Optimized preparation of DNA-chitosan nanoparticles with high transfection efficency through a central composition design

SHAO Shuai; CUI Guanghua; ZHOU Xu; GAO Zhonggao; HUANG Wei

doi:10.3969/j.issn.1006-0111.2014.06.006

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Objective This study aimed to optimize the preparation condition of DNA-chitosan nanoparticles with high transfection efficency through a central composition design. Methods The DNA-chitosan nanoparticles were prepared by complex coacervation between pEGFP and chitosan. We selected the concentrations of chitosan and plasmid as two experimental factors, and a central composite design with two factors and five levels was used to optimize the preparation condition of DNA-chitosan nanoparticles for high transfection efficency. The concentrations of chitosan and plasmid were selected as the independent variables, respectively. The dependent variables included average particle size and transfection efficiency. The morphology of DNA-chitosan nanoparticles was observed using a transmission electron microscope. The size and zeta potential of nanoparticles were measured by dynamic light scattering (DLS) and electrophoretic light scattering (ELS), respectively. The stability of plasmids in the process of nanoparticles preparation was investigated through the agrose gel electrophoresis. The expression of plasmids delivered by nanoparticles was observed under an inverted fluorescence microscope. The transfection efficiency of DNA-chitosan nanoparticles was assayed by flow cytometry. Results The preparation condition of DNA-chitosan nanoparticles with high transfection efficency was optimized successfully. Under the optimum preparative conditions, the DNA-chitosan nanoparticles were almost spherical. The average size of nanoparticles was 217.6nm, and distributed in a narrow range with a polydispersity index of 0.241. The zeta potential was +22.4 mV, which suggested that a den-sity of positive charge exist onto the surface of nanoparticles and consequently enhanced the stability of nanoparticles suspension. The results of gel electrophoresis showed that plasmids were not destroyed in the process of nanoparticles preparation. The cell transfection of nanoparticles was very highly efficient. The nanoparticles could effectively deliver the pEGFP plasmids into cells to express the green fluorescent protein at a high level. Conclusion The established mathematic models have the good predictive function. Under the optimum preparative conditions, the DNA-chitosan nanoparticles have the high potential of cell transfection.

Optimized preparation of DNA-chitosan nanoparticles with high transfection efficency through a central composition design

1. Department of Pharmaceutics, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China;Department of Pharmaceutics, Yanbian University, Yanji 133000, China

2. Department of Pharmaceutics, Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Beijing 100850, China

3. 302 Military Hospital of China, Beijing 100039, China

4. Department of Pharmaceutics, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China

Received Date: 2013-12-15

Rev Recd Date: 2014-06-13

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Abstract: Objective This study aimed to optimize the preparation condition of DNA-chitosan nanoparticles with high transfection efficency through a central composition design. Methods The DNA-chitosan nanoparticles were prepared by complex coacervation between pEGFP and chitosan. We selected the concentrations of chitosan and plasmid as two experimental factors, and a central composite design with two factors and five levels was used to optimize the preparation condition of DNA-chitosan nanoparticles for high transfection efficency. The concentrations of chitosan and plasmid were selected as the independent variables, respectively. The dependent variables included average particle size and transfection efficiency. The morphology of DNA-chitosan nanoparticles was observed using a transmission electron microscope. The size and zeta potential of nanoparticles were measured by dynamic light scattering (DLS) and electrophoretic light scattering (ELS), respectively. The stability of plasmids in the process of nanoparticles preparation was investigated through the agrose gel electrophoresis. The expression of plasmids delivered by nanoparticles was observed under an inverted fluorescence microscope. The transfection efficiency of DNA-chitosan nanoparticles was assayed by flow cytometry. Results The preparation condition of DNA-chitosan nanoparticles with high transfection efficency was optimized successfully. Under the optimum preparative conditions, the DNA-chitosan nanoparticles were almost spherical. The average size of nanoparticles was 217.6nm, and distributed in a narrow range with a polydispersity index of 0.241. The zeta potential was +22.4 mV, which suggested that a den-sity of positive charge exist onto the surface of nanoparticles and consequently enhanced the stability of nanoparticles suspension. The results of gel electrophoresis showed that plasmids were not destroyed in the process of nanoparticles preparation. The cell transfection of nanoparticles was very highly efficient. The nanoparticles could effectively deliver the pEGFP plasmids into cells to express the green fluorescent protein at a high level. Conclusion The established mathematic models have the good predictive function. Under the optimum preparative conditions, the DNA-chitosan nanoparticles have the high potential of cell transfection.

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Reference (12)

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Optimized preparation of DNA-chitosan nanoparticles with high transfection efficency through a central composition design

doi: 10.3969/j.issn.1006-0111.2014.06.006

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通讯作者: 陈斌, [email protected]

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