A Study On Labeling Reaction Of Chitosan-calcium Alginate Microsphere With A Fluorescein

The technology of good biocompatibility and biodegradation chitosan- alginate microspheres vector has become the forefront of the development of protein drugs for oral administration. To study the chitosan-calcium alginate microspheres transfer, absorption and distribution in vivo, we can use the liver, spleen biopsy and blood test for the qualitative and quantitative changes inspection. However chitosan and sodium alginate have no obvious color, it is very difficult to detect microspheres in vivo. Therefore, microspheres are labeled by a fluorescent compound. Then microspheres are detected by standard fluorescence-detection equipment to achieve the detection of chitosan- calcium alginate microspheres in vivo.In this paper using isothiocyanate (FITC) as fluorescence marker, nitrogen atom on 2-NH₂ of chitosan under alkaline conditions react with carbon atom on isothiocyano of FITC. After that, using calcium alginate microspheres as drug vector, in acidic conditions 5-COO^- on calcium alginate and 2-NH₃⁺ on FITC-labeled chitosan interact for polyelectrolyte complexation reactions. The surface of the microsphere forms control release semipermeable membrane bonding FITC. Thus microspheres are labeled by fluorescence. The effect of relevant factors on fluorescent labeling system will be carry on. And the optimum labeling conditions will be obtained. Using expansion rate as the characterization of membrane intensity, the effect of chitosan properties, reaction conditions on the membrane intensity of fluorescent microspheres in the oral medium will be discussed. Experimental results show that:1. The pH value of the reaction liquid play a decisive role in fluorescent labeling reaction. When pH of the solution >pKa (chitosan), fluorescent labeling efficiency of the chitosan will be improved significantly.2. Both the carbon atom on FITC and 5-COO^- on calcium alginate react with 2-NH₂ on chitosan. So the fluorescent labeling reaction of chitosan have a certain extent negative effect on the labeling reaction of microsphere. Meanwhile molecular weight, degree of deacetylation, concentration of chitosan, membrane reaction time and particle size of calcium alginate gel beads have the significant impact on microsphere labeling reaction. Chitosan, molecular weight of 50,000, 90% degree of deacetylation, concentration 0.01g/mL, reacts with FITC. And then the above FITC-labeled chitosan react with calcium alginate gel beads whose diameter is small for 10min. The higher labeling efficiency will be reach.3. Study shows that: the factors, low molecular weight, low deacetylation of chitosan, a higher liquid concentration and extending reaction time, will improve the stability of fluorescent microspheres in the oral medium.