IISc researchers have drastically improved (up to 98%) the cellular uptake of fluorescent probe naphthalimide by substituting two of its hydrogen atoms with iodine. The iodine atom forms a halogen bond with the MCT8 receptor that ferries the molecule across the cell membrane. The probe molecule with iodine will also help in studying the uptake of thyroid hormone.
By substituting two hydrogen atoms of a commonly used fluorescent probe naphthalimide with iodine, researchers at the Indian Institute of Science (IISC) Bengaluru have been able to remarkably increase the cellular uptake of the probe molecule from 5-8% to 98%. The probe molecule with two substituted iodine atoms was found to be safe.
Fluorescent probes absorb light of a specific wavelength and emit light of a different, typically longer, wavelength (a process known as fluorescence), and are used to study biological samples. Fluorescent probes are routinely used for studying drug delivery, membrane activity and cellular functions.
The very low ability of the probe molecules to cross the cell membrane has been a challenge. Despite the limitations, naphthalimide has been routinely used as it can be easily synthesised in large quantities and its fluorescence can be altered. By substituting two hydrogen atoms with iodine, the IISc researchers led by Prof. Govindasamy Mugesh have effectively addressed the problem of uptake.
The halogen bonding is better for iodine compound compared with chlorine and bromine.
The researchers substituted two hydrogen atoms of the probe molecule with chlorine, bromine and iodine. The size of the probe molecule increased when the hydrogen atoms were replaced with halogen atoms. Compared with iodine, the chlorine and bromine atoms are smaller in size. “We would expect the smaller molecules to get into cells easily. But to our surprise, we observed the uptake or transport of the iodine-containing molecule, which is bigger than the other two, was better. It was almost 100%,” says Prof. Mugesh from the Institute’s Department of Inorganic and Physical Chemistry and corresponding author of a paper published in the journal Angewandte Chemie.
The iodine forms a halogen bond with the receptor (MCT8) found on the cell surface that ferries the molecule across the membrane. “The halogen bonding is better for iodine compound compared with chlorine and bromine. This could be the reason why the iodine-containing molecule was taken up better despite its bigger size,” says Harinarayana Ungati from the Department of Inorganic and Physical Chemistry, IISc and first author of the paper.
The MCT8 receptor is known to transport thyroxine, which is a thyroid hormone containing iodine, into the cells. “Our study suggests that halogen bonding may play a key role in the transport of thyroid hormones across the cell membrane,” says Prof. Mugesh.
When the researchers substituted only q hydrogen atom with one iodine atom the uptake was 14-38%. It reached up to 98% when two iodine atoms were substituted with. In contrast, the percentage transport was only 15% and 22% for chlorine and bromine, respectively, when two atoms were substituted with.
Heavier halogen atoms are known to quench fluorescence, and there was reduction in fluorescence when the probe molecule containing two iodine atoms was used. “Since there is large uptake of the molecule containing iodine, we did not see a reduction in the overall fluorescence inside the cell,” says Prof. Mugesh.
The iodine-substituted probe molecule was studied using three mammalian cell lines and endothelial cells isolated from the human umbilical cord. The uptake of iodine-containing probe was similar in all instances.
“The probe molecule with iodine will help in studying the uptake of thyroid hormone, which is generally difficult to monitor. Traditionally, radio-labelled hormone is used for studying thyroid uptake in mammalian cells,” says Dr. Vijayakumar Govindaraj from the Department of Inorganic and Physical Chemistry, IISc and coauthor of the paper.