Preparation And Evaluation Of CSK And DEX-modified Mucus-penetrating And Intestinal-targeting Exenatide Nanoparticles

Proteins and peptides are medicinal bioactive macromolecules,which are very close to normal physiological substances in vivo,so they have unique biological activity and few side effects,especially for the treatment of some chronic disease,but the low bioavailability of oral delivery system is urgently needed to be solved.Therefore,the active targeting of nano-delivery carriers which can improve the oral bioavailability of polypeptide drugs has attracted extensive interest of researchers and become a hot topic in modern pharmacology.Exenatide is a type II diabetes treatment drug that helps patients control both blood sugar and body weight.Exenatide shares 53%homology with human glucagon like peptide?GLP-1?,when blood sugar levels are higher than normal.Exenatide promoted insulin secretion by promoting pancreatic?cell proliferation,inhibiting apoptosis and promoting insulin secretion.Blood glucose levels are also effectively controlled by reducing glucagon secretion.There is no oral preparation for exenatide on the market.In this paper,we have designed and synthesized CSK-dextran-poly?lactic acid-glycolic acid??CSK-DEX-PLGA?as a multifunctional nano-carrier with mucus pene-tration and intestinal targeting.In order to solve the problem of low bioavailability of exenatide in oral administration,an oral delivery system of exenatide-Zn²⁺nano-composite was constructed by using the system to solve the problem of low bioavaila-bility of exenatide.By neutralizing the positive property of DEX and the negative property of PLGA,the whole nano-particles are approximated to neutral,which is beneficial to the penetration of the mucus layer.Further functionalized modification of DEX-PLGA by CSK makes it easier for CSK-DEX-PLGA-NPs?CDPs?to reach intestinal tissue by means of affinity between CSK peptide and goblet cells,so as to improve the efficiency of transmembrane transport.The main contents of this study include the preparation and characterization of exenatide CDPs,in vitro and in vivo targeted correlation evaluation and pharmaco-dynamic evaluation,which provide some experimental and theoretical basis for the establishment of an efficient and safe delivery system for oral polypeptide delivery.The main contents and conclusions are as follows:1.Design and synthesis of the block polymer CSK-DEX-PLGA.Using CSK,DEX and PLGA as a raw material,and the aldehyde group was obtained by the isomerization?ring opening?of glucose at the end of dextran,and the Schiff base?imine?was obtained by the reaction of aldehyde group with hexanediamine.Then it was transformed into a structurally stable amine.The-COOH end of PLGA reacted with succinimide?similar to esterifcation?to obtain activated PLGA?PLGA-NHS?.Finally,the-COOH of PLGA reacted with-NH₂ of DEX to form amide bond.The end of-COOH of CSK is also activated with hexanediamine,and then reacts with-OH of DEX-PLGA copolymer to form amide bond.The reaction process was detected by HPLC,combined with the results of ¹H-NMR and GPC,and the results showed that the prepared polymer was CSK-DEX-PLGA.2.Preparation and in vitro characterization of NPsNPs were prepared by S/O/W re-emulsion method.The optimal prescription was obtained by single factor investigation,which was evaluated by particle size,PI value,entrapment efficiency and drug loading.The optimal formula was as follows:the ultrasonic power of colostrum was 300 w.The external aqueous phase volume is 4 mL,the concentration of PVA is 3%?w/v?,the ratio of polymer concentration 20 mg/mL,DMSO to dichloromethane is 1/3,and the theoretical dosage is 1 mg.The optimal particle size of CDPs was 136.3±8.9 nm,zeta potential,0.15±0.019 mV,entrapment efficiency was71.64±3.21%,drug loading was 4.62±1.37%.Under the transmission electron microscope,it is spherical,uniformly dispersed and has no adhesion phenomenon.The stability of the NPs was investigated by simulated gastric juice?SGF,pH 1.2?and simulated intestinal juice?SIF,pH 7.4?containing and without digestive enzymes,respectively.The results showed that digestive enzyme was an important factor affecting the drug stability.The addition of Zn²⁺can improve the stability of exenatide.The release behavior of simulated gastrointestinal juice without digestive enzymes was investigated in vitro.The results showed that the inclusion of exenatide in NPs could improve the stability of the exenatide,and the release of the NPs was nonsudden and showed a significant sustained release