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>
		<li>Dry the mixed lipids to remove chloroform using nitrogen. Using an appropriate length of tubing connected to house nitrogen, lightly flow nitrogen gas over the headspace of the vial while mixing. Continue until no chloroform is observed, and the remaining lipid film starts to turn white. Use polypropylene screw caps, cover the sample while introducing nitrogen in the headspace.</li>
		<li>Place vials under vacuum overnight using a vacuum desiccator to remove any residual chloroform. A thin translucent film will remain that coats the bottom of the vial.</li>
		<li>Store vials at -20 &#176;C until needed.</li>
	</ol>
	</li>
</ol>
2. Generating microbubbles from lipid films
<ol>
	<li>To make the microbubbles, add 10 mL of 1x phosphate buffer saline (PBS) containing 20% v/v Propylene Glycol and 20% v/v Glycerol (final pH 7.2-7.4) to a dry lipid film.</li>
	<li>Re-cap the sample and warm sample to 65 &#176;C for 30 min on a heating block (or heated water bath).</li>
	<li>While the sample is warming, prepare the bath sonicator by increasing the bath temperature to 65 &#176;C. 
	NOTE: This process is faster if the water is preheated in a microwave or hotplate before placing in the bath sonicator.</li>
	<li>Place the scintillation vial containing the warmed sample in the bath sonicator so that only the portion of the vial containing the lipid solution is submerged in the water bath.</li>
	<li>Sonicate the warm lipid solution for a minimum of 15 min. Ensure that the water temperature remains at 65 &#176;C. Continue to sonicate in intervals of 10-15 min until the solution is completely clear (Figure 2). 
	&#8203;NOTE: If a bath sonicator is not available, the solution can be tip sonicated at 10% power until clear. However, the microtip will wear out quicker and is more expensive to replace.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62203/62203fig02.jpg" /> 
Figure 2: Example of hydrated lipid films. Example of hydrated lipid film (A) before and (B) after bath sonication to form uni-lamellar vesicles. Following bath sonication, the lipid solution should shift from a more opaque to translucent solution. <a href="https://www.jove.com/files/ftp_upload/62203/62203fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="6">
	<li>While still warm, remove the cap and clamp the vial into the sonicator&#39;s soundproof enclosure so that the microtip attachment of the sonicator is submerged just beneath the air/liquid interface (Figure 3).</li>
	<li>Place the tank of decafluorobutane next to the soundproof enclosure of the sonicator.</li>
	<li>Prepare an ice-bath and place it next to the soundproof enclosure. This will be used later in Step 2.14.</li>
	<li>Turn on the power switch for the sonicator.</li>
	<li>After the system starts, set the power level to 70%. Do not exceed 70% amplitude with the microtip attachment. Do not start the sonicator at this time.</li>
	<li>Attach an appropriate length of tubing to guide the gas from the DFB tank outlet into the warm lipid solution held in the enclosure. The tube should be placed just into the neck of the vial to allow gas to flow into the headspace during sonication (Figure 3).</li>
	<li>Open the tank valve slowly until the gas can be seen flowing over the lipid solution. This will cause slight ripples on the surface of the liquid. If the gas flow is too high, the solution will overflow during microbubble formulation.</li>
	<li>Start the sonicator and run for 10 s continuously to generate microbubbles. If the bubble solution starts to overflow during sonication, immediately stop the sonicator.</li>
	<li>Turn off the sonicator and immediately close the DFB tank valve.</li>
	<li>Quickly cap the microbubble solution and submerge the vial in the ice bath to cool the sample below 55 &#176;C (glass transition temperature of DSPC)</li>
	<li>Leave the microbubble samples in the ice bath until needed.</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62203/62203fig03.jpg" /> 
Figure 3: Placement of probe tip into lipid solution to optimize microbubble formation. Take care to not allow the tip of the probe to touch the glass. <a href="https://www.jove.com/files/ftp_upload/62203/62203fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Preparing extruder for microbubble condensation
<ol>
	<li>Assemble high-pressure extruder as detailed in the user&#39;s manual using a 200 nm ceramic filter (supplied from manufacturer).</li>
	<li>Place the extruder in the center of a watertight container so that the sample outlet tube is not pressed against the side or crimped.</li>
	<li>Couple the extruder to the nitrogen gas tank using the adaptor supplied by the manufacturer.</li>
	<li>Make a -2 &#176;C salted ice bath in the watertight container around the extruder using 400 mL of water and 10 g of sodium chloride.</li>
