Copolymerization Procedures

Acrylonitrile can be easily copolymerized with a variety of comonomers (Krasteva et al. 2002). Copolymerizations of hydrophilic/functional monomers such as maleic acid (Nie et al. 2004a,b), N-vinyl-2-pyrrolidone (Groth et al. 2002; Krasteva et al. 2002; Wan et al. 2005), a-allyl glucoside (Xu et al. 2003, 2004), 2-hydroxyethyl methacrylate (HEMA; Huang et al. 2005a,b), acrylamides (Musale and Kulkarni 1996,1997), and N-vinylimidazol (God-jevargova et al. 2000) with acrylonitrile have been performed to improve the properties of PAN-based membranes. Modifications through copolymerization are capable of improving the bulk property of polyacrylonitrile to some extent. Furthermore, the surface properties of the membrane can also be greatly improved, because the amount of incorporated comonomers can be accurately tuned, and the hydrophilic moieties of the comonomer may migrate to the membrane surface to minimize the surface energy when used in an aqueous environment.

Carbohydrates exist in many forms and play important roles in natural living systems. Their highly hydrophilic characteristics together with their innate compatibility with biomolecules may meet the demands of protein resistance and have led to considerable interest in the synthesis of their polymers. We have synthesized the copolymer of acrylonitrile with a-allyl glucoside using the process of water-phase precipitation copolymerization (Xu et al. 2003). Copolymer membranes with different contents of a-allyl glucoside were prepared using a phase inversion process (Xu et al. 2004). The formula of a-allyl glucoside and the water contact angle of the membranes are shown in Figs. 1 and 2, respectively. BSA was chosen as a model protein to investigate the protein resistance of these copolymer membranes and typical results are shown in Fig. 3. It is well known that the hydropho-bic interaction between the membrane surfaces and proteins plays a very important role in the nonselective adsorption of protein onto those membrane surfaces. Materials that possess a hydrophilic surface usually show relatively low nonselective adsorption for proteins or cells. Carbohydrate-containing polymers are highly hydrophilic materials, a property that can be confirmed by water-contact angle measurements; however, some recognize the biomolecules or cells because of the "cluster effect". Therefore, the hydrophilicity and recognition function of the carbohydrate moieties have different effects on the adsorption of proteins or cells.

It can be seen from Fig. 1 that the adsorbed amount of BSA decreases almost linearly with increases in a-allyl glucoside content in the copolymer: the higher BSA concentration may lead to the larger amount of BSA adsorbed. The decrease in BSA adsorption can be ascribed mainly to the improvement in hydrophilicity by the carbohydrate moieties for the membrane surface. Macrophage adhesion was also performed on these membranes to evaluate their biocompatibility; the results are shown in h2c=ch z I

Fig. 1. Chemical structure of a-allyl glucoside

25 -|—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i— 0 5 10 15 20 25 30 35 40 45

AG content in AN/AG copolymer film (wt.%)

Fig. 2. Relationship between the contact angle and the content of a-allyl glucoside (AG) in the acrylonitrile/a-allyl glucoside (AN/AG) copolymer membranes

Fig. 2. Relationship between the contact angle and the content of a-allyl glucoside (AG) in the acrylonitrile/a-allyl glucoside (AN/AG) copolymer membranes

AG content in the copolymer membrane (wt.%)

Fig. 3. Adsorption of bovine serum albumin (BSA) onto AN/AG copolymer membranes

Fig. 4. Relative macrophage number versus AG content in AN/AG copolymer membranes

Fig. 4. It was clearly demonstrated that the number of macrophages that adhered onto the copolymer membranes decreased sharply when compared with that on the polyacrylonitrile membranes. However, it seems that the macrophage adhesion number increased again when the content of a-allyl glucoside exceeded around 20wt%. This might be due to the "cluster effect" of the carbohydrate moieties in the copolymer chains. It was reported that the carbohydrate density strongly affects the specific interactions between carbohydrate moieties and cells, and that the binding affinity is drastically enhanced by multivalent carbohydrate ligands (the "cluster effect").

Poly(N-vinyl-2-pyrrolidone) has excellent biocompatibility with living tissues. N-vinyl-2-pyrrolidone is also a potential comonomer for the chemical modification of polyacrylonitrile membranes by copolymerization. Groth and coworkers (Groth et al. 2002; Krasteva et al. 2002) synthesized copolymers of acrylonitrile with N-vinyl-2-pyrrolidone using a solution copolymerization process. They used this type of copolymer membrane as culture substrate for human skin fibroblasts for the development of an artificial skin. The attachment, morphology, and growth of hepatocytes on this copolymer membrane were also investigated. They proposed that there was no simple relationship between the wettability of the membrane and its ability to support cell adhesion and function. It was also suggested that this copolymer containing N-vinyl-2-pyrrolidone can be considered as an interesting substrate if sufficient numbers of hepatocytes are seeded to promote sufficient functionality for a biohybrid liver support system.

