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Nanofibrous Heparin and Heparin-mimicking Multilayers as Highly Effective Endothelialization and Antithrombogenic Coatings Chuanxiong Nie, Lang Ma, Chong Cheng, Jie Deng, and Changsheng Zhao Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm501882b • Publication Date (Web): 10 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Nanofibrous

Heparin

and

Heparin-mimicking

Multilayers as Highly Effective Endothelialization and Antithrombogenic Coatings Chuanxiong Nie,a Lang Ma,a Chong Cheng,a,* Jie Deng,a Changsheng Zhaoa,c,* a

College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials

Engineering, Sichuan University, Chengdu, 610065, China b

National Engineering Research Center for Biomaterials, Sichuan University, Chengdu, 610064,

China * Corresponding author. Tel: +86-28-85400453, Fax: +86-28-85405402, E-mail: (C. Cheng) [email protected] or [email protected]; (C.S. Zhao) [email protected] or [email protected]

ABSTRACT: Combining the advantages of the fibrous nanostructure of carbon nanotubes (CNTs) and the bioactivities of heparin/heparin-mimicking polyanions, functional nanofibrous heparin or heparin-mimicking multilayers were constructed on PVDF membrane with highly promoted endothelialization and antithrombogenic activities. To construct the multilayers,

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oxidized CNT (oCNT) was firstly functionalized with water-soluble chitosan (polycation), then enwrapped with heparin or a typical sulfonated heparin-mimicking polymers (SHP, poly (sodium 4-styrenesulfonate-co-sodium methacrylate)). Then, the surface deposited multilayers were constructed via electrostatic layer-by-layer (LbL) assembly of the functionalized oCNTs. The scanning electron microscope and atom force microscope images confirmed that the coated multilayers exhibited nanofibrous and porous structure. The live/dead cell staining and cell activity assay results indicated that the coated nanofibrous multilayers owned excellent compatibility with endothelial cells. The cell morphology observation further confirmed the promotion ability of surface endothelialization due to the coated heparin/heparin-mimicking multilayers. Further systematical evaluation on blood compatibility revealed that the surface heparin/heparin-mimicking multilayers coated membranes also owned significantly improved blood compatibility including restrained platelet adhesion and activation, prolonged blood clotting times, and inhibited activation of coagulation and complement factors. To sum up, the proposed nanofibrous multilayers integrated endothelialization and antithrombogenic properties, meanwhile, the heparin-mimicking coating validated comparable performances as heparin coating. Herein, it is expected that the surface coating of nanofibrous multilayers, especially the facilely constructed heparin-mimicking coating, may own great application potential in biomedical fields.

KEYWORDS: heparin and heparin-mimicking, nanofibrous multilayers, layer-by-layer assembly, surface endothelialization, antithrombogenic coating

1. INTRODUCTION

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Until now, one of the most vital challenges for developing advanced blood or tissue contacting artificial materials is to simultaneously achieve high biocompatibility and multiple biofunctionalities.1-3 When contacting with blood or tissues, series of bio-responses might happen at the blood(tissue)/materials interfaces, such as cytotoxicity, carcinogenicity and teratogenicity of implants,4, 5 blood components activation and thrombus generation,6 inflammation response, and also immunologic rejection,7 these potential side-effects were harmful even lethal for patients. The above raised problems were mainly caused by the unfavorable material or structure of biointerfaces. Till now, considerable methods had been developed for the surface modification of synthetic biomaterials, for instance, bulk blending,6 surface plasma treatment,8 surface coating or grafting of functional (bio)polymers,9, 10 and many other biological mimicking methods.11, 12 Recently, surface endothelialization has been gradually considered to be one of the most important approaches to fulfill high biocompatibility and extended bio-functionality, especially for biomedical membranes and implantable devices (such as vascular grafts and coronary stent systems).13 Earlier in vitro and in vivo studies indicated that endothelium had numerous vital biofunctions: (1) selective permeable barrier (2) preventing blood coagulation (3) regulating blood pressure and (4) promoting angiogenesis. Inspired from the bio-functionalities of endothelium, it is expected that the rapid and full cover of endothelia cells (ECs) on biomaterial surfaces might effectively decrease materials induced cytotoxicity, suppress thrombus formation and inflammation responses, limit intimal hyperplasia, and thus prolong the usage of materials.14 Herein, many approaches, attempting to accelerate the endothelialization on biomedical material surfaces, have been proposed.15-19 For instance, Hu et al.15 immobilized vascular endothelial growth factor (VEGF) on titanium surface via either covalent bond or heparin-VEGF interaction; they found that the heparin-VEGF modified titanium showed superior performance

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in EC growth and proliferation. However, the use of growth factor or antibody is not practical for large-scale application due to the high cost and their easy degeneration and inactivation in vitro. Thus, improving the endothelialization on material surface without introducing growth factors or antibodies has attracted great attention gradually.20-23 Heparin, one of the most commonly used clinical anticoagulant reagents, was also found to have positive effects on ECs growth and proliferation by binding and stabilizing cell growth factor; moreover, the introduced heparin could also suppress the formation of thrombus remarkably.24 Recently, heparin has been extensively used for designing bio-functional coatings to improve the cell compatibility of artificial materials, inspiring results were acquired by Huang et al. and other researchers, and the heparin modified biomaterials showed superior performances in ECs growth and proliferation.2529

However, heparin is expensive with potential side effects (internal bleeding, fast

biodegradation, and activities in other non-target biological pathways), thus it may not be suitable for the large-scale biomedical applications. In a substitutable approach, various heparinmimetic compounds had been synthesized and exhibited with lower cost, better-defined chemical structures and more specific bioactivity when compared with heparin.30-32 Furthermore, it has been demonstrated that sulfonated polymers, such as poly (sodium 4-styrenesulfonate) and poly (styrenesulfonate-co-poly (ethylene glycol) (PEG) methacrylate), are also capable of stabilizing growth factors, promoting angiogenesis and also acting as anticoagulant.33-35 Recent studies also indicated that the heparin-mimetic compounds modified biomaterials exhibited comparable blood and cell compatibilities as those of the heparin modified biomaterials.24, 27 However, using the heparin or heparin-mimicking polymers for surface coating mainly obtained 2D compact coating layers, which might limit the bio-function of the molecules in the inner layers and result in insufficient bio-functionality and bioactivity. Recent studies indicated