	<li>Place the end of the outlet tube in a scintillation vial to collect the extruded sample. 
	&#8203;NOTE: Secure the tube to the container with tape if it does not lay flat or stay within the vial.</li>
</ol>
4. Priming the extruder for microbubble condensation
<ol>
	<li>Open and close the release valve to make sure there is no pressure within the extruder.</li>
	<li>Remove the chamber lid and add 5 mL of 1x PBS to the extruder chamber.</li>
	<li>Replace the lid making sure that it clicks securely back into place.</li>
	<li>Open the nitrogen gas tank so that the pressure gauge reads 250 psi. Make sure the pressure control valve is in the closed position.</li>
	<li>Close the gas tank and open the extruder chamber inlet valve. The PBS solution will be pushed through the system and out the sample outlet tube into the scintillation vial.</li>
	<li>When only gas is exiting the tubing, open the release valve and allow the pressure to fall to 0 psi.</li>
	<li>Remove the scintillation vial.</li>
</ol>
5. Pre-cooling microbubbles for extrusion
<ol>
	<li>Open and close the release valve to make sure there is no pressure within the extruder. Place a new scintillation vial at the end of the outlet tube.</li>
	<li>Fill a steel container with 2-methyl butane and add dry ice to bring the temperature down to -18 &#176;C.</li>
	<li>Insert the microbubble solution into the chilled 2-methyl butane so the sample is submerged for 2 min. Move the scintillation vial throughout the 2 min to gently mix the bubbles. Add dry ice as needed to maintain the temperature between -15 and -18 &#176;C. Be careful not to exceed -20 &#730;C or the excipient solution will freeze and destroy the bubble sample. 
	NOTE: Steps 5.2 and 5.3 can also be done by cooling the bubble sample in a laboratory freezer over a more extended time period. However, caution should be used to carefully monitor the temperature of the freezer and avoid freezing the sample.</li>
	<li>After 2 min, remove the microbubbles from the chilled 2-methyl butane, gently shake the vial to mix the microbubbles and use a chilled 10 mL syringe to transfer the solution to the extruder.</li>
	<li>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 back in place.</li>
	<li>Verify that the pressure control valve and the release valve of the extruder are in the closed position.</li>
	<li>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.</li>
	<li>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 0 psi.</li>
	<li>Place the scintillation vial in an ice bath or fridge for storage.</li>
	<li>For long-term storage and minimizing spontaneous vaporization, store the sample in a standard freezer. Ensure the temperature is -20 &#176;C or higher to avoid freezing the sample (the excipient solution of 20% PPG and 20% Glycerol will keep the sample from freezing in most laboratory freezers).</li>
</ol>
6. Separating droplets from liposomes by centrifugation
<ol>
	<li>Transfer 10 mL of the extruded droplet solution to a 15 mL centrifuge tube.</li>
	<li>Centrifuge the extruded sample at 1,500 x g for 10 min at 4 &#176;C. A pellet comprised of DFB nanodroplets will be apparent at the bottom of the tube (Figure 4). Spontaneously vaporized droplets will appear at the top of the solution and should be discarded.</li>
</ol>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62203/62203fig04.jpg" /> 
Figure 4: Example of phase-shift DFB droplets pelleting after centrifugation. DFB nanodroplets are more dense than liposomes and will collect at the bottom of the centrifuge tube in a pellet, (red box). <a href="https://www.jove.com/files/ftp_upload/62203/62203fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Remove the supernatant&#160;and resuspend the&#160; pellet in 2&#160;mL of 1x PBS with 20% glycerol and 20% propylene glycol.</li>
	<li>Mix the tube gently to obtain a homogeneous solution and transfer the droplets to a smaller 2 mL centrifuge tube.</li>
	<li>Wash sample two more times in 2 mL centrifuge tube.</li>
	<li>After the last wash, resuspend the pellet in 100 &#181;L of 1x PBS with 20% glycerol and 20% propylene glycol and store on ice or in the freezer until needed.</li>
</ol>
7. Microscopy verification of droplet vaporization
<ol start="1">
	<li>Make a diluted droplet solution by adding 2.5 &#181;l of concentrated droplets to 7.5 &#181;l of 1x PBS.</li>
	<li>Prepare a microscope slide with 10 &#181;l of the diluted sample. Using a 40x objective, observe the sample and save images.</li>
	<li>Remove the slide from the microscope and place it on a 65 &#730;C heat plate for 1 min to vaporize nanodroplets into microbubbles.</li>
	<li>Use the same 40x objective to observe the sample after heating to verify droplet&#160;vaporization.</li>
</ol>

