Orally active nanoparticles formed with lactoferrin-glycol chitosan conjugate with alginate for insulin formulation

Abstract

Type 1 diabetes mellitus(T1DM) is a chronic disease caused by the destruction of β cells in the pancreas by autoimmune reaction, which is deficient in the secretion of insulin. Therefore, the glucose level of blood should be maintained by continuous insulin administration from the outside. Currently, abdominal pen-type insulin administration is the main treatment. However, it is difficult for patients to adhere and can cause serious side effects. For example, a patient can easily fall into hypoglycemia when overdose of insulin or more exercise than usual. In addition, lipoatrophy appears in the injection region during prolonged administration. Therefore, it is essential to develop oral insulin products that are non-toxic and biocompatible without side effects. However, it is necessary to overcome the limitations of denaturation or instability in acidic environments when insulin is delivered to oral pathway. Recently, according to a reference, there are effective particles to improve oral formulation, such as nanoparticle, liposome, nanogel, and superporous hydrogel. Above all, I selected a nanoparticle based on bio-compatible, less toxic polymer. The existing references suggest that chitosan and alginate can combine into electrostatic interactions to form nanoparticles. However, these nanoparticles are not specifically delivered to the intestinal epithelial cells. Therefore, I have developed receptor-specific nanoparticles that can specifically deliver the insulin. Lactoferrin receptor allows nanoparticles to pass through the intestinal epithelial cells and be absorbed. Accordingly, I made polymers that cross-linked lactoferrin and glycol chitosan, which can be absorbed well in the body due to low molecular weight (Lf-PEG-GC). Through FT-IR analysis, changes in the spectrum were observed to confirm that lactoferrin and glycol chitosan were synthesized by PEG coupling. In addition, the pre-gelated alginate and insulin mixture in response to calcium chloride (CaCl2) is coated with Lf-PEG-GC(Alginate/Lf-GC). At this time, different pH was used to determine the pH that could be best coated, and all reactions were conducted at the most suitable pH. The ninhydrin test allowed the quantitative of glycol chitosan of nanoparticles to be obtained according to the ratio of GC or Lf-GC. The synthesized nanoparticles were determined by the potential and size of the particles through the zeta potential & particle size analysis. The size and properties of the alginate/GC and alginate/Lf-GC were also compared using TEM imaging and AFM imaging. I have also confirmed that the loading efficiency of insulin in the two nanoparticles by human insulin ELISA. In this study, I conducted an in vivo experiment which confirmed effectiveness of nanoparticles in reducing blood glucose level in type 1 diabetes mouse model. Alginate/GC and alginate/Lf-GC administration showed a continuous reduction effect of blood glucose more than free insulin. However, alginate/Lf-GC did not do much to lower blood glucose levels than alginate/GC. The reason is that aggregation occurred in the synthesis process of nanoparticles, which does not appear to have resulted in the distribution and release of insulin. If the dispersion of alginate/Lf-GC is controlled well, it is expected that the long-term administration of the nanoparticles will have reduction effects of blood glucose without any side effects. Collectively, Lf-GC-alginate nanoparticle could be used as alternative strategy for development of oral insulin formulation.