Angelika Rück

In vivo photoproduct formation during PDT with ALA-induced endogenous porphyrins

The administration of 5-aminolevulinic acid (ALA) in tumor-bearing nude mice leads to the formation of the fluorescent, photounstable photosensitizer protoporphyrin IX in tumor tissue. On-line fluorescence spectroscopy during photodynamic therapy (PDT) shows the in vivo formation of chlorintype photoproducts of protoporphyrin. The fluorescence of protoporphyrin as well as its photoproducts is bleached completely at the end of the PDT (100 J cm-2, 630 nm). These findings were also verified using ultrashort laser pulses and time-correlated single-photon counting. A photinduced shortening of the decay times and decrease in the integral fluorescence intensity were measured in vivo due to the photodestruction of the endogenous photosensitizer protoporphyrin IX in the tumor.

Methylene blue mediated photodynamic therapy in experimental colorectal tumors in mice.

Methylene blue (MB+) is a well-known dye in medicine and has been discussed as an easily applicable drug for the topical treatment during photodynamic therapy (PDT). The therapeutic response of MB+ was investigated in vivo by local injection of MB+ in a xenotransplanted subcutanous tumor (adeno-carcinoma, G-3) in female nude mice. MB+ in a concentration of 1% was applied both undiluted and diluted to 0.1 and 0.01% with isotonic sodium chloride. Treatment with 1% MB+ and subsequent irradiation at 662 nm with 100 J/cm2 led to complete tumor destruction in 79% of the treated animals. A decrease of the fluence rate from 100 to 50 mW/cm2 increased the phototoxic response as well as fractionated light application. Small sensitizer concentrations reduced the PDT effect significantly. It seems that the light induced reaction of MB+ could be correlated with the rapid production of reactive oxygen species. Below a threshold dose of MB+ oxidative damage of the tissue is prevented. However, above this dose, as a point of no return, MB+ acts as an extremely potent oxidant.

Fluorescence formation during photodynamic therapy in the nucleus of cells incubated with cationic and anionic water-soluble photosensitizers

The variations of fluorescence during light exposure of the cationic sensitizers methylene blue (MB) and meso-tetra(4N-methylpyridyl)porphyrin (T4MPyP) as well as the anionic meso-tetra(4-sulphonatophenyl)porphyrin (TPPS4) were measured at different intracellular sites using video-intensified microscopy in combination with microspectrofluorometry. Before light exposure the sensitizers were localized in distinct parts of the cytoplasm, especially in fluorescent organelles. During irradiation a drastic fluorescence formation and increase in the cytoplasm and nucleus, which was most pronounced in the nucleoli, could be observed for the cationic sensitizers as well as TPPS4. In the case of MB the increase in fluorescence was concomitant with a spectral shift in the emission spectra. For TPPS4 and T4MPyP the formation of a second species with a Soret band shifted towards longer wavelengths was observed and correlated with the fluorescence increase in the nucleoli. Cell deformations also took place.

Time-resolved in-vivo fluorescence of photosensitizing porphyrins☆

Various components of photosensitizing porphyrins (e.g. monomers, aggregates, ionic species) have been recently localized in single cells by time-resolved fluorescence microscopy. Novel time-resolving techniques, based on picosecond laser diodes, a frequency-doubled Nd:YAG laser and time-gated microscopic equipment, were used for in-vivo measurements of the chick chorioallantoic membrane (CAM) exhibiting a pronounced vasculature. Changes of the fluorescence decay kinetics after light exposure were correlated with the formation of a photoproduct (Photosan, aminolaevulinic acid) or changes of the intracellular binding sites (tetraphenyl-porphyrins). Fluorescent components with different decay times were shown to be distributed differently within the tissue.

Light-induced Apoptosis Involves a Defined Sequence of Cytoplasmic and Nuclear Calcium Release in AlPcS4-photosensitized Rat Bladder RR 1022 Epithelial Cells¶

Abstract Oxidative stress induced by light activation of photosensitizers is regarded to have a role in triggering cell death pathways during photodynamic therapy (PDT). Reactive oxygen species have been proposed to act as signal transduction molecules activating downstream reactions that lead to apoptosis. Mainly debated is the cooperating role of other signaling systems like calcium or pH. The present work contributes to this discussion by studying PDT effects in cell cultures of rat bladder epithelial cells for the hydrophilic tetrasulfonated aluminum phthalocyanine (AlPcS4). Cells were coincubated with the photosensitizer and the calcium-sensitive probe Fluo-3. The light-induced reactions were analyzed with a confocal laser scanning microscope. The dynamics of the process during light activation was observed with subcellular resolution. A transient calcium elevation during the irradiation process was detected, especially in the cells nuclei, followed by a more sustained increase. The evaluation of the energy-dose–dependent phototoxicity after an incubation time with the photosensitizer of 1 and 24 h, showed enhanced phototoxicity when the drug was present for 24 h. Surprisingly, stimulation of cell proliferation was observed at very low light doses (at 0.2 J/cm2) when the drug was incubated for 24 h (cell viability 160%). Induction of apoptosis could be observed after irradiation with fluences between 1 and 3 J/cm2. Apoptotic cells were identified with fluorescein isothiocyanate–labeled Annexin V, which binds to phosphatidylserine after its translocation to the outer plasma membrane. In the presence of the antioxidant pyrrolidinedithiocarbamate the transient calcium elevation was totally inhibited, as was the subsequent translocation of PS. In contrast, N-acetyl-l-cysteine did not suppress the transient calcium increase. Our data might be consistent with calcium regulated processes during AlPcS4-PDT and the involvement of oxygen radicals.

