Introducing multiple bio-functional groups on the poly(ether sulfone) membrane substrate to fabricate an effective antithrombotic bio-interface

Lingren Wang ab, Min He b, Tao Gong a, Xiang Zhang b, Lincai Zhang a, Tao Liu a, Wei Ye a, Changjiang Pan *a and Changsheng Zhao *b
aJiangsu Provincial Key Laboratory for Interventional Medical Devices. Huaiyin Institute of Technology, Huaian 223003, China
bCollege of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu 610065, China

Received 13th September 2017 , Accepted 19th October 2017

First published on 25th October 2017


It has been widely recognized that functional groups on biomaterial surfaces play important roles in blood compatibility. To construct an effective antithrombotic bio-interface onto the poly(ether sulfone) (PES) membrane surface, bio-functional groups of sodium carboxylic (–COONa), sodium sulfonic (–SO3Na) and amino (–NH2) groups were introduced onto the PES membrane surface in three steps: the synthesis of PES with carboxylic (–COOH) groups (CPES) and water-soluble PES with sodium sulfonic (–SO3Na) groups and amino (–NH2) groups (SNPES); the introduction of carboxylic groups onto the PES membrane by blending CPES with PES; and the grafting of SNPES onto CPES/PES membranes via the coupling of amino groups and carboxyl groups. The physical/chemical properties and bioactivities were dependent on the proportions of the additives. After introducing bio-functional groups, the excellent hemocompatibility of the modified membranes was confirmed by the inhibited platelet adhesion and activation, prolonged clotting times, suppressed blood-related complement and leukocyte-related complement receptor activations. Furthermore, cell tests indicated that the modified membranes showed better cytocompatibility in endothelial cell proliferation than the pristine PES membrane due to the synergistic promotion of the functional groups. To sum up, these results suggested that modified membranes present great potential in fields using blood-contacting materials, such as hemodialysis and surface endothelialization.


1. Introduction

Mounting studies reveal that blood-contacting materials are widely applied in the biomedical field for hemodialysis, cardiopulmonary bypass, and other procedures that require disposable clinical instruments.1–3 Even with the current advances, hemocompatibility is still a major challenge in the preparation of blood-contacting materials. Growing lines of evidence show that the practical utilization of biomaterials to substitute or operate in contact with blood, live tissues and organs is highly dependent on the appropriate physical and chemical interface design/modification to achieve favourable biological responses or biocompatibility and functional groups on biomaterial surfaces play important roles in hemocompatibility.4–8 Among the blood-contacting materials, poly(ether sulfone) (PES) and PES-based materials, which display good oxidative, thermal, and hydrolytic stabilities, as well as good mechanical and film-forming properties, have been successfully applied in the biomedical field for hemodialysis, endothelialization, and artificial organs for over decades. However, PES cannot be used alone as a biomedical material because of its hydrophobicity and lack of bio-functional groups.1 In general, when coming into contact with blood, plasma proteins will be rapidly adsorbed at the blood/PES interface, and the adsorbed protein layer may lead to further undesirable results, such as platelet adhesion, aggregation and coagulation, which may cause severe injuries.9,10 In order to avoid these undesirable responses and thus improve the biocompatibility, the development of new materials and methods to create the next generation of blood-contacting materials with improved capabilities or modification of PES materials by various treatments to increase the hemocompatibility, cytocompatibility and other specific functions is of high interest. Although alternative materials can present several advantages that common materials fail to exhibit, they may also prove to be more vulnerable in terms of chemical and physical stability and require further modifications.11,12 Therefore, the modification of PES or PES-based materials would be a better way.

Recent studies indicated that polymers with carboxylate, sulfonate and amino as pendant groups possessed some biofunctions, such as anticoagulant ability and cell affinity. Generally, the sodium carboxylic (–COONa) group could inhibit the coagulation cascade by binding calcium ions in the blood;13–15 the sodium sulfonic (–SO3Na) group could prolong the clotting time by preventing the proteolytic conversion of fibrinogen to fibrin, and inhibit the polymerization of the fibrin monomer, once fibrin has formed;16–18 the amino (–NH2) group might be conducive to the adsorption of bovine serum albumin (BSA), which might have a positive influence on anti-platelet adhesion/aggregation and cell adhesion/proliferation, thus promoting blood compatibility and surface endothelialization.19 Therefore, the introduction of sodium carboxylic (–COONa), sodium sulfonic (–SO3Na) and amino (–NH2) groups onto the PES surface was anticipated to endow these materials with antithrombotic and endothelialization properties.

