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Nov 10, 2015 - In this work, we designed a robust and heparin-mimetic hydrogel thin film coating via combined layer-by-layer (LbL) self-assembly and ...
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Substrate-Independent Robust and Heparin-Mimetic Hydrogel Thin Film Coating via Combined LbL Self-Assembly and Mussel-Inspired Post-Cross-linking Lang Ma,† Chong Cheng,*,† Chao He,† Chuanxiong Nie,† Jie Deng,† Shudong Sun,† and Changsheng Zhao*,†,‡ †

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

ABSTRACT: In this work, we designed a robust and heparin-mimetic hydrogel thin film coating via combined layer-by-layer (LbL) self-assembly and mussel-inspired post-cross-linking. Dopamine-grafted heparin-like/-mimetic polymers (DA-g-HepLP) with abundant carboxylic and sulfonic groups were synthesized by the conjugation of adhesive molecule, DA, which exhibited substrate-independent adhesive affinity to various solid surfaces because of the formation of irreversible covalent bonds. The hydrogel thin film coated substrates were prepared by a three-step reaction: First, the substrates were coated with DA-g-HepLP to generate negatively charged surfaces. Then, multilayers were obtained via LbL coating of chitosan and the DA-g-HepLP. Finally, the noncovalent multilayers were oxidatively cross-linked by NaIO4. Surface ATR-FTIR and XPS spectra confirmed the successful fabrication of the hydrogel thin film coatings onto membrane substrates; SEM images revealed that the substrateindependent coatings owned 3D porous morphology. The soaking tests in highly alkaline, acid, and concentrated salt solutions indicated that the cross-linked hydrogel thin film coatings owned high chemical resistance. In comparison, the soaking tests in physiological solution indicated that the cross-linked hydrogel coatings owned excellent long-term stability. The live/dead cell staining and morphology observations of the adhered cells revealed that the heparin-mimetic hydrogel thin film coated substrates had low cell toxicity and high promotion ability for cell proliferation. Furthermore, systematic in vitro investigations of protein adsorption, platelet adhesion, blood clotting, and blood-related complement activation confirmed that the hydrogel film coated substrates showed excellent hemocompatibility. Both the results of inhibition zone and bactericidal activity indicated that the gentamycin sulfate loaded hydrogel thin films had significant inhibition capability toward both Escherichia coli and Staphylococcus aureus bacteria. Combined the above advantages, it is believed that the designed heparin-mimetic hydrogel thin films may show high potential for applications in various biological and clinical fields, such as long-term hemocompatible and drug-loading materials for implants. KEYWORDS: heparin-mimetic hydrogel film, mussel-inspired chemistry, LbL self-assembly, hemocompatible and antimicrobial coating

1. INTRODUCTION Surface modification of biomaterials has become increasingly important for developing advanced artificial biomedical devices that contact with blood and tissue.1−3 When implants contact human cells, blood, and tissues, a series of activations and responses may occur at the biointerfaces, such as cytotoxicity and carcinogenicity,4,5 thrombus generation, and blood component activation,6 bacterial infection, and immunologic rejection.7 These potential undesirable responses could endow harmful, even lethal, side effects for patients. Recent studies indicate that improving the biocompatibility and designing favorable properties to biointerfaces may greatly reduce the potential side effects of biomaterials. Until now, considerably effective methods, such as bulk blending,6 irradiation or plasma treatment,8 self-assembled monolayer,9 surface grafting or coating of functional (bio)polymers,10,11 and Langmuir− Blodgett deposition,12 have been developed for the surface © XXXX American Chemical Society

modification of artificial biomaterials, but none of these methods are ideal and function as the universal protocol for the modification of a broad range of biointerfaces. Recent advances in macrohydrogels and nanotechnology have led to increased interest in designing hydrogel thin films as functional coatings to modify the surface properties of substrates.13,14 Hydrogel thin films have shown great advantages both on biofunctionality and structures. Hydrogel thin films that consist of cross-linked and water-swollen hydrophilic networks are potentially functional materials for biomedical and biotechnology applications because of the biophysical similarity to living tissues and extracellular matrix,15−17 including drug and gene delivery,18−21 tissue Received: July 5, 2015 Accepted: November 10, 2015

