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

Summary

Phase-shift nanoemulsions (PSNE) can be vaporized using high intensity focused ultrasound to enhance localized heating and improve thermal ablation in tumors. In this report, the preparation of stable PSNE with a narrow size distribution is described. Furthermore, the impact of vaporized PSNE on ultrasound-mediated ablation is demonstrated in tissue-mimicking phantoms.

Abstract

High-intensity focused ultrasound (HIFU) is used clinically to thermally ablate tumors. To enhance localized heating and improve thermal ablation in tumors, lipid-coated perfluorocarbon droplets have been developed which can be vaporized by HIFU. The vasculature in many tumors is abnormally leaky due to their rapid growth, and nanoparticles are able to penetrate the fenestrations and passively accumulate within tumors. Thus, controlling the size of the droplets can result in better accumulation within tumors. In this report, the preparation of stable droplets in a phase-shift nanoemulsion (PSNE) with a narrow size distribution is described. PSNE were synthesized by sonicating a lipid solution in the presence of liquid perfluorocarbon. A narrow size distribution was obtained by extruding the PSNE multiple times using filters with pore sizes of 100 or 200 nm. The size distribution was measured over a 7-day period using dynamic light scattering. Polyacrylamide hydrogels containing PSNE were prepared for in vitro experiments. PSNE droplets in the hydrogels were vaporized with ultrasound and the resulting bubbles enhanced localized heating. Vaporized PSNE enables more rapid heating and also reduces the ultrasound intensity needed for thermal ablation. Thus, PSNE is expected to enhance thermal ablation in tumors, potentially improving therapeutic outcomes of HIFU-mediated thermal ablation treatments.

Protocol

1. Preparation of Phase-shift Nanoemulsion (PSNE)

1. Dissolve 11 mg DPPC and 1.68 mg DSPE-PEG2000 in chloroform
2. Evaporate the organic solvent to form a dry lipid film in a glass round-bottom flask
3. Dessicate the lipid film overnight
4. Rehydrate the lipid film with 5.5 ml of phosphate-buffered saline (PBS)
5. Heat solution in a 45 °C water bath until lipid film dissolves, vortexing periodically
6. Transfer lipid solution into 7 ml vial
7. Sonicate lipid solution for 2 min at 20% amplitude
8. Divide solution into two vials of 2.5 ml each (discard remaining 0.5 ml)
9. Add 2.....

Discussion

High-intensity focused ultrasound (HIFU) is used clinically to thermally ablate tumors.¹ To enhance localized heating and improve thermal ablation in tumors, lipid-coated perfluorocarbon droplets have been developed which can be vaporized by HIFU. The vasculature in many tumors is abnormally leaky due to their rapid growth.² Thus, nanoparticles are able to penetrate the fenestrations and passively accumulate within tumors, a process known as the enhanced permeability and retention (EPR) effect.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DPPC	Avanti Lipids, Alabaster, AL, USA	850355P	1,2-dipalmitoyl-sn-glycero-3-phosphocholine
DSPE-PEG2000	Avanti Lipids, Alabaster, AL, USA	880120P	1,2-distearoyl-sn-glycero-3-phosph–thanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt)
DDFP	Fluoromed, Round Rock, TX, USA	CAS: 138495-42-8	Dodecafluoropentane (C5F12)
PBS	Sigma-Aldrich, St. Louis, MO, USA	P2194	Phosphate-buffered saline
Chloroform	Sigma-Aldrich, St. Louis, MO, USA	372978	Chloroform
Acrylamide	Sigma-Aldrich, St. Louis, MO, USA	A9926	40% 19:1 acrylamide/bis-acrylamide
Tris buffer	Sigma-Aldrich, St. Louis, MO, USA	T2694	1M, pH 8, trizma hydrochloride and trizma base
BSA	Sigma-Aldrich, St. Louis, MO, USA	A3059	Bovine serum albumin
APS	Sigma-Aldrich, St. Louis, MO, USA	A3678	Ammonium persulfate solution
TEMED	Sigma-Aldrich, St. Louis, MO, USA	87689	N,N,N',N'-Tetramethylethylenediamine
Equipment
Sonicator (3 mm tip)	Sonics Materials, Inc., Newtown, CT, USA	Vibra-Cell
Water bath	Thermo Fisher Scientific, Waltham, MA, USA	Neslab EX-7
Extruder	Northern Lipids, Burnaby, BC, Canada	LIPEX
Extruder Filters	Whatman, Piscataway, NJ, USA	Nuclepore #110605 and #110606
Extruder Drain Disc	Sterlitech Corporation, Kent, WA, USA	#PETEDD25100
Plastic chamber	U.S. Plastic Corporation, Lima, OH, USA	#55288, 1 3/16"x1 3/16"x2 7/16"