Optically-Triggered Nanodroplets for Enhanced Ultrasound and Photoacoustic Imaging Alexander Hannah
Department of Biomedical Engineering University of Texas at Austin
Medical imaging continues to revolutionize clinical medicine
• • • • • • •
•
More effective surgical treatment Shorter hospital stays Elimination of exploratory surgery Earlier and more accurate diagnosis and treatment of cancer More efficient treatment after surgery Better treatment of strokes Better treatment of cardiac conditions Rapid diagnosis of vascular conditions
American Cancer Society
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Noninvasive imaging modalities
Betz, C. S., et al. OA Publishing London (2013)
• Ultrasound’s greatest weakness is difficulty in interpreting images, particularly discerning various structures in the scan • Developing ultrasound contrast agents would have a broad impact due to the modality’s clinical prevalence
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Imaging contrast agents
1) Provide high signal against background 2) Targeting helps identify source of disease
Shields, A. F. et al, Nature Medicine (1998).
Wu, A. M. et al, PNAS (2000).
PET imaging is an example of high contrast because the body does not have any endogenous signal -All of the signal comes from the radioactive tracers
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Microbubbles improve ultrasound contrast
5 mm Microbubbles scatter sound due to an acoustic impedance mismatch
Z
= ρ
x
c
Acoustic impedance (Rayl)
Density (kg m-3)
Speed of sound (m s-1)
Tissue (water)
~1.5 x 106
1000
1500
Microbubbles (perfluorocarbon gas)
~1.2 x 103
3.5
340
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Limitations of current clinical microbubbles Limitations:
Ultrasound
PET
• Contrast of US is low, even with microbubbles
• Large size (> 5 um) limits them to vasculature, so molecular info is difficult • Short half life (minutes) results in short imaging time 5
Photoacoustic imaging complements ultrasound Ultrasound imaging probe
Optical absorption coefficient (m-1)
Pulsed laser
Photoacoustic pressure wave (Pa)
𝑝 = Γ𝜇𝑎 𝐹
Laser fluence (mJ cm-2)
Gruneisen coefficient (dimensionless) 6
Photoacoustic contrast is most commonly from thermal expansion
PA Imaging
Contrast Agents Dyes
Nanoparticles
50 nm
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Limitations of photoacoustic contrast agents Contrast Agents
Limitations:
Fluorescent dyes
• Shallow imaging depth • Molar extinction coefficient of dyes is low (105 cm-1 M-1)
Nanoparticles
• Agents made of metals need regulatory approval before clinical use
Fluorescent dyes Nanoparticles
• Contrast is based on thermal expansion • Low amplitude PA signal
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Photoacoustic nanodroplets utilize three contrast mechanisms Before irradiation
Stabilizing surfactant shell
1 First laser pulse
Liquid perfluorocarbon core
2 Subsequent laser pulses
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Subsequent laser pulses
Anechoeic Hyperechoic
Encapsulated photoabsorber
Liquid nanodroplet
• High PA contrast from droplet vaporization • US contrast from bubbles • Dynamic contrast for coregistration of signal
Pulsed laser Vaporization Photoacoustic wave
Gas Microbubble
Thermal expansion
1
2
3
1
2
3
US
PA
Wilson et al. Nature Communications (2013).
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Nanodroplets target leak from vessels and are activated Cancer cells
Targeted nanodroplets
• • • •
Gas microbubbles
Pulsed laser irradiation
Sub-micron size allows for extravasation due to EPR Remotely triggered by pulsed laser irradiation Provide both photoacoustic and ultrasound contrast May encapsulate therapeutics for tumor treatment
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Overall Goal & Specific Aims The overall goal is to design and implement a robust, optically triggered dual contrast perfluorocarbon nanodroplet, with improved properties over current contrast agents, to assist this dynamic diagnostic platform for the early detection of cancers.
