Immobilization of sodium alginate sulfates on polysulfone ultrafiltration membranes for selective adsorption of low-density lipoprotein

Acta Biomaterialia 10 (2014) 234–243

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Immobilization of sodium alginate sulfates on polysulfone ultrafiltration membranes for selective adsorption of low-density lipoprotein Wei Wang a,⇑, Xiao-Jun Huang b, Jian-Da Cao a, Ping Lan a, Wen Wu a a

College of Materials and Textile Engineering, Jiaxing University, Jiaxing 314001, China Key Laboratory of Macromolecular Synthesis and Functionalization, Ministry of Education, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China b

a r t i c l e

i n f o

Article history: Received 26 April 2013 Received in revised form 15 August 2013 Accepted 26 August 2013 Available online 3 September 2013 Keywords: Polysulfone Sodium alginate sulfates Low-density lipoprotein Selective adsorption Surface modification

a b s t r a c t A novel method for the immobilization of sodium alginate sulfates (SAS) on polysulfone (PSu) ultrafiltration membranes to achieve selective adsorption of low-density lipoprotein (LDL) was developed, which involved the photoinduced graft polymerization of acrylamide on the membrane and the Hofmann rearrangement reaction of grafted acrylamide followed by chemical binding of SAS with glutaraldehyde. The surface modification processes were confirmed by attenuated total reflectance Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy characterization. Zeta potential and water contact angle measurements were performed to investigate the surface charge and wettability of the membranes. An enzyme-linked immunosorbent assay was used to measure the binding of LDL on plain and modified PSu membranes. It was found that the PSu membrane immobilized with sodium alginate sulfates (PSuSAS) greatly enhanced the selective adsorption of LDL from protein solutions and the absorbed LDL could be easily eluted with sodium chloride solution, indicating a specific and reversible binding of LDL to SAS, mainly driven by electrostatic forces. Furthermore, the PSu-SAS membrane showed good blood compatibility as examined by platelet adhesion. The results suggest that the PSu-SAS membranes are promising for application in simultaneous hemodialysis and LDL apheresis therapy. Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction Cardiovascular and cerebrovascular diseases have become a major health hazard for human beings. It is now believed clinically that the elevated level of low-density lipoprotein (LDL) in human blood is one of the key causative elements for the progression of atherosclerosis and raises the risk of heart attacks [1–3]. LDL is the major cholesterol transporter in blood, so the higher LDL concentration in plasma, the more cholesterol accumulation on the inner wall of blood vessel, which may lead to the gradual formation of atherosclerotic plaque and progress to various cardiovascular or cerebrovascular diseases [4]. Therefore, the reduction of LDL level in human blood is one of the most important interventions to prevent and cure cardiovascular diseases [5,6]. Most cases with routinely elevated LDL levels can be effectively treated using diet and drugs, while, in some familial hypercholesterolemia instances and severely affected patients who fail medical management, extracorporeal treatment by means of LDL apheresis is most widely used for its strong effect in reducing LDL cholesterol and clinical end-points [7–9]. At present, there are several common LDL apher⇑ Corresponding author. Tel.: +86 573 83640663; fax: +86 573 83643022.

esis technologies for routine clinical service, such as double-filtration plasmapheresis [10], immunoadsorption [11], heparininduced extracorporeal LDL precipitation [12,13], adsorption on dextran sulfate–cellulose [14] and direct adsorption of lipoprotein from whole blood using polyacrylamide beads coated with polyacrylic acid [15]. The therapeutic effects of these techniques are largely dependent on the performance of the applied adsorbents for LDL. Therefore, much attention has been attracted to developing more economical and effective adsorbents for LDL apheresis [16– 20]. For fabrication of LDL adsorbents, a major area of concern is the selection of an appropriate ligand and matrix. Heparin, a highly sulfated glycosaminoglycan, is regarded as one of the most effective ligands, which can interact with apolipoprotein B-100 of LDL via electrostatic attractions [21–23]. For this reason, heparin has been introduced to different matrix polymers to prepare LDL adsorbents, such as heparin-coupled Sepharose [23] and heparinmodified poly(vinyl alcohol) granules [24]. In our previous work, heparin was covalently immobilized on polysulfone (PSu) films to obtain a dialysis membrane with selective adsorption of LDL [25–27]. It was found that PSu film immobilized with heparin (PSu-Hep) exhibited excellent blood compatibility and could selectively but reversibly bind LDL from protein mixtures. However,

E-mail address: [email protected] (W. Wang). 1742-7061/$ - see front matter Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.actbio.2013.08.032

