Preparation And Properties Of New Affinity Adsorbents For Endotoxin Removal

Now with the research development in field of biomedicine and bioengineering, more and more importance are attached to the clearance of trace hazardous substances possibly exist in the product. Endotoxin is a kind of potential contaminant broadly existing everywhere and may cause serious pyrogenic and shock reactions. So the removal of endotoxin from system especially from the bio-preparations is becoming increasingly crucial and has remained an important research aim. Among these the ET removal from macromolecular substance-containing solutions is rather difficult. Till now only a little method and material has gained practical application which, however, suffers from low removal efficiency and high expense. So the study in this field is still valuable and significative.The thesis of this dissertation comes from a Med-X fund cooperative program of Fudan University and Zhongshan Hospital. According to the problems of the present ET affinity adsorbent, some novel affinity adsorbents for ET removal were developed in this dissertation starting with matrix and ligand, which were proven to have high adsorption capacity, selectivity and compatibility to blood. And the study on preparation and ET adsorption properties of these adsorbents could provide reference for the further design of ET removal materials.As for the matrix, the porous silica gel was mainly selected and the influence of its pore size and specific surface area on the property of the resultant adsorbent was studied. His was used as ligand here, and was grafted on the KH550 modified silica gel through the link with glutaraldehyde (Glu). The grafting yield of the functional group in each step were studied by means of IR, thermogravimetric analysis (TGA), elemental analysis and chemical titration method. The results show that the grafting yield of each reactant on silica gel is affected by the pore size and specific surface area of the silica gel matrix, and during the successional reactions the pore size continually decreases. Since ET has a large molecule, it is better to choose the matrix with a larger pore size to allow the diffusion of ET into the internal pore walls and to be catched by the ligand on it.As for the ligand, three ligands were investigated in this dissertation. They are: Histidine (His), which has been largely reported in literature; vinylimidazole (VI), selected here from the elicitation of the affinity mechanism of imidazole group in His; and the epoxy group-containing compound, discovered during our experiment. The active site of His and VI are both imidazole group, but His also has reactive amido and -COOH with negative charge. The ligand obtained from VI graft polymerization on silica gel is a kind of polymeric form. Through comparison between the ET removal performance of the affinity adsorbent with His and PVI as ligand respectively, a much higher capacity, efficiency and much better applicability in terms of pH and ionic strength of PVI ligand was found, which proves not only the high affinity of the active imidazole group toward ET, but also the leading function of ionic interaction between ligand and ET molecule. The epoxy-containing compound such as KH560 or GMA for use as ET ligand has never been reported yet. It was found that once the epoxy group was opened, the resultant adsorbent couldn't show ET removal ability any longer, which verifies the function of epoxy group. This finding extends the scope of ET ligand selection.Although PVI as a polymeric ligand showed high ET removal capacity, it was still affected by the too high graft density and the strong forces between its molecules. By the copolymerization of VI and methacrylate monomers, the distribution of VI chains on the matrix could be adjusted and improved. Since the reactivity ratios of VI and the methacrylates are widely discrepant, almost each VI unit in the copolymer chain will be isolated by the methacrylate segment, thus the ET removal capacity of per VI unit was increased as a result of the presence of polymeric spacer arm.As for the graft route of ligand, besides KH550, another method using KH560 for the activation of silica gel to introduce His ligand on it was studied. Two kinds of reactions were then conducted to introduce His on the epoxy-silica gel. The ET removal tests show that the direct opening of epoxy group to react with His is superior to the oxidation-addition method which use -CHO as the media reactive group.In the preparation process of these affinity adsorbents, the reaction conditions such as temperature, time, pH, concentration of reactant for His introduction on KH550 or KH560 modified silica gel and for PVI graft polymerization on KH570 modified silica gel were studied in detail. For example, the reaction of Glu with amido-silica gel was found to be greatly influenced by temperature and pH value of the reaction medium. At temperature lower than 50℃and in neutral pH, the highest content of active -CHO could be obtained. At higher temperatures than 50℃and more basic conditions, self-polymerization of Glu will occur, and the content of -CHO decreased despite the high grafting yield of Glu by TGA. Again, in the graft reaction of PVI, a series of adsorbents with PVI content (grafting degree) from 1.1% to 16% could be obtained by changing the conditions, which showed different ET removal efficiency. So the control of reaction conditions is important.The ET removal properties of the above affinity adsorbents prepared by different method or with different ligands were studied. The adsorption isotherm of His-Glu-KH550-silica gel adsorbent showed good accordance with the Langmuir model, the maximum adsorption capacity q_m and apparent dissociation constant K_d were calculated to be 1194 g/g and 1350 g/L. The adsorbent had little nonspecific adsorption of protein and other components in a serum-endotoxin solution, with a ET removal efficiency of above 85%. The isotherms of PVI-silica gel displayed well fit for Freundlich isotherm model, which is indicative of surface heterogeneity of the sorbent. The Freundlich constants K_f and n were calculated to be 109.6 EU/mg and 1.35. The adsorption kinetics was well accorded with the second order equation. The equilibrium ET adsorption efficiency increased slightly with the temperature.The study of this dissertation reveals that silica gel is a good matrix for ET affinity adsorption preparation, and PVI-silica gel adsorbent is promising in ET removal.