Recently, numerous techniques have been carried out to incorporate bio-functional groups into the PES membrane to improve the blood compatibility, such as surface grafting, surface coating, blending, and bulk modification.20–22 Each of these techniques showed some inherent drawbacks. For example, blending antithrombogenic polymers into the membrane matrix is one of the simplest methods. However, the poor miscibility between the hydrophilic/amphiphilic polymers and the hydrophobic PES matrix usually resulted in serious phase separation, and thus limited the amounts of incorporated polymers and reduced the membrane mechanical stability.23 For surface coating, the lack of stability restricted the further application to obtain desired bio-functions.24,25 Surface grafting, which is considered as the most effective method for modification, usually needs complex physical or chemical treatments to obtain active functional groups, which might not only increase the cytotoxicity (e.g., the ATRP method), but also decrease the mechanical stabilities (e.g., the blending method). In our previous studies, bulk modification of the PES matrix was addressed in an effort to carboxylate, sulfonate or aminate the PES chain, and the modified PES showed good blood compatibility.19,26,27 However, these methods might not only reduce the mechanical and thermal stabilities, but also result in a lack of control in the degree of reaction and the chemical structure of the material. More than anything else, the material was incapable of introducing other functional groups, which limited the application of the PES matrix.28,29 Recently, we have synthesized a commercial affordable heparin-like PES (HLPES) with a similar chemical structure to PES by the polycondensation method. The synthesized HLPES could be directly mixed with pristine PES without phase separation at any ratios since the synthesized HLPES has the same backbone as PES.30 So the HLPES was anticipated to improve the hemocompatibility as an additive by blending. Although the synthesized HLPES showed many advantages, its application as a membrane material was limited owing to its low molecular weight, which would reduce the mechanical properties of the membranes.

In the present study, to address these problems, we designed a kind of PES membrane with a surface containing sodium carboxylic (–COONa), sodium sulfonic (–SO3Na) and amino (–NH2) groups for the first time by the combination of blending and surface-grafting methods. Both blending PES with carboxylic (–COOH) groups (CPES) and grafting water-soluble PES with sodium sulfonic (–SO3Na) groups and amino (–NH2) groups (SNPES) were performed via condensed polymerization. A PES membrane with a carboxylated surface (CM) was prepared by blending CPES and PES at different ratios. Since the prepared CPES has a similar backbone and molecular weight to the PES matrix, CPES could be directly mixed with pristine PES without phase separation at any ratios. Moreover, the blending ratio had no influence on the mechanical properties of the membranes and the number of sodium carboxylic (–COONa) groups could be controlled. Then, the surface grafting modification was performed by immobilizing SNPES onto CM, and the resulting surface of the membrane with sodium carboxylic (–COONa) groups, sodium sulfonic (–SO3Na) groups and amino (–NH2) groups (GCM) may have good hemocompatibility and cytocompatibility, which would allow it to have much better prospects in the modification of PES membranes than the other synthesized biocompatible macromolecules. The chemical composition, cross-sectional morphology, and water contact angle of the modified membranes were explored in detail. The blood compatibility of the modified membranes was confirmed by plasma protein adsorption, blood coagulation time and platelet adhesion. Meanwhile, the concentrations of thrombin–antithrombin (TAT), platelet factor 4 (PF-4), complement component (3a, 5a) and the leukocyte activation (CD11b) in blood were measured to investigate the activation degree of blood components. Moreover, the cell compatibility was investigated by using vein endothelial cells.

2. Experimental

2.1 Synthesis of CPES and SNPES

Materials and reagents are shown in the ESI. CPES was prepared on the basis of our earlier literature with a minor modification,30 and SNPES was prepared according to a reported study.8 The detailed information is provided in the ESI.

2.2 Preparation of PES/CPES blended membranes

PES/CPES blended membranes were prepared by using a phase-inversion technique, and the detailed procedures are shown in the ESI. In this work, the prepared membranes with PES/CPES ratios of 10/0, 9/1, 8/2, 7/3 and 6/4 were termed PES, CM-1, CM-2 and CM-3, and CM-4, respectively.

2.3 Preparation of SNPES grafted membranes

The process of modifying the PES membrane by grafting SNPES is shown in Scheme 1. N-Hydroxysuccinimide (NHS) and carbodiimide (EDC) were firstly dissolved in 0.1 M 2-(N-morpholino) ethane sulfonic acid (MES) (pH 4.5–5, adjusted with NaOH) buffer solution (the concentrations of NHS and EDC are 10 and 10 mg mL−1, respectively). Then, the blended CMs were immersed in the solution for 45 min at 4 °C to activate the carboxylic (–COOH) groups. After washing three times, the activated CMs were then immersed in SNPES solution in PBS (pH 7.2, adjusted with HCL) for 6 h and subsequently rinsed with PBS solution. Subsequently, the SNPES grafted CMs were immersed in 0.1 M NaCl solution for 2 h before rinsing with DI water thoroughly to remove the residual impurity. The grafted CMs with PES/CPES ratios of 10/0, 9/1, 8/2, 7/3 and 6/4 were termed PES, GCM-1, GCM-2, GCM-3, and GCM-4, respectively.
image file: c7bm00673j-s1.tif
Scheme 1 The preparation of CPES and SNPES and the process of SNPES grafting.

