In vivo and in vitro characterisation of a protoporphyrin IX-cyclic RGD peptide conjugate for use in photodynamic therapy.

Increasing treatment specificity is one of the major aims of cancer research. Photodynamic therapy is a clinically proven treatment for some cancers and certain other diseases. Photosensitisers generally have little intrinsic selectivity for tumours and any accumulation is dependent upon the type of tumour involved. Increasing tumour selective accumulation could improve the efficacy of PDT and reduce any risk of side effects caused by photosensitiser accumulation in non-target tissue. In order to target photosensitisers to tumours, a cyclic peptide, cRGDfK (arginine-glycine-aspartic acid-phenylalanine-lysine) has been synthesised using solid phase peptide chemistry and conjugated to the porphyrin photosensitiser, protoporphyrin IX. The arginine-glycine-aspartic acid (RGD) motif has been shown to specifically bind alphavbeta3 integrins, heterodimeric glycoproteins upregulated on the surface of proliferating endothelial cells such as those in tumour neovasculature. This study reports the synthesis, in vitro and in vivo characterisation of this novel compound and compares its properties to the free photosensitiser. The individual components in our system, protoporphyrin IX and cRGDfK retain their respective photodynamic and integrin binding activity following the coupling step and produce a conjugate of high purity. The PpIX:cRGDfK conjugate is shown to be a good photosensitiser in vitro in the integrin positive human SiHa cell line and in vivo in a mouse CaNT tumour model. Moreover, pharmacokinetic analysis of PpIX:cRGDfK treated mice shows significant retention and accumulation of photosensitiser in tumour tissue with higher tumour : normal tissue ratios than the free photosensitiser. However, although the conjugate shows this higher accumulation and improved tumour : non-target tissue ratios, the overall in vivo PDT effect, between dose-light intervals of 0 and 6 h, is not significantly better than for free protoporphyrin IX This is possibly due to differences in the target environment or in the subcellular localisation of the compounds.     

Photofrin--factor of photodynamic therapy induces apoptosis and necrosis

Photodynamic therapy (PDT) causes irreversible photodamage of tumor and other malignant tissues. The effect of reactive oxygen species generation in the presence of photofrin (HpD) was studied. The studies were performed on endothelial cell line from foetal aorta of calves and on normal fibroblasts cell line (3T3 -Balb) and also on malignant line (A431). The cells were grown in presence of photofrin at different time intervals. Time of interaction of photosensitiser with cells was very important. Short time of exposure of the cells to photofrin induced mostly apoptosis in normal cells and apoptotic or necrotic changes in malignant cells. Longer effect of these factors on cells provoked necrosis. The factors of PDT influence dynamic changes of SOD and CT activity. It was dependent on the intensity of factors. These results strongly suggest that HpD has an effect on generation of ROS, which are a signal for development of morphological changes (apoptosis or necrosis) in normal and malignant cells.     

The influence of photodynamic reaction on R2C cells

Photodynamic therapy (PDT) represents a therapeutic approach in which photosensitised neoplastic cells undergo destruction under effect of light. In this study we have attempted to define effects of photochemotherapy on R2C cells, sensitised with protoporphyrin IX (PpIX) and to find out whether inhibition of gene expression by cycloheximide affects development of lesions in the cells. The photosensitised cells were exposed to visible light and development of apoptotic and necrotic lesions was followed in the cells, using the fluorescent staining with propidium iodide and Hoechst 33342. The experiments demonstrated that PpIX and light, acting in parallel, induce development of apoptotic and necrotic lesions in R2C cells. Intensity of the lesions correlated with concentration of the applied photosensitiser and with duration of light exposure. Using cycloheximide, we also inhibited protein expression in cells photosensitised with protoporphyrin before they were exposed to light. In the latter case, development of apoptosis was clearly intensified which might be explained by inhibition of anti-apoptotic protein synthesis in the cells.     

