Construction Of Starch-based Nanocarriers And Its Oral Delivery Of Insulin

In recent years,with the breakthrough of more and more clinical research of protein peptide drugs and even successful marketing,the research of oral insulin has been more extensive and in-depth.Nanodelivery systems can greatly improve the oral bioavailability of insulin,but it is still difficult to achieve large-scale production due to the complex preparation process and use of organic solvents in the drug loading process.Therefore,it is of practical significance to develop a delivery system with green and simple preparation process and improved oral bioavailability.In this paper,firstly,the complexation behavior between carboxymethyl starch(CMS)and2-Hydroxypropyltrimethylammonium chloride chitosan(HACC),the formation conditions and stability of nanocomposites were systematically explored.Secondly,insulin-loaded nanoparticles were constructed based on the formation conditions of nanocomposites,and the physicochemical properties of the nanoparticles were analyzed.Next,the biocompatibility of nanoparticles was investigated,and the hypoglycemic effects of nanoparticles were explored based on animal models.Subsequently,a Caco-2 cell transport model was constructed,and the transport pathway and mechanism of nanoparticles promoting insulin endocytosis were explored.Finally,the influences of nanocarriers on animal biochemical indexes and histopathology were explored,and the safety of nanocarriers in vivo was clarified.The main research contents are as follows:(1)CMS with two kinds of substitution degree(C0.1 and C0.2)were prepared by using waxy corn starch as raw material,and then compounded with HACC to construct nanocomposites.The etherification modification of CMS was verified by the results of fourier transform infrared spectroscopy and nuclear magnetic resonance hydrogen spectroscopy.The effects of p H on zeta potential showed that the net charge of CMS was strongly p H-dependent,and its value of CMS decreased from-9.0 m V to-20.9 m V as the p H increased from 3.0 to 7.0.The result of turbidity,fluorescence resonance spectroscopy and isothermal calorimetric titration revealed that the stability of CMS/HACC complexes depended on the mass ratio of CMS to HACC,the substitution degree of CMS,p H,and ionic strength.Mass ratio and p H jointly drove the formation and stability of CMS/HACC complexes.Sample C0.2 could interact strongly with HACC at p H 3.0,while C0.1 showed a considerable level of effect at p H 4.0;The nanocomposites formed by C0.2 and HACC at a mass ratio of 3:2 showed great p H and salt stability.Complexation behavior between CMS and HACC was spontaneous,and electrostatic interactions,hydrophobic interactions,and hydrogen bonds were all involved in the process.With the p H increasing from 3.0 to 7.0,the complexation behavior between CMS and HACC changed from enthalpy-driven exothermic to entropy-driven endothermic.(2)The nanocomposites formed by CMS with degree of substitution of 0.1(C0.1)and 0.2(C0.2)and HACC were used as carriers,and insulin was used as a drug model to construct insulin-loaded nanoparticles C0.1/HACC and C0.2/HACC.The results showed that the size of nanoparticles prepared under different mass ratios were all in the range of 200 nm to 300 nm.As the mass ratio increased,the ionic strength resistance ability of nanoparticles decreased.Compared to C0.1/HACC,C0.2/HACC showed higher particle size stability in both simulated intestinal fluid and simulated body fluid;The insulin loading capacity of C0.2/HACC was higher than that of C0.1/HACC,while increasing the p H,the insulin loading capacity of the two nanoparticles were increased to 7.84% and 12.67%,respectively.The release of C0.1/HACC and C0.2/HACC in simulated intestinal fluid were both lower than that in the simulated body fluid,showing long-term release property.The microscopic morphology of the two nanoparticles were spherical,and mannitol with the 0.5%(m/v)addition dose was used as a lyoprotectant to prevent nanoparticle from drug precipitation,carrier dissociation and other problems during lyophilization process.The freeze-dried nanoparticles showed better particle morphology and resolubility.In addition,nanoparticles coated by hydroxypropyl methylcellulose phthalate with the addition dose of 0.75%(m/v)could maintain a stable particle size in simulated gastric fluid.(3)The biocompatibility and hypoglycemic effect of nanoparticles C0.1/HACC and C0.2/HACC were investigated in this section.The exogenous fluorescence spectrum showed that the nanoparticles could embed insulin well and keep the integrity of the particles.The results of hemocompatibility and cytotoxicity studies showed that the hemolysis rates of C0.1/HACC and C0.2/HACC were both lower than 5.0%,and the cell viability were higher than 80% at a nanoparticle concentration of 1 mg/m L,indicating that nanoparticles exhibited good biocompatibility.Results of in vivo imaging of animals confirmed that insulin nanoparticles after oral administration could reduce the release of insulin in the stomach while increase release in the intestine,meanwhile promoting the accumulation of insulin in the liver.The hypoglycemic effect showed that nanoparticles could effectively reduce blood glucose levels in diabetic mice,and enteric-coated nanoparticles could further improve the bioavailability of insulin,up to 10.6%.Long-term repeated administration results showed that oral loading insulin nanoparticles could effectively alleviate the symptoms of "overeating and overeating" in diabetic mice.(4)The Caco-2 cell model was used to explore the cellular uptake and transport mechanism of insulin promoted by C0.1/HACC and C0.2/HACC.It was found that free insulin was difficult to be taken up by cells,while C0.1/HACC and C0.2/HACC could promote the uptake of insulin by cells through clathrin-mediated endocytosis and macropinocytosis;The endocytic behavior of cells did not show a significant difference between C0.1/HACC and C0.2/HACC;The results of insulin transport across the cell monolayer membrane showed that,compared with free insulin,both C0.1/HACC and C0.2/HACC could promote insulin across the cell monolayer membrane from the apical side to the basal side by opening the tight junction of cells,so that the apparent permeability coefficient was increased to 13.23 times and 13.41 times of free insulin;Further analysis of the integrity of nanoparticles showed that C0.2/HACC might transport insulin in the form of intact nanoparticle,while mainly promoted endocytosis in the form of insulin.(5)Based on the animal model,the effect of nanocarrier dosage on the safety in mice were explored.During the 28-day repeated administration,the mice were in a normal state of mind,without death or abnormal behavior.Different doses of nanocarriers had no significant effects on the body weight,food intake,organ weight coefficient ratio,and biochemical indicators of female and male mice.Low dose nanocarrier administration reduced the hemoglobin and hematocrit levels in male mice,while there were no significant changes in mean hematocrit and mean hemoglobin concentration.Compared with the control group,each dose of nanocarriers did not cause pathological changes and gastrointestinal irritation in mice,which further confirmed that CMS/HACC nanocarriers had the good biocompatibility and in vivo safety.