Studies On The Mucoadhesive Nanoparticulate Delivery System For Oral Insulin

In this study, a novel mucoadhesive polymeric nanoparticulate delivery system based on the thiolated conjugates were introduced and used for oral insulin delivery. Various physical and chemical properties of thiolated NPs were studied, hypoglycemia effect was evaluated in diabetes and healthy rats, and the mechanism of thiolated delivery system enhancing the absorption of insulin was studied.Firstly, Eul-cys conjugates were prepared by an amide polymerization reaction involving Eudragit L100 and cysteine hydrochloride mediated by EDC and NHS. The structure of conjugates were characterized by HNMR, FT-IR and DSC. The thiol group was determined by Ellman’s reagent and the highest free thiol content was 390.3±13.4 μmol/g. Eul-cys conjugates appeared as white, odorless powder with a fibrous structure after lyophilization and spherical, smooth and uniform particles were observed by SEM. The conjugates can hardly dissolve in water, methanol, ethanol and trichloromethane, but only dissolve in DMSO very slightly. The stability of thiol was influenced by pH, which was stable when the pH below 5.0 and oxidation would happen above 7.0. The effect of pH and ion strength on the swelling ratio of conjugates was found that the swelling ratio increased with the pH rising, and was propotional to the thiol content, while the result was adverse with the ion strength. Rheological study demonstrated that the mechanism of mucoadhesive properties of thiomers was realized by the formation of disulfide bond between the thiol on the backbone of thiomers and mucus glycoprotein.The HPLC method was developed to determine the drug loading and in vitro release. With the pH lowering, the particle size of conjugates decreased and the zeta potential increased. A similar result was observed by TEM, the particle swelled when the pH was high and a gradually low expansivity was obtained when the pH decreased. Utilizing the reversible property of swelling and shrinking, insulin pre-dissolved in HCl was added into Eul-cys solution dropwise. The drug diffused into the polyion complex core via the network structure on the surface and nanoparticles formed following self-assembly of oppositely-charged polyanionic nanopheres (Eul-cys) and insulin through electrostatic interactions. The same method was used for insulin loaded Eul nanoparticles. The Eul-cys and Eul NPs had a mean particle size of 324.2 ± 39.0 nm and 308.8 ± 35.7 nm, with a negative zeta potential of -3.1 ± 0.8 and -2.9 ± 0.8 mv, respectively. The trehalose was used as a cryoprotectant, the appearance of the resulting freeze-dried NPs was good, and the change of particle size of freeze-dried NPs resuspended in water was little. Drug release from two types of nanoparticles in vitro showed typical pH-dependent characteristics. Drug had a slow release in acid mediate and exhibited a fast release in PBS in neutral. The secondary structure and bioactivity of insulin after loading and release from the NPs were established by far-UV CD spectra and mice and there was no significant difference compared with native insulin. The mucoadhesion of Eul-cys and Eul nanoparticles to freshly excised rat intestine, including duodenum, jejunum, ileum and colon was evaluated and Eul-cys NPs exhibited stronger mucoadhesion than Eul NPs in various segments of the intestinal tract. There was lower mucoadhesion in the upper part of the small intestine than the posterior segments of the intestine, such as the ileum and colon. Hypoglycemia after oral administration of the insulin-loaded Eul-cys NPs produced a higher and prolonged reduction in blood glucose levels compared with Eul NPs, The pharmacological bioavailability of the two polymeric NPs was 7.33 ± 0.33% and 2.65 ± 0.63%, respectively. Histological studies showed no significant changes in the structure of the intestine and thiolated delivery system did not induce any adverse effects on the intestine.In order to increase the oral bioavailability of insulin, reduced glutathione (GSH) was introduced in the nanoparticulate drug system. Duo to its influence on the mucoadhesive properties of NPs, the mount of GSH added was investigated and the result revealed that there had no change for the mucoadhesive of NPs when the ratio of Eul-cys to GSH was higher than 4:1. The Eul-cys/GSH NPs showed a mean particle size of 260.0±17.1 nm with a negative zeta potential. There was no effect on the particle size, loading efficiency after the process of lyophilization. In the in vitro release, GSH was found to have an effect on insulin release. Due to their similar charge, a competitive release from the pores of polymeric material occurred. In the in situ release, Eul-cys and Eul-cys/GSH NPs releasing the majority of FITC-insulin into the intestinal mucus was observed, which differed from the insulin solution mainly existed in the lumen. In the transport experiment, NPs exhibited enhanced permeation ability for insulin than the solution, a higher Papp was found when the NPs were used in jejunum and ileum than that in duodenum and colon. In the closed-loop intestinal absorption studies, both Eul-cys and Eul-cys/GSH NPs produced a reduction in blood glucose levels while a better result was observed for GSH was added.The state of insulin existed in the NPs was studied by X-ray and XPS, and the interaction of insulin and the carrier was investigated by DSC and FT-IR. The result indicated that insulin in the NPs was either molecularly dispersed or amorphous form. The interaction between Eul-cys and insulin was physical, but not chemical.The mechanism of thiolated delivery system enhancing the absorption of insulin was studied and three possible reasons were included:cation binding, enzymatic inhibition and cell permeation. Eul-cys displayed their potential in inhibiting proteases from the GI tract and protecting peptides from proteolytic degradation. One possible mechanism is enzyme incorporation by polymer and Ca2+ deprivation for enzyme inactivation. In the MTT assay, NPs containing GSH at all concentrations produced no obvious suppression of Caco-2 and HT29-MTX cells viability compared with SC. Incubation of the Eul-cys NPs containing GSH with cells produced a TEER reduction but was slower and less effective compared with that of SC. However, after removal of the incubated NPs, In the case of Eul-cys NPs containing GSH, the TEER in all groups mainly returned to the initial value, but the high concentration of SC was associated with an irreversible lowering of the TEER. It appeared that although the GSH produced less permeability enhancement than the SC, it is a safer permeation enhancer for the delivery of macromolecules. In the transport studies, the apparent permeability of insulin in the presence of nanoparticles with two types of absorption enhancers increased by 1.7- to 9.6-fold compared with the control, and the effect was concentration dependent. SC had greater permeation-enhancing potential than GSH in insulin transport across the test cells, and the added HT29-MTX cells had a more marked effect on GSH than SC in facilitating insulin transport. After removal of mucus led to a 2.4 fold increase in insulin transport in the co-culture for GSH (p < 0.001), while this was not significant for SC. Thus, it appears that the mucus layer may obstruct the absorption of thiol functional drug delivery systems. GSH and SC had no effect on the ZO-1 tight junction protein, however, form the TEER results and CLSM images, we inferred that thiolated NPs were able to enhance the permeability of macromolecule drugs via paracellular pathways. In addition, the NPs might also be absorbed across the enterocytes via M cells.