In the PDT practice for tumor patients, the dose and irradiation time for the treatment are chosen by experience and not
by real need. To establish advanced PDD-PDT model system for patients, we developed a method for monitoring the
cell-death based on a spectrophotometric real-time change in fluorescence in HeLa-tumors during Photofrin®-PDT and
ALA-PDT. Here, we describe the results of application of the new PDD-PDT system to human tumors. The fluorescence
spectra obtained from human tumors were analyzed by the differential spectral analysis. The mass-spectral changes of
tumor tissues during PDD-PDT were also examined by MALDI-TOF-MS/MS. The first author's seborrheic keratosis was
monitored with this system during the PDD-PDT with a topically applied ALA-ointment. The changes in fluorescence
spectrum were successfully detected, and the tumor regressed completely within 5 months. The differential spectral
analysis of PDD-PDT-fluorescence monitoring spectra of tumors and isolated mitochondria showed a marked decrease of
three peaks in the red region indicative of the PDD (600 - 720 nm), and a transient rise followed by a decline of peaks in
the green region indicative of the PDT (450 - 580 nm). The MALDI-TOF-MS analysis of PDD-PDT HeLa-tumors
showed a consumption of Photofrin-deuteroporphyrin and ALA-PpIX, and decreases in protein mass in the range of
4,000 - 16,000 Da, m/z 4929, 8564, 10089, 15000, and an increase in m/z 7002 in a Photofrin® PDD-PDT monitoring
tumor.
The photodynamic therapy (PDT) on tumors is quite effective and widely applied but usually carried out without an immediate evaluation of results. We measured the tumor fluorescence in mice with a fiber probe connected to a linear array spectral analyzer (PMA-11, Hamamatsu Photonics). The spectrum showed a transient change in fluorescence color from red to green during Photofrin-mediated PDT. In order to examine the source of green fluorescence, the mitochondria were accessed under a Nipkow disk-scanning confocal microscope in the HeLa cell in culture after labeling them with a red fluorescent protein (DsRed1-mito) and staining the cell with Photofrin (Axcan Scandipharm). Changes in fluorescence color from red to green were observed in the area of mitochondria upon their swelling during irradiation. This finding in vitro provided clear evidence that the change in fluorescence color from red to green observed in vivo was due to the mitochondrial destruction associated with the cell-death by PDT. This technique of spectral monitoring in tumor may be useful for detection of the cell-death signal during PDT in patients.
KEYWORDS: Confocal microscopy, Microscopes, In vivo imaging, Luminescence, Objectives, Signal detection, Real time imaging, Photodynamic therapy, Tumors, Clinical research
To study cellular morphology and functions in vivo in realtime, we developed a fiber-coupled confocal microscope (FCM), and observed fluorescently-labeled cells inside the body of anesthetized rat. We developed an imaging fiber bundle (IFB), which consisted of an objective lens and a multi-fiber assembly (unit fiber: NA > 0.4, 3 micron in diameter). By combining the IFB with a real-time confocal scanner, we detected intracellular signals of the molecular messenger, and the death signals in the form of fluorescence changes even from cells located deep (> 2 mm) inside the solid organs. The FCM we developed is very promising for detailed studies in both the cell-based researches and clinical researches.
By rotating the optical axis of a nonlinear optical crystal ((beta) -BaB2O4), a tunable laser beam could be obtained from an optical parametric oscillator (OPO) laser. When the crystal was optically pumped by the third harmonics of the 1064 nm Nd:YAG laser, we had a coherent beam from 410 nm through 2550 nm continuously without changing the optical cavity. We compared photodynamic therapy (PDT) effects of two photosensitizers, phenophorbide a(Phd) and Photosan-3(Ph-3, hematoporphyrin-polyester), on Wistar rat liver. Twenty-four hours after sensitization (5 mg/kg i.v.), 670 nm and 630 nm light (75 mW/cm2) was irradiated for Phd and Ph-3 respectively at energy doses of 25, 50, and 100 J/cm2. The rats were sacrificed 24 hours after laser irradiation and analyzed pathologically. Phd produced more severe necrosis than Ph-3. Twenty-five J/cm2 of Phd was identical with 100 J/cm2 of Ph-3. Next, we treated HeLa cell tumors of nude mice by Phd 670 nm PDT and Ph-3 630 nm PDT. The PDT effects of the two photosensitizers on HeLa cell tumors were similar to those on normal liver tissue. In conclusion the OPO laser could make it possible to compare PDT effects of photosensitizers by activating them with their matched wavelengths.
A new cancer-treatment model, photodynamic therapy (PDT) combined with a type I topoisomerase inhibitor, camptothecin derivative (CPT-11), against HeLa cell tumors in BALB/c nude mice has been developed using a wide-band tunable coherent light source operated on optical parametric oscillation (OPO parametric tunable laser). The Photosan-3 PDT and CPT-11 combined therapy was remarkably effective, that is the inhibition rate (I.R.) 40 - 80%, as compared to PDT only in vivo. The analysis of HpD (Photosan-3) and CPT-11 effects on cultured HeLa cells in vitro has been studied by a video-enhanced contrast differential interference contrast microscope (VEC-DIC). Photosan-3 with 600 nm light killed cells by mitochondrial damage within 50 min, but not with 700 nm light. CPT-11 with 700 - 400 nm light killed cells within 50 min after nucleolus damage appeared after around 30 min. The localization of CPT-11 in cells was observed as fluorescence images in the nucleus, particularly the nucleoral area produced clear images using an Argus 100.
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