Name: production of membrane-filtered phase-shift decafluorobutane nanodroplets from preformed microbubbles
Uploaded: 2021-03-23T14:00:00
Description: Watch this Scientific Journal Video about Production of Membrane-Filtered Phase-Shift Decafluorobutane Nanodroplets from Preformed Microbubbles at JoVE.com

We would like to thank Dominique James in Dr. Ken Hoyt&#39;s lab for providing TRSP analysis of vaporizable phase-shift nanodroplets

1. Making lipid films
<ol>
	<li>Prepare lipid films for microbubble generation using 90% DSPC and 10% DSPE-PEG2K by mixing the lipids at the correct ratio using the following directions:
	<ol>
		<li>Make stock lipids of DSPC and DSPE-PEG2K in chloroform. Weigh 50 mg of each lipid powder in separate vials. Add 1 mL of chloroform to each vial using a 1 mL glass syringe.</li>
		<li>Add 287 &#181;L of DSPC stock and 113 &#181;L of DSPE-PEG2K stock (both 50 mg/mL) into a 20 mL scintillation vial using a glass syringe.</li>
...

<ol>
	<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 Science Engineering and Technology. 1 (1-2), 3-17 (2009).</li><li>Sheeran, P. S., Dayton, 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=Phase-change+contrast+agents+for+imaging+and+therapy.">Phase-change contrast agents for imaging and therapy.</a> Current Pharmaceutical Design. 18 (15), 2152-2165 (2012).</li><li>Mountford, P. A., Smith, W. S., Borden, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorocarbon+nanodrops+as+acoustic+temperature+probes.">Fluorocarbon nanodrops as acoustic temperature probes.</a> Langmuir: The ACS Journal of Surfaces and Colloids. 31 (39), 10656-10663 (2015).</li><li>Mountford, P. A., Thomas, A. N., Borden, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+activation+of+superheated+lipid-coated+perfluorocarbon+drops.">Thermal activation of superheated lipid-coated perfluorocarbon drops.</a> Langmuir: The ACS Journal of Surfaces and Colloids. 31 (16), 4627-4634 (2015).</li><li>Sheeran, P. S., Luois, S., Dayton, P. A., Matsunaga, T. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formulation+and+acoustic+studies+of+a+new+phase-shift+agent+for+diagnostic+and+therapeutic+ultrasound.">Formulation and acoustic studies of a new phase-shift agent for diagnostic and therapeutic ultrasound.</a> Langmuir: The ACS Journal of Surfaces and Colloids. 27 (17), 10412-10420 (2011).</li><li>Sheeran, P. S., Dayton, 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=Improving+the+performance+of+phase-change+perfluorocarbon+droplets+for+medical+ultrasonography:+current+progress,+challenges,+and+prospects.">Improving the performance of phase-change perfluorocarbon droplets for medical ultrasonography: current progress, challenges, and prospects.</a> Scientifica. 2014, 579684 (2014).</li><li>Sheeran, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+generating+submicrometer+phase-shift+perfluorocarbon+droplets+for+applications+in+medical+ultrasonography.">Methods of generating submicrometer phase-shift perfluorocarbon droplets for applications in medical ultrasonography.</a> IEEE Transactions on Ultrasonics, Ferroelectrics, and Frequency Control. 64 (1), 252-263 (2017).</li><li>Sheeran, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decafluorobutane+as+a+phase-change+contrast+agent+for+low-energy+extravascular+ultrasonic+imaging.">Decafluorobutane as a phase-change contrast agent for low-energy extravascular ultrasonic imaging.