| Oral delivery of hypoglycemic protein and polypeptide drugs such as insulin and glucagon-like peptide 1(GLP-1)is a hot issue in the field of drug delivery carriers.Although the first oral preparation,semaglutide tablet(Rybelsus),has been approved by Food and Drug Administration(FDA),most of preclinical or clinical oral preparations of hypoglycemic protein and polypeptide drugs were limited.There exist two main issues for oral delivery carriers of such drugs.On the one hand,it is how to circumvent the absorption barriers thus improving oral bioavailability of drugs to lower the basal(fasted)hyperglycemia effectively.The gastrointestinal absorption barriers comprised of strong stomach acid,protease degradation,mucus layer and the underlying intestinal epithelial cells have severely limited the absorption of drugs following oral administration,making it hard to reach the appropriate drug concentration in systemic circulation to exert hypoglycemic effects effectively on basal hyperglycemia.On the other hand,it is how to release insulin intelligently in response to the dynamic blood glucose levels in vivo,thus achieving the reasonable glucose disposal and utilization.After overcoming the absorption barriers,carriers should deliver therapeutics to the right places(targeted tissues)and release them at the right time(postprandial hyperglycemia)to exert the insulin actions on glucose metabolism efficiently,which is necessary to acquire the precise glycemic control and prevention of diabetic complications.Aimed at solving above issues,we have designed and constructed two kinds of smart insulin delivery carriers,and their mechanisms on glycemic control were further investigated.Faced with the issue that the gastrointestinal barriers have severely limited oral absorption of hypoglycemic protein and polypeptide drugs,and according to the fact that protein corona absorbed on the surface of carriers could influence their physiochemical properties,in vivo fate et al,this project firstly designed and constructed an enzyme-degradable protein corona coated cationic liposomes,named Pc CLs,to explore the approach to overcome mucus layer and intestinal epithelial cells.Cationic liposomes(CLs)were prepared as the core of Pc CLs and bovine serum albumin(BSA)was absorbed on the surface to form protein corona layer.The sizes of Pc CLs were around 190 nm,and their surface charges were-10 m V.The charges of Pc CLs were weakly negative which contributed to their excellent mucus-penetrating ability.Importantly,during the process of mucus penetration,BSA corona was gradually degraded under the existence of trypsin;and CLs were exposed when they reached the surface of intestinal epithelial cells.Subsequently,the positive-charged and lipophilic CLs could significantly increase the cellular uptake,and transepithelial transport efficiency of insulin which was 7.91 times that of free insulin group.Finally,using type I diabetic rats as animal model,the pharmacokinetic characters were evaluated.The results showed that Pc CLs remarkably increased the serum insulin concentration,and the relative bioavailability of insulin was elevated up to 11.9%;pharmacodynamic results exhibited that Pc CLs not only induced good hypoglycemic effects on basal hyperglycemia,as the blood glucose levels were reduced to be 40% of original ones,but also sustained the hypoglycemic effects at least for 7 h.By means of enzyme-degradable ability of protein corona with the aid of trypsin in the gastrointestinal tract,our designed Pc CLs achieve the intelligent conversion of surface properties to overcome mucus layer and intestinal epithelial cells sequentially,further playing the relative effects.The implementation of this project has given us a much more clear understanding of the properties of double barrier.However,since the intact carriers can not transport through the intestinal epithelial cells to reach the blood,the problem that delivering insulin to the specific targeted tissues and releasing them at the right time can’t be solved yet.Thus,such carriers are only suitable for lowering basal blood glucose levels,it remains challenge to exert sufficient control for dynamically changeable postprandial hyperglycemiaThis project further designed and developed smart insulin nanoparticles(p NPs)in order to perform multi-tissues and precise insulin delivery accompanied with glucoseresponsive release of insulin,thus controlling diabetic postprandial hyperglycemia strictly and normalizing diabetic glucose disposal and utilization on the basis of overcoming absorption barriers effectively.It is reported that,there existed abnormal glucose disposal and utilization for both type I and type II diabetes,as the glucose can’t be transformed into glycogen and stored within liver,meanwhile,the overactive hepatic and renal gluconeogenesis produce excessive glucose,resulting in basal and postprandial hyperglycemia;except for this,the significantly