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Method Article

Formulation and Acoustic Modulation of Optically Vaporized Perfluorocarbon Nanodroplets

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DOI:

10.3791/62814

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July 16th, 2021

 ,  , 
1Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 2School of Electrical Engineering, Georgia Institute of Technology, 3Department of Biomedical Engineering and School of Electrical Engineering, Georgia Institute of Technology and Emory University

In This Article

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 synthesized at sizes as small as 200 nm and are designed to take advantage of "leaky" 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 µm 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.

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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 lipids/dye to prevent contamination of the stock.
    NOTE: Infrared dyes are light sensitive, and work should be done in dim conditions or flasks should be covered in aluminum foil.
  3. Remove the solvent utilizing a rotary evaporator. Ensure that the vacuum is slowly adjusted to 332 mbar to prevent bumping. After 5 min, reduce the pressure to 42 mbar to remove any water that may have entered the solution.
    NOTE: The lipid cake can be stored overnight in a round bottom flask covered with parafilm at 4 °C.
  4. Suspend the lipid cake in 1 mL of phosphate buffered saline (PBS) and sonicate or vortex at room temperature for 5 min or until all of the lipid cake has been suspended and dissolved in the solution. Sonicate for an additional 2 min to homogenize the solution.
  5. Transfer the solution to a 7 mL glass vial and place the vial in a glass dish filled with ice to allow the solution to cool down for 5 min before adding 50 µL of perfluorohexane using a gas tight glass syringe. Remember to rinse out the syringe with perfluorohexane before dispensing it into the vial.
  6. Place the glass vial containing the lipids and ice bath in the probe sonicator enclosure and submerge the probe tip below the miniscus. Ensure that the sides of the sonicator probe does not touch the lip of the glass vial.
  7. Probe sonicate the mixture with the following settings: Amplitude 1, Process time: 20 s, Pulse-On: 1s, Pulse-off: 5s. Then sonicate at the following settings: Amplitude: 50, Process time: 5 s, Pulse-on: 1 s, Pulse-off: 10 s.
  8. Transfer the nanodroplet solution to a 1.5 mL centrifuge tube and centrifuge at 300 x g for 3 min to separate out the larger droplets (>1 µm) from smaller droplets.
  9. Discard the pellet and transfer the supernatant to another 1.5 mL centrifuge tube. Wash the supernatant by centrifuging at 3000 x g for 5 min to pellet all the droplets in solution. Resuspend the PFCnDs in 1 mL of PBS by pipetting the pellet up and down and then sonicate in a bath sonicator for 1 min.
  10. Measure the size of the droplets using dynamic light scattering (DLS). Dilute the stock PFCnDs by 100-fold (10 µL of PFCnD stock in 990 µL of PBS) and bath sonicate to disperse the PFCnDs before measuring. Representative results are shown in Figure 1.
  11. Determine the concentration of the PFCnDs utilizing the nanoparticle tracking analyzer (see Table of Materials). Dilute the PFCnDs by 100-1000-fold to ensure accurate measurement of the concentration. The protocol typically yields droplets at a concentration on the order of 1010 particles/mL.
  12. Prepare 10 mL of ultrasound coupling gel in a 50 mL centrifuge tube and add 1% (v/v) or 100 µL of PFCnDs to make solution of ~108 particles/mL. Vortex the solution to mix. Centrifuge the mixture at 4000 x g for 3 min to remove bubbles.

2. Polyacrylamide phantom preparation

  1. Degas water by filling a 500 mL vacuum flask with 400 mL of deionized water, seal with a rubber cork, and connect the flask to the vacuum line. Open the vacuum line and submerge the bottom of the flask in the bath sonicator. Sonicate for 5 min or until no gas bubble formation is visible.
  2. Prepare 10% ammonium persulfate (APS) solution by dissolving 500 mg in 5 mL of degassed water. Gently swirl the solution if the ammonium persulfate does not fully dissolve.
  3. In a 400 mL beaker with a stir bar on a stir plate, add 150 mL of degassed water and 50 mL of 40% (w/v) acrylamide-bisacrylamide solution to form 200 mL of 10% acrylamide-bisacrylamide solution. Stir the mixture at 200 rpm to allow for the proper mixing without introducing bubbles.
    CAUTION: Acrylamide is a carcinogen, and all work should be done in a fume hood with gloves, especially if working with acrylamide in powder form.
  4. Weigh out 400 mg of silica to and add it to the 10% acrylamide-bisacrylamide solution from step 2.3 to form a 0.2 %(w/v) of silica and acrylamide solution.
    CAUTION: Silica when inhaled can be a carcinogen. All work including weighing should be performed in a fume hood.
  5. Prepare a 58 mm x 58 mm x 78 mm square mold with a cylindrical inclusion by cutting off the tips of from a plastic transfer pipette and supporting it in the mold with lab tape. See Figure 2.
  6. Add 2 mL of 10 % APS solution to the beaker to make a final concentration of 0.1% APS and add 250 µL of Tetramethylethylenediamine (TEMED) to the phantom solution. Allow the solution to stir briefly (less than a min).
  7. Quickly pour the solution into the mold, while being careful not to introduce air bubbles into the solution. The solution should polymerize within 10 min. Remove the phantom by running the flat end of a lab spatula around the edge of the mold and inverting the mold.
    ​NOTE: These phantoms can be reused multiple times and should be submerged in water and stored at 4°C.

