This protocol is an easy method of reducing polydispersity of low boiling point vaporizable droplets for use in biomedical applications. This technique condenses preformed gas microbubbles and filters the resulting liquid droplets by size in a way that is controllable, scalable, and relatively cost-effective. This method can be applied to a variety of lipid microbubbles with different shell and gas material using different filters to control final droplet size.
Demonstrating the procedure will be Darrah Merillat, an undergraduate researcher from Dr.Sirsi's laboratory. Turn on the power switch for the sonicator, and set the amplitude to the maximum allowed using the microtip attachment, and ensure sonication time set to 10 seconds. Place the warm hydrated lipid solution in the sound enclosure with the microtip just beneath the surface.
Attach an appropriate length of tubing to the neck of the vial to guide the gas from the DFB tank outlet into the warm lipid solution held in the the enclosure. Open the tank valve slowly until the gas can be seen flowing over the lipid solution, causing slight ripples on the surface of the liquid. If the gas flow is too high, the solution will overflow during microbubble formation.
Start the sonicator, and run it for 10 seconds continuously to generate microbubbles. After sonication is over, immediately close the DFB tank valve. Quickly cap the microbubble solution, and submerge the vial in an ice bath to cool the sample below 55 degrees Celsius.
Use a 200-nanometer ceramic filter to assemble the high-pressure extruder according to the user's manual, and place it in the center of a watertight container, so that the sample outlet tube is not pressed against the side, or crimped. Couple the extruder to the nitrogen gas tank using the adapter supplied by the manufacturer. Place the end of the outlet tube in a scintillation vial to collect the extruded sample, securing the tube to the container with tape to stay within the vial.
Open and close the release valve to ensure that there's no pressure within the extruder. Remove the chamber lid. And add five milliliters of PBS to the extruder chamber.
Replace the lid, making sure that it clicks securely back into place. Open the nitrogen gas tank, so that the pressure gauge reads 250 PSI, making sure the pressure control valve is in the closed position. Close the gas tank, and open the extruder chamber inlet valve, causing the PBS solution to be pushed through the system, and out the sample outlet tube into the scintillation vial.
When only gas is exiting the tubing, open the release valve, and allow the pressure to fall to zero PSI. Then remove the scintillation vial. Open and close the release valve to make sure there is no pressure within the extruder, and place a new scintillation vial at the end of the outlet tube.
Fill a steel container with 2-methylbutane, and add dry ice to bring the temperature down to minus 18 degrees Celsius. Insert the microbubble solution into the chilled 2-methylbutane, submerging the sample for two minutes. Move the scintillation vial throughout the two minutes to gently mix the bubbles.
Add dry ice as needed to maintain the temperature between minus 15 and minus 18 degrees Celsius. After two minutes, remove the microbubbles from the chilled 2-methylbutane. Gently swirl the vial to mix the microbubbles, and transfer bubbles into a chilled 10-milliliter syringe.
Remove the extruder chamber lid, and add the microbubble solution to the chamber by slowly pushing the plunger on the syringe. Replace the extruder cap, making sure it clicks securely in place. Verify that the pressure control valve and the release valve of the extruder are in the closed position.
Open the nitrogen gas tank until the pressure gauge reads 250 PSI. Close the gas tank, and turn the pressure control valve to the open position. When the solution has filled the scintillation vial at the exit tubing and only gas is exiting the tube, slowly open the pressure release valve, and allow the pressure to fall to zero PSI.
Transfer 10 milliliters of the extruded droplet solution to a 15-milliliter centrifuge tube. Centrifuge the extruded sample at 1, 500 times G for 10 minutes at four degrees Celsius. Discard the supernatant and the spontaneously vaporized droplets appearing at the top of the solution.
Re-suspend the pellet comprising DFP nanodroplets in 10 milliliters of PBS with 20%glycerol and 20%propylene glycol. The size distribution of condensed bubble solutions with and without extrusion shows that the condensed only sample has a much wider distribution centered near 400 nanometers, whereas the extruded sample has a narrower distribution centered at 200 nanometers. Tunable resistance pulse sensing analysis used to analyze phase shift droplets as they have been washed by centrifugation to remove excess liposomes shows that the droplet sizes are near 200 nanometers.
The microscopy data of nanodroplet vaporization when heated shows that some spontaneously vaporized microbubbles are apparent in the field of view before heating, and a greater number of gas microbubbles are observed after heating. The microscopy images of nanodroplets inserted into the extruder directly without pre-cooling, condensed at zero degree Celsius, and at minus 18 degrees Celsius are shown here. Representative images of condensed octafluoropropane droplets before and after vaporization also show a greater number of gas microbubbles after heating, similar to the DFB droplets.
The most important thing to remember during this procedure is that the droplet yield is highly dependent on the temperature and pressure during condensation, and slight variations will impact results. After generating vaporizable droplets, they can be used to optimize in vivo imaging and drug delivery using ultrasound, as well as other in vivo and ex vivo applications. After developing this technique, the effects of nanodroplet size and gas core content on in vivo vaporization thresholds have been explored using slight modifications to this protocol.
