Name: Introduction to the Ultrasound Targeted Microbubble Destruction Technique (Video) | JoVE Journal
Uploaded: 2011-06-12T16:00:00.000+00:00
Duration: 8 min 24 s
Description: Learn ultrasound targeted microbubble destruction for targeted gene delivery to the heart and liver using acoustic cavitation. This technique enhances transfection efficiency and enables precise therapeutic gene expression monitoring.

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5 minutes

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°C for up to 6 months.</li>
</ol>
2. Microbubble Preparation
<ol>
	<li>Take a 250 μl of the prepared microbubble stock solution and incubate at 40°C for 15 minutes. </li>
	<li>The pre-warmed microbubble solution is then transferred to a 1.5 mL microtube containing 50 μl of glycerol. </li>
	<li>1-2 mg of purified plasmid DNA encoding an expression construct for the gene of interest (purified in this example by Qiagen Endotoxin free MegaPrep kit, Qiagen, Germantown, MD, with a optimal concentration of 4mg/ml). 2.4) Phosphate-buffered saline is added to a final volume of 500 μl. Endotoxin free Qiagen maxipreps are used, as well as sterile PBS in order to ensure sterility.</li>
	<li>The air in the microtube is then replaced with Octafluoropropane gas. </li>
	<li>The microtube is then shaken vigorously in a dental amalgamator for 20 seconds. </li>
	<li>The subnatant containing residual DNA and buffer that has not bound to the microbubbles is then carefully removed and the microbubble layer is washed three times with sterile PBS to remove unattached DNA, and placed on ice between each wash cycle. We typically achieve a binding efficiency of 30-40%. All reagents are sterile and care is made to minimize contamination.</li>
	<li>The plasmid DNA-bound microbubbles are then placed on ice for up to two hours until use.</li>
	<li>The subnatant removed from the microtube after mixing and the PBS washes, can be used to determine the concentration of unbound DNA, and likewise the amount bound based upon the known initial concentration, by measuring the optical density of this solution at a wavelength of 260nm using a spectrophotometer. </li>
</ol>
3. Equipment Calibration
<ol>
	<li>Prior to first use, a 1 MHz cavitation transducer needs to be calibrated to ensure proper mechanical index and pulse repetition. A submergible 1 MHz, 13mm, unfocused transducer is connected to a 20 MHz Function/Arbitrary Waveform Generator through a power amplifier.</li>
	<li>The transducer is placed in a plastic container full of water, aimed directly at a hydrophone, which has been connected to a 500 MHz oscilloscope via a charge amplifier. </li>
	<li>Waveform, frequency, amplitude, burst cycle, and power amplification can all be modified to obtain the proper duty cycle and mechanical index optimal to cavitate the microbubbles. For this particular experiment, we have calibrated the system to a mechanical index equivalent to ~1.3 at 1 MHz. </li>
</ol>
4. Microbubble Delivery &amp; UTMD
<ol>
	<li>Prior to microbubble delivery and UTMD, C57BL/6 mice were anesthetized with 100mg/kg ketamine and 5mg/kg xylazine through IP injection.</li>
	<li>Microbubble delivery was administered intravenously through a direct injection into the left ventricle of the heart or through tail vein catheterization. For the direct cardiac injection, a volume up to 100 μl of the plasmid DNA-loaded microbubble solution is injected through a 30 gauge needle inserted at the anterior 4th intercostal space under ultrasonic visualization into the left ventricle of the heart. </li>
	<li>The microbubble solution bolus resulting from the left interventricular injection is visualized using Visual Sonics’ 38 MHz high frequency ultrasound transducer placed in a stationary position on the thorax of the mouse in a long-axis view using VisualSonics’ Vevo 2100 Imaging System. All syringes and needles are sterile, insuring the most sterile environment possible for these injections.</li>
	<li>Immediately following the injection, microbubble destruction is carried out for ~5 minutes using a second, smaller sized low frequency 1.0 MHz transducer held directly over the desired organ, targeting destruction to this region. In this example, the ultrasound was administered to the liver at a pulse repetition frequency of 1.0 MHz, with a mechanical index equivalent to approximately 1.3-1.5, and pulse repetition period of 100 ms for every 20 cycles. Alternatively, the pulse can be gated to the ECG of the mouse (not shown in this experiment) to a burst of 3 frames of ultrasound, every 4-6 cardiac cycles. We obtain greater efficiency of transfection with protocols that allow the capillary bed to refill with bubbles between bursts of ultrasound. </li>
</ol>
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.
Animal use
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.
<img src="/files/ftp_upload/2963/2963fig1.jpg" alt="UTMD treated mice IVIS imaging; cardiac/liver targeting; fluorescence intensity; comparative data." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
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.
<img src="/files/ftp_upload/2963/2963fig2.jpg" alt="Immunohistochemistry liver UTMD, anti-luciferase in red, microscopy image, plasmid comparison." data-altupdatedat="1788235205000" data-alt="Figure 2"> 
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.

