groups were completely consumed, so the peak at 3300 cm−1 disappeared. With the immobilization of the hydrogel thin layers, a new peak at 1038 cm−1 appeared, which was assigned to the stretching vibrations of the sulfonate groups from poly(sulfobetaine methacrylate) (PSBMA). EDS elemental analysis was also used to detect the surface elemental compositions (data not shown), and the results demonstrated the existence of elemental Ag. The loading amounts increased with increasing AANa proportion in the hydrogel layers, which was consistent with the results observed by the SEM images. The presence of the Ag nanoparticles in the hydrogel layers was further confirmed by XRD, as shown in Figure 2B. The diffraction peaks appearing at 38.5, 44.8, 64.7,

and 77.8° were assigned to the (111), (200), (220), and (311) crystalline planes of Ag, respectively. The morphology and chemical compositions of the membranes confirmed that the Ag nanoparticle-loaded hydrogel thin layers were covalently attached onto the membrane surfaces. However, the thickness and long-term stability of the hydrogel layers are two critical factors for when the PES membranes are applied in practice. In this study, the hydrogel layers were formed between two quartz glass sheets; therefore, the thickness of the hydrogel layers could be effectively controlled by regulating the spacer thickness or the concentrations of the hydrogel precursors. The dependence of the thickness on hydrogel precursor concentration with fixed spacer thickness was studied using the cross-sectional SEM images (Figure 3). As shown in Figure 2C, it was found that the thicknesses of the hydrogel thin layers had a positive correlation with the precursor concentrations. The thinnest thickness of the layer was about 0.94 μm, as the surface polymerization could hardly be initiated when the precursor concentration was too low. Delamination of the attached hydrogel layers is a great 15966


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Figure 5. Fluorescence microscopy images for PES, PES-OH, PES-10-0, PES-8-2, PES-6-4, and PES-4-6 surfaces after exposure to E. coli (Gram negative) and S. aureus (Gram positive), respectively.

threat for long-term applications; thus, the stability of the layers was evaluated by calculating the residual masses of the samples after each cycle of soaking−drying treatment. The dried samples were immersed into PBS solution for 12 h, and then dried at room temperature. Both the swelling state and dried state masses of the membranes were weighed (the influence of salts in PBS was beyond the detection limit of the analytic balance). The soaking−drying treatment was repeated for seven cycles, and no obvious mass change was observed, as shown in Figure 2D. The results indicated that the long-term stability of the hydrogel layers was reliable, and that no delamination occurred. The swelling degrees of the attached hydrogel layers at different pH values were also investigated (Figure S2). For the hydrogel layers containing carboxylate groups, the swelling degree increased with increasing pH value, which was caused by the increased dissociation degree and the electrostatic repulsion

of the carboxylate groups at high pH values. Additionally, the mechanical properties of the modified membrane were evaluated (Figure S3). Compared with that of the pristine PES membrane, the tensile strength of the PES-OH membrane slightly decreased from around 3.4 to 2.7 MPa, which might be caused by the microphase separation of the PHEMA. After attaching the hydrogel layer, the tensile strength further decreased to 1.5 MPa, which might be attributed to the destruction of the matrix’s microstructure during the swelling− collapse process of the hydrogel layer. WCA is widely used to characterize the hydrophilicity of a membrane surface. The WCAs of the membranes are shown in Figure 4. The WCA of the pristine PES membrane was 78.3°. After attaching the hydrophilic hydrogel layers, the WCA slightly decreased to around 72°, which was inconsistent with a previous report.34 The static WCA value is affected by the surface hydrophilicity as well as the surface roughness. Even 15967


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Figure 6. (A) Inhibition zone images of the modified membranes at different time release intervals for S. aureus (Gram positive); the optical degrees of the modified membranes after different release times for S. aureus (Gram positive) (B) and E. coli (C).

