Gerd C. Beck

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.

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.

Nonlinear Dynamics of Intracellular Methylene Blue During Light Activation of Cell Cultures

Abstract— Methylene blue (MB+) is a well‐known dye in medicine and has been discussed as an easily applicable drug for topical treatment in photodynamic therapy (PDT). Methylene blue can potentially be used as a redox indicator to detect the important redox reactions that are induced during PDT. The kinetics of this process was analyzed on a subcellular level with confocal laser scanning microscopy. BKEz‐7 endothelial cells were incubated 4 h with 1 μM MB+. The fluorescence dynamics of MB+ during irradiation with 633 nm light was observed with subcellular resolution. Images were acquired at 0.5 s intervals (frame rate 1 image/0.5 s). Fluorescence was observed in the red channel of the laser scanning microscope. Synchronously, the phase‐contrast image was visualized with the green channel. Morphological changes could therefore be correlated with the dynamics of MB+. In addition, the light‐dose‐dependent phototoxicity at 633 nm irradiation was determined by viable cell counting. After an induction period (phase I), fast fluorescent spikes could be observed in the whole cytoplasm, which decayed with a time constant of about 20 s (phase II), followed by a period of nearly constant fluorescence intensity (phase III) and exponential photobleaching (phase IV). Phase II exhibits highly nonlinear kinetics, which is hypothesized to correlate probably with a nonlinear quantal production of reactive oxygen species (ROS). Morphological cell changes were not observed during phase II. During phase III, a pycnotic cell nucleus developed. From the determination of viable cells we can conclude that a light dose applied within phase II was only sublethal in correlation with morphological observations. Overproduction of ROS leading finally to cell killing during phases III and IV is discussed.

Developing optimized tissue phantom systems for optical biopsies

We present a scheme by which tissue phantom systems can be designed rapidly and systematically according to individual demands. For the optical biopsies, organ structures of biological tissues have to be modeled which requires a solid host material determining the modeling potentialities. Complex structures of biological tissues can be modeled by phantom systems based on a solid host material which determines the modeling potentialities. Mie theory shows that scattering and absorption of particles depends strongly on their material constants and size distribution. According to these predictions particles can be selected or produced. Particles are not only useful to induce scattering but can also be an interesting alternative to absorbing and fluorescent dyes. We present organic, metallic and mineralic particles, their relevant properties and outlines of their characterization. We discuss how the predictions of theory and mutual interactions between components can be checked and report on problems frequently encountered with dyes and particles as components of tissue phantom systems.

Confocal observation of hydrophilic and lipophilic photosensitizers in endothelial cells, lumen of the vessels, interstitium, and tumor cells using the chicken chorioallantoic membrane

The dynamic behavior of lipophilic and hydrophilic sensitizers in cell cultures and non animal in vivo systems with varying incubation but also during the photodynamic therapy will be summarized within the presentation. As an appropriate in vivo system we used the chorioallantoic membrane (CAM) of fertilized eggs, which served as a substrate for tumor cells. Because the CAM is a transparent membrane it is possible to view individual blood vessels and to examine tumor cells as well as structural changes of the supplying vasculature. To adapt this system to high magnification microscopy, we established a new technique for in vivo observation of the CAM tissue. This technique enables online investigations of alterations at cellular level induced by drugs with confocal laser scanning microscopy. The localization of the drugs with clinical importance was observed after different application times in the lumen of the vessels, the endothelial cells and the tumor cells. In addition light induced subcellular Ca2+-changes were observed and correlated with the photodynamic process.

Depth-sensitive fluorescence detection of dyes in tissue phantoms

It is well known that tumors can be detected by fluorescence imaging after administration of suitable photosensitizers. The tumors thickness, however, an important diagnostic parameter, cannot be assessed by optical methods up to now. We show that depth resolution can be achieved in the case of a fluorescent dye distributed homogeneously in a superficial region of a light scattering medium. Diffusion theory shows that the ratio of the fluorescence measured at different illumination angles is over a wide range sensitive to the thickness of the dye containing layer only, regardless of the dye concentration. For the experimental proof, a new temporally and mechanically stable tissue phantom system was developed. The experimental results on tissue phantoms confirm the predictions of diffusion theory.

