Surface modifying of microporous PTFE capillary for bilirubin removing from human plasma and its blood compatibility

Materials Science and Engineering C 28 (2008) 1480-1488

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Materials Science and Engineering C j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m s e c

Surface modifying of microporous PTFE capillary for bilirubin removing from human plasma and its blood compatibility Gu Jin a,⁎, Qizhi Yao a, Shanzi Zhang a, Lei Zhang a,b a b

Department of Chemistry, University of Science and Technology of China, HeFei, 230026, PR China AnHui Entry Exit Inspection and Quarantine Bureau, HeFei, 230001, PR China

A R T I C L E

I N F O

Article history: Received 22 October 2007 Received in revised form 31 March 2008 Accepted 8 April 2008 Available online 22 April 2008 Keywords: Polyvinylalcohol Surface modification Polytetrafluoroethylene Endotoxin (bilirubin) Blood compatibility

A B S T R A C T In this study, human serum albumin (HSA) was covalently immobilized onto the inner surface of microporous poly(tetrafluoroethylene) (MPTFE) capillaries for direct bilirubin removal from human plasma. To obtain active binding sites for HSA, the MPTFE capillaries were chemically functionalized by using a coating of poly(vinyl alcohol) (PVA)–glycidyl methacrylate (GMA) copolymers. Characterization of grafted MPTFE capillaries was verified by XPS, Fourier transform infrared spectroscopy (FT-IR), scanning electronic microscopy (SEM). Non-specific adsorption on the PVA–GMA coated capillary remains low (b 0.38 mg bilirubin/g), and higher affinity adsorption capacity, of up to 73.6 mg bilirubin/g polymer was obtained after HSA is immobilized. Blood compatibility of the grafted MPTFE capillary was evaluated by SEM and platelet rich plasma (PRP) contacting experiments. The experimental data on blood compatibility indicated that PVAcoated and PVA–GMA–HSA coated PTFE capillary showed a sharp suppress on platelets adhesion. The proposed method has the potential of serving in bilirubin removal in clinical application. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Bilirubin (BR) is normally conjugated with albumin to form a watersoluble complex [1,2]. The free bilirubin is a lipophilic endotoxin and may bind to cellular and mitochondria membranes. If untreated, deposition of excess bilirubin in various tissues especially the brain leads to kernicterus, which may result in permanent brain damage, further, may cause infant deaths [3,4]. Studies on the removing of bilirubin from human serum albumin become important for the patients suffering from hyperbilirubinemia. At the present time, hemoperfusion is becoming the most promising technique, for it uses an adsorbent with affinity, high adsorption capacity and good blood compatibility. Consequently, affinity adsorption by using a biomaterial has developed into a powerful tool in the biomedical field, especially for the removal of toxins from human plasma. Many studies have aimed to improve hemocompatibility and affinity of materials by surface modification [5,6], such as chemically immobilizing anticoagulant and increasing hydrophilicity so on. Now, heparin, albumin, polyethylene glycol (PEG) are considered as outstanding anticoagulant materials to prevent platelet adhesion, and thrombus formation. However, these polymers alone lack the ability to form a stable matrix and therefore must be attached to a support with suitable mechanical properties, such as cellulose [7] and PTFE [8] etc.

⁎ Corresponding author. Tel.: +86 551 3601596. E-mail address: [email protected] (G. Jin). 0928-4931/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2008.04.008

At present time, these support carriers are in form of microbeads [9] and membranes [10] because it is difficult to modify the inner surface of tube, especially like capillary with small inner diameter. However, tube has both the advantage of membrane and microbeads, and it has mass transfer of higher velocity, better adsorption capacity, less fouling, longer useful life and can be connected to recirculation flow system directly without any auxiliary equipment. In our previous works, the PVA-coated MPTFE capillaries were used as support carrier for bilirubin removal [8]. PVA is one of the important biomaterial for its chemical stability, membrane-forming ability etc. [10]. However, poor reaction activity hinders the extensive usage in the field of medical application [11]. Acrylic acid based polymers have inherent antimicrobial properties [12], and that acrylic acid has good reaction activity so as to graft active biomolecules or affinity ligand expediently. Therefore, acrylic acid based polymers have been widely used in various biomedical applications [13]. From this point of view, we developed a new method that could modify the surface of MPTFE capillary by using a PVA–GMA copolymer to improve hydrophilicity of MPTFE and to introduce reactive epoxy groups (i.e. GMA). Finally HSA was covalently immobilized onto the copolymer as affinity ligand and anticoagulant to make a novel coating to prevent or suppress unwanted or uncontrol reactions between blood and material surface. The affinity bioadsorbent (MPTFE capillary) was used for bilirubin adsorption in a flow injection system. And the performance of the affinity MPTFE capillary in human blood was evaluated by blood compatibility tests. The similar method has not been reported before.

