Preparation of polyethylenimine-chitosan/DNA nanoparticles for transfecting articular chondrocytes in vitro / 中国组织工程研究

ABSTRACT

BACKGROUND:Chitosan is wel known as good biocompatibility and biodegradability;however, its extensive use in biomedical applications is restricted due to its poor transfection efficiency. OBJECTIVE:To prepare the polyethyleneimine-chitosan/DNA nanoparticles loading enhanced green fluorescent protein gene, and to detect their physicochemical properties and gene transfection efficiency towards chondrocytes in vitro. METHODS:Low molecular weight polyethyleneimine was covalently linked to chitosan backbone to construct chitosan-graft-polyethyleneimine;then the chitosan-graft-polyethyleneimine was mixed with DNA nanoparticles, which loaded enhanced green fluorescent protein gene, by a complex coacervation method. The nanoparticle morphology was observed under a scanning electron microscopy. The sizes and zeta-potentials of the nanoparticles were measured by a Marven-nano laser diffractometer. The binding capacity of plasmid DNA was evaluated by agarose gel electrophoresis analysis. The gene transfection experiments in vitro were performed towards rabbit’s chondrocytes. The gene transfection efficiency was measured with flow cytometry and under fluorescence microscope. How marked DNA entered into the nucleus of chondrocytes mediated by the nanoparticles was detected by laser scanning confocal microscopy. RESULTS AND CONCLUSION:The prepared nanoparticles were mainly spherical, with an average size of (154.6±18.6) nm, and zeta-potential of (24.68±6.82) mV. The agarose gel electrophoresis analysis confirmed that the nanoparticles could effectively protect plasmid DNA from DNase Ⅰ-induced degradation. Gene transfection in vitro proved that the nanoparticles were efficient in transfecting rabbit’s chondrocytes and the expression of green fluorescent proteins was observed under fluorescent microscope, with a transfection efficiency of (23.80±1.74)%that was significantly higher than that of the naked plasmid DNA and chitosan/DNA nanoparticles (P<0.05). But no significant differences were observed between polyethyleneimine-chitosan/DNA nanoparticles and LipofectamineTM 2000. These findings indicate that the polyethyleneimine-chitosan/DNA nanoparticles can effectively protect plasmid DNA from nuclease degradation, and exhibit the favorable transfection ability towards articular chondrocytes.