**3.1.2.1 Protein adsorption**

Non-specific protein adsorption is a dominant factor for membrane fouling. When membrane is used for blood purification, protein adsorption is the first stage of the interactions of membrane and blood, which may lead to further undesirable results. Protein adsorption has some relationship with the blood compatibility. There are many factors which affect the interaction between membrane surface and protein, such as surface charged character, surface free energy and topological structure, solution environment (e.g. pH, ionic strength), and protein characters (Leng et al., 2003; Okpalugo et al., 2004). The hydrophilic/hydrophobic character of membrane material plays a relatively important role in the interaction between protein and membrane. Since hydrophilic surface preferentially adsorbs water rather than solutes, many researchers have followed the idea of increasing the hydrophilicity of a membrane material with the goal of reducing protein fouling and/or protein adsorption (Mockel et al., 1999). Herein, the surfaces of the PES and some typical modified PES membranes (Copolymer of poly (acrylonitrile-co-acrylic acid), PAN-AA, modified PES membranes with the ratios of the copolymer to PES of 0/16, 0.4/16 and 0.6/16, respectively; and BSA grafted membranes following the copolymer/PES blended membranes) were studied in relation to the adsorption of BSA and FNG in vitro, data are shown in Fig. 5.

### **3.1.2.2 Platelet adhesion**

72 Progress in Hemodialysis – From Emergent Biotechnology to Clinical Practice

the reduction ratio of small molecules (urea, creatinine, phosphate) during hemodialysis for

The protein adsorption experiments were made with BSA and FNG solutions. The concentrations of BSA and FNG were 4.0 g/dl and 0.3 g/dl in phosphate buffered saline (PBS, pH=7.4), respectively. The membrane with an area (for hollow fiber, it's the total surface areas of inside surface and outer surface) of 1 cm2 was incubated in distilled water for 24 h, washed 3 times with PBS solution, and then immersed in the protein solution for 2 h. After protein adsorption, the membranes were carefully rinsed 3 times with PBS solution and then rinsed with distilled water. The adsorbed proteins were quantitatively eluted with 1.0 ml 2% SDS solution for 6 h. The amount of protein in the SDS solution was quantified by

The platelet adhesion experiments were carried out using platelet-rich plasma (PRP). Healthy human fresh blood was collected using vacuum tubes (7 ml, Venoject II, Terumo, Co.), containing citrate/phosphate/dextrose/adenine-1 mixture solution (CPDA-1) as an anticoagulant (anticoagulant to blood ratio, 1:7). The blood was centrifuged at 1000 rpm for 10 min to obtain platelet-rich plasma (PRP) or at 2800 rpm for 15 min to obtain platelet-poor plasma (PPP). The fresh PRP sample was used for the platelet adsorption experiments. The PES membranes (11 cm2 each piece, always flat-sheet membranes) were immersed in PBS solution and equilibrated at 37 C for 1 h. The PBS solution was removed and then 1ml of fresh PRP was introduced. The membranes were incubated with PRP at 37 C for 2 h. PRP was decanted off and the membranes were rinsed 3 times with PBS solution. Finally, the membranes were treated with 2.5 wt% glutaraldehyde in saline for 2 days at 4 C. The samples were washed with PBS solution, subjected to a drying process by passing them through a series of graded alcohol-saline solutions (0%, 25%, 50%, 75% and 100%) and then dried at room temperature. The dried membranes after gold coating were examined using a S-2500C scanning electron microscope (SEM, Hitachi, Japan). The number of adhering platelets on the membranes was calculated from four SEM pictures at a 500 magnification from different places on the same membranes. These procedures were performed on each membrane using four independent membranes (totally n=16), and the number was finally

Non-specific protein adsorption is a dominant factor for membrane fouling. When membrane is used for blood purification, protein adsorption is the first stage of the interactions of membrane and blood, which may lead to further undesirable results. Protein adsorption has some relationship with the blood compatibility. There are many factors which affect the interaction between membrane surface and protein, such as surface charged

the PES membrane in vitro are investigated.

