Design Of Novel Oral Insulin Nanocarriers To Achieve Highly Efficient Delivery In Vivo And The Underlying Mechanism

In the recent years,several protein and peptide drugs-loaded oral formulations have obtained positive outcomings in the clinical trials,which means oral delivery of protein and peptide drugs is promising.Among them,oral delivery of insulin has drawn a lot of attention.There are so far two major problems about oral insulin delivery.One is how to efficiently overcome the oral absorption barriers of insulin,including the chemical barrier,enzymatic barrier,mucus barrier and intestinal epithelium barrier in the gastrointestinal tract.Among these,the intestinal epithelium is considered as the most formidable barrier.Besides the apical membrane,nanocarriers or drugs have to complete the intracellular traffick and exocytosis from basolateral membrane to improve the transepithelial absorption of insulin.The other is how to achieve the intelligent in vivo release of insulin carriers to tightly regulate the blood glucose levels.The glycemic rhythm of the diabetic patients closely correlates with the body and dietary state.After overcoming the oral absorption barriers and entering the blood,insulin carriers must be delivered to the specific target organ and intelligently release the drugs through a glucose-responsive manner so that insulin can efficiently regulate the blood glucose levels.Therefore,to address these two major problems,we rationally designed two kinds of novel oral insulin nanocarriers and further investigated the highly efficient delivery mechanisms in vivo.In the first part of this work,in order to address the oral absorption barriers,deoxycholic acid-modified active-transport nanoparticles?DNPs?inspired from the intestinal bile acid transport pathway have been devised to improve the oral absorption of the model drug insulin.First,deoxycholic acid-modified chitosan was synthesized through the amidation reaction.Then DNPs were prepared by self-assembly of deoxycholic acid-modified chitosan,insulin and?-PGA.The mean particle size of DNPs is 226.1 nm and the zeta potential is+9.4 mV,with spherical shape observed by TEM.The particle size,zeta potential,morphology,encapsulation efficiency and loading capacity were not significantly changed after lyophilization.In vitro studies showed that DNPs could significantly decrease the insulin degradation by proteases.Also,DNPs were able to keep stable and just a small amount of insulin would be released in simulated intestinal medium with pH 5.0-7.4.The enteric capsules loaded with freeze-dried DNPs could resist the acidic environment in the stomach and release DNPs in the small intestine.Moreover,the secondary structure of insulin was remained.Caco-2 cell monolayers after cultured for three weeks were demonstrated to express apical sodium-dependent bile acid transporters?ASBT?.The internalization,intracellular fate and exocytosis of DNPs on Caco-2 cell monolayers were investigated.It was verified that the cellular uptake amount of DNPs was 2.72-fold higher than that of unmodified nanoparticles?NPs?.When ASBT inhibitor was added or the experiment was performed at 4°C,the cellular uptake amount of DNPs markedly reduced which meant the cellular internalization of DNPs was achieved through the ASBT-mediated endocytosis pathway.DNPs were found to be outside of endosomes and lysosomes observed by STED microscopy.Therefore,the insulin degradation by lysosomes significantly decreased.What is more,DNPs escaping from endolysosomes could bind with IBABP to facilitate the basolateral transport.At last,insulin from DNPs was able to exit the monolayer in molecular form.The P_app value of DNPs was 18.13×10^-7 cm/s,which is 3.49-fold higher than that of NPs.Intravital two-photon microscopy was employed to visualize the real-time transport of nanoparticles into the intestinal villi.Along with the villi sections imaging,the amount of DNPs in the villi was much higher than that of NPs.It was also demonstrated that in vivo absorption of DNPs was dependent on the bile acid pathway.Additionally,ex vivo intestinal permeation studies showed that DNPs could significantly enhanced the permeability of insulin.The permeability was strongest in the ileum,where ASBT expression was highest.Finally,in vivo pharmacodynamic and pharmacokinetic studies were performed on STZ-induced type I diabetic rats.The results showed that the hypoglycemic effect of freeze-dried DNPs-loaded enteric capsules was better than NPs.The blood glucose decreased to 45%of the initial level.The relative bioavailability of DNPs was 15.9%,which was 12.26 and 2.22-fold higher than that of insulin and NPs,respectively.The active-transport nanoparticles aiming at overcoming the oral absorption barriers in the gastrointestinal tract especially the multiple barriers of the intestinal epithelium can improve the oral absorption of free-form insulin via the bile acid transport pathway.Furthermore,in order to ensure the absorption of intact insulin nanocarriers and be able to simulate the secretion and target properties of endogenous insulin after absorption,that is,released in high blood glucose levels and then largely accumulating in the liver,we carried out the studies in the second part.In the second part of this work,in addition to overcoming the oral absorption barriers,the distribution in the target organ and glucose-responsive intelligent release behavior of insulin nanocarriers after oral absorption was highlighted.The regulation and utilization of blood glucose can be thus achieved via simulating the secretion of endogenous insulin.Inspired from the transcytosis pathway of cholera toxin B subunit?CTB?across the intestinal