Researchers create new class of fluorescent dyes to detect reactive oxygen species in vivo

December 15, 2008
Confocal fluorescent images of rat aortic smooth muscle cells (a) incubated with hydro-Cy3, (b) treated with angiotensin II and incubated with hydro-Cy3, and (c) incubated with angiotensin II and TEMPOL before adding hydro-Cy3. Credit: Georgia Tech Image: Courtesy of Kousik Kundu

Researchers have created a new family of fluorescent probes called hydrocyanines that can be used to detect and measure the presence of reactive oxygen species. Reactive oxygen species are highly reactive metabolites of oxygen that have been implicated in a variety of inflammatory diseases, including cancer and atherosclerosis.

"We've shown that the hydrocyanines we developed are able to detect the reactive oxygen species, superoxide and the hydroxide radical, in living cells, tissue samples, and for the first time, in vivo," said Niren Murthy, assistant professor in the Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University.

A new family of reactive oxygen species (ROS) probes -- called hydrocyanines -- is synthesized by reducing a cyanine dye with sodium borohydride. Reacting with an ROS oxidizes the hydrocyanines into fluorescent cyanine dyes. Georgia Tech Image: Courtesy of Kousik Kundu

Details of the hydrocyanine synthesis process and experimental results showing the ability of the dyes to detect reactive oxygen species in cells, tissues and mouse models were reported on December 8 in the online version of the journal Angewandte Chemie International Edition. This research is supported by the National Institutes of Health and the National Science Foundation.

The researchers have created six hydrocyanine dyes to date – hydro-Cy3, hydro-Cy5, hydro-Cy7, hydro-IR-676, hydro-IR-783 and hydro-ICG – but say that there are potentially 40 probes that could be created. The dyes vary in their ability to detect intracellular or extracellular reactive oxygen species and by their emission wavelength – from 560 to 830 nanometers.

Fluorescing at higher wavelengths allows the hydrocyanine dyes to be used for deep tissue imaging in vivo, a capability that dihydroethidium (DHE), the current "gold standard" for imaging reactive oxygen species, does not have. The dyes also have other advantages over DHE.

"When DHE comes into contact with reactive oxygen species, it oxidizes into ethidium bromide, a common mutagen, which means it's toxic and can't be injected inside the body," explained Murthy. "DHE also auto-oxidizes in the presence of aqueous solutions, which creates high levels of background fluorescence and interferes with reactive oxygen species measurements."

Hydrocyanines are also simple and quick to synthesize, according to Coulter Department postdoctoral fellow Kousik Kundu. Sodium borohydride is added to commercially available cyanine dyes and the solvent is removed – the one-step process takes less than five minutes.

W. Robert Taylor, a professor in the Coulter Department and Emory's Division of Cardiology, and Emory postdoctoral fellow Sarah Knight, tested the ability of the dyes to detect reactive oxygen species inside of cells and animals.

For their first experiment, they tested the ability of hydro-Cy3, which has an emission wavelength of 560 nanometers, to detect reactive oxygen species production in the aortic smooth muscle cells of rats. They incubated the cells with hydro-Cy3 and angiotensin II, which is a stimulator of reactive oxygen species that is implicated in the development of atherosclerosis and hypertension.

Results showed that cells incubated with angiotensin II and hydro-Cy3 displayed intense intracellular fluorescence, whereas control cells incubated with hydro-Cy3 and phosphate buffer saline displayed significantly lower fluorescence. When they introduced TEMPOL, a molecule that intercepts the reactive oxygen species so that they cannot interact, the cells treated with angiotensin II and hydro-Cy3 displayed a dramatic decrease in fluorescence.

"This test demonstrated that the cellular fluorescence was due to intracellular reactive oxygen species production," said Murthy. "What was even more exciting was that we saw that once the hydrocyanine dye was oxidized, it stayed in the cell and the fluorescence was not extinguished by cellular metabolism, which is what happens with DHE."

The researchers also investigated the ability of hydro-Cy3 to image reactive oxygen species production in live mouse aorta tissue, which exhibit a physiological environment that closely resembles in vivo conditions. Explants were incubated with hydro-Cy3 and either lipopolysaccharide endotoxin (LPS), an inflammatory molecule that binds to aortic cells and causes reactive oxygen species to be produced, or the control saline solution.

Samples treated with hydro-Cy3 and LPS showed fluorescence intensity almost four times greater than explants treated with hydro-Cy3 and saline. Once more, adding TEMPOL to the sample with hydro-Cy3 and LPS decreased the fluorescence to a level comparable to the control saline explants.

After the successful cell culture and tissue experiments, the researchers progressed to in vivo mouse imaging studies. Hydro-Cy7 was selected for the in vivo tests because of its higher emission wavelength of 760 nanometers. LPS-treated mice showed twofold greater fluorescence intensity in the abdominal area than those treated with saline.

"Given their ability to detect reactive oxygen species in living cells, tissue samples and in vivo, we believe these dyes will enhance the ability of researchers to measure reactive oxygen species," noted Murthy.

The researchers' ultimate goal, though, is to use the dyes in clinical applications.

"We want to use these hydrocyanine dyes to detect overproduction of reactive oxygen species at an early stage inside the body so that we can identify patients who are more likely to suffer from these inflammatory diseases," added Murthy.

Source: Georgia Institute of Technology

Explore further: Steel byproducts trap phosphorous in agricultural drainage water

Related Stories

Photosynthesis gene can help crops grow in adverse conditions

September 18, 2015

A gene that helps plants to remain healthy during times of stress has been identified by researchers at Oxford University. Its presence helps plants to tolerate environmental pressures like drought—and it could help create ...

Scientists identify protein at death's door of cells

September 17, 2015

A protein embedded in the surface of mitochondria - the energy-producing batteries of living cells - opens the door to cell death, causing cells to experience severe power failures, according to new work by researchers at ...

Combustion's mysterious "QOOH" radicals exposed

August 11, 2015

Researchers can now discriminate between the previously unidentified hydroperoxyalkyl radicals found in the early stages of the combustion process from similar compounds, thanks to data from the Advanced Light Source at Lawrence ...

Recommended for you

Most EU nations seek to bar GM crops

October 4, 2015

Nineteen of the 28 EU member states have applied to keep genetically modified crops out of all or part of their territory, the bloc's executive arm said Sunday, the deadline for opting out of new European legislation on GM ...

The dark side of Nobel prizewinning research

October 4, 2015

Think of the Nobel prizes and you think of groundbreaking research bettering mankind, but the awards have also honoured some quite unhumanitarian inventions such as chemical weapons, DDT and lobotomies.

Internet giants race to faster mobile news apps

October 4, 2015

US tech giants are turning to the news in their competition for mobile users, developing new, faster ways to deliver content, but the benefits for struggling media outlets remain unclear.

Fusion reactors 'economically viable' say experts

October 2, 2015

Fusion reactors could become an economically viable means of generating electricity within a few decades, and policy makers should start planning to build them as a replacement for conventional nuclear power stations, according ...

1 comment

Adjust slider to filter visible comments by rank

Display comments: newest first

1 / 5 (1) Dec 16, 2008
Long, long ago a doctor won a Nobel prize for "Cancer Cause And Cure" involving adverse
forms of Oxygen. I believe his name was "Walberg"
I thought he had something then, and still do!
Outer orbit electron speed & spin are suspect!

Please sign in to add a comment. Registration is free, and takes less than a minute. Read more

Click here to reset your password.
Sign in to get notified via email when new comments are made.