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Versatile and Rapid Postfunctionalization from Cyclodextrin Modified Host Polymeric Membrane Substrate Jie Deng,† Xinyue Liu,† Shuqing Zhang,† Chong Cheng,*,† Chuanxiong Nie,† and Changsheng Zhao*,†,‡ †

College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu 610065, China ‡ National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, China S Supporting Information *

ABSTRACT: Surface modification has long been of great interest to impart desired functionalities to the bioimplants. However, due to the limitations of recent technologies in surface modification, it is highly desirable to explore novel protocols, which can advantageously and efficiently endow the inert material surfaces with versatile biofunctionalities. Herein, to achieve versatile and rapid postfunctionalization of polymeric membrane, we demonstrate a new strategy for the fabrication of β-cyclodextrin (β-CD) modified host membrane substrate that can recognize a series of well-designed guest macromolecules. The surface assembly procedure was driven by the host−guest interaction between adamantane (Ad) and β-CD. β-CD immobilized host membrane was fabricated via two steps: (1) epoxy groups enriched poly(ether sulfone) (PES) membrane was first prepared via in situ cross-linking polymerization and subsequently phase separation; (2) mono-6-deoxy-6-ethylenediamine-β-CD (EDA-β-CD) was then anchored onto the surface of the epoxy functionalized PES membrane to obtain PES-CD. Subsequently, three types of Ad-terminated polymers, including Adpoly(styrenesulfonate-co-sodium acrylate) (Ad-PSA), Ad-methoxypoly(ethylene glycol) (Ad-PEG), and Ad-poly(methyl chloride-quaternized 2-(dimethylamino)ethyl methacrylate (Ad-PMT), were separately assembled onto the β-CD immobilized surfaces to endow the membranes with anticoagulant, antifouling, and antibacterial capability, respectively. Activated partial thromboplastin time (APTT), thrombin time (TT), and prothrombin time (PT) measurements were carried out to explore the anticoagulant activity. The antifouling capability was evaluated via protein adsorption and platelet adhesion measurements. Moreover, Staphyllococcous aureus (S. aureus) was selected as model bacteria to evaluate the antibacterial ability of the functionalized membranes. The results indicated that well-regulated blood compatibility, antifouling capability, and bactericidal activity could be achieved by the proposed rapid postfunctionalization on polymeric membranes. This approach of versatile and rapid postfunctionalization is promising for the preparation of multifunctional polymeric membrane materials to meet with various demands for the further applications.

1. INTRODUCTION Development of polymeric membrane surface capable with desired biofunctions such as anticoagulant, antifouling, and antibacterial characteristics is a key issue in the wide range of biomedical applications,1−3 which includes the applications such as clinical hemodialysis membrane, cardiopulmonary bypass device, blood contacting materials, and other biomedical implants.4,5 Particularly, the design of integrated biocompatibility and biofunctionality has attracted great attention for implantable and blood contacting membranes. In recent years, numerous methods (including physical blending, surface grafting to/from, surface layer-by-layer (LbL) self-assembly and coating, etc.) have been developed to impart specific bioactivity and favorable biocompatibility to inert membranes.6−9 By the aid of these approaches, different kinds of functional groups have been successfully introduced onto membrane surfaces; therefore, the surface performance can be © 2015 American Chemical Society

remarkably altered. However, among these methods, the blending method usually changes the mechanical property of the bulk matrix. Meanwhile, the chemical processes of grafting to/from are usually achieved by multiple steps that are tedious and time-consuming. Besides, a large amount of homopolymers may be generated during some grafting processes, which makes this method uncontrollable and inapplicable in industry. In addition, the procedures of LbL self-assembly or coating on the membrane surface may be time-consuming or not stable enough to maintain long-term functionalities, which thus restrict their potentials in application. Cyclodextrins (CDs) are recognized as a series of cyclic oligosaccharides consisting of 6, 7, or 8 glucose units linked by Received: June 3, 2015 Revised: August 14, 2015 Published: August 24, 2015 9665