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ACS Applied Materials & Interfaces engineering, regeneration medicine,22−27 and implantable devices.28,29 Furthermore, the 3D hydrophilic and cross-linked network structure is much more stable at the biointerfaces compared with that of deposited multilayers because the polymer network in the hydrogel thin films is linked to the surface by multiple anchoring sites or multivalent bonds. The thicker and freestanding hydrogel thin films can be easily fabricated by spin-casting method, whereas the surface-coated ultrathin hydrogel films are usually constructed by layer-bylayer (LbL) assembly that gives the possibility of controlling and tailoring the coating thickness, interfacial properties, and versatile functions on virtually any substrate.30,31 However, most of the recent achievements are focused on polyelectrolyte multilayer films (PEM) driven by hydrogen bonding, charge transfer, electrostatic interactions, and metal−ligand interactions,32 which limits the stability of the films because they are not covalently linked and can be influenced by the factors such as the pH value, solvent, and electrolytic solution. Most recently, fabrication of covalently linked hydrogel thin films by post-cross-linking of PEM garners particular interest because of its facile chemical process and stable physical networks.32 In 2006, Caruso was the first to fabricate LbL cross-linked thin films using a CuAAC “click” chemistry method.33 Likewise, to reduce the usage of hazardous catalysts and achieve more facile chemical process, many appropriate methods have been explored, such as the thiol−ene “click” reactions,34 dynamic covalent bonds,32 host−guest interaction,32,35 and musselinspired chemistry.36 Earlier literatures indicate that the mussel-inspired chemistry may become one of the most remarkable post-cross-linking methods to construct hydrogel thin films from LbL assembled PEM. 37,38 Dopamine and 3,4-dihydroxyphenyl-L-alanine (DOPA) as well as their derivatives, which mimic the compositions of mussel foot proteins (mfps), can construct surface-adherent films onto almost any material surfaces.39 In nature, with mfp-rich byssus as the holdfast, mussels can adhere to solid surfaces because the mfps contain abundant DOPA and lysine.40 When adhering to substrates, the DOPA-containing mfps can not only form vigorous covalent or noncovalent interactions41 but also covalently cross-link themselves or the amine groups in lysines, which causes the adhesion and solidification of the byssus.42 Inspired by the adhesive capability of mussel byssus, investigators have proved that DA-grafted (bio)polymers can mimic the mussel byssus and display stable adhesive capability to solid surfaces. Kim et al. have demonstrated that chitosan−catechol conjugates can develop self-assembled hydrogels with excellent biocompatibility.43 Xu et al. have verified that DA-containing copolymers can bind vigorously to Ti surface via coordination interaction of the catechol moieties for molecular recognitions.44 We have synthesized DA-grafted heparin for the surface modification of microscale (graphene oxide) and macroscale (membrane sheets) substrates, and the modified materials exhibit greatly improved hemocompatibility and ultralow cell toxicity.10,45 In this work, inspired by the excellent adhesive properties of mfps in mussels, we developed chemically stable polyelectrolyte films via combined LbL self-assembly and mussel-inspired postcross-linking. To construct the PEM films, heparin-like/mimetic polymers (HepLP), e.g., sulfonated components contained polymers, are used as the model polyanion because they revealed excellent blood and cell compatibility after coating on substrates such as that of heparin-immobilized surfaces.46,47 For the polycation layer, chitosan (CS) has been

chosen as the model polymer because it can mimic lysine to provide an abundance of amino groups for the covalent crosslinking with DA. Subsequently, DA-grafted HepLP (DA-gHepLP) was synthesized by conjugating DA with the carboxylic segments of HepLP via carbodiimide coupling protocols. After conjugating with DA, DA-g-HepLP was endowed with substrate-independent adhesive affinity to various solid surfaces because of the formation of irreversible covalent bonds. To construct hydrogel thin film coatings, the substrates were first coated with DA-g-HepLP in basic solution to generate a negatively charged surface; then, a facile LbL self-assembly method was undertaken by alternative deposition of CS and DA-g-HepLP. After the thin film noncovalent multilayers were constructed, the substrates were immersed in NaIO4 aqueous solution for oxidative cross-linking to generate covalently crosslinked hydrogel thin films with improved chemical stability. Subsequently, to confirm the structure, biocompatibility, and biofunctionality of the fabricated hydrogel thin film coatings, the surface chemical composition and morphology, long-term stability, water contact angle, protein adsorption, cell viability, thrombotic potential, and drug/protein loading abilities for the heparin-mimetic hydrogel thin film coatings were systematically investigated.

2. EXPERIMENTAL SECTION 2.1. Mussel-Inspired Coating for Substrate-Independent Surface Modification. The materials used in this work are shown in the Supporting Information. HepLP (poly(sodium 4-vinylbenzenesulfonate-co-sodium methacrylate), PSSNa-co-PMAANa) with abundant carboxylic and sulfonic groups, was synthesized by atom-transfer radical polymerization. Likewise, the DA-g-HepLP was synthesized using carbodiimide chemistry by grafting DA with the backbone of MAA segments. The detailed synthesis methods of HepLP and DA-gHepLP were described in the Supporting Information. For the substrate-independent surface modification, 200 mg of DA-g-HepLP was dissolved in 20 mL of PBS buffer (10 mM, pH 8.5); then glass, polystyrene sheets, Al sheets, and homemade poly(ether sulfone) (PES) membranes were immersed in the above solution and continuously stirred at 30 °C for 48 h. Then, the produced DAHepLP/Glass, DA-HepLP/PS sheets, DA-HepLP/Al sheets, and DAHepLP/PES membranes were taken out from the solution, followed by continuously rinsing with deionized (DI) water for 5 min to ensure that the free ions and polymers were eliminated absolutely. 2.2. Preparation of LbL Assembly Hydrogel Thin Films. The concentrations of DA-g-HepLP aqueous solution and CS-acetic acid (1 wt %) solution were both 5.0 mg/mL. The pH values of the CS and DA-g-HepLP solutions were adjusted to 7.5 and 3.5 by 0.5 M NaOH and 1 M HCl aqueous solutions, respectively. The majority of the carboxylic and sulfonic groups in DA-g-HepLP and the amine groups in CS are nearly neutralized under these pH values. The slightly charged polyelectrolytes can easily diffuse to the substrate surface, which causes rapid growth during LbL assembly.48,49 As shown in Scheme 1, the DA-HepLP-coated substrates were then immersed in CS and DA-g-HepLP solutions (15 min for each solution) alternately, and the washing process was undertaken after each coating process with three rinses in DI water (1 min) to eliminate the excess polyelectrolytes. To make sure that the coated multilayer can maintain its original state, no drying step was performed after each coating or washing procedure; finally, the multilayer-coated substrates were freeze-dried after the assembly. Afterward, the film-coated substrates were cross-linked by oxidizating in a 1 mM NaIO4 aqueous solution for 12 h. Then, the cross-linked hydrogel thin films were rinsed completely with DI water and freeze-dried again. In this study, the PES membrane was used as the model substrate for the chemical and biological evaluation of the hydrogel thin film coatings. 2.3. Characterization of Hydrogel Thin Film Coated Substrates. The composition and morphology of the hydrogel thin B