1. Expand the photoacoustic nanodroplets’ capabilities -Clinical translation, sensitivity to vaporization, image contrast, stability, and targeting
2. Characterize the properties of the particles in US and PA imaging applications -Size, absorption, encapsulation, image contrast, vaporization threshold, recondensation
3. Image photoacoustic nanodroplets in a biological environment -Ex vivo and in vivo validation using modified particle formulations
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Specific Aim 1
1. Expand the photoacoustic nanodroplets’ capabilities -Clinical translation, sensitivity to vaporization, image contrast
2. Characterize the properties of the particles in US and PA imaging applications -Size, absorption, encapsulation, image contrast, vaporization threshold, recondensation
3. Image photoacoustic nanodroplets in a biological environment -Ex vivo and in vivo validation using modified particle formulations
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Nanodroplets with indocyanine green dye for clinic Stabilizing surfactant shell
• Synthesized with clinicallyapproved materials • Average diameter 600 nm
Liquid perfluorocarbon core
• Absorb light at 800 nm
Encapsulated indocyanine green dye
Rodriguez, Victoria B., et al. Journal of biomedical optics (2008)
Extinction Coefficient (cm -1)
400 350 300
Count Count
250 200 150 100
10 µm
1 0.8 0.6 0.4 0.2
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600 Diameter (nm)
800
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800 1000 Wavelength (nm)
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Dye is encapsulated in the particle core • Only dye encapsulated in particle is retained after washing
• Confocal microscopy identifies location of dye in droplet
• Encapsulation efficiency = 85-90% Confocal fluorescence
Brightfield
ICG-loaded droplets in water Blank ICGloaded droplets in droplets aqueous ICG in water
ICGBlank loaded droplets in droplets aqueous in water ICG
10 µm
Blank droplets in aqueous ICG
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Droplets may be responsive to 1064 nm light Stabilizing shell
Liquid perfluorocarbon core Encapsulated dye or gold nanorods
aq AuNR
50 nm
PFC
water
AuNR in PFC
Activating with 1064 nm light has advantages: 1. PA contrast is improved • Tissue absorption is weaker at 1064 nm 2. Operational cost is reduced • Inexpensive Nd:YAG laser can be used Homan, Kimberly, et al. Optics letters (2010)
Gorelikov, Ivan, et al. Langmuir (2011)
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Perfluorocarbon boiling point changes droplet properties Stabilizing shell Perfluorocarbon core Encapsulated photoabsorber
Volatile
Particle Boiling point (oC)
Stable
* Perfluorobutane boiling point (-1o C)
Perfluoropentane boiling point (29o C)
Body temperature (37o C)
Perfluorohexane boiling point (56o C)
Low boiling point
High boiling point
-Higher US contrast (vaporization efficiency)
-Longer circulation time
-Longer shelf life
-Deeper imaging due to increased sensitivity
-Facile synthesis
-Repeatable vaporization Asami, Rei, and Kenichi Kawabata, 2012.
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Linear Ultrasound Intensity (a.u.)
BLInking nanoCapsules can be activated repeatedly BLInking nanoCapsules (BLInCs) Fluorosurfactant shell
0
0.75 Time (s)
1.5
Hyperechoic Anechoic
Anechoic Laser pulse
Liquid perfluorohexane core
Near-infrared absorbing dye
• Stable at body temperature • Can be activated repeatedly • Undergo unique blinking behavior, causing spikes in US signal
Liquid nanodroplet
Recondensing
Gas microbubble
Liquid nanodroplet
Ultrasound Imaging 0 dB
1 mm -25 dB Optical Microscopy
20 µm
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Aim 1 Summary 1. Synthesized ICG-loaded droplets for clinical translation 2. Made droplets with high aspect ratio gold nanorods for high sensitivity activation (1064 nm) and high contrast imaging
Normalized Extinction Coefficient (a.u.)
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 400
3. Synthesized blinking nanocapsules for repeatable vaporization
Aqueous PEGylated Nanorods Nanorod-Loaded Droplets
1 0.9
500
600
700
800 900 1000 Wavelength (nm)
1100
1200
1300
Hyperechoic Anechoic
Anechoic Recondensing
Laser pulse
Liquid nanodroplet
Gas microbubble
Liquid nanodroplet 18
Specific Aim 2
1. Expand the photoacoustic nanodroplets’ capabilities -Clinical translation, sensitivity to vaporization, image contrast, stability, and targeting
2. Characterize the properties of the particles in US and PA imaging applications -Size, absorption, encapsulation, image contrast, vaporization threshold, recondensation
3. Image photoacoustic nanodroplets in a biological environment -Ex vivo and in vivo validation using modified particle formulations
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Samples are imaged using ultrasound and photoacoustics 3 samples of droplets were imaged: 1) Blank droplets in water
2) Dye-loaded 3) Blank droplets in droplets in water aqueous dye
