Sonja Fickweiler

INDOCYANINE GREEN: INTRACELLULAR UPTAKE AND PHOTOTHERAPEUTIC EFFECTS IN VITRO

Indocyanine green (ICG; absorption peak in human plasma 805 nm) was investigated for ICG-mediated phototherapy in vitro. The cellular uptake of ICG (1 microM-50 microM) into HaCaT keratinocytes after an incubation period of 24 h increased up to an intracellular ICG concentration of 12.1 +/- 1.3 nmol per 10(6) cells. To examine dose dependent phototoxic effects in vitro, keratinocytes were incubated with 0 microM-50 microM ICG for 24 h and irradiated by a diode laser (805 nm) with different energy densities (0, 12, 24, 48 J cm-2). All applied ICG concentrations except for 5 microM yielded a cell killing effect in combination with irradiation depending significantly on ICG concentration and light dose. Cell viability for dark control and cells incubated with 50 microM ICG and irradiated with 48 J cm-2 was 0.82 +/- 0.15 and 0.07 +/- 0.02, respectively. Sodium azide (100 mM), a quencher of reactive oxygen species, inhibited significantly the cell killing using 50 microM ICG and 24 J cm-2. Taken together, photoactivation of ICG by irradiation with a diode laser was shown to induce effectively cell killing of HaCaT keratinocytes. Moreover, this effect was inhibited by sodium azide, thus irradiation of ICG might induce a photodynamic reaction.

Indocyanine green (ICG) and laser irradiation induce photooxidation

Abstract The cellular uptake and subcellular localization of indocyanine green (ICG; absorption band 700– 850 nm), and cell survival and ultrastructural changes following ICG-mediated phototherapy were investigated in vitro in four different cell lines derived from human skin (SCL1 and SCL2 squamous cell carcinoma, HaCaT keratinocytes and N1 fibroblasts). The cellular uptake of ICG (1–50 μ M , incubation times 1, 4, 24 h) was saturable, highly cumulative and could be inhibited by the addition of 250 μ M bromosulphophthalein indicating the involvement of the organic anion transporting polypeptide (OATP). For HaCaT cells, the maximum cellular uptake (V max ) and the Michaelis constant (K m ) were 9.9 ± 1.1 m M and 47 ± 16 μ M , respectively, following a 24-h incubation with ICG. Fluorescence microscopy revealed a cytoplasmic distribution of ICG, probably bound to glutathione S -transferase. Following irradiation with a cw-diode laser (805 nm, 80 mW/cm 2 ) at doses of 24 or 48 J/cm 2 , the phototoxicity was determined using the MTT assay as a measure of cell viability. For all cell lines, ICG concentrations above 25 μ M produced a significant phototoxic effect. The EC 50 of ICG for HaCaT cells following irradiation at 24 J/cm 2 was 20.1 ± 3.9 μ M . Growth curves showed that even HaCaT cells treated at the EC 50 were killed within a week following treatment. Electron microscopy 1 h after ICG-mediated phototherapy revealed cytoplasmic vesiculation, dilation of the rough endoplasmic reticulum, the Golgi complex and the perinuclear cisternae and the beginning of chromatin condensation in the nucleus. These ultrastructural findings are not consistent with a photothermal action of ICG-mediated phototherapy. Taken together with those of previous studies by our group these results support photooxidation as a major cell-killing mechanism.

Cell-type Specific Protoporphyrin IX Metabolism in Human Bladder Cancer in vitro¶

Abstract 5-Aminolevulinic acid (ALA)–supported fluorescence endoscopy of the urinary bladder results in a detection rate of bladder cancer superior to that of white light endoscopy. The different accumulation of the metabolite protoporphyrin IX (PPIX) in tumor cells after ALA instillation is poorly understood; however, it is crucial to optimize diagnosis and potential phototherapy. For systematic analysis of cell-type specific PPIX accumulation and metabolism two human bladder carcinoma cell lines (RT4 and J82), a normal urothelial cell line (UROtsa), and a fibroblast cell line (N1) were chosen, and grown in two different growth states to model important tissue components of the urinary bladder, i.e. tumor, normal epithelium and stroma. To quantitate PPIX content, fluorescence intensities measured by flow cytometry were matched with cellular PPIX extraction values, and related to relative ferrochelatase activity, cellular iron content, number of transferrin receptors per cell and porphobilinogen deaminase (PBGD) activity. For in vitro experiments, the initial correlation of relative flow cytometric and spectrometric measurements of PPIX provides a calibration curve for consequent flow cytometric PPIX quantification. Lower fluorescence of normal cells could be explained by significant differences of ferrochelatase activity and iron content in comparison to tumor cells. However, the content of iron was not related to transferrin receptor content. PBGD activity seemed to play a minor role for the differential accumulation of PPIX in urothelial cells. In conclusion, the in vitro culture of urothelial cells and fibroblasts indicates that the most important metabolic step for PPIX accumulation in the urinary bladder is the transition from PPIX to heme. Further investigation of PPIX metabolism does support the validation of photodynamic diagnosis, and might also lead the way to a highly specific tumor related molecule.