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and thrombin time (TT) were measured by a semiautomated blood coagulation analyzer CA-50 (Sysmex Corporation, Kobe, Japan). The detailed processes are described in the Supporting Information. 2.6.4. Complement Activation in Human Blood System. The complement activation (C3a and C5a) evaluation for the membranes was measured with an enzyme-linked immunosorbent assay (ELISA) method (BD, Becton, Dickinson and Company, USA). The detailed test processes were according to the instructions provided by the manufacturer and are described in the Supporting Information. 2.7. Drug Loading Test and Bactericidal Activity. To confirm the versatile ability of the hydrogel thin films, doxorubicin (DOX) and gentamycin sulfate (GS) were chosen as the model drugs. The experiments were carried out by immersing 10 pieces of the pristine and hydrogel film coated PES membranes into 1 mg/mL DOX and 4 mg/mL GS aqueous solutions to allow the hydrogel thin films to load with DOX and GS, respectively. After slightly stirring for 48 h at room temperature, the drug loading could reach equilibrium, and the loading amounts and releasing amounts could be estimated by using the absorbance of the drug solutions (at 480 nm for DOX and 254 nm for GS) with an UV−vis spectrometer (UV-1750, Shimadzu Co., Ltd., Japan).21,22 Finally, the DOX and GS loaded samples were rinsed slightly with PBS solution for 2 min to wash out the unstable DOX and GS molecules. The antibacterial ability of the released GS was used to examine the efficiency of the drug-loaded hydrogel thin film coatings by the measurement of the inhibition zone and bactericidal efficiency.50 The detailed processes are described in the Supporting Information.

Scheme 1. Schematic Pictures for the Construction of Heparin-Mimetic Hydrogel Thin Film by Combining LbL Interfacial Assembly and Mussel-Inspired Oxidative CrossLinking

film coatings were characterized by X-ray photoelectron spectroscopy (XPS, XSAM800, Kratos Analytical, UK) and field-emission scanning electron microscopy (FE-SEM, JSM-7500F, JEOL, Japan), respectively. The hydrophilicity and zeta potentials of the hydrogel thin film coating surface were characterized by a contact angle goniometer (DSA100, KRUSS GmbH, Germany) and a Beckman Coulter, Delsa NanoC Particle Analyzer, respectively. The detailed procedures are shown in the Supporting Information. 2.4. Stability of LbL-Assembled Hydrogel Thin Films. To investigate the stability of the hydrogel thin films, the hydrogel film coated substrates were immersed in 0.1 M NaOH, 0.1 M HCl, or 5 M NaCl aqueous solution for 2 h; then, the samples were freeze-dried and observed by SEM. To explore the influences of the DA grafting ratio on the coating stability of the hydrogel thin films, DA-g-HepLP5% and DA-g-HepLP-15% were also applied for the construction of surface hydrogel thin films and then tested using the same protocol as that of DA-g-HepLP-30%. Note that in the following study the uncross-linked films/cross-linked films were prepared using DA-gHepLP-30% unless otherwise stated. Furthermore, to test the long-term stability of the coated hydrogel thin films, the substrates were immersed in PBS for varied periods (from 0 to 32 days), then washed with DI water and freeze-dried. The changes of the compositions and morphologies of the substrate surfaces were characterized by using XPS spectra and SEM images, respectively. 2.5. Cell Proliferation Activity. Human osteosarcoma cell line (MG-63) was chosen to investigate the cell compatibility of the hydrogel thin film coatings. The details of MG-63 cell culture and cell growth and morphology are shown in the Supporting Information, including the MTT assay for the viability of MG-63 cells, live/dead cell staining observed by a fluorescence microscope (Olympus IX53, Japan), and the cell morphology observed by a confocal laser scanning microscopy (CLSM, Leica TCS SP8, Germany). 2.6. Blood Compatibility. 2.6.1. Protein Adsorption. Protein adsorption tests were measured with BSA and BFG solutions under static condition by using the Micro BCA Protein Assay Reagent Kit (PIERCE). The detailed processes are described in the Supporting Information. 2.6.2. Platelet Adhesion. Platelet adhesion was studied by immersing the membranes in the platelet-rich plasma (PRP) and then drying by alcohol−PBS solutions and isoamyl acetate−alcohol solutions. The adhered platelets on the membranes were observed using SEM. The detailed procedures are shown in the Supporting Information. 2.6.3. Clotting Time. To investigate the anticoagulant properties for the DA-g-HepLP, HepLP, heparin, and the mussel-inspired LbL assembly membranes, activated partial thromboplastin time (APTT)