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that various nanoscale building blocks such as nano/microgels,36, 37 nanotubes38 and nanosheets39, 40

might be able to maximize the utilization of the functional molecules, thus reducing the

amount of heparin or heparin-mimicking polymers required for the construction of coating multilayers. Meanwhile, benefiting from their nanostructures, the nanomaterials assembled 3D multilayers could endow the bio-interfaces with high density of functional groups, large surface area and even drug or protein loading ability. Thus, it is highly necessary to explore and compare the detailed performances of heparin or heparin-mimicking polymer functionalized nanomaterials on promoting endothelialization and many other biological benefits. In this study, aiming to search for a green, scalable and facile method to enhance the endothelialization efficiency of biomedical materials, we developed 3D nanofibrous heparin and heparin-mimicking multilayers on PVDF membranes substrates by combining the advantages of nanofibrous CNTs and bioactivities of heparin/heparin-mimicking polyanions, and then validated the comparable performances of heparin-mimicking coating as heparin to achieve scalable fabrication. To construct the nanofibrous multilayers, oxidized CNT (oCNT) was firstly prepared by treating of multiwall carbon nanotube in H2SO4/HNO3; followed by the functionalization with water-soluble chitosan (CS, polycation, owns good biocompatibility and low economic cost), and then coated with heparin or a typical sulfonated heparin-mimicking polymers (SHP, poly (sodium 4-styrenesulfonate-co-sodium methacrylate), P(SSNa-co-MAANa), which was low-cost and suitable for scalable fabrication and synthesized via atom transfer polymerization as reported in our earlier research9). The nanofibrous multilayers were fabricated by LbL assembly of chitosan functionalized oCNT and the heparin or heparin-mimicking polymer functionalized oCNT on the commercial available PVDF membrane substrates. The constructed functional nanofibrous multilayers were systemically investigated using attenuated total reflection-Fourier

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transform infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), scanning electron microscope (SEM) and atomic force microscope (AFM) observations. Then, the ECs attachment and growth on the modified PVDF membrane were systemically studied. Live/dead cell staining and MTT assay were applied to evaluate the cyto-compatibility of the assembled multilayers; meanwhile, SEM and confocal laser scanning microscopy (CLSM) were applied to observe the cell morphology. In addition, to evaluate the application potential as implant, we further evaluated the blood compatibility of the nanofibrous multilayers in terms of plasma clotting time, coagulant activation, and platelet adhesion and activation. The bloodcontacting immune response was preliminary studied based on generated C3a and C5a factors using enzyme-linked immuno sorbent assay kits (ELISA). 2. MATERIALS AND METHODS 2.1 Materials. CuBr (AR, Aladdin) was purified by thoroughly washing via glacial acetic acid to remove the Cu2+, and then dried under vacuum. Multi-wall carbon nanotubes (outside diameter about 10 nm, length 15 µm, purity >90%) were purchased from Times Nano. Ltd. (China) and washed with 10 wt. % hydrochloric acid to remove the impurities before use. Chitosan (degree of deacetylation>95%, 100-200 mPa.s) was purchased from Aladdin Industrial Corporation (China) and treated with H2O2 for 4 h at 70 °C to be water-soluble. Heparin sodium salt (average Mw: 12000 Da) was obtained from Xiya reagent Co. Ltd. (China) and stored in a 4 °C fridge. Bovine serum albumin (BSA), bovine serum fibrinogen (BFG), 4',6-diamidino-2phenylindole

(DAPI),

Rhodamine-Phalloidin

and

3-(4,5-Dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide (MTT), fluorescein diacetate (FDA), and propidium iodide (PI) were purchased from Sigma Aldrich(USA). Micro BCATM protein assay kits were purchased

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from PIERCE and blood clotting kits were obtained from SIEMENS. Other reagents if not mentioned were obtained from Aladdin reagent Co. Ltd (China). 2.2 Preparation of heparin or SHP functionalized carbon nanotubes. To improve watersolubility and introduce negatively charged groups, multiwall carbon nanotube (CNT) was oxidized with H2SO4/HNO3 (3/1, v/v) for 4 h in a 70 °C oil bath.41 The oxidized CNT (oCNT) was thoroughly dialyzed against distilled water to remove the residual acid. 20 mL 2 mg/mL H2O2 degraded CS solution (the degradation of chitosan was carried out as reported42) was added drop-wisely into 100 mL 0.4 mg/mL oCNT solution under ultrasonic bath. The solution was ultra-sonicated for another 30 min and then centrifuged to remove the residual chitosan. The collected black products were re-dissolved to 0.4 mg/mL using distilled water. 20 mL 2 mg/mL heparin or SHP was then dropped in the oCNT/CS solution under ultra-sonication. The solution was ultra-sonicated and centrifuged, then re-dissolved to prepare 0.4 mg/mL oCNT/CS/Hep and oCNT/CS/SHP solutions, respectively.

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Scheme 1. Preparation of chitosan, heparin and SHP functionalized carbon nanotubes; and the TEM images of oxidized CNT (a) and functionalized CNT (b). Scale bar: 50 nm. The Zeta potentials of the prepared solutions at the concentration of 0.4 mg/mL were measured by using Zetasizer ZS90 (Malvern instruments). Transmission electron microscope (TEM) was used to detect the morphology change of the CNTs and acquired using a Tecnai G2 F20S-TWIN transmission electron microscope (FEI Ltd., USA). The solutions were dipped onto silica wafer and then the XPS spectra were detected on XSAM800 X-ray photoelectron spectroscopy (Kratos Analytical, UK). 2.3 LbL assembly of surface multilayers on PVDF membrane. The nanofibrous multilayers were constructed by the electrostatic LbL assembly method. A typical procedure was as follows: pristine PVDF membrane was immersed in 1 wt.% PEI solution overnight to generate a positively charged surface. The membrane was washed and dried for 2 min, and then immersed into 0.4 mg/mL oCNT/CS/Hep solution or oCNT/CS/SHP solution for 15 min. The membrane was washed to remove the unbonded oCNT/CS/Hep or oCNT/CS/SHP and air dried, followed by the immersion into the positively charged oCNT/CS solution. The LbL assembly cycle was repeated several times to get different numbers of coating bilayers. The heparin and heparinmimicking polymer functionalized CNT were used as the outside layer to achieve targeted biofunctionality. The membrane with n bilayers of oCNT/CS/Hep and oCNT/CS was named as Hep-n and so did the SHP-n.