W. Wang et al. / Acta Biomaterialia 10 (2014) 234–243

heparin from animal sources has the potential to induce disease affecting mammals, such as the avian influenza virus and bovine spongiform encephalopathy [28]. These concerns strongly motivate the development of a new ligand to replace heparin for LDL adsorbents. Sodium alginate (SA), a linear anionic polysaccharide, has been widely used in the biomedical, pharmaceutical, cosmetic and edible fields for decades due to its excellent biocompatible, non-toxic, non-immunogenic and biodegradable properties [29]. There is currently a trend to create ‘‘value-added’’ SAs, by performing derivatization reactions on the polysaccharide backbone. For example, sulfated modification may enable SAs to achieve heparin-like anticoagulation properties and improved cell-surface interactions [30]. The structures of sodium alginate sulfate (SAS) and heparin are very similar, due to the presence of sulfonic and carboxyl groups on a polysaccharide backbone. Therefore, we suppose that SAS may be used as a ligand to replace heparin for preparing adsorbents applied in LDL apheresis. In 2009, World Kidney Day called for worldwide attention to the inseparable relationship between chronic kidney disease (CKD) and cardiovascular disease [31]. CKD is associated with a significant increase in cardiovascular risk, and most CKD patients die of a cardiovascular cause, with the majority induced by atherosclerosis. Thus LDL reduction is also an important intervention in the management of CKD. For patients with chronic renal failure and LDL-induced coronary heart disease, a simultaneous LDL apheresis and hemodialysis procedure is specifically required [32]. As PSu is currently the most widely applied polymer for hemodialysis membranes and enables the efficient removal of small- to medium-sized molecules [33,34], it was chosen as a matrix for SAS immobilization. Herein, a method to immobilize SAS on PSu ultrafiltration membranes with the aim of preparing LDL adsorbent for simultaneous LDL apheresis and hemodialysis is conceived, as schematically described in Scheme 1. First, acrylamide (AAm) groups were grafted onto membrane surface by ultraviolet (UV) irradiation-induced graft polymerization and then transformed into primary amine (Am) groups by the Hofmann rearrangement reaction. Then the formed amine groups were coupled with the hydroxyl groups of SAS in the presence of glutaraldehyde. The surface properties of plain and modified films were analyzed by

235

attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), zeta potential (ZP), water contact angle (WCA), scanning electron microscopy (SEM) and a platelet adhesion experiment. Enzymelinked immunosorbent assays (ELISAs) were performed to investigate the ability of membranes to bind LDL selectively.

2. Experimental section 2.1. Materials PSu ultrafiltration membranes were supplied by Fresenius Medical Care, Bad Homburg, Germany. All the membranes used in this study were cut into circles with a diameter of 2.5 cm (area = 4.91 cm2). Before use, the membranes were dipped in ethanol (analytical grade) for 1 h and then rinsed with water for several times to remove any impurity adsorbed on the surface. After drying in a vacuum oven at 40 °C for 4 h, these membranes were stored in a desiccator. SA, with low molecular weight (Mw = 10,000–12,000) for pharmaceutical purposes, was purchased from Alfa Aesar company. Sodium hydroxide (analytical grade), sodium hypochlorite (5 wt.% aqueous solution), glutaraldehyde (25 wt.% aqueous solution) and hydrogen peroxide (30% aqueous solution) were commercial products and were used without further purification. LDL (4.6 mg ml1, Millipore), 3,30 ,5,50 -tetramethylbenzidine (TMB; Care Roth GmbH) and flat ELISA plates (Greiner Bio-One, Germany) were purchased and used as received. Human serum albumin (HSA), bovine serum albumin (BSA), primary antibody anti-b-lipoprotein, secondary antibody anti-chicken IgY (IgG) and Tween 20 were purchased from Sigma–Aldrich and were used as received. All water used was deionized and ultrafiltrated to 18 MO with a TKA MicroPure Water system.

2.2. Sulfation of sodium alginate SASs with a degree of substitution of 1.13 were prepared from SA through the sulfation reaction with chlorosulfonic acid in formamide (see Supporting information S1).

Scheme 1. Schematic diagram illustrating the immobilization of SAS on PSu membrane.

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2.3. Preparation of PSu-Am

2.5. Characterization

An AAm layer on the membrane surface was generated by UV irradiation-induced graft polymerization (see Supporting information S2). The grafted AAm groups were converted to Am groups by the Hofmann rearrangement reaction to produce a chemically activated membrane surface, as described in Scheme 1. Detailed processes can be found elsewhere [35,36]. Briefly, 5 ml of sodium hypochlorite aqueous solution was added into 95 ml sodium hydroxide solution (32 mmol l1) dropwise at 0 °C to give a reaction reagent. PSu film tethered with AAm (PSu-AAm) was immersed in this mixture and stirred for 1 h at 0 °C. After stirring for another hou at 25 °C, the membrane was rinsed with water and dried under a vacuum at 40 °C. For quantificational analysis of the modified sample, the density of AAm (DAAm, lmol cm2) grafted onto the membrane surface was defined as

