BINDING OF HEMATOPORPHYRIN DERIVATIVE TO BRAIN TUMOR CELLS—A FLUORESCENCE SPECTROSCOPIC STUDY

The binding of hematoporphyrin derivative (HpD) to brain tumor cells and their photosensitivity was studied as a function of HpD concentration, time of incubation and growth phase of cells. Upon binding to cells, HpD showed three fluorescence bands at 616, 636 and 678 nm. In plateau phase cells a fluorescence band at 636 nm was predominant, which was further enhanced by increasing HpD concentration and/or increasing incubation time. In exponential phase cells the maximum fluorescence was exhibited at 616 nm. After 1 h incubation of exponential phase cells with increasing HpD concentration an overall intensity enhancement occurred with no change in the distribution of bands, whereas longer incubation time caused an increase in relative intensity of the 636 nm band similar to that observed in plateau phase cells. After 1 h incubation with HpD plateau phase cells were more photosensitive than exponential phase cells, although cell bound HpD was much less in the former case. Incubation of cells for 24 h drastically enhanced the photosensitivity irrespective of the growth phase. Our results suggest a relationship between the fluorescence emission band of HpD at 636 nm and photosensitivity of cells.     

Variation in the fluorescence decay properties of haematoporphyrin derivative during its conversion to photoproducts

Haematoporphyrin derivative (HpD) photoproducts are formed in aqueous solutions during light exposure in the presence of oxygen. The evaluation of the fluorescence decay of the photoproduct-enriched HpD solution shows an increase in the short-lived components, especially about 2 ns, in comparison with HpD without photoproducts. The bleaching of the HpD fluorescence and the photoproduct formation by the fluorescence-exciting radiation has to be taken into account in the evaluation of stationary as well as time-resolved fluorescence measurements.     

Spectroscopic and Biological Testing of Photobleaching of Porphyrins in Solutions*

The photobleaching of protoporphyrin IX (PP IX) and hematoporphyrin derivative (HpD) solutions was followed using three different methods: spectrophotometry, fluorometry and photodynamically induced cytotoxicity. The latter entails photoirradiation of HT29 human colon adenocarcinoma cells in the presence of preirradiated solutions of HpD and PP IX (λ 415 nm). The highest cytotoxicity was observed in the presence of unirradiated dye and decreased with the time of preirradiation. This decay in photocytotoxicity was further used to determine the porphyrin photobleaching kinetics in solution. For both sensitizers, quantum yields of photobleaching obtained by matching fluoresence were higher than that obtained from absorbance measurements (10 and 11 times for HpD and PP IX, respectively). This difference reflects preferential photobleaching of photolabile monomeric forms compared to aggregates. The highest quantum yield was obtained in the biological test (decay in cytotoxicity) which was 14 times higher for HpD and 30 times higher for PP IX than the quantum yield obtained from absorbance measurements. The absence of correlation between biological and fluorescence measurements has to be taken into account in the in vivo situation. Dark storage of preirradiated sensitizers (37°C, 24 h) completely restored photocytotoxity for PP IX but only partially for HpD, whereas fluorescence patterns were partially restored for both sensitizers.     

SUBCELLULAR LOCALIZATION OF HEMATOPORPHYRIN DERIVATIVE IN BLADDER TUMOR CELLS IN CULTURE

Mitochondria have been implicated as a primary subcellular site of porphyrin localization and photodestruction. However, other organelles including the cell membrane, lysosomes and nucleus have been shown to be damaged by hematoporphyrin derivative (HpD) photosensitized destruction as well. In this study we attempted to follow the translocation of the fluorescent components of HpD in human bladder tumor cells (MGH-U1) in culture to determine whether specific subcellular localization occurs over time. Following a 30 min exposure to HpD the cellular fluorescence was examined immediately and 1, 2, 4, and 24 h after HpD removal using fluorescence microscopy and an interactive laser cytometer. The in vitro translocation of dye appeared to be fairly rapid with fluorescence present at the cell membrane and later (1-2 h) within a perinuclear area of the cytoplasm. To determine whether HpD had become concentrated into a specific subcellular organelle, these fluorescence distribution patterns were compared with fluorescent marker dyes specific for mitochondria, endoplasmic reticulum and other membranous organelles. The HpD fluorescence did not appear to be as discrete as the dyes specific for mitochondria or endoplasmic reticulum but appeared similar to the diffuse cytomembrane stain. Finally, the interaction between the fluorescent components of HpD and the cellular constituents was evaluated using a "fluorescence redistribution after photobleaching" technique. The results indicated that the mean lateral diffusion for HpD in MGH-U1 cells was 1.05 x 10(-8) cm2/s, a rate closer to that of lipid diffusion (10(-8)) than that of protein diffusion (10(-10)).(ABSTRACT TRUNCATED AT 250 WORDS)     