2.4 Characterization of the modified membranes

The prepared SNPES and CPES were characterized by Fourier transform infrared (FT-IR) and nuclear magnetic resonance (1H NMR) spectroscopy, and gel permeation chromatography (GPC). The modified membranes were investigated by scanning electron microscopy (SEM), surface X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), filtration test and water contact angle (WCA) measurement; the detailed characterization procedures are shown in the ESI.

The grafting yield of the SNPES was calculated using the following methods: a given mass of the SNPES was dissolved in PBS solutions (pH 7.2, adjusted with HCl), then the activated CMs were immersed in the SNPES/PBS solution. After the grafting process, the membranes were rinsed with DI water thoroughly to remove the adherent SNPES. Then, the used SNPES/PBS solution and rinsing DI water were collected and purified by dialysis against DI water for 3 days, and small molecular impurities could be removed. The rest of the SNPES was finally collected and dried, and the grafting yield (GY, mg cm−2) was calculated using eqn (1):

 
image file: c7bm00673j-t1.tif(1)
where ms (g) and mf (g) represented the weight of the dried SNPES before and after the grafting process, respectively, and A (cm2) is the area of the membrane. The experiments were repeated three times, and the data are expressed as mean ± SD.

2.5 Protein adsorption

Bovine serum albumin (BSA) and bovine serum fibrinogen (BFG) were used to study the protein adsorption. The detailed information is shown in the ESI.

2.6 Blood compatibility and endothelialisation evaluation of GCMs

The hemocompatibility of the modified membranes was evaluated by platelet adhesion and activation, clotting times, thrombin activation and blood-related complement activations. The endothelialization was investigated by the MTT method. The detailed information is provided in the ESI.

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This article does not contain any studies with animals performed by any of the authors.

3. Results and discussion

3.1 Characterization of SNPES and CPES

The chemical compositions of SNPES and CPES were characterized by FT-IR and 1H-NMR, as shown in Fig. S1 and S2. All the characteristic peaks of the SNPES (FT-IR: 1 241, 1 087, 1 025, and 3360 cm−1; 1H NMR: 7.994, 7.973, 7.773, 7.276, 7.061, 6.913, and 5.600 ppm) and the CPES (FT-IR: 3450 cm−1; 1H NMR: emerged peaks, 10.38 ppm) were observed in the FT-IR and 1H NMR spectra, indicating that SNPES and CPES had been successfully synthesized.8,31 The detailed analyses are presented in the ESI.

The molecular weight of polymers synthesized by the polycondensation method was affected by the polymerization temperature; a high molecular weight could be achieved under high temperature conditions. However, in the present study, gelation might occur when the reaction temperature is over 190 °C under the designed polymerization conditions. This finding was similar to that in a previous study.32 Considering the allowance of temperature fluctuation, the reaction was conducted at 180 °C, and CPES with an appropriate Mw of 53[thin space (1/6-em)]262 g mol−1 (SNPES, Mw = 47[thin space (1/6-em)]814 g mol−1) and a PDI of 1.28 (SNPES, PDI = 1.31) was obtained.

It is well-known that the molecular weight of polymers played an important part in mechanical properties.33 Previous studies using kinds of methods to elevate the hemocompatibility of the PES membranes have yielded mixed evidence for the importance of the molecular weight.20–27 The reduced mechanical and thermal stabilities of the modified PES membranes can be attributed to the cleavage of the molecular chain during the modification. In this study, it should be noted that the MW of CPES was similar to that of PES (∼54[thin space (1/6-em)]000 g mol−1), therefore, there was no significant effect on the mechanical properties and film-forming capabilities for the PES membrane after blending. Furthermore, the mechanical properties also might be affected by the structure of the modified membranes.

3.2 Composition and structure of the GCMs

3.2.1 ATR-FTIR and XPS. The ATR-FTIR spectra of GCM-4 and CM-4 are shown in Fig. S3, and the XPS spectrum of the GCM-4 surface is presented in Fig. S4. The results demonstrated that sodium carboxylic (–COONa), sodium sulfonic (–SO3Na) and amino (–NH2) groups were successfully introduced onto the modified membrane surfaces. The detailed analyses are presented in the ESI.
3.2.2 Morphology of the membranes. Fig. 1 shows the cross-sectional SEM micrographs for the membranes. As shown in the figure, the characteristic morphology of the asymmetric membrane consisting of a dense top-layer and porous sub-layer with a finger-like structure was observed. For the GCMs (GCM-1 to GCM-4), the morphology of finger-like structures became irregular and finally disappeared with the increase of CPES. The change of the morphology was due to the addition of the hydrophilic macromolecules, which may result in the changes of water flux.
image file: c7bm00673j-f1.tif
Fig. 1 Cross-sectional SEM images of the pristine PES membranes, GCM-1, GCM-2, GCM-3 and GCM-4.

