| Objective: A new oral dosage form was designed to contain the GLP-1 drug(Exenatide),which could improve the oral absorption of exenatide in order to effectively reduce blood glucose and relieve insulin resistance in type 2 diabetic rats.Methods: Soybean phospholipid S100 was used as an amphiphilic material and exenatide was encapsulated in medium chain oil to form reversed micelles.Tween 80 and ethanol were added as surfactants to prepare exenatide-reversed micelles by emulsification(Reverse micelle/self emulsifying drug delivery system,RM/SEDDS),further charge-adsorbed chitosan and sodium alginate to prepare exenatide-reverse micelles-selfemulsion with chitosan and sodium alginate(RM/O/W E-CA).The particle size,zeta potential,electron microscopy image,encapsulation efficiency,in vitro release profiles and peptide conformation stability of exenatide nano-formulations were investigated and evaluated.Established a Caco-2 single cell model and a Caco-2/HT-29/Raji B three-cell model to investigate the cytotoxicity of exenatide nano-formulations in Caco-2,HT-29 and Raji B cells.Confocal microscopy was used to investigate the effects of exenatide nano-formulations on cell membrane fluidity and cell tight junctions;flow cytometry was used to study the cellular uptake of exenatide nano-formulations and FITC-Nile Red resonance energy transfer fluorescence.The mechanism of intestinal absorption and transport of nanoparticles were studied in detail.Subsequently,the oral absorption of exenatide nano-preparations were investigated;a rat model of type 2 diabetes was constructed,and pathological indicators of pharmacodynamics were analyzed.Results: RM/SEDDS and RM/O/W E-CA nano preparations were successfully prepared.The particle size of RM/SEDDS was 24.84 ± 0.34 nm,similar to the size indicated by the TEM image.The electric potential of RM/SEDDS was-2.65 ± 0.35 m V.After superscribed with chitosan and ALG,the particle size of the RM/O/W E-CA increased to 89.70 ± 3.18 nm with the potential of-2.65 ± 0.51 m V.The encapsulation efficiency of RM/SEDDS and RM/O/W E-CA reaches 80% or more which with certain slow-release effect.Caco-2 single-cell model and Caco-2/HT-29/Raji B co-incubation model were successfully constructed.Cytotoxicity experiments showed that RM/SEDDS and RM/O/W E-CA nano preparations were biocompatible on cells.Both of them have better safety,and can significantly promote the transmembrane of exenatide.Photobleaching method showed that exenatide nano preparations could slow down the cell membrane fluidity,and immunofluorescence staining displayed that exenatide nano preparations could break down.Tight junctions between cells to promote transmembrane transport of exenatide;flow cytometry and fluorescence confocal results indicated that nano-formulations have a significant effect on promoting the uptake of nano-formulations;FITC-NR fluorescence in vivo uptake results indicated that the overall nano-formulations uptake into the cell.Pharmacokinetic results in rats revealed that both the RM/SEDDS nano preparation and the RM/O/W E-CA nano preparation have a sustained release effect.The oral absorption availability of the RM/SEDDS nano preparation is 11%,and the RM/O/W E-CA The oral absorption utilization of the nano-formulation reached 12.47%,which has a good application prospect.A type 2 diabetes model was successfully established,and the pharmacodynamic results showed that both the RM/SEDDS nano preparation and the RM//O/W E-CA nano preparation had good blood glucose lowering effects,and the long-term blood glucose showed an overall downward trend and to a certain extent It can protect β cells and improve insulin secretion in patients with type 2 diabetes,and the effect is significant;H&E staining of main organs in experimental rats,found no damage to tissues and organs,indicating that RM/SEDDS nano preparations and RM/O/W E-CA nano preparations are safe and effective,and have good application prospects.Conclusion: RM/SEDDS and RM/O/W E-CA nano preparations have been successfully prepared,which can effectively improve the oral absorption of exenatide,reduce blood glucose and relieve insulin resistance in type 2 diabetic rats,and can be used as a carrier for peptide drugs.It has extremely important research value and application prospect. |
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