| Objective: To investigate whether ultrasound mediated microbubbles destruction could enhance the gene transfection to cultured tumor cells in vitro, and to explore the suitable energy for ultrasound irradiation. To investigate whether local ultrasound irradiation could enhance the gene delivery to mice livers after intravenous injection of microbubbles in vivo. Methods: 1. In vitro experiment: cultured HepG2 cells were devided into three groups. Group 1 was taken as simple microbubble transfection group: a mixture of microbubbles and GFP plasmid (5μg) was added to the cells; Group 2 was taken as liposome transfection group: 5μg plasmid was used for liposome transfection as normal. Group 3 was taken as ultrasound irradiation plus microbubble group: after added a mixture of microbubbles and plasmid to the cells, ultrasound with the intensity of 0.25 W/cm2, 0.5W/cm2, 1.0 W/cm2 was applied under the bottom of the plate for 10s. 24 hours later, the expression of GFP in the cells was observed by fluorescence microscope, the numbers of positive expression cells in every high power field (400×) were counted. 2. In vivo experiment: 24 Kunming mice were randomly divided into four groups, each group had 6 mice. The first group was taken as simple plasmid DNA delivery group: 2ml of pcDNA 3.1/HBV (20μg) was quickly injected through the tail vein. The second group was taken as simple microbubbles delivery group: 2ml of the mixture of microbubbles and plasmid (20μg) was quickly injected through the tail vein. The third group was taken as simple ultrasound delivery group: after injection of 2ml plasmid through the tail vein, ultrasound with the frequency of 1MHz, and the intensity of 0.5 W/cm2 was applied on the livers of mice for 1 min. The last group was taken as ultrasound and microbubbles delivery group: after injection of 2ml mixture of microbubble and plasmid (20μg), ultrasound with the same energy was applied on the livers for 1 min. 7 days later, the mice were killed and the livers, kidneys, lungs, hearts, spleen, skeletal muscles were harvested for immunochemical staining of HBcAg. The numbers of positive expression cells in every high power fields were measured. Results: In vitro experiments, there was no positive expression cell in group 1; There were different quantity of positive expression cells in group 3. The highest GFP expression was seen in the group in which ultrasound with intensity of 0.5W/cm2 was applied. The mean number of positive expression cells under each high power field were 20.7±3.2/hp, higher than that in other subgroup (0.25 W/cm2 or 1.0 W/cm2) of group 3, and there was no significant difference compared to group 2(P>0.05). In vivo experiments, There were few positive expression of pcDNA3.1/HBV cells in livers and kidneys in group 1 and group 4, while no positive cells in lungs, hearts, spleens, skeletal muscles. In group 3, there were a few positive cells in the livers and no positive cell in the kidneys, lungs, hearts, spleens, skeletal muscles. The number of positive cells in group 3 were 26.5±3.9/hp. The expression of pcDNA3.1/HBV was the highest in group 4, the number of positive cells were 84.2±4.4/hp. It was 3.2 folds higher than that in group 3. There was no expression in kidneys, lungs , hearts, spleens, skeletal muscles in this group.Conclusion: Ultrasound and microbubbles could highly enhance the gene transfection in tumor cells in vitro. The condition of ultrasound irradiation was a important factor for gene transfection. There was no significant difference between the suitable ultrasound energy and liposome transfection method. Local ultrasound irradiation after intravenous injection of plasmid adhered on the microbubble could highly enhance the local gene transfection and expression. This method may provide a safe, high effective local gene delivery technique in vivo.Object: To develop and investigate a novel biodegradable ultrasound contrast agent made of artificial high molecular polymer---poly lactic-co-glycolic acid (PLGA), and to observe its effects o...
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