<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/pubmed?term=((Ultrasound-targeted+microbubble+destruction+can+repeatedly+direct+highly+specific+plasmid+expression+to+the+heart%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">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/pubmed?term=((Therapeutic+use+of+ultrasound+targeted+microbubble+destruction%3A+a+review+of+non-cardiac+applications%5BTitle%5D)+AND+%22Ultraschall+Med%22%5BJournal%5D)">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/pubmed?term=((Regeneration+of+pancreatic+islets+in+vivo+by+ultrasound-targeted+gene+therapy%5BTitle%5D)+AND+%22Gene+Ther%22%5BJournal%5D)">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/pubmed?term=((Ultrasound+enhances+gene+delivery+of+human+factor+IX+plasmid%5BTitle%5D)+AND+%22Hum+Gene+Ther%22%5BJournal%5D)">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/pubmed?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%5BTitle%5D)+AND+%22Diabetologia%22%5BJournal%5D)">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/pubmed?term=((Echocardiographic+destruction+of+albumin+microbubbles+directs+gene+delivery+to+the+myocardium%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">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/pubmed?term=((Microbubble+Compositions%2C+Properties+and+Biomedical+Applications%5BTitle%5D)+AND+%22Bubble+Sci+Eng+Technol%22%5BJournal%5D)">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/pubmed?term=((Ultrasound-targeted+microbubble+destruction+enhances+AAV-mediated+gene+transfection+in+human+RPE+cells+in+vitro+and+rat+retina+in+vivo%5BTitle%5D)+AND+%22Gene+Ther%22%5BJournal%5D)">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/pubmed?term=((Microbubbles+in+medical+imaging%3A+current+applications+and+future+directions%5BTitle%5D)+AND+%22Nat+Rev+Drug+Discov%22%5BJournal%5D)">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/pubmed?term=((Gene+therapy+progress+and+prospects%3A+ultrasound+for+gene+transfer%5BTitle%5D)+AND+%22Gene+Ther%22%5BJournal%5D)">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/pubmed?term=((Myocardial+injury+induced+by+ultrasound-targeted+microbubble+destruction%3A+evidence+for+the+contribution+of+myocardial+ischemia%5BTitle%5D)+AND+%22Ultrasound+Med+Biol%22%5BJournal%5D)">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>

No conflicts of interest declared.

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><tbody><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></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></td></tr><tr><td>1 MHz, 13mm, unfocused transducer</td><td>Olympus Corporation</td><td>A303S-SU</td><td></td></tr><tr><td>20 MHz Function/Arbitrary Waveform Generator</td><td>Agilent Technologies</td><td>33220A</td><td></td></tr><tr><td>Power Amplifier</td><td>Krohn-Hite Co.</td><td>Model 7500</td><td></td></tr><tr><td>Hydrophone</td><td>Bruel and Kjaer</td><td>Type 1803</td><td></td></tr><tr><td>Charge Amplifier</td><td>Bruel and Kjaer</td><td>Type 2634</td><td></td></tr><tr><td>500 MHz Oscilloscope</td><td>LeCroy</td><td>9354L</td><td></td></tr><tr><td>VisualSonics&amp;rsquo; Vevo 2100 Imaging System with 34 MHz transducer</td><td>VisualSonics, inc.</td><td>2100</td><td></td></tr><tr><td>27G one inch tail vein catheters</td><td>VisualSonics, inc.</td><td>N/A</td><td></td></tr><tr><td>Genie Plus infusion pump</td><td>Kent Scientific</td><td>GENIE</td><td></td></tr></tbody></table>

introduction to the ultrasound targeted microbubble destruction technique

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.

