ABSTRACT
Quantification of fluorescence in vivo is complicated by the influence of tissue optical properties on the collected fluorescence signal. When tissue optical properties in the measurement volume are quantified, one can obtain the intrinsic fluorescence, which equals the product of fluorophore absorption coefficient and quantum yield. We applied this method to in vivo single-fiber fluorescence spectroscopy measurements on mouse tongue, skin, liver, and oral squamous cell carcinoma, where we detected intrinsic fluorescence spectra of the photosensitizers chlorin e6 and Bremachlorin at t=[3,4.5,6,24,48] h incubation time. We observed a tissue-dependent maximum of 35% variation in the total correction factor over the visible wavelength range. Significant differences in spectral shape over time between sensitizers were observed. Although the wavelength position of the fluorescence intensity maximum for ce6 shifted to the red, Bremachlorin showed a blue shift. Furthermore, the Bremachlorin peak appeared to be broader than the ce6 fluorescence peak. Intrinsic fluorescence intensity, which can be related to photosensitizer concentration, was decreasing for all time points but showed significantly more Bremachlorin present compared to ce6 at long incubation times. Results from this study can be used to define an optimal treatment protocol for Bremachlorin-based photodynamic therapy.
Subject(s)
Chlorophyll/analogs & derivatives , Photosensitizing Agents/chemistry , Animals , Carcinoma, Squamous Cell/pathology , Chlorophyll/chemistry , Chlorophyllides , Female , Fluorescence , Green Fluorescent Proteins , Liver/pathology , Mice , Mice, Inbred BALB C , Mice, Nude , Microscopy, Fluorescence , Mouth Neoplasms/pathology , Normal Distribution , Optics and Photonics , Photochemotherapy , Porphyrins/chemistry , Skin/pathology , Spectrometry, Fluorescence , Spectrophotometry , Tongue/pathologyABSTRACT
BACKGROUND AND OBJECTIVE: The effect of photodynamic therapy (PDT) is dependent on the localization of photosensitizer in the treatment volume at the time of illumination. Investigation of photosensitizer pharmacokinetics in and around the treatment volume aids in determining the optimal drug light interval for PDT. MATERIALS AND METHODS: In this paper we have investigated the distribution of the photosensitizers chlorin e6 and Bremachlorin in the oral squamous cell carcinoma cell-line OSC19-Luc-Gfp in a tongue tumor, tumor boundary, invasive tumor boundary, and normal tongue tissue by the use of confocal microscopy of frozen sections. Tongues were harvested at t = [3, 4.5, 6, 24, 48] hours after injection. RESULTS: Both photosensitizers showed a decreasing fluorescence with increasing incubation time, and at all time points higher fluorescence was measured in tumor boundary than in tumor itself. For short incubation times, a higher fluorescence intensity was observed in the invasive tumor border and normal tissue compared to tumor tissue. Bremachlorin showed a small increase in tumor to normal ratio at 24 and 48 hours incubation time. Ce6 was undetectable at 48 hours. We did not find a correlation between photosensitizer localization and the presence of vasculature. CONCLUSION: The modest tumor/tumor boundary to normal selectivity of between 1.2 and 2.5 exhibited by Bremachlorin 24 and 48 hours after administration may allow selective targeting of tongue tumors. Further studies investigating the relationship between Bremachlorin concentration and therapeutic efficacy PDT with long incubation times are warranted.