</a> Ultrasound in Medicine &amp; Biology. 37 (9), 1518-1530 (2011).</li><li>de Gracia Lux, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+method+for+the+formation+of+monodisperse+superheated+perfluorocarbon+nanodroplets+as+activatable+ultrasound+contrast+agents.">Novel method for the formation of monodisperse superheated perfluorocarbon nanodroplets as activatable ultrasound contrast agents.</a> RSC Advances. 7 (77), 48561-48568 (2017).</li><li>Mountford, P. A., Sirsi, S. R., Borden, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Condensation+phase+diagrams+for+lipid-coated+perfluorobutane+microbubbles.">Condensation phase diagrams for lipid-coated perfluorobutane microbubbles.</a> Langmuir: The ACS Journal of Surfaces and Colloids. 30 (21), 6209-6218 (2014).</li><li>Feshitan, J. A., Chen, C. C., Kwan, J. J., Borden, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbubble+size+isolation+by+differential+centrifugation.">Microbubble size isolation by differential centrifugation.</a> Journal of Colloid and Interface Science. 329 (2), 316-324 (2009).</li><li>Wu, S. -. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focused+ultrasound-facilitated+brain+drug+delivery+using+optimized+nanodroplets:+vaporization+efficiency+dictates+large+molecular+delivery.">Focused ultrasound-facilitated brain drug delivery using optimized nanodroplets: vaporization efficiency dictates large molecular delivery.</a> Physics in Medicine and Biology. 63 (3), 035002 (2018).</li><li>Li, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+Nucleation+of+stable+perfluorocarbon+emulsions+for+ultrasound+contrast+agents.">Spontaneous Nucleation of stable perfluorocarbon emulsions for ultrasound contrast agents.</a> Nano Letters. 19 (1), 173-181 (2019).</li><li>Sheeran, P. S., Luois, S. H., Mullin, L. B., Matsunaga, T. O., Dayton, 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=Design+of+ultrasonically-activatable+nanoparticles+using+low+boiling+point+perfluorocarbons.">Design of ultrasonically-activatable nanoparticles using low boiling point perfluorocarbons.</a> Biomaterials. 33 (11), 3262-3269 (2012).</li><li>Kawabata, K., Sugita, N., Yoshikawa, H., Azuma, T., Umemura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticles+with+multiple+perfluorocarbons+for+controllable+ultrasonically+induced+phase+shifting.">Nanoparticles with multiple perfluorocarbons for controllable ultrasonically induced phase shifting.</a> Japanese Journal of Applied Physics. 44 (6), 4548-4552 (2005).</li><li>Shakya, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vaporizable+endoskeletal+droplets+via+tunable+interfacial+melting+transitions.">Vaporizable endoskeletal droplets via tunable interfacial melting transitions.</a> Science Advances. 6 (14), 7188 (2020).</li><li>Kopechek, J. A., Zhang, P., Burgess, M. T., Porter, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+phase-shift+nanoemulsions+with+narrow+size+distributions+for+acoustic+droplet+vaporization+and+bubble-enhanced+ultrasound-mediated+ablation.">Synthesis of phase-shift nanoemulsions with narrow size distributions for acoustic droplet vaporization and bubble-enhanced ultrasound-mediated ablation.</a> Journal of Visualized Experiments: JoVE. (67), e4308 (2012).</li></ol>

