Recently, chitosan has been extensively investigated as a promising carrier in the field of drug delivery. To overcome its poor solubility at physiological pH and the cytotoxicity of trimethyl chitosan, PEGylated trimethyl chitosan copolymers were synthesized and characterized systemically for the first time and their potential as insulin carriers for intranasal administration were studied.
The use of chitosan as an absorption enhancer and gene delivery vehicle is molecular weight (MW) dependent. Therefore, we utilized an oxidative depolymerization method to prepare and investigate different MW chitosans. The MW of the depolymerized chitosan was influenced by the initial concentration and source of chitosan. The solubility of different MW chitosans was assayed as a function of pH. The cytotoxicity of different MW chitosan was studied with MTT assay using L929 cell line recommended by USP 26, showing that the cytotoxicity was concentration dependent, but virtually molecular weight independent.
In an attempt to improve the solubility of chitosan at physiological pH, a two-step method was used to synthesis a series of trimethyl chitosans (TMC) with substitution degree of 40% using the different MW chitosans as starting materials. However, TMC was demonstrated to be toxic. Therefore, PEGylated TMC copolymers were synthesized to improve the biocompatibility of TMC. Solubility experiments demonstrated that PEG-g-TMC (400) copolymers were completely water-soluble over the entire pH range regardless of the PEG MW, even when the graft density was as low as 10%.
Furthermore, the in vitro biocompatibility of PEGylated TMC copolymers was studied and compared with that of TMC. Using the MTT assay, the effect of TMC MW, PEGylation ratio, PEG and TMC MW in the copolymers, and complexation with insulin on the cytotoxicity of TMC was examined and the IC50 values were calculated accordingly with the L929 cell line. All of the polymers exhibited a time- and dose-dependent cytotoxic response that increased with MW. PEGylation decreased the cytotoxicity of TMC, to a great extent in the case of low MW TMC. According to cytotoxicity results, PEG 5 kDa is preferable for PEGylation compared to PEG 550 Da at similar graft ratio. Complexation with insulin increased cell viability after 24 h incubation. Additionally we performed a LDH assay to measure the membrane damaging effects of the copolymers on the basis of the results of the MTT assay. Moreover, examining the morphological changes of the cells under inverted phase contrast microscopy corroborated the safety of the copolymers.
Polyelectrolyte-protein complexes (PEC) formation process between chitosan derivatives and insulin were studied and factors influencing the processes were investigated systemically. Turbidimetric titration in combination with dynamic light scattering (DLS) and laser doppler anemometry were applied to study the preferential binding between insulin and chitosan derivatives. Morphology of the complexes was observed with atomic force microscopy (AFM). It was demonstrated that the complex formation process was pH dependent. Binding between chitosan derivatives and insulin took place only above critical pH (pHc). Soluble complexes in the size range of 200-500 nm, which displayed a spherical or subspherical shape with smooth surface, could be obtained at optimized polymer/insulin charge ratio when the final system pH was in the range of 6.5-8.0, depending on polymer structure. Stability of the complex was polymer chain length dependent. Increasing the ionic strength of the medium accelerated complex dissociation. Conversely, high temperatures facilitated complex formation and compaction. Chitosan trimethylation and PEGylation significantly improved the stability of the complexes. The complexes could be lyophilized without affecting the complex properties.
The uptake and transport of chitosan derivatives-insulin complexes in Caco-2 cells was studied and the mechanisms were delineated. Insulin uptake was enhanced by nanocomplex formation, and was incubation time, temperature and concentration dependent. Complex uptake in Caco-2 cells was inhibited significantly by cytochalasin D and marginally inhibited by metabolic inhibitors. The uptake mechanism was assumed to be adsorptive endocytosis. Additionally, the cell uptake efficiency was influenced by a combination of polymer molecular weight, viscosity and positive charge density. Complex internalization was further demonstrated by confocal microscopy. However, none of the nanocomplexes displayed improved transport properties when compared to insulin transport data after 2 h incubation with Caco-2 monolayers. In contrast, the complexes considerably enhanced insulin uptake or adhesion, with approximately 50% of insulin being attached or internalized in the cells after 2 h incubation, 3.5 fold higher compared with free insulin.