Introduction to the Ultrasound Targeted Microbubble Destruction Technique

Summary

Ultrasound Targeted Microbubble Destruction (UTMD) can be used to direct site-specific delivery of bioactive molecules, including therapeutic genes, to target organs accessible to ultrasound, such as the heart and liver^1-6.

Abstract

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 agents^7-9. 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.

Protocol

1. Microbubble stock preparation

1. In 10 mls of PBS mix 200 mg 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine and 50 mg 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine with 1 g glucose.
2. Heat the mixture in boiling water bath 20-30 minutes, pipette mixing every 5 minutes.
3. The solution can be stored at 4°C for up to 6 months.

2. Microbubble Preparation

1. Take a 250 μl of the prepared microbubble stock solution and incubate at 40°C for 15 minutes.
2. The pre-warmed microbubble solution is then transferred to a 1.5 mL microtube containing 50 μl of glycerol.
3. 1-2 mg of pu....

Discussion

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 tech.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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
phosphate-buffered saline	Sigma-Aldrich	P5368
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