**3.1.1 Experimental** 

**3.1.1.1 Protein adsorption** 

**3.1.1.2 Platelet adhesion** 

averaged to obtain reliable data.

**3.1.2 Results and discussion 3.1.2.1 Protein adsorption** 

**3.1 Protein adsorption and platelet adhesion** 

protein analysis (Micro BCA protein assay reagent kit).

The adhesion of platelets to blood-contacting medical devices is a key event in thrombus formation on material surface. After the platelet adhesion and activation, a series of actions could produce the thrombins which led further coagulant. Therefore, in vitro platelet adhesion assay could reflect the blood compatibility of material surface. To study the platelet adhesion, the morphology of the adhering platelet and the amounts of platelet adhesion on the membrane surfaces are always investigated through scanning electron microscopy (SEM).

Figure 6 shows the typical morphology of the platelets adhering to the PES and modified PES membranes. Herein, the membranes were modified by blending sulfonated PES and a terpolymer of poly (acrylonitrile-acrylic acid-N-vinyl pyrrolidinone) (P(AN-AA-VP)). To prepare the membranes, PES, SPES and P(AN-AA-VP) were dissolved in solvent NMP. The solution was vigorously stirred until clear homogeneous solution was obtained. The concentration of all the solute was 16 wt. %. In the experiment, different kinds of membranes were prepared by changing the ratios of PES, SPES and P(AN-AA-VP) in the casting solutions, and the ratios of PES, SPES and P(AN-AA-VP) were 16:0:0, 15:0:1, 14:0:2, 10:6:0,10:5:1, 10:4:2, respectively. After vacuum degassing, the casting solutions were prepared into membranes by spin-coating coupled with a liquid-liquid phase separation technique at room temperature. The obtained membranes were washed with distilled water thoroughly to remove the residual solvent, which were confirmed by UV scanning. All the prepared membranes were in a uniform thickness of about 60~70 μm, and the membranes were termed M-16-0-0, M-15-0-1, M-14-0-2, M-10-6-0, M-10-5-1, and M-10-4-2, respectively.

As shown in Figure 6, when compared the pictures in the same amplification multiple, it was observed that a large amount of platelets were adhered and aggregated on the PES membrane surface and the platelets formed circular or "pan-cake" shape, which suggested that the platelets were activated and already retracted the pseudopods. However, for the modified membranes, very sparse platelets were found; and the platelet expressed a rounded morphology with nearly no pseudopodium and deformation.

Figure 7 shows the amounts of the adhering platelets on the membranes from platelet-rich plasma. It could be observed that much lower number of the adhering platelets on the modified membranes compared with the PES membrane. Furthermore, the platelet

Polyethersulfone Hollow Fiber Membranes for Hemodialysis 75

Fig. 6. Scanning electron micrographs of the platelets adhering to the membranes.

was attributed to the sulfonic acid group provided by SPES.

adhesion on the terpolymer modified membranes decreased with the increase of the content of the terpolymer P(AN-AA-VP). These results were consistent with those obtained from the protein adsorption, which demonstrated that the platelet adhesion had some relation with the carboxylic groups which were supplied by P(AN-AA-VP). It could also be observed that the platelet adhesion of the SPES modified membrane was significantly depressed, which

Han et al. (Han et al., 1996) suggested that the sulfonic acid groups exhibited high adsorption of albumin and low adsorption of FBG, which might improve the blood compatibility. Thus, the platelet adhesion demonstrated the enhanced blood compatibility of SPES modified membrane. Furthermore, as the ratios of P(AN-AA-VP) to SPES changing, the different amounts of the platelet adhesion could be obtained, and no adhering platelet was found on the surface of the modified membrane M-10-4-2. The reduction of the platelet adhesion on the modified membranes was considered to be the introduction of the sulfonic acid and carboxylic groups which were supplied by SPES and P(AN-AA-VP), respectively.