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Scheme 2. (A) LbL assembly of Hep or SHP functionalized CNTs on PVDF membrane. (B) Components for the coated samples. (C) Surface SEM morphologies of the PVDF membrane before (left) and after (right) assembly. The chemical structures after the coating of surface multilayers were investigated by using ATR-FTIR and XPS. The ATR-FTIR analysis was performed on Nicolet-560 spectrophotometer (Nicol, US) (range: 4000 to 500 cm-1; resolution: 2 cm-1). XPS measurements were performed on XSAM800. SEM images were obtained using JSM-7500F scanning electron microscope (JEOL, Japan). AFM images were taken on a Multimode Nanoscope V scanning probe microscopy system (Bruker, USA). Water contact angles for the samples were measured using the sessile drop method with a digital optical contact angle meter DSA100 (KRUSS GmbH, Germany) with 3 µL of water at 25 °C and relative humidity of 80%.

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2.4 Protein adsorption. A typical BCA method was applied to study the protein adsorption amounts on membranes as reported.9 The detailed procedures for protein adsorption are shown in Supporting Information. 2.5 Endothelial cell culture. Human umbilical vein endothelial cells (HUVECs) were selected to investigate the endothelial cell response of the membranes. The details about HUVECs culture and the evaluation methods are presented in Supporting Information, which includes the MTT assay for the viability of HUVECs, Living/dead cell staining by fluorescence microscopy (DMIRE2, Leica),43 cell morphology by a JSM-7500F SEM

and CLSM images (Leica,

Switzerland). 2.6 Blood compatibility. Healthy human fresh blood (adult, male) was collected using vacuum tubes containing heparin sodium as anticoagulant (anticoagulant to blood ratio, 1:9), and the blood used in all the blood tests were from the same donor. The obtained blood was centrifuged at 1000 rpm for 15 min to obtain platelet-rich plasma (PRP) and another 4000 rpm for 15 min to obtain platelet-poor plasma (PPP). 2.6.1 Platelet adhesion. Platelet adhesion was carried out by immersing the membrane in the PRP and then observed using SEM as reported.9 The procedures are shown in the Supporting Information. 2.6.2 Clotting times. The activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) for the samples were measured using CA-50 blood coagulation analyzer (Sysmex, Japan). The test procedures were described in our previous report in detail.33 Every test was repeated six times to reduce the error and the data were expressed as mean ± SD. 2.7. Enzyme-linked immune sorbent assays (ELISA). Commercial ELISA were used to investigate the platelet activation (Human Platelet Factor 4 (PF-4), Cusabio Biotech Co. Ltd.,

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China), coagulation activation (thrombin-antithrombin III complex (TAT), Enzygnost TAT micro, Assay Pro, USA) and complement activation (Human Complement Fragment 3a (C3a) and Human Complement Fragment 5a (C5a), Cusabio Biotech Co. Ltd., China). The detailed procedures could be found in supporting information. 3. RESULTS AND DISCUSSIONS 3.1 Preparation of heparin and heparin-mimicking polymer functionalized carbon nanotubes. In the previous studies, it was found that sulfonated polymers, such as PSS, P(SSNaco-EGMA) and P(SSNa-co-MAANa), could mimic the chemical structure and bio-functions of heparin, such as the anticoagulant, antithombotic, and promoting bio-adhesion properties.9, 35, 44 In this study, a typical heparin-mimicking linear polymer contains sulfonic and carboxyl groups, P(SSNa-co-MAANa), was synthesized via ATRP polymerization as reported.9 The clotting times for the sulfonated heparin-mimicking polymer (SHP) were measured to characterize its heparinoid activity. The results are shown in Fig. S1 (Supporting Information). To endow the carbon nanotubes with favorable ECs affinity and blood compatibility, the negatively charged heparin and SHP were assembled on the positively charged oCNT/CS as described in Scheme 1, respectively. The high resolution TEM data in Fig. 1A showed the typical morphological differences between the oxidized and functionalized CNTs: the oxidized CNT had a characteristic tubular shape with a diameter of about 10 nm; after the assembly, a transparent layer around CNT was observed, indicating that thin layers of chitosan and heparin/SHP were coated onto the CNT surface. To confirm the assembly of the biomolecules on CNT, the Zeta potentials of the prepared solutions were measured and the results are shown in Fig. 1B. The oCNT exhibited negatively charged surface with a Zeta potential of about -23.4 ± 0.15 mv; after CS modification, the Zeta potential turned to about 20.6 ± 1.8 mv for the

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oCNT/CS. Then, after the assembly with Hep and SHP, the surface Zeta potential changed to 35.9 ± 1.0 mv for the oCNT/CS/Hep and -39.4 ± 2.1 mv for oCNT/CS/SHP, respectively. The results proved that the functional polymers were wrapped on the oCNT surfaces by electrostatic adsorption successfully, which would facilitate the construction of multilayer nanostructure via LbL assembly. XPS was applied to study the chemical structure of the functionalized CNT. Fig. 1C shows the results of XPS C1s spectra. The C1s spectrum of the oxidized CNT showed four characteristic carbon moieties: the peak at 287.8 ev and 286.5 ev were assigned to the carboxyl groups (C=O) and the ether linkage and C-N bond, respectively; while the binding energies at 285.6 ev and 284.6 ev were ascribed to the C-OH bond and carbon skeleton (C-C), respectively. In the spectra for the functionalized CNT, the peak at 288.3 ev was assigned to the carbonyl groups (C=O) of the immobilized heparin and chitosan; the signal around 286.5 ev was ascribed to the overlapped peaks of the C-N, C-S and C-O bonds. Moreover, the spectra of O1s, N1s and S2s in the wide XPS scan (Fig. S2, Supporting Information) should be attributed to the introduced polymers (chitosan, heparin and SHP). The S2p spectra also confirmed that the sulfonated polymers (heparin and SHP) were enwrapped on the oCNT successfully.