To further investigate the surface morphology of the membranes, AFM was utilized with a tapping mode at a scan rate of 0.8 Hz over an area of 10 × 10 μm2, as shown in Fig. 2. The pristine PES membrane was relatively smooth, while CM-4 was rough, and the cuspidal outshoots on the CM surfaces could be attributed to the addition of hydrophilic CPES. When the SNPES was grafted onto the surfaces of the CMs, the modified membranes became much rougher and the density of the cuspidal outshoot increased with the increase of CPES proportions in the matrix. The additional cuspidal outshoots, belonging to the aggregation of the grafted SNPES chains, appeared on the surfaces, indicating that the SNPES was successfully grafted onto the GCM surface.


image file: c7bm00673j-f2.tif
Fig. 2 AFM images for the pristine PES membranes, CM-4, GCM-1, GCM-2, GCM-3 and GCM-4.
3.2.3 SNPES grafting degree. The grafting yields of SNPES were evaluated, as shown in Fig. 3. The grafting yield increased from 0.893 to 3.245 mg cm−2 with the increase of the CPES ratios in the matrix. The values were larger than those grafted onto the membranes by other methods, such as the photochemical technique.34 These results suggested that the amount of sodium carboxylic (–COONa) groups on the PES membrane surface could be well-controlled.
image file: c7bm00673j-f3.tif
Fig. 3 Amounts of SNPES grafted onto the CM surface. (Values are expressed as means ± SD, n = 3.)
3.2.4 Water contact angles of the membranes. Water contact angle (WCA) analysis is a convenient method to assess the hydrophobic/hydrophilic property of the membrane surface, and the WCAs of the pristine PES and modified membranes are shown in Fig. 4. The pristine PES membrane possessed the highest contact angle of about 77.6°, corresponding to the lowest surface hydrophilicity. For the CMs, the WCA decreased from 77.6° to around 50° with the increase of CPES proportions from 0% to 40%. These findings indicated that the hydrophilicity of the PES membrane could be improved by blending the hydrophilic CPES. After grafting SNPES, the WCA further decreased for the reason of introducing hydrophilic functional groups onto the surface, and the reduced degree showed a positive correlation with the proportions of CPES in the matrix. The improvement of hydrophilicity was owing to the introduction of the hydrophilic sodium carboxylic (–COONa), sodium sulfonic (–SO3Na) and amino (–NH2) groups and the rougher surface which was demonstrated by the AFM images. The enhanced hydrophilicity of membranes could improve the flux and anti-protein-fouling ability of the membranes, which would be further examined by the water flux test and protein adsorption analysis.
image file: c7bm00673j-f4.tif
Fig. 4 Water contact angles of the pristine PES membranes, CMs and GCMs. (Values are expressed as means ± SD, n = 3.)
3.2.5 Water flux. The water flux test was carried out to determine the filtration property of the membranes. The water fluxes of the pristine PES membrane, CM-1, CM-2, CM-3 and CM-4 were 23.3, 47.5, 69.4, 81.8 and 96.2 mL (m2 h mmHg)−1, respectively. The CMs exhibited a higher water flux than the pristine PES membrane, which was probably due to the increased hydrophilicity of the membrane surface depending on the carboxylic acid (–COOH) groups from the blending CPES. As indicated by SEM, a regular finger-like asymmetric structure was formed in the preparation of the pristine PES membrane when DMAc/water is used as the solvent/non-solvent pair. After blending with hydrophilic CPES, the regularity of the finger-like asymmetric structure decreased, and the pore diameter of the closed cells increased with the increase of the CPES content. This process effectively enhanced the pore area of the cross-sectional morphologies; and subsequently increased the water flux to a certain extent. After grafting SNPES, the water fluxes were 152.6, 386.1, 618.4 and 894.3 mL (m2 h mmHg)−1 for GM-1, GM-2, GM-3 and GM-4, respectively. It could be noted that the water flux for the PES/CPES blended membranes was significantly affected by the grafted SNPES, which was owing to the enhanced hydrophilicity of the membrane surface.

3.3 Protein adsorption

Protein adsorption is an important parameter to evaluate the hemocompatibility of biomaterials, since growing lines of evidence showed that the adsorption of nonspecific plasma proteins occurs on the surface as soon as the blood comes into contact with a biomaterial, and some specific proteins (e.g., fibrinogen) in the blood might trigger platelet adhesion and activation of coagulation pathways, leading to thrombus formation.35 Thus, the amount of protein adsorbed on the material surface is one of the most essential factors in evaluating the blood compatibility of the materials. There are many factors that affect the interaction between the membrane surface and proteins, such as the surface charge character, surface free energy, topological structure, solution environment, and protein characteristics; and the hydrophilicity/hydrophobicity of the membrane plays a relatively important role in the interaction between the protein and membrane. Thus, many studies have followed the idea of increasing the hydrophilicity of membrane materials.36–38 In this study, protein adsorption on the membrane surface was evaluated in relation to the adsorption of BSA and BFG in vitro, since BSA and BFG are the most typical plasma proteins that might have a direct relationship with blood compatibility and cell attachment.