Fig. 5. Adsorption of BSA at different concentrations (2 g/l open triangles; 5 g/l filled squares) on acrylonitrile/N-vinyl-2-pyrrolidone copolymer films with various NVP content

Acrylonitrile/N-vinyl-2-pyrrolidone copolymers with different contents of N-vinyl-2-pyrrolidone were also synthesized by us using a water-phase precipitation copolymerization process. It was found that the introduction of N-vinyl-2-pyrrolidone into PAN did not change the water contact angles much, and this is in accord with the findings of Groth et al. (2002). However, as shown in Fig. 5, BSA adsorption was remarkably depressed with increasing content of N-vinyl-2-pyrrolidone in the copolymer. The results of platelet adhesion also confirmed the improvement in the biocompati-bility of PAN afforded by the incorporation of N-vinyl-2-pyrrolidone. One can envisage that the protein fouling on this copolymer membrane surface might be reduced or even partly eliminated.

How do polymers containing N-vinyl-2-pyrrolidone achieve excellent biocompatibility? Hayama et al. (2004) insisted that the biocompatibility of membranes containing N-vinyl-2-pyrrolidone was dependent on both the number of N-vinyl-2-pyrrolidone units and their surface structures. They proposed that the higher the regularity of the polymer particle structure in the wet condition, the lower the wet:dry ratio surface roughness and the larger the wet:dry ratio of the polymer particle diameter. That is, the more the polymer particles swell as a result of wetting, the greater the biocompatibility.

We have synthesized the copolymers of acrylonitrile with maleic acid using the water phase precipitation copolymerization process (Nie et al. 2004c), which were denoted as PANCMA. Ultrafiltration hollow-fiber membranes of these copolymers with different molecular weights and contents of maleic acid were also prepared by a dry-wet phase inversion process. The carboxyl groups on the membrane surfaces were then converted into anhydride groups by refluxing the membranes in acetic anhydride; the resultant membrane was denoted as PANCMAn. Although the amount of protein adsorbed onto both PANCMA and PANCMAn membranes were large, it was obvious that the former, with a BSA adsorption of 2.31 g/m2, was more protein resistant than the latter, with a BSA adsorption of 4.02 g/m2. Based on the fact that the water contact angle on the PANCMA membrane was 55 °C, which was smaller than that on the PANCMAn membrane (65 o C, this different adsorption behavior of BSA might be ascribed to differences in the surface hydrophilicity.

Musale and Kulkarni (1996) investigated the fouling reduction in membranes fabricated from PAN and acrylonitrile-based copolymers with increasing acrylamide content. They suggested that membranes containing acrylamide were more hydrophilic, had a smaller dispersion force component of the surface energy and a smaller negative zeta potential than those prepared from PAN. The effect of the surface chemistry of these membranes with similar pore sizes was studied through the ultrafiltration of BSA as a function of feed pH. It was found that the PAN membrane exhibited relatively low BSA transmission rates throughout the pH range 4.0-7.5. However, the acrylamide-containing membranes exhibited marked increases in BSA transmission at pH values above the protein isoelectric point (pH 4.8). This might be ascribed to the combined effects of improved hydrophilicity/reduced dispersive surface energy and less electrostatic repulsion between the copolymer membranes and BSA. On the contrary, at pH values below the isoelectric point (pH 4.8), all of the membranes had a low BSA transmission due to strong adsorption resulting from attractive electrostatic interactions. They also found that the permeate flux and flux recovery increased for all membranes with increasing feed pH. This might be partly attributed the decreasing electrostatic attraction and increasing electrostatic repulsion between BSA and the membrane surface. At any given pH, the data comparison of permeate fluxes and flux recoveries indicated less fouling of the surface in the case of the acrylamide-containing membrane. In other words, the acrylamide-containing PAN membrane had a greater protein resistance.

Hemoglobin, a protein with a similar molecular weight to BSA but that is more hydrophobic, was also used by Musale and Kulkarni (1997) as a model protein in the ultrafiltration of PAN membranes containing acrylamide. In this case, because the hydrophobic interactions were strong, the rejection was relatively constant and the flux exhibited a minimum at the isoelectric point, while it increased monotonically through the copolymer membranes. It was also proposed that hemoglobin would be adsorbed preferentially on the membrane surface during the ultrafiltration of mixed proteins of hemoglobin and BSA.

A copolymer of acrylonitrile and N-vinylimidazol was synthesized by Godjevargova et al. ( 2000) and its ultrafiltration membrane was used for glucose oxidase immobilization. They found that the enzyme bound on the copolymer membrane had high relative activity (91%). One can envisage that the membrane containing N-vinylimidazol provided the immobilized enzyme with a good microenvironment and could optimize the conformation of enzyme for catalysis.

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