Monitoring Concentration Of Photosensitizer By Fluorescence Spectroscopy During Photodynamic Therapy

Objective: By applying fluorescence spectroscopy, the study aims to establish a fluorescence spectra collection and analysis system, and to monitor the photosensitizer concentration and the generation of photoproduct in normal saline solution, in the locally irradiated skin of normal mice and in the lesion of patients with port wine stains during photodynamic therapy(PDT). The study also aims to investigate the rule of photosensitizer concentration variation, and to determine the distribution of photosensitizer in the local skin tissue of normal mouse by mathematic simulation.Methods: The fluorescence of hematoporphyrin monomethyl ether (HMME) in normal saline solution was tested. The effect of excitation wave-length on fluorescence detection was investigated. 532 nm double-frequency Nd: YAG laser worked as excitation light source. The fluorescence of photosensitizer was monitored by optical multichannel analyzer (OMA), and fluorescence spectrum processing and analyzing was applied to get the fluorescence intensity of photosensitizer. The results collected by the fluorescence system was compared with that by fluorescence spectrophotometer to validate the fluorescence system. 532 nm laser also worked as the light source for PDT treatment. The established fluorescence system was applied to monitor the photosensitizer fluorescence, which included: (1) HMME in normal saline solution: The concentration of HMME was 1 μg/ml, 10 μg/ml, and 25 μg/ml, respectively. The laser power density was 100 mW/cm~2 (2) HMME in the skin of BALB/c mouse: The dose of HMME for intravenous injection was 10 mg/kg. The laser power density was 50 mW/cm~2 (3) PsD-007 in the lesion of patients with port wine stains: The dose of PsD-007 for intravenous injection was 4-5 mg/kg. The laser power density was 80-100 mW/cm~2. The variation of photosensitizer fluorescence and photoproductwere achieved by fluorescence spectrum processing and analyzing. And mathematic simulation was applied to analyze the experiment results further.Results: (1) 532 nm laser was more advantageous than 405 nm when it was used to excite photosensitizer fluorescence. The HMME concentration in the range of 1-25 μg/ml was linear to its fluorescence intensity. The results achieved by this system consisted with that by fluorescence spectrophotometer. (2) The photobleaching rate of 10 μg/ml HMME was more rapidly than that of 1 μg/ml. A new fluorescence peak emerged at 639 nm during the photobleaching process of HMME. The fluorescence intensity of background was invariant during the period of 0-45 min, and it became to increase during the period of 45-90 min with the prolonging of PDT. At the end of irradiation the intensity was 3 times higher than the initial intensity. By mathematic simulation the predictable curve of HMME photobleaching at any concentration of 1-25 μg/ml was established. In normal saline solution the photobleaching rate of HMME induced by continue-wave mode laser irradiation was demonstrated to be a little faster than that by quasi-continue-wave mode laser. (3) Photosensitizer concentration in local mouse skin decreased with the prolonging of PDT treatment. The fluorescence intensity at 4 min was 50% level of that before irradiation, while it decreased to 12% level after 20 min. The results of mathematical simulation were consistent with that of animal experiment. Further investigation showed that the photosensitizer in microvasculature of the upper layer of dermis is much higher than that in the adjacent tissue. (4) Although there was a considerable lesion-to-lesion variation among all the lesions tested, the photosensitizer fluorescence intensity rose at the beginning of treatment, and got peak fluorescence intensity after about 5 to 15 minutes, and began to decrease with time prolonging. The peak fluorescence intensity of the first light irradiation area was higher than that of the second one.Conclusions: (1) By the validation of fluorescence spectrophotometer the fluorescence system is demonstrated to be sensitive and reliable for monitor photosensitizer concentration. The light source of PDT irradiation works as the light source for fluorescence excitation, which can make the experiment procedure more simple and accurate. It is reliable to investigate the photosensitizer concentration variation in complex system applying this mathematical simulation. (2) The photobleaching of HMME is a very complicated process, and it can beaffected by the initial concentration of HMME and the mode of laser. Some kind of photoproduct is generated during this process, and it can also be photobleached. The predictable curve of HMME photobleaching at any concentration of 1-25 μg/ml can be applied to predict the process of its photobleaching. (3) The mathematic simulation results show that the photosensitizer concentration in vessels is much higher than that in the adjacent tissue, which may play an important role in the selective vascular damage induced by PDT. (5) The fluorescence spectroscopic method established in this study can provide an efficient way to monitoring photosensitizer concentration and photoproduct generation in the lesion of patient during PDT. It provides a useful tool for the dosimetry study of PDT, and it also will be helpful to optimize the clinical protocol and to improve the treatment efficiency of photodynamic therapy.