Synthesis of Phase-shift Nanoemulsions with Narrow Size Distributions for Acoustic Droplet Vaporization and Bubble-enhanced Ultrasound-mediated Ablation

The overall goal of the following experiment is to observe the effect of bubbles generated by vaporizing, lipid coated nano emulsions acoustically on ultrasound mediated ablation. This is achieved by first dispersing lipid coated nano emulsions throughout albumin loaded poly acrylamide hydrogels. As a second step, a focused transducer transmits short high amplitude pulses into the hydrogel, which vaporize nano emulsions locally at the focus next, the resultant bubbles are driven continuously with ultrasound, and the acoustic energy radiated by the bubbles is rapidly absorbed by the hydrogel, which accelerates the rate of ultrasound mediated heating.

The results show that the presence of bubbles dramatically reduces the time and energy required to heat the hydrogel beyond the threshold temperature for protein denaturation resulting in faster and more efficient ultrasound mediated ablation. The size of the phase shift nano emulsion is a critical determining factor in the pressure threshold. For acoustic vaporization, we've developed a protocol for producing stable phase shift nano emulsions with narrow size distributions and well-defined vaporization thresholds, as well as an in vitro system for evaluating impact on ultrasound media ablation.

While an in vitro system is described, the face of nano emulsions are designed to see extravascular tissue and solid tumors with particles that can be vaporized acoustically. Bubbles that are formed can then be used for diagnostic or therapeutic applications in the management of cancer. To begin the procedure for preparing a phase shift nano emulsion or PSNE first mix the lipids together dissolve 11 milligrams of DPPC and 1.7 milligrams of DSPE.

Peg 2000 in chloroform in a glass round bottom flask. Place the round bottom flask in a 50 degrees Celsius water bath in the chemical fume hood, and use a stream of Argonne gas to evaporate the organic solvent. To form a dry lipid film, place the flask in a desiccate and leave under vacuum overnight to desiccate the lipid film on the following morning.

Rehydrate the lipid film with 5.5 milliliters of PBS. Heat the solution in a 45 degree Celsius water bath until the lipid film dissolves for texting. Periodically transfer the lipid solution into a seven milliliter vial and then sonicate with a probe sonicate for two minutes at 20%amplitude.

Divide the solution into two vials of 2.5 milliliters each. The remaining 0.5 milliliters is discarded. Add 2.5 milliliters of PBS to each vial, and then place both vials in a zero degree Celsius ice water bath.

Add 50 microliters of doca fluoro pentane, or DDFP to each file. Son of the DDFP lipid mixture. In an ice water bath is the most critical part of this procedure.

The ice water bath is used to prevent evaporation of the liquid per fluorocarbon. During this process, Sonica each vial in the ice water bath using the following settings, 25%amplitude pulsed mode, and 60 seconds total time. After sonication transfer the PSNE solution to 20 milliliters scintillation vials at five milliliters of PBS to each vial, resulting in a final volume of 10 milliliters per vial.

Assemble the extruder following the manufacturer's directions to prime the extruder. Pipette 10 milliliters of deionized water into the top sample port. Cap the opening and tighten the vent valve.

Slowly open the nitrogen gas line to increase the pressure, forcing the sample through the membranes. Collect the sample from the outlet tubing, extrude the PSNE 16 times through a 100 nanometer or 200 nanometer filter to obtain a narrow size distribution. Begin this procedure by preparing a poly acrylamide hydrogel containing PSNE.

First, prepare a 24%bovine serum albumin or BSA solution by dissolving 2.4 grams of BSA powder in 10 milliliters of deionized water. Next, prepare a 10%ammonium per sulfate or a PS solution by diluting one gram of a PS powder in 10 milliliters of deionized water, mix these reagents in a plastic mold in the following order, 2.1 milliliters of acrylamide solution, 1.2 milliliters of tris buffer, 0.1 milliliters of 10%a PS 4.5 milliliters of 24%BSA solution, and 3.6 milliliters of deionized water. Heat the solution to 40 degrees Celsius in a water bath and then place under vacuum for one hour to degas the liquid.

This is required to avoid unwanted bubble formation within the hydrogel after an hour. Remove from the vacuum, add 480 microliters of PSNE suspension and thoroughly mixed by gently swirling the plastic chamber. In a chemical fume hood, add 12 microliters of tetraethyl, ethylene diamine or T-E-M-E-D to polymerize the acrylamide and place the chamber in a 12 degree Celsius water bath for two hours.

Polymerization is exothermic, so a cool bath is needed to avoid PSE vaporization during polymerization. Once the acrylamide has polymerized position, a focused power transducer and imaging probe in an acrylic tank filled with DGAs deionized water so that the imaging plane overlaps with the transducer focal volume. Next position, the PSNE loaded poly acrylamide hydrogel at the focus of the power transducer vaporize the PSNE within the hydrogel with short acoustic pulses, followed by a 15 second continuous wave exposure for bubble enhanced ablation.

As the BSA is denatured, the hydrogel becomes opaque due to the absorption of ultrasound and heat generation within monitor vaporization and bubble formation with a diagnostic ultrasound scanner. In this example image, the arrow indicates the focal region where a bubble cloud was formed by PSNE vaporization. Repeat acoustic exposure at a new location in the hydrogel without paper raising the PSNE and to compare the ablation time and volume.

This protocol results in lipid coated per fluorocarbon droplets with a narrow size distribution. The size distribution measured with dynamic light scattering is shown here for PSNE extruded using 100 and 200 nanometer filters. The units of the ordinate axes are based on the intensity of scattered light from particles of a certain size relative to the total scattered light intensity from the sample.

When the mean diameter and standard deviation of PSNE at one and seven days after extrusion with 100 and 200 nanometer filters are measured using dynamic light scattering, the results demonstrate that PSNE are stable for at least a week. This figure shows B mode images of PSNE before and after vaporization. In a poly acrylamide hydrogel, the arrow indicates the focal region where a bubble cloud was formed by PSNE vaporization.

These are images of a poly acrylamide hydrogel containing albumin and PSNE before and after vaporization and sonication with high intensity focused ultrasound or Hy Fu.A lesion is formed by 15 seconds of Hy Fu mediated heating. The asymmetric shape of the lesion is a result of pre focal heating that occurs due to the presence of the bubble cloud in the ultrasound path. Pre focal heating and lesion formation due to scatter from bubbles can be minimized by reducing the transmitted acoustic power After its development.

This method for producing stable phase shift nano emulsions will pave the way for researchers in the field of ultrasound mediated therapy to explore bubble enhanced HIFU ablation in solid tumors. After watching this video, you should have a good understanding of how to produce stable phase shift nano emulsions with narrow size distributions and study bubble nucleation and enhanced ultrasound mediated ablation.