Overview: The talk focused on the synthesis, characterization and use of a novel contrast agent composed of indocyanine green dye for NIR-I photoacoustic (PA) imaging. The contrast agent can be easily tuned to different sizes without enclosure in nanocarriers, has strong optical absorption and PA signal at 895 nm, can be easily functionalized with different targeting molecules and can be imaged for 120 minutes in vivo. The presentation explained details on the genesis of the idea for building a biocompatible contrast agent and give details on its easy synthesis protocols, touch upon a functionalization scheme for adding targeting molecules and demonstrate its use as a PA contrast in mice using the TriTom small animal imaging system. Photoacoustic imaging (PAI) is a noninvasive imaging modality that relies on absorption of laser light and thermal expansion of biological tissues, which generate ultrasonic waves. These ultrasound waves are then used to reconstitute an image of the tissues with anatomical details and functional information. To increase imaging depth and resolution, PAI requires exogenous molecular contrast agents with high optical absorption in the near infrared (NIR). However, the current repository of NIR dyes that are suitable for PAI is extremely limited. The FDA-approved indocyanine green (ICG) is the only commercially available contrast agent with NIR absorbance that is already used for PAI. However, ICG dyes suffer from poor photostability and high clearance rate. In this webinar, Dr. Shrishti Singh presented a synthesis method for clinically translatable ICG-JA whose mean size can be finely tuned from 200 nm to 1000 nm and that does not require encapsulation in a nanocarrier. The talk will also detail complete characterization of the agent and steps for functionalization with targeting peptides or antibodies. Additionally, the webinar also provided details about the PA properties of the contrast in vitro in different conditions including whole blood, followed by details on the photoacoustic imaging in vivo using the TriTom system. Learning Objectives: Get details on the synthesis of a NIR contrast without the need of a nanocarrier. Learn in detail about what characteristics a contrast agent should possess to qualify as a clinically translatable technology. Become familiar with methods to create a targeted contrast agent.

1. Designer and Targeted Contrast Agent for
Photoacoustic Imaging
Dr. Shrishti Singh
Post-Doctoral Researcher,
Bioengineering Department at George Mason University

2. Common imaging modalities
An emerging imaging modality: Photoacoustic Imaging (PAI)
Beyer, T., Bidaut, L., Dickson, J. et al. What scans we will read: imaging instrumentation trends in clinical oncology. Cancer Imaging 20, 38 (2020).
Weber, J., Beard, P. & Bohndiek, S. Contrast agents for molecular photoacoustic imaging. Nat Methods 13, 639–650 (2016).
2

3. Introduction to Photoacoustic Imaging (PAI)
Emma Brown, Joanna Brunker, Sarah E. Bohndiek; Photoacoustic imaging as a tool
to probe the tumour microenvironment. Dis Model Mech 1 July 2019; 12 (7)
3
Wang, L., Yao, J. A practical guide to photoacoustic tomography in the life sciences. Nat Methods 13, 627–638 (2016).

4. Endogenous Chromophores Exogenous Chromophores
• Limitation on the range of biological processes that can
be studied
• Act as a strong background masking out molecules of
interest
Organic cyanine dyes like
Indocyanine Green (ICG)
Metallic nanoparticles
Gold nanoparticles: interesting optical properties and recent
applications in cancer diagnostics and therapy
Huang, X., Jain, P., El-Sayed, I., and El-Sayed, M.
Nanomedicine 2007 2:5, 681-693
4
Types of chromophores or contrast agents
• Increased sensitivity of imaging
• Can help study tumors devoid of high-contrast
endogenous agents

5. PAI can benefit from exogenous contrast agents
Limitations of current contrast agents:
- Size cannot be tuned for different imaging applications.
- Biocompatibility.
- Requires nanocarrier encapsulation for adding targeting
moieties.
- Low photostability of certain agents such as cyanine dyes.
- Low photothermal conversion.
5

6. Liposomes
Metallic Nanoparticles
Potential issues with current ICG-JA formulations:
• Need of a nanocarrier to add targeting moieties
• Complex preparation methods
• No intrinsic control over the size of formed JA
• Additional downstream processing required
6
Exogenous contrast agents (contd.)
Indocyanine Green (ICG)
J-aggregates of Indocyanine Green (ICG-JA)

7. 7
Singh. et al, Size-tunable ICG-based contrast agent platform for targeted near-infrared photoacoustic imaging, Photoacoustics, 29(2023)
ICG J-aggregates with azide (JAAZ)

8. Scale bar: 2 µM
8
Characterization of JAAZ particles
Singh. et al, Photoacoustics, 29(2023)

9. Scale bar: 250 nm
9
Size-tuning properties
Singh. et al, Photoacoustics, 29(2023)

10. Audience Poll
10

11. Tailoring with different targeting molecules
11

12. 12
Singh. et al, Photoacoustics, 29(2023)
Addition of targeting peptide

13. Sample Size (nm) Zeta Potential (mV)
Before Drying After Drying Before Drying After Drying
JAAZ 1,083 ± 52 1,030 ± 256 -52.1 ± 2 -55.0 ± 5
N-JAAZ 281.8 ± 37 360.9 ± 38 -54.7± 1 -57.1± 3
Sample size is n=3 for DLS measurement; p=0.8470=ns for JAAZ; p=0.4622=ns for
N-JAAZ (Statistics are for DLS measurement only); n=2 for zeta potential
measurement.
Stability of the particles in physiological media
Optical Stability in Whole
Blood
13
In-vitro Stability
JAAZ N-JAAZ
Singh. et al, Photoacoustics, 29(2023)

14. 14
In-vitro PA studies
Singh. et al, Photoacoustics, 29(2023)

15. PA signal compared to whole blood
In-vitro Cell Targeting Studies
15
In-vitro PA studies (contd.)
Singh. et al, Photoacoustics, 29(2023)

16. a. Superficial thoracic vessels
b. Liver
c. Spleen
d. Intestines
e. Thoracic vertebrae
16
In-vivo PA studies
Tri-Tom Imaging System
Singh. et al, Photoacoustics, 29(2023)

17. 17
In-vivo PA studies
Singh. et al, Photoacoustics, 29(2023)
Red: OxyHb; Blue: DeoxyHb; Green: RGD-JAAZ

18. Tunable sizes
from micron to
nanosized.
Robust and
Easy synthesis
method.
Composed of
FDA approved
dye
High optical absorption in NIR-I
region.
Can be multiplexed
with different
targeting moieties.
18

19. Rémi Veneziano,
Assistant Professor
Parag Chitnis,
Associate Professor
Esra Oktay
Sean Hu
Joshua Bush
Merlyn Vargas
Dylan Scarton
Leandro Soto Cordova
Amber Hartman
Sally Farang
Graduate Student
Giovanni Giammanco
Dr. Dylan Lawrence, Photosound Inc.
Acknowledgement

20. WWW.SCINTICA.COM
Q&A Session
WWW.SCINTICA.COM
INFO@SCINTICA.COM
Please enter your questions
in the Q&A section.
Thank You!
20