2.5.1. FTIR and XPS ATR-FTIR measurements were carried out on a Vector 22 FTIR spectrometer (Brucker Optics, Switzerland) equipped with an ATR cell (KRS-5 crystal, 45°). For each spectrum, 16 scans were taken at a normal resolution of 2 cm1. XPS spectra were performed on a PHI-5000C ESCA system (Perkin-Elmer, USA) with Al Ka radiation (hv = 1486.6 eV). Binding energies were calibrated using the containment carbon (C1s, 284.7 eV). Survey spectra (from 0 to 1200 eV) with a normal resolution of 1 eV and core–lever spectra with much higher resolution (0.1 eV) were both recorded.

DAAm ¼

W1  W0 71Sm

and the density of Am (DAm, lmol cm2) on the membrane surface was defined as

DAm ¼

W1  W2 ð71  43ÞSm

where W0, W1 and W2 are the masses of the nascent membrane, the membrane after graft polymerization and the membrane after Hofmann rearrangement reaction, respectively. Sm represents the surface area of the membrane. The molecular weight of the repeat unit of grafted AAm chains is 71, and 43 represents the molecular weight of the repeat units (–CH2CHNH2–, seen in Scheme 1) on the grafted chains after the Hofmann rearrangement reaction. The efficiency of Hofmann rearrangement reaction (RHofmann) was defined as the ratio of the resulted primary Am groups to the grafted AAm:

RHofmann ¼

DAm DAAm

2.4. Immobilization of SAS on PSu membrane SAS was immobilized onto membrane surface by the reaction between hydroxyl and primary amine groups with glutaraldehyde as bifunctional linker molecules. PSu film activated with Am (PSuAm) was put in a glutaraldehyde solution (5 ml, 5 wt.%) and the mixture was shaken in a flask for 3 h at 25 °C. The membrane was then rinsed with water 6–8 times and immersed in a SAS solution (5 ml, 3 wt.%). After shaking for another 3 h at 25 °C, the membrane was washed vigorously with a vibrator and dried under a vacuum at 40 °C. As a control, another PSu-Am sample was treated following the same procedures but without the addition of SAS. The binding density of SAS (DSAS, lg cm2) to the membrane surface was calculated as:

DSAS ¼

W4  W3 Sm

where W4 is the mass of the membrane after SAS binding. W3 is the mass of the control sample without SAS binding. Each result was the average of three parallel experiments. For comparison purposes, nascent sodium alginate was also immobilized on the membrane surface and the binding density (DSA, lg cm2) was calculated according to the same method.

2.5.2. Zeta potential and water contact angle measurement ZP measurements were performed with a zeta potential analyzer (Beckman Coulter, America). The cell voltage was set at about 21.6 V, then the ZP was monitored at 25 ± 0.5 °C using 103 M KCl solution (pH 7.4). Each reported value was an average of at least five independent measurements. The WCA was estimated by the sessile drop method with a CTS200 contact angle system (Cellcons Controls, China) equipped for video capture at room temperature. Briefly, a drop (2 ll) of deionized water was lowered onto the dry membrane surface with a microsyringe and then the static contact angle was recorded. At least five WCA results were averaged to give one mean value. 2.5.3. ELISA for adsorption and desorption of LDL LDL adsorption and desorption of the membranes was ascertained by ELISA, as described in our previous work [25–27]. All the solutions were freshly prepared before the measurement. LDL or HSA was dissolved in PBS (137 mM NaCl, 1.4 mM KH2PO4, 4.3 mM Na2HPO4, 2.7 mM KCl, adjusted to pH 7.4 with 1 M NaOH) to prepare single or binary protein solutions with different concentrations. Blocking solution was prepared with 1 wt.% BSA solution in PBS (pH 7.4). The primary and secondary antibodies were diluted by 0.1 wt.% BSA solution in PBS (pH 7.4) to 1:5000 and 1:10,000, respectively. For the TMB substrate solution, 400 ll of TMB (0.5 wt.%, dissolved in DMSO) and 2 ll of hydrogen peroxide (30% aqueous solution) were added to 10 ml of substrate buffer solution (0.2 M Na2HPO4 and 0.1 M citric acid, pH 5.0). Membrane samples were cut into discs with a diameter of 1.2 cm and placed in 24-well tissue culture plates, then 500 ll of single or binary protein solution was added. The plates were incubated for 1 h at 37 °C and then washed three times with 1 ml of PBS (pH 7.4). Next, 500 ll of blocking solution was added and the plates incubated for a further 0.5 h at 37 °C. After rinsing with PBS (pH 7.4) a further three times, the primary and secondary antibodies were added and the plates incubated for 1 h at 37 °C. Each subsequent step was followed by washing three times with Trisbuffered saline (137 mM NaCl, 1.4 mM KH2PO4, 4.3 mM Na2HPO4, 2.7 mM KCl, pH 7.4, 0.1% Tween 20). Subsequently, the samples were transferred to a new 24-well plate followed by the addition of 500 ll of TMB substrate solution. After keeping at room temperature for 10 min, 500 ll of 1 M H2SO4 solution was added to stop the chromogenic reaction. Part of the dye solution (500 ll) was transferred to another 24-well plate and the optical density was measured at 450 nm with a plate reader (Anthos reader, Graz, Austria). For the LDL desorption from membranes, samples were washed with 1 ml of sodium chloride solution (0–2 M, PBS, pH 7.4) three times following incubation in a single LDL solution (10 lg ml1) for 1 h at 37 °C. After washing another three times with PBS (pH 7.4), the membranes were blocked with blocking solution and the remained LDL on the surface was determined using the ELISA method, as described above.