Generation of singlet oxygen induces phospholipid scrambling in human erythrocytes

Maintenance of phospholipid asymmetry of the plasma membrane is essential for cells to prevent phagocytic removal or acceleration of coagulation. Photodynamic treatment (PDT), which relies on the generation of reactive oxygen species to achieve inactivation of pathogens, might be a promising approach in the future for decontamination of red blood cell concentrates. To investigate whether PDT affects phospholipid asymmetry, erythrocytes were illuminated in the presence of 1,9-dimethyl-methylene blue (DMMB) as photosensitizer and subsequently labeled with FITC-labeled annexin V. This treatment resulted in about 10% annexin V positive cells, indicating exposure of phosphatidylserine (PS). Treatment of erythrocytes with N-ethylmaleimide (NEM) prior to illumination, to inhibit inward translocation of PS via the aminophospholipid translocase, resulted in enhanced PS exposure, while treatment with H(2)O(2) (previously shown to inhibit phospholipid scrambling) greatly diminished PS exposure, indicating the induction of phospholipid scrambling by PDT. Only erythrocytes illuminated in the presence of DMMB showed translocation of NBD-phosphatidylcholine (NBD-PC), confirming scrambling induction. Double label experiments indicated that PS exposure does not occur without concurrent scrambling activity. Induction of scrambling was only moderately affected by Ca(2+) depletion of the cells. In contrast, scavengers of singlet oxygen were found to prevent phospholipid scrambling induced by PDT. The results of this study show that phospholipid scrambling is induced in human erythrocytes by exposure to singlet oxygen.     

Differential effects of glucose deprivation on the cellular sensitivity towards photodynamic treatment-based production of reactive oxygen species and apoptosis-induction

Photodynamic treatment (PDT) employs a photosensitizer and the light-induced formation of reactive oxygen species--antagonized by cellular antioxidant systems--for the removal of harmful cells. This study addresses the effect of altered carbohydrate metabolism on the cellular antioxidant glutathione system, and the subsequent responses to PDT. It is shown that glucose-deprivation of 18 h prior to PDT causes a reduced level of intracellular glutathione and an increased cytotoxicity of PDT. These effects can be mimicked by inhibitors of glutathione synthesis (buthionine-sulfoximine) or its regeneration (1,3-bis-(2-chlorethyl)-1-nitrosourea). Inhibited glutathione metabolism shifts the apoptotic window to lower fluences, while glucose deprivation abolishes apoptosis as a result of ATP deficiency. Our results prove evidence for manipulation of the outcome of PDT through internal metabolic pathways.     

Studies on the effect of photodynamic therapy on in vitro cultured neoplastic cells

Photodynamic therapy (PDT) represents a therapeutic approach in which photosensitised neoplastic cells undergo destruction under the effect of light. In this study we have attempted to define effects of PDT on CHO cells, sensitised with protoporphyrin IX (PpIX). The photosensitised CHO cells were exposed to a visible light and development of apoptotic and necrotic lesions was followed in the cells, using the fluorescent staining with propidium iodide and Hoechst 33342. The experiments demonstrated that PpIX and light, acting in parallel, induce development of apoptotic and necrotic lesions in the cells. Intensity of the lesions was correlated with the concentration of the applied photosensibiliser and with the duration of exposure to light. The control experiments suggest that development of apoptosis in the applied model probably reflect mitochondrial damage, while processes developing close to the cell membrane are responsible for necrosis. In order to corroborate the obtained results, ultrastructural studies were performed on experimental groups in which evident apoptotic lesions were observed in the cells.     

Bactericidal effect of photodynamic therapy against methicillin-resistant Staphylococcus aureus strain with the use of various porphyrin photosensitizers

Photodynamic therapy (PDT) is based on photosensitizers activated by light of appropriate wavelength. Their activation leads to generation of singlet oxygen and free radicals responsible for the cytotoxic effect. The aim of this project was to compare the bactericidal effect of PDT using different porphyrin photosensitizers against a methicillin-resistant Staphylococcus aureus strain. Exogenous sensitizers (protoporphyrin IX and newly synthesized derivative, protoporphyrin diarginate) induced a 3 log10-unit reduction in bacterial viable counts. With the use of endogenous, ALA-induced porphyrins, a 1.6 log10-unit reduction was obtained. The sensitizers tested executed their antibacterial activity with no essential change in the antibiotic resistance pattern of the studied strain.     

