Immobilization of heparin on polysulfone surface for selective adsorption of low-density lipoprotein (LDL)

Acta Biomaterialia 6 (2010) 1099–1106

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Immobilization of heparin on polysulfone surface for selective adsorption of low-density lipoprotein (LDL) Xiao-Jun Huang a,b, Deepak Guduru a, Zhi-Kang Xu b, Jörg Vienken c, Thomas Groth a,* a

Biomedical Materials Group, Department of Pharmaceutics and Biopharmaceutics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Halle (Saale) 06120, Germany Key Laboratory of Macromolecular Synthesis and Functionalization (Ministry of Education), Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China c Fresenius Medical Care Deutschland GmbH, BioSciences Department, Bad Homburg 61352, Germany b

a r t i c l e

i n f o

Article history: Received 30 June 2009 Received in revised form 11 August 2009 Accepted 31 August 2009 Available online 3 September 2009 Keywords: Polysulfone Surface modification Heparin LDL adsorption Apheresis

a b s t r a c t A versatile method was developed to immobilize heparin covalently on polysulfone sheets (PSu) to achieve selective adsorption of low-density lipoprotein (LDL). This was achieved by activation of PSu with successive treatments of chlorodimethyl ether and ethylenediamine, and subsequent chemical binding of heparin with bifunctional linker molecules. A heparin density up to 0.86 lg cm2 on a dense PSu film was achieved. The modified PSu films were characterized by attenuated total reflectance Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. The hydrophilicity of the PSu film was improved greatly by covalent immobilization of heparin. The water contact angle of PSu film was decreased from 86.6 ± 3.7° to 50.5 ± 3.2° after binding of 0.36 lg cm2 heparin. An enzyme-linked immunosorbent assay was used to measure the binding of LDL on plain and modified PSu films. It was found that the heparin-modified PSu film could selectively recognize LDL from binary protein solutions. Furthermore, it was possible to desorb LDL from heparinized PSu, but not from plain PSu, with heparin, sodium chloride or urea solution, which indicates a selective but reversible binding of LDL to heparin. The results suggest that heparin-modified PSu membranes are promising for application in simultaneous hemodialysis and LDL apheresis therapy. Ó 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction The abnormal elevated level of cholesterol in human blood has been confirmed to be a main risk factor for the process of coronary heart diseases, atherosclerosis and cerebral thrombosis. Cholesterol is carried in plasma by a series of lipoproteins, such as very low-density lipoproteins (VLDL), low-density lipoproteins (LDL), intermediate density lipoproteins (IDL) and high-density lipoproteins (HDL). It is now believed that LDL is one of the major causative elements in the development of atherosclerosis [1,2]. High levels of LDL, especially coupled with low HDL concentrations, can promote cholesterol accumulation in the intra- and extracellular arterial wall that in turn leads to plaque formation causing various heart and vascular diseases [2–4]. Therefore, the reduction of LDL level in blood is used therapeutically to lower the risk of cardiovascular/cerebrovascular diseases [5–8]. In recent years, LDL has been efficiently eliminated by extracorporeal LDL apheresis systems [6–8]. Various general LDL apheresis procedures exist for routine clinical LDL elimination, such as unse-

* Corresponding author. Tel.: +49 3455528461; fax: +49 345 5527 379. E-mail address: [email protected] (T. Groth).

lective plasma exchange [9], semiselective double-filtration plasmapheresis (DFPP) [10], and there are other more selective techniques based on immunogenic or electrostatic interactions such as immunoadsorption using anti-LDL antibodies (LDL-Therasorb, Miltenyi Biotec, Bergisch Gladbach, Germany) [11], heparininduced extracorporeal LDL precipitation (HELP, B Braun, Melsungen, Germany) [12,13], adsorption on dextran sulfate–cellulose (Liposorber, Kaneka Corporation, Osaka, Japan) [14], and direct adsorption of lipids from whole blood using polyacrylamide beads coated with polyacrylic acid (DALI-system, Fresenius, Bad Homburg, Germany) [15]. Normally, these apheresis systems, except for DALI, require the separation of blood cells and plasma by plasma filtration into a secondary circuit as a first step. For the HELP system, the process is even more complex and costly and a filtration is needed to remove the precipitated LDL complexes. Furthermore, a simultaneous LDL apheresis and hemodialysis procedure is required especially in treatment of patients with chronic renal failure and LDL-induced coronary heart disease [16]. In these cases, the membrane filtration must be applied during LDL apheresis procedures. Overall, procedures to carry out LDL apheresis are complicated by the additional burden for the patient. Simultaneous treatments of ESRD patients to continuously remove LDL during hemodialysis sessions without resorting to a combination of