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Langmuir α-1,4-linkages, named as α-, β-, or γ-CD, respectively.10 The hydrophobic cavity structure of CDs enables it to form inclusion complexes with a great number of guest molecules such as poly(ethylene glycol) (PEG), 11 ferrocene,12,13 azobenzene,14 adamantane (Ad),15 and their derivatives.16 Over the past several years, numerous CD-based host−guest reactions have been carried out to construct multifunctional materials and smart/responsive interfaces.17 Plenty of works on the vesicle membrane modification have been reported by the groups of Zhou,18,19 Ravoo,20,21 and Liu22 via diverse chemical approaches; especially, Zhou et al. reported that the membranes of hyperbranched polymer vesicles are advantageous in constructing multivalent intravesicular interactions to realize large-scale, highly efficient, reversible, and stable aggregation, which can be potential nanocompounds for designing of multifunctional biointerfaces. Inspired by these earlier pioneering researches on CD-based host−guest reactions, CD molecules have been successfully anchored to some polymeric membrane surfaces, such as nylon-6,23 poly(vinylidene fluoride) (PVDF),24 and poly(tetrafluoroethylene) (PTFE),25 which enable these membrane substrates to recognize some guest molecules. Among these host−guest complexations, due to the near-perfect size match between β-CD and Ad, the interaction between β-CD and Ad is extremely robust with an association constant of about 3 × 104 M−1;26 thus, the β-CD/ Ad inclusion complex is a much more powerful method to promote the surface functionalities. Moreover, Zhao et al.27 have reported the surface modification of cellulose fiber via the immobilization of polymer chains with the driving force of βCD and Ad complex; consequently, the surface properties of the cellulose fibers can be fine-tuned by the appropriate choice of guest polymers with certain intrinsic properties and chain length. Very recently, Ji et al.28 have developed a method for dual functionalization of poly(ε-caprolactone) (PCL) film surface by means of the inclusion complex between β-CD and Ad, for which two types of Ad-terminated molecules are assembled onto a β-CD anchored PCL surface to endow the surface with dual functionalities. Although, some pioneering works have been focused on the surface modification via this βCD and Ad-based host−guest interaction. Few of them could be recognized as a universal, facile, and robust approach to achieve surface functionalization via β-CD and Ad interaction. Meanwhile, these works also did not carry out systematical investigations to design a more adaptable method that could be used to immobilize a series of different functional guest macromolecules to meet different challenges. Moreover, the chemical routes that anchoring of the β-CD onto the membrane surfaces in these works needed multisteps, which were complicated and tedious. Thus, a more universal, low-cost, and convenient method should be developed for the modification of host membranes via the β-CD and Ad-based host−guest interaction. Until now, although there were reports on the fabrication of β-CD modified membrane via a convenient in situ immobilization method for separation applications,29−32 most of these procedures used a method of co-cross-linking of the polymer matrix and β-CD molecules to achieve a considerable amount of embedded functional β-CD molecules in the membrane matrix, which would also restrict its potential for further surface modification. In our earlier reports, we have found that in situ cross-linking copolymerization can be an efficient way to fabricate high-performance and robust ultrafiltration membrane with the advantage of effectively reducing the elution of the

additive functional polymers. Thus, we suggest that a large amount of functional β-CD groups could be immobilized onto membrane surfaces by using the proposed in situ cross-linking copolymerization,33,34 which may become a highly universal and robust method for the design of β-CD and Ad interaction based functional membrane. In this study, a novel β-CD anchored host membrane was fabricated via in situ cross-linking copolymerization method by using the poly(ether sulfone) (PES) as model membrane substrate. In a typical procedure, glycidyl methacrylate (GMA) and N,N′-methylenebis(acrylamide) (MBA) were copolymerized with PES matrix in N,N′-Dimethylacetamide (DMAC). Afterward, the GMA functionalized PES membranes (PES-GMA) were prepared via a phase transition method in water.34 Subsequently, mono6-deoxy-6-ethylenediamine-β-CD (EDA-β-CD) was anchored onto the PES-GMA surfaces via the reaction between the epoxy and amino groups in water to obtain a β-CD enriched surface (PES-CD), which have the capability to recognize a series of Ad-terminated polymers for postfunctionalization of the membrane substrates. Then, three types of Ad-terminated functional polymers were assembled onto the PES-CD host membrane surface to achieve the postfunctionalization of the PES membrane. Ad-poly(sodium 4-vinylbenzenesulfonate-cosodium acrylate) (Ad-PSA, anticoagulant) and Ad-poly([2(methacryloyloxy)ethyl] trimethylammonium chloride) (AdPMT, bactericidal) were synthesized via atom transfer radical polymerization (ATRP); Ad-poly(ethylene glycol) (Ad-PEG, antifouling) was synthesized via the esterification reaction between adamantaneacetic acid and methoxypoly(ethylene glycol). The negatively charged Ad-PSA with numerous amount of sulfonic acid and carboxyl acid groups has shown heparin-mimetic bioactivity, such as anticoagulant, antithombotic, and promotion of cell adhesion,9 while the PEG segments (Ad-PEG) has been widely applied in the field of construction of antifouling surface.35 Moreover, the positively charged Ad-PMT with quaternary ammonium groups has been known as one of most widely used bactericidal polymers.36 Herein, by taking the advantage of β-CD and Ad interaction and in situ cross-linking copolymerization, we have designed a series of anticoagulant, antifouling, and antibacterial membranes via the host−guest self-assembly of Ad-PSA, Ad-PEG, and Ad-PMT with the PES-CD membranes, respectively. The surface chemistry and morphology of these postfunctionalized membranes have been carefully investigated. Then, measurements of APTT, TT, PT, protein adsorption, platelet adhesion, and bactericidal capability have been carried out in detail to demonstrate the efficiency of the proposed method for membrane postfunctionalization. It is believed that this new strategy for the fabrication of β-CD modified host membrane is convenient and efficient, which can be recognized as a universal approach to achieve surface immobilization of various welldefined guest biomacromolecules to satisfy different biomedical application fields.

