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Novel Class of Turn-ON Near-Infrared Probes for Diagnosis and Imaging of Inflammation and Cancer

A novel class of Turn-ON probes with Near-Infrared (NIR) fluorescence mode of action was developed. The NIR probes are designed for activation by specific overexpressed enzymes.

This novel and simple strategy introduces a Turn-ON mechanism in molecules with cyanine spectroscopic characteristics. The approach is based on a new design of a distinctive fluorochrome, which can be modularly derivatized to generate different imaging probes for biological research and clinical needs.

The Need and Potential Application
Despite the significant progress made in research and treatment in recent years, cancer is still one of the main causes of death in developed countries. Currently, many cancer patients are diagnosed when their disease is too far advanced to be cured. As a result, there is an urgent medical need for improved early detection methods and novel targeted delivery systems.

Our technology establishes the basis for the design and synthesis of a new class of NIR molecular probes with a Turn-ON mode of operation. Such probes could be utilized in a straightforward manner as research tools for (i) in vitro applications such as cellular uptake, trafficking and proof of concept related to enzymatic levels activating the fluorescent system, and most importantly for (ii) imaging and monitoring cancer inoculated in laboratory animals. In the next phase, the NIR probe could be used for early detection of cancer in patients and to obtain clinical information for non-invasively monitoring cancer progression and efficacy of treatment.

Simplicity - compared to most Turn-ON probes of cyanines that are forced to use a fluorophore and a quencher (or two fluorophores which quench each other), QCy7 can be simply masked with an applied trigger.
Increased signal-to-noise ratio as compared to FRET-based probes.
The novel NIR probe demonstrates high quantum yield in water and simple synthesis.
QCy7 can be modularly derivatized with various substrates to generate the corresponding probes.

Stage of Development
QCy7, synthesized with UV-Vis spectrum, exhibits two absorption peaks with the maximum at 460 nm and 570 nm. As expected, the fluorescence spectrum shows an emission peak in the NIR region at a wavelength of 715 nm. The obtained large Stokes shift between the excitation and the emission wavelengths of about 150 nm is a desirable phenomenon in fluorescence probe design, which assists in increasing the signal-to-noise ratio.

To demonstrate the in vivo potential of QCy7 for imaging of biological factors, we synthesized a probe that was designed for activation by H2O2. The results indicate detection of hydrogen peroxide by full conversion of the sulfo-QCy7 within 30 minutes. No NIR fluorescence is observed in the absence of hydrogen peroxide. The sensitivity of the probe towards detection of hydrogen peroxide was evaluated.  The probe can straightforwardly detect hydrogen peroxide concentrations below 1 mM.

To demonstrate the imaging capabilities of our probe in vivo, we used a model of acute inflammation in mice, induced by lipopolysaccharide (LPS). The signal-to-noise ratio of the NIR fluorescence intensity observed by the hydrogen peroxide imaging probe in mice was about ten-fold higher than in the control group. Such a ratio should be adequate to obtain a strong contrast image. As far as we know, this is the highest ratio ever obtained by a Turn-ON probe for an in vivo hydrogen peroxide imaging probe.

Provisional patent application submitted, October 2010. 

Supporting Publications
Karton-Lifshin, N., Segal, E., Omer, L., Portnoy, M., Satchi-Fainaro, R., Shabat, D., “A Unique Paradigm for a Turn-ON Near-Infrared Cyanine-Based Probe: Non-Invasive Intravital Optical Imaging of Hydrogen Peroxide”, J. Am. Chem. Soc., 2011, 133, 10960-5.