NIR dyes for Bioimaging Applications: A Comprehensive Research

Near-infrared (NIR) dyes are the category of molecules that absorb light in the near-infrared region of the electromagnetic spectrum (typically 700-900 nm) and emit light at a slightly longer wavelength. Upon absorption of light at a specific wavelength, these molecules briefly enter an excited state and then return to their ground state by emitting light at a lower energy (longer wavelength, emission). This distinctive characteristic enables them to “glow” when exposed to NIR light, making them useful for imaging.

NIR Dyes for Bioimaging

NIR light can penetrate deeper into tissues compared to visible light, enabling visualization of structures and processes within living organisms. Also, biological tissues produce less background fluorescence in the NIR range, resulting in clearer images with higher contrast. NIR dyes can thus serve as valuable tools for in-vivo imaging offering advantages such as – being non-invasive, allowing dynamic monitoring of biological processes, targeted imaging as NIR dyes can be conjugated to specific molecules, like antibodies, to highlight particular cells or tissues of interest.

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Classification of NIR dyes

Based on Chemical Structure

Cyanine Dyes

Structure: These dyes are characterized by a polymethine chain that connects two heterocyclic nitrogen-containing rings.
Properties: Cyanine dyes offer bright fluorescence, tunable emission wavelengths, and good photostability.
Examples: Indocyanine Green (ICG), IRDye 800CW
Applications: Angiography (blood vessel imaging), tumor imaging, and lymphatic mapping.

Squaraine Dyes

Structure: Squaraine dyes contain a central squarylium ring with electron-donating groups attached.
Properties: These dyes are known for their intense absorption and emission in the NIR region, as well as their high quantum yield.
Examples: IRDye 800RS, SQ-212
Applications: Tumor imaging, cell tracking, and diagnostics

Porphyrin-Based Dyes

Structure: These dyes are based on the porphyrin ring system, which is also found in hemoglobin and chlorophyll.
Properties: They exhibit strong absorbance in the visible and NIR range, making them versatile for various imaging modalities.
Examples: Protoporphyrin IX (PpIX), Chlorin e6
Applications: Tumor imaging, photodynamic therapy, and angiography.

Other Dyes

Xanthene Dyes
D-A-D Dyes
BODIPY Dyes
Fluorescent Proteins
Quantum Dots

Based on Wavelength

NIR-I dyes

These dyes absorb and emit light in the shorter near-infrared region, typically between 700-900 nm. While they provide acceptable resolution for imaging, their penetration depth in tissues is restricted, due to greater light scattering at these wavelengths. Examples include Indocyanine Green (ICG), IRDye 800CW, etc.

NIR-II dyes

NIR-II dyes absorb and emit light in the longer near-infrared region, ranging from 900-1700 nm. Because there is less dispersion at longer wavelengths, deeper tissue penetration is possible. Examples include CH1055-PEG (a modified cyanine dye), IR-1061, and other experimental dyes.

Feature	NIR-I dyes (700-900 nm)	NIR-II dyes (1000-1700 nm)
Tissue Penetration	Moderate	Deep
Resolution	Good	Excellent
Autofluorescence	Low	Very Low

Applications of NIR Dyes in-vivo Imaging

NIR Dyes have paved the way for a range of possibilities for visualizing and understanding biological processes within living organisms. Some of the common in-vivo applications where NIR dyes are used, are as follows –

Sentinel Lymph Node Mapping (SLNM)

https://www.cancer.gov/about-cancer/diagnosis-staging/staging/sentinel-node-biopsy-fact-sheet

The sentinel lymph nodes are the first lymph nodes to which cancer cells may spread from the original tumor. Sentinel lymph node mapping is a medical procedure used to identify if cancer has spread from the primary tumor to the lymphatic system.

How NIR Dyes Help? A NIR dye, such as indocyanine green (ICG), is injected near the tumor site. It travels through lymphatic vessels and accumulates in the sentinel lymph nodes. Surgeons can then easily identify and remove these nodes for biopsy, aiding in cancer staging and treatment planning.