PICOSECOND FLUORESCENCE SPECTROSCOPY ON INCORPORATION PROCESSES OF HEMATOPORPHYRIN DERIVATIVE INTO MALIGNANT TUMOR CELLS in vitro

Abstract— By using a highly sensitive streak-camera technique, we investigate incorporation processes of HpD into malignant tumor m-KSA cells in vitro. The picosecond decays of the total fluorescence spectra, the wavelength-resolved fluorescence decays and the time-resolved fluorescence spectra from HpD in the cells are measured as a function of the incubation time. The results show that the aggregate component of HpD which has a fast fluorescence lifetime of 100 ps and a red-shifted band of ∼ 660 nm selectively accumulates more and more in the cells with the increase of the incubation time.     

CELLULAR BINDING OF HEMATOPORPHYRIN DERIVATIVE IN HUMAN BLADDER CANCER CELL LINES: KK-47

Abstract— The fluorescence lifetime and degree of fluorescence polarization of hematoporphyrin derivative (HpD) have been investigated using different solutions: organic and micellar solutions. Ham's F12 medium, and KK-47 cell suspension. The lifetime and polarization degree in organic and micellar solutions did not change with increasing incubation time, but the polarization degree in the cell suspensions temporarily increased at the initial incubation time and then decreased 4 h after incubation. The lifetime in the cell suspensions exhibited a bi-phasic exponential decay. The results obtained suggested that mainly dimeric HpD may bind weakly to the cell membrane, and then slowly be distributed throughout the cytoplasm. The polarity and viscosity of the intracellular loci containing HpD were evaluated from the fluorescence polarizations of HpD in MeOH-H₂O mixtures and ethylene glycol(EG)-MeOH mixtures. The dielectric constant and viscosity of the loci containing HpD were 35 and 11 cp, respectively. Accordingly, the intracellular location of HpD were considered relatively hydrophilic loci of the cells.     

FEMTOSECOND STUDIES OF HEMATOPORPHYRIN DERIVATIVE IN SOLUTION: EFFECT OF pH AND SOLVENT ON THE EXCITED STATE DYNAMICS

Abstract We report direct femtosecond measurements of the excited state dynamics of hematoporphyrin derivative (HpD) in solution. The dynamics are found to be very sensitive to the solvent and pH of aqueous solutions. The decay of the excited singlet states is much faster in acidic and pH 7 buffer aqueous solutions (<230 ps) than in basic aqueous solutions or organic solvents (> 10 ns). The dynamical results show strong correlation with static fluorescence measurements: weaker fluorescence in acidic and pH 7 buffer solutions corresponding to shorter-lived excited states. A new fast decay component with a time constant around 5 ps is identified both in acidic aqueous solutions and in organic solvents such as acetone and attributed to internal conversion from the second to the first excited singlet state of aggregates or certain oligomers in HpD, in accord with the observation that the fast decay component is larger at a higher concentration. Oxygen is found to have no effect on the dynamics on the time scale investigated, 1 ns, indicating that oxygen quenching of the singlet excited states is insignificant on this time scale. The sensitive solvent and pH dependence of the excited state dynamics has important clinical implications in the use of HpD as a photosensitizing agent.     

Cellular uptake,localization and photodynamic effects of haematoporphyrin derivative in human glioma and squamous carcinoma cell lines

Uptake, intracellular concentration, localization and photodynamic effects of a haematoporphyrin derivative (HpD, Photosan-3) were compared in human glioma (BMG-1, wild-type p53) and squamous carcinoma (4451, mutated p53) cell lines. Concentration and time dependence of cellular uptake of HpD was assayed from methanol extracts and whole cell suspension spectroscopy, while localization was studied by fluorescence microscopy-based image analysis. Colony-forming ability, apoptosis, cell-cycle progression and cytogenetic damage (micronuclei formation) were investigated as parameters of photodynamic response following irradiation with red light. BMG-1 cells were more sensitive to the photodynamic treatment than 4451 cells, although the 4451 cells accumulated a higher amount of HpD and did not differ significantly from BMG-1 cells with respect to intracellular localization. Photodynamically-induced cytogenetic damage and apoptosis were considerably higher in BMG-1 cells as compared to 4451 cells. The present results strongly suggest that manifestation of the photodynamically-induced lesions in the form of cytogenetic damage and apoptosis are among the important determinants of cellular sensitivity to HpD-PDT besides the photodynamic dose (intracellular concentration of the photosensitizer and the light dose).     