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2. Materials and methods 2.1. Chemicals and apparatus MPTFE capillaries, kindly provided by Professor K. Watanabe, were used for the present study. These capillaries had an internal diameter of 2 mm, a wall thickness of approximately 0.5 mm. Scanning electron microscopy of MPTFE capillary inner walls has been presented in Fig. 1(a) and (b). PVA (average Mr 14,000, 100% hydrolysed) was purchased from the chemical reagent company of Shanghai, China. HSA (human serum albumin) was purchased from Sigma. The blood samples having different bilirubin initial concentrations were directly provided by Anhui Province People Hospital (Hefei, China). Reagents such as GMA, terephthaldehyde,

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phosphate buffer saline (PBS, pH 7.4), etc. were analytical reagent grade and were obtained from local drug store. The fresh piranha solution was prepared by mixing concentrated sulfuric acid and hydrogen peroxide (1:1, v/v). All water used in the experiment was deionized water. A flow injection system (Model FI-2100, Beijing Haiguang Instrument Co., China) was used for the feeding of human plasma. The concentrations of bilirubin and albumin in the plasma samples were determined by 7060 automated analyzer (HITACHI, Japan). BPC (Blood Platelet count) was determined by ABBOTT CELL-DYN 1600 Hematology Analyzer (HITACHI, Japan). The FT-IR spectra of PVA–GMA copolymer were recorded on a 360 FT-IR spectrometer (Nicolet, America). The PVA-coated MPTFE capillary inner surface was characterized by SEM (JEOL JSM-6700F, America) and XPS (ESCALAB-250).

Fig. 1. The SEM micrographs of MPTFE and PVA–GMA coated MPTFE capillary. a: Unmodified MPTFE inner wall; b: the partly magnified a; c: PVA–GMA coated MPTFE inner wall, PVA: 20 mg/ml; d: PVA–GMA coated MPTFE inner wall, PVA: 40 mg/ml; e: PVA–GMA coated MPTFE inner wall, PVA: 50 mg/ml; f: the partly magnified e, PVA: 50 mg/ml.

