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.     

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.     

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).     

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.     

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.     

Comparison of phototoxicity mechanism between pulsed and continuous wave irradiation in photodynamic therapy

A study has been conducted in which HeLa cells are incubated with hematoporphyrin derivative (HpD) for 1 h (1 microgram/ml of HpD in PBS) to compare the use of continuous wave (CW) and pulsed laser (10 Hz repetition rate and 7-9 ns pulse width) light for photodynamic therapy. Cytotoxic effects on the cells are evaluated by the 3-(4,5-dimethyl-2-thiazolyl)-2-5-diphenyl-2H-tetrazolium bromide (MTT) assay and the fluorescein diacetate (FDA)/propidium iodide (PI) stain method using a flow cytometer. The type of cell death is estimated by analysis of the DNA content and observation of the nuclear morphology. The cytotoxicity ratio of cells irradiated by pulsed laser light is estimated to be lower than that for CW laser light. The viability of cells that received pulsed laser light gradually decreases, whereas no significant changes are found in the cells irradiated with CW laser light with the elapse of post-irradiation time. The type of cell death differs between the pulsed and CW laser light irradiations. These findings suggest that the cytotoxic efficacy of the excitation light source is displayed by the difference in the type of cell death, namely apoptosis or necrosis.     

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.     

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.     

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.     

LOCALIZATION OF MONO-L-ASPARTYL CHLORIN e6 (NPe6) IN MOUSE TISSUES

Abstract It is known that HpD is retained longer by malignant tissue than normal tissue and is therefore a useful material for photodynamic therapy (PDT). Currently, vigorous research is being conducted throughout the world to discover a new material which can have greater cancer cell affinity than hematoporphyrin derivative (HpD) and will be used effectively for PDT. Investigation has been conducted to determine the spectral characteristics and cancer cell affinity of NPe6, a recently developed material.
Structurally, a double bond on the D-ring of the porphyrin ring of mono-L-aspartyl chlorin e6 (NPe6) has been reduced, thereby changing its spectral properties from that of HpD. This difference accounts for the stronger absorption bands in wavelengths longer than those of HpD. Furthermore, NPe6 in tumor showed stronger absorption at 660 nm than HpD. Absorption by hemoglobin (Hb) in the blood occurs at wavelengths in the range 500-600 nm, thereby lowering light transmittance. A compound which has a strong absorption band at wavelengths longer than 600 nm and consequently is not affected by Hb will naturally be activated by light at a greater depth in tissue than compounds which do not share this characteristic. The localization of NPe6 in sarcoma and various internal organs was examined with an endoscopic spectrophotometer using an excimer dye laser. After 72 h i.v. NPe6 injection, the results indicate that NPe6 has 10 times greater uptake in malignant tissue cells than in normal organs. Based on the above observations, it was concluded that NPe6 could be effective for PDT if toxicity is low and that this compound has a high malignant tissue affinity.     

THE EFFECT OF PORPHYRIN PHOTOSENSITIZERS ON THE TOXICITY OF l,3-BIS(2-CHLOROETHYL)-1- NITROSOUREA IN C57BL/6J MICE

Abstract The most widely used agents for photodynamic therapy are the porphyrin photosensitizers. It has been shown that hematoporphyrin derivative (HpD) can cause murine marrow hypercellularity and splenic hypertrophy. We have examined the effect on survival and marrow cellularity of high dose l,3-bis(2-chloroethyl)-l-nitrosourea (BCNU) after HpD or dihematoporphyrin ether (DHE) pretreatment in C57BL/6J mice.
The lethal toxicity of the LD_S0+ 10% dose of BCNU (60 mg kg⁻¹) was significantly reduced by pretreatment with HpD when the HpD was administered at least 3 days prior to the BCNU. HpD administered 1 or 2 days prior to BCNU or after BCNU had no effect. The percent death rate was reduced from 80 to 0% when HpD was administered 7 and 5 days prior to BCNU.
No alteration of the lethal toxicity rate of BCNU at doses of 80 mg kg⁻¹ were identified with DHE pretreatment although some increase in median survival was noted in two groups. Some reduction in lethal toxicity was noted when 60 mg kg⁻¹ BCNU was used and the pretreatment dose of DHE was 10 or 25 mg kg⁻¹ given twice 3 days apart. Furthermore, a significant reduction of BCNU induced marrow cell depletion was found when low doses of DHE were used as pretreatment. High doses of DHE resulted in marrow depletion. Both HpD and DHE altered the toxicity of BCNU.
Should porphyrin photosensitizers, which alone have little toxicity, prove to protect against nitrosourea toxicity then an important dose limiting factor (myelotoxicity) could be altered if not reduction in the tumouricidal activity occurs.     