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 ...

UTMD stands for 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. In UTMD bioactive molecules such as negatively charged plasmid, DNA vectors encoding a gene of interest are added to the cation shells of lipid microbubble contrast agents, the vector bound microbubbles can be administered intravenously or directly to the left ventricle of the heart.In larger animals, they can also be delivered by an trac coronary catheter with or without a dwell period. The subsequent delivery from the circulation to a target organ occurs optimally through 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 micro vasculature of the targeted region.As a result of this sauna parion effect, the transfection efficiency into and across the endothelial cells is increased and transgene encoding vectors can be deposited into the surrounding tissue of interest. Plasma DNA remaining in the circulation is rapidly degraded by nucleases in the blood microbubble preparation. An aliquot of a prepared microbubble stock solution containing glucose phosphate buffered saline, diol glycerol three phosphatidylcholine and diol glycerol three.Phosphatidyl ethanolamine is first incubated at 40 degrees Celsius for 15 minutes. The warmed microbubble solution is then transferred to a 1.5 milliliter micro tube containing glycerol and between one and two milligrams of purified plasma DNA encoding an expression construct for the gene of interest. Phosphate buffered saline is added to a final volume of 500 microliters.The air in the micro tube is then replaced with Octa fluoro propane gas. The micro tube is then shaken vigorously in a dental amalgamator for 20 seconds. Equipment calibration prior to first use the one megahertz UTMD cavitation transducer needs to be calibrated to ensure proper mechanical index and pulse repetition.A submerge one megahertz transducer is connected to a waveform generator through a power amplifier. The transducer is placed in a plastic container full of water aimed directly at a hydrophone, which has been connected to an oscilloscope via charge amplifier, waveform frequency amplitude, burst cycle, and power amplification can all be modified to obtain the proper duty cycle and mechanical index optimal to cavitate the microbubbles. In this setup, we have calibrated the system to a mechanical index equivalent to approximately 1.3 at one MEGAHERZ MICROBUBBLE delivery and UTMD prior to microbubble delivery and U-T-M-D-C 57 BL six mice are anesthetized with 100 milligrams per kilogram ketamine, and five milligrams per kilogram xylazine through IP injection.Microbubble delivery is administered intravenously through a direct injection into the left ventricle of the heart or through tail vein catheterization, which will be shown later for the direct cardiac injection. A volume up to 100 microliters of the plasma DNA loaded microbubble solution is injected through a 30 gauge needle inserted at the anterior fourth intercostal space under ultrasonic visualization into the left ventricle of the heart. The microbubble solution bolus resulting from the left interventricular injection is visualized using visual sonic's high frequency ultrasound transducer placed in a stationary position on the thorax of the mouse in a long axis view.Immediately following the injection, microbubble destruction is carried out for approximately five minutes, using a second smaller size low frequency, 1.0 megahertz transducer held directly over the desired organ targeting destruction to this region. The ultrasound is administered to the liver at a pulse repetition frequency of 1.0 megahertz with a mechanical index equivalent to approximately 1.3 to 1.5, and pulse repetition period of 100 milliseconds for every 20 cycles, or the pulse can be gated to the ECG of the mouse to a burst of three frames of ultrasound every four to six cardiac cycles. 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 run along the tail of the mouse. The syringe containing the microbubbles is placed in an infusion pump that will automatically administer a uniform preset volume of solution over a preset period of time.Bioluminescence analysis of UTMD, the effectiveness of the UTMD mediated plasma DNA delivery can be evaluated through a variety of methods depending upon the genes encoded in the construct. In particular, in vivo bioluminescence imaging is one method that allows you to monitor the presence and duration of gene expression. In mice transfected with the plasmid encoding a bioluminescence reporter gene such as luciferase.The Enogen in vivo imaging system is used for bioluminescence. Imaging images are typically taken of all mice the first day after UTMD mediated transfection and is repeated every three to four days until bioluminescence gene expression is no longer visually detectable through the system. To prepare mice for bioluminescent imaging, mice first receive an IP injection of the luciferase reporter probe Lucifer.The mice are then anesthetized. Approximately three minutes later, bio distribution of the Lucifer substrate is allowed to proceed for approximately 10 minutes before the animal or animals are placed in the Xeno Gen imaging chamber and a full body image scan is taken during the acquisition, the photons emitted from the fireplace. Luciferase de Lucifer photochemical reaction are determined by the system after each image is acquired.Using the Xeno in vivo imaging system, the system software is used to measure and quantify the visible luminescent region of interest.