A comparative analysis of phenothiazinium salts for the photosensitisation of murine fibrosarcoma (RIF-1) cells in vitro.

Photodynamic therapy (PDT) is a treatment combining a photosensitiser, molecular oxygen and visible light of characteristic wavelength to produce cytotoxic reactive oxygen species (ROS). Within our centre, a series of phenothiazinium salts were synthesised and initial characterisation studies performed to determine any potential use for PDT. All photosensitisers within the series were shown to have useful spectral properties for PDT, with absorbance lambdamax above 667 nm. The Log P values of the compounds were shown to range from -0.9 to > +2.0. Furthermore, Log P values were shown to be important in determining the site of subcellular localisation and as such the site of photooxidative damage. Derivatives with a Log P value of greater than +1.0 were shown to initially localise to the lysosomes then relocalise throughout the cytoplasm following illumination, whereas compounds with intermediate Log P values (-0.7 to +1.0) all remained lysosomal. Only methylene blue (Log P-0.9) was shown to redistribute to the nucleus upon illumination. Following treatment of RIF-1 cells with each phenothiazinium salt for 1 h and subsequent exposure to 665 nm laser light at a fluence rate of 10 mW cm(-2)(18 J cm(-2)), it was determined that the most potent photosensitiser was 260-fold more potent than methylene blue. Furthermore, the PDT efficacy of the photosensitisers was shown to be related to the level of mitochondrial damage induced directly following illumination.     

Release of regulators of angiogenesis following Hypocrellin-A and -B photodynamic therapy of human brain tumor cells

Photodynamic therapy (PDT) is an innovative strategy for the treatment of solid neoplasms of the brain. Aside from inducing cell death in tumor cells, PDT induces endothelial cell death and promotes formation of blood clots; however, exact mechanisms that trigger these phenomena remain largely unknown. We now used Western blotting to analyze secretion of regulators of angiogenesis to the supernatants of one glioma, one macrophage, and one endothelial cell line following Hypocrellin-A and -B photodynamic therapy. We observed induction of proangiogenic VEGF (vascular endothelial growth factor) and of antiangiogenic sFlt-1, angiostatin, p43, allograft inflammatory factor-1, and connective tissue growth factor. Release of thrombospondin-1 was diminished in a glioma cell line supernatant. Endostatin release was induced in glioma cells and reduced in macrophages and endothelial cells. These data show that a wide range of antiangiogenic factors are secreted by brain tumor cells following Hypocrellin photochemotherapy. However, VEGF release is also induced thus suggesting both favorable and deleterious effects on tumor outgrowth.     

Mechanism of cell death by 5‐aminolevulinic acid‐based photodynamic action and its enhancement by ferrochelatase inhibitors in human histiocytic lymphoma cell line U937

Photodynamic therapy (PDT) for tumors is based on the tumor‐selective accumulation of a photosensitizer, protoporphyrin IX (PpIX), followed by irradiation with visible light. However, the molecular mechanism of cell death caused by PDT has not been fully elucidated. The 5‐aminolevulinic acid (ALA)‐based photodynamic action (PDA) was dependent on the accumulation of PpIX, the level of which decreased rapidly by eliminating ALA from the incubation medium in human histiocytic lymphoma U937 cells. PDA induced apoptosis characterized by lipid peroxidation, increase in Bak and Bax/Bcl‐xL, decrease in Bid, membrane depolarization, cytochrome c release, caspase‐3 activation, phosphatidylserine (PS) externalization. PDT‐induced cell death seemed to occur predominantly via apoptosis through distribution of PpIX in mitochondria. These cell death events were enhanced by ferrochelatase inhibitors. These results indicated that ALA‐based‐PDA induced apoptotic cell death through a mitochondrial pathway and that ferrochelatase inhibitors might enhanced the effect of PDT for tumors even at low concentrations of ALA. Copyright © 2009 John Wiley & Sons, Ltd.     