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2.2. Surface modifying of MPTFE capillaries 2.2.1. Preparation of PVA–GMA copolymer PVA solution with a concentration of 1 wt.% was prepared by dissolving 0.5 g of PVA in 50 ml of deionized double-distilled water for 2 h at 90 °C, then the 0.8 ml of GMA and sulfuric acid were put into the solution of PVA in a 250 ml two-necked round bottom flask respectively, and nitrogen gases were pre-gassed for 30 min, finally ceric ammonium sulfate was then added carefully to initiate the reaction between PVA and GMA. The concentrations of cerium (IV) ions and hydrogen ions are 7.0×10− 3 M and 0.012 M, respectively. The mixed solution was kept at room temperature for 4 h, and nitrogen atmosphere was maintained throughout the reaction period, then the solution was transferred into 250 ml beaker, and 1,4dioxane was added to the solution until the white precipitate emerges. The solution was filtered using suction flask, washed with 70% 1,4-dioxane, dipped in tetrahydrofuran for 24 h for removing the homopolymers, and dried under vacuum at 50 °C for more than 24 h to a constant weight. 2.2.2. Coating of MPTFE capillaries with PVA and PVA–GMA MPTFE capillaries were firstly activated by fresh piranha solution [14]. Then, MPTFE capillaries were modified with PVA–GMA by a twostep procedure. In the first step, PVA was deposited on the surface of capillary by a simple adsorption process carried out in an aqueous medium [8]. In the second step, PVA–GMA copolymers adsorbed on the MPTFE capillaries were chemically cross-linked to give a stable PVA– GMA coating on the surface. After adsorption of PVA from solution, the final acid concentration of the medium was adjusted to 0.1 M by adding HCl, a 10-mg amount of terephthaldehyde (tere) was dissolved in 10 ml of water and this solution was added to the previous medium, then mixed solution was laid aside, the temperature of solution was increased to 80 °C, the cross-linking reaction was completed in 2 h, finally the capillaries were taken out from the solution and washed several times with hot distilled water, 100 mM phosphate buffer (pH = 8.0) and 10 mM phosphate buffer (pH = 7.0), respectively. The PVA–GMA coated MPTFE capillaries were stored at 4 °C. 2.2.3. Immobilization of human serum albumin For immobilization of HSA, the MPTFE capillary was preconditioned with water and incubation buffer (100 mM phosphate, pH 7.0) [15]. 50 ml of 0.5 mg/ml HSA in incubation buffer was pumped into the capillary by using a FIA system, then capillary was incubated with 5 ml fresh HSA solution for 18 h, rinsing with incubation buffer followed, before applying the washing solution (incubation buffer supplemented with 0.1% (w/w) Tween-20, 150 mM NaCl) to remove the non-covalently bound protein fraction. Capillary was shaken in or perfused three times with 10 ml washing solution for at least 1 h, finally capillary was washed with the buffer used for application. The Ponceau S staining method, described in Borcherding et al. [15] and Ulbricht et al. [16], was adapted to determine the amount of bound protein on the capillary.

bilirubin in the initial (before adsorption) and in the final solution (after adsorption) (mg/l), respectively; V is the volume of the human plasma; and m is the mass of the capillary (g). The concentration of bilirubin and albumin in the plasma samples was determined using HITACHI 7060 automated analyzer. In a typical flow injection system, 50 ml of the plasma was recirculated through the modified capillary for 2 h. Bilirubin adsorption from human plasma containing 2.12 mg/l was studied at different temperatures (4, 25, and 37 °C), pH values (3–9.5) and ionic strengths (adjusted by added NaCl to the plasma). Adsorption rates were obtained both in the continuous recirculation flow injection system. Human plasma samples containing different amounts of bilirubin were used in these experiments. The changes in the bilirubin concentration with time were followed to obtain the adsorption rate curves. The flow rate was 1 ml/min. These studies were performed at a constant temperature of 25 °C. 2.4. Regeneration of the modified capillary The bilirubin-saturated capillary was regenerated with BSA and sodium hydroxide. The bilirubin-saturated capillary was eluted with recirculating the BSA (300 mg/l) solution. Then the absorbed BSA on the capillary was eluted with 5 M NaSCN eluant, and the capillary regenerated successively with 1% Tween 80 and distilled water. The elution process by the alkaline solution included immersing the bilirubin absorbed capillary in 0.1 M NaOH aqueous solution, followed by the procedure of washing with large volume of distilled water and phosphate buffer (pH 7.4). And we also used combined methods mentioned above. The regenerated capillary was then reused for bilirubin equilibrium adsorption. 2.5. Blood compatibility Plasma recalcification time (PRT) is an indicator of intrinsic coagulation cascade activation, and therefore a useful marker of biomaterial induced coagulation activation. The PRT was measured referring to references [8,17]. The analysis of hemolysis was performed using the unmodified capillary, the PVA–GMA coated capillary and the PVA–GMA–HSAimmobilized capillary, respectively. Hemolytic activity was assessed with determining hemoglobin release under static conditions using the two-phase ISO/TR 7405-1984 (f) hemolysis test [18]. In order to determine the potential blood compatibility of the materials, platelet adhesion studies were conducted since platelet adhesion is one of the most important steps during blood coagulation on artificial surfaces [19]. The platelet adhesion test was performed by referring to our previous work [20]. 3. Results and discussion 3.1. Surface modification of MPTFE capillary