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2.5.4. SEM and platelet adhesion experiment The morphology of membrane surface was observed using a JSM 6400F field-emission scanning electron microscope (Jeol, Japan). For this purpose, membrane samples were washed with water/ethanol sequence and dried at room temperature. Thereafter, the samples were sputtered with gold for SEM observation. Platelet-rich plasma (PRP, 200,000 platelets ll1), anticoagulated with sodium citrate (supplied by the First Affiliated Hospital of Medical School of Zhejiang University), was used to perform the platelet adhesion experiment, as described in our previous work [26,27]. Briefly, 20 ll of PRP was dropped onto the membrane surface and incubated for 30 min at room temperature. Thereafter the membrane was gently washed with PBS (pH 7.4) and then the adhered platelets were fixed with 2.5 wt.% glutaraldehyde in PBS (pH 7.4) for 30 min. Finally, the sample was washed again with PBS (pH 7.4) and dehydrated with a series of ethanol/water mixtures of increasing ethanol concentration (10, 30, 50, 70, 96 and 100 vol.% ethanol), for 10 min in each mixture. The membranes were air dried, coated with gold and imaged by SEM.

3. Results and discussion

Fig. 2. Effects of primary amine group density on the SA and SAS binding density.

3.1. Effect of reaction condition on the immobilization of SAS Fig. 1 shows the density of primary amine group (DAm) and the efficiency of Hofmann rearrangement reaction (RHofmann) obtained with different AAm grafting densities. PSu grafted with AAm (PSuAAm) samples with different DAAm were achieved by changing the monomer concentration from 50 to 250 g l1 but with a constant photoinitiator solution (benzophenone in n-heptane) of 20 g l1 and a UV irradiation time of 30 min (see Supporting information S2). According to the data in the literature [36], all experiments were carried out under the same reaction conditions as described in Section 2.3, which gave a fast reaction and a relatively acceptable yield. As expected, the Hofmann rearrangement reaction of these PSu-AAm membranes showed relatively high efficiency. When the DAAm of PSu-AAm was increased from 5.24 to 21.89 lmol cm2, RHofmann was decreased slightly from 92.94 to 82.28%, while DAm was increased almost linearly from 4.87 to 18.01 lmol cm2.

Fig. 1. Influences of AAm grafting density on the density of primary amine group and the efficiency of the Hofmann rearrangement reaction.

Fig. 2 shows the effects of primary amine group density (DAm) on the binding density of SA and SAS. The amount of attached polysaccharide moieties increased with the DAm. This phenomenon was because, with the increase in DAm, more binding sites were available on the surface, which favored the reaction between the primary amine group and the polysaccharide. However, the SAS binding density on the surface was always lower than SA under the same reaction conditions. This could be ascribed to some of the hydroxyl groups being substituted by sulfonic groups, causing a decrease in the number of SAS coupling sites. 3.2. Chemical changes of the membrane surfaces ATR-FTIR and XPS spectra were employed to follow the chemical changes of the modified membranes. Fig. 3 shows the ATR-FTIR spectra of the studied membranes. It can be seen that, compared with the nascent PSu, the PSu-AAm surface has two new adsorption peaks, at 1665 and 1615 cm1, assigned to amide I and amide II of AAm, respectively. After the Hofmann rearrangement reaction, the characteristic peak of amide II at 1615 cm1 almost disappeared, and the peak of amide I at 1665 cm1 shifted to a lower wavenumber (1654 cm1) with obviously decreased intensity. This could be ascribed to most of the AAm groups being converted to Am groups. For the membranes coupled with SA, another new adsorption peak appears at 1725 cm1, almost certainly arising from the carbonyl group (C@O) of the immobilized SA. Meanwhile, the adsorption at 1654 cm1 is obviously decreased, which is probably due to the coupling reaction. However, the spectra of PSu-SA and PSu-SA membranes are very similar, thus a further chemical structure analysis is required for confirmation. XPS spectra were employed to analyze the chemical structure of the nascent and modified PSu membrane surface to a higher sensitivity. The survey scan spectra of the studied samples are presented in Fig. 4 and the elemental compositions calculated from the spectra are listed in Table 1. Fig. 4 demonstrates that, compared with the plain PSu, all the modified membranes show an additional peak at 402.8 eV, corresponding to the binding energy of N1s. The nitrogen content of the PSu-AAm surface was increased from 0 to 13.87% in comparison with that of the nascent PSu surface, as listed in Table 1, indicating that AAm groups had been produced after graft polymerization. The difference in nitrogen content between PSu-AAm and PSu-Am was only marginal. After