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The authors have nothing to disclose.

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><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 ...

production-membrane-filtered-phase-shift-decafluorobutane

Research

JoVE Journal

Bioengineering

2.7K Views.  The University of Texas at Dallas. Dit protocol is een eenvoudige methode om polydispersiteit van verdampbare druppels met een laag kookpunt te verminderen voor gebruik in biomedische toepassingen. Deze techniek condenseert voorgevormde gasmicrobubbels en filtert de resulterende vloeistofdruppels op grootte op een manier die controleerbaar, schaalbaar en relatief kosteneffectief is. Deze methode kan worden toegepast op een verscheidenheid aan lipide microbubbels met verschillende schaal- en gasmateriaal met behulp van verschil...

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

Synthesis of Phase-shift Nanoemulsions with Narrow Size Distributions for Acoustic Droplet Vaporization and Bubble-enhanced Ultrasound-mediated Ablation

High-intensity focused ultrasound (HIFU) is used clinically to thermally ablate tumors. To enhance localized heating and improve thermal ablation in tumors, lipid-coated perfluorocarbon droplets have been developed which can be vaporized by HIFU. The vasculature in many tumors is abnormally leaky due to their rapid growth, and nanoparticles are able to penetrate the fenestrations and passively accumulate within tumors. Thus, controlling the size of the droplets can result in better accumulation within tumors. In this report, the preparation of stable droplets in a phase-shift nanoemulsion (PSNE) with a narrow size distribution is described. PSNE were synthesized by sonicating a lipid solution in the presence of liquid perfluorocarbon. A narrow size distribution was obtained by extruding the PSNE multiple times using filters with pore sizes of 100 or 200 nm. The size distribution was measured over a 7-day period using dynamic light scattering. Polyacrylamide hydrogels containing PSNE were prepared for in vitro experiments. PSNE droplets in the hydrogels were vaporized with ultrasound and the resulting bubbles enhanced localized heating. Vaporized PSNE enables more rapid heating and also reduces the ultrasound intensity needed for thermal ablation. Thus, PSNE is expected to enhance thermal ablation in tumors, potentially improving therapeutic outcomes of HIFU-mediated thermal ablation treatments.

Phase-shift nanoemulsions (PSNE) can be vaporized using high intensity focused ultrasound to enhance localized heating and improve thermal ablation in tumors. In this report, the preparation of stable PSNE with a narrow size distribution is described. Furthermore, the impact of vaporized PSNE on ultrasound-mediated ablation is demonstrated in tissue-mimicking phantoms.

High-intensity  focused ultrasound (HIFU) is used clinically to thermally ablate tumors. To  enhance localized heating and improve thermal ablation in ...

Engineering

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Manufacture of Concentrated, Lipid-based Oxygen Microbubble Emulsions by High Shear Homogenization and Serial Concentration

Gas-filled microbubbles have been developed as ultrasound contrast and drug delivery agents. Microbubbles can be produced by processing surfactants using sonication, mechanical agitation, microfluidic devices, or homogenization. Recently, lipid-based oxygen microbubbles (LOMs) have been designed to deliver oxygen intravenously during medical emergencies, reversing life-threatening hypoxemia, and preventing subsequent organ injury, cardiac arrest, and death. We present methods for scaled-up production of highly oxygenated microbubbles using a closed-loop high-shear homogenizer. The process can produce 2 L of concentrated LOMs (90% by volume) in 90 min. Resulting bubbles have a mean diameter of ~2 &#956;m, and a rheologic profile consistent with that of blood when diluted to 60 volume %. This technique produces LOMs in high capacity and with high oxygen purity, suggesting that this technique may be useful for translational research labs.

We describe methods for the manufacture of large volumes of lipid-based oxygen microbubbles (LOMs) designed for intravenous oxygen delivery using high-shear homogenization and serial concentration.

Gas-filled microbubbles have been developed as ultrasound contrast and drug delivery agents. Microbubbles can be produced by processing surfactants using ...