Fig. 5 shows the adsorbed amounts of BSA and BFG onto the pristine PES membrane, GCM-1, GCM-2, GCM-3 and GCM-4 surfaces. As shown in the figure, it was found that about 17 μg cm−2 BFG was adsorbed on the surface of the pristine PES membrane, and the amounts of adsorbed BFG decreased with the addition of SNPES for the GCMs. However, for BSA adsorption, it was found that the BSA adsorbed amounts increased with increasing the proportions of CPES in the matrix.


image file: c7bm00673j-f5.tif
Fig. 5 BSA and BFG adsorbed amounts on pristine PES membranes and GCMs. (Values are expressed as means ± SD, n = 3.)

According to the results in the protein adsorption experiment, the lower BFG adsorption amounts might be attributed to the synergistic influence of the hydrophilic sodium carboxylic (–COONa), sodium sulfonic (–SO3Na) and amino (–NH2) groups from CPES and SNPES. It is well known that the increased membrane hydrophilicity plays a relatively important role in reducing BFG adsorption and/or protein fouling, and the reduced BFG adsorption was in good agreement with the increased hydrophilicity of the membrane surfaces.39 Furthermore, negatively charged groups exhibited relatively low BFG adsorption amounts. Therefore, the hydrophilicity and the negatively charged groups of the membrane surfaces should be responsible for the decrease of the BFG adsorption. In general, it is believed that the adsorption of BFG onto a membrane usually provides a good indication of hemocompatibility, since the fibrinogen in blood plasma is particularly important for blood platelet adhesion.40 For BSA, the adsorbed BSA did not present a significant decrease; considering the increased hydrophilicity of the GCMs, these results were different from the early report.41 However, some researchers reported that sulfonic acid groups exhibited certain adsorption capacity to BSA through an exothermal process,42 meanwhile, it is well known that biomaterials modified with amino (–NH2) groups are in favor of BSA adsorption.19 Therefore, the increased BSA adsorption on the GCM surface might be caused by the sodium sulfonic (–SO3Na) and amino (–NH2) groups, and the results suggested that the membrane surface enriched with sodium sulfonic (–SO3Na) and amino (–NH2) groups might have high binding affinity toward BSA, which might have positive influence on cell attachments.43,44

3.4 Blood compatibility

3.4.1 Platelet adhesion and activation. For blood-contacting biomaterials, the extent of platelet adhesion and platelet aggregation on the material surface is considered to be a key step in thrombus formation. After platelet adhesion, a series of actions could cause platelet activation, and the activated platelets then accelerated thrombin formation and led to further coagulation. Platelet factor 4 (PF-4) is a small cytokine belonging to the CXC chemokine family that is also known as chemokine ligand 4 (CXCL-4), which is released from the alpha-granules of activated platelets during platelet aggregation, and promotes blood coagulation by moderating the effects of heparin-like molecules.45 Therefore, in this study, the morphologies of the adherent platelets were observed by SEM and the level of platelet activation was evaluated via the concentration of PF-4. The results are presented in Fig. 6.
image file: c7bm00673j-f6.tif
Fig. 6 (A) Scanning electron micrographs of the platelets adhering onto the pristine PES membrane, GCM-1, GCM-2, GCM-3 and GCM-4. (B) The generated concentrations of PF-4 for the samples with whole blood. (Values are expressed as means ± SD, n = 3. P < 0.05.)

Fig. 6A shows the SEM micrographs of the platelets adhering onto the pristine PES membrane and GCMs. As shown in the figure, numerous platelets adhered and aggregated on the pristine PES membrane surface. The platelets spread into irregular shapes, and lots of pseudopodium can be seen clearly. Platelet aggregation indicated that thrombus formation, which is a life-threatening phenomenon for patients, might occur at the surface of the pristine PES membrane. However, for GCMs, very few platelets were found, and the platelets expressed a round morphology with nearly no pseudopodium and deformation. In addition, the amounts of the adhering platelets decreased remarkably with increasing proportions of CPES in the matrix, and the floccule observed on the GCM surface in the analysis of SEM was the adsorbed plasma proteins.

Fig. 6B shows the concentrations of PF-4 for the membranes with whole blood. As shown in the figure, the results of platelet activation showed a slight increase in the concentration of PF-4 for the pristine PES membrane, and with an increase in the amounts of sodium carboxylic (–COONa), sodium sulfonic (–SO3Na) and amino (–NH2) groups on the surface of membranes, the PF-4 concentration decreased. For GCM-4, the concentration of PF-4 showed no difference from that of the control samples (P > 0.05). These results indicated that the platelet activation for the modified membranes with sodium carboxylic (–COONa), sodium sulfonic (–SO3Na) and amino (–NH2) groups was significantly suppressed compared to the pristine PES membrane.

The platelet adhesion and activation results were consistent with the previous WCA analysis and BSA/BFG adsorption, which suggested that these phenomena were caused by the introduction of the hydrophilic groups and the resulting increase in hydrophilicity and the relatively high BSA and low BFG adsorption. In this work, introducing sodium carboxylic (–COONa) and sodium sulfonic (–SO3Na) groups endowed the PES matrix with an anionic surface, and the platelet (anionic surface, too) adhesion could be minimized owing to the electrostatic repulsion of the negatively charged surface. Furthermore, plasma protein adsorption occurs as soon as the blood is in contact with the biomaterial and has an important influence on platelet adhesion and activation. The attached BSA molecules are able to form a natural barrier resisting non-specific protein (e.g., BFG) adsorption, and the platelet aggregation is mediated by the binding of the BFG to a platelet cell surface receptor.46,47 As a result, surface-induced platelet activation was depressed, and platelet adhesion and aggregation were minimized.