The antibacterial adhesion property of the membranes was firstly evaluated by the live/dead two-color fluorescence method,38 as shown in Figure 5. After co-culturing with solutions of either E. coli or S. aureus for 12 h, a large number of viable bacteria (the green fluorescence) aggregated into clusters on the pristine PES membrane surface, while some dead bacteria (the red fluorescence) were observed. After attaching the Ag nanoparticle-loaded hydrogel thin layers, the number of attached bacteria obviously decreased, and some live bacteria were found for both E. coli and S. aureus. The modified membranes without the loaded Ag nanoparticles were also used to evaluate the antiadhesion property (Figure S4). Combining the results of Figures 5 and S4, it was observed that the Ag nanoparticle-loaded membranes exhibited lower amounts of attached live bacteria than that of those membranes without the loaded Ag nanoparticles. For the modified membranes with/ without loaded Ag nanoparticles, the hydrogel layers significantly reduced bacterial adhesion compared with that of the pure PES and PES-OH membranes. Additionally, with increased proportions of AANa in the hydrogel layers, the number of attached bacteria slightly increased due to the decreased surface hydrophilicity. The results indicate that the hydrogel layers could effectively inhibit bacteria adhesion. The loaded Ag nanoparticles can be released to kill bacteria and inhibit bacteria growth. The loading amounts and release

though the attached hydrophilic hydrogel layers should enhance the surface hydrophilicity of the PES membrane, the increased roughness may offset its influence on the WCA. To better monitor the wettability of the porous hydrogel layers, the WCAs of the membranes versus contact time were also investigated. As shown in Figure 4, the hydrogel thin layers exhibited a significant decrease in WCA under a drop age from 0 to 20 s. Compared with those of PES and PES-OH, the constant WCAs (after 20 s) of the hydrogel-attached membranes significantly decreased. As the proportion of AANa was increased from 0 to 60%, the final WCAs increased from 15 to around 40°, because the hydrophilicity of PSBMA was superior to poly(sodium acrylate) (PAANa). 3.2. Antibacterial Property. PSBMA is well-known for its strong resistance to protein adsorption, cell attachment, and bacteria adhesion.7,35 Ag+ ions may connect to the negatively charged bacterial cell wall and rupture it.36,37 We hypothesized that the Ag nanoparticle-loaded PSBMA/AANa hydrogel layers could not only resist bacteria adhesion, but also inhibit bacteria growth, finally suppressing the formation of biofilms. The antibacterial property of the modified membranes was evaluated via the live/dead two-color fluorescence, inhibition zone, and optical degree of co-cultured solution methods using E. coli and S. aureus. 15968


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Figure 7. (A) Adsorbed amounts of BSA and BFG on the membrane surfaces; (B) clotting times of the membranes; (C) hemolysis ratios of human red blood cells after incubation with the membranes for 3 h; (D) SEM images of platelet adhesion on the membranes.

antibacterial capacity by combining two antibacterial mechanisms into one system: the hydrogel layers could effectively resist the bacteria adhesion due to their excellent hydration capacity, and the loaded Ag nanoparticles could not only kill the adhered bacteria, but also be released into the surrounding area to suppress the bacteria growth. In addition, the effective release of Ag ions could last for more than 5 weeks. Therefore, it is believed that the designed hydrogel layers can significantly improve the antibacterial properties of the PES membrane. 3.3. Blood Compatibility. PES-based membranes are widely used in blood purification, thus the blood compatibility of the modified membranes is also important.40 Plasma protein adsorption amounts, platelet adhesion, clotting times, hemolysis ratio, and blood-related complement activation were tested to evaluate the hemocompatibility of the hydrogel layerattached membranes. It is well-known that nonspecific protein adsorption is the first interaction event that occurs at the interface between material surfaces and blood.41 Some specific proteins in blood, such as fibrinogen, serum albumin, and clotting enzymes, play an important role in material-induced clotting. For example, fibrinogen plays a critical role in causing final blood coagulation; even a small amount of adsorption on a material surface may induce a multistep and interlinked process, including platelet adhesion and activation, and clot formation.42 In this study, the BSA and BFG adsorption amounts were evaluated, as shown in Figure 7A. The pristine PES membrane had high protein adsorption amounts of 19.3 μg/cm2 for BSA and 18.5 μg/cm2 for BFG, which were attributed to the

