Preparation Of Liraglutide-loaded Nanoparticles For Type 2 Diabetes Mellitus

Type-2 diabetes mellitus have been the prevalent type of diabetes,accounting for more than 90% patients with diabetes.Glucagon-like peptide?GLP-1?is a natural incretin hormone released from intestinal L-cells,which can physiologically regulate the reduction of blood glucose,the proliferation of pancreatic ?-cells the prevention of?-cell apoptosis and the elongation of gastric emptying,rendering a good prospect in the treatment of type-2 diabetes mellitus.However,GLP-1 is prone to be degraded by dipeptidyl peptidase-4?DPP-4?,resulting in its half-life less than two minutes and limiting its clinical application.Thus,the development of stable GLP-1 analogues or its sustained-releasing preparations have become a hot topic for researchers in recent years.Liraglutide is an acylated human GLP-1 receptor agonist,with a 97% homology to human GLP-1?7-37?,which has a good hypoglycemic effect.The half-life of liraglutide can reach up to 13 hours and is it still administered once a day.In order to improve patient compliance and the efficacy of liraglutide,it is necessary to develop long-acting liraglutide drugs.In this work,we aimed to prepare the liraglutide nanoformulations.First,the precursor of liraglutide was prepared by recombinant peptide technology followed by chemical modification to prepare liraglutide.Then,the liraglutide was encapsulated by PLGA nanoparticles.The precursor of liraglutide,an Arg34 GLP-1?7-37?mutant,currently produced by peptide solid phase synthesis method.In our study,a novel process was explored,including the lactose-induced expression of fusion protein MFH-GLP-1^R34 as the inclusion bodies in Escherichia coli,the purification of GLP-1^R34 and the conjugation of palmitic acid side chain at the position 26 of GLP-1^R34 through the glutamate-mediated.The carrier protein contributed to the over-expression of the precursor.The lactose-induced expression indicated that non-toxic effect on the cell compared with IPTG induced expression,with the considerable expression level.The fusion protein was incubated in fermenter after optimizing the concentration of lactose,the induction time,the initial induction of OD600 and the culture medium,which the 10 g/L cell amounts were obtained by batch feeding fermentation.The liraglutide precursor was successfully purified by ion exchange chromatography.We obtain a purity of 90% or more liraglutide precusor and the precusors were isolated by HPLC.Our results revealed that thepreparation of liraglutide precursors by ion exchange was economical and effective.Then PLGA nanoparticles were prepared by emulsification solvent evaporation method.The process of preparing PLGA nanoparticles was optimized,including solvent,emulsifier concentration and dilution ratio.Finally,nanoparticles with uniformity,small particle size and high stability were obtained for subsequent biological activity experiments.In conclusion,Escherichia coli was used as the host strain for the expression of liraglutide precursors.Compared with the traditional method,the expression of liraglutide in Saccharomyces cerevisiae was more economical,and the ion exchangemethod was used to purify liraglutide.It is more simple and efficient,and explores the preparation process of PLGA nanoparticles,which lays the foundation for the development of long-acting GLP-1 analogues.