A comprehensive body of literature is available that discusses the formulation, physics, and potential applications of microbubbles and phase-shift droplets for in vivo imaging and therapy. This discussion pertains explicitly to generating lipid microbubbles and converting them into sub-micron phase-shift droplets using a low boiling point DFB gas and high-pressure extrusion. The method outlined here is meant to provide a relatively simple method of producing large amounts of lipid microbubbles and DFB phase-shift drople...

Ultrasound contrast agents (UCAs) are rapidly growing in popularity for imaging and therapy applications. Microbubbles, the original UCAs, are currently the mainstream agents used in clinical diagnostic applications. Microbubbles are gas-filled spheres, typically 1-10 &#181;m in diameter, surrounded by lipid, protein, or polymer shells1. However, their size and in vivo stability can limit their functionality in many applications. Phase-shift nanodroplets, which contain a superheated liquid core, can overcome some of these limitations due to their smaller size and improved circulation-life2. When exposed to heat or acoustic energy, the superheated liquid core vaporizes to form a gas microbubble2,3,4,5. Since the vaporization threshold is directly related to droplet size5,6, formulating droplet suspensions with uniform size would be highly desirable for achieving consistent activation thresholds. Formulation methods that produce uniform droplet sizes are often complex and costly, whereas more cost-effective approaches result in polydisperse solutions7. Another limitation is the ability to generate stable phase-shift droplets with low-boiling point perfluorocarbon (PFC) gases, which is critical for efficient activation in vivo8. In this manuscript, a protocol is described for generating stable filtered low-boiling point vaporizable phase-shift droplets for in vivo imaging and therapy applications.
There are many methods of producing monodispersed submicron phase-shift droplets7. One of the most robust methods of controlling size is the use of microfluidic devices. These devices can be costly, have slow rates of droplet production (~104-106 droplets/s)7, and require extensive training. Microfluidic devices also generally require high-boiling point gases to avoid spontaneous vaporization and clogging of the system7. However, a recent study by de Gracia Lux et al.9 demonstrates how cooling a microfluidizer can be used to generate high concentrations of sub-micron phase-shift (1010-1012/mL) using low-boiling point decafluorobutane (DFB) or octafluoropropane (OFP).
In general, low-boiling point gases such as DFB or OFP are easier to handle using preformed gas bubbles. Vaporizable droplets can be produced from precursor lipid-stabilized bubbles by condensing the gas using low temperatures and elevated pressure5,10. The concentration of droplets produced using this method depends on precursor microbubble concentration and efficiency of conversion of bubbles to droplets. Concentrated microbubbles have been reported from tip sonication approaching &#62; 1010 MB/mL11, while a separate study has reported droplet concentrations ranging from ~1-3 x1011 droplets/mL from condensed OFP and DFP bubbles12. When monodispersed droplets are not a concern, condensation methods are the most straightforward and lowest-cost methods of generating lipid-stabilized phase-shift droplets using low-boiling point PFCs. Methods of generating uniform size bubbles before condensing can help create more monodisperse populations of droplets. However, generating monodisperse precursor bubbles is also difficult, requiring more costly approaches such as microfluidics or repeated differential centrifugation techniques11. An alternative approach to producing DFB and OFB nanodroplets has recently been published using spontaneous nucleation of droplets in liposomes13. This method, utilizing an &#34;Ouzo&#34; effect, is a simple way to generate low-boiling point PFC droplets without needing to condense bubbles. The size-distribution of the PFC droplets can be controlled by delicate titration and mixing PFC, lipid, and ethanol components used to initiate nucleation of the droplets. It is also worth noting that mixing of perfluorocarbons can be used to control stability and activation thresholds of nanodroplets14,15. More recent work by Shakya et al. demonstrates how nanodroplet activation can be tuned by emulsifying high boiling-point PFCs within a hydrocarbon endoskeleton to facilitate heterogenous nucleation within the droplet core16, which is an approach that can be considered along with other forms of droplet size filtration.
Once formed, phase-shift droplets can be extruded after formation to create more monodisperse populations. In fact, a similar protocol to the method described here has been published previously by Kopechek et al.17 using high boiling-point dodecofluorpentane (DDFP) as the droplet core. Readers seeking to use phase-shift droplets with high-boiling point perfluorocarbons (stable at room temperature) should reference the article above instead. Generating and extruding droplets with low boiling point gasses, such as DFB and OFP, is more complicated and is best approached by condensing preformed gas bubbles.