increased renal gluconeogenesis generate local hyperglycemia causing diabetic nephropathy(DN).In this study,p NPs were prepared through self-assemble method by neonatal Fc receptor binding peptide(Fc Bp)-conjugated polymers,poly(lactic-co-glycolic acid)-copolyethylene glycol-Fc binding peptide(PLGA-PEG-Fc Bp),and glucose-responsive polymers,methoxy polyethylene glycol-co-polymethionine(m PEG-Poly Met),whose sizes were around 140 nm and their charges were-3 m V.p NPs could encapsulate insulin efficiently with the drug loading capacity of 7.15%.By micro thermophoresis,it was confirmed that the modification of Fc Bp endowed p NPs with higher binding affinity with Fc Rn as the value of Kd reached to 21.0 μM.In vitro studies showed that p NPs could release insulin in response to glucose with high concentration,and had the ability to release drugs in response to different glucose concentrations for multiple times,which was related to the morphological changes of p NPs from large vesicles to small vesicles and further dissolvement during those processes.Through the cellular experiments,it was found that p NPs could effectively penetrate the mucus layer and be endocytosed on the apical side of intestinal epithelial cells;after entering the cells,carriers were transported to the basolateral side through the early endosomes and the recycling endosomes successively,finally they ere excreted in the intact form,avoiding the lysosomal degradation effectively;finally,p NPs could significantly promote the transepithelial transport efficiency of insulin,which was 15.43 times that of the free insulin group.In vivo biodistribution studies on mice illustrated that p NPs could overcome mucus layer and intestinal epithelial cells barriers efficiently,and further accumulated in liver and kidney in intact form.Utilizing the advanced fluorescence micro-optical sectioning tomography(f MOST)technology,we further investigated the characters of precise distribution of p NPs within the tissues.It was clear that p NPs mainly deposited in the Space of Disse of liver and Bowman’s capsule of kidney.In vivo imaging experiments verified that postprandial hyperglycemia could trigger the dissociation of p NPs accumulated in the liver and kidney to release insulin.Subsequently,DN mice were selected as animal model,results of pharmacokinetic research showed that consistent with the secretion pathway of endogenous insulin,p NPs were mainly absorbed through the hepatic portal vein,where the serum insulin concentration were 18.9 times and 3.0times higher than those from the subcutaneous insulin and NPs,respectively;pharmacodynamic results exhibited that after a single oral administration,p NPs could not only lower the basal hyperglycemia,as the blood glucose levels were reduced to be35% of original ones,but also reduce the postprandial hyperglycemia to normal levels rapidly.During the one-month administration,we found that p NPs could significantly reduce the urinary protein within 24 h and improve the renal functions while maintaining postprandial normoglycemia effectively.By examining the renal morphologies,it was further confirmed that p NPs could reverse the progression of DN.The study on the mechanisms of such effects further revealed that the precise distribution of p NPs in the liver and kidney and the glucose-responsive drug release behaviors enabled the fully utilization of glucose by the liver as glucose was stored in the liver in the form of glycogen,quantitative results showed that the glycogen content in liver was 8.15 times that of DN group;at the same time,the harmful renal glycogen accumulation was reduced by inhibiting renal gluconeogenesis as the glycogen content in kidney was 0.69 times that of DN group through the quantitative analysis.Such reasonable glucose disposal and utilization mode was beneficial to effectively control the postprandial hyperglycemia and reverse DN.So far,faced with the two key issues for oral delivery carriers of hypoglycemic protein and polypeptide drugs,those projects have designed and constructed enzymedegradable protein corona coated cationic liposomes and smart insulin nanoparticles.Both of them can overcome the double barriers,while latter one can deliver insulin to the specific tissue precisely,and further releasing insulin in response to hyperglycemia to achieve the tight regulation of postprandial hyperglycemia and DN reversal.On the one hand,those projects provide certain fresh ideas for the rational development of oral delivery carriers of hypoglycemic protein and polypeptide drugs to effectively overcome mucus layer and intestinal epithelial cells;On the other hand,those projects investigate the importance of precise delivery accompanied with glucose-responsive insulin release for the good glycemic control following oral administration,thus providing a novel strategy for this kind of disease. |
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