epithelium and the secretion and target behavior of endogenous insulin,smart polymersomes?Pep-PMS?were designed.Pep-PMS were able to target the ganglioside GM1 and equipped with glucose-responsive release property.First,functional polymer mPEG-Polymethionine was synthesized through amine-initiated ring opening polymerization and GM1-targeting peptide-modified PEG-PLGA was synthesized through amidation and click chemistry reaction.The polymerization degree of methionine was 30,determined by ¹H NMR and gel permeation chromatography.Subsequently,insulin and glucose oxidase?GOx?loaded Pep-PMS were prepared using a solvent evaporation method.The particle size of Pep-PMS was 154.0 nm,the zeta potential was+5.22 mV.Pep-PMS were spherical,with hydrophobic vesicle membrane and inner chamber observed by Cryo-TEM.In vitro studies showed that Pep-PMS could dissociated and release insulin in the presence of H₂O₂.When Pep-PMS were incubated in the high glucose medium,large amounts of H₂O₂ were produced by GOx to induce the rapid release of insulin from Pep-PMS,while only a little insulin was released in low glucose medium.Pep-PMS were transformed into smaller polymersomes and finally dissolved in the high glucose medium observed by Cryo-TEM and STED.Caco-2 cells were used to investigate the uptake,intracellular fate and transcellular transport of Pep-PMS.The results showed that the cellular uptake amount of Pep-PMS was 7.13 and 2.72-fold higher than that of insulin and unmodified polymersomes?PMS?,respectively.When Caco-2 cells were not incubated with GM1 or GM1 was inhibited with excess Pep,the cellular uptake amount of Pep-PMS significantly declined.It indicated that Pep-PMS was internalized through GM1-mediated active endocytosis.Moreover,Pep-PMS remained intact inside the cells.Pep-PMS exhibited no colocalization with lysosomes in 2 h,while most Pep-PMS were colocalized with Golgi apparatus.It demonstrated that Pep-PMS were transferred to Golgi apparatus rather than lysosomes due to GM1-mediated intracellular transport pathway.The P_app value of insulin from Pep-PMS was 17.52×10^-7 cm/s,which was 14.02 and 4.85-fold higher than that of insulin and PMS,respectively.Furthermore,intact Pep-PMS from the basolateral medium were observed by Cryo-TEM.STZ-induced type I diabetic rats were used to study the blood glucose control of Pep-PMS after subcutaneous injection.Pep-PMS were confirmed to decrease the blood glucose level in hyperglycemic conditions but not induce hypoglycemia in normal conditions.This is because Pep-PMS are able to intelligently release insulin in response to the elevated blood glucose level.In vivo intestinal villi absorption studies further demonstrated more Pep-PMS than PMS were transported into the villi.Also,Pep-PMS remained intact in the villi.In vivo biodistribution studies verified that Pep-PMS were able to largely accumulate in the liver in intact form.The studies on precision-cut liver slices and a type I diabetic mouse exhibited that Pep-PMS could release insuin in the liver in response to the increased glucose concentration.Subsequently,the pharmacodynamic and pharmacokinetic studies were performed on type I diabetic rats.The results showed that Pep-PMS induced markedly stronger hypoglycemic effect than PMS and large dose of Pep-PMS could intelligently release insulin twice to maintain the blood glucose level.The relative bioavailability of Pep-PMS was only 3.9%calculated from the peripheral serum insulin levels.However,because Pep-PMS largely accumulated in the liver,there might be bias in using the peripheral serum insulin concentration to calculate the relative bioavailability.Therefore,we further measured the portal serum insulin levels.The oral absorption of Pep-PMS was 20.3 and 4.12-fold higher than that of insulin and PMS,respectively.At last,the in vivo glucose uptake and hepatic glycogen production of diabetic rats treated with different formulations were investigated.Glucose was barely transported into the liver of the untreated diabetic rat and a low hepatic glycogen content was obtained.After treating with subcutaneous injection of insulin,a higher glucose uptake was observed in the liver and the hepatic glycogen content was increased.After treating with oral administration of Pep-PMS,both the hepatic glucose uptake and hepatic glycogen content were the highest,which is similar with that of healthy rats.In summary,this work focuses on the two major scientific problems limiting oral insulin delivery.On one hand,based on the bile acid transport pathway,active-transport nanoparticles were designed and developed to efficiently overcome the oral absorption barriers of insulin.The nanopartilces were able to escape from the endolysosomes after entering the intestinal epithelium.Thus,the intracellular lysosomal degradation of insulin was avoided.Finally,free-form insulin molecules could exit the cells from the basolateral side.On the other hand,inspired from the transepithelial transport pathway of CTB,smart polymersomes were designed and prepared to overcome the oral absorption barriers and achieve the transepithelial delivery in intact form.Moreover,after traversing the intestinal epithelium,smart polymersomes could further accumulate in the liver to simulate the target and glucose-responsive release behavior of endogenously secreted insulin.Hence,this work provides some new directions for designing insulin nanocarriers to efficiently overcome the oral absorption barriers.Also,a novel strategy is proposed for designing orally delivered smart insulin carriers with highly efficient in vivo delivery,which is mimicking the secretion of endogenous insulin to achieve the optimal glycemic control.