Fig. 5. (a). BSA adsorption on the membrane surfaces with the blending ratios of PES to PANAA as 16/0.2, 16/0.4, 16/0.6. (■) For the blended membranes; (□) for the BSA grafted membranes (each point represents the means±S.D. of three independent measurements.). (b) BFG adsorption on the membrane surfaces with the blending ratios of PES to PANAA as 16/0.2, 16/0.4, 16/0.6. (■) For the blended membranes; (□) for the BSA grafted membranes (each point represents the means±S.D. of three independent measurements.). (From reference, Fang et al., 2009)

Fig. 5. (a). BSA adsorption on the membrane surfaces with the blending ratios of PES to PANAA as 16/0.2, 16/0.4, 16/0.6. (■) For the blended membranes; (□) for the BSA grafted membranes (each point represents the means±S.D. of three independent measurements.). (b) BFG adsorption on the membrane surfaces with the blending ratios of PES to PANAA as 16/0.2, 16/0.4, 16/0.6. (■) For the blended membranes; (□) for the BSA grafted membranes

(each point represents the means±S.D. of three independent measurements.).

(From reference, Fang et al., 2009)

Fig. 6. Scanning electron micrographs of the platelets adhering to the membranes.

adhesion on the terpolymer modified membranes decreased with the increase of the content of the terpolymer P(AN-AA-VP). These results were consistent with those obtained from the protein adsorption, which demonstrated that the platelet adhesion had some relation with the carboxylic groups which were supplied by P(AN-AA-VP). It could also be observed that the platelet adhesion of the SPES modified membrane was significantly depressed, which was attributed to the sulfonic acid group provided by SPES.

Han et al. (Han et al., 1996) suggested that the sulfonic acid groups exhibited high adsorption of albumin and low adsorption of FBG, which might improve the blood compatibility. Thus, the platelet adhesion demonstrated the enhanced blood compatibility of SPES modified membrane. Furthermore, as the ratios of P(AN-AA-VP) to SPES changing, the different amounts of the platelet adhesion could be obtained, and no adhering platelet was found on the surface of the modified membrane M-10-4-2. The reduction of the platelet adhesion on the modified membranes was considered to be the introduction of the sulfonic acid and carboxylic groups which were supplied by SPES and P(AN-AA-VP), respectively.

Polyethersulfone Hollow Fiber Membranes for Hemodialysis 77

curve. Phosphate was determined using the molybdate blue method: phosphate reacts with ammonium molybdate and is then reduced by stannous chloride to form a blue complex,

Fresh swine blood was collected using a glass tank, containing citrate/phosphate/dextrose/ adenine-1 mixture solution (CPDA-1) as an anticoagulant (anticoagulant to blood ratio, 1:7). The dialysis procedure was the same as the section **3.2.1.1**, and the solute clearance was calculated using the same formula as described in the section 2.2. The concentrations of urea, creatinine and phosphate were determined using an Auto Biochemistry Analyzer

Table 1 summarizes the clearance data and the reduction ratio after the dialysis for small molecules in vitro. It was clearly that the clearances and the reduction ratios for all the solutes were larger using the simulated solution than that for blood. The removal of small molecules during dialysis is governed by hydrodynamic conditions within the dialyser rather than membrane structure since the major resistance to transport from the blood into the dialysis fluid lies not in the membrane but boundary layers adjacent to the membrane. Thus, the data of clearance and the reduction ratio (Table 1) for the simulated solution were higher than that for the blood due to the proteins in the blood, which may induce

Hemolysis ratio was determined for the swine blood in vitro and for the goat blood in vivo. Data showed that there was only a slightly hemodialysis phenomena (about 1.7%) in vitro.

solution 174.06.0 169.05.0 170.06.0 94.33.8 92.44.1 Blood in vitro 157.57.4 143.66.8 144.57.2 71.23.9 69.94.0 Blood in vivo 153.69.4 141.68.2 142.57.3 69.24.5 68.95.2

The biocompatibility and separation performance of PES-based hemodialysis membranes in vivo are also discussed. Animal experiments are carried out to evaluate the PES hollow fiber membranes firstly, and goat was selected as the experimental animal. Experiments were performed to evaluate the solute clearance and the blood compatibility. The blood compatibility and performance of the PES-based high-flux hemodialysis membrane in hemodialyzation were also clinically evaluated, and compared with those of two conventional high-flux membranes, polysulfone (PSF) and polyamide (PA) membranes. The PES and PSF membranes showed similar blood compatibility and solute clearance, and the

Table 1. Small molecular clearance at a blood (or simulated solution) flow rate of 180

blood compatibility for PES and PSF might be better than that of the PA membrane.

