Abstract
An oral route is the most convenient and comfortable means of administering protein drugs, and eliminates pain caused by an injection, the stress association with multiple daily injections and possible infections. Oral administration has a higher patient compliance rate than injections. However, oral administration of protein drugs also has certain obstacles, as drugs must overcome various significant barriers in the gastrointestinal (GI) tract prior to delivery to the blood stream. First, protein drugs are rapidly degraded by the low pH of gastric medium in the stomach. Secondly, some digestive enzymes in the stomach and small intestine may lead to degradation of protein drugs. Finally, the intestinal epithelium is a major barrier to the absorption of protein drugs, as they cannot diffuse across the cells through the lipid-bilayer cell membranes. The study presents a novel nanoparticle (NP) delivery system which is able to provide a protection from the GI environment and enhances absorption of protein drugs in the intestinal epithelium. This novel delivery system was prepared using a simple and mild ionic-gelation method to which a poly-γ-glutamic acid (□-PGA) solution was added to a low molecular-weight chitosan (low-MW CS) solution. The particle size and the zeta potential value of the prepared NPs can be controlled under various weight ratios of CS to γ-PGA. The diameters of the prepared NPs were 110-150 nm and these NPs had a negative or positive surface charge depending on the relative concentrations of CS to □-PGA utilized. X-ray diffractograms demonstrated that the crystal structure of CS was disrupted when combined with □-PGA. The ionized CS and γ-PGA formed polyelectrolyte complexes via electrostatic interactions. The FT-IR spectra showed that CS and □-PGA were ionized at pH 2.5-6.6 and the prepared NPs remained intact in the pH range of 2.5-6.8. By TEM examinations, the proposed NPs have a matrix structure and are spherical in shape. The ability of the prepared NPs to enhance paracellular transport was investigated in vitro in Caco-2 cell monolayers. The NPs with a positive surface charge (or shelled with CS) effectively reduced the transepithelial electrical resistance (TEER) of Caco-2 cell monolayers. After removal of the incubated nanoparticles, a gradual increase in TEER was occurred. Confocal laser scanning microscopy observations verified that the NPs transiently opened the tight junctions between Caco-2 cells and facilitated transport of the NPs via the paracellular pathways. Furthermore, orally administered NPs were co-localized with the ZO-1 proteins at cell-cell contact sites in the small intestine of rats. This suggested that these NPs were able to interact and modulate the function of ZO-1 proteins, thus allowing transport of the NPs through the intestinal epithelium. In the protein drug (insulin) loading process, increasing the amount of insulin used led to a larger size of the NPs together with a significant increase in their insulin loading efficiency and loading content. The insulin concentration used was 84.0 □g/ml, the loading efficiency and loading content of the NPs were approximately 55% and 15%, respectively. Insulin release profiles were significantly affected by their stability at distinct pH environments. No significant conformational change was observed for insulin released from the NPs at pH 7.4, as compared with that of the standard insulin. Moreover, in vivo results clearly indicated that oral administration of insulin in NPs in diabetic rats achieved a sustained effect in decreasing the blood glucose level over at least 10 h, suggesting that the effect of the proposed NPs enhanced absorption of fully functional insulin. These experimental results indicated that the prepared NPs increased absorption of insulin in the intestine and provided a protection from the GI environment. This novel NP system, which is composed of CS and γ-PGA, may be a suitable carrier for oral insulin delivery.