Department of Medicine, JABSOM, University of Hawaii
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Walton, C. B., Anderson, C. D., Boulay, R., Shohet, R. V. Introduction to the Ultrasound Targeted Microbubble Destruction Technique. J. Vis. Exp. (52), e2963, doi:10.3791/2963 (2011).
In UTMD, bioactive molecules, such as negatively charged plasmid DNA vectors encoding a gene of interest, are added to the cationic shells of lipid microbubble contrast agents7-9. In mice these vector-carrying microbubbles can be administered intravenously or directly to the left ventricle of the heart. In larger animals they can also be infused through an intracoronary catheter. The subsequent delivery from the circulation to a target organ occurs by acoustic cavitation at a resonant frequency of the microbubbles. It seems likely that the mechanical energy generated by the microbubble destruction results in transient pore formation in or between the endothelial cells of the microvasculature of the targeted region10. As a result of this sonoporation effect, the transfection efficiency into and across the endothelial cells is enhanced, and transgene-encoding vectors are deposited into the surrounding tissue. Plasmid DNA remaining in the circulation is rapidly degraded by nucleases in the blood, which further reduces the likelihood of delivery to non-sonicated tissues and leads to highly specific target-organ transfection.
1. Microbubble stock preparation
2. Microbubble Preparation
3. Equipment Calibration
4. Microbubble Delivery & UTMD
5. Alternative Delivery Method
We chose to highlight the interventricular injection due to the complexity of the procedure, but in many instances, such as prolonged infusion of microbubbles, a tail vein injection is the preferred method. For the tail-vein method of microbubble delivery, the mouse is anesthetized the same way. A syringe containing the plasmid DNA-bound microbubbles is connected to a 27 gauge needle/tail vein catheter. The tail vein catheter is inserted into the distal third of either the right or left lateral veins that along the tail of the mouse. The syringe containing the microbubbles is placed in an infusion pump that automatically administers a uniform preset volume of solution over a preset period of time. We typically infuse 200-300μl at a rate of 3ml/hour.
All animals were handled in accordance with good animal practice as defined by the relevant national and/or local animal welfare bodies, and all animal work was approved by the appropriate committee (University of Hawaii Institutional Animal Care and Use Committee, approval number 07-100-3). Appropriate anesthesia (ketamine/ zylazine) was used and analgesics (Bupivicaine and Buprenorphine) were available, though not required.
6. Representative Results:
The effectiveness of the UTMD-mediated plasmid DNA delivery can be evaluated through a variety of methods depending upon the genes encoded in the construct, such as, but not limited to; luciferase in vivo imaging, B-gal ex vivo staining, and/or immunohistology. In particular, in vivo bioluminescence imaging allows one to monitor the presence and duration of gene expression serially in mice transfected with a plasmid encoding a bioluminescent reporter gene (luciferase). The Xenogen In Vivo Imaging System (IVIS) (Caliper Life Sciences, Hopkinton, MA) is used for bioluminescent imaging. Images are typically taken of all mice the first day after UTMD mediated transfection and is repeated every three to four days until bioluminescent gene expression is no longer visually detectable through the system (Figure 1). To prepare mice for bioluminescent imaging, mice first receive an IP injection of the luciferase reporter probe D-luciferin (Caliper Life Sciences) and are then anesthetized ~3 minutes later. Biodistribution of the D-luciferin substrate is allowed to proceed for ~10 minutes before the animal is placed in the IVIS imaging chamber and a full body image scan is taken. During the acquisition, the photons emitted from the firefly luciferase/D-luciferin photochemical reaction are measured. Figure 1 also illustrates similar IVIS bioluminescent imaging of the liver following UTMD, and Figure 2 is an epifluorescence (100X) image of the transfected liver using an anti-luciferase primary antibody (Sigma-Aldrich) and AlexFluor-568 conjugated secondary antibody (Invitrogen). It is clear to see that the UTMD mediated liver transfection has affected not only the endothelial cells, but the hepatocyes as well.
Figure 1. Xenogen/IVIS imaging of cardiac UTMD treated mice. (A) Negative Control Mouse: Plasmid + PBS followed by cardiac directed UTMD, (B) Treatment Mouse: Plasmid + Microbubbles followed by cardiac directed UTMD, and (C) Treatment Mouse: Plasmid + Microbubbles followed by liver directed UTMD.
Figure 2. Immunohistochemistry of liver UTMD (anti-luciferase in red). (A) Plasmid with no UTMD, and (B) Plasmid with UTMD. Confocal image (100X); nuclei are DAPI stained blue.
UTMD represents a novel approach to gene delivery. As a platform technology it can be combined with any of the many potential gene therapy strategies, to deliver a myriad of bioactive molecules when a high degree of tissue specificity is desired. The main biological limitation of the technique is the low efficiency of transfection. Another important consideration is the accessibility of the target organ to ultrasound, which can be markedly diminished by intervening bone or air. The technique requires optimization of technique based on target tissue11 as well as a basic understanding of acoustics, to limit tissue damage11 and increase efficiency. However, it provides an inexpensive, rapid, and repeatable approach to evaluating the effect of tissue-specific transgene expression, and may deliver effective therapy for disorders where low levels of transgene expression could be useful.
No conflicts of interest declared.
Grant support has included NHLBI HL080532, NHLBI HL073449, NCRR RR16453, and an AHA National Grant-in Aid Award (to RVS). A special thanks is extended to the Distance Course Design and Consulting (DCDC) group, dcdcgroup.org, for their assistance with video production and to the US Department of Education Grant No. P336C050047 that founded the DCDC.
|1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine||Sigma-Aldrich||P-5911||component of the microbubble lipid shell|
|1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine||Sigma-Aldrich||P-3275||component of the microbubble lipid shell|
|glucose||Sigma-Aldrich||G5400||thought to stabilize the microbubbles|
|glycerol||Sigma-Aldrich||G5516||believed to prevent microbubbles from coalescing|
|Octafluoropropane gas||Airgas||N/A||inert gas used in clinical applications|
|VialMix dental amalgamator||Bristol-Myers Squibb||N/A|
|1 MHz, 13mm, unfocused transducer||Olympus Corporation||A303S-SU|
|20 MHz Function/Arbitrary Waveform Generator||Agilent Technologies||33220A|
|Power Amplifier||Krohn-Hite Co.||Model 7500|
|Hydrophone||Bruel and Kjaer||Type 1803|
|Charge Amplifier||Bruel and Kjaer||Type 2634|
|500 MHz Oscilloscope||LeCroy||9354L|
|VisualSonics’ Vevo 2100 Imaging System with 34 MHz transducer||VisualSonics, inc.||2100|
|27G one inch tail vein catheters||VisualSonics, inc.||N/A|
|Genie Plus infusion pump||Kent Scientific||GENIE|