Preparation Of Chitosan And Its Derivatives And Relaxation Property In Vitro And In Vivo Of Modified Magnetic Resonance Imaging Contrast Agent

Chitosan is the deacetylated product of chitin, which is the second largest high-yield resource in nature. Chitsan is one of the important raw materials in food industry. The further development and application of chitosan processing products is one of the ways to avoid wastage of natural resources and expand the application of chitosan, especially in the biomedical field with high value. The common contrast agent Gd-DTPA (Magnevist(?)) was found that the contrast time in body was short and the relaxivity was relatively low. In addition, Gd-DTPA is non-targeted and has toxicity, even lead to nephrogenic systemic fibrosis (NSF) of organism renal cells. The commercial contrast agent was modified with chitosan with low degree of polymerization, which was non-toxic, biodegradable and biocompatible. The final goal was to improve the relaxivity of contrast agent and the safety in body. The contents of this paper are divided into the following three parts.1. Water-soluble low molecular weight chitosan with various degrees of polymerization were prepared with the method of microwave-assisted hydrogen peroxide degradation. The three derivatives were also synthesized. The potential of low molecular weight chitosan and its derivatives as hydrophilic drug carrier model was studied.1.1The structures of low molecular weight chitosan and its derivatives were characterized through’H NMR, Gel filtration chromatography (GFC), IR, etc. The results showed that only β-1,4glucoside bond was fractured in the degradation process. There was no side reaction such as ring-opening. The polydispersity index was greatly lowed when the degree of polymerization of low molecular weight chitosan was below20.1.2The UV and fluorescence spectra of bovine serum albumin (BSA), which was in low molecular weight chitosan and its derivatives solution, were studied. The results showed that the main binding force was hydrophobic and hydrogen bonding effect. The conformation of BSA was not changed in the microenvironment of low molecular weight chitosan and its derivatives solution. Besides, the natural activity of BSA was not affected. It was concluded that they have the potential to be good hydrophilic drug carrier model.1.3The thermodynamic effect between low molecular weight chitosan and its derivatives and active pharmaceutical BSA was studied by isothermal titration calorimetry (ITC). The results indicated that the binding process was exothermic and spontaneous. The main binding force between low molecular weight chitosan and BSA was hydrogen bonding effect. The main binding force between its derivatives and BSA was hydrogen bonding and hydrophobic effect. The conclusion was consistent with the results of UV and fluorescence spectra.2. The new ligand was modified with low molecular weight chitosan at various degrees of polymerization and then chelated with paramagnetic Mn(Ⅱ) as well as Gd(Ⅲ) to prepare new functional complexes as potential MRI contrast agents. The relaxation property of Mn(Ⅱ) and Gd(Ⅲ) complexes was studied.2.1The longitudinal relaxivity of Mn(Ⅱ) and Gd(Ⅲ) complexes modified with low molecular weight chitosan was measured with inversion recovery sequence. The results showed that the relaxivity of Gd(Ⅲ) complex was not only higher than that of commercial Gd-DTPA but also Mn(Ⅱ) complex. Under certain condition, the longitudinal relaxivity was increased with the increase of degree of polymerization of low molecular weight chitosan. The longitudinal relaxivity in BSA solution was higher than that in aqueous solution, which suggested that the interaction between the complexes and BSA occurred. The compound model of complexes-BSA was formed.2.2Ti-weighted imaging in vitro of Mn(Ⅱ) and Gd(Ⅲ) complex was measured with multislice spin echo sequence. It was found that there was a correlation between lightness and imaging contrast of FLASH images and the concentration of the complexes. The results of Ti-weighted imaging in vitro of Gd(Ⅲ) complex were optimal at the equivalent concentration. It was also found that the imaging result was related to the degree of polymerization of low molecular weight chitosan.2.3The thermodynamic effect between the Mn(Ⅱ) and Gd(Ⅲ) complexes and BSA was measured with ITC. The results showed that the binding process was both spontaneous and entropy-driven. The main binding force was hydrophobic effect. 3. The Gd(Ⅲ) complex modified with low molecular weight chitosan was chosen as a main study object. The safety in mice and MR imaging in vivo in the rat of Gd(Ⅲ) complex were studied.3.1The results of acute toxicity study showed that the mice remained healthy one week after the injection of Gd(Ⅲ) complex, and normal weight was gained. There was no significant toxicity.3.2The micronucleus result of polychromatic erythrocyte (PCE) in mice marrow was negative, which indicated that there is no harm on mice chromosome. Gd(Ⅲ) complex would not lead to mutagenesis side effects.3.3The results of Gd(Ⅲ) residual analysis showed that Gd(Ⅲ) residue in mice liver and kidney was firstly increased and then decreased. The residues were promptly decreased after one day. However, Gd(Ⅲ) in other organs were gradually decreased, and Gd(Ⅲ) was metabolized out of the body.3.4The results of MR imaging in vivo showed that the length of retention time of Gd(Ⅲ) complex persisted in rat liver, and the target site were related to the degree of polymerization of low molecular weight chitosan for modification. The retention time of Gd(Ⅲ) complex modified with low molecular weight chitosan with11degree of polymerization in rat liver was longest and the signal strength was not reduced within a certain time, which was advantageous for liver target. However, the Gd(Ⅲ) complex modified with low molecular weight chitosan with6degree of polymerization had no effect in the rat liver, and it reached the kidney and metabolized out of the body.