The Establishment Of GLP-1 Analogues Receptor Analysis Method And Preliminary Exploration Of Its Oral Administration

Glucagon like peptide-1?GLP-1?is an endogenous molecule secreted by L cells in the human intestinal tract for the treatment of type 2 diabetes mellitus.It has an"incretin effect"and the advantage of glucose dependent regulation is special.The effect of GLP-1 analogues for the treatment of diabetes is outstanding,but the current route of administration is injection.the patient's compliance is poor,and the drugs need to be chilled at low temperatures,not easy to carry.Based on the above reasons,our research group is prepared to build a GLP-1 analogue nanoparticles drug delivery system based on nanoparticles technology.This study will explore the preliminary conditions of drug delivery system of nanoparticles from the following two aspects:?1?establishing a CHO model for the screening and activity verification of GLP-1 analogues.?2?exploring the feasibility of drug-mediated carrier transport model.In order to screen and activity verification of GLP-1 analogues,a eukaryotic expression vector pEGFP-N1/hGLP-1R-EGFP was constructed and transfected into Chinese hamster ovary?CHO?cells.After limited selection by G418,the combination selection of flow cytometry and limiting dilution was performed and a stable and highly expressed monoclonal cell line was obtained.Inverted fluorescence analysis,flow cytometry analysis and RT-PCR results showed that the gene was transcribed and translated,and the expressed receptor protein was located on the cell membrane side.Liraglutide and Exendin-4 were used as a model drug for the activity assay,respectively.The results show that the cell model has high drug sensitivity,relatively stable activity assay with different model cell generations and initially explores the receptor expression of model cells,fetal bovine serum and its single component BSA has a significant effect on the drug EC₅₀measurements.A Caco-2 monolayer cell model was constructed and three indicators were observed,including cell transmembrane resistance values,differences in alkaline phosphatase expression,and ultrathin section morphology,to verify the successful construction of the model.The phenol red permeability experiment further demonstrates the integrity of the Caco-2 monolayer cell model.The Caco-2cytotoxicity test was performed on six gastrointestinal highly-absorbed drugs,which were selected from Pharmacopoeia and other reference materials and the results showed that there is no apparent toxicity to Caco-2 cells when the drug concentration was lower than 120?M.The indicamethine,mexiletine and procainamide with relatively high relative transport efficiency were screened by the Caco-2 cell model,in which indomethacin and mexiletine can be stably accessed to the surface of gold nanoparticles via structural modification.The drug-modified gold nanoparticles was used to explore the transmembrane efficiency of Caco-2 cells.The results showed that the gold nanoparticles can promote the adhesion of gold nanoparticles to the Caco-2 cell layer,but whether to be absorbed by transmembrane need further verified.