UCSD Researchers Develop TRACE Technology to Enhance Targeted Drug Delivery and Reduce Side Effects

University of California San Diego researchers have developed a breakthrough technology that could revolutionize targeted drug delivery and diagnostic imaging by addressing one of medicine's most persistent challenges: the indiscriminate nature of potent therapeutics that harm healthy cells alongside diseased ones.

The new method, called TRACE (tetrazine release and activation by cellular enzymes), uses molecular cages to lock tetrazine molecules in an inert state until they encounter specific cellular enzymes that unlock the cage and activate the therapeutic payload. The research, led by Professor Neal K. Devaraj from UC San Diego's Department of Chemistry and Biochemistry, was published in Nature Chemical Biology.

Addressing Drug Delivery Precision

Current drug delivery systems, particularly for potent medications like chemotherapy, face a fundamental limitation: they often lack specificity, affecting both target cells and healthy tissue. "Potent drugs like chemotherapy can be life-saving, but often with life-threatening side effects," the researchers noted. "Notably, they can be indiscriminate, killing both cancer cells and healthy cells in one swoop."

The TRACE system builds upon bioorthogonal chemistry, a process that allows chemical reactions to occur in living systems without interfering with native biochemical processes. Tetrazine molecules, which Devaraj helped introduce to bioorthogonal chemistry in 2008, react quickly with partner molecules and are already being used in human clinical trials as drug-delivery mechanisms.

However, traditional tetrazine reactions can be indiscriminate, reacting across different cell types in complex biological systems. "In humans, this means imaging may lack precision or drug therapeutics may act on healthy cells in addition to diseased ones," according to the research team.

Molecular Caging Technology

To overcome this limitation, Devaraj's laboratory developed molecular cages that encase tetrazine molecules, preventing them from "clicking" with other molecules until activated by specific cellular enzymes. Once the cage encounters the target enzyme, the tetrazine is rapidly released and can trigger chemical reactions inside target cells.

"What we've shown is that you can, essentially, program the chemistry in specific cell types," stated Devaraj, who also holds the Murray Goodman Endowed Chair in Chemistry and Biochemistry. "You want this to work in a cell type that's over-expressing a particular enzyme, like a cancer cell, but not in other cells — that's what we've figured out."

The research team studied different tetrazine structures to identify those with the fastest uncaging rates and quickest reaction times. They also employed a competing tetrazine-reactive scavenger to suppress activation outside target cells, further improving spatial precision and essentially programming the chemistry to work in specific cell types.

Proof-of-Concept with Doxorubicin

In proof-of-concept testing, the researchers used real enzymes that are over-expressed in certain diseases in conjunction with doxorubicin (DOX), a potent chemotherapy drug with limited clinical applications due to its high cell toxicity. When comparing the tetrazine cages to a control group, doxorubicin was only deployed when the cages came into contact with specific enzymes.

This targeted approach could potentially expand the therapeutic window for doxorubicin and similar potent drugs by concentrating their effects on diseased cells while sparing healthy tissue.

Diagnostic Imaging Applications

Beyond drug delivery, the TRACE technology also enables precise diagnostic imaging. The team developed fluorescent probes that only activate after TRACE activation, demonstrating that only cells expressing both the target enzyme and the molecular tag fluoresced.

In one application, researchers created a probe to label cells with high alkaline phosphatase (ALP) activity, a marker often elevated in certain tumors. The probe attached to a cell-surface "handle" and turned fluorescent only where ALP was active, allowing precise visualization of enzyme activity on live cells.

Future Directions

Devaraj, who has been researching tetrazines for nearly 20 years, continues to explore ways to improve selectivity, which may lead to increased drug efficacy with fewer side effects. "I am very interested in the idea that you could rethink how you deliver drugs and imaging agents, and that you can do these things in the human body," he stated. "That's what led us to develop tetrazine reagents a long time ago. It's turned out to be a really rich space, and, all these years later, they're still offering surprises."

The research team included Caroline H. Knittel, Stormi R. Chadwick, Jacob A. Vance, Cedrik Kuehling, and Neal K. Devaraj, all from UC San Diego. Funding was provided by the National Institutes of Health (R35GM141939) and the German Research Foundation (KN 1447/1-1).