The fluorescent dye indocyanine green (ICG) is clinically approved and has been applied for ophthalmic and intraoperative angiography, measurement of cardiac output and liver function, or as contrast agent in cancer surgery. Though ICG is known for its photochemical effects, it has played a minor role so far in photodynamic therapy or techniques for targeted protein-inactivation. Here, we investigated ICG as an antibody-conjugate for the selective inactivation of the protein Ki-67 in the nucleus of cells. Conjugates of the Ki-67 antibody TuBB-9 with different amounts of ICG were synthesized and delivered into HeLa and OVCAR-5 cells through conjugation to the nuclear localization sequence. Endosomal escape of the macromolecular antibodies into the cytoplasm was optically triggered by photochemical internalization with the photosensitizer BPD. The second light irradiation at 690 nm inactivated Ki-67 and subsequently caused cell death. Here, we show that ICG as an antibody-conjugate can be an effective photosensitizing agent. Best effects were achieved with 1.8 ICG molecules per antibody. Conjugated to antibodies, the ICG absorption peaks vary proportionally with concentration. The absorption of ICG above 650 nm within the optical window of tissue opens the possibility of selective Ki-67 inactivation deep inside of tissues.
Fluorescence microscopy has the potential to study the spatial distribution of photosensitizers in tissue samples with cellular or subcellular resolution. A fluorescence microscope was developed to study the distribution of photosensitizer in tissue samples by acquiring fluorescence images in various spectral ranges and spatially resolved fluorescence spectra both from identical samples. Both methods provide complementary information, since the fluorescence images show the distribution of the sensitizers with a high spatial resolution whereas spatially resolved fluorescence spectra can identify the sensitizers and separate their fluorescence from background light emission by the spectral shape of the fluorescence. Protoporphyrin IX (PPIX) distribution induced by 5-aminolevulinic acid (ALA) was studied by fluorescence microscopy in basal cell carcinoma (BCC) and in cervical intraepithelial neoplasia (CIN). In an attempt to understand the varying success in treating BCC with topically applied ALA the PPIX distribution was studied in BCC samples of 10 patients. A strong fluorescence was observed in tumor cells as well as in epidermis, sebaceous glands, and hair follicles. The depth of PPIX sensitization of the BCCs ranged from 0.4 to 3 mm and the ratio of tumor versus epidermal fluorescence of uninvolved skin was near one. In the BCCs an uneven sensitization with a lower fluorescence in the center of the tumor was often observed. Samples of the cervical mucosa also showed PPIX fluorescence in the endothelial layer, the malignant tissues and the glands. No increased fluorescence of the dysplastic lesions compared to the epithelium was observed.
Arthroscopic synovectomy, which is limited today to the large joints, is an important early treatment of rheumatoid arthritis (RA). Photodynamic therapy (PDT) is potentially to be a less invasive method of removing the synovial membrane. Therefore, in a rabbit model of RA, the accumulation of the photosensitizer Protoporphyrin IX (PPIX) after intra-articular and systemic application of ALA into arthritic rabbit knee joints was studied in skin, patella, synovial tissue, and meniscus by fluorescence microscopy. PPIX fluorescence was measured in biopsies taken at different times after application of neutral and acid ALA solutions. Significant PPIX fluorescence was observed in the synovial membrane and skin 2 and 4 hours after application. Using intra-articular application, ALA solutions prepared with pH 5.5 were at least as efficient as neutral solutions in sensitizing the synovial membrane. Skin also showed PPIX within 4 hours after application. After 24 hours, a marginal PPIX fluorescence was detected in these tissues. On the other hand, in cartilage and meniscus significant PPIX accumulation was still observed 24 hours after ALA injection. Systemic application of ALA also showed a good accumulation of PPIX. Further experiments are needed to show whether accumulation of the photosensitizer and tissue selectivity are sufficient for a successful treatment of rheumatoid synovitis.
Five-aminolevulinic acid (ALA) is being used to induce formation and accumulation of endogenous protoporphyrin IX both in preclinical and clinical studies on photodynamic therapy and diagnosis. At the high concentrations needed for clinical application, ALA is not stable in aqueous solutions in the neutral to basic pH range. This could be of critical importance for the clinical use of ALA. The chemical instability of ALA was studied by using UV-Vis absorption spectroscopy. Our results show that ALA undergoes a chemical reaction, which is a nonreversible condensation process yielding two different products -- a hydropyrazine and a pyrazine, the latter formed by oxidation of the hydropyrazine. Preliminary kinetic studies of this reaction showed a complex pH-, concentration and temperature-dependency of the reaction rate. On the basis of these studies we optimized conditions for the preparation of ALA solutions to be used in clinical trials, leading to the expected accumulation of protoporphyrin IX in tumor cells.
The only early therapy of rheumatoid arthritis in orthopedic surgery is a synovectomy, which is restricted to more or less big joints. A laser-synovectomy of small joints is ineffective yet. An alternative method may be photodynamic therapy. In our study we describe the photodynamic effect of Photosan 3 in a cell culture study.