Dynamics of photosensitizers in cell cultures and nonanimal in-vivo systems

The biochemical background of photodynamic therapy is only partly resolved. The biodynamic, which is shown to be different for lipophilic and hydrophilic photosensitizers influences the photodynamic efficiency. A complex cellular and subcellular localization and relocalization of the drugs is not only observed with varying incubation time but also during the light induced process. The dynamic behavior of lipophilic and hydrophilic phthalocyanines in cell cultures and non animal in vivo systems will be summarized within the presentation. As in vivo system the chorioallantoic membrane (CAM) of fertilized eggs was used. The dynamic behavior was observed with a confocal laser scanning microscope.

Dynamic fluorescence changes of hydrophilic and lipophilic phthalocyanines during PDT in vivo and in vitro

The fluorescence emission of hydrophilic tetrasulphonated aluminum phthalocyanine (AlPcS4) and hydrophobic zinc phthalocyanine (ZnPc), bound to the membrane of liposomes were investigated in vivo in an appropriate tumor model of the rat bladder and in addition in RR 1022 epithelial cells of the rat. The sensitizers were administered systemically to the rats and PDT was performed 24 h later. During PDT treatment the fluorescence was measured every 15 s. Fluorescence was excited with the 633 nm light of a HeNe laser and the fluorescence spectra were recorded with an optical multichannel analyzer system. PDT was performed in the case of both sensitizers with 672 nm light from an Ar+-Dye laser. Fluorescence changes during PDT were significantly different for the two phthalocyanines. In the case of AlPcS4 an initial fluorescence intensity increase followed by subsequent photobleaching could be observed. In contrast, ZnPc fluorescence showed an exponential decrease and no increase in the beginning of the treatment. Tumor necrosis 24 h after PDT was significant only for ZnPc. RR 1022 cells incubated 24 h with AlPcS4 revealed a granular fluorescence pattern, whereas ZnPc was localized diffusely in the cytoplasm of the cells. In correlation to the measurements in vivo subcellular relocalization and fluorescence intensity increase could be detected exclusively in the case of AlPcS4.

Nonlinear intracellular dynamics of photosensitizers during PDT

The dynamic of hydrophilic photosensitizers was investigated in single cells during light activation. Laser scanning microscopy, steady-state and time-resolved microspectrofluorometry were used to serve the subcellular localization of the drugs and to analyze the fluorescence kinetic during continuous irradiation. The quantitative phototoxic response of the cells was determined at low light doses as well as higher light doses. Morphological changes of the cells as well as phototoxic response were correlated with the fluorescence kinetics of the sensitizers. In detail, meso-tetra porphyrine, tetra-sulfonated aluminum phthalocyanine and Methylene Blue showed a nonlinear kinetic behavior at low light doses which was dependent on the growth phase of the cells. A pycnotic cell nuclei was correlated with a change in the kinetic behavior and observed at higher light doses. In the case of hydrophilic dyes, light induced stimulation of the cells and phototoxic damage are dose dependent and are influenced by the nonlinear kinetics of the photosensitizers.

Estimation of the parameters of dye distributions by optical probing

The observable parameters remittance, transmittance and fluorescence emission of layered systems are determined from Monte Carlo simulations and the diffusion approximation. We calculate the effect of additional absorption caused by a fluorescing dye, depending on dye- bearing layer thickness. The results of the simulations are compared to the diffusion approximation, establishing a range of accuracy of the diffusion approximation. We show that, provided the basic optical properties of the system are known, additional absorption and layer thickness can be calculated from non-invasive measurements of remittance and fluorescence emission. We give an estimation of the effect of experimental uncertainty on the results for dye absorption and layer thickness.