Name: formulation and acoustic modulation of optically vaporized perfluorocarbon nanodroplets
Uploaded: 2021-07-16T15:00:00
Description: Watch this Scientific Journal Video about Formulation and Acoustic Modulation of Optically Vaporized Perfluorocarbon Nanodroplets at JoVE.com

The work was supported in part by the Breast Cancer Research Foundation under grant BCRF-20-043.

1. Perfluorocarbon nanodroplet formulation
<ol>
	<li>Rinse out a 10 mL round-bottom flask with chloroform and wash out a 10 &#181;L 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.</li>
	<li>Using the syringes, add 200 &#181;L of DSPE-mPEG2000 (25 mg/mL), 6.3 &#181;L of 1,2-distearoyl-sn-glycero-3-...

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 &#34;leak&#34; 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...

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 &#181;m), 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 synthesized at sizes as small as 200 nm and are designed to take advantage of &#34;leaky&#34; vasculature or pores and open fenestrations found in tumor vasculature. While these disruptions are tumor dependent, this permeability allows for extravasation of nanoparticles from ~200 nm - 1.2 &#181;m depending on the tumor3,4. In their initial form, these particles produce little to no ultrasound contrast. Upon vaporization - induced acoustically or optically - the core phase changes from liquid to gas, inducing a two and half to five-fold increase in diameter5,6,7 and generating photoacoustic and ultrasound contrast. While acoustic vaporization is the most common activation method, this approach creates acoustic artifacts that limits the imaging of the vaporization. Additionally, most perfluorocarbons require focused ultrasound with a mechanical index beyond the safety threshold to vaporize8. This has led to the development of lower boiling point PFCnDs, which can be synthesized by condensing microbubbles into nanodroplets9. However, these droplets are more volatile and subject to spontaneous vaporization10.
Optical droplet vaporization (ODV), on the other hand, requires the addition of an optical trigger such as nanoparticles11,12,13 or dye6,14,15 and can vaporize higher boiling point perfluorocarbons using fluences within the ANSI safety limit11. PFCnDs synthesized with higher boiling point cores are more stable and will recondense after vaporization, allowing for background free imaging16, multiplexing17, and super-resolution18. One of the major limitations of these techniques is the fact that high boiling point PFCnDs are echogenic after vaporization for only a short timeframe, on the scale of milliseconds19, and are relatively faint. While this issue can be mitigated through repeated vaporizations and averaging, detection and separation of droplet signal remains a challenge.
Taking inspiration from pulse inversion, the duration and contrast can be enhanced by modifying the phase of the ultrasound imaging pulse19. By starting the ultrasound imaging pulse with a rarefaction phase (n-pulse), both the duration and contrast of the vaporized PFCnDs increases. In contrast, starting the ultrasound imaging pulse with a compression phase (p-pulse), results in reduced contrast and shorter in duration. This article will describe how to synthesize optically triggerable perfluorocarbon nanodroplets, polyacrylamide phantoms commonly used in imaging, and demonstrate contrast enhancement and improved signal longevity through acoustic modulation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ammonium Persulfate (APS)</td>
			<td>VWR</td>
			<td>97064-592</td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)</td>
			<td>Avanti Polar Lipids</td>
			<td>850365C</td>
			<td>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.</td>
		</tr>
		<tr>
			<td>1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG)</td>
			<td>Avanti Polar Lipids</td>
			<td>880120C</td>
			<td>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.</td>
		</tr>
		<tr>
			<td>Acrylamide : Bisacrylamide solution (19:1) 40% (w/v), OmniPur&#174;</td>
			<td>VWR</td>
			<td>EM-1300</td>
			<td>acrylamide solution, lower concentration/ powder</td>
		</tr>
		<tr>
			<td>IR-1048</td>
			<td>Sigma</td>
			<td>405175</td>
			<td>Infrared dye</td>
		</tr>
		<tr>
			<td>L11-4v</td>
			<td>Verasonics</td>
			<td>-</td>
			<td>ultrasound linear array transducer</td>
		</tr>
		<tr>
			<td>Microtip 1/8&#34;</td>
			<td>Qsonica LLC</td>
			<td>4418</td>
			<td>microtip for probe sonicator</td>
		</tr>
		<tr>
			<td>N, N, N&#8242;, N&#8242; -Tetramethylethylenediamine (TEMED)</td>
			<td>VWR</td>
			<td>97064-902</td>
			<td>Used to polymerize polyacrylamide by forming free radicals in the presence of ammonium persulfate</td>
		</tr>
		<tr>
			<td>Nova II</td>
			<td>Ophir-Spiricon</td>
			<td>7Z01550</td>
			<td>laser power meter</td>
		</tr>
		<tr>
			<td>Perfluorohexane</td>
			<td>Fluoromed</td>
			<td>APF-60M</td>
			<td>perfluorocarbon liquid</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS) tablets</td>
			<td>VWR</td>
			<td>97062-732</td>
			<td>Tablets used to make PBS</td>
		</tr>
		<tr>
			<td>Q500</td>
			<td>Qsonica LLC</td>
			<td>Q500-110</td>
			<td>Probe sonicator</td>
		</tr>
		<tr>
			<td>Silica gel</td>
			<td>Sigma-Aldrich</td>
			<td>288500</td>
			<td>2-25 &#956;m particle size</td>
		</tr>
		<tr>
			<td>Tempest 30</td>
			<td>New wave research</td>
			<td>-</td>
			<td>Pulsed laser system</td>
		</tr>
		<tr>
			<td>Vantage 128</td>
			<td>Verasonics</td>
			<td>-</td>
			<td>research ultrasound imaging system</td>
		</tr>
		<tr>
			<td>Zetasizer Nano ZS</td>
			<td>Malvern Instruments Ltd</td>
			<td>-</td>
			<td>Makes size measurements based on dynamic light scattering</td>
		</tr>
	</tbody>
</table>

formulation and acoustic modulation of optically vaporized perfluorocarbon nanodroplets

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.

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 &#181;m. 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 ripening21,22 (<strong class="xfig...

Watch this Scientific Journal Video about Formulation and Acoustic Modulation of Optically Vaporized Perfluorocarbon Nanodroplets at JoVE.com

Formulation and Acoustic Modulation of Optically Vaporized Perfluorocarbon Nanodroplets

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.

Microbubbles are the most commonly used imaging contrast agent in ultrasound. However, due to their size, they are limited to vascular compartments. These ...

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