Ultrasound-based gene delivery methods are becoming more widely used based on the critical attributes of small animal research. These attributes include, but are not limited to, the important benefits of a non-invasive, non-immunogenic, longitudinal and reproducible approach of delivering genes, drugs or proteins at the cellular level.
The introduction of the SoniGene™ Gene & Drug Delivery System provides a powerful and promising approach for gene delivery applications. More importantly, the combination of these products with the Vevo High-Resolution Imaging System provides a complete solution for small animal gene delivery applications.
Gene and/or Drug Delivery are now possible with the combinations of the following products:
- Vevo Micro-Imaging System
- SoniGene System
The combination of these products, along with microbubbles, provides an effective mechanism for the targeted delivery of genes or drugs at the cellular level.
The Vevo system is used to identify the region of interest and provide longitudinal assessment of the effect of the gene or drug delivered. Image-Guided Needle Injection can also be used for regional delivery of the gene/drug solution.
The SoniGene system is a low-frequency ultrasound device that is integrated with the Vevo system. The SoniGene system delivers a low-frequency/high-powered ultrasound pulse sequence. When used in conjunction with the contrast agents, SoniGene system will cause a sonoporation of the targeted cells and allow the gene or drug to transfect to the cellular level. Contrast agents can also act as a carrier agent for the gene or drug to the targeted site.
Gene Delivery and Microbubble Contrast Agent configurations:
Gene mixed in suspension with Non-Targeted contrast
Sonoporation: Q & A
What is Sonoporation?
Sonoporation refers to the effect of low-frequency (1 to 3MHz) ultrasound on living cells. The application of ultrasound in the presence of cavitation nuclei (microbubbles) can create transient pores in a cell membrane, allowing drug molecules, proteins, or foreign DNA to enter the cell.
What are microbubbles?
A microbubble is a microscopic (1 to 3 micron) bubble containing an inert gas. The shell of the microbubble is composed of lipids.
Is it necessary that I use microbubbles?
No, but the application of ultrasound in the presence of microbubbles and naked DNA has been found to be one of the most simple and effective methods of gene transfer available. If your laboratory already has a protocol in place using a lipid based transfection reagent you can use ultrasound in conjunction with your existing procedures. The application of ultrasound in conjunction with lipid based transfection reagents has been shown to increase gene expression1, as well as to possibly allow a reduction in the amount of reagent used.
How does sonoporation compare to other transfection methods in terms of cell viability?
When all parameters are optimized, sonoporation causes little irreversible cell damage in most cell lines. Cell membrane recovery time has been shown to be less than 10 seconds2. The factors that must be controlled to prevent irreversible cell damage and death are:
1. Concentrations of transfection reagent or microbubbles3
2. Ultrasound power output
3. Ultrasound application time
4. Ultrasound duty cycle
In the published literature, everyone seems to be using different frequencies, which is best?
Much experimentation was/is done using diagnostic ultrasound systems, since many medical research labs already have these instruments on hand. Because these systems vary in frequency based on their intended application, the result is a wide range of frequencies in the literature. Recent research has found a frequency of 1MHz to be most effective when used in conjunction with microbubbles. Application of 1MHz ultrasound results in the greatest relative expansion of the microbubbles prior to bursting4, apparently causing the greatest effect. The SoniGene system is pre-configured for use at 1MHz.
How much output power do I need?
Though not an easy question, the answer is the minimum needed to do the job. Experimenters who have tried various ranges of power output and application times have found there is a point where additional time and power no longer result in increased gene expression. Increasing power and exposure time beyond this point will only result in a decrease in the number of viable cells. Typically, in vitro sonoporation uses 0.5 to 1 W/cm2 while in vivo sonoporation works well with 2 W/cm2 or more. The SoniGene system can provide power outputs from 0.1 to 5.0 W/cm2 and duty cycles of 10, 20, 50, and 100% are standard but custom duty cycles and power outputs are available.
How do I apply ultrasound to my experiment?
Since 1MHz ultrasound does not travel through air, it is necessary for the probe to contact the medium directly or indirectly using a coupling gel. The SoniGene probe may be placed directly into the culture medium. Alternately, an ultrasound coupling gel may be placed between the probe and the bottom of the culture dish. A third method involves floating a well plate in a water bath with the ultrasound transducer in the water below the plate.
Ideally, the SoniGene transducer should be mounted in plane with the Vevo ultrasound transducer to ensure the correct target is being affected.
How does it work?
Research has yet to yield a conclusive theory on the mechanism of transfer and so the debate continues. The current theory for microbubble-enhanced transfection is the destruction of each microbubble causes a chain reaction of cavitation events2. This cavitation opens transient pores in cell membranes, allowing the entry of foreign DNA into the cell.
Select Sonoporation References:
- Combined use of Ultrasound and Acoustic Cationic Liposomes Results in Improved Gene Delivery into Smooth Muscle Cells. Shaoling Huang, et al., American Society of Gene Therapy, Annual Meeting. June 2002.
- Gene Transfer with Echo-enhanced Contrast Agents: Comparison between Albunex, Optison and Levovist in Mice-Initial Results. Tiell Li, PhD et al., Radiology. November 2003.
- Threshold of fragmentaion for ultrasonic contrast agents. James Chomas, Paul Dayton, Donovan May, Kathy Ferrara., Journal of Biomedical Optics. (2001), 6, 141-150.
- Ultrasound-Induced Membrane Porosity. Cheri X. Deng, Fred Seiling, Hua Pan, and Jianmin Cui. Ultrasound in Medicine and Biology. (2004) 30, 519-526.
- Selective clinical ultrasound signals mediate differential gene transfer and expression in two human prostate cancer cell lines: LnCap and PC-3, Biochem. Tata, D. B. et.al. (1997). Biophys. Res. Comm. 234 (1), 64– 67.
Commonly used terminology:
transfection