Laser picosecond microspectrofluorometry of haematoporphyrin in cells and liposomes.

The results of a laser picosecond microspectrofluorometric study of the spectral and kinetic characteristics of haematoporphyrin (Hp) fluorescence at various sites in cultured SPEV cells and phosphatidylcholine liposomes are presented. The computer-controlled detection system is based on the single-photon counting method with picosecond time resolution. In aqueous medium, the Hp fluorescence spectrum is characterized by two bands at 615 and 675 nm. In living cells and liposomes, Hp fluorescence is red shifted to 630 and 690 nm. In addition a new band at 665 nm is detected. The dependence of this band on the incubation time and Hp concentration was investigated. The fluorescence decay kinetics of Hp in a culture medium, liposome and a cell nuclear membrane were measured. Possible Hp aggregate formation in the lipid bilayer and its implications are discussed.     

GIANT CELL FORMATION IN BLADDER TUMOR CELLS FOLLOWING HEMATOPORPHYRIN DERIVATIVE-SENSITIZED PHOTOIRRADIATION

Abstract— Giant cell induction was studied in cultured bladder tumor cells (MGH-Ul; commonly referred to as "EJ") following hematoporphyrin derivative (HpD)-sensitized photoirradiation. Eight days after treatment, control colonies (no treatment, HpD alone, light alone) were generally uniform in size and density and contained few giant cells. Colony heterogeneity and giant cell frequency increased with light dose in the presence of HpD. The morphology and frequency of giant cell formation, with respect to survival levels, were comparable to those observed in 280 kVp X-irradiation of the same cells.     

Time-gated fluorescence spectroscopy of porphyrin derivatives and aluminium phthalocyanine incorporated in vivo in a murine ascitic tumour model.

The effect of systemic administration on drug uptake at cellular level was evaluated using time-gated fluorescence spectroscopy performed on a murine ascitic tumour model. Mice bearing L1210 leukaemia were injected intraperitoneally or intravenously with 25 mg per kg body weight hematoporphyrin derivative (HpD), 12.5 mg per kg body weight photofrin II (PII), 25 or 5 mg per kg body weight disulphonated aluminium phthalocyanine (AlS2Pc). Every 2 h and for up to 22 or 30 h, mice were sacrificed, leukaemic cells extracted from the peritoneum, washed, and resuspended in buffer for fluorescence measurements. HpD and PII emission spectra were almost identical 12 h after intraperitoneal injection with main peaks at 630 nm and no appreciable changes afterwards. In the first 12 h, the PII fluorescence spectrum was constant, while in the case of HpD a shoulder at 615 nm was detectable. Similar fluorescence behaviour was observed after intravenous administration of porphyrin derivatives. These results seem to confirm that the tumour localizing fraction is the part actually retained by the cells. The AlS2Pc spectrum peaked at 685 nm and did not change in any of our experiments. AlS2Pc is incorporated more rapidly with respect to porphyrins, as was clearly observed in the case of intravenous administration, where the AlS2Pc fluorescence was readily detectable after 2 h, whereas the PII emission became apparent only after 4-6 h.     

TIME-GATED FLUORESCENCE IMAGING FOR THE DIAGNOSIS OF TUMORS IN A MURINE MODEL

A system for time-gated fluorescence imaging was used to perform measurements on tumor-bearing mice treated with hematoporphyrin derivative (HpD). The aim of the study was to define the potential of this technique in the diagnosis of tumors by taking advantage of the long fluorescence lifetime of the exogenous dye with respect to the decay times of the natural fluorescence. After the administration of three different drug doses (5, 10 and 25 mg/kg body weight), fluorescence images were acquired at various uptake times (from 2 h to 10 d), to determine the best instrumental conditions and experimental procedure for the detection of tumors in the murine model considered. The optimal fluorescence contrast between the tumor area and the surrounding healthy tissue was found at 12 h after the administration of either 5 or 10 mg/kg HpD and was anticipated at 8 h for the highest drug dose. In this optimum condition, the tumor region could be identified even after the injection of 5 mg/kg HpD. A better fluorescence contrast was always obtained in 15 ns-delayed images with respect to synchronous ones.     