3. RESULTS AND DISCUSSION 3.1. Compositions and Morphologies of Hydrogel Thin Films. In this work, HepLP (PSSNa-co-PMAANa) was synthesized by atom-transfer radical polymerization, and DA-gHepLP was synthesized using carbodiimide coupling protocols, as shown in Figure S1A. Figure S1B shows the 1H NMR spectra for HepLP and DA-g-HepLP. The calculated substitutions of DA were approximately 30, 15, and 5% from the 1H NMR spectra. After conjugating with DA, DA-g-HepLP is endowed with strong affinity to various solid surfaces because of the formation of irreversible covalent bonds.38 DA-g-HepLP is not stable under basic conditions, and the DA groups can be easily oxidized by oxygen in alkaline aqueous solution to generate oquinone and cross-linking structures, thus adhering to solid substrates.39,41 In our work, PES membranes were used as the model substrates for the chemical and biological evaluation of the hydrogel thin film coatings, which were first coated with DA-g-HepLP to generate a negatively charged surface. Then, a facile LbL assembly method was undertaken by alternately soaking the substrates in the solutions of CS (polycation) and DA-g-HepLP (polyanion). After the thin film noncovalent multilayers were constructed, the membranes were immersed in NaIO4 aqueous solution for oxidative cross-linking to generate covalently cross-linked hydrogel thin films with improved chemical stability. The LbL assembly procedure was monitored by measuring the UV−vis absorption spectra after the deposition of each coating layer. As shown in Figure 1A, the even numbers of bilayers (0, 2, 4, 6, ...) that assembled by DA-g-HepLP and CS were formed on a quartz slide. Figure S2 shows the UV−vis absorption spectra for 5 mg/mL DA-g-HepLP, HepLP, and CS aqueous solutions. As shown in Figure 1B, with increasing numbers of bilayers, the approximatively linear increase in the absorbance at 226 nm indicated a gradual assembly process with nearly an equal amount of the polyelectrolytes formed in each cycle. The results indicated that the self-assembly C

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procedures were executed in a purposefully and orderly manner. To investigate the influence of the DA grafting ratio on the process of the hydrogel thin film coating, the UV−vis absorption spectra of the deposition of DA-g-HepLP-5% and CS were also measured and shown in Figure S3. The coating with 5% DA grafting ratio showed curves and tendency similar to that of the 30% DA grafting ratio. The results indicated that the DA grafting ratio had limited influence on the LbL assembly process. The surface chemical compositions of the hydrogel thin film coated substrates were analyzed by XPS. Figure 2A,B shows the XPS wide spectra for PES and DA-HepLP/CS-20/PES membrane surfaces, respectively. After coating the hydrogel thin film, an intensive characteristic peak of N element emerges, which indicates the existence of CS. Figure 2a,b shows the XPS C 1s spectra for PES and DA-HepLP/CS-20/PES membrane surfaces, respectively. Obviously, two unique carbon moieties are shown in the PES C 1s spectrum in Figure 2a: The peak at 284.6 eV is for the carbon skeleton (C−C/CC); the peaks at 286.2 and 285.1 eV are for C−O and C−S bonds of PES, respectively.51 In Figure 2b, the peak of C−S bond at about 285.1 eV is ascribed to the C−N bond (from amide bond). The peak intensity at 285.1 eV increases significantly owing to the overlap of the C−S bond and C−N bond compared to PES; in comparison, the peak intensity at 286.3 eV of C−O bond also increases dramatically. Furthermore, the new peak at 287.3 eV (CO) can be assigned to the carbonyl of the MAA segments of DA-g-HepLP. The ATR-FTIR spectra, as shown in Figure S4, also indicated that all the characteristic peaks are observed. Combined with the results of ATR-FTIR, it can be confirmed that the hydrogel thin films have been successfully constructed on the membrane surfaces.

Figure 1. (A) UV−vis absorption spectra for multilayer films assembled on quartz with increasing bilayer numbers by DA-gHepLP-30%. (B) Absorbance as a function of bilayer number monitored at 226 nm.

Figure 2. XPS wide and C 1s spectra, respectively, for the (A and a) PES and (B and b) DA-HepLP/CS-20/PES membrane surfaces. D

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Figure 3. (A) Surface zeta potential for each LbL-coated layer with alternating DA-HepLP and CS. The first measurement (layer 0) is the surface zeta potential of DA-HepLP/PES membrane. Typical SEM images of (B) the surface morphologies for the pristine PES membrane and (C) the hydrogel thin films, including DA-HepLP/CS-5/PES, DA-HepLP/CS-10/PES, and DA-HepLP/CS-20/PES membranes. The surface morphologies of hydrogel thin film coated glass, PS sheet, and Al sheet are shown in Figure S9.

Figure 4. SEM images of the un-cross-linked (top row) and cross-linked (bottom row) DA-HepLP/CS-20/PES membranes after immersing in (A and D) 0.1 M NaOH, (B and E) 0.1 M HCl, and (C and F) 5 M NaCl solutions, respectively. The immersion times for the un-cross-linked and cross-linked films are both 2 h.