2.3. Bilirubin removal from human plasma The bilirubin adsorption from human plasma with the unmodified and HSA-immobilized MPTFE capillary was carried out in a flow injection system [8]. The affinity MPTFE capillary connected to the flow injection system directly without any auxiliary equipment. The blood samples were obtained from patients with hyperbilirubinemia. Recirculation of human plasma was achieved by changed rotation direction of pump. In which an adsorption system equipped with a water bath apparatus for temperature control was used. The amounts of bilirubin removed from the human plasma (or adsorption capacity) were described by the following equation: T ¼ ðC0  C ÞV=m

ð1Þ

Where T is the amount of bilirubin adsorbed onto unit mass of the capillary (mg/g polymer); C0 and C are the concentrations of the

As we know microbeads adsorbent systems and microporous membrane adsorbent systems need complex packed procedure and/or auxiliary instrument. Compared with those absorbents mentioned above, the capillaries are easy to be connected to transport system and without any packed steps. Starting from this point, we selected PTFE capillaries as the carrier and flow injection system as continuous recirculation system for the feeding of human plasma in bilirubin removal experiments. And as we know, PTFE is an extremely inert and hydrophobic matrix, which has poor blood compatibility, so it must be modified before used in purpose of biological or medical treatment. At present, the modification of PTFE microbeads and PTFE membranes has been performed by methods of irradiation (i.e., γ-radial, laser and plasma) [21–23]. However, these methods need special equipments and destroyed the surface structure. Moreover, the modification of microtube, especially PTFE capillary, is a very difficult job, not only because of

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while the strong absorption bands at 1095 cm− 1 and 1717 cm− 1 disappeared. These provide substantial evidence of grafting GMA onto PVA. Moreover, the SEM micrographs and XPS of unmodified/ modified MPTFE capillaries confirmed that PVA–GMA coating was attached onto the inner surface of MPTFE capillaries [see Figs. 1(c), (d) and 2 (a), (b), ]. The mechanism of reaction between PVA and GMA may be as following:

Fig. 2. The XPS of unmodified and modified MPTFE capillary. a: Unmodified MPTFE capillary. b: Modified MPTFE capillary.

its chemical stability, but also because of the shape of capillary [24]. LÖhbach first modified PTFE vessels with fresh piranha solution [14]. Watanabe modified the PTFE capillary with NaOH solution [25]. However, it is not enough because the surface of PTFE remained hydrophobic and the active site was quite low. In our previous paper, the PVA was coupled to the surface of MPTFE, which can be as an intermediate for further attachment of an adhesion molecule [8]. However, PVA is not an active intermediate, and it can combine directly covalently with a few of biomolecules or ligands. For a successful affinity adsorbent, the ligand should be attached to the adsorbent matrix in a way that its binding to the target protein would not be seriously disturbed. The employment of spacer arm may set the ligand away from the solid matrix to make it more accessible to the protein. For solving this problem, in this paper, GMA, a non-toxic and high reaction active molecule, is coupled with PVA to form a copolymer in order to immobilize HSA. Graft copolymerization is one of the techniques employed for improving the chemical and physical properties of a polymer. A wide range of monomers has been successfully grafted to PVA, nylon, chitosan, respectively, by using Ce(IV) [26] or potassium persulphate as initiators [27,28]. In this article, GMA was coupled with PVA to form PVA–GMA copolymer, then PVA–GMA is coated onto MPTFE capillary, and its epoxide functional groups were used to immobilize HSA. The spectrum of the grafted PVA exhibits strong absorption bands at 1095 cm− 1, 1333 cm− 1, 1451 cm− 1, 1717 cm− 1, 2924 cm− 1 and 3423 cm− 1. These absorption peaks are due to the presence of νO–H [– CH(OH)–], δO–H [–CH(OH)–], δC–H [–CH2–], νCfO [no alcoholysis CfO in PVA], νC–H [–CH2–] and νO–H [–OH–], respectively. The spectrum of the grafted PVA–GMA exhibits some different strong absorption bands with PVA are for νCfO [–CO–CH(CfO)–] at 1733 cm− 1; for νC–O–C (epoxy) at 1266 cm− 1; for δC–H [–CHfCH–] at 993 cm− 1 and 907 cm− 1