9-Acetoxy-2,7,12,17-tetrakis(β-methoxyethyl)-porphycene (ATMPn), a novel photosensitizer for photodynamic therapy: uptake kinetics and intracellular localization

The optimal photosensitizer for topical or systemic photodynamic therapy (PDT) has not yet been found. A promising new second-generation sensitizer is 9-acetoxy-2,7,12,17-tetrakis-(beta-methoxyethyl)-porphycene (ATMPn) whose time- and temperature-dependent uptake and intracellular localization were investigated in two human-skin-derived cell lines (HaCaT keratinocytes and dermal fibroblasts). Flow cytometry analysis (0-800 s) revealed an immediate increase in fluorescence in the cells after start of incubation with 100 ng ml-1 ATMPn (in cell culture medium). At longer incubation periods (0-24 h) a constant increase in fluorescence up to 12 h, with a steady state up to 24 h, was observed. Keratinocyte showed a faster rate of ATMPn uptake than fibroblasts within the first 12 h. Temperature-dependent ATMPn uptake was measured at 4 and 37 degrees C. An increase in fluorescence was observed even at 4 degrees C, suggesting that cellular uptake of ATMPn is partially based on passive diffusion. Confocal laser scan miscroscopy showed spotty, granular fluorescence inside the cytoplasm after incubation with ATMPn, similar to the pattern of rhodamine 123 which stains mitochondria. These results demonstrated an unusually fast intracellular, probably intramitochondrial, uptake of ATMPn in vitro. Therefore the use of ATMPn in photodynamic therapy might allow a reduction of the time span between administration of drug and irradiation.

Photosensitization of human skin cell lines by ATMPn (9-acetoxy-2,7,12,17-tetrakis-(beta-methoxyethyl)-porphycene) in vitro: mechanism of action.

9-Acetoxy-2,7,12,17-tetrakis-(beta-methoxyethyl)-porphycene (ATMPn) is a promising new photosensitizer characterized by high absorption around 640 nm and high singlet oxygen yield. To study the mechanism of action in vitro we have investigated uptake, intracellular localization, cell survival and ultrastructural changes following photodynamic treatment in human cell lines derived from the skin (SCL1 and SCL2, squamous cell carcinoma; HaCaT keratinocytes; N1 fibroblasts). Using flow cytometry we have determined the cellular fluorescence as a marker for the uptake of ATMPn after incubation for 60 min. Co-staining with ATMPn and fluorescent dyes specific for cell organelles reveals an intracellular localization of ATMPn in lysosomes. Following irradiation using an incoherent light source (580-740 nm) and a light fluence of 24 J cm-2, phototoxicity is determined by means of the 3-4.5 dimethylthiazol-2,5 diphenyl tetrazolium bromide (MTT) assay. For all cell lines ATMPn concentrations above 15 nM yield a significant phototoxic effect. The 50% effective concentration, EC50, for SCL1 cells is 11.2 +/- 2.9 nM ATMPn. ATMPn uptake and phototoxicity are more effective for HaCaT and SCL1 as compared to SCL2 and N1 cells. Growth curves confirmed the results of the MTT assay. Because of the high lysosomal accumulation of ATMPn, already low photosensitizer concentrations without dark toxicity yield a high photodynamic effect. Immunofluorescence and electron microscopy reveal damage to tonofilaments, plasma membrane and mitochondria, indicating a mechanism unrelated to apoptosis. A dose yielding complete cell killing, as needed for oncological indications, might lead to necrosis, whereas lower sub-lethal doses result in induction of apoptosis.

Cell-type-specific response to shock waves of suspended or pelleted cells as analysed by flow cytometry or electrical cell volume determination

Shock-wave-induced cell damage of suspended or pelleted bladder cancer cells was analysed with the flow cytometric propidium iodide (PI)/fluorescein diacetate assay, and electrical volume determination using the CASY 1 analyser system and growth curves. The CASY system revealed a smaller fraction of suspended RT4 cells with impaired membrane integrity than the flow cytometric assay. No differences were found for pelleted RT4 cells and suspended J82 cells. The discrepancies of the two viability assays indicated a different response of the cell membrane to shock waves which was dependent on the exposure system and the cell type. Growth curves indicated delayed cell death for suspended RT4 cells and exclusively immediate cell death for pelleted RT4 cells and suspended J82 cells. PI positive suspended RT4 cells were morphologically intact while pelleted RT4 cells and suspended J82 cells were mainly disrupted. From these data it can be concluded that intracellular or membrane alterations seem to be correlated with the occurrence of cavitational effects while cell disruption can likewise occur by the direct impact of the shock wave.