1742-7061/$ - see front matter Ó 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2009.08.039

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different methods would not only reduce costs, but probably also provide more safety and comfort for the patients. For fabrication of LDL adsorbents, selection of an appropriate ligand and matrix is presently a major area of concern. Heparin, a highly sulfated polysaccharide, is known as one of the most efficient LDL ligands, which can interact with apolipoprotein B of LDL via electrostatic interactions [17,18]. For this reason different heparin-based adsorbents had been suggested, such as heparin-coupled Sepharose [8] and heparin-modified poly(vinyl alcohol) (PVA) granules [19]. Furthermore, heparin was used as ligand to prepare sensors for the measurement of LDL in blood [20,21]. Based on the complexity of the contemporary LDL apheresis procedures and the obvious advantage of a simultaneous LDL apheresis–hemodialysis treatment, we suppose that direct immobilization of a highly selective LDL ligand such as heparin on hemodialysis membranes may be useful for selectively adsorbing LDL in ESRD patients with hypercholesterolemia. As hydrophilized polysulfone (PSu) is currently the most widely used polymer for hemodialysis membranes, and enables efficient removal of small to medium-sized molecules [22,23], PSu was used here as matrix for heparin immobilization. The objective of this study was to explore different chemical routes to immobilize heparin on PSu films covalently with a high yield. Herein, a method to tether heparin onto a PSu surface is described, and the surface properties were analyzed by Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) and water contact angle measurements. The ability of modified PSu to bind LDL selectively was studied by enzyme-linked immunosorbent assays (ELISAs). It was found that modification of PSu with heparin results in a material surface that can bind selectively and reversibly high quantities of LDL. 2. Materials and methods 2.1. Materials Pure PSu granules were supplied by Fresenius Medical Care. Tetrahydrofuran (THF), tin(IV) chloride (SnCl4), chlorodimethyl ether, ethylenediamine (EDA), 1-ethyl-3-(dimethyl-aminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), toluidine blue (TB), bovine serum albumin (BSA), human serum albumin (HSA, 99%), primary antibody: anti-b-lipoprotein (LDL, antibody produced in goat whole antiserum), secondary antibody: anti-goat IgG (peroxidase antibody produced in rabbit), blocking buffer and Tween 20 were purchased from Sigma–Aldrich and used as received. LDL (5.8 mg ml1, Millipore), 3,30 ,5,50 -tetramethylbenzidine (TMB, Care Roth GmbH), heparin (sodium salt, 166.0 U mg1, VWR International GmbH) and flat ELISA plates (Greiner Bio-One, Germany) were purchased and used as received. Water used in all experiments was deionized and ultrafiltered to 18 MX with a TKA MicroPure Water system. 2.2. Fabrication of PSu dense film Pure PSu was dissolved in THF at about 25 °C for 24 h with vigorous stirring to form a 10 wt.% homogeneous solution. After air bubbles were removed completely, the solution was cast onto a clean glass plate using a casting knife with a 100 lm gate opening. The glass plate with the nascent film was directly dried for 24 h at 60 °C under vacuum. The dense film was peeled off and then dried for another 24 h at 80 °C under vacuum to ensure dryness before surface modification was conducted. 2.3. Immobilization of heparin on PSu surface PSu was chemically activated according to the method of Higuchi et al. [24]. The PSu film was immersed briefly in a solution of