3.4.2 Clotting time. The measurement of blood clotting time has already been proved to be applicable to estimate the blood compatibility of a biomaterial/blood interface. In this study, APTT and TT were used to evaluate the blood compatibility of the membranes. In general, APTT was used to measure the inhibition efficacy of both the intrinsic and the common coagulation pathways including factors II, V, X, XII or fibrinogen.48 TT was used to measure the clot formation time taken for the thrombin converted fibrinogen into fibrin.

Fig. 7 shows the APTT and TT test results of the membranes. From Fig. 7A, it is found that the APTTs for the CMs were slightly prolonged compared with that of the pristine PES membrane (P < 0.05). With an increase in the content of the CPES, the APTT of the PES/CPES blended membranes increased, resulting in nearly 48% higher than that of the pure PES membrane when the blending ratio was 40%. However, when the SNPES was grafted on the CMs, the APTTs were significantly prolonged, which might be attributed to the hydrophilicity of the grafted sodium sulfonic (–SO3Na) groups. The GCM-4 shows clotting times approximately three times as long as APTT compared with the pristine PES membrane (P < 0.05). Nevertheless, the contrary phenomenon could be found in the TT results, and its increment range was higher before grafting and lower after the process (Fig. 7B). The results suggested that sodium sulfonic (–SO3Na) had more influence on the endogenous pathway of coagulation and the sodium carboxylic (–COONa) groups might be more effective in prolonging the TTs.


image file: c7bm00673j-f7.tif
Fig. 7 (A) Activated partial thromboplastin times (APTTs) of the poor platelet plasma (PPP), pristine PES membranes, CMs and GCMs. (Values are expressed as means ± SD, n = 3. P < 0.05.) (B) Thrombin times (TTs) of the poor platelet plasma (PPP), pristine PES membranes, CMs and GCMs. (Values are expressed as means ± SD, n = 3. P < 0.05.)

The improvement of anticoagulant activity was in good agreement with the increased hydrophilicity. In this case, we propose that these phenomena result from more differences in the surface energy of the respective substrates. The GCMs could be considered as heparinized membranes owing to the existence of functional groups (–COONa, –SO3Na) similar to heparin. Rhodes et al. studied the intrinsic pathway by observing the rate of clotting diminish by virtue of factor XIIa and reported that heparinized materials could be distinguished from non-heparinized materials.49 In another way, Nagahara and co-workers found that carboxylic acid could be considered as one of the most promising factors as an Xa inhibitor.50 Furthermore, the role of Ca2+ in coagulation has been known since the late 19th century, and its depletion (e.g., using –COOH chelation) prevents blood coagulation as Ca2+ is required at several stages of the coagulation cascade.51 Apart from these, Silver et al. demonstrated that sulfonated polymers do not directly inhibit the fibrin assembly, but rather complex free Ca2+ or interfere with factor XIIIa.52 It is well known that factor XIIa plays a critical role in the intrinsic coagulation cascade, factor XIIIa can mediate the cross-linking of fibronectin into stable collagen, and factor Xa, whose major practical role is the generation of thrombin by the limited proteolysis of prothrombin, holds a central position that links the intrinsic and extrinsic activation mechanisms in the final common pathway of coagulation. As such, the synergistic influence of sodium carboxylic (–COONa) and sodium sulfonic (–SO3Na) groups was of vital importance for enhancing the blood compatibility. In addition, these also might help to explain why sodium sulfonic (–SO3Na) had more influence on the endogenous pathway of coagulation and the sodium carboxylic (–COONa) groups had more influence on TTs. Moreover, the improvement of anticoagulant activity was also in good agreement with the decreased BFG adsorption, suppressed platelet adhesion and activation.

In our earlier report,30 the APTTs and TTs of blending heparin-like (–SO3Na, –COONa groups) surface membranes with PES/HLPES ratios of 15/5, 10/10 and 5/15 were 60s/21s, 104s/22s, and 118s/23s, respectively. Comparing the results of the clotting time in this study, it was found that the APTT and TT for the membrane surface with –SO3Na, –COONa and –NH2 groups were longer than those for the surface with –SO3Na and –COONa groups. This might be due to the adsorption of BSA by the –NH2 groups in the blood compatible modification of PES membranes and the amount of the functional groups.19,53 Different characteristics of the –COONa, –SO3Na and –NH2 groups led to different surface properties. These results suggested that the synergistic influence of sodium carboxylic (–COONa), sodium sulfonic (–SO3Na) and amino (–NH2) groups was of vital importance for enhancing the blood compatibility.

