Folic Acid-modified Reverse Micelles Lipid Nanocapsules For Oral Delivery Study To Overcome The Intestinal Epithelial Barriers

Protein peptide drugs are widely used in clinical therapeutic areas due to their high selectivity and efficacy.Most peptide drugs are currently administered by injection,but frequent administration results in poor patient compliance.Oral administration is a preferred method of drug delivery as it does not directly damage the skin or mucous membranes and can be delivered to the bloodstream via the gastrointestinal tract.Exenatide is a peptide used in the clinical treatment of type II diabetes,and although it is very effective,its low bioavailability when administered orally has limited its widespread use.Therefore,it is important to find a delivery system that can deliver protein peptide drugs orally and efficiently.In this study,we designed and prepared folic acid-modified exenatide reverse micelle lipid nanocapsules(FA-RM-ELNC)by modifying DSPE-PEG2000-FA on the surface of nanoparticles,and evaluated their physicochemical properties and in vivo and ex vivo delivery effectiveness.The aim is to provide an experimental basis and a theoretical basis for the establishment of an efficient and safe oral peptide drug delivery system.The main findings of the study are as follows:1.Formulation process study and in vitro evaluation of exenatide lipid nanocapsulesLipid nanocapsules(LNC)and reverse micelle lipid nanocapsules(RM-LNC)were prepared using the phase inversion temperature method.The functional modification was achieved by grafting DSPE-PEG-FA onto the surface of the LNC particles by postinsertion method.The optimum prescription and preparation process of the lipid nanocapsules were obtained by screening seven factors using the single-factor method with the particle size,polydisperse index(PDI),zeta potential and encapsulation rate as the evaluation indicators: the amounts of oil,water and surfactant were 0.269 mg,0.734 m L and 0.206 mg,respectively;the amount of exenatide aqueous solution was 30 μL(200mg/ml);the speed of preparation of the micelles was 1200 rpm;the amount of preformed micelles was 300 μL;the amount of end-of-formulation condensate was 3.625 m L;and the end-of-formulation stirring time was 10 minutes;The amount of DSPE-PEG2000-FA used for functional modification of 1 m L of exenatide reverse micelle lipid nanocapsules(RMELNC)was 0.75 m L(10 mg/m L).The resulting FA-RM-ELNC had a particle size of130.34±1.98 nm,a PDI of 0.068±0.033,a zeta potential of-0.31±0.42 m V and an encapsulation rate of 79.12±0.25%.The in vitro stability,drug conformational stability and in vitro release properties of lipid nanocapsules were investigated.The physical stability of the reverse micelle lipid nanocapsules and folic acid-modified reverse micelle lipid nanocapsules(FA-RM-LNC)was investigated in four different simulated media.The PDI and particle size of both nanoparticles did not change significantly over time in the simulated media.Circular Dichroism analysis showed that exenatide in lipid nanocapsules prepared in different formulations could maintain its original conformation.After 6 h of action in the simulated intestinal fluid,the exenatide solution group released 71% and both the RM-ELNC and FARM-ELNC groups released approximately 60%,with no significant difference in release trends between the two nanopreparation groups.2.In vitro cellular evaluation and intestinal absorption studies of exenatide lipid nanocapsulesIn vitro cellular evaluation was performed by using Caco-2 cells as a model.RM-LNC and FA-RM-LNC were co-incubated with cells and cell viability ranged from 80% to 120%when particle polymer concentrations ranged from 0.5 to 8 mg/m L.Cell uptake was observed at different time points,the fluorescence intensity was found to be higher in both the FA-RM-LNC group than the RM-LNC group.Examination of the particle uptake mechanism under different inhibitor conditions showed that mainly caveolin-and lipid raftmediated endocytosis were involved in the endocytosis of RM-LNC,the caveolin-and macropinocytosis-mediated endocytosis pathways were involved in the endocytosis of FARM-LNC.Folic acid facilitated the epithelial internalization of functional LNC by altering the FA-RM-LNC uptake pathway through specific ligand-receptor interactions.Papp was1.23-fold higher in the FA-RM-LNC group than in the RM-LNC group in transmembrane transport experiments,and the increase in FA-RM-LNC transport was mediated by the specific affinity of DSPE-PEG-FA for Caco-2 cells.Co-incubation of the fluorescent DID-labelled RM-LNC and FA-RM-LNC with rat small intestinal mucus revealed that DID-RM-LNC showed significant aggregation in rat intestinal mucus,whereas DID-FA-RM-LNC showed no significant aggregation.Specific target binding of folic acid and enterocytes was found under uptake imaging at different intestinal sites resulting in significantly increased absorption of FA-RM-LNC in the small intestine compared to the RM-LNC group,particularly in the duodenal and jejunal sites.After 2.5 hours of oral administration of the preparation in an ex vivo flipped intestinal loop assay,FA-RM-LNC transported approximately 1.24,1.37 and 1.19 times more than RMLNC in the duodenum,jejunum and ileum,respectively.3.Pharmacokinetics and pharmacodynamics of exenatide lipid nanocapsulesThe study examined the pharmacokinetics of exenatide lipid nanocapsules in SD rats.The results showed that the DSPE-PEG-FA modified lipid nanocapsules greatly improved the bioavailability of exenatide via oral delivery.Using subcutaneous injection as a control,the bioavailability of oral exenatide group was only 3.7%,that of FA-RM-ELNC group was5.87%,while that of oral FA-RM-ELNC group was 7.53%,which was 1.28 times higher than that of RM-ELNC group.The hypoglycaemic effects of RM-ELNC,FA-RM-ELNC and exenatide solutions were examined at the animal level using db/db mice as a model of type 2 diabetes.The results showed that in a single dose RM-LNC reduced blood glucose to 72% after 6 hours,while FA-RM-LNC had a more pronounced hypoglycaemic effect(66%)at the same time point.Furthermore,24 hours after the administration of FA-RM-LNC,the blood glucose level remained at 64% of the initial value.The RM-ELNC group maintained blood glucose levels between 80% and 90% in multiple doses,while the FA-RM-ELNC group was able to maintain blood glucose levels that could be between 65% and 90%.The results suggest that the effect of folic acid-modified exenatide reverse micelle lipid nanocapsules on glycaemic control in the diabetic model rats is significant.In summary,folic acid-modified reverse micelle lipid nanocapsules were designed and prepared for the oral delivery of exenatide using their intestinal targeting properties.Compared with the reverse micelle lipid nanocapsules,the folic acid-modified reverse micelle lipid nanocapsules significantly improved the intestinal absorption of exenatide and improved the low bioavailability of exenatide for oral administration.This study also provides an experimental and theoretical basis for the establishment of an efficient and safe oral peptide drug delivery system.