Fig. 1(a) and (b) shows the inner wall structure of MPTFE capillary with netty porosity (forming grooves). Moreover, from Fig. 1(c) and (d), we know that average pore aperture at PVA–GMA coating is about 60– 180 nm for 20 mg/ml PVA and 30–100 nm for 40 mg/ml PVA, respectively. When the initial concentration of PVA was over 40 mg/ml, the pores of surface disappeared (see Fig. 1e and f). However the surface of modified MPTFE capillary still reserved grooves. Compared to common PTFE capillaries [20], we can consider that the netty grooves and the surface of node with having pores in lower PVA concentration as well as the ordered groove form without pores in higher PVA concentration ensures the effective adsorption of bilirubin. The formed porous surface including the pore size (see Fig.1c and d) or non-porous surface (see Fig. 1e and f) relates to the coagulation rate. In general, macrovoids were formed when the coagulation is fast, whereas the slow coagulation rate results in a porous sponge-like structure [29]. However, the authors also state some exceptions, viz. macrovoid formation can be suppressed when the polymer concentration exceeds a certain minimum value [30]. As we know, the coagulation rate is relative to the concentration of PVA and terephthaldehyde, and the temperature of solution. When the PVA concentration was low (≤40 mg/ml), the porous surface could be formed as long as the conditions (e.g. temperature) were well controlled during coagulation process. But the porous surface disappeared when the PVA is over 40 mg/ml, the conclusion is consistent with result of literature [30]. Table 1 listed the effect of PVA concentration on the behaviors of modified MPTFE capillaries. As we knew from Table 1, the capacities of grafted HSA and adsorbed bilirubin in high concentration PVA solution were higher than that in low concentration PVA solution. However, when the PVA concentration is over 40 mg/ml, it is very difficult to prepare the PVA coating for gelation of PVA solution, and to flow by on-line system. So the suitable amount of PVA favors preparing efficient coating with suitable pore size. In this paper, 40 mg/ml is an experimental condition. 3.2. Human serum albumin immobilization In this study, HSA was used as the affinity ligand for specific binding of bilirubin molecules. For high loading capacity and activity of the immobilized HSA onto the inner surface of MPTFE, direct multipoint covalent attachment of HSA to the epoxide groups of PVA– GMA-activated was carried out. The introduced epoxy groups allowed a stable covalent immobilization of proteins mainly through their Table 1 The effect of the PVA initial concentration on the behaviors of modified MPTFE capillaries PVA initial concentration

Pore size (nm)

HSA grafted capacity (mg/g polymer)

Bilirubin adsorbed (mg/g polymer)

20 mg/ml 40 mg/ml 50 mg/ml

60–180 30–100 –

83.2 125.1 136.6

51.5 73.6 78.5

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Fig. 3. Adsorption rates of bilirubin from human plasma with time in the continuous recirculation systems: flow rate: 1.0 ml/min; temperature: 25 °C; bilirubin initial concentration: 2.12 mg/l and total plasma volume: 20 ml.

exposed amino groups. The result for HSA immobilized onto PVA– GMA-activated capillaries is 125.1 mg/g. 3.3. Bilirubin removal from aqueous solution and human plasma 3.3.1. Adsorption rate Fig. 3 gives the non-specific and specific adsorption of bilirubin onto MPTFE capillaries. The amount of bilirubin adsorption grafted onto the PVA–GMA coated and HSA-based MPTFE capillaries were about 0.38 mg and 73.6 mg bilirubin/g polymer, respectively. The non-specific adsorption of bilirubin on the MPTFE capillaries is very low. As expected, immobilization of HSA on PVA–GMA coated MPTFE capillaries results in an increase in adsorption capacity of the affinity capillaries. Fig. 4 indicates that the extent of adsorption of bilirubin increases with the increase of initial concentration of bilirubin in the adsorption medium up to 73.6 mg/l. Adsorption rates increased with increasing bilirubin concentration. That is because of a high driving force, which is the bilirubin concentration difference between the human plasma and the solid (i.e., the capillary) phases, in the case of high bilirubin concentration.