The LbL assembly process was also monitored by measuring the surface zeta potential after the deposition of each coating layer. As shown in Figure 3A, the zeta potential of pristine PES membrane was about −5.5 mV. After coating with DA-gHepLP, the zeta potential was about −17.9 mV for the DAHepLP/PES membrane. After that, the surface zeta potential fluctuated between −30 and 35 mV with the alternative depositions of CS and DA-g-HepLP. Combined with the results of UV−vis absorption spectra, the reproducible and periodical change demonstrated the multilayer growth of the charged

polyelectrolytes on the substrate surfaces. For the coating layer detection, as shown in Figure 3B,C, the SEM observations showed the typical morphologies for the pristine PES and hydrogel thin film coated membrane surfaces. The “20” in “DAHepLP/CS-20/PES” means that the hydrogel thin films contained 20 bilayers of DA-g-HepLP and CS (likewise 5 and 10 layers for DA-HepLP/CS-5/PES and DA-HepLP/CS-10/ PES, respectively), and all the outermost layers were the DA-gHepLP. It was noted that the pristine PES substrate presented even surface morphology; however, the hydrogel thin film E

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Figure 5. (A and B) XPS wide scans and N 1s high-resolution spectra for the hydrogel thin films on PES membrane surfaces after immersing in PBS solution for 32 days. (C and D) Relative amounts and SEM images of the hydrogel thin films retained on the PES membrane surfaces after soaking in PBS solution for various periods (represented by N atom %, with 0 day set as 100%). Cross-linked film-5%-32D and 15%-32D represent the hydrogel films forming by DA-g-HepLP-5% and -15%, respectively. Note that when there is no specific illustration the (un-)cross-linked films means that DA-g-HepLP-30% is used for the surface coating.

damage was observed for the hydrogel films after the handling with the etching solutions for 2 h (Figure 4D−F). Furthermore, the mass residual was calculated, and the data are shown in Figure S6. These results indicated that the structure stability of the cross-linked multilayers has been improved after oxidative cross-linking of DA even in harsh environments. Therefore, it was expected that the DA-HepLP/CS hydrogel thin film coated substrates can withstand the cyclic cleaning process after each usage. Earlier reports had revealed that compared to physical and electrostatic adsorption the covalently immobilized multilayer films could have improved stability and persistence and avoid the burst release of the immobilized biomolecules.52 Herein, XPS spectra are applied to examine the long-term stability and degradation of the coated hydrogel thin films by detecting the remained amount of N atoms. Figure 5A,B shows the XPS wide spectra and N 1s spectra (insert images) for pristine crosslinked film-0D (DA-HepLP/CS-20/PES, 0 day), cross-linked film-32D, un-cross-linked film-32D, cross-linked film-5%-32D (DA-HepLP-5%/CS-20/PES, 32 days), and cross-linked film15%-32D (DA-HepLP-15%/CS-20/PES, 32 days) surfaces. The characteristic peaks of nitrogen (binding energy: 398.8 eV (N 1s)), carbon (binding energy: 284.9 eV (C 1s)), oxygen (binding energy: 531.9 eV (O 1s)), and sulfur (binding energy:

coated substrates exhibit 3D porous surface structures, especially DA-HepLP/CS-20/PES. Furthermore, with increasing the coating bilayers from 5 to 20, the 3D surface structures became more and more dense and homogeneous. The same results of the DA-HepLP-5%/CS-20/PES and DA-HepLP15%/CS-20/PES are shown in Figure S5. For the surface morphologies of other hydrogel thin film coated substrates (DA-HepLP/CS-10/Glass, DA-HepLP/CS-10/PS, and DAHepLP/CS-10/Al), as shown in Figure S9, similar dense and homogeneous 3D porous structures were observed with some minor differences on different substrates. 3.2. Stability of Hydrogel Thin Films. The structure stability of the coated hydrogel thin films is of critical importance for the safe and long-term performance. As shown in Figures 4 and 5, the stability of the coatings was evaluated by soaking the samples in highly alkaline, acid, and concentrated salt solutions, and the long-term stability was studied by soaking in PBS solution. The substrates assembled with un-cross-linked and cross-linked DA-HepLP/CS multilayers were immersed in 0.1 M NaOH, 0.1 M HCl, and 5 M NaCl aqueous solutions. The coated un-cross-linked multilayers were obviously destroyed after soaking in etching solutions for 2 h (Figure 4A−C). However, the cross-linked hydrogel thin films maintained fine coating morphology, and no detectable F

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Figure 6. (a) Representative pictures of water contact angles (taken at 10 s) from independent experiments. (b) Static WCAs of pristine PES, DAHepLP/CS-5/PES, DA-HepLP/CS-10/PES, and DA-HepLP/CS-20/PES membranes. (c) Measurements of water contact angles with increasing contacting time. (d) Protein-adsorbed amounts for the pristine PES, DA-HepLP/CS-5/PES, DA-HepLP/CS-10/PES, and DA-HepLP/CS-20/PES membranes.