Dynamic fluorescence changes during photodynamic therapy in vivo and in vitro of hydrophilic A1(III) phthalocyanine tetrasulphonate and lipophilic Zn(II) phthalocyanine administered in liposomes.

The fluorescence emission of hydrophilic tetrasulphonated aluminium phthalocyanine (AlPcS4) and hydrophobic zinc phthalocyanine (ZnPc), bound to the membrane of liposomes, was investigated in vivo in an appropriate tumour model of the rat bladder and in RR 1022 epithelial cells of the rat. The sensitizers were administered systemically to the rats and photodynamic therapy (PDT) was performed 24 h later. During PDT treatment, the fluorescence was measured every 30 s. The fluorescence was excited with 633 nm light from an HeNe laser and the fluorescence spectra were detected with an optical multichannel analyser system. PDT was performed for both sensitizers using 672 nm light from an Ar+ dye laser. The fluorescence changes during PDT were significantly different for the two phthalocyanines. For AlPcS4, an initial fluorescence intensity increase, followed by subsequent photobleaching, was observed. In contrast, ZnPc fluorescence showed an exponential decrease and no increase at the start of treatment. Tumour necrosis 24 h after PDT was significant only for ZnPc. RR 1022 cells incubated for 24 h with AlPcS4 revealed a granular fluorescence pattern, whereas ZnPc was localized diffusely in the cytoplasm of the cells. In agreement with the in vivo measurements, subcellular relocalization and a fluorescence intensity increase were detected exclusively in the case of AlPcS4. Morphological changes at this time were significant only for ZnPc. The subcellular localization and fluorescence kinetics were obtained using a confocal laser scanning microscope.

Intracellular distribution of photosensitizing porphyrins measured by video-enhanced fluorescence microscopy

The intracellular distribution of photosensitizing porphyrins with different amounts of aggregates was measured in a fluorescence microscope using video-intensified detection and image processing. Porphyrin fluorescence was localized in the plasma membrane, cytoplasm, nuclear membrane and nucleoli. The fluorescence intensity in the plasma membrane decreased with increasing aggregation of the photosensitizers. In addition, a redistribution of the porphyrin molecules from the plasma membrane to the nuclear membrane and adjacent intracellular sites was observed with increasing incubation time. Photobleaching of porphyrin fluorescence was most pronounced in the plasma membrane and least efficient within the nucleoli.

Intracellular fluorescence behaviour of meso-tetra(4-sulphonatophenyl)porphyrin during photodynamic treatment at various growth phases of cultured cells

Meso-tetra(4-sulphonatophenyl)porphyrin (TPPS4) taken up by cells is mainly localized in lysosomes as previously shown by fluorescence microscopical and fluorescence spectroscopical investigations. In the present study the intracellular fluorescence behaviour and the intracellular amount of this dye at various growth periods of cells were examined. For cells irradiated in the growth phase a relocalization of TPPS4 from the lysosomes into the cytoplasm and finally into the nucleus was observed. In contrast, for cells irradiated in the stationary phase no redistribution could be detected and therefore no evidence for severe damage of the lysosomal membranes and subsequently for the release of lytical enzymes is given. In both cases lethal damage of the cells was achieved as examined using the trypan blue exclusion test. This indicates that damage of the lysosomes is less important in the photodynamic inactivation of cells sensitized by TPPS4.

Characterization of the chick chorioallantoic membrane model as a short-term in vivo system for human skin.

Abstract We report on the cultivation and characterization of human skin on the chorioallantoic membrane of chicken eggs with the aim of replacing animals in short-term investigations in dermatology. Adult human split-thickness skin was grafted onto the chorioallantoic membrane of 5-day chick embryos. Grafts and surrounding host tissue were examined daily by in vivo stereomicroscopy and in histological sections and were characterized using a panel of monoclonal antibodies. The skin grafts were completely incorporated into the chorioallantoic membrane 2 days after transplantation. A remarkable angiogenesis occurred towards the grafts. Skin tissues revascularized within 2 or 3 days by reperfusion of the existing graft vasculature. Anastomosis of host and graft blood vessels occurred and the transplanted skin was nourished by the host blood supply as indicated by nucleated chick erythrocytes in the skin vessels. The skin grafts on the chorioallantoic membrane preserved an almost entire human phenotype. Besides a fully differentiated human epidermis and dermis containing all the cellular and extracellular constituents such as skin immune cells, capillary vessels composed of human endothelial cells were enclosed by a basement membrane of human origin. The integrin expression pattern formed in human skin transplants 5 days after grafting was identical to that of human skin controls before grafting.

Time-resolved pH-dependent fluorescence of hydrophilic porphyrins in solution and in cultivated cells

Fluorescence decay kinetics and time-gated (nanosecond) emission spectra of the hydrophilic photosensitizers meso-tetra(4-sulfonatophenyl)porphyrin (TPPS4) and uroporphyrin III (UP III) are reported. These substances are characterized by low aggregation, preferential accumulation within lysosomes and a pH-dependent composition of unprotonated and protonated species. A comparison of TPPS4 and UP III in buffer solutions and in confluently growing RR 1022 epithelial cells showed that the intracellular pH value of the environment of both photosensitizers was about 4.7. A slight decrease by 0.10-0.15 pH units occurred after light exposure which (in the case of TPPS4) was concomitant with a lethal damage of the cells. A photoproduct at 640 nm with a characteristic fluorescence lifetime of 4.3 +/- 0.8 ns was detected for UP III in buffer solutions at pH values above 5. The absence of this photoproduct in epithelial cells again indicated that UP III was located within lysosomes.