In this protocol, a common method of generating preformed lipid microbubbles with a DFB gas core using probe tip sonication is described. Next, a commercial extruder is used to condense preformed microbubbles into submicron phase-shift nanodroplets (Figure 1). The resulting droplets are then activatable by heat and ultrasound. This method can produce larger volumes of nanodroplet solution than conventional condensation methods with narrower size-distributions without the need for expensive microfluidic devices. The production of nanodroplet solutions with narrow size distributions can likely generate more uniform vaporization thresholds. This will maximize their potential for numerous applications such as imaging, ablation, drug delivery, and embolization1,3,4,6.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62203/62203fig01.jpg" /> 
Figure 1: Schematic of high-pressure extrusion setup for condensing preformed microbubbles into phase-shift nanodroplets. Microbubble solution is added to and contained in the extruder chamber, and 250 psi, from the nitrogen tank, is applied through the chamber inlet valve. The nitrogen gas will push the microbubble solution through the filter at the base of the chamber, condensing the sample to nanodroplets. Solution is finally pushed out of extruder through the sample outlet tube and collected. <a href="https://www.jove.com/files/ftp_upload/62203/62203fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 mL Centrifuge Tubes</td>
			<td>Falcon</td>
			<td>352095</td>
			<td>Collecting and centrifuging droplets</td>
		</tr>
		<tr>
			<td>200 nm polycarbonate filter</td>
			<td>Whatman</td>
			<td>110606</td>
			<td>Extruder filters</td>
		</tr>
		<tr>
			<td>2-methylbutane</td>
			<td>Fisher Chemical</td>
			<td>03551-4</td>
			<td>Rapid precooling of microbubble solution prior to extrusion</td>
		</tr>
		<tr>
			<td>3-prong clamps X2</td>
			<td>Fisher</td>
			<td>02-217-002</td>
			<td>Holding scintilation vials in place for probe tip sonication</td>
		</tr>
		<tr>
			<td>400W Analog Probe Tip Sonicator with Horn</td>
			<td>Branson</td>
			<td>101-063-198R</td>
			<td>Used to generate lipid microbubbles from lipid solution</td>
		</tr>
		<tr>
			<td>Bath Sonicator</td>
			<td>Fisher Scientific</td>
			<td>15337402</td>
			<td>Used to help breakdown liposomes into unilamellar vesicles</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Bioreagents</td>
			<td>C298-4</td>
			<td>Used to make lipid film for microbubble preperation</td>
		</tr>
		<tr>
			<td>Decafluorobutane (Perfluorobutane) Gas</td>
			<td>FluoroMed L.P.</td>
			<td>1 kg</td>
			<td>generating microbubbles via probe tip sonication</td>
		</tr>
		<tr>
			<td>Dry Ice</td>
			<td>-</td>
			<td>-</td>
			<td>Rapid precooling of microbubble solution prior to extrusion</td>
		</tr>
		<tr>
			<td>DSPC Lipid Powder</td>
			<td>NOF America</td>
			<td>COATSOME MC-8080</td>
			<td>Component of lipid film</td>
		</tr>
		<tr>
			<td>DSPE-PEG-2K Lipid Powder</td>
			<td>NOF America</td>
			<td>SUNBRIGHT DSPE-020CN</td>
			<td>Component of lipid film</td>
		</tr>
		<tr>
			<td>General Thermometer</td>
			<td>-</td>
			<td>-</td>
			<td>Used to measure ice bath temperature and 2-methylbutane temperature ( needs to accommodate -20C temperatures)</td>
		</tr>
		<tr>
			<td>Glass Syringes</td>
			<td>Hamilton</td>
			<td>81139</td>
			<td>Used to mix lipids in chloroform</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Bioreagents</td>
			<td>BP229-1</td>
			<td>Reduces freezing temperature of PBS solution</td>
		</tr>
		<tr>
			<td>Heating Block</td>
			<td>VWR Scientific Products</td>
			<td></td>
			<td>Heating lipid films and vaporizing droplets</td>
		</tr>
		<tr>
			<td>Lipex 10 mL Extruder</td>
			<td>Evonik</td>
			<td></td>
			<td>Commercial high-pressure extrusion system</td>
		</tr>
		<tr>
			<td>Mini Vortex Mixer</td>
			<td>Fisher brand</td>
			<td>14-955-151</td>
			<td>Used to remove excess chloroform from lipid films</td>
		</tr>
		<tr>
			<td>Nitrogen Tank</td>
			<td>-</td>
			<td>-</td>
			<td>Used to operate extruder</td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td>Hydrate lipid films and washing droplets</td>
		</tr>
		<tr>
			<td>Polyester Drain Disk</td>
			<td>Whatman</td>
			<td>230600</td>
			<td>Provides support for polycarbonate filter</td>
		</tr>
		<tr>
			<td>Polypropylene Caps</td>
			<td>Fisher Scientific</td>
			<td>298417</td>
			<td>Used for solution storage</td>
		</tr>
		<tr>
			<td>Propylene Glycol</td>
			<td>Fisher Chemical</td>
			<td>P355-1</td>
			<td>Reduces freezing temperature of PBS solution</td>
		</tr>
		<tr>
			<td>Scintiliation Vials</td>
			<td>DWK Life Sciences Wheaton</td>
			<td>986532</td>
			<td>Used for lipid films and microbubble generation</td>
		</tr>
		<tr>
			<td>Small hammer</td>
			<td>-</td>
			<td>-</td>
			<td>Used to break apart dry ice for cooling methylbutane</td>
		</tr>
		<tr>
			<td>Sonicator Microtip Attachment</td>
			<td>Branson</td>
			<td>101148070</td>
			<td>Used to generate microbubbles from lipid solution</td>
		</tr>
		<tr>
			<td>Steel Container</td>
			<td>Medegen</td>
			<td>79310</td>
			<td>Rapid precooling of microbubble solution prior to extrusion ( any container rated to -20C will work)</td>
		</tr>
		<tr>
			<td>Vacuume Dessicator</td>
			<td>Bel-Art SP Scienceware</td>
			<td>08-648-100</td>
			<td>Removes excess chloroform from lipid films</td>
		</tr>
		<tr>
			<td>2mL Centrifuge Tube</td>
			<td>Fisher</td>
			<td>02682004</td>
			<td>Used for concentrating nanodroplets </td>
		</tr>
	</tbody>
</table>

