A subscription to JoVE is required to view this content. Sign in or start your free trial.
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 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.
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 µm 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 > 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 "Ouzo" 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.
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. Please click here to view a larger version of this figure.
1. Making lipid films
2. Generating microbubbles from lipid films
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
3. Preparing extruder for microbubble condensation
4. Priming the extruder for microbubble condensation
5. Pre-cooling microbubbles for extrusion
6. Separating droplets from liposomes by centrifugation
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). Please click here to view a larger version of this figure.
7. Microscopy verification of droplet vaporization
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...
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...
The authors have nothing to disclose.
We would like to thank Dominique James in Dr. Ken Hoyt's lab for providing TRSP analysis of vaporizable phase-shift nanodroplets
Name | Company | Catalog Number | Comments |
15 mL Centrifuge Tubes | Falcon | 352095 | Collecting and centrifuging droplets |
200 nm polycarbonate filter | Whatman | 110606 | Extruder filters |
2-methylbutane | Fisher Chemical | 03551-4 | Rapid precooling of microbubble solution prior to extrusion |
3-prong clamps X2 | Fisher | 02-217-002 | Holding scintilation vials in place for probe tip sonication |
400W Analog Probe Tip Sonicator with Horn | Branson | 101-063-198R | Used to generate lipid microbubbles from lipid solution |
Bath Sonicator | Fisher Scientific | 15337402 | Used to help breakdown liposomes into unilamellar vesicles |
Chloroform | Fisher Bioreagents | C298-4 | Used to make lipid film for microbubble preperation |
Decafluorobutane (Perfluorobutane) Gas | FluoroMed L.P. | 1 kg | generating microbubbles via probe tip sonication |
Dry Ice | - | - | Rapid precooling of microbubble solution prior to extrusion |
DSPC Lipid Powder | NOF America | COATSOME MC-8080 | Component of lipid film |
DSPE-PEG-2K Lipid Powder | NOF America | SUNBRIGHT DSPE-020CN | Component of lipid film |
General Thermometer | - | - | Used to measure ice bath temperature and 2-methylbutane temperature ( needs to accommodate -20C temperatures) |
Glass Syringes | Hamilton | 81139 | Used to mix lipids in chloroform |
Glycerol | Fisher Bioreagents | BP229-1 | Reduces freezing temperature of PBS solution |
Heating Block | VWR Scientific Products | Heating lipid films and vaporizing droplets | |
Lipex 10 mL Extruder | Evonik | Commercial high-pressure extrusion system | |
Mini Vortex Mixer | Fisher brand | 14-955-151 | Used to remove excess chloroform from lipid films |
Nitrogen Tank | - | - | Used to operate extruder |
Phosphate Buffer Saline | Fisher Scientific | Hydrate lipid films and washing droplets | |
Polyester Drain Disk | Whatman | 230600 | Provides support for polycarbonate filter |
Polypropylene Caps | Fisher Scientific | 298417 | Used for solution storage |
Propylene Glycol | Fisher Chemical | P355-1 | Reduces freezing temperature of PBS solution |
Scintiliation Vials | DWK Life Sciences Wheaton | 986532 | Used for lipid films and microbubble generation |
Small hammer | - | - | Used to break apart dry ice for cooling methylbutane |
Sonicator Microtip Attachment | Branson | 101148070 | Used to generate microbubbles from lipid solution |
Steel Container | Medegen | 79310 | Rapid precooling of microbubble solution prior to extrusion ( any container rated to -20C will work) |
Vacuume Dessicator | Bel-Art SP Scienceware | 08-648-100 | Removes excess chloroform from lipid films |
2mL Centrifuge Tube | Fisher | 02682004 | Used for concentrating nanodroplets |
Request permission to reuse the text or figures of this JoVE article
Request PermissionThis article has been published
Video Coming Soon
Copyright © 2025 MyJoVE Corporation. All rights reserved