Researchers Create New Photodynamic Molecular Beacon to Help Target Tumors and Minimize Side Effects

May 15, 2007— Photodynamic therapy for cancer uses light to activate drugs to kill cancer cells. A light-sensitive drug is injected into the body and moves through the patient's bloodstream to accumulate in a tumor. Doctors then shine a laser on the area to activate the drugs.

Canadian doctors say photodynamic therapy works best with cancer of the esophagus, lungs, skin, brain and bladder, if caught in early stages.

In the USA, photodynamic therapy is approved by the Food and Drug Administration to treat or relieve the symptoms of esophogal cancer and non-small cell lung cancer.

One of the problems with photodynamic therapy is that patients may remain over-sensitive to light for weeks afterwards, or even suffer from burns in tissue close to the target area.

Now, research scientists in Canada have taken a step forward by using an enzyme found in specific cancer cells to help light a "photodynamic molecular beacon" inside the cell. The method may make it easier to target tumors without damage to surrounding healthy tissue.

In a study published today in the Proceedings of the National Academy of Sciences (PNAS), Dr. Gang Zheng, a senior scientist now at Ontario Cancer Institute who specializes in prostate cancer research and molecular beacons and Dr. Brian Wilson, a medical biophysicist at Ontario Cancer Institute and the University of Toronto describe their method and present results from tests on mice. They have been collaborating with one another for over three years, and in 2006 OCI recruited Dr. Zheng from the University of Pennsylvania.

"Photodynamic therapy," they write, "is an emerging cancer treatment modality involving the combination of light, a photosensitizer (PS), and molecular oxygen." It offers "unique control" because "the key cytoxic agent, singlet oxygen (¹O₂)," is produced only where the light source shines on it. The therapy can be controlled in three ways to achieve selective tissue targeting:

1. Control how light is delivered to the diseased tissue by positioning, for example by using advanced fiber optics to insert light into the prostate. However, this method cannot achieve high selectivity between diseased and healthy tissue, so it is relatively toxic..
2. Control of how the photosensitizer is delivered to the tumor tissue. This method "has resulted in improvement" but "is still vulnerable to collateral damage to surrounding normal tissues."
3. "A new direction is to exert control of the [photosensitizer's] ability to produce ¹O₂. This third level of control has the potential to acheive ultimate PTD selecivity to cancer cells while leaving normal cells unharmed."

Molecular beacons (which can be used simply for microscopic imaging of molecules) are known technically as FRET-based target activable probes (FRET stands for "fluorescence resonance energy transfe"). By combining the principals of FRET and photodynamic therapy, Dr. Zheng and Dr. Wilson say, their aim is to overcome the problem of collateral damage by achieving "unprecedented selectivity."

To this end they set out to make a photodynamic molecular beacon comprised of "a disease-specific linker," a photosensitizer (PS), and a ¹O₂ quencher, "so that the PS's photoactivity is silenced until the linker interacts with a target molecule, such as a tumor-associated protease." Their first shot, in 2004, used a cell apoptosis marker, caspase-3. Although it apparently worked, it was difficult to validate, since it was hard to measure light-toxicity (i.e. burn damage) in the dead cells. They decided they needed to find "a specific cleavable peptide linker to target tumor-associated proteases."

For this they turned to one of the matrix mellaproteinases (MMPs), a family of pro-enzymes that are thought to play an important role in normal tissue remodeling and repair as well as in degenerative conditions of tissue overgrowth such as fibrosis and arthiritis and in the spread of cancer cells (metastasis). In tumors, MMPs break down the matrix around cells and stimulate the cells to travel and invade surrounding (healthy) tissue. Zheng and Wilson focused on MMP7, which is high in "pancreatic, colon, breast, and nonsmall-cell lung cancer." MMP7 has been identified also as "a key factor in prostate cancer-induced bone destruction and a potential therapeutic target" (Cancer Cell, 2005).