2. EXPERIMENTAL SECTION Materials. β-Cyclodextrin (β-CD, >98%), p-toluenesulfonyl chloride (TsCl, 99%), glycidyl methacrylate (GMA, 97%), 2,2′azobis(2-methylpropionitrile) (AIBN, 98%), α-bromoisobutyryl bromide (BIBB, 98%), sodium acrylate (AANa), sodium 4-vinylbenzenesulfonate (SSNa, 90%), [2-(methacryloyloxy)ethyl]trimethylammonium chloride solution (MT, 75 wt % in water), N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDETA, 99%), N,N′-dicyclohexylcarbodiimide (DCC, 99%), 4-(dimethylamino)pyridine (DMAP, 99%), 1-adamantanol (AR, 99%), 1-adamantane9666

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Langmuir acetic acid (AdAc, 98%), N,N′-methylenebis(acrylamide) (MBA, 99%), and ethylenediamine (EDA, >99%) were purchased from Aladdin (Shanghai, China) and used without further purification. Cyclic ligand 5,7,7,12,14,14-hexamethyl-1,4,8,11-tetraazacyclotetradecane (Me6TATD) was synthesized according to a previous paper.37 Methoxypoly(ethylene glycol) (mPEG, Mn = 2000) was purchased from Sigma-Aldrich (USA). Poly(ether sulfone) (PES, Ultrason E6020P, BASF) was used as received. CuBr (AR, Aladdin) was purified by stirring in glacial acetic acid overnight, filtered off, washed with ethanol, and then dried in a vacuum oven at 30 °C for 24 h. N,N′-Dimethylacetamide (DMAC, AR), triethylamine (TEA, AR), and tetrahydrofuran (THF, AR) from Kelong Chemical Reagent were distilled before use. All the other reagents (analytical grade) were purchased from Kelong Chemical Reagent Co., Ltd., and used without further purification. Synthesis of Mono-6-dexoy-6-(p-tolysulfonyl)-β-CD (β-CDOTs) and Mono-6-deoxy-6-ethylenediamine-β-CD (EDA-β-CD). The synthesis procedures were similar to the previous report with some modifications.14 Briefly, β-CD (60 g, 52.8 mmol) was suspended in 500 mL of ultrapure water, and then NaOH (6.57 g, 164.1 mmol) in 20 mL of ultrapure water was added dropwise, after which the solution became homogeneous. The solution was immersed into an ice−water bath, and TsCl (15.12 g, 79.2 mmol) in 30 mL of acetonitrile was then added dropwise over 30 min, causing the formation of white precipitates. After further stirring at room temperature for 2 h, the suspension was refrigerated overnight at 4 °C. Afterward, the precipitate was recovered by suction filtration and recrystallized three times from hot water. The product was dried under vacuum at 50 °C for 24 h. Then, 5 g of as-prepared β-CD-OTs was dissolved in 50 mL of EDA (excess amount) and refluxed at 80 °C for 4 h. Then, the solution was cooled down, and the unreacted EDA was removed via rotary evaporation. For further purification, EDA-β-CD was dissolved in water and precipitated in alcohol and washed with a copious amount of alcohol. The product was dried under vacuum at 50 °C for 24 h. Fabrication of PES-CD Host Membrane. First, PES-GMA was prepared according to our previous report.34 8 g of PES (16 wt %) was dissolved in 39.4 g of DMAC to get a homogeneous solution, following with the addition of GMA (2.5 g, 17.6 mmol), MBA (0.027 g, 0.176 mmol), and AIBN (0.0289 g, 0.176 mmol). The reaction was sealed in a N2 atmosphere and carried out at 70 °C for 24 h. Subsequently, the resultant mixture was cooled to room temperature and vacuum degassed. The PES-GMA membranes, 55 ± 5 μm, were prepared via spinning coating and liquid−liquid phase separation. The obtained membranes were immersed into DI water for at least 2 days with refreshing the water several times to remove any residual solvent. Afterward, the PES-GMA membranes were immersed into 50 mL of ultrapure water containing 1 g of EDA-β-CD. The reaction was carried out at 60 °C for 24 h with stirring under a N2 atmosphere to obtain PES-CD membranes. Then the membranes were washed with a copious amount of ultrapure water to remove any physically attached EDA-β-CD molecules. Synthesis of 1-Adamantyl α-Bromoisobutyrate (ABIB). The procedure for the synthesis of the ATRP initiator, ABIB, was according to a previous work.38 1-Adamantanol (0.761 g, 0.005 mol) and TEA (0.607 g, 0.006 mol) were dissolved in 10 mL of CH2Cl2. The mixture was cooled via an ice−water bath; then, BIBB (0.742 mL, 0.006 mol) in CH2Cl2 (5 mL) was added dropwise. The reaction was maintained at 0 °C with stirring for 2 h and then at room temperature for another 24 h. The purification procedure was the same as that in the literature.38 Synthesis of Ad-PSA and Ad-PMT via ATRP. ABIB (0.075 g, 0.25 mmol) was dissolved in a Schlenk tube containing 25 mL of DMSO (40 mL of DMSO/H2O, v/v: 2:3 for Ad-PSA instead), followed with the addition of MT (6.67 g, 75 wt % in water, 24.07 mmol) (4.186 g of SSNa and 0.814 g of AANa for Ad-PSA instead). Then, PMDETA (0.0171 g, 0.88 mmol) (Me6TATD, 0.17 g, 0.6 mmol for Ad-PSA instead) and CuBr (0.0715 g, 0.5 mmol) were added under a N2 atmosphere. The Schlenk tube was closed by a three-way stopcock. After three freeze−pump−thaw cycles, the tube was sealed

