This protocol is significant because it provides the complete procedure for creating droplets from the lipid film to lipid solution to microbubbles and then to droplets. The main advantage of this technique is the simplicity for condensing microbubbles into droplets. Just by cooling and swirling, droplets can be made without the need for pressurization.
A visual demonstration is critical because a simple test to see if the microbubbles have been successfully condensed is by visually checking the change in translucency. Demonstrating the procedure will be Kimoon Yoo, a master's student in the Gang Zheng laboratory. Using a decapper, remove the aluminum seal on the serum vial and transfer one milliliter of the lipid solution to a 1.85 milliliter borosilicate glass sample vial with a phenol screw cap by letting the lipid solution flow down the interior wall without creating bubbles.
With the 1.85 milliliter sample vial prepare to flow in decafluorobutane gas into the sample vial headspace using the gas exchanger. Ensure that all the valves on the gas exchanger are properly closed and the pump is turned off. Open the manifold valve and carefully unsheathe the corresponding needle from the manifold.
Open pressure valve A and pressure valve B and turn the gas cylinder valve approximately 1/16 to 1/8 counter-clockwise to partially open it. Open the T handle valve and uncapped the sample vial with the lipid solution. Place the manifold needle above the vial's liquid air interface, slowly turn the air regulator valve clockwise until the air regulator gauge needle moves slightly from its resting position and let the perfluorocarbon gas gently flow into the vial headspace for 30 seconds, taking care to not create bubbles.
Adjust the air regulator valve if necessary. After 30 seconds, carefully and quickly kept the sample vial without moving the vial too much. Both the gas cylinder valve, the T handle valve, air regulator valve, pressure valve A, pressure valve B, and manifold valve.
Carefully sheath the needle, then labeled the sample vial and seal the neck with wax film going clockwise. Store the sample vial in the dark and at four degrees Celsius for at least 10 minutes or up to 24 hours. Place about 100 grams of dry ice in an insulated container in place regular ice in another insulated container.
Retrieve a previously prepared decafluorobutane serum vial to 1.5 inch 20 gauge needles, a one milliliter plastic syringe, a 200 milliliter container, metal tongs, and a thermometer. Place the sample vial with the lipid solution in the mechanical agitator and agitate for 45 seconds. There should be a change in color and translucency.
After the mechanical agitation, stand the sample vial right side up shielded from light and start a 15 minute countdown to cool down the vial and size select the microbubbles. When the countdown has reached 10 minutes, fill a container with about 200 milliliters of isopropanol and cool it to minus 20 degrees Celsius with dry ice using metal tongs. After the microbubbles have been sized selected for 15 minutes, look for the size selected partition inside the sample vial.
Keeping the sample vial right side up, carefully uncapped the sample vial and withdraw about 0.7 milliliters of the bottom partition with a 1.5 inch 20 gauge needle attached to a one milliliter plastic syringe. Ensure none of the top partition is withdrawn and do not flick the syringe to remove air pockets. Aiming at the center circle on the rubber stopper, insert a different 20 gauge needle into a decafluorobutane serum vial while keeping the needle near the top of the serum vial to vent and then insert the needle with the size selected microbubbles.
Slowly transfer the size selected microbubbles. Let the liquid gently slide down the interior wall of the decafluorobutane serum vial. Once all the size selected microbubble solution has been transferred, remove the needle with the syringe, but keep the venting needle in to relieve negative pressure.
Add small amounts of dry ice or room temperature isopropanol to the isopropanol bath to ensure the bath temperature is between minus 15 to minus 17 degrees Celsius. With the 20 gauge venting needle inserted near the top of the serum vial, place the serum vial and the isopropanol bath, keeping the microbubble level below the level of the isopropanol, but the vial neck above it and intermittently swirled the serum vial for two minutes to condense the microbubbles. Do not swirl the serum vial continuously in the isopropanol and do not let the solution freeze.
Swirl for about five seconds and lift the serum vial out of the isopropanol. Check for ice nucleation, then resume swirling in isopropanol. If there is ice formation, swirl the serum vial in the air until it dissipates.
After the two minute condensation remove the serum vial from the isopropanol bath and remove the venting needle. The microbubbles should have condensed into droplets as indicated by the change in translucency. Wipe the serum vial, label it, and place it on regular ice in a dark insulated container until ready for use.
Unopened droplets with an intact aluminum seal should be stable for up to six hours as long as the melted ice gets replaced as needed. When ready to use, remove the aluminum seal with the decapper. After using this protocol, pre-condensed, size selected microbubbles and post-condensed droplets were sized on a Coulter counter with a 10 micron aperture.
The sizing data for the 30%pyro-lipid formulation is shown. The statistics based on the sizing data are shown here. Using a ratio of pre-and post-condensed mean diameters, the results showed that as the pyro-lipid content increased, the concentration decreased, and the mean diameter increased.
The representative absorbents measurements of the 30%pyro-lipid droplets sample is shown. This showed that the intact assemblies have different optical properties as reflected by different peak compared to the individual unassembled lipid components. Representative florescence measurements of the pre-condensed microbubble, post-condensed droplet sample with 30%pyro-lipid are shown here, demonstrating different fluorescence peaks for intact samples in the disrupted form.
Representative ultrasound images of the 30%pyro-lipid droplets sample imaged at different pressures are shown. At low pressures, only background signal from air bubbles stuck from the agar synthesis was observed. At a slightly higher power, a few microbubbles were generated, which is demonstrated by the appearance of bright speckles.
More microbubbles were generated as the pressure increased. It is important to remember that the condensation temperature here is optimized for this specific droplet shell formulation. Different shell formulations may require a different temperature.
