JEFF HARFORD, LI-COR BIOSCIENCES
Targeted therapeutics offer hope for future treatments for a wide range of human diseases. In response to the need for safe and effective treatments, the field of targeted therapeutics has grown substantially in recent years — and optically driven technology is pointing to the conditions in which these treatments will be most beneficial.
Targeted therapeutics are defined as unlabeled molecules designed to act specifically on a biomolecule of interest, or target. NIR fluorescent technology is an invaluable tool for developing both labeled and unlabeled targeted therapeutics.
Unfortunately, this technique has been held back by a lack of awareness of its benefits. NIR detection allows high signal versus background to enable accurate protein expression results even when the expression is low or subtle. This contrasts with visible or chemiluminescence detection, in which visualization or quantification of the expression may be less effective or accurate.
Fluorescent dyes provide a very stable signal — compared to chemiluminescence (a chemical reaction that produces light) — that is unaffected by timing. So, whether expression levels are measured in the minutes, days, or weeks following completion of the blot, the results remain the same. Data captured by an NIR imaging system demonstrates excellent sensitivity, resulting in images with low background and a high signal-to-noise ratio. With high sensitivity, even subtle changes between samples can be observed in a series of images.
When using two spectrally distinct fluorescent dyes, multiple proteins can be detected simultaneously. This ensures that all proteins, such as a target of interest and a normalization agent, are being observed under the exact same experimental conditions.
NIR detection enables plate-based assays to be used with high sensitivity, to allow for a variety of assay parameters, which in turn enables running more samples and controls, ultimately allowing for more experimental conditions. These conditions are imperative for therapeutic development.
Various cell-based assays leverage the advantages of NIR fluorescence to efficiently identify, validate, and characterize targets and leads within their cellular context, to enable the creation of targeted therapeutics.
NIR detection allows high signal versus background to enable accurate protein expression results even when the expression is low or subtle.
NIR fluorescent cell-based assays are well suited for many experiments commonly used in the development of targeted therapeutics, such as the competition assay. This assay can be used to probe receptor-ligand interactions in vitro. Fluorophore-labeled ligands may be used to determine binding affinities to their target receptors. Unlabeled ligands would then be added to compete in binding to the same target, to provide data on ligand specificity.
Various genetic manipulation techniques, such as CRISPR-Cas9 and RNAi knockdowns, can be used to create important experimental controls, study target biology, and assess drug specificity, and they can even be applied as a therapeutic themselves. NIR fluorescent cell-based assays can be used to examine the effect of gene manipulation and to quantify changes in protein expression for pathway characterization.
Therapeutic efficacy may be determined by observing changes in phosphorylation of target proteins following treatment. For example, dose-response assays can be performed to find the IC50 or EC50 (concentration level) of a therapeutic. NIR fluorescent cell-based assays provide the exceptional quality, consistency, and throughput necessary for the development of targeted therapeutics.
Once a therapeutic has been fully characterized in vitro, typically the next step is to validate the therapeutic in vivo. In vivo validation allows for the evaluation of absorption, distribution, metabolism, excretion, and toxicity — to better understand the behavior of a therapeutic.
After delivering a fluorescently labeled therapeutic into an animal, NIR imaging can be used to detect the signal from within the body. The deeper light penetration and reduced tissue autofluorescence from NIR light results in exceptional sensitivity and high signal-to-noise ratios for target detection and quantification.
Further understanding of a therapeutic can be achieved ex vivo using immunohistochemistry. A dye-labeled therapeutic allows for confident determination of localization, biodistribution, and specificity of a therapeutic within excised tissue and organs.
The versatility of NIR fluorescence, along with its many advantages, makes it an excellent tool throughout the development of a targeted therapeutic. This technology merits serious consideration by both the research and medical communities to further realize its potential.
Meet the author
Jeff Harford is senior product marketing manager for LI-COR Biosciences. His more than 30 years of biotech experience include being on the team that launched the first near-infrared DNA sequencer and IRDye near-infrared dyes. He is currently active in introducing novel ways to use near-infrared technologies for therapeutic development.
Views on how to advance biophotonics
NIR detection allows high signal versus background to enable accurate protein expression results even when the expression is low or subtle.
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