Formulation and Acoustic Modulation of Optically Vaporized Perfluorocarbon Nanodroplets

Summary

Optically activated perfluorocarbon nanodroplets show promise in imaging applications outside of the vascular system. This article will demonstrate how to synthesize these particles, crosslink polyacrylamide phantoms, and modulate the droplets acoustically to enhance their signal.

Abstract

Microbubbles are the most commonly used imaging contrast agent in ultrasound. However, due to their size, they are limited to vascular compartments. These microbubbles can be condensed or formulated as perfluorocarbon nanodroplets (PFCnDs) that are small enough to extravasate and then be triggered acoustically at the target site. These nanoparticles can be further enhanced by including an optical absorber such as near infrared organic dye or nanoparticles (e.g., copper sulfide nanoparticles or gold nanoparticles/nanorods). Optically tagged PFCnDs can be vaporized through laser irradiation in a process known as optical droplet vaporization (ODV). This process of activation enables the use of high boiling point perfluorocarbon cores, which cannot be vaporized acoustically under the maximum mechanical index threshold for diagnostic imaging. Higher boiling point cores result in droplets that will recondense after vaporization, resulting in "blinking" PFCnDs that briefly produce contrast after vaporization before condensing back into nanodroplet form. This process can be repeated to produce contrast on demand, allowing for the background free imaging, multiplexing, super-resolution, and contrast enhancement through both optical and acoustic modulation. This article will demonstrate how to synthesize optically-triggerable, lipid shell PFCnDs utilizing probe sonication, create polyacrylamide phantoms to characterize the nanodroplets, and acoustically modulate the PFCnDs after ODV to improve contrast.

Introduction

Microbubbles are the most ubiquitous ultrasound contrast agent owing to their biocompatibility and excellent echogenicity in comparison to soft tissues. This makes them valuable tools for visualizing blood flow, organ delineation, and other applications¹. However, their size (1-10 µm), which makes them exceptional for imaging based on their resonant frequency, restricts their applications to the vasculature².

This limitation has led to the development of PFCnDs, which are nano-emulsions composed of a surfactant encased around a liquid perfluorocarbon core. These nanoparticles can be synth....

Protocol

1. Perfluorocarbon nanodroplet formulation

1. Rinse out a 10 mL round-bottom flask with chloroform and wash out a 10 µL and 1 mL gas tight glass syringe with chloroform by repeatedly aspirating the full syringe volume and expelling it for a total of three times.
   CAUTION: Chloroform is volatile and can be toxic if inhaled. All work with this solvent should be performed in a fume hood.
2. Using the syringes, add 200 µL of DSPE-mPEG2000 (25 mg/mL), 6.3 µL of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, 25 mg/mL) and 1 mL of IR 1048 (1 mg/mL in chloroform) into the round-bottom flask. Remember to clean out the syringes between li....

Results

Successful formulation and centrifugal separation of the PFCnDs should yield droplets around the size of 200-300 nm in diameter (Figure 1A). Improperly separated droplets may show small peaks around 1 µm. These solutions can be further bath sonicated to break up the larger droplets. The size of the droplets will increase over time due to coalescing and/or diffusion in a process known as Ostwald ripening^21,22 (Fig.......

Discussion

Probe sonication is a relatively simple and easy to learn method to fabricate PFCnDs. There are a few steps where care must be taken. When handling chloroform, it is imperative that a positive displacement pipette or glass syringes is used, as it is volatile and will "leak" from standard air displacement pipettes. Furthermore, if using a positive displacement, ensure that an appropriate tip is used as chloroform will dissolve most plastic tips, which can introduce contaminants into the solution. A positive displa.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ammonium Persulfate (APS)	VWR	97064-592
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)	Avanti Polar Lipids	850365C	Lipids, these can be purchased suspended in chloroform or in powder form. For long term storage, powder form is the best but chloroform is more practical.
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG)	Avanti Polar Lipids	880120C	Lipids, these can be purchased suspended in chloroform or in powder form. For long term storage, powder form is the best but chloroform is more practical.
Acrylamide : Bisacrylamide solution (19:1) 40% (w/v), OmniPur®	VWR	EM-1300	acrylamide solution, lower concentration/ powder
IR-1048	Sigma	405175	Infrared dye
L11-4v	Verasonics	-	ultrasound linear array transducer
Microtip 1/8"	Qsonica LLC	4418	microtip for probe sonicator
N, N, N′, N′ -Tetramethylethylenediamine (TEMED)	VWR	97064-902	Used to polymerize polyacrylamide by forming free radicals in the presence of ammonium persulfate
Nova II	Ophir-Spiricon	7Z01550	laser power meter
Perfluorohexane	Fluoromed	APF-60M	perfluorocarbon liquid
Phosphate buffered saline (PBS) tablets	VWR	97062-732	Tablets used to make PBS
Q500	Qsonica LLC	Q500-110	Probe sonicator
Silica gel	Sigma-Aldrich	288500	2-25 μm particle size
Tempest 30	New wave research	-	Pulsed laser system
Vantage 128	Verasonics	-	research ultrasound imaging system
Zetasizer Nano ZS	Malvern Instruments Ltd	-	Makes size measurements based on dynamic light scattering