chlorodimethyl ether, hexane and SnCl4 at 25–30 °C for various reaction times (10 min–24 h) and then washed in methanol for 2 h. Thereafter, the chloromethylated PSu (PSu-CH2Cl) was immersed into EDA at 25 °C to obtain amino groups for the subsequent chemical binding of heparin. Finally, EDA-modified PSu (PSu-NH2) was submerged into heparin and EDC/NHS solution (5 mg ml1 of heparin and EDC in citrate buffer solution: 0.2 M Na2HPO4 and 0.1 M citric acid, adjusted to pH 4.7 with 1 M NaOH, molar ratio of EDC to NHS = 1:1) for 24 h at 25–30 °C to bind heparin covalently. The modified PSu was taken out and washed three times with phosphate-buffered saline (PBS, 0.03 M Na2HPO4, 0.02 M KH2PO4, and 0.137 M NaCl, adjusted to pH 7.0 with 1 M NaOH). 2.4. Surface characterization 2.4.1. Heparin density on the modified PSu surface The quantity of heparin bound to PSu surface was assayed by the toluidine blue (TB) colorimetric method according to the literature [25,26]. The assay is based on the fact that TB will irreversibly bind to a polyanion substrate (e.g. heparin-modified PSu). The amount of immobilized heparin can be calculated by comparison with a standard curve using soluble heparin of known concentration. For a calibration curve, TB was dissolved in 0.01 mol l1 hydrochloric acid containing 0.2 wt.% NaCl to prepare 0.005% TB solution. A series of heparin solutions with concentrations varying from 0 to 25 lg ml1 were prepared by dissolving heparin in an aqueous 0.2 wt.% NaCl solution. Heparin standard solution (0.5 ml) was added to TB solution (0.5 ml) and then agitated for 30 s. Next, n-hexane (1 ml) was added, and the mixture was shaken well so that the heparin–TB complex was extracted into the organic layer. The non-extracted TB remaining in the aqueous phase was determined by measuring the absorption at 631 nm. A linear correlation between the concentration of heparin in the aqueous solution and the absorbance at 631 nm caused by the residual TB was obtained and used as a calibration curve to determine the quantity of immobilized heparin. Accordingly, the heparin-modified PSu sheet film was cut into round size with a diameter of 1.35 cm and immersed in TB solution and incubated for 30 min. Subsequently, n-hexane was added and the mixture shaken well to ensure uniformity in treatment. After removing the PSu from the solution, the absorbance of aqueous layer at 631 nm was measured by UV–visible spectrophotometry. The quantity of immobilized heparin was calculated from the above constructed calibration curve. Each value was an average of five independent measurements. 2.4.2. FTIR and XPS Attenuated total reflectance (ATR)-FTIR measurements were carried out on a Vector 22 FTIR (Brucker Optics, Switzerland) equipped with an ATR cell (KRS-5 crystal, 45°). Sixteen scans were taken for each spectrum at a normal resolution of 2 cm1. XPS spectra were recorded on a PHI-5000C ESCA system (Perkin-Elmer, USA) with Al Ka excitation radiation. The pressure in the analysis chamber was maintained at 106 Pa or lower during measurements. To compensate for the surface charging effect, all survey and core-level spectra were referenced to the C1s hydrocarbon peak at 284.6 eV. 2.4.3. Water contact angle measurement The hydrophilicity of the PSu film was characterized on the basis of water contact angle. Static contact angle was measured at room temperature on a contact angle goniometer (OCA20, Dataphysics, Germany) equipped with video capture. In a typical sessile drop method, a total of 2 ll of deionized water was dropped onto a

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dry sample with a microsyringe in an atmosphere of air. The contact angle image was recorded and determined from the image with imaging software. At least five contact angles were averaged to obtain a value.

2.5. LDL adsorption on the PSu films surface measured by ELISA LDL adsorption on the surface of PSu film was investigated by ELISA, as described previously [27,28]. Protein and antibody solutions were freshly prepared before the measurement. For the primary antibody, anti-b-lipoprotein was added to 0.1 wt.% BSA solution at a dilution of 1:10,000. For the peroxidase-conjugated secondary antibody, anti-goat IgG was diluted 1:20,000 with 0.1 wt.% BSA solution. For the substrate solution (freshly prepared), 400 ll of TMB (0.5%, dissolved in DMSO) and 2 ll of hydrogen peroxide (30%) were added to 10 ml substrate buffer solution (0.2 M Na2HPO4 and 0.1 M citric acid, adjusted to pH 5.0 with 1 M NaOH). LDL or human serum albumin (HSA) was dissolved in PBS (137 mM NaCl, 1.4 mM KH2PO4, 4.3 mM Na2HPO4, 2.7 mM KCl, pH 7.4). PSu sheet films were cut into discs with a diameter of 0.5 cm and placed in 96-well tissue culture plates, then 100 ll of protein solution, and PBS (pH 7.4) without protein (blank), were added. The plates were incubated for 1 h at 37 °C and then washed three times with 200 ll PBS (pH 7.4). Blocking solution (100 ll, Sigma–Aldrich) was added and incubated for 0.5 h at 37 °C. After rinsing with PBS (pH 7.4), the primary antibody and peroxidaseconjugated secondary antibody were respectively added and incubated for 1 h at 37 °C. Each subsequent step was followed by washing with Tris-buffered saline (TBS, 137 mM NaCl, 1.4 mM KH2PO4, 4.3 mM Na2HPO4, 2.7 mM KCl, pH 7.4, 0.1% Tween 20). Subsequently, the films were transferred to another 96-well plate, followed by addition of TMB substrate solution at room tempera-

