Title:Inducing Tumor Cell Apoptosis: Mediated Survivin miRNA by Ultrasound and Cationic Lipid Contrast Agent
Volume: 21
Issue: 9
关键词:
超声、阳离子造影剂、基因治疗、PEI、肿瘤细胞凋亡、存活蛋白 miRNA。
摘要:
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 strong
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(CUCA)
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), PEI/DNA ratio equal to a nitrogen/phosphate ratio of 7 for transfection
and cationic liposomal ultrasound contrast agent 5%; and (6) Blank group, no
treatment), 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 C-UCA 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 CUCA
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.