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(May 3, 2023) Webinar: Exploring a Novel NIR-2 Photoacoustic Agent to Improve Image Contrast for Deep Tissue Applications
The document introduces a novel biodegradable and biocompatible semiconductor nanocrystal called bornite that could improve photoacoustic imaging contrast for deep tissue applications. Experiments show bornite generates a 5x stronger photoacoustic signal than gold nanorods and indocyanine green. It also allows 2-3x deeper imaging of up to 5cm in tissue phantoms and provides around 2x better contrast in vivo. Bornite could be a safer and more effective photoacoustic contrast agent compared to existing alternatives.
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(May 3, 2023) Webinar: Exploring a Novel NIR-2 Photoacoustic Agent to Improve Image Contrast for Deep Tissue Applications
- 1. Exploring a NovelNIR-2 Photoacoustic Agent to Improve Image Contrast for Deep Tissue Applications Vinoin Devpaul Vincely PhD Candidate, Department of Biomedical Engineering, Tulane University
- 2. Photoacoustic Imaging (PAI) •PA imaging uses nanosecond laser pulses to generate detectable acoustic waves • The hybrid nature allows for imaging beyond the limits of optical diffusion (1-2 cm). • The unique nature of PAI allows probing of anatomical, functional and molecular imaging applications. Choi et. al. Journal of Applied Physics, 2020 Liu et. al. Chem Soc Rev, 2019 2 Nanosecond Laser Pulse Transducer Acoustic Waves
- 3. Optical Chromophores • Opticalchromophores facilitate this conversion through thermoelastic expansion. • Optical chromophores can be endogenous or exogenous in nature. Choi et. al. Journal of Applied Physics, 2020 Liu et. al. Chem Soc Rev, 2019 3 Nanosecond Laser Pulse Transducer Acoustic Waves
- 4. • Endogenous chromophoresare naturally occurring molecules intrinsically found in biological systems. • Eg: Hemoglobin, Melanin & Lipids • Optical contrast predominantly in visible- first near infrared window (NIR-I) • High tissue scattering & laser safety limitations in this window. Endogenous Contrast Upputuri et. al., Journal of Biomedical Optics, 2019 4
- 5. Imaging in NIR-IIwindow 5 • Advantages: – Higher laser exposure thresholds – Reduced tissue optical scattering – Minimal autofluorescence • Disadvantages: – Low optical contrast from endogenous chromophores – High absorption by water Upputuri et. al., Journal of Biomedical Optics, 2019
- 6. Exogenous Contrast • ImprovesPA image contrast significantly compared to endogenous contrast. • Tunability of peak wavelength of absorption. • Needed for targeted molecular imaging and drug delivery. • Some of these agents – Small molecule dyes – Metallic nanoparticles – Semiconductor nanocrystals
- 7. Exogenous Contrast Small Molecule DyesMetallic Nanoparticles Semiconductor Nanocrystals Examples Indocyanine Green (ICG) Gold Nanorods (AuNRs) Metal sulfides (CuS) Advantages • FDA-approved for in vivo applications • Improved optical extinctions due to localized surface plasmon resonance (LSPR) • Tunable while being chemically inert • Exhibit high LSPR at ultrasmall size (< 10 nm) • Good photostability Limitations • Optical absorption peak overlaps with hemoglobin, making in vivo distinction difficult • Poor photostability • Limited retention due to rapid clearance, limiting targeted delivery • Large Sizes (100-200 nm) • Slow clearance with concerns of toxicity • Heavy metal containing compositions • Non-biodegradable • Concerns of toxicity
- 8. Novel Biodegradable andBiocompatible Semiconductor NC • Bornite (Cu5FeS4) with a peak LSPR at ~1100 nm • Ultrasmall size distribution (2 – 6 nm) • Photothermally efficient and stable • Biocompatible composition with low cytotoxicity • Uniquely biodegradable Kay et. al. “Controlled Synthesis and Exploration of CuxFeS4 Bornite Nanocrystals” Chemistry of Materials, 2021 Normalized Absorbance (a.u.)
