| Oral delivery of protein drugs has attractive worldwide attention due to its convenience of administration and good patient compliance.In recent years,nanocarriers show great potential for the oral delivery of protein drugs that have very limited oral bioavailability.Orally administered nanocarriers always encounter the rigorous defenses of the gastrointestinal.To achieve effective absorption of the protein drugs into blood circulation,nanocarriers are required to overcome the intestinal mucus and epithelial barriers.However,overcoming these two barriers need different or diametrically opposite surface properties of nanocarriers.Therefore,how to precisely regulate the surface properties of the nanocarriers which enable them sequentially through the intestinal mucus and epithelial barriers is the key to improve the oral bioavailability of drugs.Based on the acid microclimate on the surface of jejunal epithelial cells(pH 5.3-6.0),we construct an oral drug delivery system which could facilitate both of these two processes.Through intelligent biological response,the surface properties of nanocarriers could be accurately regulate from hydrophilicity and electric neutrality to hydrophobicity or electro-positivity.1.Preparation and characterization of nanocarriers responding to the acid microclimate on the surface of jejunal epithelial cells.Mucus penetrating material was synthesized by connecting polyethylene glycol and poly(lactic-co-glycolic acid)with hydrazine bond which is pH sensitive.And its structure was confirmed by FT-IR and 1H-NMR.Using insulin as a model protein drugs.The insulin-loaded PEG-Hyd-PLGA nanoparticles were produced utilizing double emulsion solvent evaporation techniques.The optimized nanoparticle showed an average size of 139.6±1.10 nm,PDI of 0.169± 0.02 and zeta potential of-32.8±0.72 mV.The encapsulation efficiency and drug loading capacity were 48.03±3.30%and 4.65 ± 0.30%,respectively.In vitro release profiles of insulin from nanoparticles showed that the cumulative release rate in PBS(pH 6.8)for 12 h was 43.06%,and it presented a sudden release and slow release followed by.As the freeze-drying protective agent,5%PEG4000 was preferred which could remain the properties of nanoparticles after freeze-drying.When incubated in a weak acidic microenvironment,PEG-Hyd-PLGA nanoparticles could gradually shed the PEG sell and then expose PLGA core,resulting in an increase of zeta potential.It was demonstrated that PEG-Hyd-PLGA nanoparticles have great potential for improving the oral absorption of insulin.2.Study of oral absorption mechanisms of nanocarriers responding to the acid microclimate on the surface of jejunal epithelial cells.Fluorescent dye DiD was used to label the nanoparticles.Since the acid-sensitive hydrazine bond of the PEG-Hyd-PLGA remained stable under the condition of intestinal liquid(pH 6.8),the reserved PEG layer greatly reduced the affinity between nanoparticles and mucin.In an acidic environment,the nanoparticles exposed the hydrophobic core gradually,which leaded to an increase of 10%of aggregation rate within half an hour.The results of mucin affinity experiment manifested that PEG-Hyd-PLGA nanoparticles have a stronger ability of mucus penetration,and their surface properties could gradually change to high cell affinity when they reach the acid microclimate on the surface of jejunal epithelial cells.Caco-2 cell model was used to evaluate the cytotoxicity and cellular uptake of nanoparticles.Cell viabilities of nanocarriers in various concentrations(0.02-2 mg/mL)were higher than 90%,which indicated the polymer materials had not obvious cytotoxicity.The cellular uptake of PEG-Hyd-PLGA nanoparticles increased with the decrease of pH.When mucin existed,the PEG-Hyd-PLGA nanoparticles were internalized by caco-2 cells more efficiently than PEG-PLGA and PLGA nanoparticles.These results showed that PEG could reduce the interaction with mucin,and the PEG-Hyd-PLGA nanoparticles gradually exposed the hydrophobic core in the acidic environment,which increased the cellular uptake.The mucus penetration results showed that PEG-Hyd-PLGA nanoparticles were mucus inert,so that they can reach depth of mucous layer.Enterocytes absorption studies indicated that PEG-Hyd-PLGA nanoparticles could quickly and evenly distribute around the intestinal villi,and gradually break PEG shell in the jejunal mucus microenvironment,which making better absorbed by epithelial cells.The results of enzyme degradation experiment showed that the prepared nanoparticles could protect the loaded insulin from being degraded by trypsin.Based on the above results,PEG-Hyd-PLGA nanoparticles had both the ability to penetrate the mucous layer and be effectively absorbed by epithelial cells.The nanoparticles had an huge advantages in the oral treatment of diabetes.3.Study of the hypoglycemic effect of the insulin-loaded PEG-Hyd-PLGA nanoparticles in normal and diabetic rats.Type Ⅰ diabetic rat model was established by intraperitoneally injecting the streptozotocin.The results showed that PEG-Hyd-PLGA nanoparticles have more pronounced hypoglycemic effect following the jejunum segment administration.In normal rats,the blood glucose levels of PEG-Hyd-PLGA group(50 IU/kg)could reduce to 58.89%at 6 h,and remained 60%until 10 h.Its pharmacological availability(PA)relative to subcutaneous injection was 2.66%,which was 1.55 times and 1.29 times higher than that of PEG-PLGA group and PLGA group,respectively.In diabetic rats,the blood glucose levels of PEG-Hyd-PLGA group(100 IU/kg)could reduce to about 60%,and its hypoglycemic effect lasted longer.Its PA relative to subcutaneous injection was 4.81%,which was 2.41 times and 1.62 times higher than that of PEG-PLGA group and PLGA group,respectively.In conclusion,the drug delivery system constructed in this project had both the ability to penetrate the mucous layer and be effectively absorbed by epithelial cells.Because of the responsive release of PEG shell,the surface properties of PEG-Hyd-PLGA nanoparticles could smart transform from hydrophilicity to hydrophobicity when they got to the weak acid microenvironment of the apical side of jejunal epithelial cells.The drug delivery system has greatly potential for the oral administration of protein drugs.And it provides theoretical basis and experimental basis for the design of oral delivery system of protein drugs. |