Photodynamic therapy: an update

Photodynamic therapy (PDT) is a promising local treatment modality based on the selective accumulation of a photosensitizer in malignant tissues and the subsequent irradiation with laser light. Photodynamic therapy of malignant tumors includes biological, photochemical and photophysical processes. These processes involve: (a) absorption of photosensitizing agent; (b) selective retention of the photosensitizer in tumors and (c) irradiation of sensitized tumor by laser radiation. This report provides a review of photosensitizers, photochemistry, subcellular targets, side effects and laser involved in photodynamic therapy. In addition, gradual increase in knowledge related to in vitro and in vivo mechanisms of action of PDT, as well as some clinical applications of photodynamic therapy are presented.     

Isolation and characterization of squamous carcinoma cells resistant to photodynamic therapy

Photodynamic therapy (PDT) employing methyl δ-aminolevulinic acid (Me-ALA), as a precursor of the photosensitizer protoporphyrin IX (PpIX), is used for the treatment of non melanoma cutaneous cancer (NMCC). However, one of the problems of PDT is the apparition of resistant cell populations. The aim of this study was to isolate and characterize squamous carcinoma cells SCC-13 resistant to PDT with Me-ALA. The SCC-13 parental population was submitted to successive cycles of Me-ALA-PDT and 10 resistant populations were finally obtained. In parental and resistant cells there were analyzed the cell morphology (toluidine blue), the intracellular PpIX content (flow cytometry) and its localization (fluorescence microscopy), the capacity of closing wounds (scratch wound assay), the expression of cell-cell adhesion proteins (E-cadherin and β-catenin), cell-substrate adhesion proteins (β1-integrin, vinculin and phospho-FAK), cytoskeleton proteins (α-tubulin and F-actin) and the inhibitor of apoptosis protein survivin, in the activated form as phospho-survivin (indirect immunofluorescence and Western blot). The results obtained indicate that resistant cells showed a more fibroblastic morphology, few differences in intracellular content of the photosensitizer, higher capacity of closing wounds, higher number of stress fibers, more expression of cell-substrate adhesion proteins and higher expression of phospho-survivin than parental cells. These distinctive features of the resistant cells can provide decisive information to enhance the efficacy of Me-ALA applications in clinic dermatology.     

Targeted inhibition of p38alpha MAPK suppresses tumor-associated endothelial cell migration in response to hypericin-based photodynamic therapy

Photodynamic therapy (PDT) is an established anticancer modality and hypericin is a promising photosensitizer for the treatment of bladder tumors. We show that exposure of bladder cancer cells to hypericin PDT leads to a rapid rise in the cytosolic calcium concentration which is followed by the generation of arachidonic acid by phospholipase A2 (PLA2). PLA2 inhibition significantly protects cells from the PDT-induced intrinsic apoptosis and attenuates the activation of p38 MAPK, a survival signal mediating the up-regulation of cyclooxygenase-2 that converts arachidonic acid into prostanoids. Importantly, inhibition of p38alpha MAPK blocks the release of vascular endothelial growth factor and suppresses tumor-promoted endothelial cell migration, a key step in angiogenesis. Hence, targeted inhibition of p38alpha MAPK could be therapeutically beneficial to PDT, since it would prevent COX-2 expression, the inducible release of growth and angiogenic factors by the cancer cells, and cause an increase in the levels of free arachidonic acid, which promotes apoptosis.     