behavior are important for the antibacterial properties of the modified membranes. For PES-9-1, PES-8-2, PES-7-3, PES-6-4, and PES-4-6, the Ag loading amounts were 9.6, 16.7, 28.4, 37.3, and 49.8 μg/cm2, respectively. To investigate the long-term bactericidal efficiency, the modified membranes were immersed into DI water for different time intervals, the solutions were taken out to test the release profiles (Figure S5), and the membranes were evaluated for their antibacterial properties by the inhibition zone method,39 and the results are shown in Figure 6A. Obvious bacterial inhibition zones were observed for all the membranes, and no significant difference in size was observed among the membranes loaded with different amounts of Ag nanoparticles. It is noteworthy that the inhibition zones of the three samples clearly remained, even after 5 weeks, indicating that the membranes still maintain strong bactericidal ability. The results were consistent with the Ag release profiles, where the Ag release could last for more than 5 weeks. To further confirm the long-term antibacterial properties, the modified membranes after releasing Ag+ for different time intervals were used to measure the optical degrees of the bacterial membrane co-cultured solutions (after culturing for 12 h). As shown in Figure 6B,C, for both S. aureus and E. coli, the growth rates were fast for the control sample, whereas the modified membranes effectively inhibited the growth of the bacteria even after 5 weeks of Ag+ release. However, the antibacterial properties slightly decreased after 3 weeks of Ag+ release. In summary, the above results indicate that the Ag nanoparticle-loaded hydrogel thin layers showed strong 15969


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Figure 8. Generated concentrations of C3a (A), C5a (B), PF4 (C), and TAT (D) after incubation of the membranes with whole blood.

biomaterials.43 APTT is an indicator of the efficacy of the intrinsic and common plasma coagulation pathway; and TT is used to observe the clot formation time taken for the thrombin conversion of fibrinogen into fibrin.44 As shown in Figure 7B, compared with that for the pristine PES membrane, the APTT value increased from 47 to 75 s for the PES-10-0 membrane, and this was mainly caused by the strong resistance to plasma protein adsorption. With increasing AANa proportion in the hydrogel layers, the APTT value further increased to 110 s, which was attributed to the increase of the carboxylate groups that could bind Ca2+ to inhibit the activation of Factor IX−IXa, Factor X−Xa, and prothrombin to thrombin.2 Meanwhile, the TT value remained unchanged for all the membranes. The results indicate that the modified membranes had an effect on the endogenous pathway of coagulation, which was attributed to the binding of Ca2+ by the carboxylate groups and the strong resistance to plasma protein adsorption. The hemolysis test was also conducted to evaluate the blood compatibility of the modified membranes. Some earlier reports revealed that Ag nanoparticles can cause serious hemolysis when the concentrations increased to some extent.45,46 In this study, the amount of Ag nanoparticles adsorbed in the hydrogel thin layers was controlled by the density of the carboxylate groups. As shown in Figure 7C, the membranes without Ag nanoparticles (PES and PES-10-0) showed very low hemolysis ratios of around 1%. When the mole ratios of carboxylate groups in the hydrogel layers were below 30%, the hemolysis ratios were below 5%. With further increases in the mole ratios of the carboxylate groups, severe hemolysis was observed, which was caused by the high concentrations of Ag nanoparticles. Therefore, when the Ag nanoparticle-loaded hydrogel thin layers are used for blood-contacting devices, the amount of

