Preparation And Characterization Of Insulin-loaded Thiolated Hyaluronic Acid Nanoparticles

The research of the oral route for the administration of peptide drugs has been a hotspot for pharmacy researchers at home and abroad. Oral bioavailability of these drugs is very low to their own physicochemical properties and other characteristics. Among these drugs, researchers are particularly concerned about the insulin. Insulin is clinically suspected first-line drug for the treatment of Type1diabetes. Patients usually need long and frequent injections because of the short half-life period of insulin. Oral insulin with easy application and good patient compliance conforms with endogenous insulin secretion pattern. However, gastrointestinal enzymatic degradation and poor membrane permeability largely impede the appliance of insulin’s oral administration. Use of polymer nanoparticles as carriers for oral administration of insulin can improve the oral bioavailability by prolonging drug residence time and preventing enzymatic degradation.In this study, we prepared thiolated hyaluronic acid (HA-Cys) by connecting hyaluronic acid and L-cysteine through an amide bond.The resulting conjugates exhibited(225.7±4.4) μmol immobilized free thiol groups per gram polymer. Its structure was confirmed by1H-NMR and IR. Adhesion experiments in vitro showed a good adhesion property of HA-Cys.Insulin-loaded thiolated hyaluronic acid nanoparticles(Ins-HA-Cys-NPs) were prepared by using HA-Cys as carriers by ultrasound-emulsification method. An oil phase/aqueous phase volume ratio, HA-Cys dosage and Insulin dosage were determined for Ins-HA-Cys-NPs preparation by single factor experiment. In addition, central composite design-response surface methodology was introduced for optimizing the preparation technology of nanoparticles according to entrapment efficiency, drug loading, particle size and polydispersity index(PDI) of Ins-HA-Cys-NPs. The optimal composition of oil/aqueous phase volume ratio was1:4.12. The best dosage of HA-Cys and insulin was25.95mg and8.04mg respectively. The size of nanoparticles was178.5±0.75nm, the PDI was0.214±0.01, and zeta potential was-38.47±0.46mV. The insulin encapsulation efficiency (EE) was (48.85±0.66)%and drug loading capacity (DL) was (4.79±0.13)%.The morphology, adhesion properties, release behavior in vitro and antienzymic effect of Ins-HA-Cys-NPs, as well as pharmacodynamics in vivo, were studied. The results showed that Ins-HA-Cys-NPs were spherical solid particles with a good polydispersity index. The zeta potential changed from (-17.32±0.38) mV to (-36.06±0.62) mV after mixing Ins-HA-Cys-NPs with mucin showed that Ins-HA-Cys-NPs had good adhesion properties in vitro. Ins-HA-Cys-NPs prolonged drug release time when compared to Ins-HA-NPs. Moreover, Ins-HA-Cys-NPs had good resistance to enzymatic degradation including pepsin and trypsin enzyme. Diabetic rats showed gently hypoglycemic effect after oral administration of the nanoparticles for1-12h. The relative bioavailability of the nanoparticles was6.9%.