However, separate static contact angles were not sufficient to evaluate the wettability. To better monitor the wettability of the porous hydrogel films, the changes in water contact angle with increasing the contacting time were also investigated. As shown in Figure 6c, the hydrogel thin films exhibited significant decrease of WCA under the drop age from 0 to 10 s. 3.4. Protein Adsorption. The protein adsorbed amount on a material surface is reckoned to be one of the important factors in the evaluation of biocompatibility of biomaterials.53 In the present work,54−56 protein adsorption was investigated by measuring the BSA and BFG adsorbed amounts in vitro, and the results are shown in Figure 6d. We note that the hydrogel thin films with 5 bilayers showed significantly decreased protein adsorption for both BSA and BFG, whereas when the coated bilayers further increased, the protein adsorption would further increase. It is well-known that a material with a more hydrophilic surface generally shows a lower amount of protein adsorption;57 in comparison, it is found that the 3D porous structure of hydrogel film also plays great influence on the protein adsorption. Furthermore, the cationic polyelectrolyte CS that existed in the hydrogel thin films would also contribute to the protein adsorption. Thus, as the bilayers increased, a slightly increase of protein adsorption was observed. In a conclusion, the decreased plasma protein adsorption might be to the benefit of the improvement in blood compatibility; also, the slightly increased protein adsorption of DA-HepLP/CS-20/ PES may benefit to cell adhesion. 3.5. Cell Viability and Morphology. The cell viability was determined by using the MTT colorimetric assay at different culture time intervals (2, 4, and 6 days). As shown in Figure S7,

164.1 eV (S 2p)) were observed and used for the atom ratio calculation. The intensity of the nitrogen for the un-crosslinked film-32D obviously decreased compared to those for the pristine cross-linked film-0D. Simultaneously, there was only a little difference between the intensity of the nitrogen for the cross-linked film-32D and that for the pristine cross-linked film0D, likewise for those of the cross-linked film-5%-32D and cross-linked film-15%-32D. After soaking in PBS solution for various periods of time (presented by N atom %, rated at 0 day as 100%), the detailed changes of the N concentrations for the substrates were measured and calculated by XPS spectra, and the data are shown in Figure 5C,D. After continuous soaking in PBS, the un-cross-linked multilayer showed a gradually decreased N content and finally retained less than 50% of the total N content, whereas the cross-linked hydrogel thin films could retain more than 80% of the total N content, which indicated that the cross-linked DA-HepLP/CS coating films owned more excellent stability and showed highly potential as long-term coating materials for various biomedical devices. 3.3. Water Contact Angle Analysis. Water contact angle (WCA) measurement was utilized to estimate the wettability of the as-prepared membranes. As shown in Figure 6a,b, the contact angle of the pristine PES membrane was 71.1°. However, the hydrogel thin film surfaces possessed much lower WCAs, ranging from 25 to 1.4°, and the modified surfaces became more hydrophilic with increasing bilayers. It was interesting to find that the DA-HepLP/CS-10/PES and DAHepLP/CS-20/PES membranes presented high wettability which could be ascribed to the hydrophilic nature of DAHepLP/CS and the homogeneous 3D porous hydrogel films. G

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Figure 7. (a) Fluorescence images of the MG-63 cells obtained on the pristine PES, DA-HepLP/CS-5/PES, DA-HepLP/CS-10/PES, and DAHepLP/CS-20/PES substrates by live/dead staining. (b) CLSM images of MG-63 cells growing on the pristine PES, DA-HepLP/CS-5/PES, DAHepLP/CS-10/PES, and DA-HepLP/CS-20/PES substrates. MG-63 cells were incubated on each type of substrate for 5d, then F-actin was stained with Rhodamine-Phalloidin (red), and the cell nucleus was counterstained with DAPI (blue).

it was observed that there were no obvious cytotoxicity for the hydrogel thin film coated substrates on the 2, 4, and 6 days because all the samples exhibited absorbance similar to or slightly lower than that of the control sample. The cell morphologies of the MG-63 cells cultured for 5 days on the substrate surfaces were observed and shown in Figure 7. As shown in Figure 7a, the MG-63 cells were first evaluated by using the live/dead staining with FDA (green) and PI (red), respectively. Fluorescence images showed that most of the cells were alive on the pristine and hydrogel thin film coated substrates; almost no dead cells can be detected. To better observe the cell morphologies cultured on the hydrogel thin films, CLSM observation was carried out with a double staining of cytoplasm and nuclei. The MG-63 cells cultured on pristine PES membrane exhibited no significant intercellular network of cytoplasm. For the heparin-mimetic hydrogel thin films, the MG-63 cells spread with ruffling of peripheral cytoplasm; in comparison, distinct regional aggregations were also observed. The well-organized intercellular F-actin structures and cell aggregations were intensely advocated as a highly useful culture mode for MG-63 cell proliferation and differentiation over a long period.58,59 The results confirmed that the heparinmimetic hydrogel thin films could promote cell adhesion and growth. 3.6. Blood Compatibility. 3.6.1. Platelet Adhesion. Platelet adhesion on the interfaces of blood-contacting biomaterials is a key procedure in thrombus formation.60 The adhered and activated platelets may accelerate thrombosis and then lead to further coagulation; thus, in this work, the platelet morphologies and the adhered platelet number were explored to evaluate the antithrombotic ability of the heparin-mimetic hydrogel thin films. Figure 8 shows the SEM images of the platelets adhering on pristine and coated substrates. It could be observed that there were numerous platelets aggregated and accumulated on the pristine PES membrane surface. These platelets spread in flattened and irregular shapes, and a lot of pseudopodia were observed, which revealed that activated platelets might be generated on the PES membrane surface. However, for the heparin-mimetic hydrogel thin film coated substrates, the adhered platelets were seldom observed, and the platelets showed nearly round shape and no pseudopodia or deformation. From the SEM images and the calculated platelet adhering numbers (Figure 8F), it could be concluded that platelet adhesion was obviously suppressed, and vast numbers of platelets maintained their discoid shape on the hydrogel thin films, indicating that no activation happened. As the bilayers

