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.     

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.     

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)     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Studies on photophysics and photochemistry of N-salicylidene-p-(N,N-dimethylamino)aniline

The fluorescence spectra of N-salicylidene-p-(N,N-dimethylamino)aniline have been investigated in various solvents and three kinds of fluorescence were found; they were that of excited intermediate, exciplex and excited dimer. According to the transient absorption spectra and decay kinetic data of photoproducts of the title compound, it has been found that the photoproducts in cyclohexane are a zwitterion and a mixed dimer formed by a zwitterion and an enol; in acetonitrile the photoproducts are a zwitterion, a mixed dimer formed by a zwitterion and an enol and a dimer formed of zwitterion. Photochromic and luminescence mechanisms of the title compound are discussed as well.     

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.     

INVESTIGATION OF PROTRIPTYLINE PHOTOPRODUCTS WHICH CAUSE CELL MEMBRANE DISRUPTION

Abstract— Protriptyline (PTL; N-methyl-5H-dibenzo[a,d]cycloheptene-5-propylamine) hydrochloride is a skin photosensitizing agent in humans. The fluorescence and photochemical behavior of PTL varies with the solvent. In water, 40% ethanol and ethanol in the hydrochloride salt of PTL has a fluorescence quantum yield of 0.81. The fluorescence quantum yield of PTL free base in selected organic solvents is between 0.41 and 0.17; in ethanol it remains at 0.81. Photolysis of PTL in acidic aqueous solution yields at least five photoproducts which were separated by high-performance liquid chromatography. Three of these photoproducts lysed red blood cells. One of the photoproducts has been identified as a cyclobutyl photodimer of PTL based on its mass spectrum and UV absorption and its ability to undergo photore-versal with 254 nm irradiation. The others were not cyclobutyl dimers. The yield of lytic products decreased as the ethanol content was increased and were not formed from PTL free base in any solvent.     

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.     

PHOTOCHEMICAL PROPERTIES OF PORPHYRIN c: AN AGENT FOR USE IN TUMOR PHOTOTHERAPY

Abstract— The absorption and fluorescence properties of porphyrin c (P c ), the porphyrin chromophore present in cytochrome c , have been determined in several solvents and micellar environments. In aqueous buffer solutions at pH 7.5 Pc may exist in both a fluorescent monomeric form with quantum yield of fluorescence, (Φ_f,) ∼ 0.03, and fluorescence lifetime, (τ_f) ∼ 8 ns, and as a non-fluorescent aggregate. The proportion of monomeric form is higher in organic solvents and micelles but is reduced with increasing porphyrin concentrations in aqueous solutions. Porphyrin c readily complexes with Zn²⁺ to produce a fluorescent chelate (Zn-P c ) with Φ_f, ∼ 0.02 and τ_f, ∼ 2 ns at pH 7.5. The yields of singlet excited oxygen formation from Pc and the Zn-P c complex are higher than observed for hematoporphyrin derivative (HpD). Both P c and Zn-P c are effective agents in tumor phototherapy and do not induce the prolonged cutaneous photosensitivity observed with the use of HpD.     

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).     

Enhancement of the non-tryptophan fluorescence of human lens proteins after near-UV light exposure

Abstract. In this work, the non-tryptophan fluorescence (360 nm excited; 440 nm emitted) of human lens proteins was found to be intensified by exposing whole lens homogenates to near-UV light in the presence of tryptophan photoproducts. The induced fluorescence accumulates mainly in the soluble phase proteins, whereas in aging and brown cataractous lenses, the major fluorescence is found in the insoluble proteins. Using SDS-polyacrylamide gel electrophoresis with densitometric and fluorescence scanning techniques, the polypeptide chains of the three major protein fractions were analyzed for their specific non-tryptophan fluorescences. The same chains were found in all fractions. Two chains (11,000 and 45,000 daltons) were found to accumulate most of the induced fluorescence. These also contained the greatest intrinsic fluorescence initially. The data indicates that specific polypeptide chains in the lens proteins are most sensitive to modifications due to their exposure to near-UV light in the presence of tryptophan photoproducts.     

LOCAL NEUROTOXICITY OF HEMATOPORPHYRIN DERIVATIVE FOLLOWING INTRACEREBRAL INJECTION

The present study reports on toxicity of hematoporphyrin derivative (HpD) for normal brain tissue in vivo without the addition of light. Hematoporphyrin derivative was injected by slow infusion in rat brains. Histological examination was carried out for intervals after HpD administration, ranging from 0 h to 15 days. Ultrastructural changes were examinated with transmission electron microscopy. The extent of the necrosis was determined for different HpD concentrations and compared with control animals infused with 0.9% saline. Leukocytic infiltration was observed at day 5. Transmission electron microscopy showed that nuclei of neurons were completely disintegrated 4 h after HpD administration. Furthermore disruption of myelin sheaths was observed. The extent of the necrosis decreased with lower HpD doses. Injection of 2 μg HpD in a volume of 4 μL (0.5 mg/mL) resulted in a virtually equal extension of the tissue damage, as compared to the mechanical damage in the control animals caused by the infusion procedure.     

Photosensitizers in dermatology

Systemic injection of hematoporphyrin derivative (HpD) in contribution with visible light (red or blue-green) delivered by laser was used to treat a patient with psoriasis. The psoriatic lesions responded vigorously to laser treatments, forming eschars by 1 week post irradiation. In contrast, only minimal erythema was observed in the noninvolved, clinically normal appearing skin. Two approaches for localized HpD administration were investigated in the guinea-pig and minipig models as a means of achieving local photodynamic effects. Intracutaneous injection of HpD produced localized cutaneous photosensitization with either UVA or red light. Azone increased percutaneous penetration of HpD in human skin in vitro. Topical application of HpD and irradiation with UVA produced localized cutaneous photosensitivity and inhibition of epidermal DNA synthesis.     

Time-resolved fluorescence spectroscopy of hematoporphyrin, mesoporphyrin, pheophorbide a and chlorin e6 in ethanol and aqueous solution

The fluorescence decay I(t) and time-resolved spectra I(lambda, t) of some porphyrins and chlorins in ethanol and phosphate-buffered aqueous solution were investigated with a time-correlated single-photon-counting apparatus with a mode-locked Ar+ laser (514.5 nm) as the excitation source. The fluorescence of hematoporphyrin, mesoporphyrin and pheophorbide aa is considerably influenced by the conditions of aggregation (these compounds undergo aggregation in phosphate-buffered solution but not in ethanolic solution). The fluorescence decay of chlorin e6 which remains monomeric in both solvents is single exponential in all cases. The fluorescence spectra of hematoporphyrin, mesoporphyrin and pheophorbide a in phosphate-buffered solution are shifted with respect to the spectra obtained in ethanol; moreover, a new emission band (X band) appears, whose intensity increases on increasing the amount of equilibrium aggregates and shows a fast fluorescence decay. For hematoporphyrin and mesoporphyrin the appearance of the X band emission appears to be correlated with irreversible photoprocesses leading to fluorescent photoproducts. Analysis of the reported fluorescence spectra of cancer cells after incubation with hematoporphyrin derivative suggests that the fluorescent photoproducts might be formed also in vivo.