effect.3.Correlation evaluation of cell and mucus in vitro.Caco-2 cells were used as model cells to evaluate the cell level in vitro.MTT assay was used to investigate the cytotoxicity of NPs.The results showed that both DPs and CDPs had no obvious cytotoxicity to Caco-2 cells.The uptake of coumarin-6-labeled NPs?100 ng/mL?in vitro was investigated by flow cytometry and laser confocal microscopy.The results showed that the uptake ability of CDPs was stronger than that of DPs,but all of them entered the cells through active transport.The uptake inhibition experiment showed that the uptake of coumarin-6-labeled CDPs was involved in the energy-dependent pathway mediated by reticular protein,fossa protein and lipid raft.The results of transmembrane transport experiment showed that the transporter amount of FITC-exenatide-CDPs was significantly higher than that of FITC-exenatide-DPs;the presence of free CSK decreased the amount of FITC-exenatide-CDPs transporter,which indicated that CSK and CDPs were competitive with targeted binding sites.The increase of CDPs transport was mainly mediated by CSK peptide and the specific affinity of small intestinal cup-like cells.The mucus model was constructed by laying the mouse mucus in a Transwell chamber,and the mucus penetration of the NPs was qualitatively and quantitatively evaluated by a cell microtiter plate imager.The results showed that CDPs and DPs had similar rate of mucus aggregation.The CDPs have the advantage of negatively charged CSK on the surface,which is slightly weaker than that of the negative mucus.Therefore,the penetration rate of the CDPs was 2.1 times that of the DPs,showing the desired degree of mucus penetration.There was significant difference in statistical signify cance.4.Correlation evaluation of in vivo distribution.The target absorption and distribution of Dir-labeled CDPs in vivo were evaluated qualitatively by small animal in vivo imaging.The results of observation in vivo and fluorescence intensity changes of isolated organs?heart,liver,spleen,lung,kidney,gastrointestinal?showed that the absorption and distribution of CDPs group were faster and had a higher targeting to the small intestine.The results of intestinal epithelial cell permeability test and intestinal ring ligation in vitro showed that the fluorescence intensity of Nile red labeled CDPs in duodenum,jejunum and ileum was higher than that in DPs.CDPs,which was about 1.5 times higher than that of DPs.These results suggest that CDPs binds specifically to small intestinal goblet cells mediated by CSK and promotes its absorption in the small intestine and prolongation of circulation time in vivo.5.Evaluation of pharmacokinetic properties.The drug metabolism of different dosages of exenatide in Sprague Dawley rats was investigated.To establish a method for the determination of drug content in vivo,the plasma concentration of exenatide enzyme-linked immunosorbent assay?Elisa?was determined in rats and the pharmacokinetic parameters were calculated.The results showed that compared with subcutaneous injection group,the bioavailability of exenatide-Zn²⁺-CDPs was 9.2%.Compared with the oral administration of exenatide solution,the bioavailability of exenatide-Zn²⁺-DPs was increased by 4 times,and the bioavailability was 1.61 times higher than that of exenatide solution.These data suggest that CSK can promote the targeting recognition between CDPs and small intestinal goblet cells and enhance the oral bioavailability of exenatide.6.Pharmacodynamic evaluation in vivoDb/db mice were used as the model of type 2 diabetes.The pharmacological effects of different doses of exenatide on lowering blood glucose were investigated at animal level.The results showed that CDPs modified by CSK showed obvious and persistent hypoglycemic effect in both single and multiple administration.In enzymelinked immune-sorbent assay?Elisa?,the area of insulin staining in CDPs group was higher than that in oral exenatide solution group and DPs group,indicating that oral administration of exenatide-loaded CDPs could improve the function of pancreatic?-cells,and promote the secretion of insulin by?cells.In this study,we successfully constructed CDPs,carrying exenatide and demons-trated by cell and animal experiments that functionalized exogenous CDPs can improve the permeability of mucus and intestinal targeting,and then improve the oral bioavai-lability of exenatide,and showed a good hypoglycemic effect.It provides a theoretical basis for the preparation of exenatide as an oral preparation for treatment of type 2diabetes mellitus.