production of membrane-filtered phase-shift decafluorobutane nanodroplets from preformed microbubbles

There are many methods that can be used for the production of vaporizable phase-shift droplets for imaging and therapy. Each method utilizes different techniques and varies in price, materials, and purpose. Many of these fabrication methods result in polydisperse populations with non-uniform activation thresholds. Additionally, controlling the droplet sizes typically requires stable perfluorocarbon liquids with high activation thresholds that are not practical in vivo. Producing uniform droplet sizes using low-boiling point gases would be beneficial for in vivo imaging and therapy experiments. This article describes a simple and economical method for the formation of size-filtered lipid-stabilized phase-shift nanodroplets with low-boiling point decafluorobutane (DFB). A common method of generating lipid microbubbles is described, in addition to a novel method of condensing them with high-pressure extrusion in a single step. This method is designed to save time, maximize efficiency, and generate larger volumes of microbubble and nanodroplet solutions for a wide variety of applications using common laboratory equipment found in many biological laboratories.

Representative results of the size distribution are included using dynamic light scattering (DLS) and tunable resistive pulse sensing (TRSP) analysis. Figure 5 shows the size distribution of condensed bubble solutions with and without extrusion. Without extrusion, the protocol ends at step 5.3. The chilled bubbles are condensed by venting the sample to atmospheric pressure while cold. The condensed only sample has a much wider distribution centered near 400 nm. The extruded sample has a narr...

Watch this Scientific Journal Video about Production of Membrane-Filtered Phase-Shift Decafluorobutane Nanodroplets from Preformed Microbubbles at JoVE.com

Production of Membrane-Filtered Phase-Shift Decafluorobutane Nanodroplets from Preformed Microbubbles

This protocol describes a method of generating large volumes of lipid encapsulated decafluorobutane microbubbles using probe-tip sonication and subsequently condensing them into phase-shift nanodroplets using high-pressure extrusion and mechanical filtration.

There are many methods that can be used for the production of vaporizable phase-shift droplets for imaging and therapy. Each method utilizes different ...