Clearance (ml/min) Reduction ratio (%)

Urea creatinine phosphate Urea creatinine

and then measured at 670nm with the UV-VIS spectraphotometer U-200A.

**3.2.1.2 Hemodialysis using swine blood in vitro** 

7170A (Hitachi Co., Ltd., Tokyo, Japan)

**3.2.2 Results and discussion** 

concentration polarization.

Data are expressed as the meansSD, n =3

**4. Performance evaluation in vivo** 

ml/min and dialysate flow rate of 500 ml/min

Simulated

The platelet adhesion results were consistent with FBG adsorption. It is well known that FBG adsorption from plasma onto a material surface might promote the adhesion of the platelets because it had the ability to bind specifically to the platelet membrane glycoprotein, GP IIb-IIIa (Phillips et al., 1988). Thus, the observed decreasing amounts of platelet adhesion might be attributed to the increased hydrophilicity, and decreased FBG adsorption. These results indicated that the surface heparin-like PES membranes modified by SPES and P(AN-AA-VP) had good blood compatibility for using as blood contacting devices.

Fig. 7. The number of the adhering platelets on the membranes

### **3.2 Ultrafiltration and solute clearances 3.2.1 Experimental**

### **3.2.1.1 Hemodialysis using a simulation solution**

The test solutions were prepared according to the international standard ISO 8637. The molar concentrations of urea, creatinine and phosphate in the simulation solution were 15mmol/l, 500mol/l and 1mmol/l, respectively. The test procedure was accordant to the procedure in ISO 8637. The ultrafiltration coefficient was calculated as the unit ml/mmHg.h. The clearance (K) of small molecules (urea, creatinine, phosphate) were established by sampling from the inlet and outlet segments of the extracorporeal circuit 1 h after the initiation of the treatment, and was calculated using the following formula.

$$K = \left(\frac{\mathbf{C}\_{BI} - \mathbf{C}\_{BO}}{\mathbf{C}\_{BI}}\right) \mathbf{Q}\_{BI} + \frac{\mathbf{C}\_{BO}}{\mathbf{C}\_{BI}} \mathbf{Q}\_{FA}$$

where CBI is the solute concentration in the blood (here is the simulation solution); I and O refer to the inlet and the outlet to the device, respectively; QBI is the blood flow rate at the dialyser inlet; QF is the filtration rate.

Urea was determined by a reagent Kit for Urea Determination (Diethyl-Monoxime, Beijing chemical regent factory, China); creatinine was quantified by the absorption at 235nm using an UV-VIS spectraphotometer U-200A (Hitachi Co., Ltd., Tokyo, Japan) through a standard curve. Phosphate was determined using the molybdate blue method: phosphate reacts with ammonium molybdate and is then reduced by stannous chloride to form a blue complex, and then measured at 670nm with the UV-VIS spectraphotometer U-200A.

### **3.2.1.2 Hemodialysis using swine blood in vitro**

Fresh swine blood was collected using a glass tank, containing citrate/phosphate/dextrose/ adenine-1 mixture solution (CPDA-1) as an anticoagulant (anticoagulant to blood ratio, 1:7). The dialysis procedure was the same as the section **3.2.1.1**, and the solute clearance was calculated using the same formula as described in the section 2.2. The concentrations of urea, creatinine and phosphate were determined using an Auto Biochemistry Analyzer 7170A (Hitachi Co., Ltd., Tokyo, Japan)