Scheme 1. Procedures for the Preparation of Host PES-CD Membrane and Postfunctionalization of PES-CD Surface with Three Different Types of Ad-Polymers

off in a N2 atmosphere. The polymerization was carried out at 70 °C for 24 h and then terminated by exposing to air. Afterward, the resultant mixture was purified via dialysis (MWCO = 3500 Da) against ultrapure water and ethanol alternatively for 1 week. The obtained solution was freeze-dried to obtain the product. Synthesis of Ad-PEG. The synthetic procedure was similar to an earlier literature.39 Briefly, AdAc (0.43 g, 2.2 mmol), mPEG (1 g, 0.5 mmol), and DMAP (0.05 g) were dissolved in 30 mL of dry THF; subsequently, DCC (0.55 g, 2.66 mmol) was added. The reaction was carried out at room temperature for 24 h. The white precipitate was removed via filtration, and the Ad-PEG was obtained and purified via precipitation from THF to diethyl ether for three times. Postfunctionalization of the Host PES-CD Membrane via Ad/CD Complex. Ad-PSA (0.2 g), Ad-PEG (0.2 g), and Ad-PMT (0.2 g) were separately dissolved in 20 mL of ultrapure water to obtain the aqueous solutions of these guest functional polymers. Then, the host PES-CD membranes were individually immersed into the AdPSA, Ad-PEG, and Ad-PMT solutions for 12 h, followed by washing with ultrapure water to obtain PES-PSA, PES-PEG, and PES-PMT, respectively. Characterization. 1H NMR spectra were recorded on a Bruker spectrometer (400 MHz). Fourier transform infrared (FT-IR) spectrometer (Nicolet 560, USA) and X-ray photoelectron spectrometer (XSAM800, Kratos Analytical, UK) were applied to confirm the surface compositions of the membranes. Atomic force microscopy (AFM) images were obtained via a Multimode Nanoscope V scanning probe microscopy system (Bruker, USA). Field emission scanning electron microscope (FE-SEM) images were acquired by using a scanning electron microscope (JSM-7500F, JEOL, Japan). The surface contact angles of double-distilled (DI) water on the membranes were measured via a contact angle goniometer (OCA20, Dataphysics, Germany). Permeability. PBS buffer flux was measured at room temperature by a dead-end ultrafiltration (UF) cell with an effective membrane area of 3.9 cm2 according to our previous report. Each time for the test, the membranes were precompacted at 0.07 MPa for 15 min to get steady filtration, following with the flux test at 0.06 MPa. The water flux was calculated using the equation34

flux (g/m 2 ·h·mmHg) = M /SPt where M (g) is the mass of the permeated solution, S (m2) is the effective membrane area, t (h) is the time for collecting the solution, and P (mmHg) is the pressure during the flux test. Clotting Time. APTT, TT, and PT measurements have been widely used to evaluate the in vitro antithrombogenicity of blood 9667

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concentration of 108 cells/mL. Then, the sterilized samples were put in a 24-well cell-culture plate, test surface up, followed with the addition of 2 mL of the above bacterial suspension, and incubated at 37 °C for 2 h without stirring. Then, the samples were scrupulously rinsed with normal saline to remove loosely attached cells. A live/dead staining assay was performed using the BacLight viability kit to evaluate the biocidal activity of the membrane surfaces. For the examination, the membranes were immersed in 1 mL of staining solution containing a 1:1 mixture of propidium iodide and SYTO9 in normal saline, incubated in the dark for 15 min, and then rinsed with normal saline, followed with the examination by fluorescence microscopy. Three separate samples were analyzed for each kind of membranes to get the reliable data.