Specific fluorescent tracers. Imaging and applications for photodynamic therapy

Our main objective is to enlarge the fluorescence use in biosciences, with especially the photodynamic therapy (PDT) used for cancer treatment as one of the target applications. Meta-tetra(hydroxyphenyl)chlorin (m-THPC) is a second-generation photosensitiser, applied in photodynamic therapy. The localisation of this sensitiser as well as its induced cell death mechanisms in human breast cancer cells (MCF-7 and its resistant subline MCF-7DXR, DXR: doxorubicin) were evaluated using fluorescence microscopy. In addition, we will present two additional routes, whose aims are to create new features to respond to the PDT questioning: firstly, the synthesis of fluorescent tracers, with a particular attention to the presence of hydrophilic groups (glucosamine ring) on the basic fluorophore structure to orientate the localisation of the probe and, secondly, the use of scanning near-field optical microscopy to reach a better resolution for the fluorescence microscopy analysis.     

The outcome of 5-ALA-mediated photodynamic treatment in melanoma cells is influenced by vitamin C and heme oxygenase-1

Photodynamic therapy (PDT) is an important clinical approach for cancer treatment. It involves the administration of a photosensitizer, followed by its activation with light and induction of cell death. The underlying mechanism is an increased production of reactive oxygen species (ROS) leading to oxidative stress, which is followed by cell death. However, effectiveness of PDT is limited due to an initiation of endogenous rescue response systems like heme oxygenase-1 (HO-1) in tumor cells. In recent years, consuming of antioxidant supplements has become widespread, but the effect of exogenously applied antioxidants on cancer therapy outcome remains unclear. Thus, this study was aimed to investigate if exogenous antioxidants might decrease ROS-induced cytotoxicity in photodynamic treatment. Lycopene, β-carotene, vitamin C, N-acetylcysteine, trolox, and N-tert-butyl-α-phenylnitrone in different doses were administered to human melanoma cells prior exposure to photodynamic treatment. Supplementation with vitamin C resulted in a significant decrease of the cell death rate, whereas the other tested antioxidants had no effect on cell viability and oxidative stress markers. The simultaneous application of vitamin C with the HO-1 activity inhibitor zinc protoporphyrine IX (ZnPPIX) caused a considerable decrease of photodynamic treatment-induced cytotoxicity compared to ZnPPIX alone. It can be summarized that exogenously applied antioxidants do not have a leading role in the protective response against photodynamic treatment. However, further studies are necessary to investigate more antioxidants and other substances, which might affect the outcome of photodynamic treatment in cancer therapy.     

Photodynamic and non-photodynamic action of several porphyrins on the activity of some heme-enzymes

The action of porphyrins, uroporphyrin I and III (URO I and URO III), pentacarboxylic porphyrin I (PENTA I), coproporphyrin I and III (COPRO I and COPRO III), protoporphyrin IX (PROTO IX) and mesoporphyrin (MESO), on the activity of human erythrocytes delta-aminolevulinic acid dehydratase, porphobilinogenase, deaminase and uroporphyrinogen decarboxylase in the dark and under UV light was investigated. Both photoinactivation and light-independent inactivation was found in all four enzymes using URO I as sensitizer. URO III had a similar action as URO I on porphobilinogenase and deaminase and PROTO IX exerted equal effect as URO I on delta-aminolevulinic acid dehydratase and uroporphyrinogen decarboxylase. Photodynamic efficiency of the porphyrins was dependent on their molecular structure. Selective photodecomposition of enzymes by URO I, greater specificity of tumor uptake by URO I and enhanced porphyrin synthesis by tumors from delta-aminolevulic acid, with predominant formation of URO I, underline the possibility of using URO I in detection of malignant cells and photodynamic therapy.     