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Fig. 1. (A) TEM images for the oCNT and functionalized CNTs. Scale bars for all the images: 50 nm. (B) Zeta potentials for the oCNT and functionalized CNTs solutions at a concentration of 0.4 mg/mL. Each test was repeated for at least 6 times to get a reliable value, and the values are expressed as mean ± SD. (C) XPS C1s spectra for the oCNT, oCNT/CS, oCNT/CS/SHP and oCNT/CS/Hep, respectively. 3.2 Construction of nanofibrous heparin and heparin-mimicking multilayers on PVDF membrane surface. Facile electrostatic LbL assembly method was applied to construct the functional nanofibrous multilayers on PVDF membrane. As described in Scheme 2, PVDF membrane was firstly coated with PEI to generate a positively charged surface. The PEI coated membrane was then immersed in negatively charged oCNT/CS/Hep (or oCNT/CS/SHP) solution and positively charged oCNT/CS solution alternatively. The procedure was repeated several times to construct a nanofibrous multilayer structure on PVDF membrane surface.

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The chemical structure of the assembled multilayers was investigated in terms of ATR-FTIR and XPS. Fig. 2A shows the ATR-FTIR spectra for the samples, an enhanced adsorption peak around 1578 cm-1 has been detected, which can be ascribed to the amino groups of the chitosan. The peak around 1217 cm-1 in the spectra for SHP-10 was assigned to the aromatic groups in SHP; meanwhile, the peaks around 1110 cm-1 in the spectra for both SHP-10 and Hep-10 were attributed to the –SO3- symmetric vibration. XPS was applied to further detect the chemical compositions of the assembled multilayers. The XPS C1s spectra for pristine, Hep-10 and SHP10 are shown in Fig. 2B, 2C and 2D, respectively. The C1s spectrum of pristine PVDF membrane showed three characteristic carbon moieties: the peak at 290.7 ev and 286.4 ev were assigned to the carbon-fluorin bond (-CF2-) and carbon-carbon (fluorin) bond (-C-C(F)-), respectively; while the peak at 284.6 ev was ascribed to the polymer skeleton (C-C). In Fig. 2C and Fig. 2D, a new peak at 288.3 ev was observed, which can be assigned to the carbonyl groups of the immobilized heparin and chitosan; the signal around 286.5 ev could be ascribed to the overlapped peaks of the C-N, C-S and C-O bonds. Moreover, the spectra of O1s, N1s and S2s in the wide XPS scan (Fig. 2E) should be attributed to the introduced polymers (chitosan, heparin and SHP). From the element analysis, obtained from XPS spectra and shown in Fig. S3 in Supporting Information, it was also found that after the surface assembly of the functionalized CNTs, the contents of F elements decreased dramatically and the contents of O elements increased; in addition, the N and S elements were detected on Hep-10 and SHP-10 membranes. These results indicated that the nanofibrous heparin and heparin-mimicking multilayers were immobilized on PVDF membrane surface successfully via surface LbL assembly.

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Fig. 2. (A) ATR-FTIR spectra for PVDF, PVDF/PEI, SHP-10 and Hep-10. The XPS C1s spectra for PVDF (B), Hep-10 (C) and SHP-10 (D), and XPS wide spectra for SHP-10, Hep-10 and pristine PVDF (E). The XPS N1s spectra and element analysis are presented in Supporting Information.

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To further study the construction of the nanofibrous multilayers on PVDF membrane surface, the morphologies of membrane surface before and after the assembly were observed using FESEM and AFM (Fig. 3). It was observed that the pristine PVDF membrane presented a potholed but smooth surface morphology; while, the multilayers coated membranes exhibited nanofibrous structure with covered functionalized CNTs. Furthermore, with the number of the bilayers increased from 1 to 10, the coating thickness increased gradually. The AFM images showed identical results to the SEM observation that the nanofibrous 3D porous structures were obtained on the membrane surfaces after the assembly of the Hep and SHP functionalized CNT. The hydrophilic/hydrophobic properties are usually characterized through water contact angle (WCA) measurement, which is convenient and efficient to estimate the wettability property of biointerface. As indicated by the surface chemical analysis and morphology observation, there are abundant Hep or SHP functionalized CNT coated on the PVDF substrate, which may alter the super-hydrophobic and antifouling PVDF membrane into more hydrophilic and bioadhesive substrate. It was found from Fig. 3D that the pristine PVDF membrane exhibited superhydrophobic character with the water contact angle of about 121.9°. The WCAs for the multilayers deposited membranes decreased notably to 66.2° and 72.1° for SHP-10 and Hep-10, respectively. While, the static water contact angle measurements alone were not adequate to evaluate the hydrophilicity. To further investigate the hydrophilicity of the nanofibrous multilayers, the changes of the water contact angle with increased drop age were also carried out, and the results indicated that the nanofibrous multilayers revealed slightly decreased contact angles when time extended (Fig. S4, Supporting Information). The results confirmed that the membranes become much more hydrophilic after the deposition of the heparin or heparinmimicking multilayers.

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Fig. 3. FE-SEM images of the surfaces for pristine PVDF membrane (A), heparin functionalized membranes (B-1, Hep-1; B-2, Hep-5; and B-3, Hep-10), and SHP functionalized membranes (C1, SHP-1; C-2, SHP-5; and C-3, SHP-10). AFM images of the surfaces for pristine PVDF membrane (a), heparin functionalized membranes (b-1, Hep-1; b-2, Hep-5; and b-3, Hep-10), and SHP functionalized membranes (c-1, SHP-1; c-2, SHP-5; and c-3, SHP-10). (D) Water contact angles for the membranes and the representative pictures of water contact angles (taken at 10 seconds) from independent experiments, values are expressed as mean ± SD, n=9. Based on the results of ATR-FTIR, XPS and surface morphology observations, it could be concluded that functional multilayers were coated onto PVDF membranes successfully. The constructed heparin and heparin-mimicking 3D nanofibrous multilayers might influence the bioresponse of the membranes such as protein adsorption, ECs attachment and growth, blood