Irradiation with pulsed lasers can change mechanisms and efficacy of photodynamic therapy (PDT) depending on the laser pulse parameters. Since most photosensitizers have a relatively high triplet quantum yield and triplet lifetimes of tens of microseconds, even moderate power densities below 100 kW/cm2 can lead to a saturation of the singlet oxygen production, thereby reducing the PDT effect. A simple quantitative model is developed to estimate this effect. According to this, for laser pulses not longer than the triplet lifetime, the PDT efficacy depends on the product of single pulse energy, irradiation wavelength, and the extinction coefficient of the photosensitizer. Peak irradiance and pulse width have minor influence on the efficacy of pulsed irradiation, which decreases as the triplet quantum yield reaches one. The model is supported by in vitro experiments with Photosan 3 and in vivo and in vitro experiments with Photosan 3, Photofrin, aluminum sulphonated phthalocyanine (AlSPc) and benzoporphyrin derivative monoacid ring A (BPD-MA) reported in the literature.
Benzoporphyrin derivative, monoacid ring A (BPD-MA) is a second generation porphyrin photosensitizer, with a significant absorption at 692 nm. The ability of two different lasers (a high-intensity pulsed ruby laser, and a continuous wave (cw) argon-ion laser pumped dye laser) in producing photodynamic damage to human bladder carcinoma cells in vitro under similar conditions was compared. Cells incubated in 0.14 (mu) M BPD-MA for 3 hours were irradiated with 1 or 3 J/cm2 with either pulsed or cw irradiation at 694 nm. Cell survival was determined using an MTT assay. With the ruby laser essentially no phototoxicity was observed at the high intensity pulsed irradiances used, whereas 38% and 6% survival rates were observed for 1 and 3 J/cm2, respectively, using cw irradiation. Possible explanations for the lack of BPD-MA phototoxicity using the ruby laser are: rapid photodegradation, saturation and excitation into higher excited states of the sensitizer. No BPD-MA photodegradation was observed in 1.4 (mu) M BPD-MA in 10% fetal calf serum solutions using the ruby laser. However, an oxygen-dependent photodegradation with the formation of a chlorin-type photoproduct was observed in these solutions using cw irradiation. A simple calculation indicated that the high pulse irradiances used in this study (4.4 X 107 W/cm2) were approximately 3 orders of magnitude greater than required for the onset of saturation. If higher excited states (Sn or Tn) are populated, they do not undergo any photochemistry resulting in phototoxicity or in photoproduct formation. These results show that with the low saturation threshold of BPD-MA, the choice of source and irradiance are important considerations in planning a therapeutic regime.
Benzoporphyrin derivative monoacid ring A (BPD-MA) is a potent new photosensitizer with a significant extinction coefficient at 690 nm. In this study, the photosensitizing efficacy of BPD-MA irradiated at 694 nm with continuous wave (cw) radiation from an argon ion laser pumped dye laser and high intensity pulsed radiation from a ruby laser was compared. Human bladder carcinoma cells in vitro were treated with 0.14 (mu) M BPD-MA for 3 h, washed, then irradiated with either cw or pulsed irradiation. A cumulative cw radiant exposure of 1 or 3 J/cm2 led to cellular survival rates of 38% and 6% respectively compared to untreated controls. Essentially no phototoxicity was observed at the high intensity pulsed powers used in this study.
Multidrug resistance in cancer chemotherapy is a well established phenomenon. One of the most common phenotypical changes in acquired or intrinsic multidrug resistance in human tumor cells is the overexpression of the mdrl gene product P-glycoprotein, which acts as an active efflux pump. Increased levels of P-glycoprotein are associated with resistance to a variety of anticancer drugs commonly used in tumor chemotherapy like anthracyclins, vinca- alcaloids, epipodophyllotoxins or actinomycin D. We investigated the efficacy or photodynamic therapy in the treatment of tumor cells expressing the multidrug resistance phenotype. Our data show that multidrug resistant cells are highly cross resistant to the phototoxic stain rhodamine 123 but exhibit only low degrees of cross resistance (2 - 3 -folds) to the photosensitizers Photosan-3, Clorin-2, methylene blue and meso-tetra (4- sulfonatophenyl) porphine (TPPS4). Resistance is associated with a decrease in intracellular accumulation of the photosensitizer. Verapamil, a membrane active compound known to enhance drug sensitivity in multidrug resistant cells by inhibition of P-glycoprotein, also increases phototoxicity in multidrug resistant cells. Our results imply that tumors expressing the multidrug resistance phenotype might fail to respond to photochemotherapy with rhodamine 123. On the other hand, multidrug resistance may not play an important role in photodynamic therapy with Photosan-3, Chlorin-2, methylene blue or TPPS4.
Previous biophysical investigations1 demonstrated photosensitive
propertieB of EPIRUBICIN with sufficient photochemical stability
significant radical production and sufficient maintanance of
pharmacological properties like intercalation potency
Previous in-vitro investigations2 (human bladder cancer line ATCC
5637) demonstrated after drug treatment an enhancement of cytotoxic activity with continoüs irradiation (100 mW/cm2, 20 mm)
performed at the wavelength of maximal absorption of the drug
Because of a high tissue absorption of this short wavelength combined with decreasing photochemical reactions in deeper tissue,
we investigated whether the reaction rate can be increased using
pulsed irradiation with an averaged light power of also
100 mW/cm2. As light sources we used an Ar2-laser and an Excimer
dye laser system.
To simulate different tissue depths we exposed monolayer cells in
a first step with different averaged intensities (100, 10, 1
mW/cm2 ) and in a second step we additionally varied drug concentrations (50, 5 ug/ml).
24 h after EPIRUBICIN treatment using a concentration of
50 ug/ml, viability of pulsed irradiated cells was 2 fold less
compared to irradiated cells.
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