Preparation and characterization of chitosan nanoparticles loaded with pcDNA3.1(-)/ MAGE-3-HSP70

Jilin WU; Yangde ZHANG; Jian LI; Hong ZHANG

ABSTRACT

BACKGROUND:

Naked plasmid DNA cannot transfect cells effectively because of negative charge and the degradation of nuclease. Chitosan is a biodegradable natural cationic polysaccharide. It can provide effective protection against DNases and enhance transfection efficiency.

OBJECTIVE:

To prepare chitosan nanoparticles loaded with pcDNA3.1(-)/MAGE-3-HSP70 by complex coacervation method and to research its characteristics.DESIGN, TIME AND

SETTING:

A controlled experiment was performed at the Key Laboratory of Nano-biotechnology, National Ministry of Public Health of China from February to August 2009.MATERIALS pcDNA3.1 (-)/MAGE-3-HSP70 was constructed at the Key Laboratory of Nano-biotechnology, National Ministry of Public Health of China. Chitosan, deacetylation degree > 90.0%, viscosity < 100 cps, batch number 060306, was obtained from Shanghai Bio Life Science & Technology Co., Ltd. B16 cells were presented by the Institute of Oncology, Central South University.

METHODS:

Chitosan nanoparticles loaded with pcDNA3.1(-)/MAGE-3-HSP70 were prepared by complex coacervation method. Then the naparticles were transfected into B16 cells, and the level of MAGE-3-HSP70 mRNA was tested using reverse transcription-polymerase chain reaction (RT-PCR) technology. The in vitro cytotoxicity of the nanoparticles was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl (MTT) assay.MAIN OUTCOME

MEASURES:

Particle diameter and Zeta potential were determined by ZetaSizer 1000HSA. Efficiency of the encapsulation was measured with a spectrophotometer. The combination manner was observed by gel retardation test. The ability to protect plasmid DNA from Dnase I degradation was evaluated by DNase Ⅰ protection test.

RESULTS:

The mean diameter of chitosan plasmid DNA nanoparticles was 223 nm, its zeta potential was 16 mV. The encapsulation efficiency of DNA was 92.3%. The transfection efficiency of chitosan plasmid DNA nanoparticles by B16 cells was about equivalent to that of the Lipofectamine 2000 reagent. Chitosan plasmid DNA nanoparticles were much less cytotoxlc when compared with Lipofectamine 2000-pDNA complexes.

CONCLUSION:

Chitosan plasmid DNA nanoparticle were nontoxic to cultured cells and plasmid DNA can be efficiently transferred into B16 cells by chitosan nanoparticles.