Cody L. Martin and Dr. William Pitt, Chemical Engineering
Gene delivery has been an important topic of medical and scientific research today, due to its potential in aiding the fight against cancer, heart disease and many genetic disorders. By transfecting appropriate genes into diseased cells, specific proteins can be produced that will alleviate problems at the cellular level. In addition, if the gene delivery is done by a targeted non-viral approach, such as ultrasound combined with contrast agent bubbles, the efficiency of transfection always increases. The targeted non-viral approach consists of applying focused ultrasound to a specific tissue in the presence of microbubbles. The application of ultrasound will cause cavitation, the oscillatory expansion and contraction of bubbles. As the ultrasonic intensity is increased, the bubbles will collapse, and the resulting shock wave and fluid motion will perturb the membranes of nearby cells, actually creating transient holes and allowing direct uptake of the genetic material into the cells. Therefore, the gene delivery is non-invasive; the transducer is set on the skin and there are no incisions. Also, the gene delivery is targeted—cavitation and the collapse of the bubbles will only occur where the ultrasound is directed.
The use of gas bubbles is ideal for gene delivery in vitro or for gene delivery in vivo to endothelial cells in the circulation system. However, the problem with using conventional gas bubbles is that they are too large to pass through the gaps in endothelial cell boundary of the vascular system to cells beyond this barrier. Therefore, the tissues beyond the endothelial layer cannot receive the gene delivery. However, the key to this project was to determine the transfection efficiency of nanosized emulsion droplets that can be made small enough to pass through the gaps (22 nm) between the endothelial cells lining the circulatory system. The idea is that once the emulsion droplets are past the endothelial layer, focused ultrasound can be used to transform the liquid droplets to gas and create bubbles. Then, the bubbles will follow the same collapse mechanism that the conventional bubbles do with ultrasound—cavitate and perturb (shear) the cell membrane, which enhances the delivery of genes and the expression of the needed proteins.
The nanoemulsion used in this project did not create a droplet size small enough to pass the endothelial gaps. However, Dr. Pitt’s research group is studying different emulsion mixtures with different components in order to minimize the droplet size to 22 nm. The nanoemulsion used in this project was a perfluoropentane-perfluorooctanoic acid mixture, and the average droplet size was around 250 nm. This size is small enough to pass through gaps in the capillaries of some cancerous tumors; so this could be used for gene delivery to tumors.
In order to determine if nanoemulsion liquid droplets would enhance gene transfection, experiments were performed on a monolayer of MCF7 breast cancer cells in 24-well tissue culture plates. Ultrasound was delivered to the cells with a 500 kHz focused power transducer from Sonic Concepts (Woodville, WA). The frequency of the ultrasound was maintained at 500 kHz while the duty cycle (the fraction of time that the ultrasound is active—100% for continuous ultrasound), the duration of sonication, and the intensity of the ultrasound varied in each experiment. These parameters were varied to arrive at optimal parameters for maximum transfection efficiency. Each experiment tested a different constraint.
Before insonation, a plasmid containing a gene for green fluorescent protein (GFP) was introduced into the well. After exposure to ultrasound for various time spans, the wells were incubated for 48 hours to allow expression of the GFP gene. The results were determined by visualizing the cell monolayer in an IX70 Olympus fluorescent microscope at 200X. Five or more random areas on each well were visualized and pictures were taken with both white light and fluorescent light. Those cells that were transfected with the GFP plasmid transcribed the gene and translated the resulting mRNA into the GFP protein, which emits green fluorescence when illuminated with light of about 510 nm. Therefore, the number of transfected cells was counted using the green fluorescent signal, and the total number of cells was counted using white light (see Figures 1 and 2 to visualize a field illuminated by fluorescent light and white light respectively). The percent of cells transfected was calculated by dividing the total number of fluorescent cells in all viewing areas by the total number of cells in all of the viewing areas.
The results of this project were conclusive in that gene transfection efficiency did increase with the application of ultrasound in the presence of nanoemulsion droplets. However, the transfection efficiency was quite low, only 0.2% transfection with the optimal ultrasound parameters, which were 23.3 W/cm2 at a 25% duty cycle for 90 seconds.
Though the transfection efficiency was low, the project was still successful in that there was some transfection. Further research can be developed and performed in order to test other nanoemulsion contrast agents and different constraints that are part of using ultrasound. This project was a great learning experience, and hopefully, it can be a stepping stone for further research and discovery.