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ture for 10 min. The reaction was stopped by adding 100 ll of 1 M H2SO4. Part of the dye solution (100 ll) was transferred to a new 96-well plate. The optical density (OD) was measured at 450 nm vs. 620 nm with a plate reader (Anthos reader, Graz, Austria). 2.6. Desorption of LDL from PSu films After incubation in protein solution, PSu films were respectively immersed in sodium chloride (0.15–2.0 M, PBS, pH 7.4), heparin (10–1000 lg ml1, PBS, pH 7.4) or urea (0–8.0 M) solution at room temperature for three times, each time using 100 ll solution for 10 min. After washing with PBS (pH 7.4), the films were blocked with blocking solution and the amount of LDL remained on the films was determined using the ELISA method described above. 3. Results and discussion 3.1. Chemical activation of and heparin immobilization on PSu Surface modification is an attractive and efficient approach to endow polymeric membranes with tailored surface properties in a defined, selective way while preserving their original bulk structure. In recent years, several kinds of methods have been developed to activate PSu and immobilize different functional groups or ligands on PSu membrane surface [24,29,30]. For example, Higuchi et al. described a simple Friedel–Crafts reaction to chemically activate PSu for the further covalent attachment of affinity ligands on the surface of the hydrophobic PSu membrane [24]. It was found that the degree of substitution on PSu surface could be easily controlled over a wide range. In this work, as schematically described in Fig. 1, heparin was tethered on the PSu surface by

Fig. 1. Scheme of the chemical activation of PSu film.

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ular weight ranging from 3 to 50 kDa limits its tethering on the film surface [32,33]. Similar heparin densities on various surfaces were reported in the literature. For example, Chen et al. [25] covalently immobilized heparin on silicone surface with a density of 0.68 lg cm2 and Kang et al. [26] reported a heparin density of approximately 1.1–1.3 lg cm2 on polyurethane surfaces.

3.2. Characterization and hydrophilicity of the heparin-modified PSu surface

Fig. 2. Effect of chloromethylation time on the heparin density on PSu dense film. A higher heparin density on PSu dense film can be achieved by prolongation of the primary activation with chlorodimethyl ether.

covalent chemical bonds, which include the chemical activation of PSu with chlorodimethyl ether and EDA, and subsequently chemical binding of heparin in the presence of EDC/NHS. For this purpose, PSu was first activated under the optimum conditions according to the method of Higuchi et al. [24]. It was found that the primary activation of PSu by chlorodimethyl ether was the main factor affecting the degree of immobilization of heparin. Fig. 2 shows that the heparin density on dense PSu films initially increased markedly with the chloromethylation time and then almost reached a plateau when the chloromethylation time exceeded 1 h. This is reasonable given that the reactions on PSu film surface in this work are heterogeneous and the steric hindrance of reactants is significant during solid-surface reaction. Furthermore, it is well known that, given the steric constraints that exist, carrying out chemical reactions directly between polymer surface and macromolecules is relatively difficult to achieve with high efficiency [31]. The steric hindrance of heparin with a molec-

