The impact of physical and biological factors on intracellular uptake, trafficking and gene transfection after ultrasound exposure
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We used megahertz pulsed ultrasound and studied gene transfection with a human prostate cancer cell line. We first studied the compromise of cell viability and uptake efficiency and found out that increasing sonication temperature or changing US contrast agents could improve drug/gene delivery mediated by US exposure. We also found that accounting for cell debris after sonication was important to correctly determine cell viability. Next, we verified the capability of US to deliver DNA into the cell nuclei, which is necessary for successful gene transfection. Under the optimal sonication conditions, ~ 30% of cells showed DNA uptake right after US exposure and most had a portion of DNA already localized in the cell nuclei. The maximum transfection efficiency was ~ 12% at 8 h post US exposure. From the DNA perspective, ~ 30% of DNA was localized in the cell nuclei immediately after US exposure and ~ 30% was in the autophagosomes/ autophagolysosomes with the rest ¡°free¡± in the cytoplasm. At later time up to 24 h, DNA continued to be distributed ~ 30% in the nuclei and most or all of the rest in autophagosomes/autophagolysosomes. Our results showed that US was able to deliver DNA into the cell nuclei shortly after the treatment and that the rest of DNA was mostly cleared by autophagosomes/autophagolysosomes. To further increase transfection efficiency, we then studied the differences between live cells with DNA uptake and those with successful gene transfection post US exposure using cell sorting, cell cycle and microarray analysis. Cells with gene transfection were found to accumulate at the G1 phase of cell cycle and associate with the up-regulation of 32 genes (e.g., GADD45¦Á) and the down-regulation of 46 genes (e.g., TOP2¦Á). Drugs that regulate the expression levels of GADD45¦Á and TOP2¦Á were found to further enhance the transfection mediated by US. A maximun increase of ~ 2 fold in transfection efficiency was observed when cells were sonicated with 0.6 mg/mL ethyl methanesulfonate to up-regulate GADD45¦Á. These results suggestted that using drugs that regulate certain introcellular processes could further enhance US-mediated gene transfection. Over a broad range of US conditions, the integrity of three common gene delivery vectors, plasmid DNA, siRNA and adeno-associated virus, were not affected by US exposure. This thesis verified that US was able to delivery DNA into the cell nuclei to facilitate rapid gene transfection, and provided a proof of princible that by modulating certain intracellular processes, the efficiency of US-mediated gene transfection could be further increased. US could potentially be a safe and efficient method for gene therapy.