Lyophilisation Improves Bioactivity and Stability of Insulin-Loaded Polymeric-Oligonucleotide Nanoparticles for Diabetes Treatment.

The oral bioavailability of therapeutic proteins is limited by the gastrointestinal barriers. Encapsulation of labile proteins into nanoparticles is a promising strategy. In order to improve the stability of nanoparticles, lyophilisation has been used to remove water molecules from the suspension. Although various cryoprotections were employed in the preparation of lyophilised nanoparticles, the selection of cryoprotectant type and concentration in majority of the developed formulation was not justified. In this study, nanoparticles were fabricated by cationic chitosan and anionic Dz13Scr using complex coacervation. The effect of cryoprotectant types (mannitol, sorbitol, sucrose and trehalose) and their concentrations (1, 3, 5, 7, 10% w/v) on physiochemical properties of nanoparticles were measured. Cellular assays were performed to investigate the impact of selected cryoprotectant on cytotoxicity, glucose consumption, oral absorption mechanism and gastrointestinal permeability. The obtained results revealed that mannitol (7% w/v) could produce nanoparticles with small size (313.2 nm), slight positive charge and uniform size distribution. The addition of cryoprotectant could preserve the bioactivity of entrapped insulin and improve the stability of nanoparticles against mechanical stress during lyophilisation. The gastrointestinal absorption of nanoparticles is associated with both endocytic and paracellular pathways. With the use of 7% mannitol, lyophilised nanoparticles induced a significant glucose uptake in C2C12 cells. This work illustrated the importance of appropriate cryoprotectant in conservation of particle physiochemical properties, structural integrity and bioactivity. An incompatible cryoprotectant and inappropriate concentration could lead to cake collapse and formation of heterogeneous particle size populations.