109 droplets/mL
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Dye-loaded droplets vaporize upon irradiation
Dye-loaded droplets in water
Blank droplets in aqueous dye
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Ultrasound and photoacoustic imaging confirms vaporization
• Photoacoustic signal is emitted only from 1st pulse • Only ICG-loaded droplets emit signal
• Over 20 dB increase in ultrasound signal upon irradiation • Only dye-loaded droplets enhance signal 2 mm
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Droplet vaporization observed with microscopy and US Brightfield light microscope
Nanodroplets on microscope slide
Nanodroplets embedded in a tissuemimicking phantom Laser for droplet vaporization Before irradiation
Ultrasound image
Laser scan area 100
200
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400
500
600
After irradiation
700
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900
5 mm 50
~10 µm
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*Only a fraction of droplets vaporize
400
450
500
20 μm
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Signal increases with vaporization efficiency • Identical samples of nanodroplets imaged at three different temperatures
• Different boiling point droplets irradiated
100
100
200
200
Droplet boiling point (stability) 300
300
400
400 500
500
600
600
700
700
Perfluorobutane Perfluoropentane Perfluorohexane 800 (b.p. = -1oC) (b.p. = 56oC) (b.p. = 29oC) 900 800
900
50
50
100
150
100
200
150
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250
250
Increasing US/PA signal is likely due to increasing droplet vaporization efficiency
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Droplets can be modified to provide increased US contrast • Samples of droplets prepared with increasing numbers of gold nanorods
Nanorods per Droplet
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Laser induces vaporization at 2-4 mJ/cm2
• Droplet-embedded phantom is imaged with ultrasound before and after laser irradiation (λ = 1064
5 mm
nm)
• Change in US signal increases with fluence
• First observable signal is at ~2 mJ/cm2
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Phantom imaging of BLInCs shows specificity Phantom Schematic
Phantom Imaging
• Irradiated with 10 Hz pulsed laser (40 mJ/cm2) • Imaged with high frame rate US(670 frames/s)
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US imaging of BLInCs inclusion in phantom Phantom Imaging
1 mm * 20x slowed 28
0
0.15
0.3
0.45
0.6
0.75
0.9
1.05
1.2
Blinking pixel Static pixel
0
0.15
0.3
0.45
0.6
0.75
0.9
1.05
1.2
0.1
Blinking pixel Static pixel
.08
A
.06
.04
.02
0
.02
-1.2
Time (s)
1 mm -50
-0.6
-0.3
0
0.3
0.6
0
Nanodroplet Map
Ultrasound Signal (dB)
Ultrasound Signal (dB)
0
For each pixel: 1. Linear US signal over time 2. Differentiated US signal 3. Autocorrelate differential signal 4. Display ratio of peaks of autocorrelation A/B
-0.9
0.9
1.2
Delay (s)
Time (s)
Ultrasound Image
B
0.03
Nanodroplet signal (a.u.)
Blinking pixel Static pixel
Ultrasound Intensity Autocorrelation (a.u.)
Differential Ultrasound Intensity (a.u.)
Linear Ultrasound Intensity (a.u.)
Phantom imaging of BLInCs shows specificity
1 mm -50
0.01
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Aim 2 Summary 1. Imaged ICG-loaded nanodroplets in solution for quantification of image contrast 2. Measured influence on droplet vaporization of temperature, laser fluence, photoabsorber loading, and perfluorocarbon boiling point
100 200 300 400 500
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3. Imaged blinking nanocapsules in phantom and processed images for high contrast map of the particles
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150
Specific Aim 3
1. Expand the photoacoustic nanodroplets’ capabilities -Clinical translation, sensitivity to vaporization, image contrast, stability, and targeting
2. Characterize the properties of the particles in US and PA imaging applications -Size, absorption, encapsulation, image contrast, vaporization threshold, recondensation
3. Image photoacoustic nanodroplets in a biological environment -Ex vivo and in vivo validation using modified particle formulations
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Nanodroplets provide dual contrast ex vivo
5 mm
5 mm
Laser on
(λ = 1064 nm)
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Lymph node high contrast imaging of BLInCs
30 min
Ultrasound Signal (dB)
0
Off
On
Lymph node Liquid nanodroplet
Gas microbubble
-50
Injected BLInCs + pulsed laser + image processing
0.1
Lymph node
1 mm
-50
Nanodroplet Signal (a.u.)
Luke, G. P. et al, Cancer Research (2014).
Ultrasound Signal (dB)
0
No injected BLInCs + pulsed laser + image processing
0.01
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Imaging of BLInCs in brain shows microvasculature 0
0 0.3
0.1
0
-0.1
-0.2
-0.3
-50
0.2
0.1
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-0.1
-0.2
-0.3
-50
Nanodroplet Signal (a.u.)