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compatibility and immune response, which would be investigated and discussed in the following parts. 3.3 Protein adsorption. Among the intricate bio-reactions and bio-interactions between the biomaterials and living system, protein adsorption is the first event happens at the interface and plays a crucial role in materials associated cell attachment and growth.45-48 Fig. 4 shows the BSA and BFG adsorption results for samples before and after surface coating. It was noticed that only a small amount of proteins adsorbed on pristine PVDF membrane, since PVDF membrane was super-hydrophobic and exhibited excellent anti-fouling property.49 After the coating of nanofibrous multilayers, the amounts of the adsorbed protein increased significantly; meanwhile, with the increase of the coated layers, the adsorbed protein also increased dramatically for both BSA and BFG. The increased protein adsorption for the f-CNTs modified membranes might be caused by the nanofibrous structure and the heparin and heparin-mimicking polymers. Though, the coated multilayers are negatively charged, heparin and SHP are reported to be able to bind with a wide variety of proteins.50-52 Meanwhile, earlier reports revealed that the nanoporous structure had significant impact on protein adsorption.53 Overall, the results indicated that the constructed nanofibrous multilayers revealed increased serum protein adsorption than the pristine membrane, which might endow great benefits to the cell attachment, thus enhancing the endothelialization on membrane surface.54

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Fig. 4. Protein adsorption amounts on materials surface. Values are expressed as mean± SD, n=6, #p, *p< 0.05 compared with pristine PVDF membrane, respectively. 3.4 Endothelial cells culture. One major aim of this study is to search for an approach that could accelerate the ECs growth and proliferation on materials surface. It has been known that ECs are negatively charged and the cells communicate with the substrate through the adsorbed protein layers via specific recognition and binding sites between adsorbed proteins and cell membranes. The negatively charged surface might repulse the anchorage of the negative ECs to the materials surface.55 However, in this study, it was found that the nanofibrous multilayers were able to enhance the adsorption of protein, which might also increase the affinity of ECs onto the material surfaces through the adsorbed proteins. Meanwhile, the heparin and SHP modified surfaces are expected to be favorable for cell growth and proliferation by the bonding and stabilizing of cell growth factors such as VEGF and fibroblast growth factor 2 (FGF-2), as established in earlier studies.35, 56, 57 Chitosan was also reported to be able to enhance the affinity between cells and materials. HUVEC was selected to evaluate the endothelialization ability on the nanofibrous multilayer coated substrates in this study.

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3.4.1 Cell viability and affinity. Fig. 5A shows the FDA/PI staining results observed by using fluorescence microscope. No dead cell was observed on pristine PVDF, Hep-10 and SHP-10, which implied that all the membranes owned low toxicity towards HUVECs. While, much more living cells were observed on the Hep-10 and SHP-10 membranes compared to the pristine PVDF membrane. The average numbers of the attached HUVECs were also estimated from the images, and the results are shown in Fig. 5B. The tissue culture polystyrene (TCPS) cell plate was used as the control. Only a small number of HUVECs (about 18 cells per mm2) could be counted on pristine PVDF membrane, which was even lower than bare TCPS. After the deposition of the heparin functionalized multilayers, the numbers of the adhered HUVECs increased significantly (about 215 cells per mm2 for Hep-10), indicating that the heparin mimicking coatings could improve the attachment of HUVECs significantly. The enhancement of cell attachment should be resulted from the nanofibrous structure and the functionality of the immobilized biomacromolecules (chitosan, heparin or SHP), which could improve the affinity of HUVECs to the substrates. In addition, MTT assay has been applied to study the cell viability on the samples, and Fig. 5C shows the results of MTT assay. It was noticed that the pristine PVDF membrane had nearly the same absorbance as the control. The cells cultured on the functional nanofibrous multilayers deposited membranes owned much higher viability than the pristine PVDF membrane and the control. Literatures had reported that the HUVECs growth could be enhanced by surface roughness at nanometer scale;58 meanwhile, other factors, including surface charge, surface wettability and free energy and the existence of bioactive factors, could influence the cellmaterial surface interaction.59 Thus, the changing tendency of MTT absorbance for these samples might be influenced by these complicated factors. The MTT values for the SHP functionalized

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membranes were slightly lower than those of the heparin functionalized membranes, but still much higher than that of the pristine PVDF membrane, which suggested that the sulfonated polymers could mimic the biofunctionality of heparin and dramatically promote HUVEC growth. The results of MTT assay were consistent with the results of living/dead cells staining and cell number counting; thus, it could be concluded that the deposited functional nanofibrous multilayers could effectively increase HUVEC attachment on the substrate.

Fig. 5. (A) FDA/PI staining for the cells cultured on pristine PVDF, Hep-10 and SHP-10. Scale bar: 250 µm. (B) HUVEC adhesion on bare TCPS, pristine and modified PVDF membranes. (C) MTT assay of the cell cultured membranes. Formazan absorbance is expressed as a function of time for the HUVECs seeded onto different membranes. TCPS was used as the control sample. The results are expressed as means ± SD, n= 12, *p, #p, &p < 0.01 compared with pristine PVDF membrane.

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3.4.2 Cell morphology observations. When contacting with materials, mammal cells will undergo morphological changes to attach and stabilize on the biointerface. This cell adhesion consists of sequential processes of contact, filopodial growth, cytoplasmic webbing, cell mass flattening and the peripheral cytoplasm ruffling .60 The morphology of the cells cultured for 7 days was observed by using FE-SEM, and the images are shown in Fig. 6A. It was noticed that the HUVECs cultured on pristine PVDF tended to be a typical flattened shape. As observed, only a few HUVECs were spread on the pristine PVDF membrane. The cells cultured on the functionalized PVDF membranes tended spread with peripheral cytoplasm ruffling and fully covered on the material surface with a dense ECs layer. With the increase of the layer numbers, the endothelial cell layer became much thicker and the cells displayed a shuttle-like shape. To get a better view of the cell structures cultured on the functional nanofibrous multilayers, CLSM observation with a double staining of cytoplasm and nuclei was carried out. The CLSM images of the cells cultured on the Hep-10 and SHP-10 membranes are shown in Fig. 6B. As observed, the HUVESs seeded onto the functional nanofibrous multilayers showed distinct regional aggregations. A dense intercellular network of ECs cytoplasm was observed. The results of the cell morphology observation indicated that the functional nanofibrous multilayer deposited surfaces could efficiently enhance cell attachment and promote cell growth, since the HUVECs might be able to bind to the sulfonic groups of the multilayers. Concluding the results of living/dead cell staining, MTT assay and cell morphology observation, the nanofibrous heparin and SHP functionalized PVDF membranes showed positive effect on EC attachment and growth. After 7 days of cell culture, a dense ECs layer was observed on the functionalized PVDF membrane, which was much better than the pristine PVDF

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membrane. In general, the endothelialization on PVDF membrane was enhanced by the deposition of heparin/heparin-mimicking nanofibrous multilayers onto the surface.

