Chitosan Surface Modification With Cell Membrane Mimetic Phosphorylcholine Functionality

Bilirubin is the metabolite of hemoglobin in senescent red blood cells. Liver damage and related malfunctions can result in hyperbilirubinemia, especially among newborn infants, and may cause jaundice, a yellow discoloration of skin and other tissues. Excess free bilirubin (unconjugated bilirubin) tends to deposit in tissues, especially in the brain. Human body cannot removal the excess free bilirubin by metabolism. Hemoperfusion is an attractive treatment of blood purification. Successful hemoperfusion requires the adsorbents to be specific; high capacity of adsorption; blood compatible; and not being poisonous. Nevertheless, the current adsorbents show poor hemocompatibility, such as promoting plasma protein adsorption, platelet adhesion and activation, and thrombus development, and consequently not satisfactory for use in hemoperfusion. In this thesis, we prepared a new kind of adsorbent by modifying chitosan with phosphorylcholine dichloride. The modification process and the performance of the modified surfaces were investigated systematically.The main work of the thesis includes the following three parts:(1) Phosphorylcholine dichloride was prepared with phosphoryl chloride and choline chloride under anhydrous condition in a single step reaction. After investigating the relationship between the content of phosphorylcholine dichloride and the molar feed ratio of phosphoryl chloride to choline chloride, we found increasing the molar ratio can increase the content of phosphorylcholine dichloride. Meanwhile the content of phosphorylcholine dichloride can be increased by exhausting the excess phosphoryl chloride from the product. The content of the phosphorylcholine dichloride was determined by a modified potentiometric titration method. The result showed that the content of the phosphorylcholine dichloride reaches 88 % as the molar ratio of phosphoryl chloride and choline chloride in feed is 3:1 and after exhausting the excess phosphoryl chloride from the product, the content increases to 96 %.(2) We prepared the crosslinked chitosan (CS-GA) film with glutaraldehyde. The results of X-ray Photoelectron Spectroscopy (XPS) and ATR-FTIR indicated that the chitosan (CS) was successfully crosslinked by glutaraldehyde and the degree of cross linking was approximately 17 %. The degree of deacetylation of chitosan was also calculated about 92 %. The phosphorylcholine modified CS-GA film (CS-GA-PC) was prepared by grafting CS-GA with PC groups in chloroform. The change of hydrophilicity and the grafted PC density were characterised by dynamic contact angle (DCA) and X-ray Photoelectron Spectroscopy (XPS) measurements respectively. The results of ATR-FTIR and DCA confirmed that the modification process was successful. According to the data of XPS, we estimate the grafted PC ratio was approximately 28 %. Platelet adhesion assay revealed that the adhered platelets on CS-GA-PC surface shows random distribution with the size of individual platelets (2-4μm). On the contrary, the adhered platelets on CS-GA surface displays remarkable aggregation of the platelets and finally form agglomerate, suggesting the activation of platelets. Protein absorption experiments revealed that the adsorbed amounts of bovine serum albumin (BSA) and fibrinogen (Fg) on the CS-GA-PC surface are much less than that on CS-GA. After the PC modification, the adsorbedamounts of BSA and Fg decrease from 0.74μg/cm² and 0.87μg/cm² to 0.21 ug/cm and 0.35μg/cm² respectively. The reductions reach 72 % and 60 % for BSA and Fg respectively. These results demonstrated that surface modification with phosphorylcholine dichloride is a promising route to improve the hemocompatibility of chitosan.(3) CS-GA microspheres were prepared by emulsification-crosslink method and phosphorylcholine modified CS-GA microspheres were synthetized by grafting CS-GA microsphere with PC groups in chloroform. The result of ATR-IR showed that the peak in 2950 cm-1 was stretching vibration of methylene and the peak in 1646 cm^-1 was the characteristic absorption peak of -C=N. That indicated crosslinking process was successful. Compared with CS-GA microspheres, the enhanced absorption around 1041, 1076 cm^-1 (symmetric O=P—O stretch) and 1450 cm^-1 (CH₃ bending from N⁺(CH₃)₃) of CS-GA-PC is the evidence of the successful grafting of PC groups. The result of scanning electron microscope (SEM) revealed that particle diameters of CS-GA microspheres and phosphorylcholine modified CS-GA microspheres are not affected during the modification; both are about 100μm with a narrow size distribution. After modification, the morphology structure of the surface was not changed. Protein absorption experiments showed that the adsorptive capacity of BSA on the CS-GA-PC microspheres is not reduced obviously compared with that on the CS-GA microspheres. Bilirubin adsorption experiment demonstrated that the absorption reaches equilibrum in 2 h, and 90 % of the bilirubin is removed (adsorbed) in the adsorption condition.