Preparation Of ALG-CHI Microcapsules Containing IL1Ra And Its In-situ Treatment Of Inflammatory Bowel Disease

Inflammatory bowel disease is a recurring chronic inflammatory disease of the gastrointestinal tract.The clinical manifestations are abdominal pain,diarrhea,and even bloody stools.Due to its repeated attacks and prolonged unhealed,it seriously affects the quality of life of patients.Its etiology and pathogenesis have not yet been clarified,but studies have shown that it is closely related to the IL-1? increase in the intestinal mucosa.Endogenous interleukin 1 receptor antagonist,which can inhibit IL-1?-mediated signaling and reduce the biological effects caused by IL-1?,has the ability to inhibits the production of related secondary inflammatory factors,thus it has the potential to relieve or treat inflammatory bowel disease.As a protein,IL1Ra's structural integrity and biological activity are easily damaged by the harsh pH environment and protease in the digestive tract.Meanwhile,simultaneous injection IL1 Ra administration brings some other problems such as short half-life and allergic reaction at the injection site.Alginate-chitosan(ALG-CHI)microcapsules is pH-sensitive therefore it can protect the activity of IL1 Ra while it pass through the digestive tract,and release protein drugs in a higher pH environment in the colon.Therefore,it can achieve the purpose of in-situ treatment of IBD by taking IL1 Ra orally and improve the medication compliance of patients.In this study,a recombinant Escherichia coli BL21(DE3)which can express IL1 Ra was constructed,and the induction conditions were optimized to improve the protein expression.IL1 Ra protein was affinity-purified by Ni column.After that,IL1Ra-ALGCHI microcapsules were prepared by electrostatic spray method,and their pH responsiveness was tested in vitro,while the IBD mouse model was employed to estimate the therapeutic effect in vivo.In the second chapter,the nucleic acid sequence encoding IL1 Ra protein was inserted into pET30 a to construct the recombinant plasmid pET30a-IL1 Ra,and the plasmid was transformed into E.coli BL21(DE3)and cultured in solid LB medium.After IPTG induction,the expression pattern of the target protein was identified by SDS-PAGE,and the results showed that IL1 Ra was expressed as a soluble protein in host bacteria.The recombinant protein was confirmed to conform to the molecular weight and sequence of the human IL1 Ra protein by Western Blot and Nano LC-ESIMS/MS.In the third chapter of this thesis,the time point of IPTG addition was determined by measuring the growth curve of BL21(DE3)engineered bacteria,and the induction conditions of engineering bacteria were optimized to improve the expression.The optimal induction process was as follows: 0.1 mmol/L IPTG was added at the fourth hour after inoculation,and lasted for 14 hours at 15°C.The target protein was obtained by affinity chromatography on Ni-IDA medium.In the fourth chapter,the IL1Ra-ALG-CHI microcapsules were prepared by electrostatic spray method.The optimal preparation parameters determined by single factor and orthogonal experiments were: application voltage 24 KV,injection propulsion speed 0.2 mL/h,liquid surface height 11 cm,sodium alginate viscosity 450 mPa·S.The microcapsules under the optical microscope and the scanning electron microscope are spherical and uniform in particle size.The swelling ability and cumulative release rate of the microcapsules in the simulated gastric fluid,physiological saline,and simulated intestinal fluid were measured.The results show that the IL1Ra-ALG-CHI microcapsules have pH responsiveness,which is beneficial to the in-situ drug release of the protein.In the fifth chapter,the dextran sodium sulfate(DSS)induced IBD mouse model was constructed for estimate the IL1Ra-ALG-CHI microcapsules relief and treatment effects.For grouping,the animals were divided into 5 groups: normal group,model group,IL1Ra-ALG-CHI microcapsule group,BSA-ALG-CHI microcapsule group and unencapsulated IL1 Ra group.The results showed that compared with the other treatment groups,the IL1Ra-ALG-CHI microcapsule group had lower DAI score,better colon integrity and tissue morphological,lower histological damage degree,as well as lower level of colon tissue MPO activity and inflammation-related cytokine concentrations(TNF-?,IL-1?).These results implied that IL1 Ra can be delivered to the colon site by microcapsules and released to achieve the effect of relieving and treating IBD.By constructing an engineering bacterial strain BL21(DE3),the IL1 Ra protein can be expressed and purified,and the prepared pH-responsive IL1Ra-ALG-CHI microcapsule can relieve and treat IBD.This study suggested that the in-situ IBD treatment by IL1 Ra oral administration could be realized,which provides new ideas and ways for the development of protein drugs for IBD treatment.