PDT with methylene blue monitored microscopically

David Shenkenberg

Although we know the chemistry of photodynamic therapy (PDT), we do not fully understand how it occurs on the microscopic scale, specifically within the cell membrane. In the process of PDT, cells ultimately die when the cell membrane ruptures, so the membrane is key to understanding the procedure. Therefore, researchers at Institut Charles Sadron in Strasbourg, France, and Universidade de São Paulo, Brazil, studied how PDT destroys the cell membrane.

For the PDT agent, they used methylene blue, a widely studied photosensitizer that has the potential to be approved for clinical use. They used membrane vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) because it was easier to study their physical properties than it would have been with living cells. In particular, they used “giant unilamellar vesicles,” large vesicles with a single lipid bilayer, because they could view photo-oxidation within the 50-Å membrane with an optical microscope.

To examine vesicle properties, the researchers observed the entities with the 100× objective of a Nikon microscope in transmission and reflection interference contrast microscopy modes. They stimulated PDT with green light, obtained by filtering the emission of the microscope’s mercury lamp.

As reported in the Nov. 29 online publication of Langmuir, the scientists determined that they needed to use at least 25 μmol/l of methylene blue to destroy the membrane vesicles. They also discovered that only a few thousand joules of green light per square centimeter could induce a photodynamic effect.

Considering a single vesicle, the diameter decreased at a rate of approximately 5 μm/min, suggesting that PDT destroyed 1 percent of phospholipids in the membrane per second. With the assumption that cutting one of DOPC’s two phospholipid chains is necessary for membrane destruction, 4 percent of singlet oxygen molecules efficiently cut the lipid chains. If cutting both is necessary, the efficiency would be 8 percent. The scientists believe that this information bridges the gap between the molecular and microscopic mechanisms of PDT.

They noted that this report may have implications for inflammation and for diseases that involve membrane disruption such as cancer, atherosclerosis, Parkinson’s and Alzheimer’s.