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Fig. 4. XPS spectra of nascent PSu, PSu-AAm (DAAm = 20.34 lmol cm2), PSu-Am (DAm = 17.49 lmol cm2), PSu-SA (DSA = 119.48 lg cm2) and PSu-SAS (DSAS = 107.03 lg cm2). Fig. 3. ATR-FTIR spectra of nascent PSu, PSu-AAm (DAAm = 20.34 lmol cm2), PSuAm (DAm = 17.49 lmol cm2), PSu-SA (DSA = 119.48 lg cm2) and PSu-SAS (DSAS = 107.03 lg cm2).

Table 1 The chemical compositions of membranes as calculated from XPS survey scans. Samples

binding with polysaccharide, the nitrogen content on membrane surfaces decreased significantly, from 14.15% for PSu-Am to 3.35% for PSu-SA and 3.14% for PSu-SAS, respectively, which was consistent with the intensity change of peak at 402.8 eV in the XPS spectra, as shown in Fig. 4. Meanwhile, the oxygen content was increased from 18.66% on PSu-Am to 30.03% on PSu-SA and 33.27% on PSu-SAS, respectively. This is due to the fact that both the immobilized SA and SAS possess a relatively high oxygen content. It can be also seen from Table 1 that the sulfur content of PSuAAm, PSu-Am and PSu-SA was decreased from 2.25% to 0.22%, 0.38% and 0.31%, respectively, in comparison with that of nascent PSu membrane. After binding with SAS, the sulfur content on the membrane surface increased again to 1.49%. This is certainly attributable to the sulfate groups in SAS. The chemical change of membrane surface can also be detected from the high-resolution core-level spectra of C1s, as depicted in Fig. 5. The C1s core-level spectrum of PSu-AAm could be resolved into two peaks. One peak, at 284.7 eV, is ascribed to C–H or C–C. Another peak, at a binding energy of 287.8 eV, is attributed to the C atoms in the amide groups of grafted AAm (O@C–NH2). After the Hofmann rearrangement reaction, the C1s core-level spectrum of PSu-Am showed a much more symmetrical peak than that of PSu-AAm. With the formation of primary amine groups on the membrane surface, two fitted peaks were observed, at 284.7 and 286.5 eV, assigned to C–H (or C–C) and C–NH2, respectively. As for the two fitted peaks at 284.7 and 288.4 eV in the C1s core-level spectrum of PSu-SA, they could be attributed to C–H (or C–C) and

Nascent PSu PSu-AAm PSu-Am PSu-SA PSu-SAS

Concentration (wt.%) C

O

S

N

79.53 66.74 66.31 65.56 62.05

18.19 19.16 18.66 30.03 33.27

2.25 0.22 0.38 0.31 1.49

13.87 14.15 3.35 3.14

O@C–O, respectively. These results confirmed the surface modification processes as described in Scheme 1. 3.3. Surface properties of the membranes The surface modification of PSu membrane will certainly influence the surface properties of the sample, such as the charge density and wettability, which in turn confirm the modification processes. Table 2 presents the zeta potential at pH 7.4 and the WCA data of pristine PSu, PSu-AAm (DAAm = 20.34 lmol cm2), PSu-Am (DAm = 17.49 lmol cm2), PSu-SA (DSA = 119.48 lg cm2) and PSu-SAS (DSAS = 107.03 lg cm2) membranes. It can be seen that the zeta potential was increased from 44.83 mV for nascent PSu to 18.39 mV for PSu-AAm. This increase in surface potential can be attributed to the presence of acylamino groups on the PSu-AAm. The zeta potential of PSu-Am increased slightly compared with that of PSu-AAm due to the positively chargeable amine groups [37]. In contrast, the magnitude of the surface potential was decreased to negative values after immobilizing SA onto the

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PSu-AAm and PSu-Am (DAm = 17.49 lmol cm2) was only marginal. After binding with SA and SAS, the WCA of the membrane was decreased slightly, from 68.0° for PSu-Am to 61.3° for PSu-SA (DSA = 119.48 lg cm2) and 59.1° for PSu-SAS (DSAS = 107.03 lg cm2), repectively. This is due to the presence of hydrophilic groups, such as –OH, –COO– and –OSO3–. 3.4. LDL adsorption and desorption measurements by ELISA

Fig. 5. XPS C1s core-level spectra of PSu-AAm (DAAm = 20.34 lmol cm2), PSu-Am (DAm = 17.49 lmol cm2) and PSu-SA (DSA = 119.48 lg cm2).

