Pia Steinbach

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.

CELLULAR FLUORESCENCE OF THE ENDOGENOUS PHOTOSENSITIZER PROTOPORPHYRIN IX FOLLOWING EXPOSURE TO 5-AMINOLEVULINIC ACID

Abstract— Supplying 5‐aminolevulinic acid (ALA), a precursor in the biosynthetic pathway to heme from an external source leads to an accumulation of the endogenous fluorescent photosensitizer protoporphyrin IX (PPIX). Following instillation of ALA in the urinary bladder neoplastic tissue can be discerned by fluorescence cystoscopy or treated by illumination with light of an appropriate wavelength. In order to provide a biological rationale for the clinical findings, we have analyzed the capacity of three different cell lines to accumulate PPIX by flow cytometry. Three different urothelial cell lines, normal fibroblasts and endothelial cells were exposed to ALA under varying conditions. Urothelial cell lines J82 and RT4, derived from malignancies of the bladder displayed fluorescence intensities 9‐ and 16‐fold, respectively, above the fluorescence level of the normal urothelial cell line HCV29. Human umbilical cord endothelial cells fluoresced moderately while the fibroblast cell line Nl exhibited a fluorescence level comparable to those of the cancer cells. Fluoresence increased with increasing cell density and was also dependent on the growth of cells as monolayers or multicellular spheroids. Increasing ALA concentrations led to saturation of fluorescence after 4 h of incubation at cell type‐specific fluorescence levels obtained at different ALA concentrations. Continuous incubation in medium containing serum resulted in a linear rise of fluorescence during the first 4 h, which was followed by a saturation period (8–24 h) and a renewed rise. In the case of serum depletion, fluorescence intensities were significantly higher and increased linearly during the entire 48 h incubation period. By replacing serum with albumin, it could be shown that the emission of PPIX into the medium in the presence of serum is mainly caused by this protein. The ALA‐induced fluorescence was predominantly perinuclear after 4 h of incubation and relocated toward the cell membrane after prolonged incubation. This study demonstrated the complexity of factors influencing the ALA‐induced fluorescence and should stimulate further research in this field.

In vitro investigations on cellular damage induced by high energy shock waves

Single-cell suspensions of the prostate carcinoma cell line PCA were exposed to electromagnetically generated ultrasound shock waves (source and focusing lens identical to those used in the commercially available lithotripor Lithostar Plus). Cell loss of up to 40% occurred in sample tubes containing air. To expose multicellular tumor spheroids and cells growing on a microcarrier, an experimental setup was developed that prevented motion of the specimen. Intracellular damage of intact spheroids was analyzed by laser scanning microscopy following specific fluorescence staining. Different sensitivities of individual cell components with respect to the applied energy density of the pulses were found, namely defects on cell membranes (0.12 mJ/mm2), vimentin (0.21 mJ/mm2), mitochondria (0.33 mJ/mm2) and nuclear membranes (0.5 mJ/mm2). Loss of cells growing on a microcarrier was found after application of 200 pulses with 0.21 mJ/mm2.

Induction of stress fibres and intercellular gaps in human vascular endothelium by shock-waves

Human umbilical cords were exposed to high amplitude focussed ultrasonic pulses with focal energy densities 0.6 and 0.4 mJ mm-2. The endothelium of the exposed vessel (vein) was examined by means of confocal laser scanning microscopy and scanning electron microscopy. The degree of tissue change ranged from the induction of stress fibres and intercellular gaps to the complete detachment of endothelial cells combined with damage of the basement membrane. An increased number of stress fibres may indicate an increased vessel wall permeability. This might explain the enhanced effects in experimental tumour therapy that have been found by other authors when combining shock-waves with drugs.

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.

Determination of the energy-dependent extent of vascular damage caused by high-energy shock waves in an umbilical cord model

SummaryTo determine the spatial extent of shock-waveinduced vascular damage human umbilical cords were exposed to electromagnetically generated, focused ultrasound waves of different energy densities. During treatment macroscopically visible hematoma and superficial holes appeared. Following exposure specimens were fixed and examined histologically. In addition to vessel wall necrosis and rupture, complete detachment of endothelial cells in defined regions was observed. A correlation of the extent of the damage with the energy density distribution revealed that a local energy density of 0.3 mJ/mm2 is the lower threshold for the occurrence of severe vascular damage.

Analysis of cell cycle‐related Ki‐67 and p120 expression by flow cytometric BrdUrd‐Hoechst/7AAD and immunolabeling technique

Flow cytometric multiparameter analysis of two proliferation associated antigens, Ki-67 and p120, was combined with cell cycle kinetic analysis, achieved by continuous labeling with 5-Bromodeoxyuridine (BrdUrd), followed by staining with Hoechst 33258 and 7-Aminoactinomycin D (7AAD). Exponential and plateau phase monolayer cultures of the human bladder carcinoma cell line J82 were examined. Resting cells, characterized by their absent BrdUrd incorporation, showed no reactivity with the MIB1 antibody, which was used for the detection of the Ki-67 antigen. Proliferating cells revealed a cell cycle phase dependent Ki-67 staining intensity, which was partially related to the time period spent in G1 after mitosis. In contrast to the Ki-67 antigen expression, no decrease in p120 immunofluorescence staining intensity of non-cycling cells could be observed. We could demonstrate that a dissection of the history of cell replication, obtained by the BrdUrd/Hoechst technique combined with a simultaneous immunofluorescence staining reveals detailed information, on a single cell level, about time dependent expression of proliferation associated antigens in all cell cycle compartments.