The ATR-FTIR spectra of plain and modified PSu films are shown in Fig. 3. The spectra of all films are similar at wavelengths below 1600 cm1. The 1585 and 1486 cm1 bands are due to the aromatic groups, and the 1325/1290 cm1 doublet and 1150 cm1 band are assigned to the aromatic sulfone chromophore. Fig. 3 shows that, compared to the plain surfaces, the PSu-heparin surface has a new peak appearing at 1725 cm1, almost certainly arising from the carbonyl group (C@O) of immobilized heparin. Furthermore, PSu-heparin showed a shoulder at around 1050 cm1, which was due to the symmetric stretching of the —SO3  group of immobilized heparin [25,26]. To further verify the chemical structures on the modified PSu, XPS survey scans were performed to assess the elemental composition of the film surface. Fig. 4 demonstrates that, compared to plain PSu, the chloromethylated PSu (PSu-CH2Cl) showed an additional peak corresponding to Cl-2p3 (binding energy 202 eV). In contrast to this, the peak corresponding to Cl-2p3 disappeared on the surface of PSu modified with EDA (PSu-NH2) and heparin-modified PSu (PSu-heparin). Furthermore, a new nitrogen component was observed on the surface of PSu modified with EDA (PSuNH2) and heparin-modified PSu (PSu-heparin) based on the terminal amine group of tethered ethylenediamine and the amide group of immobilized heparin [25,26]. The chemical compositions of the different surface-modified PSu, calculated from the XPS survey scans, are shown in Table 1. Compared to PSu-CH2Cl, the chlorine content decreased from 3.98% to 0% on PSu-NH2 and PSu-heparin, which is consistent with the XPS spectra shown in Fig. 4. After immobilization of heparin on

Fig. 3. ATR-FTIR spectra of the pure PSu, PSu-CH2Cl, PSu-NH2 and PSu-heparin films.

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Fig. 4. ESCA survey scan spectra of the pure PSu, PSu-CH2Cl, PSu-NH2 and PSuheparin films.

Table 1 Chemical composition of surface-modified PSu film calculated from ESCA scan spectra. Substrate

PSu PSu-CH2Cl PSu-NH2 PSu-heparin

at.% C

O

S

Cl

N

78.56 77.29 77.69 67.74

19.19 16.79 12.23 24.39

2.25 1.94 2.25 2.20

– 3.98 – –

– – 7.83 5.67

PSu-NH2 surface, the oxygen content on PSu-heparin surface increased from 12.23% to 24.39%, while the carbon, sulfate and nitrogen content was decreased from 77.69%, 2.25% and 7.83% to 67.74%, 2.20% and 5.67%, respectively. This is ascribed to the fact that immobilized heparin possesses relatively high oxygen content [25,26]. The wettability of the plain and heparin-modified PSu was estimated by water contact angle measurements. Static contact angles on the plain and heparin-modified PSu films are shown in Fig. 5. The water contact angles were obviously decreased with increased heparin density on the modified PSu films and decreased from 86.6 ± 3.7° to 50.5 ± 3.2° after binding of 0.36 lg cm2 heparin. Overall, the successful modification of PSu with heparin was demonstrated by using TB blue assay, the appearance of carbonyl and sulfate groups in ATR-FTIR spectra, new sulfur and nitrogen peaks in XPS scans and the observed changes in water contact angle after modification.

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Fig. 5. Water contact angle of the modified PSu films with different heparin densities. The higher heparin density on the modified PSu film leads to a hydrophilized surface with a lower water contact angle.

by ELISA. This indicates that this ELISA can also be applied to detect LDL adsorbed on PSu surface. The adsorption of LDL from single protein solutions on the plain and modified PSu is shown in Fig. 6. It was observed that the amount of absorbed LDL increased with concentration as was expected. The increase shows the characteristics of Langmuir type of adsorption with a plateau value as was observed for other proteins similarly adsorbing on biomaterials [34–36]. Fig. 6 shows also that LDL adsorbs on the plain and heparin-modified PSu films. In this context, many studies have shown that adsorption of proteins is increased on surfaces with lower wettability due to hydrophobic interactions between surface and protein [29,34–37]. It should be noticed that after heparin immobilization, when PSu film became quite hydrophilic, significantly higher quantities of LDL were adsorbed compared to plain PSu. This indicates dominance of the electrostatic interaction between heparin and LDL, which has been described by Cardin et al. [17] and Gigli et al. [18], is also exploited during HELP apheresis [12,13] and allows the adsorption of relatively high quantities of LDL.