This protocol is an easy method of reducing polydispersity of low boiling point vaporizable droplets for use in biomedical applications. This technique condenses preformed gas microbubbles and filters the resulting liquid droplets by size in a way that is controllable, scalable, and relatively cost-effective. This method can be applied to a variety of lipid microbubbles with different shell and gas material using different filters to control final droplet size.Demonstrating the procedure will be Darrah Merillat, an undergraduate researcher from Dr.Sirsi's laboratory. Turn on the power switch for the sonicator, and set the amplitude to the maximum allowed using the microtip attachment, and ensure sonication time set to 10 seconds. Place the warm hydrated lipid solution in the sound enclosure with the microtip just beneath the surface.Attach an appropriate length of tubing to the neck of the vial to guide the gas from the DFB tank outlet into the warm lipid solution held in the the enclosure. Open the tank valve slowly until the gas can be seen flowing over the lipid solution, causing slight ripples on the surface of the liquid. If the gas flow is too high, the solution will overflow during microbubble formation.Start the sonicator, and run it for 10 seconds continuously to generate microbubbles. After sonication is over, immediately close the DFB tank valve. Quickly cap the microbubble solution, and submerge the vial in an ice bath to cool the sample below 55 degrees Celsius.Use a 200-nanometer ceramic filter to assemble the high-pressure extruder according to the user's manual, and place it in the center of a watertight container, so that the sample outlet tube is not pressed against the side, or crimped. Couple the extruder to the nitrogen gas tank using the adapter supplied by the manufacturer. Place the end of the outlet tube in a scintillation vial to collect the extruded sample, securing the tube to the container with tape to stay within the vial.Open and close the release valve to ensure that there's no pressure within the extruder. Remove the chamber lid. And add five milliliters of PBS to the extruder chamber.Replace the lid, making sure that it clicks securely back into place. Open the nitrogen gas tank, so that the pressure gauge reads 250 PSI, making sure the pressure control valve is in the closed position. Close the gas tank, and open the extruder chamber inlet valve, causing the PBS solution to be pushed through the system, and out the sample outlet tube into the scintillation vial.When only gas is exiting the tubing, open the release valve, and allow the pressure to fall to zero PSI. Then remove the scintillation vial. Open and close the release valve to make sure there is no pressure within the extruder, and place a new scintillation vial at the end of the outlet tube.Fill a steel container with 2-methylbutane, and add dry ice to bring the temperature down to minus 18 degrees Celsius. Insert the microbubble solution into the chilled 2-methylbutane, submerging the sample for two minutes. Move the scintillation vial throughout the two minutes to gently mix the bubbles.Add dry ice as needed to maintain the temperature between minus 15 and minus 18 degrees Celsius. After two minutes, remove the microbubbles from the chilled 2-methylbutane. Gently swirl the vial to mix the microbubbles, and transfer bubbles into a chilled 10-milliliter syringe.Remove the extruder chamber lid, and add the microbubble solution to the chamber by slowly pushing the plunger on the syringe. Replace the extruder cap, making sure it clicks securely in place. Verify that the pressure control valve and the release valve of the extruder are in the closed position.Open the nitrogen gas tank until the pressure gauge reads 250 PSI. Close the gas tank, and turn the pressure control valve to the open position. When the solution has filled the scintillation vial at the exit tubing and only gas is exiting the tube, slowly open the pressure release valve, and allow the pressure to fall to zero PSI.Transfer 10 milliliters of the extruded droplet solution to a 15-milliliter centrifuge tube. Centrifuge the extruded sample at 1, 500 times G for 10 minutes at four degrees Celsius. Discard the supernatant and the spontaneously vaporized droplets appearing at the top of the solution.Re-suspend the pellet comprising DFP nanodroplets in 10 milliliters of PBS with 20%glycerol and 20%propylene glycol. The size distribution of condensed bubble solutions with and without extrusion shows that the condensed only sample has a much wider distribution centered near 400 nanometers, whereas the extruded sample has a narrower distribution centered at 200 nanometers. Tunable resistance pulse sensing analysis used to analyze phase shift droplets as they have been washed by centrifugation to remove excess liposomes shows that the droplet sizes are near 200 nanometers.The microscopy data of nanodroplet vaporization when heated shows that some spontaneously vaporized microbubbles are apparent in the field of view before heating, and a greater number of gas microbubbles are observed after heating. The microscopy images of nanodroplets inserted into the extruder directly without pre-cooling, condensed at zero degree Celsius, and at minus 18 degrees Celsius are shown here. Representative images of condensed octafluoropropane droplets before and after vaporization also show a greater number of gas microbubbles after heating, similar to the DFB droplets.The most important thing to remember during this procedure is that the droplet yield is highly dependent on the temperature and pressure during condensation, and slight variations will impact results. After generating vaporizable droplets, they can be used to optimize in vivo imaging and drug delivery using ultrasound, as well as other in vivo and ex vivo applications. After developing this technique, the effects of nanodroplet size and gas core content on in vivo vaporization thresholds have been explored using slight modifications to this protocol.

Research

JoVE Journal

Bioengineering

Production and Targeting of Monovalent Quantum Dots

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in ...

Microfluidic Production of Lysolipid-Containing Temperature-Sensitive Liposomes

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading ...

Magnetic-, Acoustic-, and Optical-Triple-Responsive Microbubbles for Magnetic Hyperthermia and Pothotothermal Combination Cancer Therapy

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has ...

Imaging and Quantification of the Area of Fast-Moving Microbubbles Using a High-Speed Camera and Image Analysis

An experimental and image analysis technique is presented for imaging cavitation bubbles and calculating their area. The high-speed imaging experimental ...

An Efficient Method for Adenovirus Production

Adenoviral transduction has the advantage of a strong and transient induction of the expression of the gene of interest into a broad variety of cell types ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

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

Microbubble contrast agents hold great promise for drug delivery applications with ultrasound. Encapsulating drugs in nanoparticles reduces systemic ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

Formulation and Acoustic Modulation of Optically Vaporized Perfluorocarbon Nanodroplets

Microbubbles are the most commonly used imaging contrast agent in ultrasound. However, due to their size, they are limited to vascular compartments. These ...