A Femtoliter Droplet Array for Massively Parallel Protein Synthesis from Single DNA Molecules

Advances in spatial resolution and detection sensitivity of scientific instrumentation make it possible to apply small reactors for biological and chemical research. To meet the demand for high-performance microreactors, we developed a femtoliter droplet array (FemDA) device and exemplified its application in massively parallel cell-free protein synthesis (CFPS) reactions. Over one million uniform droplets were readily generated within a finger-sized area using a two-step oil-sealing protocol. Every droplet was anchored in a femtoliter microchamber composed of a hydrophilic bottom and a hydrophobic sidewall. The hybrid hydrophilic-in-hydrophobic structure and the dedicated sealing oils and surfactants are crucial for stably retaining the femtoliter aqueous solution in the femtoliter space without evaporation loss. The femtoliter configuration and the simple structure of the FemDA device allowed minimal reagent consumption. The uniform dimension of the droplet reactors made large-scale quantitative and time-course measurements convincing and reliable. The FemDA technology correlated the protein yield of the CFPS reaction with the number of DNA molecules in each droplet. We streamlined the procedures about the microfabrication of the device, the formation of the femtoliter droplets, and the acquisition and analysis of the microscopic image data. The detailed protocol with the optimized low running cost makes the FemDA technology accessible to everyone who has standard cleanroom facilities and a conventional fluorescence microscope in their own place.

The overall goal of the protocol is to prepare over one million ordered, uniform, stable, and biocompatible femtoliter droplets on a 1 cm2 planar substrate that can be used for cell-free protein synthesis.

Advances in spatial resolution and detection sensitivity of scientific instrumentation make it possible to apply small reactors for biological and ...

Chemistry

Phase Change Dimethyldioctadecylammonium-Shelled Microdroplets as a Promising Drug Delivery System: Results on 3D Spheroids of Mammalian Tumor Cells

Significant improvement of phase-change perfluorocarbon microdroplets (MDs) in the vast theranostic scenario passes through the optimization of the MDs composition with respect to synthesis efficiency, stability, and drug delivery capability. To this aim, decafluoropentane (DFP) MDs stabilized by a shell of dimethyldioctadecylammonium bromide (DDAB) cationic surfactant were designed. A high concentration of DDAB-MDs was readily obtained within a few seconds by pulsed high-power insonation, resulting in low polydisperse 1 &#181;m size droplets. Highly positive &#950;-potential, together with a long, saturated hydrocarbon chains of the DDAB shell, are key factors to stabilize the droplet and the drug cargo therein. The high affinity of the DDAB shell with cell plasma membrane allows for localized chemotherapeutics delivery by increasing the drug concentration at the tumor cell interface and boosting the uptake. This would turn DDAB-MDs into a relevant drug delivery tool exhibiting high antitumor activity at very low drug doses.
In this work, the efficacy of such an approach is shown to dramatically improve the effect of doxorubicin against 3D spheroids of mammalian tumor cells, MDA-MB-231. The use of&#160;three-dimensional (3D) cell cultures developed in the form of multicellular tumor spheroids (i.e., densely packed cells in a spherical shape) has numerous advantages compared to 2D cell cultures: in addition to have the potential to bridge the gap between conventional in vitro studies and animal testing, it will improve the ability to perform more predictive in vitro screening assays for preclinical drug development or evaluate the potential of off-label drugs and new co-targeting strategies.

Decafluoropentane microdroplets developed with a shell of dimethyldioctadecylammonium bromide exhibited an exceptional colloidal stability and an actractive biointerface. DDAB-MDs proved to be efficient drug reservoirs characterized by a high affinity to plasma membranes together with enhanced uptake and antitumor activity of Doxorubicin against human triple-negative breast cancer (MDA-MB-231) 3D model.

Significant improvement of phase-change perfluorocarbon microdroplets (MDs) in the vast theranostic scenario passes through the optimization of the MDs ...