introduction-to-ultrasound-targeted-microbubble-destruction

Research

JoVE Journal

Bioengineering

Insonation of microbubbles is a promising strategy for tumor ablation at reduced time-averaged acoustic powers, as well as for the targeted delivery of therapeutics. The purpose of the present study is to develop low duty cycle ultrasound pulsing strategies and nanocarriers to maximize non-thermal microvascular ablation and payload delivery to subcutaneous C6 gliomas.

Contrast Ultrasound Targeted Treatment of Gliomas in Mice via Drug-Bearing Nanoparticle Delivery and Microvascular Ablation

Microbubble-mediated focused ultrasound disruption of the blood-brain barrier (BBB) is a promising technique for non-invasive targeted drug delivery in the brain1-3. This protocol outlines the experimental procedure for MRI-guided transcranial BBB disruption in a rat model.

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Here, we present a protocol to inject ultrasound microbubble contrast agents into living, isolated late-gestation stage murine embryos. This method enables the study of perfusion parameters and of vascular molecular markers within the embryo using contrast-enhanced high-frequency ultrasound imaging.

Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

The blood-brain barrier (BBB) can be temporarily disrupted with microbubble-mediated focused ultrasound (FUS). Here, we describe a step-by-step protocol for high-throughput BBB opening in vivo using a modular FUS system accessible for non-ultrasound experts.

A High-Throughput Image-Guided Stereotactic Neuronavigation and Focused Ultrasound System for Blood-Brain Barrier Opening in Rodents

The presented experimental protocol can be used to perform real time measurements of cavitation activity in a cell culture device with the aim of enabling investigation of the conditions required for successful drug delivery and/or other bioeffects.

Studying Cavitation Enhanced Therapy

A fast and reliable technique is proposed to control the shape oscillations of a single, trapped acoustic bubble that is based on coalescence technique between two bubbles. The steady-state, symmetry-controlled bubble shape oscillations allow analysis of the fluid flow generated in the vicinity of the bubble interface.

Induction of Microstreaming by Nonspherical Bubble Oscillations in an Acoustic Levitation System

This protocol describes the surgical and technical procedures that enable real-time in vivo multiphoton fluorescence imaging of the rodent brain during focused ultrasound and microbubble treatments to increase blood-brain barrier permeability.

Real-Time Intravital Multiphoton Microscopy to Visualize Focused Ultrasound and Microbubble Treatments to Increase Blood-Brain Barrier Permeability

The presented protocols can be used to characterize the response of fluorescently-labeled microbubbles designed for ultrasound-triggered drug delivery applications, including their activation mechanisms as well as their bioeffects. This paper covers a range of in vitro and in vivo microscopy techniques performed to capture the relevant length and timescales.

Multi-timescale Microscopy Methods for the Characterization of Fluorescently-labeled Microbubbles for Ultrasound-Triggered Drug Release

This protocol outlines the fabrication of lipid microbubbles and a compatible one-pot microbubble radiolabeling method with purification-free &gt;95% labeling efficiency that conserves microbubble physicochemical properties. This method is effective across diverse lipid microbubble formulations and can be tailored to generate radioactive and/or fluorescent microbubbles.

Fabricating and Labeling Microbubbles with Fluorescent and Radioactive Tracers

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

Learn ultrasound targeted microbubble destruction for targeted gene delivery to the heart and liver using acoustic cavitation. This technique enhances transfection efficiency and enables precise therapeutic gene expression monitoring.

Introduction to the Ultrasound Targeted Microbubble Destruction Technique

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