3. Perfluorocarbon nanodroplet imaging

  1. Turn on and warm up the pulsed laser system for ~20 min following manufacturer instructions. Ensure that the fiber optical bundle is properly connected to the laser output and the two legs are properly placed within the fiber bundle holder.
  2. Turn on the ultrasound imaging system, connect array imaging transducer (L11-4v) to the system and fix the transducer within the holder to align its imaging plane with laser cross section.
  3. Set the pulse repetition frequency of the laser system to 10Hz and place a powermeter at the end of the fiber bundle to measure energy. Tune the q-switch delay until the estimated fluence is 70 mJ/cm2.
    CAUTION: Appropriate eyewear must be worn when firing the laser and laser curtains must enclose the space.
  4. Backfill one of the channels in the polyacrylamide phantom with the ultrasound gel/PFCnD mixture using a 1 mL plastic slip tip syringe. Liberally cover the top of the channel with ultrasound gel and remove any bubbles with a 1 mL plastic slip tip syringe. Place the polyacrylamide phantom underneath the transducer and fiber bundle as shown in Figure 3.
  5. Use the combined laser ultrasound and elasticity (CLUE) imaging platform based on the software20 to image PFCnD synchronized with optical activation. Change the general user-defined parameters in Param structure for imaging: set start/end depth to 0/40mm, center frequency to 6.9MHz, and transducer name to 'L11-4v'.
  6. Define a new RunCase and design a module sequence for repeated optical activation/recondensation and US imaging of PFHnDs. This is done by listing predefined modules such as ultrafast imaging (mUF), external laser (mExtLaser) and idle(mIdle).
    1. Repeat the sequence set mExtLaser-mIdle-mUF-mExtLaser-mUF twice to acquire both n-pulse and p-pulse imaging data.
      NOTE: The first mExtLaser module in each sequence is set as a sham laser by setting ExtLaser.Enable to 0 and the 'mIdle' is included to minimize time between background US images and the n/p-pulse US images after laser activation.
  7. Set module parameters for each module placed in the current run case's module sequence. Access each module parameter by index corresponding to its order in module sequence. Modules will execute pre-defined operations with module parameters user set here.
    1. Set ExtLaser.QSdelay in external laser modules to the value of laser Q-switch delay tuned in step 3.3, in microseconds. This module waits for laser system's flashlamp trigger out and generates Q-switch trigger after the delay specified in QSdelay.
    2. In ultrafast imaging module, set Resource.numFrame to 100, set SeqControl.PRI to 200 (µs), and set TW.polarity to 1 for P-pulse and -1 for N-pulse (see Figure 4 for corresponding pulse shape). This module will transmit ultrafast 0-degree planar wave with pulse type specified in TW.polarity.
      1. Acquire full aperture imaging window of 38.8 mm wide for number of frames in Resource.numFrame, pulse repetition interval of SeqControl.PRI, then save data for off-line processing.
    3. Set SeqControl.lastPRI_Module in idle module to the length of time between laser pulses (100 ms) subtracted by Q-switch delay, imaging data acquisition time (20 ms), and a 20 µs margin for the signal to travel. This module keeps the system under 'no operation' state for the time in SeqControl.lastPRI_Module to fill the time gap between the end of imaging data acquisition and next laser pulse excitation.

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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 ripening21,22 (Fig...

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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...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

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

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Ammonium Persulfate (APS)VWR97064-592
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)Avanti Polar Lipids850365CLipids, 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 Lipids880120CLipids, 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®VWREM-1300acrylamide solution, lower concentration/ powder
IR-1048Sigma405175Infrared dye
L11-4vVerasonics-ultrasound linear array transducer
Microtip 1/8"Qsonica LLC4418microtip for probe sonicator
N, N, N′, N′ -Tetramethylethylenediamine (TEMED)VWR97064-902Used to polymerize polyacrylamide by forming free radicals in the presence of ammonium persulfate
Nova IIOphir-Spiricon7Z01550laser power meter
PerfluorohexaneFluoromedAPF-60Mperfluorocarbon liquid
Phosphate buffered saline (PBS) tabletsVWR97062-732Tablets used to make PBS
Q500Qsonica LLCQ500-110Probe sonicator
Silica gelSigma-Aldrich2885002-25 μm particle size
Tempest 30New wave research-Pulsed laser system
Vantage 128Verasonics-research ultrasound imaging system
Zetasizer Nano ZSMalvern Instruments Ltd-Makes size measurements based on dynamic light scattering

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Optical Droplet VaporizationUltrasound Contrast AgentsLipid Shell NanodropletsProbe SonicationDynamic Light ScatteringPolyacrylamide PhantomCentrifugal SeparationOstwald Ripening
Formulation and Acoustic Modulation of Optically Vaporized Perfluorocarbon Nanodroplets
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