Scheme 2. Synthetic Routes of Ad-Terminated ATRP Initiators and Ad-Terminated Functional Polymers

3. RESULTS AND DISCUSSION Synthesis of β-CD-OTs and EDA-β-CD. The synthetic procedures to obtain the β-CD-OTs and EDA-β-CD were according to our previous report.14 In the purification procedure of EDA-β-CD, ethanol was used to precipitate the product from its water solution; while, in the previous report, acetone was adopted. We found that the remanent ethylenediamine could not be easily removed by acetone. However, ethanol could efficiently remove the resultant ethylenediamine; meanwhile, the EDA-β-CD also showed excellent precipitation capability in ethanol. The characterization results of the β-CDOTs and EDA-β-CD were identical with our previous report (Figure S1). MS m/z: 1215.52 [M + K+]. Characterization of the PES-CD Host Membrane. The ATR-FTIR spectra of PES, PES-GMA, and PES-CD are shown in Figure 1. In the spectra of PES-GMA, the signal at 1728

contacting materials in recent years.40 Thus, in this study, the anticoagulant bioactivity of the postfunctionalized membranes was evaluated via APTT, TT, and PT evaluations by using platelet-poor plasma (PPP). The procedures for the evaluations are depicted in detail in the Supporting Information. Protein Adsorption Evaluation. Bovine serum albumin (BSA) and bovine serum fibrinogen (FBG) were selected as two model proteins to evaluate the protein adsorption property of the pristine PES and the postfunctionalized PES membranes. The evaluation procedure was similar to our previous report.41 Briefly, the membrane with an area of 1 × 1 cm2 was first immersed in normal saline overnight at 4 °C and then incubated at 37 °C for 1 h. Afterward, the normal saline was sucked out, and BSA (or FBG) in phosphate buffer solution with a concentration of 1 mg/mL was added and kept static for 1 h to reach an adsorption equilibrium. Then, the membrane was scrupulously rinsed with PBS solution and DI water. Subsequently, the adsorbed protein was washed down by using 2 mL of 2% sodium dodecyl sulfate (SDS) solution at 37 °C with shaking for 2 h. The adsorption and desorption times were carefully determined in preliminary experiments. The protein concentration of the washing solution was determined via the Micro BCATA Protein Assay Reagent Kit. Platelet Adhesion. Platelet-rich plasma (PRP) was utilized to evaluate the platelet adhesion property of the membranes. Healthy fresh human blood (male, 28 years old) was collected using clinical anticoagulant vacuum tubes. The blood was centrifuged at 1500 rpm for 15 min to obtain PRP. The samples were first immersed in normal saline and equilibrated at 37 °C for 1 h in a 24-well cell-culture plate. Afterward, the normal saline was sucked out, and subsequently 1 mL of the fresh PRP was added, followed with incubation at 37 °C for 2 h. Then, the membranes were scrupulously rinsed with normal saline for three times and treated with 2.5% glutaraldehyde in normal saline overnight. Afterward, the membranes were washed with normal saline and subjected to a special drying process.9 Platelet adhesion was observed via a FE-SEM. The numbers of the adherent platelets on the membranes were calculated from six SEM images at 500× magnification at different places on the same membrane. Bactericidal Activity. The method for evaluating the antibacterial efficiency was similar to a previously reported method.42 Briefly, S. aureus was first cultured in a pure culture at 37 °C on a Luria−Bertani (LB) agar plate. Then, one colony was then used to inoculate 50 mL of LB medium and incubated at 37 °C for 22 h. Afterward, the bacteria were collected via centrifugation at 8000 rpm for 10 min. The cell pellets were washed with normal saline for three times, and then suspended in normal saline to get a final suspension with a

Figure 1. ATR-FTIR spectra of PES, PES-GMA, and PES-CD.

cm−1 was assigned to the stretching vibration of the carbonyl groups of GMA. Moreover, the signals at 1012 and 906 cm−1 should be attributed to vibration of the epoxy groups of GMA.34 In the spectra of PES-CD, the signal at 3375 cm−1 should be ascribed to the stretching vibration of the hydroxyl groups of β-CD, and the new peak at 1033 cm−1 was attributed to the deformation vibration of the C−O groups of β-CD.43 The results indicated that β-CD was successfully anchored onto the PES-GMA membrane surface. Synthesis of ABIB and the Ad-Terminated Functional Polymers. The synthetic procedure of ABIB was according to a previous report.38 The Ad-terminated ATRP initiator, ABIB, was synthesized by the esterification reaction of BIBB with 1adamantanol. Figure 2A shows the 1H NMR spectrum of ABIB, which confirms the existence of the characteristic peak of the BIBB group at about 1.9 ppm. The result was identical with the 9668

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Figure 2. 1H NMR spectra of ABIB (A) in CDCl3, Ad-PEG (B) in D2O, Ad-PSA (C) in D2O, and Ad-PMT (D) in D2O.