- 9. Objective • Compare PAsignal generation by bornite against conventional PA contrast agents (ICG & AuNRs). • Explore maximum PA imaging depth that can be achieved with bornite in an ex vivo porcine tissue phantom. • Explore improvement in image contrast using bornite in an in vivo mice model. 9
- 10. Synthesis of Bornite •Protocol developed by Kays et. al. 2021 • Oxidized bornite nanocrystals were synthesized using a hot colloidal injection. • The peak LSPR develops in the NIR region using different rates of particle oxidization. • Bornite exhibits good biodegradation with up to 70% of injected dose being excreted/degraded within 2 weeks. Kay et. al. “Controlled Synthesis and Exploration of CuxFeS4 Bornite Nanocrystals” Chemistry of Materials, 2021
- 11. 2-D PA ImagingInstrumentation • Opotek Benchtop laser – Imaging ranges: 690-950 nm, 1064 nm, 1200-2400 nm • Verasonics Vantage 256 – Coupled with a L7-4 Linear array transducer • PhotoSound Legion AMP – Front-end amplifier – 40 dB amplification 11
- 12. 3-D PA ImagingInstrumentation • PhotoSound Tritom – 6 MHz Arc Transducer (96 channel) – ~36s for 3D volume acquisition 12
- 13. • Varying concentrationsof contrast agents (Bornite, AuNRs and ICG) were loaded in vinyl tubing (0.9 mm diameter). • PA imaging was performed at the respective wavelength of peak absorption – Bornite & AuNRs – 1064 nm – ICG – 780 nm • PA signals were normalized to laser energy level. 13 Phantom Design for Characterization of PA Signals from Contrast Agents 3 5 2 1 4 1 cm
- 14. 14 • The novelsemiconductor nanocrystals show a 5x increase in PA signal compared to Gold Nanorods. Characterization of PA Signals from Contrast Agents 1 cm 1 cm (b) 1 2 1 0 PA Intensity (au.)
- 15.
- 16. Imaging Protocol toExplore Maximum Imaging Depth in an ex vivo Porcine Phantom 16 Tube Light Path Transducer 4 cm 3 cm 2 cm • Slices (2 – 5cm) of ex vivo porcine tissues were held together in a gelatin mold. • A tube to carry contrast agents placed underneath the slices. • 2-D transducer was placed antiparallelly to the laser illumination. • Three solutions imaged: – ~300 OD ICG solution – ~3 OD Bornite solution – ~3 OD AuNR solution
- 17. Maximum Imaging Depthin an ex vivo Porcine Phantom 17 • Bornite showed a 2-3x increase in contrast to noise (CNR) compared to AuNRs at all imaging depths. Pork slice Tube 1 cm 1 0 Intensity (au.) 1 0 PA Intensity (au.) Tube ROI Bornite AuNRs
- 18. Imaging Protocol toTest Improvement of PA Image Contrast in an in vivo Mice Model • Pregnant CD-1 mice injected with a solution of ~7 OD of respective contrast agent (Bornite or AuNRs). • Pre-injection imaging at 808 nm for anatomical reference. • Imaging at 1064 nm for both agents, before injection and at 5 min intervals for 90 mins. 18
- 19. 19 Pre-injection Bornite AuNRs Improvementin PA Image Contrast in an in vivo Mice Model
- 20. Mammary Artery Pre-injection Bornite AuNRs Improvementin PA Image Contrast in an in vivo Mice Model - Segmentation
- 21. Ovarian Artery Pre-injection Bornite AuNRs Improvement in PAImage Contrast in an in vivo Mice Model - Segmentation
- 22. Placenta Pre-injection Bornite AuNRs Improvementin PA Image Contrast in an in vivo Mice Model - Segmentation
- 23. Improvement of PAImage Contrast in an in vivo Mice Model - Segmentation • An ~2x increase in image contrast is observed from animals injected with the bornite. Novel NC
- 24. Conclusions • Introduced auniquely biodegradable and biocompatible copper-iron based (bornite) NIR-2 semiconductor NC for PA imaging. • Provided a systematic comparison of its PA performance against other conventional agents in phantoms and an in vivo mice model. • An overall 5x increase in PA signal compared to conventional agents was explored. • Observed a 2-3x increase in CNR for imaging depths up to 5 cm. • ~2x increase in image contrast from in vivo mice model was observed. 24
- 25. Future Work • Explorea systematic analysis of the biodegradability of current formulation of bornite • Target these particles to image specific organs – such as the placenta 25
- 26. Acknowledgements • BFIL Lab –Dr. Carolyn Bayer • Dennis Lab – Dr. Allison Dennis – Xingjian Zhong • Funding: – R01 HD097466 – Research Corporation for Science Advancement, Chan- Zuckerberg Initiative 26 Biomedical and Functional Imaging Laboratory https://bfilab.org
- 27. Q&A Session WWW.SCINTICA.COM INFO@SCINTICA.COM Please enteryour questions in the Q&A section. Thank You!