Fig. 5. Effects of pH on bilirubin adsorption. Flow rate: 1.0 ml/min; bilirubin initial concentration: 2.12 mg/l; temperature: 25 °C and total plasma volume: 20 ml.

and the bilirubin are higher in neutral solution than those in acidic and basic solutions. In other words, only in neutral solution (pH 6–7) bilirubin has the smallest polarity and has the biggest affinity to the HSA-immobilized capillary. And with the increasing of pH, the hydrogen bonds in the molecular structure were destroyed gradually and the solubility of bilirubin increased gradually, which resulted in the increasing of binding capacity on the capillary. In addition, this phenomenon also can be explained by electrostatic interactions: both bilirubin and HSA molecules tendtobepositivelychargedinacidicsolution;andtobenegativelycharged in basic solution, so a strong electrostatic repulsion effects occurred between bilirubin and HSA molecules, which resulted in low adsorption capacity in strong acidic or basic solution. But only very weak electrostatic repel interactions exist in neutral solution. Therefore, the adsorption curve versus pH value behaves as the phenomena described above.

3.3.2. Effects of pH Several experiments are conducted to study the effect of pH on removal of bilirubin from aqueous solutions and the results are presented in Fig. 5. Since bilirubin possesses both amino groups and carboxylic groups, it is soluble both in the acidic and basic solutions, but not in neutral solution. For this, the interactions between the adsorbent (HSA)

3.3.3. Effects of ionic strength The adsorption capacity of HSA-based MPTFE capillaries to bilirubin was reduced to 1.85 fold with increasing salt concentration from 0.05 to 0.5 M, and it inclines to have a notable influence in high salinity solutions. It may be because the negative carboxyl ion of bilirubin in the experimental conditions could absorb antiparticles around it to form an “ionic atmosphere”. At the same time, the HSA-immobilized capillary was also negatively charged in the solution, which was also the source for “ionic atmosphere”. The existence of ionic atmosphere can weaken or destroy the interactions between bilirubin and the adsorbent (HSA) for the salt linkage provides some stability to the HSA-bilirubin [31]. Therefore, at the condition of high ionic strength the interactions between

Fig. 4. Effect of bilirubin initial concentration on adsorption flow-rate: 1.0 ml/min; temperature: 25 °C and total plasma volume: 20 ml.

Fig. 6. Effects of HSA concentration on bilirubin adsorption. Flow rate: 1.0 ml/min; bilirubin concentration: 2.12 mg/l; temperature: 25 °C; and total plasma volume: 20 ml.

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Serum albumin is the natural carrier for bilirubin in the blood. Each albumin molecule may have as many as 12 binding sites for bilirubin, but only two of the sites bind bilirubin molecules tightly. For successful use in hemoperfusion, an adsorbent should be capable competing with at least the weak binding sites of albumin for the unconjugated bilirubin. In view of reason, we selected HSA as the affinity ligand. When the human plasma flows through the HSA-immobilized affinity MPTFE capillary, both albumin and bilirubin were possible to adsorb on the capillary. A serious hypoproteinemia will damage patient if large numbers of protein adsorbed in bilirubin removal from human blood. This result usually prevents most bilirubin adsorbents in clinical application. In order to observe the interrelation between albumin and bilirubin adsorptions, we also followed the changes of albumin concentration (by adding HSA) in the plasma samples before and after each adsorption cycle. The effect of HSA concentration on bilirubin removal was shown in Fig. 6.