Fig. 6. (A) SEM images of the HUVECs cultured on the membranes for 7 days. (B) CLSM images of the HUVECs cultured on the Hep-10 and SHP-10 after 7 days. (C) Cartoon images that represent the HUVECs attachment and growth on the pristine membrane and nanofibrous multilayer deposited membranes. SEM images at the magnification of ×300 and images for PEI coated PVDF are shown in Fig. S5, Supporting Information. Detailed CLSM images are presented in Fig. S6, Supporting Information.

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3.5. Blood compatibility. Blood contacting is inevitable in many biomedical applications, for instance synthetic vascular grafts, coronary stent systems and artificial kidney;10 on the interface of blood-noncompatibile materials, a series of unfavorable blood response might occur, such as platelet adhesion and activation, blood coagulation and thus resulted formation of thrombus.61-63 All these reactions are harmful even fatal for patients; thus, the evaluation of blood compatibility for the proposed nanofibrous multilayer coated membranes is also of vital importance. 3.5.1 Platelet adhesion and activation. Platelet adhesion and activation on materials surface are considered to be one of the triggers of thrombus formation. The activated platelets can trigger other coagulation factors, which may induce thrombosis and then result in further coagulations.64 In this article, the morphologies of the platelets adhered on membrane surfaces were studied by SEM observation and the amounts were counted from at least 6 SEM images, and the results are shown in Fig. 7A and Fig. 7B, respectively. On the pristine PVDF membrane, some adhered and aggregated platelets with irregular morphology were found, which indicated that potential thrombus formation might be induced at the material interface. After the coating of heparin and heparin-mimicking multilayers, only a very small amount of platelets were observed and the adhered platelets displayed sphere-like shapes with almost no pseudopodia or deformation. The Hep-10 exhibited the best blood compatibility with nearly no platelet adhered. The restrained interface platelet adhesion might be resulted from the enrichment of the functional sulfonated groups, which could repel the adhesion of platelet; furthermore, earlier researches had also revealed that the heparin and heparin-mimicking structure on membrane surface could significantly inhibit the platelet activation and then resulted in the decrease of platelet adhesion.65, 66

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To further study the interaction between the samples and blood, platelet activation was investigated using ELISA kits. Platelet factor 4 (PF-4) , which acts an essential role for injury and coagulant responses, was used to further investigate the platelet activation for the modified biomedical membranes, since it could be generated and released from the activated human platelets.67 From Fig. 7C, it was found that the pristine PVDF membrane had the highest PF-4 concentration, which suggested that platelet activation were probably induced. With the immobilization of the heparin and heparin-mimicking multilayers on the membrane surfaces, the generated PF-4 concentration decreased, especially for the heparin functionalized samples.

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Fig. 7. (A) SEM images of platelets adhered on pristine and nanofibrous multilayer coated PVDF membranes. Note: the platelets adhered on nanofibrous multilayer coated membranes were indicated with red arrow. (B) Average amounts of platelets adhered on pristine and nanofibrous multilayer coated PVDF membranes counted from 6 SEM images. (C) The concentrations of PF4 for the samples with whole blood incubated for 2 h, values are expressed as mean ± SD, n=12, *p< 0.05 compared with pristine PVDF membrane. (D) Cartoon images represent the platelet adhesion and activation on materials surface. 3.4.2 Clotting times and coagulant activation. Anticoagulant property is another important factor in the evaluation of the material interfaces induced blood compatibility.68 APTT and PT are the typical used global screening procedures for measuring coagulation abnormalities in the intrinsic pathway and extrinsic pathway, respectively. These two indexes are also applied to evaluate functional deficiencies of Factor II, III, V, VIII, X, or fibrinogen.69 TT is another general method to detect the anticoagulation ability of the materials. The TT value is dominated by the concentration and clotting activity of fibrinogen in plasma, which reflects the fibrinolytic system level in the common pathway of the clotting cascade.70 Fig. 8A and Fig. S7 show the APTT, TT and PT values for the pristine and surface coated PVDF membranes, respectively. The APTT and TT values for the pristine PVDF increased slightly compared to the plasma, which should be attributed to its excellent antifouling property. After modification, the membranes showed significantly increased clotting time. The APTT for the SHP-5, SHP-10 and Hep-5 were 141.8s, 247.3s and 232.8s, respectively; meanwhile, the Hep-10 showed the best anticoagulant property with an APTT value exceeds 400s, which was considered as incoagulable clinically. For the TT values, all the heparin or heparin-mimicking multilayer functionalized membranes revealed exceptional increase compared with the pristine PVDF. The APTT and TT

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clotting times for the SHP functionalized membranes were slightly lower than that of the heparin functionalized membrane, but they were still much longer than that of plasma and the neat PVDF. The PT values for all the membranes were in the same level as that of plasma (data are presented in Fig.S5). The results also indicated that the anticoagulant multilayers mainly exhibited effects on the intrinsic pathway of coagulation, which might be ascribed to the reaction or combination between the coagulation factors (V and X) in plasma. When contacting with material surface, thrombin may be activated. The thrombin could be inactivated immediately when reacted with anti-thrombin III (AT-III) and then resulted in the TAT complex formation.71 Thus, the amount of generated TAT complex is also essential for the detection of blood compatibility of samples. As shown in Fig. 8B, compared with plasma, no significant increase was observed for the pristine PVDF membrane. While, a decrease of generated TAT amounts was found for the coated substrates. Furthermore, with the increase of the coated bilayers, the generated TAT values of modified membranes decreased gradually. The results suggested that the TAT generation level was successfully inhibited by the deposition of the nanofibrous multilayers. 3.5.3 Blood related complement activation. Complement activation, generated by the localized inflammatory mediator, is recognized as a trigger in the host defense mechanism. Thus, it is another important aspect for the blood compatibility evaluation. Complement activation could be studied by determining the generated anaphylatoxins C3a, C4a and C5a. In this study, the generated concentrations of C3a and C5a were detected by using ELISA kits, and shown in Fig. 8C and Fig. 8D, respectively. It was observed that the neat PVDF exhibited a slightly higher C3a concentration than plasma. The values decreased after the coating of heparin and SHP contained multilayers; the Hep-10 generated the lowest C3a concentration of 466.3 ng/mL. Similar to the

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results of C3a concentration, the pristine PVDF membrane exhibited the highest C5a value of 22.4 ng/mL and the C5a concentration decreased dramatically after the coating of functoinal multilayers. The results revealed that the deposited heparin and heparin-mimicking polymer functionalized nanofibrous multilayers might inhibit the inflammatory response, and further broaden the applications of PVDF membranes in blood contacting fields. Taking the results of platelet adhesion and activation, clotting times, coagulant activation, and complement activation together, it is believed that the blood compatibility of the modified biomedical substrate was greatly enhanced by the coating of both the heparin and heparinmimicking nanofibrous multilayers, which might confer the PVDF membrane with great potential applications in blood-contacting fields.