Table 2 Zeta potential and WCA of pristine and modified PSu membranes. Samples

Zeta potential at pH 7.4 (mV)

Water contact angle (°)

Nascent PSu PSu-AAm PSu-Am PSu-SA PSu-SAS

44.83 ± 10.21 18.39 ± 4.67 22.89 ± 5.26 9.49 ± 2.34 40.10 ± 9.16

92.2 ± 8.7 69.7 ± 8.2 68.0 ± 7.6 61.3 ± 6.9 59.1 ± 6.2

PSu-Am surface. This can be attributed to the immobilized SA molecules possessing a number of negatively ionizable carboxylic groups. Moreover, the surface potential of PSu-SAS was 40.10 mV, significantly lower than the 9.49 mV for PSu-SA. This is attributable to the fact that SAS possesses more negatively charged groups because of the presence of sulfate groups. The wettability of the plain and modified PSu membranes was investigated by WCA measurements, and the results are listed in Table 2. The WCA of PSu-AAm (DAAm = 20.34 lmol cm2) was decreased obviously, from 92.2° to 69.7°, because of the presence of hydrophilic acylamino groups. The difference of WCA between

ELISAs have been demonstrated to be reliable, sensitive and reproducible methods for measuring the amount of specific protein (e.g. albumin, fibrinogen and fibronectin) adsorbed on a biomaterial surface from complex protein mixtures or plasma [38,39]. Relative values of protein adsorption to biomaterial surfaces have been reported as optical densities from colorimetric ELISAs. Here, the LDL adsorption of the studied membranes from a single protein solution was investigated by ELISA, as shown in Fig. 6(A). It was observed that the amount of LDL adsorption increased with LDL concentration for all samples, as expected. The increase shows the characteristics of Langmuir-type adsorption with a plateau value, as has been observed for other proteins similarly adsorbing on biomaterials [40,41]. Many studies have shown that protein adsorption on a surface with high wettablity may decrease due to the hydrophobic interactions between the surface and the protein [40–43]. A similar phenomenon was also observed in the present work. As shown in Fig. 6(A), compared with plain PSu, the absorbance was obviously decreased for PSu-AAm after exposure to the same LDL solution, indicating a lower LDL adsorption. This should be ascribed to the improved hydrophilicity, as revealed by the WCA measurements. For the same reason, the amount of LDL absorption to PSu-Am was slightly less than that to PSu-AAm. However, after SA immobilization, when the membrane surface became more hydrophilic, an obviously increased amount of LDL was adsorbed on PSu-SA compared to PSu-AAm or PSu-Am. This increase can be attributed to the immobilized SA, which is a linear anionic polysaccharide with a number of negative –COO– groups. LDL, in which the structural domain B100 has a positively charged group of amino acids (lysine and arginine), could specifically bind to negative –COO– groups by electrostatic interaction [20,21]. The amount of LDL adsorption on PSu-SA being lower compared to that on plain PSu was due to the limited negative charge density on the PSu-SA surface, as revealed by zeta potential measurements. It should be noted that, after SAS immobilization, an obviously larger amount of LDL was adsorbed on PSu-SAS compared to PSu-SA. This might be attributed to the significantly increased negative charge density on the PSu-SAS surface due to the presence of –OSO3– groups, as revealed by zeta potential measurements. Apheresis procedures are usually performed with regeneration of the adsorber surfaces to allow multiple or repetitive sessions with the same adsorber to reduce the cost of treatment. This requires the repetitive washing of adsorber and the desorption of bound proteins or other agents. The specific adsorption of protein to a biomaterial surface induced by electrostatic interactions is highly dependent on the ionic strength, while the adsorption induced by hydrophobic interaction remains undisturbed [41–45]. To learn more about the dominating interaction force between the membrane surface and LDL, and to see if the studied membranes can be regenerated after exposure to LDL, NaCl solutions with different ionic strengths were introduced as eluents with the purpose of disturbing the electrostatic binding. The desorption effect at the concentration of NaCl in PBS was taken as the zero level. Fig. 6(B) presents the LDL desorption from membranes after exposure to 10 lg ml1 LDL solution and subsequent washing with NaCl solutions. It was found that LDL bound to the plain PSu was almost unaffected by the NaCl concentration up to 2 mol l1, suggesting that the LDL previously adsorbed on the membrane surface