As seen here, the adsorption capacity decreases with the increasing of HSA concentration. This decrease can be explained by two possible reasons. One is the complex of HSA conjugated with bilirubin has a larger molecular volume compared to bilirubin molecular, so it can decrease the diffusivity of bilirubin from the bulk solution to the surface of capillary; on the other hand, larger molecular volume can reduce the opportunity of bilirubin molecules to interact with the ligand molecules. Another possible reason is a competitive adsorption exists between bilirubin molecules and HSA molecules. The competitive ability of HSA molecules is improved with increasing its concentration, so the sorbents have a smaller adsorption capacity for bilirubin molecules. Each albumin molecule can offer twelve sites for bilirubin adsorption, but only two sites can bind bilirubin molecule tightly. However, the mol ratio of bilirubin molecules to albumin molecules adsorbed on the affinity capillary is significantly larger than two. The result was similar to that of other related experiments [3]. Denizli et al. [32] considered that adsorption of albumin–bilirubin conjugates might occur in the sorbents, but bilirubin molecules were preferentially adsorbed by ligands in direct interaction. We believe that it is also the case in our system. There is equilibrium between the free and albumin-conjugated bilirubin. More bilirubin molecules will be released from the albumin conjugates in order to attain this equilibrium when one removes the free form by using sorbents. This process will continuously strip bilirubin molecules from the protein conjugate until adsorption equilibrium is reached among the free bilirubin, the albumin-conjugated bilirubin and the sorbent. In addition, the protein adsorption and platelet adhesion should be critical index for evaluating blood compatibility of artificial surfaces [33]. In this study, albumin adsorption was in the range of 3.3–3.6 mg HSA/g polymer for MPTFE capillaries. This is a low HSA adsorption. Many other sorbents for bilirubin removal have good performance in phosphate solution, but very poor adsorption capacity of bilirubin in human plasma

Fig. 7. The SEM micrographs of PVA-coated MPTFE capillary after adhering natural men platelets. a: The magnified multiples 4000×. b: The magnified multiples 10,000×.

Fig. 8. The SEM photos of PVA–GMA–HSA coated MPTFE capillary after adhering natural men platelets. a: The magnified multiples 4000×. b: The magnified multiples 10,000×.

Table 2 Blood compatibility of the unmodified and modified MPTFE capillariesa Materials

Recalcification time (s)

RSD (%)

Hemolysis of material (%)

RSD (%)

BPC (109/L)

RSD (%)

Blank MPTFE capillaries PVA–GMA/MPTFE capillaries HSA/MPTFE capillaries

178.8 92.3 139.1

2.8 3.7 1.7

6.53 2.34

4.0 1.8

250 93 156

3.5 5.0 3.5

151.2

1.1

0.21

2.3

201

2.0

RSD: Relative standard deviation. a Mean values for 6 times determination.

bilirubin and the adsorbent were interfered which led to a decreasing of bilirubin adsorption on the affinity capillary. 3.4. Bilirubin versus albumin adsorption

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increased after chemical grafting. The results suggested the HSAimmobilized capillary has good anticoagulant effect. The anticoagulant mechanism of affinity capillary needs further study. 3.5.2. Analysis of hemolysis The hemolysis values for the different tested materials are compared in Table 2. No apparent hemolysis takes place on modified capillary (i.e., HSA-immobilized MPTFE capillaries), however, there is evidence that some extent of hemolysis occurs on the unmodified capillaries. Again, this confirms that better blood compatibility can be achieved by the affinity ligands (i.e., HSA) immobilized on the capillary. 3.5.3. Platelet adhesion test The blood platelet count results of different samples in platelet adhesive test were presented in Table 2. As seen here, adding PRP to unmodified capillary led to a significant decrease of BPC. But the effect of the surface-grafted materials was weak. So, we can conclude the affinity capillary has good blood compatibility from the result of above experiments. Noting this point is very important. Many sorbents do not have good blood compatibility though they have good adsorption capacity of bilirubin. So these sorbents were prevented from using in biomedical or required auxiliary methods such as envelope. Figs. 7 and 8 showed SEM photos of PVA-coated and PVA–GMA– HSA grafted MPTFE capillary after adhering natural men platelets. The results showed that only a few of platelets were adhered onto modified MPTFEE surfaces. Figs. 9 and 10 showed the situation of natural men whole blood adhered on the surface of PVA-coated and PVA–GMA–HSA grafted MPTFE capillary. It could be seen obviously from above mentioned figures that only a few of blood components (e.g. platelets, erythrocytes etc.) were adhered onto modified MPTFEE surfaces. Fig. 9. The SEM photos of PVA-coated MPTFE capillary after adhering natural men whole blood. a: The magnified multiples 4000×. b: The magnified multiples 10,000×.

