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. Microbubble stock preparation
<ol>
	<li>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.</li>
	<li>Heat the mixture in boiling water bath 20-30 minutes, pipette mixing every 5 minutes. </li>
	<li>The solution can be stored at 4&deg;C for up to 6 months.</li>
</ol>
2. Microbubble Preparation
<ol>
	<li>Take a 250 &mu;l of the prepared microbubble stock solution and incubate at 40&deg;...

<ol>
	<li>Bekeredjian, R., Chen, S., Frenkel, P. A., Grayburn, P. A., Shohet, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound-targeted+microbubble+destruction+can+repeatedly+direct+highly+specific+plasmid+expression+to+the+heart.">Ultrasound-targeted microbubble destruction can repeatedly direct highly specific plasmid expression to the heart.</a> Circulation. 108, 1022-1026 (2003).</li><li>Bekeredjian, R., Katus, H. A., Kuecherer, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+use+of+ultrasound+targeted+microbubble+destruction:+a+review+of+non-cardiac+applications.">Therapeutic use of ultrasound targeted microbubble destruction: a review of non-cardiac applications.</a> Ultraschall Med. 27, 134-140 (2006).</li><li>Chen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+pancreatic+islets+in+vivo+by+ultrasound-targeted+gene+therapy.">Regeneration of pancreatic islets in vivo by ultrasound-targeted gene therapy.</a> Gene Ther. 17, 1411-1420 (2010).</li><li>Miao, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound+enhances+gene+delivery+of+human+factor+IX+plasmid.">Ultrasound enhances gene delivery of human factor IX plasmid.</a> Hum Gene Ther. 16, 893-905 (2005).</li><li>Shimoda, M., Chen, S., Noguchi, H., Matsumoto, S., Grayburn, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+non-viral+gene+delivery+of+human+vascular+endothelial+growth+factor+improves+revascularisation+and+restoration+of+euglycaemia+after+human+islet+transplantation+into+mouse+liver.">In vivo non-viral gene delivery of human vascular endothelial growth factor improves revascularisation and restoration of euglycaemia after human islet transplantation into mouse liver.</a> Diabetologia. 53, 1669-1679 (2010).</li><li>Shohet, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Echocardiographic+destruction+of+albumin+microbubbles+directs+gene+delivery+to+the+myocardium.">Echocardiographic destruction of albumin microbubbles directs gene delivery to the myocardium.</a> Circulation. 101, 2554-2556 (2000).</li><li>Sirsi, S., Borden, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbubble+Compositions,+Properties+and+Biomedical+Applications.">Microbubble Compositions, Properties and Biomedical Applications.</a> Bubble Sci Eng Technol. 1, 3-17 (2009).</li><li>Li, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound-targeted+microbubble+destruction+enhances+AAV-mediated+gene+transfection+in+human+RPE+cells+in+vitro+and+rat+retina+in+vivo.">Ultrasound-targeted microbubble destruction enhances AAV-mediated gene transfection in human RPE cells in vitro and rat retina in vivo.</a> Gene Ther. 16, 1146-1153 (2009).</li><li>Lindner, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbubbles+in+medical+imaging:+current+applications+and+future+directions.">Microbubbles in medical imaging: current applications and future directions.</a> Nat Rev Drug Discov. 3, 527-532 (2004).</li><li>Newman, C. M., Bettinger, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+progress+and+prospects:+ultrasound+for+gene+transfer.">Gene therapy progress and prospects: ultrasound for gene transfer.</a> Gene Ther. 14, 465-475 (2007).</li><li>Vancraeynest, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+injury+induced+by+ultrasound-targeted+microbubble+destruction:+evidence+for+the+contribution+of+myocardial+ischemia.">Myocardial injury induced by ultrasound-targeted microbubble destruction: evidence for the contribution of myocardial ischemia.</a> Ultrasound Med Biol. 35, 672-679 (2009).</li></ol>

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...

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent/equipment</th>
			<th>Company</th>
			<th>Catalogue number</th>
			<th>Comments</th>
		</tr>
		<tr>
			<td>1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine</td>
			<td>Sigma-Aldrich</td>
			<td>P-5911</td>
			<td>component of the microbubble lipid shell</td>
		</tr>
		<tr>
			<td>1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine</td>
			<td>Sigma-Aldrich</td>
			<td>P-3275</td>
			<td>component of the microbubble lipid shell</td>
		</tr>
		<tr>
			<td>glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G5400</td>
			<td>thought to stabilize the microbubbles</td>
		</tr>
		<tr>
			<td>phosphate-buffered saline</td>
			<td>Sigma-Aldrich</td>
			<td>P5368</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td>believed to prevent microbubbles from coalescing</td>
		</tr>
		<tr>
			<td>Octafluoropropane gas</td>
			<td>AirGas</td>
			<td>N/A</td>
			<td>inert gas used in clinical applications</td>
		</tr>
		<tr>
			<td>VialMix dental amalgamator</td>
			<td>Bristol-Myers Squibb</td>
			<td>N/A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1 MHz, 13mm, unfocused transducer</td>
			<td>Olympus</td>
			<td>A303S-SU</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>20 MHz Function/Arbitrary Waveform Generator</td>
			<td>Agilent</td>
			<td>33220A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Power Amplifier</td>
			<td>Krohn-Hite Co.</td>
			<td>Model 7500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hydrophone</td>
			<td>Bruel and Kjaer</td>
			<td>Type 1803</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Charge Amplifier</td>
			<td>Bruel and Kjaer</td>
			<td>Type 2634</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>500 MHz Oscilloscope</td>
			<td>LeCroy</td>
			<td>9354L</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>VisualSonics&#x2019; Vevo 2100 Imaging System with 34 MHz transducer</td>
			<td>VisualSonics</td>
			<td>2100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>27G one inch tail vein catheters</td>
			<td>VisualSonics</td>
			<td>N/A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Genie Plus infusion pump</td>
			<td>Kent Scientific</td>
			<td>GENIE</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

introduction to the ultrasound targeted microbubble destruction technique

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.

Watch this Scientific Journal Video about Introduction to the Ultrasound Targeted Microbubble Destruction Technique at JoVE.com

Introduction to the Ultrasound Targeted Microbubble Destruction Technique

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 liver1-6.

In UTMD, bioactive molecules, such as negatively charged plasmid DNA vectors encoding a gene of interest, are added to the cationic shells of lipid ...

introduction-to-ultrasound-targeted-microbubble-destruction

Research

JoVE Journal

Bioengineering

15.3K vistas.  University of Hawaii. Ultrasonido dirigido destrucción de microburbujas (UTMD) se puede utilizar para dirigir el sitio de entrega específicos de moléculas bioactivas, incluyendo genes terapéuticos, para dirigirse a los órganos accesibles a la ecografía, como el corazón y el hígado 1-6.