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Nanoparticles enable two-photon PDT in living cells

Jan 2007
David L. Shenkenberg

Gliomas -- the most aggressive cancers -- occur in the brain and will likely kill a person in four to six months. There is no cure. New research, however, could allow photodynamic therapy (PDT) to destroy those tumors.

PDT involves activation of chemicals called photosensitizers that produce highly reactive singlet oxygen that kills cells when activated via a light source such as a laser. Because photosensitizers can be activated after they enter or surround cancer cells, using them also can eliminate or minimize the damage to healthy tissue.

A 630-nm laser beam used in single-photon PDT can, at best, penetrate about 5-mm deep. PDT performed with photosensitizers that can be activated with two-photon excitation may enable a 780-nm, infrared beam to travel a centimeter or deeper through tissue. Although gliomas occasionally can grow bigger than that, two-photon PDT may eradicate most of the malignancy without the need for additional surgery or radiation.

Encapsulating a photosensitizer in polyacrylamide nanoparticles enabled two-photon PDT with living rat brain cancer cells. The red cells are dead, as a result of PDT, and the green cells are alive.

However, some two-photon photosensitizers are inherently toxic and can harm healthy tissue before they reach cancer cells. By encapsulating them in polyacrylamide nanoparticles, researchers at the University of Michigan in Ann Arbor have demonstrated two-photon PDT in living cells.

The scientists chose polyacrylamide because it has the soft, flexible consistency of tissue, and it does not clump together or bind to healthy tissue. Plus, other molecules can be added to the polymer to enable it to target specific tissues.

For the photosensitizer, the research..−.ers used 5, 10, 15, 20-tetrakis(1-methyl 4-pyridinio) porphyrin tetra(p-toluenesulfonate). According to professor Raoul Kopelman, an author of the study, the porphyrin-based dye is only one of a few two-photon photosensitizers. Without encapsulation in nanoparticles, it can be pumped back out by cancer cells.

Initial experiments showed that polyacrylamide itself did not harm cells, that the photosensitizer did not contact cells while encapsulated in the nanoparticles and that the photosensitizer still produced significant reactive oxygen when it was stimulated with light.

For their primary experiment, the scientists incubated rat glioma cells with nanoparticles that contained the two-photon photosensitizer. They placed this mixture on microscope slides and positioned them on an Olympus inverted microscope. To induce PDT, they focused a 780-nm Spectra-Physics Ti:sapphire laser beam through a 40× objective.

They operated the laser in pulsed mode for 1 s at a fluence of 100 mW/cm2, and the rat glioma cells began to die 90 min later. They exposed other rat glioma cells to 1 min of laser light. These cells started perishing 30 min later, and most of them expired after 120 min.

The researchers also recently demonstrated that single-photon PDT can successfully treat gliomas in living rats. They hope that two-photon PDT will enable treatment of other tissues that remain challenging for PDT because they are more opaque than brain tissue. In the future, the group may perform the technique using nanoparticles that are targeted to specific tumors.

Nano Letters, November 2006, pp. 2383-2637.

Biophotonicschemicalsliving cellsMicroscopynanoparticlesNews & Featurestwo-photon PDT

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