Figure 3. XPS spectra for the PES-CD, PES-PSA, PES-PEG, and PES-PMT.

previous report,38 and the peak integral ratios were consistent with the chemical structure of the target product. Generally, Ad-terminated polymers can be synthesized via direct polymerization from a functional initiator or through the modification of polymers with reactive terminal moieties. In this study, both these two methods were applied to prepare the three types of Ad-terminated polymers. The Ad-PSA and AdPMT were prepared via ATRP from ABIB, while the Ad-PEG was synthesized via the esterification reaction of mPEG with AdAc to obtain an Ad terminal moiety (Scheme 2). Figures 2B, 2C, and 2D show the 1H NMR spectra for the Ad-PEG, AdPSA, and Ad-PMT, respectively. In Figure 2B, the peaks for Ad and mPEG could be clearly observed. After the reaction, the signal of the CH2 of the mPEG near the ester bond shifted from b to b′ (red letters in Figure 2B).39 The peak integral ratios were consistent with the chemical structure of the target product. For Ad-PSA and Ad-PMT, the small characteristic

signals of Ad moiety were hard to be observed due to the low content of the Ad moiety and the overlapping from the PSA or PMT polymers, while the strong signals for the PSA and PMT could be clearly observed (Figures 2C and 2D). Postfunctionalization of the Host PES-CD Membrane. The surface functionalization of the host PES membranes was performed via the supramolecular assembly between β-CD and Ad groups in a mild and environmentally friendly medium. In this study, three different functional polymers, Ad-PSA, AdPEG, and Ad-PMT, were immobilized onto the PES-CD surface individually. The chemical composition variations of the membrane surfaces after the assembly of the functional polymers were compared by XPS analysis. As shown in Figure 3, it could be observed that after the assembly of Ad-PSA the peak intensity for S element was obviously increased, and new peaks for Na element were also observed (Figure 3A-2). Meanwhile, Figure 9669

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the C 1s spectrum of PES-PMT showed a similar result as that for the PES-PSA.9,14 Moreover, we have tried to detect 1H NMR spectra of PESGMA and PES-CD as well as the 1H NOESY spectra of these postfunctionalized membranes. However, we have not got reliable results. Because of the poor solubility of these crosslinked structures of the membranes, the peaks in the 1H NMR spectra were unorderly; besides, after dissolving these membranes in DMSO, some fiberized sediments could be obviously observed. On the basis of the ATR-FTIR and XPS results, we may conclude that the CD-Ad inclusion complexes have formed. The morphology variation of the membrane surfaces after the assembly of the functional polymers was analyzed via AFM measurement. As shown in Figure 4, the PES membrane surface was flat, while the PES-GMA membrane prepared by in situ cross-linking exhibited a rough surface with some potholes. After anchoring of the β-CD molecules, the surface became smoother, and the surface root-mean-square (Rms) roughness was decreased compared to those for the PES and PES-GMA (Figure 4B). Furthermore, after the assembly of the Ad-PSA and Ad-PMT, spherule-like structures were observed. However, for the PES-PEG, the surface was quite even, which might be attributed by the lower molecular weight of the Ad-PEG compared with those of Ad-PSA and Ad-PMT. Surface Hydrophilic/Hydrophobic Performance and Membrane Permeability. The hydrophilic/hydrophobic property is a key feature for biomaterials, which can be measured via static water contact angel analysis (Figure 4C). As

3B shows the C 1s spectra of the membranes; it could also be observed that after the assembly of the Ad-PSA, the relative content of the C−O bond (binding energy: 286.2 eV) dramatically decreased compared with PES-CD. Thus, it can be concluded that the Ad-PSA has been successfully assembled onto the PES-CD surface, while after the assembly of Ad-PEG, the oxygen content increased compared to that of the PES-CD (Table 1); moreover, in the C 1s spectrum of PES-PEG (Figure Table 1. Elemental Composition on the Modified Substrate Surfaces Determined by XPS Analysis concn (at. %) PES-CD PES-PSA PES-PEG PES-PMT

O

N

C

S

27.80 26.11 28.39 26.06

1.76 1.61 1.56 2.72

69.28 69.93 69.48 69.78

1.16 2.25 0.56 1.43

3B), the relative content of C−O bond (binding energy: 286.2 eV) obviously increased compared with PES-CD. These results indicated that the Ad-PEG was readily assembled onto the PESCD surface via the host−guest interaction between the β-CD and Ad groups. Moreover, after the assembly of Ad-PMT onto PES-CD surface, the carbon and nitrogen contents were obviously increased compared to these for the PES-CD (Table 1). In the N 1s spectra (Figure 3A), a new peak of N+−C (binding energy: 402.2 eV) could be obviously observed, while