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Fig. 6. (A) LDL adsorption on PSu, PSu-AAm (DAAm = 20.34 lmol cm2), PSu-Am (DAm = 17.49 lmol cm2), PSu-SA (DSA = 119.48 lg cm2) and PSu-SAS (DSAS = 107.03 lg cm2) membranes in a single protein solution; (B) LDL desorption from samples after adsorption of 10 lg ml1 LDL solution and subsequent washing with NaCl solutions.

was predominantly induced by hydrophobic interaction. Although the plain PSu exhibits a negatively charged surface, as revealed by zeta potential measurements, it still lacks negatively ionizable groups and presents a hydrophobic surface, as revealed by WCAs. Thus the adsorption of LDL onto plain PSu is mainly induced by hydrophobic interaction. For the same reason, the amount of LDL desorbed from both PSu-AAm and PSu-Am surface was negligible and almost undisturbed with increasing NaCl concentration. However, the amount of LDL desorbed from PSu-SA and PSu-SAS was noticeable and increased steadily with NaCl concentration, as shown in Fig. 6(B). These results indicate that the LDL adsorption to plain PSu, with a hydrophobic surface, and to PSu-AAm and PSu-Am, with hydrophilic but positively charged surfaces, was mainly induced by hydrophobic interaction, whereas the LDL adsorption to PSu-SA and PSu-SAS, with hydrophilic and negatively charged surfaces, was mainly driven by electrostatic attraction. Thus, the LDL adsorption to plain PSu, PSu-AAm and PSu-Am was non-specific and irreversible, while that to PSu-SA and PSu-SAS was a specific and reversible process, allowing regeneration of the adsorber surface for subsequent therapeutic reuse. The adsorption of proteins onto biomaterial surfaces in blood or plasma usually involves competition between all the present proteins. However, it is very difficult to control all the important variables when complex mixtures of proteins are used to simulate such a competitive process. To simplify this evaluation, adsorption studies from mixtures of only two proteins are usually used to simulate the competitive effect of protein adsorption in human blood plasma [43–45]. Based on the high concentration of HSA in plasma, mixtures of LDL and HSA were prepared at a variety of concentrations. The studied membranes were then exposed to these mixtures. According to the Vroman effect [45], the composition of the adsorbed protein layer is strongly dependent on the interaction time during competitive adsorption from protein mixtures. The proteins with higher concentrations initially predominate on the surface due to the higher probability of collisions, but these can be replaced by other proteins with higher surface affinity as the interaction time progresses [44,45].

Fig. 7 shows the results of competitive adsorption from binary protein solutions composed of HAS and LDL to the plain and modified PSu membrane surfaces. As shown in Fig. 7(A), when the concentration of LDL was fixed at 10 lg ml1 while the concentration of HSA was changed from 0 to 10 mg ml1, the presence of HSA largely lowered the adsorption of LDL to the PSu, PSu-AAm and PSuAm surfaces. However, a lesser effect was observed for PSu-SA and SAS. This could be because LDL adsorption to PSu, PSu-AAm and PSu-Am is non-specific and mainly induced by hydrophobic interaction, thus the HSA with higher concentration may bind predominantly on the surface and reduce the adsorption of LDL obviously, while LDL adsorption to PSu-SA and PSu-SAS is specific and mainly driven by electrostatic attraction with high affinity. It was noted that obvious adsorption of LDL still occurred on PSu-SAS even at the high HSA concentration of 10 mg ml1. This represents a ratio of HSA to LDL of 1000:1, which is much higher than that in the plasma of healthy human (where the levels of HSA and LDL have been shown to be 30–50 g l1 and 100–120 mg dl1, respectively [5,6]). To further investigate how an increased ratio of LDL to HSA would modulate the adsorption of LDL, the concentration of HSA was fixed at 1 mg ml1 and the LDL concentration was increased from 0.5 to 10 lg ml1, as shown in Fig. 7(B). It was found that PSu, PSu-AAm and PSu-Am showed negligible increments of LDL adsorption compared with PSu-SAS, which exhibited an obviously increased adsorption with LDL concentration. The increments of LDL adsorption on PSu-SA with LDL concentration were higher than that on PSu but lower than that on PSu-SAS membranes. Moreover, the binding amount of LDL on PSu-SAS was much higher than that on plain PSu and other modified PSu. This can be attributed to the immobilized SAS, a polyanion polysaccharide with a number of negative groups, such as –OSO3– and –COO–, enhancing the electrostatic affinity between the membrane surface and the LDL. An obviously increased binding of LDL was also observed with the increasing LDL concentration, reaching a ratio of LDL to HSA at 1:100. As mentioned previously, in human plasma the ratio of LDL to