0.2
0.05 0.3
Ultrasound Signal (dB)
Ultrasound Signal (dB)
Retro-orbital injection of BLInCs
1 mm
0.03
Locating BLInCs identifies microvasculature for studying neurophysiology 34
Aim 3 Summary 1. Imaged gold nanorod-loaded nanodroplets ex vivo to show high contrast PA imaging 2. Imaged BLInCs injected into mouse tongue in vivo and identified particles in lymph node using correlation algorithm 3. Retro-orbitally injected BLInCs and imaged brain to identify microvasculature
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Future Work – characterizing tissue properties 100
• Measuring droplet behavior may identify tissue characteristics 100
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Interstitial pressure
Tissue stiffness 300
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Soft Hard
Soft phantom (healthy tissue) Stiff phantom 700(tumor) 250
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US Intensity
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20406080100 120 140
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Future Work – active molecular targeting of droplets Stabilizing surfactant shell
Cell-specific targeting antibody
Liquid perfluorocarbon core
• EGFR targeted nanodroplets
Encapsulated indocyanine green dye
Breast cancer cells under microscope
• Breast cancer cells • Incubated for 2.5 hours
Droplets without antibody
Droplets with antibody
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Future Work – BLInCs as a therapeutic agent
• Sonoporation • BLInCs may encapsulate drugs • Disrupt the bloodbrain barrier
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Conclusions Specific Aim 1: Expand the properties of photoacoustic nanodroplets
Specific Aim 2:
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Characterize the properties of the particles in US and PA imaging applications
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Specific Aim 3 Image photoacoustic nanodroplets in a biological environment 39
Acknowledgments
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Alexander Hannah
Department of Biomedical Engineering University of Texas at Austin
Publications and Presentations Peer-Reviewed Journal Publications: [1] A. Hannah, G. Luke, S. Emelianov, “Blinking phase-change nanoparticles for background-free ultrasound imaging,” to be submitted February 2015. [2] A. Hannah, G. Luke, K. Wilson, K. Homan, S. Emelianov, “Indocyanine Green-Loaded Photoacoustic Nanodroplets – Dual Contrast Nanoconstructs for Enhanced Photoacoustic and Ultrasound Imaging” ACS Nano 8.1 (2013): 250-259. [3] A. Hannah, D. VanderLaan, Y.S. Chen, S. Emelianov, “High contrast photoacoustic nanodroplets responsive to 1064 nm light,” Biomedical Optics Express 5.9 (2014): 3042-3052.
Abstracts and Proceedings: [4] A. Hannah, G. Luke, S. Emelianov, “Blinking perfluorocarbon nanoparticles for background-free optically activated ultrasound and photoacoustic imaging,” presented at the 2015 SPIE Photonics West Symposium: Photons Plus Ultrasound: Imaging and Sensing (2015). [5] A. Hannah, S. Emelianov, “Optically Activated Ultrasound Contrast Agents for Diagnostic Imaging,” poster presentation at the Biomedical Engineering Society Conference, 2014. [6] D. Santiesteban, A. Hannah, L. Suggs, S. Emelianov, “Development of a Controlled Oxygen Delivery System to Increase Adipose Stem Cell Survival,” presentation at the Biomedical Engineering Society Conference, 2014. [7] A. Hannah, G. Luke, S. Emelianov, “Imaging laser activated microbubble recondensation kinetics using high frame rate ultrasound,” IEEE Ultrasonics Conference, 2014. [8] A. Hannah, D. VanderLaan, S. Emelianov, “Optimizing properties of photoacoustic nanodroplets for ultrasound and photoacoustic image contrast” presented at the 2015 SPIE Photonics West Symposium: Photons Plus Ultrasound: Imaging and Sensing (2014). [9] A. Hannah, G. Luke, K. Wilson, K. Homan, S. Emelianov, “ICG-loaded perfluorocarbon nanodroplets for molecularly targeted photoacoustic imaging,” presented at the World Molecular Imaging Congress, 2013. [10] A. Hannah, G. Luke, K. Wilson, K. Homan, S. Emelianov, “Photoacoustic signal enhancement using optical vaporization of ICG-loaded nanodroplets,” presented at SPIE Photonics West, 2013. [11] A. Hannah, K. Wilson, K. Homan, S. Emelianov, “Ultrasound-induced cellular uptake of plasmonic gold nanorods,” SPIE BioS 2012 proceedings, Vol. 7899 (2012) [12] K. Wilson, A. Hannah, K. Homan, S. Emelianov, “Design and applications of photoacoustic nanodroplets in imaging and therapy,” presented at the Journal of the Acoustical Society of America, 129, 2642 (2011).
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