Figure 8. SEM images of platelets adhering to the (A and a) PES, (B and b) DA-HepLP/CS-5/PES, (C and c) DA-HepLP/CS-10/PES, and (D and d) DA-HepLP/CS-20/PES substrates. (E) Illustrated scheme for the activated platelets for the PES membrane and the antiplatelet adhesion of the hydrogel thin film coated membranes. (F) Average platelet adhering number estimated by four SEM images.

increased, the adhering platelet number decreased slightly. The reduced platelet adhesion could be ascribed to the enhanced surface negative charges and hydrophilicity owing to the introduction of the heparin-mimetic multilayers. Considered the results of the morphologies and the numbers of adhered platelets, we might conclude that the platelet activation did not occur or had been greatly suppressed on the heparin-mimetic hydrogel thin film coated substrates. 3.6.2. Blood Clotting Time. The clotting times (APTT and TT) were measured to evaluate the anticoagulant activities of the DA-g-HepLP, HepLP and the coated hydrogel thin films. In general, APTT is used to measure the inhibited efficacy of both the intrinsic and the common plasma coagulation pathways.61 It is an endogenous coagulation pathway. TT is used to measure the clot formation time taken for the thrombin converted H

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Figure 9. (A) Activated partial thromboplastin time (APTT) and (B) thrombin time (TT) tests for the DA-g-HepLP, HepLP, and heparin. For the control group, 5 μL of PBS was added instead. Values are expressed as means ± SD (n = 3), and the marks (*) mean that the difference attained a statistically significant increase compared with the control (plasma). *, P < 0.05. (C) APTT and (D) TT values of the PPP, pristine PES, DAHepLP/CS-5/PES, DA-HepLP/CS-10/PES, and DA-HepLP/CS-20/PES membranes. Values are expressed as means ± SD (n = 3), and the * mean that the difference attained a statistically significant increase compared with the control (plasma). *, P < 0.05.

the anticoagulation activity of the hydrogel thin films, the APTT and TT have been recorded, and the data are shown in Figure 9C,D, respectively. The APTT of the hydrogel thin film coated substrates increased significantly in contrast with that of the pristine PES membrane, and the blood was incoagulable (exceeded 600 s) when the coating bilayers exceeded 10. The TT test results also revealed that the clotting time had been significantly prolonged, similar to APTT. The improvements of anticoagulant activity combined with the enhanced hydrophilicity and suppressed platelet adhesion indicate that the heparin-mimetic hydrogel thin film coated substrates can inhibit the generation of thrombus when in contact with blood during clinical treatments. 3.6.3. Complement Activation. Complement activation is the result of the triggering of the host defense mechanism generated by the localized inflammatory mediator. In this study, the generated concentrations of C3a and C5a were detected as model indexes to evaluate the complement activation using an ELISA method; data are shown in Figure 10. It was observed that there was no significant increase in the C3a concentrations for the hydrogel thin film coated substrates, which were almost the same as that of pristine PES membrane. The C5a concentrations for the hydrogel thin film coated substrates were slightly higher than that for whole blood but were commensurate with that for pristine PES membrane. In general, the results verified that the heparin-mimetic hydrogel thin film coated substrates exhibited no significant blood-related complement activation; thus, no inflammation response would be activated when contacted with blood.

fibrinogen into fibrin in the platelet-poor plasma (PPP).62 This pathway is at the bottom of the clotting cascade. APTT and TT tests were carried out by contacting PPP with the different concentrations of polymers and the hydrogel film coated substrates. Statistical methods were used to analyze the results. A longer clotting time indicates the lower potential of thrombus generation. Figure 9A shows the APTT results. The blood-clotting times of DA-g-HepLP are similar to those of HepLP and gradually increase with the increase of the sample concentration. The blood was incoagulable (exceeded 600 s) when the concentration exceeded 25 μg/100 μL blood from the APTT results. Although the clotting times of the synthetic DA-gHepLP and HepLP were lower than that of heparin, they still showed high potential as anticoagulant reagent because of their low-cost and facile preparation process. The TT test results (Figure 9B) of the samples had the same tendency to APTT, and coagulation did not occur when the concentration exceeded 10 μg/100 μL blood. The remarkably prolonged TT values might be resulted by the aggregation and solidification of fibrinogen because of the abundant sulfonic acid groups. In summary, it can be concluded that the DA grafting has limited influence on the anticoagulant activity of HepLP; the excellent anticoagulant property of the DA-gHepLP endows it great potential for various blood-contacting applications. The heparin-mimetic hydrogel thin films have been coated on the membrane surfaces, and this might endow the membrane with excellent anticoagulant activity. To evaluate I

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Figure 11. Releasing behaviors of (A) DOX and (B) GS from the drug-loaded hydrogel thin films and PES membranes. Figure 10. (A) Generated concentrations of C3a after incubation of the membranes with whole blood; values are expressed as means ± SD, n = 3. (B) Generated concentrations of C5a after incubation of the membranes with whole blood; values are expressed as means ± SD, n = 3.

