“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.
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.Technical Contact: Niren Murthy (404-385-5145);
Abby Vogel | Newswise Science News
Water forms 'spine of hydration' around DNA, group finds
26.05.2017 | Cornell University
How herpesviruses win the footrace against the immune system
26.05.2017 | Helmholtz-Zentrum für Infektionsforschung
Staphylococcus aureus is a feared pathogen (MRSA, multi-resistant S. aureus) due to frequent resistances against many antibiotics, especially in hospital infections. Researchers at the Paul-Ehrlich-Institut have identified immunological processes that prevent a successful immune response directed against the pathogenic agent. The delivery of bacterial proteins with RNA adjuvant or messenger RNA (mRNA) into immune cells allows the re-direction of the immune response towards an active defense against S. aureus. This could be of significant importance for the development of an effective vaccine. PLOS Pathogens has published these research results online on 25 May 2017.
Staphylococcus aureus (S. aureus) is a bacterium that colonizes by far more than half of the skin and the mucosa of adults, usually without causing infections....
Physicists from the University of Würzburg are capable of generating identical looking single light particles at the push of a button. Two new studies now demonstrate the potential this method holds.
The quantum computer has fuelled the imagination of scientists for decades: It is based on fundamentally different phenomena than a conventional computer....
An international team of physicists has monitored the scattering behaviour of electrons in a non-conducting material in real-time. Their insights could be beneficial for radiotherapy.
We can refer to electrons in non-conducting materials as ‘sluggish’. Typically, they remain fixed in a location, deep inside an atomic composite. It is hence...
Two-dimensional magnetic structures are regarded as a promising material for new types of data storage, since the magnetic properties of individual molecular building blocks can be investigated and modified. For the first time, researchers have now produced a wafer-thin ferrimagnet, in which molecules with different magnetic centers arrange themselves on a gold surface to form a checkerboard pattern. Scientists at the Swiss Nanoscience Institute at the University of Basel and the Paul Scherrer Institute published their findings in the journal Nature Communications.
Ferrimagnets are composed of two centers which are magnetized at different strengths and point in opposing directions. Two-dimensional, quasi-flat ferrimagnets...
An Australian-Chinese research team has created the world's thinnest hologram, paving the way towards the integration of 3D holography into everyday...
24.05.2017 | Event News
23.05.2017 | Event News
22.05.2017 | Event News
26.05.2017 | Life Sciences
26.05.2017 | Life Sciences
26.05.2017 | Physics and Astronomy