Editor's Notes
- #2 Thanks Lawrence, for that introduction and welcome to everyone who’s joining us today. I would like to take a moment to thank Scintica for this opportunity to present some of my latest work. Today I will be talking about “A novel NIR-2 contrast agent for deep tissue photoacoustic imaging. This is a collaborative work with Dr. Allison Dennis and her group from Northeastern University. Who have provided the NIR-2 contrast agents imaged in this study.
- #3 Photoacoustic imaging uses nanosecond laser pulses to diffusely illuminate samples that then generate acoustic waves that can be detected using conventional ultrasound transducers. The hybrid optical illumination and acoustic detection allows for imaging beyond optical diffusion limits – up to 2 cm. The unique approach of PAI can provide anatomical, functional, molecular information.
- #4 Optical chromophores in the sample, convert the incident optical energy and release it as acoustic waves through a process called thermoelastic expansion, which can then be detected using conventional ultrasound transducers. These optical chromophores can be either endogenous or exogenous in nature.
- #5 Endogenous chromophores are light absorbing molecules that are intrinsically found in biological systems. These include substances like oxy- & deoxygenated hemoglobin, melanin, lipids and water. Hence a common PA application is to capitalize on the differences in the optical characteristics between oxy- and deoxy- hemoglobin by imaging at 690 nm where deoxy shows higher absorption and at 950 where oxy shows higher absorption and spectrally unmixing the concentration of each chromophore to predict local oxygenation. However, looking at this plot we see that optical contrast from endogenous chromophores is predominantly in the visible- 1st NIR window. Imaging at this window for deep tissue can be limited due to high tissue scattering and limited laser exposures available.
- #6 Moving to the second NIR window allows for some distinct advantages towards deep tissue imaging. These include a higher threshold for laser safety exposures for tissues. The exposure at 1064 nm is 1000 mW/cm2 which is roughly 3.5 times more than that allowed at 800 nm. Tissue scattering in this window is exponentially reduced. As you can see in this plot, tissue scattering by different tissue types in the 2nd NIR window is reduced anywhere between 2-10x compared to the visible-1st NIR window. Finally, there is minimized autofluresence from biological tissue within this window. However, there are some key disadvantages while using this window, namely – the low optical contrast from endogenous chromophores and high attenuation by water.
- #7 To combat this, we need exogenous contrast agents that can significantly improve PA contrast compared to endogenous chromophores. Certain agents allow for the tunability of their wavelengths of peak absorption, allowing us to preform PA imaging at desired optical excitations. And specific imaging applications, such as molecular imaging and targeted drug delivery, need exogenous contrast agents. Some of these include - small molecule dyes, metallic nanoparticles and semiconductor nanocrystals
- #8 ICG Small molecule dyes are commercially available, organic dyes such as indocyanine green (ICG). The key advantage of these dyes are that they organic with some having FDA approval for in-vivo imaging However, they exhibit optical characteristics that overlap quite well with the other endogenous chromophores like hemoglobin, making in-vivo differentiation difficult. They show poor photostability at in-vivo temperatures that diminish the photoacoustic intensities over time. Finally, ICG exhibits a limited retention of 3-5 mins due to its propensity to bind to serum album followed by rapid clearance via the liver, limiting its use for targeted delivery and functional imaging AuNRs Metallic nanoparticle, such as gold nanorods have been widely used due to their unique optical and physiochemical properties. The key advantage of metallic nanoparticles is that they show greatly improved optical extinctions compared to organic dyes, due to a phenomenon called localized plasmon resonance. Next gold-based nanoparticles shows high tunability of the wavelength of peak absorption by shape modulation, while being chemically inert. However, they do come with their fair share of limitations, namely, - large sizes (100 – 200 nm) for imaging in NIR-2 window. These large sizes show slow clearance with concerns of long-term bioaccumulation and cytotoxicity. Semiconductor nanocrystals Semiconductor nanocrystals, such as metal sulfides, have rapidly gained popularity as nano probes. They are particularly of interest due to their ability to exhibit high LSPR at ultrasmall size (that is <10 nm) – while being photostable. However, metal sulfides predominantly use heavy-metals in their composition that do not readily degrade in biological systems, leading to concerns of cytotoxicity.