3.4.3 Thrombin activation. The TAT level in plasma after the membranes came into contact with blood is shown in Fig. S4. The detailed analyses are presented in the ESI. The TAT results indicated that the generated TAT level of the PES membrane was ameliorated through the modification.
3.4.4 Complement activation. Complement activation is the triggering of the host defense mechanism generated by the localized inflammatory mediator. The complement system contains about 30 different plasma proteins that act powerfully in the body's immuno-potentiation and coagulation systems, often through the surface activation of pro-enzymes.54 The anaphylatoxins C3a and C5a are liberated as activation byproducts and are potent pro-inflammatory mediators that bind to specific cell surface receptors and cause leukocyte activation.55 In this study, the complement activation was evaluated via the concentrations of C3a and C5a, respectively, and the results are presented in Fig. 8.
image file: c7bm00673j-f8.tif
Fig. 8 (A) The concentrations of C3a for the samples with whole blood (values are expressed as means ± SD, n = 3. P > 0.05); (B) the concentrations of C5a for the samples with whole blood. (Values are expressed as means ± SD, n = 3. P > 0.05.)

As shown in Fig. 8A, the pristine PES membrane showed slightly higher C3a generation compared to the control. After introducing sodium carboxylic (–COONa), sodium sulfonic (–SO3Na) and amino (–NH2) groups on the surface of the membranes, the activated C3a concentrations decreased compared with that of the pristine PES membrane and showed no significant difference from the control sample (P > 0.05). Significantly, similar results are observed for the C5a concentrations (Fig. 8B). The pristine PES membrane showed a significant increase in C5a level, and no significant difference in C5a levels was observed for the GCMs when compared to the control sample. The results of complement activation demonstrated that the pristine PES membrane had a negative effect on the activations of C3a and C5a in the blood, and the GCMs successfully inhibited the formation of soluble C3a and C5a in blood plasma compared with the pristine PES membrane.

It can be seen that the complement activation results were consistent with the previous clotting time measurement, which indicated that the inactivation of factor XII could be achieved by constructing a heparin-like structure. Holmer and coworkers demonstrated similarities in the inhibition of factor Xa, factor XIIa and kallikrein.56 This suggests the possibility that the factor XIIa-inhibiting property of the GCMs was accompanied by anti-kallikrein and anti-factor Xa properties. Gorbet et al. demonstrated that the complement and coagulation cascades interact significantly: factor XIIa and kallikrein in plasma have the capacity to trigger the complement cascade, and thrombin activates C3, C5, C6, and factor B,57 which in turn implies that the antithrombotic properties of GCMs may be related to the ability to potentiate the inactivation of the complement system.

3.4.5 Inflammatory response. The expressions of CD11b on monocytes and granulocytes in blood are shown in Fig. S5. The detailed analyses are presented in the ESI. The results indicated that the activation of leukocytes could be significantly inhibited by GCMs.

3.5 Endothelialization

It is well known that PES membranes have been widely employed in biomedical fields such as tissue engineering, artificial organs and bio-artificial blood vessels and in medical devices used for blood purification.1 Thus, clinical field related cell proliferation played a critical role in modifying membranes. In this study, human umbilical vein endothelial cells (HUVECs) are selected for the evaluation of the cell proliferation of the membranes.
3.5.1 Fluorescence staining. Fig. 9A shows the fluorescence pictures of the endothelial cells cultured for 6 days on the pristine PES membranes, CMs and GCMs. The amount of HUVECs grown on the pristine PES membrane was the least, while the cells spread and covered almost the whole surfaces and flattened with a larger attachment area on the surfaces of the GCMs, especially on the GCM-4. For the CMs, it could be noted that the amount of HUVECs was larger than that of the PES membrane but smaller than that of the relative GCMs. The results suggested that the sodium carboxylic (–COONa), sodium sulfonic (–SO3Na) and amino (–NH2) groups were all beneficial to cell proliferation and the membrane surface enriched with sodium carboxylic (–COONa), sodium sulfonic (–SO3Na) and amino (–NH2) groups could promote HUVEC attachment and growth.
image file: c7bm00673j-f9.tif
Fig. 9 (A) Fluorescence staining (FITC) pictures of vein endothelial cells cultured on the PES, CM-1, CM-2, CM-3, CM-4, GCM-1, GCM-2, GCM-3 and GCM-4 after 6 days. (B) MTT tetrazolium assay. Formazan absorbance was expressed as a function of time from endotheliocytes seeded onto different membranes and the controls. (Values are expressed as means ± SD, n = 3, *P < 0.05, **P < 0.05 and ***P < 0.05 compared with the values for the pristine PES membrane at 2, 4 and 6 days, respectively; #P < 0.05 between days for the same sample.)
3.5.2 Cell viability tested by MTT assay. To further evaluate cytotoxicity, cell proliferation and activation of the GCMs, the MTT tetrazolium salt colorimetric assay technique was employed. Fig. 9B shows the cell viability results of the MTT assay, and the results were analysed by statistical methods (P < 0.05). As shown in the figure, the viability of the cells on each membrane increased with the increase of culture time. Moreover, it is observed that on the second, fourth and sixth days, the viability of the cells on the GCMs increased compared with that of the control sample and the pristine PES membrane. Moreover, the GCMs retained a better viability during the whole culture time, which indicated that the introduction of sodium carboxylic (–COONa), sodium sulfonic (–SO3Na) and amino (–NH2) groups might have a positive influence on the cytocompatibility of the membranes.