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.     

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)     

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.     

COMPARATIVE KINETICS OF HEMATOPORPHYRIN DERIVATIVE UPTAKE AND SUSCEPTIBILITY OF Bacillus subtilis AND Streptococcus faecalis TO PHOTODYNAMIC ACTION

Hematoporphyrin derivative (HpD) is widely used in photoradiation therapy of tumors and other diseases, and has been shown to affect the viability of gram positive bacteria. This investigation assessed the efficiency of binding of HpD to Bacillus subtilis and Streptococcus faecalis when HpD-treated organisms were exposed to red light. Kinetic studies indicated that the amount of HpD bound increased with increasing external concentration of HpD until saturation of binding sites was reached. S. faecalis had a higher affinity for HpD and was more susceptible to photoinactivation than B. subtilis. The data from this study suggest that differences in susceptibility of microorganisms to photoinactivation are directly related to the affinity of each strain for HpD.     

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.     

ACUTE SKIN RESPONSE IN ALBINO MICE FOLLOWING PORPHYRIN PHOTOSENSITIZATION UNDER OXIC AND ANOXIC CONDITIONS

Abstract Acute normal skin toxicity induced by porphyrin photosensitization has been examined using albino mice. Oxic and anoxic (clamped) skin was exposed to red light (630 nm) 24 h following administration of hematoporphyrin derivative (HpD) or Photofrin II (the active component of HpD). Experiments were also performed to determine the effect of sodium pentobarbital anesthesia on HpD and Photofrin II photosensitization of normal skin. Results from this study demonstrated that comparable levels of acute skin damage were induced by HpD and Photofrin II under oxic conditions but neither porphyrin produced any apparent phototoxicity under anoxic conditions. In addition, the level of skin damage induced by porphyrin photosensitization was not affected by sodium pentobarbital anesthesia.     

Ca2+ INFLUX INDUCED BY PHOTODYNAMIC ACTION IN HUMAN CEREBRAL GLIOMA (U-87 MG) CELLS: POSSIBLE INVOLVEMENT OF A CALCIUM CHANNEL

Abstract The plasma membrane has been implicated as a critical target of photodynamic action on cells. We have observed that the photosensitization of human cerebral glioma (U-87 MG) cells by hematoporphyrin derivative (HpD) causes a large increase in intracellular calcium [Ca²⁺]. This increase in [Ca²⁺]_i was solely due to the influx of extracellular Ca²⁺ through the plasma membrane and showed a dependence on HpD concentration, light dose and concentration of calcium in the extracellular medium. The magnitude of the Ca²⁺ influx decreased with increasing postirradiation time, which suggests that the cell membrane partially recovers from the photodynamic injury. The photoinduced Ca²⁺ influx was inhibited by the Ca²⁺ channel blocker diltiazem and the reducing agent dithioerythritol. These findings are discussed in terms of possible activation of a Ca²⁺ channel as a result of photosensitization.     

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.     

EFFECT OF PHOTODYNAMIC THERAPY ON ANTITUMOR IMMUNE DEFENSES: COMPARISON OF THE PHOTOSENSITIZERS HEMATOPORPHYRIN DERIVATIVE AND CHLORO-ALUMINUM SULFONATED PHTHALOCYANINE

The effects of the two photosensitizers chloroaluminum sulfonated phthalocyanine (ClAlSPc) and hematoporphyrin derivative (HpD) on the functional activities of macrophages and natural killer (NK) cells, two immunocyte populations implicated in the control of tumor development and spread, have been investigated. Murine peritoneal macrophages treated in vivo with ClAlSPc or HpD at 10 mg/kg body weight showed no impairment of Fc-mediated phagocytic capacity and only minor disturbances of in vitro tumoricidal/tumoristatic function. The NK cell activity of splenocytes obtained from photosensitizer-treated mice, assayed 24 or 48 h after i.v. injection of ClAlSPc or HpD at 10 mg/kg was unaffected compared to controls. However significant inhibition of NK activity was observed when splenocytes obtained from mice with or without subcutaneous Colo 26 tumors, treated with ClAlSPc plus laser therapy (675 nm) were used as effector cells. The results show that impairment of some anti-tumor activity can be observed in phthalocyanine treated or phthalocyanine + laser-treated animals but this relatively minor impairment may augur well for the use of systemic phthalocyanine administration in photodynamic therapy.