Photodynamic therapy and some clinical applications in oncology

Photodynamic therapy (PDT), a new treatment modality for localized cancers involving the selective interaction of visible light with photosensitizers, such as hematoporphyrin derivatives (HpD) or dihematoporphyrin ether/ester (DHE) (Photofrin II). Photodynamic therapy of malignant tumors includes biological, photochemical and photophysical processes. These processes involve: (i) absorption of photosensitizing agent; (ii) selective retention of photosensitizer in tumors and (iii) irradiation of sensitized tumor by laser irradiation. This paper provides a review of photosensitizers, photochemistry, subcellular targets, side effects and lasers involved in photodynamic therapy. In addition, gradual increase in knowledge related to in vivo and in vitro mechanisms of action of PDT, as well as some clinical applications of photodynamic therapy are presented.     

Light-harvesting ionic dendrimer porphyrins as new photosensitizers for photodynamic therapy

Photodynamic therapy (PDT) is a promising therapeutic modality for treatment of solid tumors. In this study, third-generation aryl ether dendrimer porphyrins (DPs) with either 32 quaternary ammonium groups (32(+)DPZn) or 32 carboxylic groups (32(-)DPZn) were evaluated as a novel, supramolecular class of photosensitizers for PDT. DPs showed a different cell-association profile depending on the positive or negative charge on the periphery, and both DPs eventually localized in membrane-limited organelles. In contrast, protoporphyrin IX (PIX), which is a hydrophobic and relatively low molecular weight photosensitizer used as a control in this study, diffused through the cytoplasm except the nucleus. Confocal fluorescent imaging using organelle-specific dyes indicated that PIX induced severe photodamage to disrupt membranes and intracellular organelles, including the plasma membrane, mitochondrion, and lysosome. On the other hand, cells treated with DPs kept the characteristic fluorescent pattern of such organelles even after photoirradiation. However, notably 32(+)DPZn achieved remarkably higher (1)O(2)-induced cytotoxicity against LLC cells than PIX. Furthermore, both dendrimer porphyrins had far lower dark toxicity as compared with PIX, demonstrating their highly selective photosensitizing effect in combination with a reduced systemic toxicity.     

Evaluation of human erythrocytes as model cells in photodynamic therapy

The role of erythrocytes as targets in photodynamic therapy is a controversially discussed topic in the literature. Therefore five different, but well known photosensitisers (three zinc phthalocyanines, tetrabenzoporphine and pheophorbide a delivered in liposomes were used for photodynamic treatment of human erythrocytes. The phototoxic effect on these cells showed pronounced differences. It was in the range: zinc phthalocyanine = pheophorbide a > tetrabenzoporphine > zinc octa-n-alkyl phthalocyanines. Data from the zinc octa-n-alkyl phthalocyanines were compared with photodynamic effects within cutaneous cell lines, treated under the same experimental conditions. The results show that erythrocytes are unlikely to make good models for predicting the efficiency of the photosensitiser in general, and the same applies to cells other than erythrocytes and in vivo. Possible reasons could be differences in dye accumulation. However, erythrocytes may well serve as model cells to explore the cellular and molecular mechanisms of photodynamic treatment.     

Apoptosis of vascular smooth muscle cells induced by photodynamic therapy with protoporphyrin IX

Photodynamic therapy (PDT) had been shown effective in the treatment of intimal hyperplasia, which contributes to restenosis, by eradicating cells in the vessel wall. This study is designed to evaluate the effects of PDT with protoporphyrin IX (PpIX) on the viability of vascular smooth muscle cells (SMCs) and to define the cell-death pathway. Fluorescence microscopy and laser-induced fluorescence spectroscopic detection showed that SMCs selectively uptake PpIX, and the intracellular PpIX concentration increases with the amount of PpIX in the incubation solution. PDT with PpIX impaired cellular viability from 93 ± 3.4% to 36 ± 3.9% when the light intensity increases from 2 to 9 J/cm² and intracellular PpIX concentration increases from 0.5 to 20 μg/ml. Although PDT induced both apoptosis and necrosis, the ratio of apoptotic cells increased with light dosage or intracellular PpIX concentration. The loss of mitochondrial membrane potential coincided with the apoptotic ratio. Our results indicated that the induction of apoptosis of SMCs may be one of the mechanisms by which PDT inhibits restenosis in vivo.