Fabricating and Labeling Microbubbles with Fluorescent and Radioactive Tracers

Microbubbles are lipid-shelled, gas-filled particles that have evolved from vascular ultrasound contrast agents into revolutionary cancer therapy platforms. When combined with therapeutic focused ultrasound (FUS), they can safely and locally overcome physiological barriers (e.g., blood-brain barrier), deliver drugs to otherwise inaccessible cancers (e.g., glioblastoma and pancreatic cancer), and treat neurodegenerative diseases. The therapeutic arsenal of microbubble-FUS is advancing in new directions, including synergistic combination radiotherapy, multimodal imaging, and all-in-one drug loading and delivery from microbubble shells.
Labeling microbubbles with radiotracers is key to establishing these expanded theranostic capabilities. However, existing microbubble radiolabeling strategies rely on purification methodologies known to perturb microbubble physicochemical properties, use short-lived radioisotopes, and do not always yield stable chelation. Collectively, this creates ambiguity surrounding the accuracy of microbubble radioimaging and the efficiency of tumor radioisotope delivery.
This protocol describes a new one-pot, purification-free microbubble labeling methodology that preserves microbubble physicochemical properties while achieving &#62;95% radioisotope chelation efficiency. It is versatile and can be applied successfully across custom and commercial microbubble formulations with differing acyl lipid chain length, charge, and chelator/probe (porphyrin, DTPA, DiI) composition. It can be adaptively applied during ground-up microbubble fabrication and to pre-made microbubble formulations with modular customizability of fluorescence and multimodal fluorescence/radioactive properties. Accordingly, this flexible method enables the production of tailored, traceable (radio, fluorescent, or radio/fluorescent active) multimodal microbubbles that are useful for advancing mechanistic, imaging, and therapeutic microbubble-FUS applications.

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.

Microbubbles are lipid-shelled, gas-filled particles that have evolved from vascular ultrasound contrast agents into revolutionary cancer therapy ...

Cancer Research

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 toxicity and increases circulation time of the drugs. In a novel approach to microbubble-assisted drug delivery, nanoparticles are incorporated in or on microbubble shells, enabling local and triggered release of the nanoparticle payload with ultrasound. A thorough understanding of the release mechanisms within the vast ultrasound parameter space is crucial for efficient and controlled release. This set of presented protocols is applicable to microbubbles with a shell containing a fluorescent label. Here, the focus is on microbubbles loaded with poly(2-ethyl-butyl cyanoacrylate) polymeric nanoparticles, doped with a modified Nile Red dye. The particles are fixed within a denatured casein shell. The microbubbles are produced by vigorous stirring, forming a dispersion of perfluoropropane gas in the liquid phase containing casein and nanoparticles, after which the microbubble shell self-assembles. A variety of microscopy techniques are needed to characterize the nanoparticle-stabilized microbubbles at all relevant timescales of the nanoparticle release process. Fluorescence of the nanoparticles enables confocal imaging of single microbubbles, revealing the particle distribution within the shell. In vitro ultra-high-speed imaging using bright-field microscopy at 10 million frames per second provides insight into the bubble dynamics in response to ultrasound insonation. Finally, nanoparticle release from the bubble shell is best visualized by means of fluorescence microscopy, performed at 500,000 frames per second. To characterize drug delivery in vivo, the triggered release of nanoparticles within the vasculature and their extravasation beyond the endothelial layer is studied using intravital microscopy in tumors implanted in dorsal skinfold window chambers, over a timescale of several minutes. The combination of these complementary characterization techniques provides unique insight into the behavior of microbubbles and their payload release at a range of time and length scales, both in vitro and in vivo.

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.

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

Preparation and Characterization of Targeted Microbubbles

Targeting of microbubbles (ultrasound contrast agents for molecular imaging) has been researched for more than two decades. However, methods of microbubble preparation and targeting ligand attachment are cumbersome, complicated, and lengthy. Therefore, there is a need to simplify the targeted microbubble preparation procedure to bring it closer to clinical translation. The purpose of this publication is to provide a detailed description and explanation of the steps necessary for targeted microbubble preparation, functional characterization and testing. A sequence of the optimized and simplified procedures is presented for two systems: a biotin-streptavidin targeting pair model, and a cyclic RGD peptide targeting the recombinant &#945;v&#946;3 protein, which is overexpressed on the endothelial lining of the tumor neovasculature.
Here, we show the following: covalent coupling of the targeting ligand to a lipid anchor, assessment of the reagent quality, and tests that confirm the successful completion of the reaction; preparation of the aqueous precursor medium containing microbubble shell components, followed by microbubble preparation via amalgamation; assessment of the efficacy of lipid transfer onto the microbubble stabilizer shell; adjustment of microbubble size distribution by flotation at normal gravity to remove larger microbubbles that might be detrimental for in vivo use; assessment of microbubble size distribution by electrozone sensing; evaluation of targeted binding of the microbubbles to receptor-coated surface in a static binding assay test (in an inverted dish); and evaluation of targeted binding of the microbubbles to receptor-coated surface in a parallel plate flow chamber test.