or albumin solution, which prevented the possibility of these sorbents in biomedical use. Since the normal value of HSA concentration is 40– 55 mg/ml, we did not attempt to work at higher concentration. From here, we can see the affinity capillary still maintains satisfactory adsorption capacity at high concentration solution of HSA. 3.5. Blood compatibility Platelet adhesion is one of the most main factors in evaluating the hemocompatibility of artificial surfaces [32]. When blood contacts a foreign material, it would always be adsorbed onto the material surfaces, and provoke the adhesion of platelets, white blood cells and some red blood cells onto the plasma protein layer and lead to the formation of fibrin. To confirm preliminary blood compatibility of PVA–GMA–HSA grafted MPTFE capillaries, a platelet adhesion test in vitro was carried out. The number of adhesion test revealed that MPTFE capillary grafted PVA–GMA–HSA coating shows excellent anti-platelet adhesion. The results may offer the possibility of using this for biomaterial devices, which are directly in contact with blood. 3.5.1. Plasma recalcification times Plasma recalcification time, a measurement of intrinsic coagulation cascade activation, indicates the time required for fibrin clot formation once calcium has been introduced into sodium citrate anticoagulated plasma. Contact activation of the intrinsic cascade in plasma will vary with the type of surface, and thus plasma recalcification can be used as an indicator of blood–biomaterial interactions. The plasma recalcification times (PRTs) of the surface-modified PTFE capillary are shown in Table 2. We selected sample blank as reference. The PRT of unmodified PTFE capillary surface was slightly

Fig. 10. The SEM photos of PVA–GMA–HSA coated MPTFE capillary after adhering natural men whole blood. a: The magnified multiples 2000×. b: The magnified multiples 4000×.

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The unmodified MPTFE capillary showed a sharp suppress on platelets and other blood components adhesion. However, the unmodified hydrophobic surface induces easily to change the morphology of those blood components. On the other hand, the hydrophilic surface is in favor of growing of cell (e.g. endotheliocyte). As we know, the hydrophilicity and swelling behavior of polymeric materials play an important role in their blood compatibility properties, and the presence of significant concentrations of bound water within the biomaterial provides a low interfacial tension with blood [34]. Many researchers have been pointed out that the water surface of the material surface is an important factor for the material to express the good blood compatibility. The low platelet adhesion on PVA-coated and PVA–GMA–HSA grafted PTFE capillaries may be related to its hydrophilicity, high water content and low interfacial tension between the hydrogel surface and the surrounding fluids which result in substantial resistance to non-specific protein adsorption, and platelet adhesion usually will not occur to a significant extent if blood protein cannot be adsorbed onto the surface.

have created a HSA-based containing PVA–GMA coating that can be used to modify the surface of otherwise thrombogenic biomaterials. We have demonstrated here that the addition of the PVA–GMA–HSA coating to the inner surface of MPTFE capillaries improves the blood compatibility of the MPTFE. Moreover, through the inclusion of HSA side chains, we effectively improve the surface hemocompatibility even further. Thus, we can exploit the desirable properties of PVA– GMA, while catering the polymer system to the desired application. Comparison of other bilirubin adsorbents shows that the human serum albumin immobilized capillaries have not only a high-affinity adsorption capacity towards bilirubin molecules but also a shorter adsorption equilibrium time. This new adsorbents have both the advantage of membrane and micro-column, and they have mass transfer of higher velocity, better adsorption capacity; less fouling, longer useful life and can be connected to recirculation flow system directly without any auxiliary equipment. This system is low cost and easy to operate, and has potential merit in clinical application for bilirubin removal.

4. Conclusions

Acknowledgements

Despite the desirable properties of PVA in relation to certain biomedical applications such as its antibacterial properties, low toxicity, and suitable physical and mechanical properties, its thrombogenicity limits its use in blood contacting applications. Therefore, we

This work was supported by the National Science Foundation of China (No.: 29405038). We are very grateful to professor Watanabe (University of Science and Technology of Tokyo) for providing the microporous PTFE capillaries.

Appendix A. FT-IR spectrum of PVA and PVA–GMA FT-IR spectrum of PVA.

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FT-IR spectrum of PVA–GMA.

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