3.8. Antibacterial Properties of the AntimicrobialDrug-Loaded Hydrogel Thin Film. To examine the versatile functionality of the heparin-mimetic hydrogel thin films in biomedical applications, the GS-loaded DA-HepLP/CS-20 coated membranes were applied to examine the antimicrobial activity of the loaded drug; the antibacterial effects were expressed by investigating the bacterial inhibition zone and viability toward Escherichia coli and Staphylococcus aureus.66 As shown in Figure 12A,B, the sizes of the inhibition zones for E. coli were 0.0 mm (PES) and 9.4 mm (DA-HepLP/CS-20/ PES). As for S. aureus, the inhibition zones were 0.0 mm (PES) and 9.7 mm (DA-HepLP/CS-20/PES). To investigate further the bactericidal efficiency of the coated substrates in physiological media, the optical density of the bacteria− membrane cocultured solutions was detected. As shown in Figure 12C,D, high bacterial viability was observed for the control sample and pristine PES membrane after coculture for 24 h. However, the optical density for the GS-loaded DAHepLP/CS-20/PES exhibited significant reduction for both E. coli (retained 3.4% viability) and S. aureus (retained 2.6% viability). The results of the inhibition zone and bactericidal activity tests indicated that the GS-loaded hydrogel thin films had significant inhibition capability toward both E. coli (Gramnegative) and S. aureus (Gram-positive) bacteria, which further suggested that the hydrogel thin films exhibited good drugloading and -releasing ability thus extending the potential applications of the heparin-mimetic hydrogel thin films in various biological and clinical fields.

3.7. In Vitro Drug/Protein Release. Small molecular drugs can be incorporated into the hydrogel thin films by conjugation or adsorption. The adsorption amounts of DOX (anticancer drug) and GS (antimicrobial drug) by the hydrogel thin films were calculated to be 8.2 and 10.1 μg/cm2, respectively. The in vitro release behaviors from the drugloaded hydrogel thin films were studied in buffered solutions (pH 7.4 and 5.3, respectively), as shown in Figure 11. The release of both DOX and GS showed two distinct stages: a relatively rapid release over the first 10 h, followed by a sustained long-term release. The DA-HepLP/CS-20/PES showed a higher drug-releasing amount than that of PES; in comparison, the release of DOX or GS at pH 7.4 was lower than that at pH 5.3, which demonstrated that the pH value could influence the release of DOX or GS as reported by earlier literatures.63−65 The protein release behavior of the hydrogel thin films has also been studied by using the BSA as the model protein, and the data are shown in Figure S8. The results for BSA release were similar to those for the DOX and GS release. It was expected that the hydrogel thin films with controllable release behaviors could be used as a carrier for growth factor and delivery of many other extracellular matrix proteins, which could benefit the adhesion and growth of cells. J

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Figure 12. Inhibition zone picture for (A) E. coli (Gram-negative) and (B) S. aureus (Gram-positive) for PES and DA-HepLP/CS-20/PES (both incubated with GS). Optical densities for (C) E. coli (Gram-negative) and (D) S. aureus (Gram-positive); the absorbance represents the bacterial viability after exposure to pristine and hydrogel-coated membranes for 24 h.

4. CONCLUSIONS In this work, robust and heparin-mimetic hydrogel thin film coatings have been constructed via combined LbL self-assembly and mussel-inspired post-cross-linking. The coated DAHepLP/CS hydrogel thin films revealed 3D porous surface morphology; the hydrogel thin film coatings not only showed high stability in highly alkaline, acid, and concentrated salt solutions but also exhibited excellent long-term stability in physiological solutions. The live/dead cell staining and cell morphology observations indicated that the heparin-mimetic hydrogel thin film coated substrates had low cell toxicity and high promotion ability for cell proliferation. Considering the results of platelet adhesion/activation, clotting times, and complement activation together, it was believed that the hydrogel thin films exhibited good blood compatibility, which might confer great application potential to the hydrogel thin film coatings in various blood-contacting fields. Furthermore, both the results of inhibition zone and bactericidal activity tests indicated that the hydrogel thin film coatings can also load antimicrobial drug for significant inhibition toward E. coli (Gram-negative) and S. aureus (Gram-positive) bacteria. Combining the above advantages, it is believed that the designed heparin-mimetic hydrogel thin films may show high potential for applications in various biological and clinical fields, such as long-term hemocompatible and drug/protein-loading materials for implants.

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Experimental procedures for formation of HepLP and DA-g-HepLP, as well as spectroscopic information and descriptions of methods used to characterize these hydrogel thin films. (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] or [email protected]. *E-mail: [email protected] or [email protected]. Tel.: +86-28-85400453. Fax: +86-28-85405402. 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), the State Key Laboratory of Polymer Materials Engineering (Grant no. sklpme2015-1-03), and the Sichuan Province Youth Science and Technology Innovation Team (no. 2015TD0001). We thank our laboratory members for their generous help and gratefully acknowledge the help of Ms. Hui Wang, of the Analytical & Testing Center at Sichuan University, for the SEM images.

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