hydrophobic interactions between the proteins and the membrane surface. However, for the hydrogel layer-attached membranes, the protein adsorption amounts were almost undetectable via the Micro BCA method when the AANa proportions in the hydrogel layers were below 30%. With further increases of the AANa proportion in the hydrogel layers, the surface hydrophilicity decreased, so PES-7-3, PES-64, and PES-4-6 showed protein adsorption amounts of 1.39, 1.51, and 1.76 μg/cm2, respectively. The strong resistance capacity to protein adsorption was attributed to the strong hydration ability of the hydrogel layers. The decreased protein adsorption amounts could improve the blood compatibility of the membranes. Adhesion and activation of platelets are considered to be the key factors in thrombus formation. Thus, the number and morphology of adhering platelets on a material surface are widely used to evaluate blood compatibility. As shown in Figure 7D, many platelets aggregated and adhered on the pure PES membrane surface. However, for the hydrogel thin-layerattached membranes, no adhered platelet could be found, demonstrating the beneficial effect of the superhydrophilic hydrogel layers on the blood compatibility of the PES membrane. Additionally, irregular surfaces were observed in Figure 7D. It is well-known that there are some positively charged compounds, such as lysozyme and low density lipoprotein. Herein, the irregular surfaces might be aggregates formed by some positively charged compounds. Blood clotting times are important indexes for evaluating the blood compatibility of materials. APTT and TT are usually used to examine the intrinsic pathway and to exhibit the bioactivity of intrinsic blood coagulation factors, and they have been widely used to evaluate the blood compatibility of 15970


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Figure 9. Viability of cells proliferated on the modified membranes: (A) optical density (OD) of L929 cells proliferated 1, 3, 5, and 7 days tested at 450 nm by using a CCK-8 assay; (B) cytotoxicity tested at 630 nm by an MTT assay; (C) cell shape in the leach liquor; (D) SEM images of L929 cells cultured on the modified membranes for 7 days.

Ag nanoparticles should be controlled below the threshold that would cause severe hemolysis. Complement activation and contact activation are also considered to be crucial indicators of the blood compatibility of materials after coming in contact with blood. Complement activation is the result of the triggering of the host defense mechanism generated by the localized inflammatory mediator, and the generated concentrations of C3a and C5a are usually detected as model indexes to evaluate the complement activation.47 Contact activation may cause blood coagulation. Platelet activation can result in platelet aggregation and coagulation cascade activation. The activated platelet can express PF4, which reflects the platelet activation level.48 Thrombin, a potent platelet activating agonist, resulting from the activation of a coagulation cascade, can combine with antithrombin III to generate thrombin−antithrombin III (TAT) complexes, which demonstrates the extent of thrombin generation.49 In this study, PF4 and TAT concentrations were used to evaluate the contact activation level. C3a, C5a, PF4, and TAT concentrations were detected via an ELISA method, as shown in Figure 8A−D. Compared with those of blood and the pristine PES membrane, the concentrations of C3a, C5a, and TAT significantly decreased after attaching the PSBMA hydrogel layers, and slightly increased with increased proportions of AANa in the hydrogel layers. There was no significant difference in PF4 concentrations for the membranes, and the values were approximately the same as those for blood and the pristine PES membrane. The results indicate that the

attached hydrogel layers effectively suppressed the complement activation, and did not show obvious platelet activation and coagulation cascade activation, compared with the pristine PES membrane. 3.4. Cell Tests. The proliferation and adhesion of L929 cells on the membranes were investigated by a CCK-8 assay. As shown in Figure 9A, the number of cells increased continuously for TCP (the control sample) and the pure PES membrane during the 7 days culture; however, no obvious cell proliferation was observed for all the hydrogel layer-attached membranes. It has been reported that PSBMA has a strong capacity to resist the attachment of proteins, bacteria, and cells due to its high hydrophilicity. Additionally, there is a risk that Ag nanoparticles can cause cytotoxicity. The poor cell compatibility of the modified membranes might be caused by the antiadhesion property of the hydrogel layers or the large amount of Ag nanoparticles. Therefore, the cytotoxicity of the modified membranes was evaluated by an MTT assay. As shown in Figure 9B, with the incubation of the cells in the leach liquor of the modified membranes for 24, 48, and 72 h, the viabilities of the cells increased continuously. The cells cultured in the leach liquor displayed a healthy shape with a high density for all samples, as shown in Figure 9C, indicating that the amounts of Ag nanoparticles adsorbed in the hydrogel layers did not approach the threshold that would cause severe cytotoxicity. SEM was also used to observe the cells’ morphology on the membrane surfaces. As shown in Figure 9D, the cells spread on the pure PES membrane surface with 15971


