Hexyl-aminolevulinate-mediated photodynamic therapy: how to spare normal urothelium. An in vitro approach.
OBJECTIVE
Photodynamic therapy (PDT) of superficial bladder cancer may cause damages to the normal surrounding bladder wall. Prevention of these is important for bladder healing. We studied the influence of photosensitizer concentration, irradiation parameters, and production of reactive oxygen species (ROS) on the photodynamically induced damage in the porcine urothelium invitro. The aim was to determine the threshold conditions for the cell survival.


METHODS
Living porcine bladder mucosae were incubated with solution of hexylester of 5-aminolevulinic acid (HAL). The mucosae were irradiated with increasing doses and cell alterations were evaluated by scanning electron microscopy and by Sytox green fluorescence. The urothelial survival score was correlated with Protoporphyrin IX (PpIX) photobleaching and intracellular fluorescence of Rhodamine 123 reflecting the ROS production.


RESULTS
The mortality ratio was dependent on PpIX concentration. After 3 hours of incubation, the threshold radiant exposures for blue light were 0.15 and 0.75 J/cm(2) (irradiance 30 and 75 mW/cm(2), respectively) and for white light 0.55 J/cm(2) (irradiance 30 mW/cm(2)). Photobleaching rate increased with decreasing irradiance. Interestingly, the DHR123/R123 reporter system correlated well with the threshold exposures under all conditions used.


CONCLUSIONS
We have determined radiant exposures sparing half of normal urothelial cells. We propose that the use of low irradiance combined with systems reporting the ROS production in the irradiated tissue could improve the in vivo dosimetry and optimize the PDT.
Vaucher
Laurent
L
Institute of Physiology, University of Lausanne, Lausanne, Switzerland. laurent.vaucher@chuv.ch
Jichlinski
Patrice
P
Lange
Norbert
N
Ritter-Schenk
Céline
C
van den Bergh
Hubert
H
Kucera
Pavel
P
D000622:Aminolevulinic Acid / Q000031:analogs & derivatives* / Q000627:therapeutic use; D000818:Animals; D002470:Cell Survival; D066298:In Vitro Techniques; D008855:Microscopy, Electron, Scanning; D009092:Mucous Membrane; D038761:Photobleaching; D010778:Photochemotherapy; D017319:Photosensitizing Agents / Q000494:pharmacology; D011524:Protoporphyrins / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism; D017695:Soft Tissue Injuries / Q000517:prevention & control; D013050:Spectrometry, Fluorescence; D013552:Swine; D001749:Urinary Bladder Neoplasms / Q000188:drug therapy; D019459:Urothelium / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology / Q000528:radiation effects*
D017319:Photosensitizing Agents; D011524:Protoporphyrins; D017382:Reactive Oxygen Species; D000622:Aminolevulinic Acid; C028025:protoporphyrin IX; C419924:5-aminolevulinic acid hexyl ester