The Interactions Between Superoxide Dismutase And Hydrophobic Modified N-(2-hydroxy)Propyl-3-trimethylammonium Chitosan Chloride

Chitosan, the only positive charged polysaccharide, is the deacetylated form of chitin. It’s well known for the biocompatibility, which allows its use in various protein drug delivery applications. Amphiphilic chitosan derivatives can assemble with protein in aqueous solution to form complex nanoparticles, which depends on the species of protein, the hydrophobic group on chitosan, the degree of substitution, and condition in solution such as pH and ionic strength.Based on the previous research in our lab, we prepared cholic acid (CA) and oleic acid (OA) modified N-(2-hydroxy) propyl-3-trimethyl ammonium chitosan chloride (HTCC-CA/HTCC-OA) with different substitution degrees to investigate their delivery of superoxide dismutase (SOD). The structures of the polymers were characterized by FTIR,1H NMR and pyrene fluorescence. UV, TEM and DLS were utilized to investigate the nanoparticles assembled in aqueous solution.HTCC-CA forms nanoparticles with SOD in aqueous solution. The binding with SOD can produce SOD/HTCC-CA complex nanoparticles with size about200nm. The loading efficiency can be as high as90%. The SOD/HTCC-CA complexes have extended SOD release in pH7.4phosphate buffer saline. After adding35%ethanol in the HTCC-CA solution, the chain stretched as the hydrophobic aggregates in HTCC-CA exposed to organic solvent, and more SOD was encapsuled during the process of removing the ethanol. Cell culture result shows HTCC-CA does not have cytotoxicity even at a concentration of100μg/mL after48h incubation. The good biocompatibility of HTCC-CA derivatives is an important property for their biomedical applications. Increasing the cholic acid substitution degree of HTCC-CA can significantly enhance the cellular uptake of the loaded SOD. The SOD activity and malonaldehyde concentration in the serum and several organs of the rats were investigated. The free HTCC-CA is effective to scavenge superoxide radicals in the blood circulation, and the SOD/HTCC-CA nanoparticles have better antioxidant efficiency than free SOD and free HTCC-CA, demonstrating SOD/HTCC-CA nanoparticles is superior to free SOD protein therapy.HTCC-OA with different chitosan molecular weight and different substitution degrees were prepared. HTCC-OA with less OA substitution degree than the CA substitution degree in HTCCC-CA can bind with SOD to form complex nanoparticles. At pH7.4and weight ratio of HTCC-OA to SOD1:1, the loading efficiency of SOD is more than80%. The SOD/polymer complexes also have extended SOD release in pH7.4phosphate buffer saline.40%ethanol can also strengthen the controlled release effect.