3.3. LDL adsorption on PSu film measured by ELISA ELISAs have been demonstrated to be reliable, sensitive and reproducible methods and have been applied to measure the amounts of specific proteins (e.g. albumin, fibrinogen and fibronectin) adsorbed on biomaterial surfaces from complex protein mixtures or plasma [27,28]. Relative values of protein adsorbing to biomaterials have been reported as optical densities from colorimetric ELISAs. The standard curves of LDL adsorption on hydrophobic plates measured by ELISA are available in Supporting Information. There was a linear correlation between the coating concentration of LDL on the plates and the absorbance measured

Fig. 6. Adsorption of LDL from single protein solution with different concentrations (0.5, 1.0, 5.0, 10 and 20 lg ml1) on the pure and heparin-modified PSu films. The adsorption of LDL on both the pure and heparin-modified PSu films surface followed the Langmuir isotherm model. A higher quantity of LDL is adsorbed on the heparin-modified PSu film surface compared to plain PSu.

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Adsorption of proteins to biomaterials from blood or plasma usually involves competition of all the proteins in the mixture for the available surface. However, because it is very difficult to control all the important variables using complex mixtures of proteins, competitive adsorption studies from mixtures of two proteins are usually used as an alternative to human blood plasma for evaluation of protein adsorption to biomaterials [35–38]. Based on the high concentration of human serum albumin (HSA) in plasma, mixtures of LDL and HSA were prepared and plain and heparin-modified PSu films exposed to these mixtures. It is assumed that this approach simulates the competitive effect of protein adsorption in (complex) mixtures of proteins. According to the Vroman effect [38], for the competitive adsorption from mixtures of proteins, the composition of the adsorbed protein layer is strongly dependent on the interaction time. The high-concentration proteins initially dominate the surface due to the higher collision rates, but can be replaced by other proteins with higher surface affinity as time passes [34–37]. Hence, in a second experiment, the adsorption of LDL was measured from binary protein solutions composed of HSA and LDL. Fig. 7A and B shows the result of this investigation. When the concentration of LDL was low (10 lg ml1) it was observed that the presence of HSA in solution reduced the LDL adsorption on the plain PSu to zero provided the concentration of HSA was higher than 10 mg ml1. In contrast to this finding, even at higher HSA concentrations of 25 mg ml1 adsorption of LDL still occurred on heparin-modified PSu. This represents a ratio of HSA to LDL of 1000:1, which is obviously much higher than that of the ratio between HSA and LDL in human plasma for healthy persons (the level of HSA and LDL was 30–50 g l1 and 100–120 mg dl1 [2–5], respectively). To test further how an increased ratio of LDL to HSA would modulate the binding of LDL, the concentration of HSA was fixed to 1 mg ml1 and LDL concentration was increased as shown in Fig. 7B. Again, the binding of LDL to heparin-modified PSu was much higher than to plain PSu. A greatly increased binding of LDL was observed with increased concentration, reaching a ratio of 1:100. As mentioned previously, in human plasma the ratio of LDL to HSA is normally 1:30–50 for healthy persons, but this ratio is much higher in hypercholesterolemic patients (LDL P 160 mg dl2) [2–6]. The ability of heparin-modified PSu to adsorb LDL from binary protein solutions means that this material could be a

potent material for adsorption of LDL under pathological conditions. The significant increase in LDL adsorption after heparin modification of PSu can be attributed to the immobilized heparin, which is a polyanion polysaccharide with high density of negative groups, such as –OSO3– and –COO–. LDL, in which the structural domain B100 has a positively charged group of amino acids (lysine and arginine), could specifically bind to heparin by electrostatic interaction [17–19]. Furthermore, the chain conformation and three-dimensional structure on the substrates’ surface have a significant effect on the protein adsorption [36,39]. For heparin-modified PSu film, the immobilized heparin with a molecular weight ranging from 3 to 50 kDa [32] can present a three-dimensional surface with more binding sites for LDL adsorption than that of the plain pure PSu with a surface that is flat in molecular terms. 3.4. Regeneration of the PSu film containing recognized LDL The specific interaction between LDL and heparin is mainly driven by electrostatic forces [17–21]. This type of interaction is significantly dependent on ionic strength [21,40], while the hydrophobic interaction between proteins and surfaces remains undisturbed [35–38]. Apheresis procedures are usually carried out with regeneration of the adsorber surfaces to allow multiple or repetitive sessions with the same adsorber while reducing the cost of treatment. This requires repeated washing of adsorber and desorption of bound proteins or other agents. To learn more about the dominating interaction force between LDL and either PSu surfaces, and to see if heparinized PSu can be regenerated after exposure to LDL, desorption experiments were carried out. Washing of PSu films after exposure to LDL was performed with heparin, NaCl and urea solutions. Heparin was used as competitive reagent; strong NaCl solutions disturb the electrostatic interaction force; and urea serves to disrupt the non-covalent bonds in proteins and is widely applied as a protein adsorber or column regeneration buffer for chromatography [41]. Fig. 8A shows the result of desorption experiments of LDL from PSu films exposed to binary protein solutions (LDL = 10 lg ml1 and HSA = 1.0 mg ml1) after addition of increasing quantities of heparin in PBS (pH 7.4). It was found that LDL bound to the plain PSu surface is unaffected by heparin concentration of up to 1000 lg ml1, but LDL could be desorbed from heparin-modified PSu surface by heparin solutions with a broad concentration, with

