The sulphonated derivatives of porphyrins (e.g. TPPS4) are hydrophilic photosensitizers and have certain advantages like fully known structures and the possibility of synthetic production. The aim of this work was to study in vitro cytotoxicity and to compare the new photosensitizer MgTPPS4 with TPPS4 and its other metal-complexes (ZnTPPS4, PdTPPS4) on human skin melanom and mouse fibroblast cell lines. A photodynamic treatment was induced by light emitting diodes with three different total doses (1, 5 and 10J/cm(2)). For proper analysis and understanding of cell behavior after the administration of sensitizers, a complex battery of in vitro tests including the production of reactive oxygen species, the MTT viability test, a comet assay, a cell cycle and a type of cell death determination were used. We discovered that the most suitable photosensitizer is ZnTPPS4 because it had the biggest lethal influence on melanoma cells and the lowest lethal influence on fibroblast cells. The second most effective photosensitizer seemed to be MgTPPS4. On the basis of our results we can also assume that there is a higher accumulation of photosensitizer in a tumorous cell line. The higher concentration of photosensitizer and light dose resulted in more reactive oxygen species production and found more cells undergoing necrosis.
- MeSH
- Apoptosis drug effects MeSH
- Cell Death drug effects MeSH
- NIH 3T3 Cells MeSH
- Fibroblasts drug effects MeSH
- Photochemotherapy MeSH
- Photosensitizing Agents pharmacology MeSH
- Comet Assay MeSH
- Coordination Complexes pharmacology MeSH
- Humans MeSH
- Metalloporphyrins pharmacology MeSH
- Mice MeSH
- Cell Line, Tumor MeSH
- Palladium pharmacology MeSH
- Reactive Oxygen Species metabolism MeSH
- Light MeSH
- Cell Survival drug effects radiation effects MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
The present work provides a proof-of-concept that the singlet oxygen-sensitized delayed fluorescence (SOSDF) can be detected from individual living mammalian cells in a time-resolved microscopy experiment. To this end, 3T3 mouse fibroblasts incubated with 100 μM TPPS4 or TMPyP were used and the microsecond kinetics of the delayed fluorescence (DF) were recorded. The analysis revealed that SOSDF is the major component of the overall DF signal. The microscopy approach enables precise control of experimental conditions - the DF kinetics are clearly influenced by the presence of the (1)O2 quencher (sodium azide), H2O/D2O exchange, and the oxygen concentration. Analysis of SOSDF kinetics, which was reconstructed as a difference DF kinetics between the unquenched and the NaN3-quenched samples, provides a cellular (1)O2 lifetime of τΔ = 1-2 μs and a TPPS4 triplet lifetime of τT = 22 ± 5 μs in agreement with previously published values. The short SOSDF acquisition times, typically in the range of tens of seconds, enable us to study the dynamic cellular processes. It is shown that SOSDF lifetimes increase during PDT-like treatment, which may provide valuable information about changes of the intracellular microenvironment. SOSDF is proposed and evaluated as an alternative tool for (1)O2 detection in biological systems.
- MeSH
- Single-Cell Analysis instrumentation methods MeSH
- Sodium Azide chemistry MeSH
- 3T3 Cells MeSH
- Equipment Design MeSH
- Fibroblasts chemistry MeSH
- Fluorescence * MeSH
- Kinetics MeSH
- Oxygen chemistry MeSH
- Microscopy instrumentation methods MeSH
- Mice MeSH
- Deuterium Oxide chemistry MeSH
- Singlet Oxygen chemistry MeSH
- Water chemistry MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
BACKGROUND: Bacterial resistance to antibiotics is a constantly growing challenge. Photodynamic therapy (PDT) offers a new approach to the treatment of bacterial and viral diseases. The aim of this study was to compare the efficacy of photosensitizers used in PDT applied to cell lines and bacterial strains. METHODS: We tested the cytotoxicity and phototoxicity of 3 photosensitizers: TPPS4, ZnTPPS4 and TMPyP applied to the NIH3T3 cell line using two established methods for measuring ROS production and, MTT viability assay. Bacterial viability was determined spectrophotometrically over 24 h following PDT. RESULTS: The most efficient photosensitiser was TMPyP as it reduced the viability of the NIH3T3 cell line by more than 85%. In general, the photosensitisers were more phototoxic to the two Gram-positive bacterial strains, Enterococcus faecalis and Staphylococcus aureus. The viability of E. faecalis was reduced to 78 % by a dose radiation 0.5 J/cm(2) and concentration of TMPyP 1.562 μmol/L. The viability of bacterium S. aureus was reduced to 23 % when exposed to a radiation dose 0.5 J/cm(2) and 100 μmol/L concentration of ZnTPPS4. The highest viability decrease (15 %) for Pseudomonas aeruginosa was caused by 0.5 J/cm(2) radiation dose and 50 μmol/L TMPyP concentration. Escherichia coli proved to be PDT resistant as the bacterial viability was higher than 90%. CONCLUSIONS: The goal of the present study was to test the efficiency of photosensitizers on the NIH 3T3 cell line and bacterial cells. Subsequently we would like to study effectiveness of photosensitizers bound to carriers (for example cyclodextrins) on other cell line and bacterial strain.