Fig. 8. (A) APTT and TT values for the membranes. (B) The generated TAT concentrations of the membranes with PRP flowing for 2 h. (C, D) Concentrations of C3a and C5a for the samples

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with whole blood incubated for 2 h, respectively. Values are expressed as mean ± SD, n=12, *p, #

p< 0.05 compared with pristine PVDF membrane, respectively.

4. CONCLUSIONS In this study, heparin and heparin-mimicking nanofibrous multilayers were constructed on PVDF membrane surfaces via LbL assembly successfully. The heparin and heparin-mimicking polymers played an important role to improve the ECs attachment and growth. A dense layer of HUVEC was fully covered on the functionalized PVDF membrane, thus demonstrating that enhanced endothelialization could be achieved by the synergistic promotion of the nanofibrous multilayers and heparin/heparin-mimicking polymers. The results of further blood compatibility investigations indicated that the functionalized PVDF membrane owned suppressed platelet adhesion and activation, prolonged clotting time, and suppressed complement activation. It is expected that the integrated bioactivities of highly effective endothelialization and antithrombogenic ability for the coated multilayers may endow them great potential in various biomedical applications. Moreover, it was notable that the synthesis of nanofibrous heparinmimicking multilayers was facile and low-cost, and it showed comparable performance to the heparin multilayers. The proposed method could also be used to construct multilayers on many other substrates, for instance, polyethersulfone membrane. Therefore, the nanofibrous heparinmimicking multilayers may be suitable for the scalable construction of cell adhesive and antithrombogenic interface on various organic and inorganic artificial materials (such as hemodialysis membrane, vascular prosthesis, titanium and stainless steel devices). Supporting Information Procedures for protein adsorption, endothelial cell culture, MTT assay, living/dead cell staining, cell morphology observation, platelet adhesion and ELISA tests; Synthesis and heparin-

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like activity of SHP; XPS wide scan and S2p spectra for oCNT and functionalized CNT; XPS N1s spectra and element analysis for pristine PVDF and modified PVDF membranes; Dynamic water contact angle for the membranes; Morphologies of the HUVECs observed by SEM at a magnification of x300; Morphologies of the HUVECs observed by CLSM; PT values for the membranes; Nano-fibrous coatings on polyethersulfone membranes. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author * Corresponding author. Tel: +86-28-85400453, Fax: +86-28-85405402, E-mail: (C. Cheng) [email protected] or [email protected]; (C.S. Zhao) [email protected] or [email protected] Notes The authors declare no competing financial interest. Acknowledgement This work was financially sponsored by the National Natural Science Foundation of China (No. 51225303 and 51433007), the Programme of Introducing Talents of Discipline to Universities (B13040), and the Sichuan Province Youngth Science and Technology Innovation Team (No. 2015TD0001). We sincerely acknowledge the financial assistance of visiting research program by the China Scholarship Council (CSC). We should also thank our laboratory members for their generous help, and gratefully acknowledge the help of Ms. Hui Wang, of the Analytical and Testing Center at Sichuan University, for the SEM observation. REFERENCES

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46. Dekker, A.; Reitsma, K.; Beugeling, T.; Bantjes, A.; Feijen, J.; van Aken, W. G., Biomaterials 1991, 12, 130-138. 47. Khan, W.; Kapoor, M.; Kumar, N., Acta Biomater. 2007, 3, 541-549. 48. Zhang, K.; Liu, T.; Li, J.-A.; Chen, J.-Y.; Wang, J.; Huang, N., J. Biomed. Mater. Res. A 2014, 102, 588-609. 49. Wei, J.; Helm, G. S.; Corner-Walker, N.; Hou, X., Desalination 2006, 192, 252-261. 50. Young, E.; Prins, M.; Levine, M.; Hirsh, J., Thromb. Haemostasis 1992, 67, 639-643. 51. Aksoy, E. A.; Hasirci, V.; Hasirci, N.; Motta, A.; Fedel, M.; Migliaresi, C., J. Bioact. Compat. Polym. 2008, 23, 505-519. 52. Capila, I.; Linhardt, R. J., Angew. Chem., Int. Ed. 2002, 41, 390-412. 53. Lord, M. S.; Foss, M.; Besenbacher, F., Nano Today 2010, 5, 66-78. 54. Woo, K. M.; Chen, V. J.; Ma, P. X., J. Biomed. Mater. Res. A 2003, 67, 531-537. 55. Zhu, Y.; Sun, Y., Colloid Surf. B-Biointerfaces 2004, 36, 49-55. 56. Martino, M. M.; Briquez, P. S.; Ranga, A.; Lutolf, M. P.; Hubbell, J. A., Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 4563-4568. 57. Mammadov, R.; Mammadov, B.; Guler, M. O.; Tekinay, A. B., Biomacromolecules 2012, 13, 3311-3319. 58. Chung, T.-W.; Liu, D.-Z.; Wang, S.-Y.; Wang, S.-S., Biomaterials 2003, 24, 4655-4661.