The goal of this protocol is to prepare, purify and characterize gas-filled microbubbles (targeted contrast agents for ultrasound molecular imaging). Two targeting systems are described: biotinylated bubbles adherent to streptavidin, and cyclic RGD peptide microbubbles that bind to &#945;v&#946;3, a known tumor neovasculature biomarker.

Targeting of microbubbles (ultrasound contrast agents for molecular imaging) has been researched for more than two decades. However, methods of ...

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 microbubbles can be condensed or formulated as perfluorocarbon nanodroplets (PFCnDs) that are small enough to extravasate and then be triggered acoustically at the target site. These nanoparticles can be further enhanced by including an optical absorber such as near infrared organic dye or nanoparticles (e.g., copper sulfide nanoparticles or gold nanoparticles/nanorods). Optically tagged PFCnDs can be vaporized through laser irradiation in a process known as optical droplet vaporization (ODV). This process of activation enables the use of high boiling point perfluorocarbon cores, which cannot be vaporized acoustically under the maximum mechanical index threshold for diagnostic imaging. Higher boiling point cores result in droplets that will recondense after vaporization, resulting in "blinking" PFCnDs that briefly produce contrast after vaporization before condensing back into nanodroplet form. This process can be repeated to produce contrast on demand, allowing for the background free imaging, multiplexing, super-resolution, and contrast enhancement through both optical and acoustic modulation. This article will demonstrate how to synthesize optically-triggerable, lipid shell PFCnDs utilizing probe sonication, create polyacrylamide phantoms to characterize the nanodroplets, and acoustically modulate the PFCnDs after ODV to improve contrast.

Optically activated perfluorocarbon nanodroplets show promise in imaging applications outside of the vascular system. This article will demonstrate how to synthesize these particles, crosslink polyacrylamide phantoms, and modulate the droplets acoustically to enhance their signal.

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

Synthesis and Characterization of Multi-Modal Phase-Change Porphyrin Droplets

Phase-change droplets are a class of ultrasound contrast agents that can convert into echogenic microbubbles in situ with the application of sufficient acoustic energy. Droplets are smaller and more stable than their microbubble counterparts. However, traditional ultrasound contrast agents are not trackable beyond acoustic feedback measurements, which makes quantifying contrast agent bio-distribution or accumulation ex vivo difficult. Researchers may have to rely on fluorescent or optically absorbent companion diagnostic particles to infer bio-distribution. The purpose of this protocol is to detail steps for creating multi-modal phase-change porphyrin droplets using a condensation method. Porphyrins are fluorescent molecules with distinct absorbance bands that can be conjugated onto lipids and incorporated into droplets to extend droplet versatility, enabling more robust bio-distribution while retaining acoustic properties. Seven formulations with varying porphyrin-lipid and base lipid contents were made to investigate microbubble and droplet size distributions. Characterizations suited to porphyrin-containing structures are also described in the protocol to demonstrate their analytic versatility in-solution. Sizing showed that the post-condensed mean diameters were 1.72 to 2.38 times smaller than precursor populations. Absorbance characterization showed intact assemblies had a Q-band peak of 700 nm while disrupted samples had an absorbance peak at 671 nm. Fluorescence characterization showed intact 30% porphyrin-lipid assemblies were fluorescently quenched (&gt;97%), with fluorescence recovery achieved upon disruption. Acoustic vaporization showed that porphyrin droplets were non-echogenic at lower pressures and could be converted into echogenic microbubbles with sufficient pressures. These characterizations show the potential for porphyrin droplets to eliminate the need for absorbance or fluorescence-based companion diagnostic strategies to quantify ultrasound contrast agent bio-distribution for delivery or therapeutic applications in vivo or ex vivo.

In this protocol, methods for synthesizing and characterizing multi-modal phase-change porphyrin droplets are outlined.

Phase-change droplets are a class of ultrasound contrast agents that can convert into echogenic microbubbles in situ with the application of sufficient ...

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

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