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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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. .......

Representative Results

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.......

Discussion

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.......

Acknowledgements

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

....

Materials

NameCompanyCatalog NumberComments
15 mL Centrifuge TubesFalcon352095Collecting and centrifuging droplets
200 nm polycarbonate filterWhatman110606Extruder filters
2-methylbutaneFisher Chemical03551-4Rapid precooling of microbubble solution prior to extrusion
3-prong clamps X2Fisher02-217-002Holding scintilation vials in place for probe tip sonication
400W Analog Probe Tip Sonicator with HornBranson101-063-198RUsed to generate lipid microbubbles from lipid solution
Bath SonicatorFisher Scientific15337402Used to help breakdown liposomes into unilamellar vesicles
ChloroformFisher BioreagentsC298-4Used to make lipid film for microbubble preperation
Decafluorobutane (Perfluorobutane) GasFluoroMed L.P.1 kggenerating microbubbles via probe tip sonication
Dry Ice--Rapid precooling of microbubble solution prior to extrusion
DSPC Lipid PowderNOF AmericaCOATSOME MC-8080Component of lipid film
DSPE-PEG-2K Lipid PowderNOF AmericaSUNBRIGHT DSPE-020CNComponent of lipid film
General Thermometer--Used to measure ice bath temperature and 2-methylbutane temperature ( needs to accommodate -20C temperatures)
Glass SyringesHamilton81139Used to mix lipids in chloroform
GlycerolFisher BioreagentsBP229-1Reduces freezing temperature of PBS solution
Heating BlockVWR Scientific ProductsHeating lipid films and vaporizing droplets
Lipex 10 mL ExtruderEvonikCommercial high-pressure extrusion system
Mini Vortex MixerFisher brand14-955-151Used to remove excess chloroform from lipid films
Nitrogen Tank--Used to operate extruder
Phosphate Buffer SalineFisher ScientificHydrate lipid films and washing droplets
Polyester Drain DiskWhatman230600Provides support for polycarbonate filter
Polypropylene CapsFisher Scientific298417Used for solution storage
Propylene GlycolFisher ChemicalP355-1Reduces freezing temperature of PBS solution
Scintiliation VialsDWK Life Sciences Wheaton986532Used for lipid films and microbubble generation
Small hammer--Used to break apart dry ice for cooling methylbutane
Sonicator Microtip AttachmentBranson101148070Used to generate microbubbles from lipid solution
Steel ContainerMedegen79310Rapid precooling of microbubble solution prior to extrusion ( any container rated to -20C will work)
Vacuume DessicatorBel-Art SP Scienceware08-648-100Removes excess chloroform from lipid films
2mL Centrifuge TubeFisher02682004Used for concentrating nanodroplets

References

  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....

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