Study On Novel Glucose-responsive Nanoparticle System For Insulin Oral Delivery

The traditional treatment for people with insulin-dependent diabetes usually requires frequent insulin injections to maintain normoglycemia,which can cause the side effects of inflammation,induration and allergy to the injection site and bring great pain and psychological burden to the patient.The disorder of the blood glucose metabolism in diabetic patients may result in a series of complications and even lead to death via insulin shock.It is necessary to relies self-regulation of blood glucose concentration in a non-invasive route.Herein,a stimulus-sensitive turnover strategy system applided for insulin oral delivery was developed by designing an intelligent glucose-responsive“switch”for blood glucose regulation,in which the insulin release behavior was rapidly switched“ON”in response to hyperglycemic state and“OFF”at normal glucose levels illustrating sustained drug release.The main work of this thesis is summarized as follows:Firstly,we have prepared a glucose-responsive nanoparticles by ultrasonic self-assembly method using 2-nitroimidazole-L-cysteine-alginate(NI-CYS-ALG)polymer modified by L-cysteine(CYS)and 2-nitroimidazole(NI).Glucose oxidase(GOx)and insulin(INS)were encapsulated into nanoparticles to obtain glucose-responsive nanoparticles(GR-NPs).The chemical structure of NI-CYS-ALG was characterized by nuclear magnetic resonance spectrum(~1H NMR),Fourier transform infrared(FTIR)spectroscopy.Morphology and Zeta potential value of GR-NPs were detected by transmission electron microscope(TEM)and dynamic light scattering(DLS),respectively.The results of drug loading/encapusulation efficiency and bioadhesion of GR-NPs(with different weight ratios of CYS/ALG)illustrated that the GR-NPs had a significant good bioadhesion ability and drug loading efficiency when the weight ratio of CYS/ALG was 2:1,which was selected as optimal formula.And then,the GR-NPs system(the weight ratio of CYS/ALG,2:1)was prepared successfully and evaluated comprehensively in vitro such as pH stability,enzyme inhibition and glucose-responsive capacity.As illustrated,the GR-NPs system was an effective and safe peroral carrier for insulin oral delivery that could overcome pH and enzyme degradation.GR-NPs showed glucose-responsive drug release behavior which could induce pH value decreasing,size of nanoparticles increasing and the amount of2-nitroimdazole reducing when exposed in high glucose concentration solution.In addition,biological activity,cell viability and other relative interactions between cells and GR-NPs were evaluated by MTT,CD and cells staining separately.The type I diabetic mice could maitain normal blood glucose level for 24 hours after oral administration presenting a long period of oral hypoglycemic effect.Finally,in vivo tests on type I diabetic rats also displayed a significant hypoglycemic effect,avoiding the risk of hyperglycemia and maintaining a normal range for a relatively long time(14 h)after oral administration.The observation of ex-vivo living imaging and confocal laser microscope(CLSM)of small intestine showed that GR-NPs could not be excreted immediately after oral administration,and could be retained in the small intestine for a long time which could further increase the absorption of drugs.Moreover,the mechanism of GR-NPs transport through epithelial cells might be caused by the high content of cysteine on GR-NPs.As described,the effects of GR-NPs on the distribution of actin microfilaments(F-actin)and tight junctional proteins(ZO-1,Occludin)were observed by confocal laser microscope using Caco-2 cells as a model.GR-NPs could change the integrity of F-actin and the redistribution of ZO-1 protein to open the tight junctional structure of cells,thus enhanced the cross-membrane absorption of GR-NPs.In conclusion,the constructed glucose-responsive nano-delivery system displays a significant hypoglycemic effect after oral administration in diabetic rats,presenting a good function of enzyme inhibition in gastrointestinal tract and showing a good bioadhesive and glucose-responsive capability.This study might broaden the development of non-invasive oral administration for type?diabetes,which could also provide a rational design of protein and polypeptide for oral delivery.