PHOTOINACTIVATION OF CHINESE HAMSTER CELLS BY HEMATOPORPHYRIN DERIVATIVE AND RED LIGHT

Abstract— The use of hematoporphyrin derivative (HpD) has previously been demonstrated to be beneficial in clinical cancer therapy. This paper describes cell culture studies used to examine HpD phototherapy in Chinese hamster ovary cells (line CHO). Survival curves have been obtained for both direct HpD toxicity and HpD induced photoinactivation. Examination of HpD induced photoinactivation as a function of stage in the cell growth cycle has also been performed, as has the quantitative measurement of HpD uptake in cells (using ³H-HpD) as a function of cellular incubation time, serum concentration in the incubation medium, and cell cycle position. In the absence of light, no toxicity was observed for HpD incubation levels of up to 400 μg/m/ when incubations times were 3 h or less. Exposure of cells to light alone (> 590 nm, 4.0 mW/cm²) for 9 min was also found to be completely nontoxic. Survival curves obtained for exponentially growing cells labeled with various concentrations of HpD and subsequently illuminated with red light exhibited a threshold or shoulder region at short exposure times followed by exponential killing at longer exposure times. The cell cycle response curves for HpD induced photoinactivation of synchronized CHO cells was nearly flat, indicating no variation in sensitivity for cells treated at time periods from 6 to 15 h after mitosis. Additon of serum to the incubation medium resulted in improved plating efficiency and reproducible survival curves but decreased cellular uptake of HpD.     

HEMATOPORPHYRIN DERIVATIVE UPTAKE BY ATHEROMA IN ATHEROSCLEROTIC RABBITS: THE SPECTRA OF FLUORESCENCE FROM HEMATOPORPHYRIN DERIVATIVE DEMONSTRATED BY AN EXCIMER DYE LASER

Abstract A new diagnostic and therapeutic endoscopic system consisting of an excimer pulse dye laser is presented. This report demonstrates the accumulation of hematoporphyrin derivative (HpD) in atheroma as shown by the fluorescence of HpD using this equipment. Atheroma was induced in the aorta of WHHL (Watanabe heritable hyperlipidemic) rabbits, 5 mg kg⁻¹ HpD was injected intravenously and the rabbits were sacrificed 24 h later. The aorta was dissected and the localization of HpD was examined. Characteristic peaks of the fluorescence of HpD at 630, 665 and 690 nm wavelength were detected in the atheromatous lesion. However, in the fatty plaque, the emission peak at 630 nm was lower and the 665 nm peak faded away. No fluorescence with peaks was detected in the normal area. The ratio of fluorescence intensity in atheroma, border zones and normal areas was 10.4 : 5.0 : 1.0. On normal rabbits made atherosclerotic by diet and balloon damage, an ultra thin endoscopic catheter was inserted from the descending aorta of atherosclerotic rabbits under anesthesia. Essentially the same data was obtained by these studies in vivo as was obtained in the in vitro studies. The above data suggests the possibility of future applications of this equipment for diagnosis of atheroma.     

MEMBRANE LYSIS IN CHINESE HAMSTER OVARY CELLS TREATED WITH HEMATOPORPHYRIN DERIVATIVE PLUS LIGHT

Abstract Chinese hamster ovary cells in exponential growth were incubated with various concentrations of hematoporphyrin derivative (HpD). Cellular porphyrin content was determined after 2 h incubation at 37°C using [³H]-hematoporphyrin derivative. Photoactivation of cell-bound HpD by red light resulted in a family of survival curves with terminal slopes proportional to cellular HpD concentration. The degree of cellular lysis, assayed 1 h after illumination using a chromium-51 labeling technique, was also found to be related to cellular HpD concentration. The amount of 51Cr released increased with post-irradiation incubation to a level parallel to cell lethality as measured by colony formation. These data suggest that lysis of the cell membrane may be largely responsible for cellular inactivation following HpD photoirradiation.     

