| Diabetes is a type of chronic metabolic disease characterized by hyperglycemia,which is mainly caused by insulin deficiency or insufficient secretion.Subcutaneous injection of exogenous insulin is a common clinical treatment mode for type I and mid to late-stage type II diabetes.Oral delivery is the most ideal administration route for insulin.It can simulate the normal physiological pathway of endogenous insulin secreted in human body to a certain extent,and reduce serious complications caused by long-term insulin injection,such as hypoglycemia,peripheral hyperinsulinemia,etc.However,due to the poor stability of insulin in the gastrointestinal tract and its low transepithelial transport efficiency,drug delivery carriers and advanced preparation technology are needed to further improve its oral bioavailability,in order to meet the requirements of clinical application.Pectin extracted from various plants is a kind of natural acidic polysaccharide with high gastrointestinal stability,good bioadhesion and biodegrade ability by colonic microbiota.Pectin-based oral delivery carriers can prevent the degradation of peptide drugs by gastrointestinal extreme p H and digestive enzymes.Furthermore,conjugation of enterocyte targeted ligands（such as folic acid）with pectin-based carriers can improve the transport efficiency of insulin.Therefore,this paper aims to develop a novel folate-pectin nanocarrier for targeted oral delivery of insulin.Firstly,folic acid as the targeting ligand was covalently conjugated to pectins with different molecular weights.Structure and folate grafting degree of the products were analyzed by nuclear magnetic resonance spectroscopy,fourier transform infrared spectroscopy and absorption spectroscopy.Insulin loaded folate-pectin nanoparticles（INS/FAN）were constructed by ionic crosslinking method using the as-prepared folate-pectin.A quantative analysis method for insulin determination was established by ultra performance liquid chromatography（UPLC）.The preparation conditions of ionic crosslinked nanoparticles were optimized by single factor experiments,and the physicochemical properties of the optimal nanoparticles were characterized.Results showed that INS/FANs were spherical or ellipsoidal particles with particle size of 173.4±3.8 nm,Zeta potential of-21.8±0.3 m V,drug loading efficiency of 22.7±0.1%and encapsulation efficiency of 98.3±0.5%.The structural stability of insulin released from nanoparticles was studied by SDS-PAGE and circular dichroism spectrum.Results indicated that the ionic crosslinking process did not change the structure of insulin.Freeze-drying stability of nanoparticles was evaluated by comparing the changes of particle size before and after freeze-drying.The results showed that adding 5%trehalose in INS/FAN solution,and the particle size of the nanoparticles changed minimally before and after freeze-drying.Insulin release behaviour from INS/FAN in simulated digestive juices and blood was determined by UPLC.It was noted that there was a significant burst release of insulin from INS/FAN in gastric juice.The cumulative release rate reached to62.1%after 2 h incubation.In comparison,the release rate of insulin became much slower in small intestine and blood,and the cumulative release rate was 76.1%after 10 h incubation.In order to improve insulin burst release in gastric juice,ionic crosslinked nanoparticles were further crosslinked by adipic dihydrazide（ADH）.Before chemical crosslinking,2,3-dimethylmaleic anhydride（DMMA）was used to protect the free amino group of insulin to avoid the inactivation of insulin during the crosslinking process.The results of UPLC and MS analysis showed that the produtcs were mainly mono-and dis-ubstituted insulin derivatives.Under acidic conditions,the DMMA protecting groups of insulin could be completely taken off.DMMA-insulin loaded dual crosslinked nanoparticles（INS/DFAN）were prepared by calcium ions crosslinking and subsequent ADH chemical crosslinking.Particle size of INS/DFAN was 167.0±3.61 nm,Zeta potential was-17.49±1.88 m V,drug loading and encapsulation efficiency were 22.9±0.1%and 99.1±0.3%respectively.Compared with INS/FAN,the cumulative insulin release of dual-crosslinked INS/DFAN in gastric juice decreased by 24.9%.Enzymes degradation behivour of INS/DFAN was investigated in simulated gastrointestinal juices containing pepsin and trypsin.Insulin degragation curves indicated that both INS/FAN and INS/DFAN could resist the enzymatic hydrolysis of insulin by digestive enzymes,and the protective effect of INS/DFAN was stronger than that of INS/FAN.The cytotoxicity,cell uptake and transport efficiency of INS/DFAN were further investigated.Results showed that INS/DFAN had no significant toxic effects on Caco-2 cells and NCM460 cells.Cell uptake experiments showed that INS/DFAN could increase insulin uptake by Caco-2 cells and insulin intake decreased after competitive inhibition of folate receptors.The apparent permeability coefficient(Papp)of INS/DFAN transported across Caco-2 cell monolayer was increased to 3.6 x 10-6 cm/s,which was 4.7 times higher than free insulin.Tissue adhesion,penetration and distribution of INS/DFAN in different segments of small intestine were investigated.The results showed that FITC-insulin loaded INS/DFAN had bioadhesion in each segment of the small intestine,but their distribution and penetration were slightly different.Tissue penetration of INS/DFAN in the jejunum was significantly deeper than that in the duodenum and ileum.INS/DFAN was mainly distributed in the small intestine within 2-4 h after oral administration and transported to the colon at 6 h.Finally,the hypoglycemic effect of INS/DFAN was evaluated in the model of type I diabetic rats.After oral administration of nanoparticles,the blood glucose level of rats decreased continuously and was significantly lower than of the control rats within 2-8 h.In conclusion,we constructed insulin-loaded pectin nanoparticles targeting folate receptor in intestinal epithelial cells by ionic and chemical dual crosslinking method.The morphology and size of the prepared nanoparticles meet the requirements of trans intestinal epithelial transport.Moreover,these nanoparticles possess good drug loading performance and high stability,and can effectively improve the trans intestinal transport efficiency of insulin,increase the adhesion and penetration of insulin in the intestinal epithelium,and improve the oral hypoglycemic effect of insulin.The results of this study provide a theoretical basis for the development of oral insulin delivery systems based on natural polysaccharides,and have important significance for improving the quality of life of diabetic patients depended on long-term insulin injection. |