The combined effects of high-energy shock waves and cytostatic drugs or cytokines on human bladder cancer cells.

The effects of shock waves generated by an experimental Siemens lithotripter in combination with cytostatic drugs or cytokines on several bladder cancer cell lines were examined in vitro. Proliferation after treatment was determined with the 3-4,5-dimethylthiazol-2,5 diphenyl tetrazolium bromide assay. Dose enhancement ratios were calculated for each drug and each shock wave application mode in order to characterise the sensitising effect of shock wave pretreatment. The influence of the time between shock wave and drug treatment as well as the effects of different sequences of shock wave and drug treatment or concomitant treatment were assessed for selected combinations of cell lines and drugs. It was found that shock wave treatment could render certain cell lines more susceptible to subsequent cis-platinum, mitomycin C or actinomycin D incubation. Cell lines sensitive to tumour necrosis factor alpha or interferon alpha were further sensitised to these cytokines by shock wave pretreatment. The enhanced sensitivity to cis-platinum and actinomycin D decreased rapidly during the first hours after shock wave treatment. The antiproliferative effect was most pronounced after concomitant shock wave and drug treatment. The sensitisation to interferon alpha diminishes more slowly after shock wave exposure. From the results presented in this study it is concluded that transient shock wave-induced permeabilisation of cell membrane not only enhances drug efficiency, but also causes damage to cell organelles and alterations in cellular metabolism.

Targeting of the tumor microcirculation by photodynamic therapy with a synthetic porphycene

9-acetoxy-2,7,12,17-tetrakis-(beta-methoxyethyl)-porphycene (ATMPn) is a chemically pure substance with fast pharmacokinetics and superior photodynamic properties in vitro as compared to Photofrin. In this study the pharmacokinetics, photodynamic efficacy and tissue localization of ATMPn were investigated in vivo. Amelanotic melanomas (A-Mel-3) were implanted in dorsal skin fold chambers fitted to Syrian Golden hamsters. Fluorescence kinetics of ATMPn (1.4 mumol kg-1 b.w.i.v.; n = 8) were monitored by intravital microscopy. Quantitative measurements of fluorescence intensity were carried out by digital image analysis. For tumor growth studies 1.4 mumol kg-1 was injected 24 h (n = 3), 3 h (n = 3), 1 min (n = 6) and 2.8 mumol kg-1 1 min (n = 6) before PDT (Laser (630 nm) or lamp (600-750 nm), 100 mW cm-2, 100 J cm-2). Tumor volume was measured for 28 d. Solid tumors (n = 3) were excised 1 min after injection of ATMPn (2.8 mumol kg-1) and cryostat sections (20 mm) were analyzed by confocal laser scanning microscopy (CLSM) for tissue localization of the dye. Maximal fluorescence (mean +/- S.E.) arose in the tumor (94 +/- 7%) and surrounding host tissue (67 +/- 5%) 30 s post injection followed by a rapid decrease. Hardly any fluorescence was detectable 12 h after administration. Only PDT 1 min after injection of ATMPn was effective yielding 3/6 complete remissions (2.8 mmol kg-1, laser) and 6/6 complete remissions (2.8 mumol kg-1, lamp), respectively. One minute after injection the dye is primarily localized in the vascular wall of normal and tumor vessels as shown by CLSM. PDT at a time, when the dye is localized primarily in the tumor microcirculation, exhibits the best tumor killing effects showing that vascular targeting is effective in treating solid malignant tumors. ATMPn in liposomes makes administration and light irradiation in one session possible due to its fast pharmacokinetics. Thus, using ATMPn as a photosensitizer may provide more flexibility to perform PDT after surgical exploration and debulking as adjuvant therapy.

Shock wave induced endothelial damage—In situ analysis by confocal laser scanning microscopy

For more than a decade, extracorporal shock wave lithotripsy has been a standard clinical method for the treatment of urinary stones. However, side effects that are likely to be correlated to vessel damage can often be observed using noninvasive diagnostic techniques, e.g., magnetic resonance imaging. To avoid side effects it is useful to understand the interaction between shock waves and the vascular system. In particular, this is important in view of new applications like gallstone lithothripsy. In the present study, we exposed human umbilical vessels to electromagnetically generated ultrasound shock waves to analyze subsequent alterations of their endothelial layer. Following en face preparation and fluorescent staining, the endothelium was examined in a confocal laser scanning microscope. Endothelial cells of the shock wave exposed vessels revealed permeabilization of plasma membranes and mitochondrial alterations as potentially lethal damage. An increase in the number of stress fibres may indicate functional changes possibly influencing vessel wall permeability.