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.     

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.     

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.     

Fluorescence of hematoporphyrin in living cells and in solution

The fluorescence properties of hematoporphyrin (Hp) and its derivative (HpD) were investigated in leukemia cells and in normal lymphocytes under a microscope, and the results were compared with those in solution. The spectra and the time behaviour of Hp (or HpD) fluorescence in living cells were found to be almost the same as those in Hp solution of very high concentration. This implies that Hp is much more concentrated in the cells than in the medium. It was also found that irradiation with intense light easily gives rise to a photoproduct which gives an additional peak in the fluorescence spectrum. Possible methods to increase the sensitivity of cancer detection and localization are discussed.     

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.     

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)     

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.     

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.     

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.     

PHOTODYNAMIC EFFECTS ON CELLS IN VITRO EXPOSED TO HEMATOPORPHYRIN DERIVATIVE and LIGHT

Abstract Several effects of hematoporphyrin derivative (HpD) and light on NHIK 3025 cells in vitro were studied. The treatment resulted in a partly repairable reduction of the rate of thymidine incorporation into DNA, a division delay, a reduced rate of protein synthesis, a reduced rate of active cellular uptake of α-aminoisobutyrate, a reduction in the colony-forming ability and an increased permeability of the cell membrane to chromate. Thymidine incorporation was by far the most sensitive parameter studied. However, comparison of the photodynamic effects after 1 and 18 h incubation with HpD prior to irradiation indicated that neither the reduced rate of DNA synthesis nor any of the other observed effects was the main primary cause of cell inactivation under all conditions. Several of the effects, such as increased permeability of the cell membrane to chromate, reduction in the rate of protein synthesis and reduction in the rate of repair of the damage to the mechanism of DNA synthesis, were clearly of secondary nature. When seen in relation to cellular survival, membrane damage was more important after short incubation times with HpD than after long incubation times.     

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).     

Fluorescence of the DNA double helix (dA)20 x (dT)20 studied by femtosecond spectroscopy--effect of the duplex size on the properties of the excited states

The fluorescence of the DNA double-stranded oligomer (dA)20 x (dT)20 is studied at room temperature by fluorescence up-conversion at times shorter than 10 ps. The profile of the up-conversion spectra is similar to that of the steady-state fluorescence spectrum, showing that the majority of the photons are emitted within the probed time scale. At all the probed wavelengths, the fluorescence decays are slower than those of the monomeric chromophores dAMP and TMP. The fluorescence anisotropy decays show strong wavelength dependence. These data allow us to conclude that energy transfer takes place in this double helix and that this process involves exciton states. The spectral and dynamical properties of the oligomer are compared to those of the polymer poly(dA) x poly(dT), composed of about 2000 base pairs, reported previously. The oligomer absorption spectrum is characterized by a smaller hypsochromic shift and weaker hypochromism compared to the polymer. Moreover, the fluorescence decays of (dA)20 x (dT)20 are twice as fast as those of poly(dA) x poly(dT), and its fluorescence anisotropy decays more slowly. These differences are the fingerprints of a larger delocalization of the excited states induced by an increase in the size of the duplex.     

Selective Examination of Plasma Membrane–Associated Photosensitizers Using Total Internal Reflection Fluorescence Spectroscopy: Correlation between Photobleaching and Photodynamic Efficacy of Protoporphyrin IX

