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

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.
   .......

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.......

Materials

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