Construction Of Autofluorescent Chitosan Microspheres For Gastric Target And Mechanism Of Autofluorescence

Rapamycin, a triene-macrolide immune inhibitor, is a secondary metabolites produced by Streptomyces hygroscopicus. Recent study found that rapamycin can control appetite, and further effectively regulate weight, but rapamycin will cause systemic side effects if it entered blood circulatory system by mesenteric vascular absorption after oral administration. However, the effective regulation for appetite could be realized only when rapamycin play drug efficiency in the stomach. In order to achieve this aim, chitosan microspheres with uniform size, pH sensitivity, strong stomach adhesion were prepared in this study, and the detail contents are as follows:1. The first part focuses on preparation of uniform-sized chitosan microspheres using premix membrane emulsification. The uniform-sized microspheres (1μm) were successfully prepared under optimal conditions. The optimal conditions for preparation uniform-sized emulsification of W/O by pressing membrane emulsification were as follows:the content of quaternized chitosan was 50%, the molar ratio of aldehyde group crosslinker and amino group of chitosan was 1:1, the mass ratio between chitosan and glycerolphosphate disodium was 1:3, the volume ratio between oil phase and water phase was 30:1, transmembrane pressure was 0.3 Mpa, and the press was 5 times. The optimal solidification conditions were as follows:the emulsion were solidified by GP into gel under 37℃for 1h. Then the temperature was increased to 50℃, and the gel was further solidified into microspheres for 4 h, the whole solidification process were stirred under 600 rpm.2. The pH sensitivity and adhesion characteristics of chitosan microspheres were qualitatively and quantitatively evaluated employing the autofluorescent characteristics of chitosan microspheres. Results showed most of microspheres adhesion in the stomach, and there are 30% microspheres could adhere on surface of stomach within lh after oral administration to rats, and the microspheres adhesion on stomach was reduced with emptying of stomach. In addition, most of microspheres adhesion on stomach were degraded in gastric environment. These results suggested the prepared microspheres hold great potential stomach targeted carrier.3. The third part was mechanism of autofluoresce of microspheres. In this part, a compounds containing—C=N—C=C—was synthesized by 2-methyl-2-crotonaldehyde and 2-amino-2-methyl propane in this study. Its structure of this was confirmed by infrared spectroscopy (FT/IR) and liquid chromatography-mass spectroscopy (LC-MS), Results showed that a relatively pure compounds was obtained. Furthermore, the fluorescent property was analyzed by fluorescence spectroscopy and Laser scanning confocal microscope (LSCM), and found the synthesis exactly showed the same fluorescent property with chitosan microspheres. Therefore, the auto-fluorescence of chitosan microspheres crosslinked with glutaraldehyde mainly contribute to conjugate structure of—C=N—C=C—This thesis constructed a new type of stomach targeted microspheres successfully which may provide targeted release system for drugs such as rapamycin, and explained chitosan microspheres auto-fluorescence mechanism in the molecular level.