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Fig. 7. LDL adsorption on PSu, PSu-AAm (DAAm = 20.34 lmol cm2), PSu-Am (DAm = 17.49 lmol cm2), PSu-SA (DSA = 119.48 lg cm2) and PSu-SAS (DSAS = 107.03 lg cm2) membranes from binary protein solutions: (A) LDL = 10 lg ml1, HSA = 0, 0.5, 1, 5, 10 mg ml1; (B) HSA = 1 mg ml1, LDL = 0.5, 1, 4, 8, 10 lg ml1.

Fig. 8. SEM images of nascent PSu (a), PSu-AAm (b, DAAm = 20.34 lmol cm2) and PSu-SAS (c, DSAS = 107.03 lg cm2) surfaces.

Fig. 9. Platelet adhesion on nascent PSu (a), PSu-SA (b, DSA = 119.48 lg cm2) and PSu-SAS (c, DSAS = 107.03 lg cm2) surfaces.

HSA is normally 1:30–50 for healthy persons, but this ratio is much higher in hypercholesterolemic patients (LDL P 160 mg dl1). The ability of PSu-SAS to adsorb LDL from binary

protein solutions means that this new type of membrane could be a potent material for selective removal of LDL under pathological conditions.

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3.5. Surface morphology and platelet adhesion of the membranes The surface morphology of nascent PSu, PSu-AAm and PSu-SAS membranes was studied by SEM and the images are displayed in Fig. 8. From the surface images it can be observed that there were small pores on the surface of the nascent PSu. With the coverage of the grafted poly(AAm) layer, the surface pores were blocked and small cracks formed. After the immobilization of SAS, the modified surface became slightly smoother. The extent of platelet adhesion and the morphology of adhered platelets are considered to be early indicators of the hemocompatibility of blood-contacting biomaterials, and these depend significantly upon the surface composition of the biomaterials [26,27]. Thus, platelet adhesion to PSu, PSu-SA and PSu-SAS was examined (Fig. 9). The extent and morphology of the platelets adhered to the modified surfaces indicated differences in blood compatibility. Numerous adherent platelets could be observed on the PSu surface. However, platelet adhesion was effectively suppressed on PSu-SA and only a very few platelets were adhered to the PSu-SAS surface. Furthermore, not only the number but also the morphology of the adhering platelets needs to be considered. The shape change of activated platelets can be classified into five stages, in the following sequence: discoid, dendritic (early pseudopodial), spread/ dendritic (intermediate pseudopodial), spreading (late pseudopodial and hyaloplasm spreading) and full spreading (hyaloplasm well spreading and no distinct pseudopodia) [46]. As can be seen in Fig. 9, on the membrane surface of PSu, the platelets are larger and show a greater spreading area. At the same time, platelets have the tendency to aggregate, and platelet fragments were also found in the sample. However, on the PSu-SAS membrane with a SAS density of 107.03 lg cm2, the platelets retained their original discoid shape, indicating an inactived state. The above results show that the blood compatibility of PSu membranes could be enhanced significantly by the immobilization of SAS employed here. 4. Conclusions SAS, a heparin analogue, was covalently immobilized onto PSu membrane by a three-step synthesis method, which involved the UV irradiation-induced graft polymerization of AAm followed by the Hofmann rearrangement reaction and then chemical binding. The plain and modified PSu membranes were extensively characterized by ATR-FTIR, XPS, ZP, WCA and ELISA. The ATR-FTIR, XPS and ZP results confirmed the success of the surface modification process. The hydrophilicity of the membrane was greatly improved by the surface modification. The ELISA results suggested that the LDL adsorption to plain PSu, PSu-AAm and PSu-Am was a nonspecific and irreversible process mainly induced by hydrophobic interaction, while the LDL adsorption to PSu-SA and PSu-SAS was a specific and reversible process mainly driven by electrostatic attraction. PSu-SAS membranes had greatly enhanced adsorption of LDL both from single protein solutions and from mixtures with HSA that closely resemble the conditions in human plasma. The LDL adsorbed on the PSu-SAS surface could be easily desorbed with sodium chloride solution. Furthermore, the PSu-SAS membranes showed good blood compatibility, as revealed by platelet adhesion. In conclusion, PSu-SAS membranes are promising for application in simultaneous hemodialysis and LDL apheresis therapy. Acknowledgements We gratefully acknowledge the financial support from the Zhejiang Provincial Natural Science Foundation of China (Grant No. LY13E030003), the Fundamental Research Funds for the Central Universities (Grant No. 2013QNA4049), and the Open

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