According to several trials and reviews reported in the literature, the sodium carboxylic (–COONa) and sodium sulfonic (–SO3Na) groups were expected to be favorable for cell proliferation by the bonding and stabilizing of cell growth factors,58 and the HUVECs might be bound to sodium carboxylic (–COONa) and sodium sulfonic (–SO3Na) groups, thus facilitating the adherence to PES substrates. Thus, the sodium carboxylic (–COONa) and sodium sulfonic (–SO3Na) group immobilized surface was expected to be favorable for cell proliferation. Meanwhile, the HUVECs, which are negatively charged, communicate with the membrane through the adsorbed protein layers via specific recognition and binding sites between the adsorbed proteins and cell membranes, as such, a large amount of adsorption of BSA might have a positive influence on the cell adhesion. Apart from this, serum albumin was a specific inhibitor of apoptosis in human endothelial cells.59 Therefore, the biomaterial surface enriched with amino (–NH2) groups might be conductive to the adsorption of BSA, which might have a positive influence on the cell adhesion, thus promoting the surface endothelialization. For these reasons, the cytocompatibility of GCMs was enhanced.

To sum up, better blood compatibility and cytocompatibility of PES membranes with HUVECs could be obtained by introducing sodium carboxylic (–COONa), sodium sulfonic (–SO3Na) and amino (–NH2) groups on the surface of the PES membrane, and the modified membranes had the potential to be used for blood purification, tissue engineering and artificial blood vessels.

It was reported that the heparin-like (–SO3Na, –COONa) structure surface had a positive effect on the cytocompatibility of membranes.30 Considering the results of the MTT assay, it could be noted that the surface with –COONa, –SO3Na, and –NH2 groups showed higher viability in comparison with the surface with –SO3Na and –COONa groups. The reason for the phenomenon might be the synergistic influence of BSA adsorption (increasing affinity) and heparin-mimicking (bonding growth factors) and the amount of the functional groups,8,53 which demonstrated that the synergistic influence of sodium carboxylic (–COONa), sodium sulfonic (–SO3Na) and amino (–NH2) groups was of vital importance for cytocompatibility.

4. Conclusions

In this paper, a kind of highly hemocompatible PES-based membrane with a surface containing sodium carboxylic (–COONa), sodium sulfonic (–SO3Na) and amino (–NH2) groups was prepared by a combination of blending and surface-grafting methods. The modified PES-based membranes were prepared through two steps: first, PES was blended with the hydrophilic CPES in the solvent of DMAc, and then the membranes were prepared by phase-inversion, using a liquid–liquid phase separation technique; then, the SNPES was grafted onto the surface of the membranes through coupling of amino groups and the carboxyl groups by using water-soluble carbodiimide and N-hydroxysuccinimide. Since the CPES can be directly blended with the PES matrix at any ratios to form a miscible polymer, there was no significant effect on the mechanical properties and film-forming capabilities for the PES membrane after the blending. The ATR-FTIR, XPS and AFM results confirmed that the SNPES was successfully grafted onto the membranes. The SEM results suggest that the existence of the CPES alters the morphology of the membranes and the water flux test results indicated that the CPES and SNPES have a considerable effect on the water flux. After the modification, BSA adsorption was increased whereas BFG adsorption was decreased, and in vitro testing of the GCMs compared to the pristine PES membranes showed improvements in the in vitro blood compatibility in terms of platelet adhesion and activation, clotting times, thrombin activation, complement activation and inflammatory response. Furthermore, the cell fluorescence staining observation and cytotoxicity assays demonstrated that the GCMs showed superior performance in endothelial cell adhesion and proliferation. The obtained results suggested that the introduction of sodium carboxylic (–COONa), sodium sulfonic (–SO3Na) and amino (–NH2) groups conferred the modified membranes with excellent hemocompatibility and cytocompatibility of PES membranes with HUVECs, and the GCMs may have a promising future for applications in the biomedical field such as blood purification, surface endothelialization and bio-artificial vessels.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was financially sponsored by the National Natural Science Foundation of China (no. 31470926, 51433007, 31500778 and 51503073) and Major Natural Science Fund for Colleges and Universities in Jiangsu Province (No. 17KJA530002). We should also thank our laboratory members for their generous help, and gratefully acknowledge the help of Ms. H. Wang of the Analytical and Testing Center at Sichuan University, for the SEM, and Ms Liang of the Department of Nephrology at West China Hospital, for the collection of fresh human blood.

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Footnote

Electronic supplementary information (ESI) available. See DOI: 10.1039/c7bm00673j

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