Research Article

ACS Applied Materials & Interfaces low cell density, demonstrating its poor cell affinity. For the hydrogel layer-attached membranes, a few cells displayed a round shape morphology, indicating that no adhesion, spreading, and proliferation had happened on the surfaces. Thus, the modified membranes exhibited excellent anti-cell adhesion property, but no cytotoxicity, which can cause poor cell compatibility of membranes. 3.5. Pure Water Flux and Antifouling Property of Membranes. The pure water fluxes for the pristine PES membrane and PES-10-0 with the thinnest hydrogel coating of around 1.0 μm were calculated. For the pristine PES membrane, the pure water flux was 27.4 mL/m2 h mmHg. After attaching the hydrogel layer, the flux decreased to 20.5 mL/m2 mmHg for the reason that some of the membrane pores were blocked by the hydrogel layer. The BSA rejection ratios of pristine PES and PES-10-0 were 91.3 and 93.4%. It was also observed that the fluxes of the pristine PES membrane and PES-10-0 decreased dramatically when the solution changed from pure water to BSA solution due to the fouling caused by the deposition and adsorption of protein molecules onto the membrane surfaces and in the membrane pores. The flux recovery ratio was 0% for the pure PES membrane, due to the adsorption of BSA molecules onto the pore surfaces. For PES10-0, the flux recovery ratio was 74.7, 68.4, and 59.3% for the first, second, and third times, respectively. The attached hydrogel thin layer can improve the hydrophilicity of the membrane surface, thus decreasing protein adsorption; the layer helps to detach the adsorbed protein, thus the recovery ratio of PES was better than that of pure PES. For the membranes prepared by the phase invasion method, the pore size on the skin layer of the membrane was significantly affected by the solvent.50 In this study, NMP was selected as solvent, and the pore size of the prepared membrane was of the nanoscale. Thus, the fluxes of the membranes were low, and the pore canal was easily blocked by the protein molecules adsorbed on the pores.51

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AUTHOR INFORMATION

Corresponding Authors

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

Changsheng Zhao: 0000-0002-4619-3499 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge that this work was financially sponsored by the National Natural Science Foundation of China (Nos. 51433007, 51503125, and 51673125), and the China Postdoctoral Science Foundation (Nos. 2015M580791 and 2016T90852).

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REFERENCES

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4. CONCLUSIONS In this study, antibacterial hydrogel thin layers with excellent blood compatibility were covalently attached onto the PES membrane surfaces. The thickness of the hydrogel layers could be effectively controlled, and the stability of the hydrogel layers was reliable. After attaching Ag nanoparticles, the membranes could not only effectively resist the adhesion of bacteria but also inhibit the growth of the surrounding bacteria. As expected, the modified membranes could effectively inhibit the formation of biofilms. Additionally, the excellent hemocompatibility of the modified membranes was confirmed by the undetectable plasma protein adsorption, antiplatelet adhesion, prolonged clotting time, low hemolysis ratio, and suppressed blood-related activation. With the attaching of the superhydrophilic hydrogel layers, the membranes showed strong antiadhesion properties toward the cells. The amounts of adsorbed Ag did not approach the threshold that would cause cytotoxicity. The designed hydrogel layers have great potential to be widely applied as coatings for many biomedical devices to endow them with strong antibacterial abilities.

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Detailed description of experiments and characterization methods; SEM images of Ag nanoparticles and the size distribution; swelling degrees of the hydrogel layers; plot of the mechanical properties of the modified membranes; fluorescence microscopy images of the modified membranes without Ag nanoparticles after exposure to E. coli (Gram negative) and S. aureus (Gram positive); Ag loading amounts; release profiles versus time (PDF)

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b03176. 15972


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Design of Antibacterial Poly(ether sulfone) Membranes via Covalently

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