EQUILIBRIUM AMONG HEMATOPORPHYRIN-DERIVATIVE COMPONENTS: INFLUENCE OF THE INTERACTION WITH CELLULAR STRUCTURES

Abstract— Hematoporphyrin-derivative (HpD) is a complex mixture of porphyrins in different aggregation states. The spectral analysis of HpD in aqueous solution shows the presence of monomeric species through the fluorescence emission spectrum and of both monomeric and aggregated species through the absorption spectrum.
The interaction with biopolymers and cellular components results in the appearance of new emission bands at 630 nm and in the640–670 nm spectral region which can be evidenced under suitable excitation conditions. Correspondingly, two new decay times (∼ 0.6 ns, and ∼ 3 ns), are observable. The new fluorescent species detected can be considered as the result of the hydrophobic effect induced by cellular structures on porphyrin aggregates.     

ACTION SPECTRUM (620–640 nm) FOR HEMATOPORPHYRIN DERIVATIVE INDUCED CELL KILLING

Abstract— Monochromatic red light generated by a tunable dye laser is currently being utilized for the treatment of solid tumors with hematoporphyrin derivative (HpD) photoradiation therapy (PRT). Experiments were performed using mammalian cells to determine the most efficient wavelength of red light (620 to 640 nm range) for HpD induced cellular photoinactivation. Decrease in the clonogenic potential of Chinese hamster ovary (CHO) cells was examined following both short (I h) and extended (12 h) HpD incubation times. Maximal photosensitization was observed with wavelengths ranging from 630 to 632.5 nm and the action spectra for cell killing matched the absorption spectra for HpD bound to cells. Similar observations were obtained following both short and extended HpD-cell incubation times. The potential relevance of these results as they relate to clinical HpD PRT are discussed.     

血卟啉衍生物(HpD)的表面增强共振拉曼光谱

近年来,采用光敏剂(H_pD)与光的协同效应,诊治癌肿疾病有了较大的进展,所以,研究其光谱特性是有其现实意义的。 用共振拉曼光谱和表面增强拉曼光谱所获得的散射强度,都比通常的拉曼光谱有几个数量级的增强,并可得到稀溶液(10~(-6)～10~(-3)mol/L)的高质量光谱,本文报道了8×10~(-3)mol/L HpD水溶液的共振拉曼光谱和5×10~(-5)mol/L HpD水溶液的表面增强共振拉曼光谱,结果表明,表面增强共振拉曼光谱比共振拉曼光谱的散射强度滴出三个量级。     

TIME-GATED FLUORESCENCE SPECTROSCOPY OF THE TUMOR LOCALIZING FRACTION OF HpD IN THE PRESENCE OF CATIONIC SURFACTANT

Time-gated fluorescence spectroscopy was performed on the tumor localizing fraction (TLF) of HpD in buffer at different concentrations of cationic surfactant. This technique obtains emission spectra with programmable delay relative to the excitation pulse. According to the measured fluorescence decay-time constants (approximately 0.7, approximately 3 and approximately 15 ns) three gates were considered, delayed by 0, 5 and 18 ns, respectively, to evaluate the contribution of the emitting molecular species to the spectra. Simultaneous to these measurements, fluorescence decay waveforms and time-integrated spectra were also detected. In buffer and in detergent micelles the fluorescence spectra are given by the superposition of the emission of the different molecular species present in the solution, and no appreciable interaction among the chromophores is observed. On the contrary, in the pre-micellar range of the surfactant, evidence for the existence of an energy transfer mechanism was found. This effect seems to be related to the configurational state of the TLF polymeric chains and depends on the relative TLF/surfactant concentration.     

Fluorescence Lifetime Imaging of Experimental Tumors in Hematoporphyrin Derivative-Sensitized Mice

Tumor detection has been carried out in mice sensitized with hematoporphyrin derivative (HpD) by measuring the spatial distribution of the fluorescence lifetime of the exogenous compound. This result has been achieved using a time-gated video camera and a suitable mathematical processing that led to the so-called “lifetime images.” Extensive experimental tests have been performed on mice bearing the MS-2 fibrosarcoma or the L1210 leukemia. Lifetime images of mice show that the fluorescence decay of HpD is appreciably slower in the tumor than in healthy tissues nearby, allowing a reliable detection of the neoplasia. The lengthening of the lifetime in tumors depends little on the drug dose, which in our experiments could be lowered down to 0.1 mg/kg body weight, still allowing a definite tumor detection. In order to ascertain the results achieved with the imaging apparatus, high-resolution spectroscopy, based on a time-correlated single photon counting system, has also been performed to measure the fluorescence lifetime of the drug inside the tumor and outside. The outcomes obtained with two techniques are in good agreement.