Fluorescence spectra, fluorescence decay kinetics, photobleaching kinetics and photodynamic efficacy of protoporphyrin IX (PP) were investigated in endothelial cells in vitro after different incubation times. Fluorescence spectra and photobleaching kinetics were determined during total internal reflection (TIR) illumination or epiillumination. Because penetration depth of the excitation light during TIR illumination was limited to about 100 nm, plasma membrane-associated PP was almost selectively examined. Spectra obtained by TIR fluorescence spectroscopy (FS) showed a very low background, where-as spectra obtained by epi-illumination exhibited considerable background by autofluorescence and scattered light. For photobleaching kinetics during TIR illumination after 1 h or 24 h incubation, a biexponential fluorescence decrease was observed with a rapidly and a slowly bleaching portion. After 1 h incubation, the rapidly bleaching portion was the predominant fraction, whereas after 24 h incubation comparable relative amounts of the rapidly and slowly bleaching portion were determined. The rapidly and slowly bleaching portion were assigned to PP monomers and aggregated species in close vicinity to the plasma membrane. Fluorescence decay measurements after epi-illumination support the decrease of PP monomers within the whole cell with increasing incubation time. In contrast to TIR illumination, photobleaching of PP during epi-illumination was characterized by slow monoexponential fluorescence decrease after 1 h or 24 h incubation. Photodynamic efficacy of PP using epi-illumination was found to depend strongly on incubation time. Considerable cell inactivation was determined for short incubation times (1 h or 3 h), whereas photodynamic efficacy was diminished for longer incubation times. Reduced photodynamic efficacy after long incubation times was assigned to the lower amount of photodynamically active monomers determined close to the plasma membrane as well as within the whole cell. In conclusion, TIRFS measurements are suggested to be an appropriate tool for the examination of the plasma membrane-associated photosensitizer fraction in living cells.     

Time-gated fluorescence spectroscopy of porphyrin derivatives incorporated into cells

Time-gated fluorescence spectroscopy was performed on the tumour-localizing fraction (TLF) of haematoporphyrin derivative (HPD) incorporated into cells. Three different cell lines were incubated with 20 and 5 micrograms ml-1 of TLF for various time periods; they were then washed and resuspended in buffer. Fluorescence decay measurements and time-integrated and time-gated spectra were then obtained from the cell suspensions. Similar experiments were repeated using HPD containing 60% of the active material. The experimental results show a modification of the emission spectra for both drugs depending on the incubation time; this modification is more significant for the TLF. In particular, the emission peak observed in aqueous solution at 615 nm is shifted to 630 nm as a consequence of incorporation into cells, and the gated spectra indicate that the fluorescence emission is mainly related to monomers and unfolded polymeric chains. The ratio between the intensities of the two peaks depends on the relative amount of the TLF; the peak at 615 nm is more pronounced for HPD. The results obtained seem to indicate that both the composition of the drug and the metabolic properties of the biological environment strongly influence the uptake process and the fluorescence behaviour of the incorporated sensitizer.     

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.     

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.     

Collective behavior of Franck-Condon excited states and energy transfer in DNA double helices

Absorption of UV radiation by DNA bases is known to induce carcinogenic mutations. The lesion distribution depends on the sequence around the hotspots, suggesting cooperativity between bases. Here we show that such cooperativity may intervene at the very first step of a cascade of events by formation of Franck-Condon states delocalized over several bases and subsequent energy transfer faster than 100 fs. Our study focuses on the double helix poly(dA).poly(dT), whose fluorescence, induced by femtosecond pulses at 267 nm, is probed by the upconversion technique and time-correlated single photon counting, over a large time domain (100 fs to 100 ns). The time-resolved fluorescence decays and fluorescence anisotropy decays are discussed in relation with the steady-state absorption and fluorescence spectra in the frame of exciton theory.     

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.     

Global and target analysis of time-resolved fluorescence spectra of Di-9H-fluoren-9-yldimethylsilane: dynamics and energetics for intramolecular excimer formation

Dynamics and energetics for intramolecular excimer formation of a diarylsilane, di-9H-fluoren-9-yldimethylsilane (DFYDMS) have been investigated by means of ps time-resolved fluorescence spectroscopy and ab initio calculation. Multiple fluorescence decay curves were globally deconvolved to generate time-resolved fluorescence spectra and decay-associated spectra (DAS), from which species-associated spectra (SAS) were obtained. It is shown in the global analysis that there are at least three excited states: Two states are the locally excited (LE) states (lambda(max) approximately 320 nm) having lifetimes of 0.70 +/- 0.04 and 1.75 +/- 0.02 ns, and another is the excimer state (lambda(max) approximately 400 nm) having a lifetime of 7.34 +/- 0.02 ns. The species which decays with 0.70 ns evolves into a species with a red-shifted spectrum, which in turn decays in 7.34 ns. The experimental and ab initio results indicate that the rise time of 0.70 ns corresponds to the conversion of the initial S(1) LE state having a near sandwich geometry to the S(1) excimer state adopting a true sandwich geometry.     

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。