The combined effects of high-energy shock waves and ionising radiation on a human bladder cancer cell line

The effects of high-energy shock waves (HESW) generated by an experimental Siemens lithotripter in combination with 137Cs gamma-rays were examined in vitro. Proliferation after treatment of immobilised pellets of either single cells or multicellular spheroids of the bladder cancer cell line RT4 was determined using colony-forming assays and cell cycle analysis. Surviving and cell cycle fractions were calculated for each shock wave and radiation application mode separately, and for sequential combination in different successions for the purpose of characterizing the interaction of both treatment modalities. Combination of HESW and ionising radiation turned out to act additively or slightly supra-additively on both biologic models.

Dosedependent photodynamic effects of 9-acetoxy-2,7,12,17-tetrakis(\-methoxyethyl)-porphycene in vitro.

Porphycenes are chemically pure photosensitizers for topical and systemic photodynamic therapy (PDT). Fast cellular uptake of 9-acetoxy-2,7,12,17-tetrakis-(sB-methoxyethyl)porphycene (ATMPn) has been shown previously.HaCaT human keratinocytes were incubated with ATMPn (1 nmol l-1 to 1 µmol l-1 in DMSO or DOPC liposomes). After 1 h, cells were irradiated with different light doses (0, 24, 48J cm-2) using an incoherent light source (580—740 nm, 40 mW cm-2). Cytotoxic effects were determined by assessing the mitochondrial activity using the MTT assay 24 h following irradiation.Cytotoxic effects were dependent on ATMPn concentration and light dose. Using 20 nmol 1-1, a 50% decrease of mitochondrial activity (EC50) after irradiation with 24 J cm-2 was achieved. Lowering the ATMPn concentration (10nmol 1-1) and increasing the light dose (48 J cm-2) yielded the same effect (EC50). Maximal decrease of mitochondrial activity (90%) was achieved using ATMPn concentrations of 50–100 nmol l-1 and a light dose of 24 J cm-2 or 25 nmol l-1 ATMPn and 48 Jcm-2.There was no difference regarding the dose-dependent cytotoxic effects using either ATMPn in DMSO or DOPC liposomes. In the control group (incubation with 1 nmol 1-1 to 1µmol 1-1 ATMPn, no irradiation), dark toxicity was not observed.Cell photosensitization with ATMPn was very efficient in vitro yielding the maximal cytotoxic effect at very low ATMPn concentrations as compared to other photosensitizers. Since ATMPn in DMSO and DOPC liposomes revealed the same cytotoxic effects without dark toxicity, theDMSO formulation, which is much easier to prepare, will be preferred in future studies.

Photosensitization of skin-derived cell lines by Dimegin [2,4-di-(α-methoxyethyl)-deuteroporphyrin IX] in vitro

The deuteroporphyrin‐IX derivative Dimegin [2,4‐di‐(α‐methoxyethyl)‐deuteroporphyrin IX] was investigated with respect to cellular uptake, intracellular localization and cell survival following photodynamic treatment in human cell lines derived from the skin (SCL1 and SCL2, squamous cell carcinoma; HaCaT keratinocytes; N1 fibroblasts). Using flow cytometry, we determined the cellular fluorescence as a marker of the uptake of Dimegin after incubation for 24 h. The intracellular localization of Dimegin was analysed using fluorescence microscopy and co‐staining with fluorescent dyes specific for cell organelles. Following irradiation with an incoherent light source (580–740 nm) using a light dose of 24 J/cm2, phototoxicity was determined by means of trypan blue dye exclusion, MTT assays and growth curves. The relative Dimegin fluorescence of the different cell lines declined as follows: SCL1>HaCat>N1>SCL2. Intracellular localization of Dimegin was found in the mitochondria. For all cell lines Dimegin concentrations above 15 mM yielded a significant phototoxic effect. The EC50 for SCL1 cells was 8.9±2.0 μM Dimegin. The EC50 for the cell lines increased as follows: SCL1

5-aminolevulinic acid (ALA) mediated photodynamic therapy of bladder cancer cell lines

Topical application of 5-aminolevulinic acid (ALA) can be effectively used for photodynamic therapy and diagnosis of superficial bladder cancer. Administration of the heme precursor ALA leads to the selective accumulation of the photosensitizer protoporphyrin IX (PPIX) in certain types of tissue. The aim of this study was to determine the cellular PPIX concentration and the effect of photodynamic therapy mediated by ALA on two bladder cancer cell lines (RT4, J82) and a fibroblast cell line (N1). Following incubation with ALA the kinetics of cellular PPIX were examined using flow cytometry combined with extraction. The cancer cell lines showed considerably higher PPIX concentrations than the fibroblast cell line: RT4 1030, J82 710, and N1 110 ng PPIX/mg protein. Photodynamic therapy was performed with an incoherent light source (580 - 740 nm, 40 mW/cm2, 30 J/cm2). In contrast to the fibroblast cell line, which was resistant to photodynamic therapy, the cancer cell lines were effectively killed following the treatment as determined by MTT assay. This study suggests that ALA-mediated photodynamic therapy may be effective in transitional cell carcinoma of the bladder. Based on these findings, this therapeutic method should be further evaluated clinically.