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An understanding of how molecules cross biological barriers is important both for pharmacological applications and basic research. Present methods, however, allow only a qualitative assessment of passage across membranes. Paul Wender and his colleagues at Stanford University hoped to go from this qualitative understanding to a more quantitative tool.

They designed a releasable luciferin−transporter system comprising an octaarginine transporter conjugated to a luciferin molecule via a disulfide linker. Upon entry into the cell, this linker is cleaved, releasing free luciferin. The uptake of this construct into cells can be measured in cells expressing luciferase, which catalyzes the oxidation of luciferin, resulting in the release of a photon. “The beauty of the system is that unlike fluorescence, we get a photon for every turnover event, so it's pristinely quantifiable,” says Wender.

In the work described in the Journal of the American Chemical Society, Wender and colleagues report the synthesis of releasable luciferin−transporter conjugates and validation of this system in prostate cancer cells stably transfected with a luciferase-encoding gene. They were able to quantify, in real time, the uptake and release of the conjugates by measuring the luminescence.

Armed with this assay, researchers now can study transport and delivery of cargo in different cell types and tissues, as well as design conjugates that transport molecules effectively and release them at the desired rates. Further, this assay can be used to quantify the expression levels of bioactivatable targets, such as proteases, in cells and tissues: researchers could design a transporter with a linker containing a specific protease cleavage site and then use the luciferase assay to determine the activity of this enzyme. Using this information, they can design conjugates that release their cargo only in specific tissues.

One important advantage of this luciferin-based system is that researchers do not have to fix cells or section tissues or even kill animals. As Wender summarizes, “It gets about as close to where you'd like to be with research. And that is, you are just observing something, you are not perturbing it. You are sending something in and something is coming out, and that is the photon that allows you to count the effect.”