Development of hybrid-type modified chitosan derivative nanoparticles for the intracellular delivery of midkine-siRNA in hepatocellular carcinoma cells

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Most of the patients with HCC lose the surgical opportunity at the time of diagnosis. Some novel therapeutic modalities, like gene therapy, are promising for the treatment of HCC. However, the success of gene therapy depends on two aspects: efficient gene materials and gene delivery vectors. The present study was to develop new chitosan-based nanoparticles for a midkine-siRNA (anti-HCC gene drug) delivery. METHODS: The novel gene delivery vector (MixNCH) was synthesized by hybrid-type modification of chitosan with 2-chloroethylamine hydrochloride and N,N-dimethyl-2-chloroethylamine hydrochloride. The chemical structure of MixNCH was characterized by FT-IR and (HNMR)-H-1. The cytotoxicity of MixNCH was determined by MTS assay. The gene condensation ability and size, zeta potential and morphology of MixNCH/MK-siRNA nanopartides were measured. The in vitro transfection and gene knockdown efficiency of midkine by MixNCH/MK-siRNA nanopartides was detected by qRT-PCR and Western blotting. Gene knockdown effect at the molecule level on the proliferation of HepG2 in vitro was determined by MTS assay. RESULTS: MixNCH was successfully acquired by aminoalkylation modification of chitosan. The MixNCH could condense MK-siRNA well above the weight ratio of 3. The average size of MixNCH/MK-siRNA nanopartides was 100-200 nm, and the surface charge was about +5 mV. Morphologically, MixNCH/MK-siRNA nanopartides were in regular spherical shape with no aggregation. Regarding to the in vitro transfection of nanoparticles, the MixNCR/MK-siRNA nanoparticles reduced MK mRNA level to 14.03%+/- 4.03%, which were comparable to Biotrans (8.94%+/- 3.77%). MixNCH/MK-siRNA effectively inhibited the proliferation of HepG2 in vitro. CONCLUSION: MixNCH/MK-siRNA nanopartides could be effective for the treatment of hepatocellular carcinoma.