Zheng and Wilson created their new MMP7 triggered photodynamic molecular beacon like an intricate firecracker that uses MMP7's cleaving power not to light a fuse, exactly, but to loose a bond. The bond, or linker, is designed to keep the photosensitizer from igniting the cytotoxic oxgen until the damage will be take place inside of cancer cells. Paradoxically, to keep ignition from occuring the two ends have to be kept close to one another, so that the quencher can "damp" the photosensitizer. To make this work the linker has to double round, or self-fold.

Photodynamic molecular beacon. Figure 1, PNAS full text, .pdf, p.8990

The new beacon (PMB), PP_MMP7B, is made from a high-grade photosensitizer (labeled PS above) at one end of a petide string and, at the other end, a "black hole" quencher (BHQ3) (labeled Q). The quencher is powerful enough to smother both fluorescence and singleton oxygen. As long as PS and Q are close together, nothing happens. The peptide linker's "self-folding" structure keeps things that way except in the vicinity of MMP7. Zheng and Wilson made their linker out of a peptide sequence that MMP7 can dissolve. Until the beacon comes in contact with MMP7 inside a tumor, it remains "optically silent" and "photodynamically inactive." When it enters MMP7-expressing cells, the MMP7 there "should induce specific cleavage of the peptide linker and remove the Pyro from the vicinity of BHQ3, restoring its fluorescence and photoreactivity." Subsequently, when a laser beam is shone -- and penetrates the cell, the tumor, or the body -- the targeted cells should light up "and produce cytotoxic ¹O₂, while leaving normal cells undetectable and unharmed."

Does it work?

After syntheiszing their new drug, Zheng and Wilson tried it out in a test tube, where MMP7 did the trick as expected. The Pyro fragment ended up in or near to the cancer cells' mitochondria (an effective target). Next, under a microscopic it worked on a type of human nasal cancer cells high in MMP7; and (as predicted) it did not work on a type of breast cancer cells low in MMP7. Finally they tried it on mice, where, again, it worked as predicted, shrinking tumors. Zheng and Wilson say most likely the shrinkage was from apoptosis.

"The process really is about controlling the drug’s ability to produce this reactive form of oxygen," says Dr. Zheng. "For the first time, using mouse models and on separate cells, we have shown that it is possible to limit the collateral damage to surrounding normal cells using this approach."

Clinical trials in patients, they say, are still a year or two away.

"This is an exciting step in the fight against cancer," said Dr. Wilson. "This process should greatly enhance the therapeutic window, making tumors much more susceptible to PDT damage than normal cells and tissues."

This study is a follow-up to Dr. Zheng’s and Dr. Wilson’s 2004 concept paper, published in the Journal of the American Chemical Society, which laid out the theory of what they have now proven in the lab.

Sources & Links

Photodynamic molecular beacon as an activatable photosensitizer based on protease-controlled singlet oxygen quenching and activation Gang Zheng, Juan Chen, Klara Stefflova, Mark Jarvi, Hui Li, and Brian C. Wilson. PNAS published May 14, 2007 OPEN ACCESS ARTICLE -- available in .pdf

Protease-Triggered Photosensitizing Beacon Based on Singlet Oxygen Quenching and Activation Brian C. Wilson, Gang Zheng, et al. Jour. Am. Chem. Soc, Aug. 2004.

Gang Zheng, PhD is Senior Scientist, Division of Biophysics and Bioimaging
Ontario Cancer Institute (OCI)

Brian Wilson, PhD is Professor in the Department of Medical Biophysics, University of Toronto.

The Ontario Cancer Institute at Princess Margaret Hospital, Toronto, Canada is one of the world leaders in terms of PDT delivery. This research was partially supported by funds from National Institutes of Health (US), the U.S. Army (DOD) Breast Cancer Research Program, the Oncologic Foundation of Buffalo, the Canadian Cancer Society, the Joey and Toby Tanenbaum/Brazilian Ball Chair in Prostate Cancer Research Princess Margaret Hospital (G.Z.), the National Cancer Institute of Canada (B.C.W.), and the Muzzo Fund of the Princess Margaret Hospital Foundation.

Photodynamic Therapy (PDT) Center,
Roswell Park Cancer Institute, USA

Photodynamic Therapy for Cancer: Questions and Answers NCI

Photodynamic therapy zaps cancer (CBC, Canada) November 10, 2000

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