Fig. 7. Adsorption of LDL on the pure and heparin-modified PSu film from binary protein solutions of LDL and human serum albumin (HSA): (A) LDL = 10 lg ml1, HSA = 0.1, 1.0, 5.0, 10, 25 mg ml1; (B) HSA = 1.0 mg ml1, LDL = 0.5, 1.0, 5.0, 10 lg ml1. The heparin-modified PSu film shows a higher ability of binging LDL from binary protein solutions.

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Fig. 8. Desorption of LDL from PSu films exposed to binary protein solutions (LDL = 10 lg ml1 and HSA = 1.0 mg ml1) after washing with heparin (A), NaCl (B) and urea (C) solutions. LDL can be eluted from heparin-modified PSu, but not from plain surface, with heparin, NaCl or urea solution.

some elution beginning at 10 lg ml1. As heparin has a high affinity for LDL [17–21], it can displace the adsorbed LDL on the heparin-modified PSu surface by competitive interaction and result in LDL elution from the heparin-modified PSu in all cases of various concentrations of heparin. However, the high cost of heparin and the large amount of washing solution required results in expensive regeneration and/ or extensive purification steps. From this point of view, salt and urea are good alternatives to heparin for an effective and economic regeneration. As shown in Fig. 8B, the adsorbed LDL was eluted when NaCl (PBS, pH 7.4) was used for elution as well as heparin solution. The amount of the desorbed LDL was increased by increasing the concentration of NaCl in PBS (pH 7.4). It is reasonable that high NaCl concentrations can reduce the surface potential by reduction of the Debye–Hückel length and hence the range of electrostatic interaction force [21,40]. Compared to heparin and NaCl, urea can more effectively remove adsorbed LDL from heparin-modified PSu (Fig. 8C). Approximately 70% of the adsorbed LDL was eluted from the heparin-modified PSu surface by 8 M urea solution, while there was no elution of the adsorbed LDL from the plain PSu. This can be ascribed to the fact that urea can interfere with stabilizing intramolecular interactions and disrupt the three-dimensional structure of proteins mediated by non-covalent forces [41]. On the other hand, there was some irreversibly

adsorbed amount of LDL on both plain and heparin-modified PSu films. Combined with the irreversibly adsorbed LDL, the heparinmodified PSu described here can participate in selective LDL adsorption and remains relatively inert to the non-specific adsorption at the same time. 4. Conclusions We have found that it is possible to covalently immobilize heparin onto the surface of a PSu film by a three-step synthesis method. A heparin density up to 0.86 lg cm2 could be achieved on dense PSu film. The hydrophilicity of the PSu surface was improved greatly by covalent immobilization of heparin. The water contact angle of PSu films was decreased from 89.6 ± 3.7° to 50.5 ± 3.2° after binding of 0.36 lg cm2 heparin. The results of protein adsorption show that the surface-bound heparin greatly enhanced the adsorption of LDL both from single protein solutions and mixtures with HSA that closely resemble the conditions in human plasma in both healthy and diseased subjects. In addition, compared to plain PSu, the adsorbed LDL could be easily desorbed from the modified PSu surface with heparin, NaCl or urea solution. In conclusion, heparin-modified PSu membranes may have great potential for simultaneous application in hemodialysis and LDL apheresis.

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