Feng Yang, Liu-Fu Chun and Zhi-Yi Chen* Pages 1 - 11 ( 11 )
Background: Non-viral delivery systems is a promising method for gene or drug delivery. Polyethyleneimine(PEI) is a double-edged sword. It internalizes itself into the cell through endocytosis and promotes gene transfer efficiency. However, the large positive charge also makes PEI highly toxic to cells. Ultrasound-targeted microbubble destruction (UTMD) is a promising non-viral method for gene and drug delivery, but its efficiency still needs to be improved.
Objective: The aim of this study was to explore a system that combines ultrasound with non-viral gene delivery for the treatment of cervical cancer HeLa cells.
Methods: In this study, we synthesized a kind of cationic ultrasound contrast agent(C-UCA)that the physical and chemical properties, gene carrying capacity and cytotoxicity were verified. On the basis of previous studies, we further optimized the following transfusion parameters including ultrasound parameters, microbubble concentration, plasmid concentration, cell density and other parameters. The experiment was designed to compare the following six groups: (1) Plasmid group (P group), plasmid 15 μg; (2) PEI + plasmid group (PEI + P group),1 μl of PEI containing 10 nmol nitrogen and 1 μg of DNA containing 3 nmol phosphate for a PEI/DNA ratio equal to a nitrogen/phosphate ratio of 7 for transfection; (3) Ultrasound + plasmid group (US + P), plasmid 15 µg; (4) Ultrasound + cationic liposomal ultrasound contrast agent + plasmid group (UTMD + P group), plasmid 15 µg and cationic liposomal ultrasound contrast agent 5%; (5) Ultrasound + cationic liposomal ultrasound contrast agent + PEI + plasmid group (UTMD + PEI + P group), The influence on Hela cells was observed under microscope, the efficiency of apoptosis was measured by flow cytometry, and cell viability was tested in CCK 8.
Results: The optimized transfection parameters can improve the transfection efficiency of ultrasound combined with CUCA to a certain extent, but its transfection efficiency is still lower than that of branched polyethyleneimine (bPEI) 25 kDa. By investigating the effect of HeLa cells apoptosis induced by UTMD in combination with PEI mediated survivin miRNA, we found that both PEI alone and ultrasound in combination with C-UCA were able to transfect cells with survivin miRNA to effectively induce HeLa cell apoptosis. However, the synergistic effect between the two methods was not significant.
Conclusion: In contrast, the combined use of ultrasound, C-UCA and PEI may significantly reduce the transfection efficiency of UTMD and PEI, and the specific mechanism remains to be further studied.
Ultrasound, Cationic contrast agent , Gene therapy, PEI, Tumor cell apoptosis, Survivin miRNA
Department of Ultrasound Medicine, Laboratory of Ultrasound Molecular Imaging, The Third Affiliated Hospital of Guangzhou Medical University. Guangzhou, Guangdong, 510000, Department of Ultrasound Medicine, Laboratory of Ultrasound Molecular Imaging, The Third Affiliated Hospital of Guangzhou Medical University. Guangzhou, Guangdong, 510000, Department of Ultrasound Medicine, Laboratory of Ultrasound Molecular Imaging, The Third Affiliated Hospital of Guangzhou Medical University. Guangzhou, Guangdong, 510000