Figure 4. (A) AFM images of the substrates for PES, PES-GMA, PES-CD, PES-PSA, PES-PEG, and PES-PMT; scanning area is 10 μm for all the samples. (B) Rms roughnesses for the membranes at different modification stages. (C) Static water contact angles for PES, PES-CD, PES-PSA, PESPEG, and PES-PMT. 9670

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respectively, which confirmed that the heparin-mimicking structure of PSA brushes with a large number of sulfonic and carboxyl groups had significant anticoagulant activity. The results indicated that the PES−PSA membrane would influence the endogenous pathway of coagulation cascade, which might be ascribed to the combination or reaction between the coagulation factors (V and X) in plasma and the hydrophilic surface of the PES−PSA membrane.41 Protein Adsorption Evaluation. Surface protein adsorption has been reported to be one of the most momentous factors of biomedical materials with respect to the blood compatibility. It has been well-known that a more hydrophilic surface usually exhibits a lower amount of protein adsorption.47 In the present study, both the BSA and FBG adsorptions were evaluated, and the results are shown in Figure 5B. The pristine PES membrane surface demonstrated the highest level of protein adsorption. After the modification, the hydrophilic hydroxyl groups on the β-CD ring endowed the PES-CD with better antifouling capability compared with the pristine PES membrane. However, the protein adsorption amount for PESPSA and PES-PMT did not show significant difference compared with the PES membrane. PEG has been approved by the FDA to be one kind of functional molecule providing nonfouling capability. In this study, we also demonstrated that the PEG brushes significantly reduced the protein adsorption amount as shown for the PES-PEG, which was probably due to its low interfacial free energy with water, steric stabilization effects, and high surface mobility. Platelet Adhesion and Activation. For blood contacting biomaterials, platelet adhesion is a key event during the thrombus formation.48 In this study, platelet adhesion on the pristine PES membrane surface and postfunctionalized surfaces were evaluated and quantified by SEM images (Figure 6). For the surfaces of PES and PES-PMT, a large number of platelets aggregated and accumulated on the surfaces, especially for the PES-PMT, for which had been previously demonstrated that the quaternary ammonium enriched surface showed adverse effects on the platelet adhesion. Meanwhile, the platelet adhesion on the PES-CD, PES-PSA, and PES-PEG had been successfully suppressed compared with the pristine PES membrane. Particularly, the PES-PEG surface demonstrated the lowest level of platelet adhesion, which should be due to the antifouling property of the PEG brushes. Combined with the protein adsorption results, it can be confirmed that the Ad-PEG has been successfully assembled onto the PES-CD surface, and antifouling brushes have formed on the membrane interface. Moreover, besides the platelet adhesion amount, the platelet morphology can also be used as an index for evaluating the thrombotic potential. It has been reported that the change of platelet shapes determines the rate at which platelet aggregation progress and therefore affects the subsequently procoagulant events occurring at the blood−material interface.49 From the SEM images in Figure 6, it could be observed that the platelets on the PES membrane presented flattened and irregular shapes with abundant amount of pseudopodia. Compared with the PES membrane surface, the aggregation of platelet on the surface of PES-PMT was adversely accelerated, and the platelet presented dendritic shape with extruding pseudopodia, while the platelets on the PES-CD, PES-PSA, and PES-PEG kept round shape with very few pseudopodia, thus indicating less platelets activation, especially for the PES-PEG. Bactericidal Activity of the Membranes. Quaternary ammonium-based polymers are recognized as antibacterial

it could be observed, the pristine PES and PES-GMA membranes were hydrophobic with the water contact angle (WCA) of about 67° (Figure S2). After anchoring the β-CD molecules, the surface wettability was found to be prominently improved (36 ± 1°), which should be due to the multiple hydroxyl groups presenting on the fringe of the β-CD. It was worth noting that when the Ad-polymers were immobilized on the PES-CD surface, the WCAs were adversely increased by the assembly of Ad-PSA and Ad-PMT. The WCA for the PMT was slightly higher than that for the PSA, which was identical with our previous report.14 For the permeability measurement, as shown in Figure S3, the flux of PBS solution for pristine PES membrane was very low. However, with the introduction of the GMA and β-CD molecules, the membrane became more porous and hydrophilic, thus resulting in a higher PBS flux. Moreover, it could be observed that the efficient and rapid postfunctionalization of the membrane with the Ad-polymers did not cause an adverse effect on the permeability of membrane. Clotting Time (APTT, TT, and PT). APTT and PT have been widely used to evaluate coagulation abnormalities in the intrinsic and extrinsic pathway, respectively, which can also be utilized to detect the functional deficiencies in factor II, III, V, VIII, X, and fibrinogen,44,45 while TT is a commonly adopted method to evaluate the anticoagulation ability of blood contact materials, which is mainly affected by the content and coagulation activity of fibrinogen in plasma. The TT value reflects the activity of fibrinolytic system in the common pathway of coagulation cascade.46 Figure 5A shows the clotting

