Newly developed fluorescent probes detect lysosomal pH

Researchers at Michigan Technological University (Houghton, MI) have developed a new set of fluorescent probes that can detect problems with lysosomes, which are known as the garbage disposals of animal cells.

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"A lot of diseases are related to problems with lysosome functions," says Jingtuo Zhang, a chemistry doctoral candidate at Michigan Tech. Zhang and his advisor, Haiying Liu, have developed the new probes, essentially chemical dyes that illuminate lysosome structures with fluorescence. The probes respond to pH, which gives a more clear idea of a cell's health, Zhang says.

Responding to different acidic conditions within lysosomes is a unique feature of the new probes. This is important because small changes in pH can reflect much bigger problems. Currently, few commercially available lysosome fluorescent probes are sensitive to pH.

The fluorescence is near-infrared (NIR), meaning that the probes emit light that can penetrate deeper than commercial dyes—making for better bioimaging of lysosome structures. The dyes also have minimal toxicity, which is possible due to the team decreasing toxicity by creating new molecules via a process called synthesis.

The molecules created by the team use a core of boron dipyrromethene (BODIPY). Rings of carbon make up the bulk of BODIPY, like a three-legged stool. The working part of the core is a section with fused carbon rings, nitrogen, boron, and fluoride. The BODIPY edges then are modified by piperazine rings. Finally, the team connected long chains. All together, the probe is a leggy molecule centered on the BODIPY rings, trailing flexible carbon chains kinked with oxygens. The oxygen-rich chains make the molecules more water-soluble, making it easier on the body and for bioimaging applications. The piperazine addition then targets lysosome cells specifically, allowing the BODIPY core to do its work in the right place and fluoresce.

In the fluorescent images, the chemicals appear as glowstick-bright colors. "There are clear dot structures when we zoom in on the images," Zhang says, pointing out where the green and red colors have concentrated in the rounded lysosome structures outside the blue nucleus.

Ideally, Zhang explains, the team wants to see orange, which shows where their probes and popular commercial dyes overlap in the lysosomes. The overlap is good, indicating the probes are indeed targeting the right cellular structure. Ashutosh Tiwari, an assistant professor of chemistry at Michigan Tech, is particularly interested in those orange zones.

Tiwari worked with Zhang and Liu to apply their synthesized probes and oversaw the cell cultures and testing. He says the team is trying to balance the chemical's impact on the cell without losing its lysosome-targeting and fluorescent functionality.

The researchers are currently looking to commercialize their product. They also plan to continue making modifications to the BODIPY fluorescent probes to further enhance the lysosome targeting and lower the toxicity even further.

Full details of the work appear in the Journal of Materials Chemistry B; for more information, please visit http://dx.doi.org/10.1039/C4TB01878H.

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