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1. Department of Medical Ultrasound, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou 510150, China.
3. Guangzhou Research Institute of Obstetrics and Gynecology, Key Laboratory for Major Obstetric Diseases of Guangdong Province, The Third Affiliated Hospital of Guangzhou Medical University, Institute of Obstetrics and Gynecology, Guangzhou 510150, China.
4. Department of Nuclear Medicine, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou 510150, China.
Objective: To enhance the transfection efficiency of the human breast cancer cell lines (MCF-7) cells by plasmid DNA, the optimized condition and synergistic role of ultrasound (US) irradiation with contrast agent and polyethyleneimine (PEI) were discussed.
Methods: MCF-7 cells were transfected with the compounds prepared by the plasmid DNA encoding luciferase (pCMV-luciferase-GL3) and PEI. Meanwhile, SonoVue microbubble was added to the cell suspension to serve as nucleation sites for acoustic cavitation before US irradiation. Then, the DNA expression of luciferase plasmid and viability of cells were evaluated through optimizing US irradiation. Furthermore, the influencing factor, such as the concentration of plasmid, incubation time, serum, the type of solvent and the volume of culture media, were analyzed.
Results: The viability of cells and US-induced enhancement of luciferase activity were influenced by the US intensity, exposure time and duty cycle. US irradiation under an appropriate condition could accelerate the permeation of the PEI/DNA complex through the cell membrane. It could enhance the transfection efficiency of plasmid DNA. The optimal US condition for the enhancement was determined to be 1W/cm2, 10% DC for 3 mins. In contrast to the PEI/DNA complex alone without US irradiation or US irradiation alone, the combination of US irradiation with contrast agent and PEI had a significantly enhanced luciferase activity (P<0.01). The 2 hrs preirradiation incubation with PEI/DNA complex for MCF-7 cells exhibited a significantly enhanced luciferase activity (P<0.01). In addition, the transfection efficiency was also affected by serum, type of solvent and the volume of culture media.
Conclusions: These results suggest that optimized parameters of US and transfection enhance the efficiency of gene expression in MCF-7 cells apparently. The combination of US irradiation with contrast agent and PEI has a synergy and it is not only a simple but also an applied promising method to increase plasmid DNA expression.
Key words: Ultrasound, contrast agent, polyethyleneimine, transfectionm cancer, cell
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