Biomacromolecule Immobilization

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Since PAN is one of the most important polymeric materials used in blood-contacting devices, efforts have been taken to build a blood-compatible surface. A well-known anticoagulant, heparin, which can catalytically increase the formation rate of antithrombin III and inhibit thrombin and some other coagulating proteases, is commonly used to treat patients who submit to hemodialysis. Heparin is a mixture of variably sulfated polysaccharide chains composed of repeating units of D-gluosamine and either L-iduronic or D-glucuronic acids, as well as some other biomacromolecules such as chitosan, insulin. Together with some proteins, heparin has been proved to be an effective agent in curtailing thrombosis and is effective when immobilized onto polymer surfaces.

In order to modify a PAN membrane to endow antibacterial activity and anticoagulation activity, Lin et al. (2004) immobilized the surface of PAN membrane with a chitosan/heparin complex, denominated as PAN-C/H. The anticoagulation activity was evaluated using protein adsorption, platelet adhesion, and coagulation time including activated partial throm-boplastin time, prothrombin time, fibrinogen time, and thrombin time. Their immobilization procedure is shown in Fig. 13.

They reported that the hydrophilicity of the membrane after bioma-cromolecule-induced immobilization increased slightly. However, the be-

Actication of PAN surface and inducing the carboxyl groups

S NaOH S

(1) Direct immobilization of heparin onto PAN-A by EDC

s edc s

M=Heparin: PAN-H; M=Collagen: PAN-PC; M=Albumin: PAN-PA

(2) Immobilization of heparin onto chitosan-grafted PAN by GA

s edc p s-COOH + NH2-Chitosan -► s-CONH-Chitosan (PAN-C)

GA p

-Chitosan-NH2 -- S-Chitosan-N=CH(CH2)3CHO

°H"Hepann. ^-Chitosan-N=CH(CH2)3COO-Heparin (PAN-C/H)

Fig. 13. Chemical scheme of biomacromolecule-immobilized PAN membranes. A Direct reaction of heparin, collagen, or albumin. B Chitosan (C)/heparin (H) conjugate reaction. EDC N,N-(3-dimethylaminopropyl)-N'-ethyl carbodiimide, GA glutaraldehyde, PAN-A activated PAN membrane, PAN-H heparin-grafted PAN membrane, PAN-PC collagen-grafted PAN membrane, PAN-PA albumin-grafted PAN membrane, PAN-C chitosan-grafted PAN membrane, PAN-C/H chitosan/heparin-complex-grafted PAN membrane havior of protein adsorption onto membranes depended significantly on the surface characteristics, hydrophilicity, roughness, surface charge and chemistry. As shown in Table 2, the adsorbed amount of human serum albumin (HSA) and human plasma fibrinogen (HPF) on PAN-C/H was reduced to 38% and 26%, respectively, of those of PAN. The isoelectric point of HSA and HPF in the blood are 4.8 and 5.5, respectively; thus these proteins carry a negative charge in the normal blood circumstance (pH 7.4). Lin et al. (2004) proposed that, after hydrolysis, PAN-A carries a negative charge (-COO-), thus the protein adsorption was reduced. On the other hand, chitosan is a weak base with a pKa of 6.5, thus PAN-C carries positive charge (-NH+) at pH 7.4. The adsorption of these two proteins was promoted because of the electrical attraction; however, when PAN-C was heparinized, the repulsive SO- and COO- on heparin reduced the adsorp-

Table 2. Platelet adhesion and plasma protein adsorption on the PAN-based membranes

surfaces

Membrane type

Platelet adhesion

HAS adsorption

HPF adsorption

numbers (cells/mm2)

(|g/cm2)

(|g/cm2)

PAN

4810±38

279.7±14.9

564.3±16.1

PAN-A

3811±28

261.7±12.5

5G1.3±15.2

PAN-C

6689±57

288.3±13.9

615.3±14.3

PAN-H

1770±15

154.4±11.9

211.3±12.7

PAN-C/H

1623±16

145.4±12.5

147.9±15.6

PAN-PA

2584±26

-

237±14

PAN-PC

6050±40

-

588±29

tion of proteins. To sum up, they considered that electrical attraction or repulsion might play a key role in the adsorption of protein.

Lin et al. (2004) found that both the protein adsorption and platelet adhesion on the heparin-immobilized PAN membrane surface had the same trend; that is, a higher heparin immobilizing density yielded a lower plasma protein adsorption and lower platelet adhesion on the surface. They also pointed out, however, that the hydrophilic PAN-C surface caused more protein adsorption and a reduction of platelet adhesion was not observed. They proposed that a hydrophilic surface did not always correspond to protein resistance and suppression of platelet adhesion.

In some cases the proteins themselves were immobilized onto the membrane surface to improve the hemocompatibility. As we known, fibrinogen activates and albumin inhibits the adhesion of platelets (Klee and Hocker 2000). Therefore, albumin was often physically adsorbed or cova-lently immobilized onto the membrane surface. Liu et al. (2005) studied the hemocompatibility of PAN membranes covalently immobilized with either collagen (denoted as PAN-PC), which promoted platelet adhesion, or HSA (denoted as PAN-PA), which inhibited platelet adhesion. It can be seen from Table 2 that both platelet adhesion and HPF adsorption were promoted on PAN-PC membrane even compared with original PAN membrane. Both the platelet adhesion and the HPF adsorption of PAN-PA membranes stood almost at the same level with those of the PAN-H membrane. They found that immobilization with HSA resulted in a rougher surface morphology of the PAN membrane, reduced platelet adhesion, fibrinogen adsorption, prolonged the blood coagulation times, and reduced leukopenia and ana-phylatoxin. Collagen-immobilized PAN membranes, however, exhibited the opposite effect when contacting blood, although they induced the least complement activation. They indicated that not all plasma proteins are capable of improving hemocompatibility.

We also immobilized heparin or insulin onto the PANCMA membrane; the procedure is illustrated in Fig. 14. Through the platelet- and macrophage-adhesion experiments, it was found that the immobilization of heparin or insulin improved the hemocompatibility of PANCMA membranes.

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