Preparation Of Protein/Peptide Loaded Microspheres And Characterization Of The Internal Component Distribution In Microspheres

The problem caused by the frequent injection of protein/peptide drugs promoted the research and development of oral dosage forms of protein/peptide drugs. How to avoid the degradation of hydrochloric acid in gastric and gastrointestinal protease in oral process and the improvement of its oral absorption is the key form of research and development. In this study, the method of co-lyophilize with PEG6000 was used to micronize the protein/peptide drugs without changed its activity and structure, and then using the solid in oil in oil(S/O/O) emulsion solvent evaporation method with the p H sensitive material Eudragit L100 to encapsulate proteins/peptides for protect the proteins/peptides. On this basis, the proteins/peptides nanoparticles were prepared, and the nanoparticles were encapsulated into microspheres. On the one hand, proteins/peptides nanoparticles could be uniformly dispersed in the organic solvent without aggregation and could solve the problem of the drug undistribution in the microspheres; on the other hand, it could avoid the problems such as low entrapment efficiency caused by the shape and particle size of protein powders. Then, a visualization method for the measurement of the distribution of protein/peptide within single microsphere was established using SR-FTIR.Using the morphology of microspheres as evaluating indexes, single factor experiments were executed. The optimal preparation technique was: the starting temperature 10 ℃, the heating rate 1 oC/min, homogenized strength 10000 rpm, mechanical stirring speed 750 rpm, dispersing agent was sucrose ester, dispersing agent added to the polymer solution. Dispersed microspheres with smooth surface were received under the optimized conditions.BSA was applied as protein/peptide model drug. BCA method was used to determine the concentration of the drug in the media. In this method, the result of the recovery test was 99.80-107.33%,RSD<3%, the drug extraction method recovery was 95.63-108.10%,RSD<4%. All the results indicated that the BCA method could meet the measurement requirement. Using the morphology, particle size, encapsulation efficiency and in vitro release as evaluation index, the main factors of polymer and drug power weight ratio, concentration of Eudragit and sucrose ester influence of the prescription of drug loaded microspheres were investigated. The results showed that increasing the weight ratio of Eudragit and drug power, the encapsulation efficiency can increase and the morphology become smoother and more rounded. The particle size increased with increasing concentration of Eudragit L100. The encapsulation efficiency as low as 66.3% when the concentration of sucrose ester were 1%. At p H 1.2, when the concentration of sucrose ester was 1%, the cumulative release was highest. The drug release in the p H 7.4 PBS was slow because the sucrose stearate’s hydrophobic structures.The BSA nanoparticles were fabricated by a modified freezing-induced phase separation method of aqueous of BSA and MβCD. Using the protein yield and particle size as evaluation index, the weight ratio of BSA and MβCD and the concentration of phosphate were studied. The results show that when the weight ratio of MβCD and BSA was 4:1 and the concentration of phosphate was 0.02 mol/L, the particle size was smallest and the protein yield was up to 90%. The size effect of nanoparticles may solve the non-uniform distribution of drugs in the internal of microspheres. Microspheres loading with BSA nanoparticles were preparaed. The microspheres compared with the PEG-BSA loaded microspheres and directly freeze dried BSA loaded microspheres. The results suggested that microspheres encapsulated BSA nanoparticles with the same method had higher encapsulationefficiency(>87%), higher drur loading(>3.9%), and low release profile in p H 1.2 HCl. So, using BSA nanoparticle was better than directly freeze dried for microsphere preparation by S/O/O method.Using the PLGA microsphere as the subjects, established the mathematical post test data processing of optical paths normalization and relative intensity ratios. Then, using the Eudragit microsphere as the subjects, verify the feasibility of these method. Matlab software was used read the matrix at specific bands of protein/peptide and polymer. SR-FTIR mapping was normalized by the optical paths and the relative intensity ratio of point by point was calculated. The relative intensity ratios eliminate the effects of optical path on the reading map and can character the distribution of protein/peptide drugs in microspheres.