Experimental Study Of Gene / Mode Of Drug Delivery And Biological Effects In The Ultrasonic Field

With the development and therapeutic applications of ultrasound techniques and advances in ultrasound contrast agents (UCAs or Microbubbles, MBs) in recent years, the synergistic effects of ultrasonic cavitation and UCAs have shown high potential in biomedicine such as gene therapy, drug delivery, thrombus lysis, diagnosis and clinic therapy for vascular diseases and tumors, and ultrasound is becoming one of the hot research fields.Human umbilical vein endothelial cells (HUVECs) and HeLa cells were used in this work. Plasmid pEGFP-N1 encoding enhanced green fluorescent protein served as exogenous gene and nucleinic acid fluorescent dye TO-PROTM-1,PI,DAPI served as mode drug. The effects of the low-frequency ultrasound with and without MBs or transfection agent lipofectamineTM 2000 on cell viability and proliferation was tested by MTT colorimetric assay; migration was evaluated by Millicell cell culture inserts combining with May-Grunwald Giemsa staining; cystoskeleton arragment was observed by Bradford method; EGFP gene expression was detected by fluorescence microscopy, and transfection efficiency of EGFP gene and delivery efficiency of mode drug were measured by flow cytometry. The results are summarized as follows:1. EGFP gene can be introduced into both HUVECs and HeLa cells by low-frequency ultrasound (20 kHz) with transfection efficiency ranging from 0.2% to 2%. The transfection efficiency is also dependent on cell types. The transfection efficiency of HeLa cell is higher than that of HUVECs.2. The transfection efficiency of HeLa cell by ultrasound and lipofectamineTM 2000 decreased with exposure time increase, but the transfection increased. It may be related to the cytotoxicity of lipofectamineTM 2000 to HeLa cell.3. The transfection efficiency by ultrasound exposure can be enhanced greatly to average 3~4 folds in the presence of MBs, with the highest 3.5 and 4.6 folds for HUVECs and HeLa cell respectively. It is also found that MBs have side effects to cell viability from our experiments. 4. The delivery efficiency of mode drugs by ultrasound exposure is relatively high comparing with transfection efficiency of EGFP gene; moreover, the delivery trendency is a little different from that of EGFP gene transfection: with exposure time increased the trasfection efficiency increased firstly, reached a peak at 6s exposure for gene transfection, then decresed gradually. However, the delivery efficiency reached the peak as 9s exposure for drug delivery.5. Cell viability decreased by excessive exposure. The migration ability also obviously declined, and cystoskeleton was disorganized and became loose from the micrographs. Excessive exposure could also make the cell shrink remarkably.