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59. Boyan, B. D.; Hummert, T. W.; Dean, D. D.; Schwartz, Z., Biomaterials 1996, 17, 137146. 60. Rajaraman, R.; Rounds, D.; Yen, S.; Rembaum, A., Exp. Cell Res. 1974, 88, 327-339. 61. Furie, B.; Furie, B. C., N. Engl. J. Med. 2008, 359, 938-949. 62. Goldberg, A. L., Nature 2003, 426, 895-899. 63. Michelson, A.; MacGregor, H.; Barnard, M.; Kestin, A.; Rohrer, M.; Valeri, C., Thromb. Haemostasis 1994, 71, 633-640. 64. Baumgartner, H. R., Microvasc. Res. 1973, 5, 167-179. 65. Nie, C.; Ma, L.; Xia, Y.; He, C.; Deng, J.; Wang, L.; Cheng, C.; Sun, S.; Zhao, C., J. Membr. Sci. 2015, 475, 455-468. 66. Nie, S.; Tang, M.; Cheng, C.; Yin, Z.; Wang, L.; Sun, S.; Zhao, C., Biomater. Sci. 2014, 2, 98-109. 67. Niewiarowski, S.; Thomas, D. P., Nature 1969, 222, 1269 - 1270. 68. Dee, K. C.; Puleo, D. A.; Bizios, R., An introduction to tissue-biomaterial interactions. John Wiley & Sons: 2003. 69. Kamal, A. H.; Tefferi, A.; Pruthi, R. K. How to interpret and pursue an abnormal prothrombin time, activated partial thromboplastin time, and bleeding time in adults, Mayo Clinic Proceedings, 2007; Elsevier: 2007; pp 864-873.

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70. Key, N.; Makris, M.; O'Shaughnessy, D.; Lillicrap, D., Practical hemostasis and thrombosis. Wiley Online Library: 2009. 71. Nie, S.; Qin, H.; Cheng, C.; Zhao, W.; Sun, S.; Su, B.; Zhao, C.; Gu, Z., J. Mater. Chem. B 2014, 2, 4911-4921.

Table of Contents Graphic

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Scheme 1. Preparation of chitosan, heparin and SHP functionalized carbon nanotubes; and the TEM images of oxidized CNT (a) and functionalized CNT (b). Scale bar: 50 nm. 88x65mm (300 x 300 DPI)

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Scheme 2. (A) LbL assembly of Hep or SHP functionalized CNTs on PVDF membrane. (B) Components for the coated samples. (C) Surface SEM morphologies of the PVDF membrane before (left) and after (right) assembly. 88x66mm (300 x 300 DPI)

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Biomacromolecules

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Fig. 1. (A) TEM images for the oCNT and functionalized CNTs. Scale bars for all the images: 50 nm. (B) Zeta potentials for the oCNT and functionalized CNTs solutions at a concentration of 0.4 mg/mL. Each test was repeated for at least 6 times to get a reliable value, and the values are expressed as mean ± SD. (C) XPS C1s spectra for the oCNT, oCNT/CS, oCNT/CS/SHP and oCNT/CS/Hep, respectively. 88x66mm (300 x 300 DPI)

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Fig. 2. (A) ATR-FTIR spectra for PVDF, PVDF/PEI, SHP-10 and Hep-10. The XPS C1s spectra for PVDF (B), Hep-10 (C) and SHP-10 (D), and XPS wide spectra for SHP-10, Hep-10 and pristine PVDF (E). The XPS N1s spectra and element analysis are presented in Supporting information. 88x129mm (300 x 300 DPI)

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Biomacromolecules

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Fig. 3. FE-SEM images of the surfaces for pristine PVDF membrane (A), heparin functionalized membranes (B-1, Hep-1; B-2, Hep-5; and B-3, Hep-10), and SHP functionalized membranes (C-1, SHP-1; C-2, SHP-5; and C-3, SHP-10). AFM images of the surfaces for pristine PVDF membrane (a), heparin functionalized membranes (b-1, Hep-1; b-2, Hep-5; and b-3, Hep-10), and SHP functionalized membranes (c-1, SHP-1; c2, SHP-5; and c-3, SHP-10). (D) Water contact angles for the membranes and the representative pictures of water contact angles (taken at 10 seconds) from independent experiments, values are expressed as mean ± SD, n=9. 88x69mm (300 x 300 DPI)

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Fig. 4. Protein adsorption amounts on materials surface. Values are expressed as mean± SD, n=6, #p, *p< 0.05 compared with pristine PVDF membrane, respectively. 88x61mm (300 x 300 DPI)

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Biomacromolecules

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Fig. 5. (A) FDA/PI staining for the cells cultured on pristine PVDF, Hep-10 and SHP-10. Scale bar: 250 µm. (B) HUVEC adhesion on bare TCPS, pristine and modified PVDF membranes. (C) MTT assay of the cell cultured membranes. Formazan absorbance is expressed as a function of time for the HUVECs seeded onto different membranes. TCPS was used as the control sample. The results are expressed as means ± SD, n= 12, *p, #p, &p < 0.01 compared with pristine PVDF membrane. 88x72mm (300 x 300 DPI)

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Fig. 6. (A) SEM images of the HUVECs cultured on the membranes for 7 days. (B) CLSM images of the HUVECs cultured on the Hep-10 and SHP-10 after 7 days. (C) Cartoon images that represent the HUVECs attachment and growth on the pristine membrane and nanofibrous multilayer deposited membranes. SEM images at the magnification of ×300 and images for PEI coated PVDF are shown in Fig. S5, Supporting information. Detailed CLSM images are presented in Fig. S6, Supporting information. 88x108mm (300 x 300 DPI)

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Biomacromolecules

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Fig. 7. (A) SEM images of platelets adhered on pristine and nanofibrous multilayer coated PVDF membranes. Note: the platelets adhered on nanofibrous multilayer coated membranes were indicated with red arrow. (B) Average amounts of platelets adhered on pristine and nanofibrous multilayer coated PVDF membranes counted from 6 SEM images. (C) The concentrations of PF-4 for the samples with whole blood incubated for 2 h, values are expressed as mean ± SD, n=12, *p< 0.05 compared with pristine PVDF membrane. (D) Cartoon images represent the platelet adhesion and activation on materials surface. 88x99mm (300 x 300 DPI)

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Fig. 8. (A) APTT and TT values for the membranes. (B) The generated thrombin–antithrombin III (TAT) concentrations of the membranes with PRP flowing for 2h. (C, D) Concentrations of C3a and C5a for the samples with whole blood incubated for 2 h, respectively. Values are expressed as mean ± SD, n=12, *p, #p< 0.05 compared with pristine PVDF membrane, respectively. 88x64mm (300 x 300 DPI)

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Nanofibrous heparin and heparin-mimicking multilayers as highly effective endothelialization and antithrombogenic coatings.

Combining the advantages of the fibrous nanostructure of carbon nanotubes (CNTs) and the bioactivities of heparin/heparin-mimicking polyanions, functi...
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