Development And Evaluation Of Oral Dosage Form Of SAK-HV/Chitosan Quaternary Ammonium Salt-PLGA Microspheres

At present,the oral delivery of protein drug was still a great challenge.It had some limitations including its instability in the pH environment,susceptibility of being degradated by digestive enzymes,difficulty in passing through mucus layer and gastrointestinal epithelial cells,which resulted in low bioavailability.In this paper,the oral dosage of SAK-HV/chitosan quaternary ammonium salt-PLGA microsphere was prepared by double emulsion method using mucoadhesive chitosan quaternary ammonium salt and PLGA(Polylactic acid-glycolic acid copolymer)with the properties of slow and controlled release.It could overcome the biochemical barrier and prolong the residence time in the small intestine,thereby improving its bioavailability.The study of SAK-HV/chitosan quaternary ammonium salt-PLGA microsphere included three parts.In the first part,the SAK-HV/chitosan quaternary ammonium salt-PLGA microsphere was prepared and characterized.First,the encapsulation efficiency(70.8±2.2)% of the drug in the microspheres was determined by high-speed centrifugation,and the drug loading was(1.1±0.02)%.The PDI of the drug-loaded microspheres was determined by a laser particle size analyzer to be(0.386±0.048),the particle size was(2.573±0.202)?m,and the potential was(26.667±2.483)mV.The results of the scanning electron microscopy(SEM)and fluorescence microscopy showed that the form of drug-loaded microsphere was spherical and uniform.In the simulated gastric and intestinal fluid,the release amount of 4h was(36.9±0.8)%,(44.1±0.7)%,respectively.The results of SDS-PAGE analysis showed that the SAK-HV extracted from the microspheres was intact structurally and stable.The SAK-HV released from the microspheres had a high thrombolytic activity,and its activity ratio was 60.6 %.In the second part,the retention time of drug-loaded microspheres in the small intestine and the pharmacokinetics in wistar rats were studied.The analysis of the retention time of drug-loaded microspheres in the small intestine of rats showed that the fluorescence intensity distribution of the SAK-HV solution group and the drug-loaded microsphere group were highest at the jejunum segment in 2,4,and 6 h after oral administration.At the jejunum segment,the fluorescence intensity and residence time of the drug-loaded microspheres group were higher than those of the SAK-HV solution group,which indicated that the drug-loaded microspheres were mainly adhered to the small intestine jejunum segment and had good mucoadhesiveness.The pharmacokinetics of SAK-HV in rats was determined by ELISA.The results showed that the half-life of the drug-loaded microspheres(5.4±1.6)h higher than that of the tail vein injection group(3.6±2.8)h.The Tmax of the oral SAK-HV group was(4.8±3.9)h,the Cmax was(18.4±15.3)?g/L,and the F was 0.4%.The Tmax of the drug-loaded microsphere group was 4 h,the Cmax was(133.8±10.4)?g/L,and the F was 3.4%,indicating that the oral bioavailability of the drug was significantly improved(p < 0.05).In the third part,the preliminary evaluation of oral administration of drug-loaded microspheres in C57BL/6J mice was carried out.The experimental results showed that on the 21 st day,the oral drug-loaded microsphere group could significantly produce more antibody against SAK-HV,which was approximately 11-fold,7.2-fold higher than the oral SAK-HV solution group and the tail vein injection SAK-HV solution group,respectively.The level of antibody produced by the oral drug-loaded microsphere group on the 21 st day was significantly increased,which was 5.2 times that of the 14 th day(p < 0.01).The level of complement in the blood was measured on the 28 th day,and the statistical analysis showed no difference among the three groups,indicating that C3 a and C5 a complement were not activated.The results of histological studies showed that there was no obvious damage of the liver,kidney and small intestine in the drug-loaded microsphere group compared with the normal control group.In summary,the chitosan quaternary ammonium salt-PLGA microspheres may be an effective carrier for oral delivery of SAK-HV protein.