JoVE Logo
Faculty Resource Center

Sign In





Representative Results






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

Published: March 23rd, 2021



1Department of Bioengineering, The University of Texas at Dallas, 2Department of Radiology, The University of Texas Southwestern

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 acoust....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

1. Making lipid films

  1. 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:
    1. 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.
    2. Add 287 µL of DSPC stock and 113 µL of DSPE-PEG2K stock (both 50 mg/mL) into a 20 mL scintillation vial using a glass syringe.
    3. .......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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


Log in or to access full content. Learn more about your institution’s access to JoVE content here

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

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

This article has been published

Video Coming Soon

JoVE Logo


Terms of Use





Copyright © 2024 MyJoVE Corporation. All rights reserved