- #9 In this, we present a novel semiconductor contrast agent with non-heavy metal containing composition with a peak LSPR at 1100 nm. Here I have attached a representative plot of the normalized absorbance spectra of bornite against other agents used in this study. These particles are ultrasmall with size distributions between 2-6 nm. They show good photothermal efficiency And have a biocompatible composition with low cytotoxicity. Furthermore, they are uniquely biodegradable
- #10 There are 3 studies that I will be presenting today. First is a comparison of the PA signal generation from the novel NIR-2 against other conventional agents – ICG and gold nanorods. We will explore the maximum PA imaging depth that can be achieved in an inhomogeneous ex-vivo porcine tissues using bornite. Finally, we will explore the improvement in image contrast using these agents in an in-vivo mice model
- #11 The synthesis protocol of the bornite nanocrystals was developed by Kays et. al. You can find more details in the following citation. Briefly, oxidized bornite nanocrystals were synthesized using a hot colloidal injection of Cu and Fe, yielding crystals with a size distribution between 2.7 – 6.1 nm. The peak LSPR is tuned to the NIR-2 window by modulating the particles oxidation. As you can see in the graph here. As we increase the time for oxidation we observe a strong peak in the NIR2 window Preliminary studies of biodegradeation show that bornite is able to clear 70% of the injected dose within 2 weeks
- #12 Next, I will provide a brief description of our imaging systems. We used an opotek benchtop laser for all optical excitations. Our laser has a capability of imaging in wavelengths ranges of 690-950 nm, 1064 and 1200-2400 nm. We perfomed 2D imaging with a Verasonics Vantage 256 data acquisition system. This was coupled to a L7-4 linear array transducer The native vantage system has limitations in broad-band signal acquisition due to the low input impedance of the data acquisition port. This results in the loss of low-frequency signals. To avoid these low frequency losses, a front-end amplifier with a high acoustic impedance, the Photosound Legion Amplifier, was used. The inclusion of the amplifier provides a 40dB boost in low signal detection.
- #13 Next we performed 3D imaging with the Photosound TriTom system.
- #15 Here, I have plotted the mean PA signal generated from the various sample dilutions on the y-axis and the respective sample optical densities in the x-axis. The green data points correspond to ICG while the black data points correspond to Bornite. Keep in mind, that with the ODs matched, we can see bornite expressing an expected PA response with increased concentrations.
- #17 Now we want to probe the maximum achievable depth using Bornite under inhomogeneous tissues. To do this an ex-vivo porcine phantom was prepared. 3-slices of the porcine tissues with varying depths (2,3,4 cm) as shown, were held together in a gelatin mold. Similar to before, a tube to carry the contrast agents was placed underneath the tissue slices. As shown here, the samples were illuminated from the bottom while the transducer was placed at the top, antiparallelly. Three solutions were imaged with this phantom – highly concentration of ICG and a 1.06 mg/ml solution of Bornite were imaged at each tissue slice. Keep in mind that the ICG solution is significantly more concentrated than bornite and we will get to why that is.
- #25 In conclusion, we have used a biodegradable, biocompatible Bornite nanocrystals to generate PA signals under inhomogeneous tissue phantoms. We have shown that imaging at NIR-2, allows for greater fluence penetration with imaging higher depths upto 4 cm in our porcine tissue model. Finally, we have also shown that significantly lower doses of bornite can be used to generate ample PA signals compared to highly concentrated doses needed for ICG. In future work, we will explore the potential of using these nanocrystals for in-vivo imaging in mice models
- #26 Preliminary degradation results were performed for a different oxidation of bornite. Hence, for future studies a systematic analysis of the biodegradability of the specific oxidized bornite will be explored. Next, a protocol for targeted delivery of bornite to imaging specific anatomy – such as the placenta, will be explored.
- #27 With that, I would like to acknowledge our team, my PI Dr. Carolyn Bayer and our collaborators, Dr. Allison Dennis from Northeastern university Finally, I would like to acknowledge our funding agencies and thank all the members of BFIL team!