Dye	Clinical Trial
Indocyanine Green	FDA approved, Widely used
SGM-101	Phase II Trials

Tumor Margin Visualization & Surgery

NIR dyes that preferentially bind to tumor cells can be used to “light up” the tumor, directing surgeons to its exact position and size. Those with a high affinity for specific tumor markers (e.g., EGFR, HER2) can clearly delineate the boundary between the tumor and healthy tissue. This real-time imaging enhances tumor removal accuracy and achieves precise resections while potentially lowering the chance of recurrence.

Dye	Clinical Trial
OTL38 (Pafalocianine)	FDA approved
LS301	Phase I/II Trials
CLR1502	Phase I/II Trials
BLZ-100	Early Phase
MM-302	Phase-I

Angiogenesis Imaging

Angiogenesis imaging focuses on observing the formation of new blood vessels (angiogenesis), which aids tumor growth and spread. NIR dyes that bind to angiogenesis markers (such as VEGF) enable non-invasive monitoring of tumor vascularity. This data can be used to track therapy progress and forecast outcomes.

The NIR dye HX005, is currently in preclinical studies with potential for trials.

Lymphatic Imaging

Lymphatic imaging involves assessing the structure and function of the lymphatic system, which plays a crucial role in immune function and cancer metastasis. NIR dyes can help visualize lymphatic vessels and track the flow of lymph, aiding in the diagnosis of lymphatic disorders and assessing the risk of cancer spread.

Dye	Clinical Trial
BLZ-150	Early Stage
OTL703	Preclinical/Early trials, focus on breast cancer

Other Applications

NIR dyes are also being investigated for:

Image-Guided Drug Delivery: NIR dyes attached to therapeutic agents allow for targeted delivery and monitoring of drug distribution within tumors.
Inflammation Imaging: NIR dyes that bind to inflammatory markers can help visualize and monitor inflammatory conditions.
Cardiac Imaging: NIR dyes can assess blood flow and perfusion in the heart, aiding in the diagnosis of heart diseases.

FDA Approved NIR Dyes

Indocyanine Green (ICG)

Indocyanine Green is a cyanine dye with peak absorption and emission in the NIR-I range, offering moderate tissue penetration. It stands as the most widely used and versatile NIR dye in clinical practice.

Applications:

Angiography: Visualizing blood vessels to assess circulation and detect abnormalities.
Tumor Surgery: Identifying tumor margins and guiding surgical resection.
Sentinel Lymph Node Mapping (SLNM): Determining the spread of cancer cells in lymphatic systems.
Other: Used in ophthalmic angiography, liver function assessment, and more.

Strengths:

Well-established safety profile
Relatively inexpensive
Versatile for multiple applications

Limitations:

Limited tissue penetration compared to NIR-II dyes
Non-specific binding, requiring conjugation for targeted imaging

Methylene Blue

Methylene Blue is a multifaceted blue colored dye which exhibits some fluorescence in the NIR-I region.

Applications:

Sentinel Lymph Node Biopsy: Used to stain lymph nodes for easier identification during surgery.
Photodynamic Therapy (PDT): When activated by light, it can generate reactive oxygen species to destroy cancer cells.

OTL38 (Pafolacianine)

Pafolacianine is a folate-targeted NIR fluorescent imaging agent. It binds to folate receptors, often overexpressed in ovarian cancer cells, allowing surgeons to visualize cancerous tissue.

Applications:

Ovarian Cancer Surgery: Aids in the intraoperative identification of malignant ovarian cancer lesions.

Strengths:

Targeted approach enhances specificity
Real-time visualization during surgery

Limitations:

Currently approved only for ovarian cancer
Requires a specific NIR fluorescence imaging system

Promising NIR Dyes in Clinical Trials

NIR-I Dyes

Bevacizumab-IRDye800CW: This targeted imaging agent combines the anti-angiogenic drug bevacizumab with the NIR dye IRDye800CW. This conjugate aims to visualize and monitor tumors more effectively during surgery and potentially improve treatment response assessment. It is currently in Phase I/II Trials.
SGM-101: Currently in Phase II trials, this cyanine-like dye is being investigated for sentinel lymph node mapping, a critical step in cancer staging.

NIR-II Dyes

CH1055-PEG: This modified cyanine dye is one of the first NIR-II dyes to be tested in humans. It has shown promising applications in tumor visualization and deep tissue imaging, offering advantages over traditional NIR-I dyes. It is currently in Phase II trials.

For a complete list of NIR dyes with their potential applications and trial phase, please click here.