Figure 5. (A) APTT, PT, and TT assays for PES, PES-CD, PES-PSA, PES-PEG, and PES-PMT; values were expressed as means ± SD, n = 3. (B) BSA and FBG adsorption amounts onto the membranes; values were expressed as means ± SD, n = 3.

times for the pristine membrane and the membranes at different modification stages. It could be observed that with anchoring of β-CD and subsequent assembly of Ad-PEG and Ad-PMT no conspicuous variations of APTT, PT, and TT were observed. However, after the assembly of Ad-PSA, the APTT, PT, and TT increased dramatically to 162, 28, and 46 s, 9671

DOI: 10.1021/acs.langmuir.5b02038 Langmuir 2015, 31, 9665−9674

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Figure 6. SEM images of platelets adhered on the PES (A), PES-CD (B), PES-PSA (C), PES-PEG (D), and PES-PMT (E); the scale bars for SEM images are 1 μm. Average numbers of the adhered platelets onto the membranes from PRP calculated from six SEM images (F).

Figure 7. Typical fluorescence microscopy images of the attached S. aureus (PES (A, a), PES-CD (B, b), PES-PSA (C, c), PES-PEG (D, d), and PESPMT (E, e); green staining indicates live bacteria, while red staining indicates dead bacteria); all the scale bars are 5 μm. (F) Calculated numbers of bacteria attachment on the PES, PES-CD, PES-PSA, PES-PEG, and PES-PMT surfaces.

E). Moreover, the average adhered numbers of the live and dead cells were calculated from fluorescent microscopy images (Figure 7F). It could be observed that a large number of live bacteria were adhered onto the pristine PES membrane. With the anchoring of β-CD, the number of the adhered bacteria was obviously decreased, which was consistent with the tendency of the protein adsorption and platelet adhesion evaluations. For

polymers that have been widely used for the construction of antibacterial materials.36 In this study, S. aureus was selected as a model bacterium to investigate the antibacterial activity of the postfunctionalized membrane surfaces. On the membrane surfaces, dead and live bacteria were stained with red fluorescent nucleic acid stain propidium iodide and green fluorescent nucleic acid stain SYTO9, respectively (Figure 7A− 9672

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the membrane of PES-PEG, there was nearly no bacterium adhered on the membrane surface, which confirmed the excellent antifouling property of the PES-PEG surface. As for the PES-PMT, although there were a few bacteria adhered on the membrane surface, no live bacterium was observed, which confirmed the high bactericidal efficiency for the quaternary ammonium polymer immobilized surface. Thus, in this study, the antifouling and bactericidal membranes had been achieved via the postassembly of Ad-PEG and Ad-PMT onto the host PES-CD surfaces.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b02038. Figures S1−S3 and Table S1 (PDF)

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REFERENCES

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4. CONCLUSIONS In this study, to achieve the postfunctionalization of membrane substrates, by taking the advantages of β-CD and Ad host− guest interaction and in situ cross-linking copolymerization, we demonstrated a new strategy for the fabrication of β-CD modified host membrane that could recognize a series of welldesigned and biofunctional guest macromolecules. The versatile and rapid surface functionalization of the host PES membranes were performed via the supramolecular assembly between βCD and Ad groups in a mild and environmentally friendly medium. Surface ATR-FTIR spectra, XPS data, and AFM images confirmed the successful anchoring of the β-CD on PES-GMA and the successful postassembly of Ad-polymers on the PES-CD surface. Typically, surface wettability, clotting times, protein adsorption, platelet adhesion, and antibacterial activity were performed to evaluate and examine the anticoagulant, antifouling, and antibacterial characteristics of the postfunctionalized membranes. The results indicated that the host−guest self-assembly of functional Ad-polymers with the PES-CD membrane could endow the inert PES membrane with improved blood compatibility (by PSA), antifouling capability (by PEG), or promoted bactericidal efficiency (by PMT). This approach of versatile and rapid postfunctionalization is also promising for the preparation of many other multifunctional polymeric membranes to meet with diverse biomedical demands for both industrial and clinical applications.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] or [email protected] (C.C.). *E-mail: [email protected] or [email protected] (C.Z.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially sponsored by the National Natural Science Foundation of China (Nos. 51225303 and 51433007) and the Sichuan Province Youngth Science and Technology Innovation Team (No. 2015TD0001). We 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 SEM observation. 9673

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