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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....
1. Making lipid films
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.......
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 |
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