- MeSH
- NIH 3T3 Cells drug effects radiation effects MeSH
- Enterococcus faecalis drug effects radiation effects MeSH
- Escherichia coli drug effects radiation effects MeSH
- Photochemotherapy * MeSH
- Photosensitizing Agents pharmacology MeSH
- Metalloporphyrins pharmacology MeSH
- Microbial Viability drug effects radiation effects MeSH
- Mice MeSH
- Porphyrins pharmacology MeSH
- Pseudomonas aeruginosa drug effects radiation effects MeSH
- Reactive Oxygen Species analysis MeSH
- Staphylococcus aureus drug effects radiation effects MeSH
- Cell Survival drug effects radiation effects MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Six common water-soluble singlet oxygen ((1)O2) photosensitizers - 5,10,15,20-tetrakis(1-methyl-4-pyridinio) porphine (TMPyP), meso-tetrakis(4-sulfonathophenyl)porphine (TPPS4), Al(III) phthalocyanine chloride tetrasulfonic acid (AlPcS4), eosin Y, rose bengal, and methylene blue - were investigated in terms of their ability to produce delayed fluorescence (DF) in solutions at room temperature. All the photosensitizers dissolved in air-saturated phosphate buffered saline (PBS, pH 7.4) exhibit easily detectable DF, which can be nearly completely quenched by 10 mM NaN3, a specific (1)O2 quencher. The DF kinetics has a biexponential rise-decay character in a microsecond time domain. Therefore, we propose that singlet oxygen-sensitized delayed fluorescence (SOSDF), where the triplet state of a photosensitizer reacts with (1)O2 giving rise to an excited singlet state of the photosensitizer, is the prevailing mechanism. It was confirmed by additional evidence, such as a monoexponential decay of triplet-triplet transient absorption kinetics, dependence of SOSDF kinetics on oxygen concentration, absence of SOSDF in a nitrogen-saturated sample, or the effect of isotopic exchange H2O-D2O. Eosin Y and AlPcS4 show the largest SOSDF quantum yield among the selected photosensitizers, whereas rose bengal possesses the highest ratio of SOSDF intensity to prompt fluorescence intensity. The rate constant for the reaction of triplet state with (1)O2 giving rise to the excited singlet state of photosensitizer was estimated to be ~/>1 × 10(9) M(-1) s(-1). SOSDF kinetics contains information about both triplet and (1)O2 lifetimes and concentrations, which makes it a very useful alternative tool for monitoring photosensitizing and (1)O2 quenching processes, allowing its detection in the visible spectral region, utilizing the photosensitizer itself as a (1)O2 probe. Under our experimental conditions, SOSDF was up to three orders of magnitude more intense than the infrared (1)O2 phosphorescence and by far the most important pathway of DF. SOSDF was also detected in a suspension of 3T3 mouse fibroblast cells, which underlines the importance of SOSDF and its relevance for biological systems.
- MeSH
- 3T3 Cells MeSH
- Spectrometry, Fluorescence MeSH
- Photosensitizing Agents chemistry MeSH
- Kinetics MeSH
- Hydrogen-Ion Concentration MeSH
- Quantum Theory MeSH
- Mice MeSH
- Singlet Oxygen chemistry MeSH
- Temperature MeSH
- Water chemistry MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Photodynamic therapy (PDT) is an alternative method of tumour treatment. It is based on a photochemical reaction of a photosensitizer, irradiation, and O(2) which converts to cytotoxic (1)O(2) and other forms of reactive oxygen species (ROS). The comet assay (also called single-cell gel electrophoresis, SCGE) is a sensitive, simple and quantitative technique for detection of DNA damage. In our study we investigated the phototoxicity of the two porphyrin photosensitizers, TPPS4 and MgTPPS4, on HeLa cells. Three different radiation doses and six different concentrations of the photosensitizers were used. Our results show that the DNA of the cells treated with the TPPS(4) and MgTPPS(4) at the concentrations higher than 5 μM was highly fragmented indicating a strong phototoxic effect resulting in a cell apoptosis. On the base of our results we can hypothesize that even the irradiation dose of 1 J cm(-2) is sufficient enough to provoke the DNA fragmentation.
- MeSH
- Apoptosis drug effects MeSH
- Radiation Dosage MeSH
- Photochemotherapy methods MeSH
- Photosensitizing Agents administration & dosage pharmacology MeSH
- DNA Fragmentation drug effects MeSH
- HeLa Cells MeSH
- Magnesium chemistry MeSH
- Comet Assay MeSH
- Humans MeSH
- Metalloporphyrins administration & dosage chemistry pharmacology MeSH
- Porphyrins administration & dosage chemistry pharmacology MeSH
- Dose-Response Relationship, Drug MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The objectives of this study was to investigate the production of reactive oxygen species (ROS) after photodynamic therapy (PDT) in vitro. We examined second generation sensitizers, porphyrines (TPPS4, ZnTPPS4 and PdTPPS4) and compared their effectivity on ROS generation in G361 cell line. Used porphyrines are very efficient water-soluble aromatic dyes with potential to use in photomedicine and have a high propensity to accumulate in the membranes of intracellular organelles like lysosomes and mitochondria. Interaction between the triplet excited state of the sensitizer and molecular oxygen leads to produce singlet oxygen and other ROS to induce cell death. Production of ROS was verificated by molecular probe CM-H2DCFDA and viability of cells was determined by MTT assay. Our results demonstrated that ZnTPPS4 induces the highest ROS production in cell line compared to TPPS4 and PdTPPS4 at each used concentration and light dose. These results consist with a fact that photodynamic effect depends on sensitizer type, its concentration and light dose.
Bylo sledováno celkem 18 nemocných (11 mužů a 7 žen), kteří byli léčeni v letech 1999-2003 nakožní klinice FN v Hradci Králové pomocí fotodynamické terapie (PDT). Při léčbě byl použitfotosenzibilizátor mezo-tetra-para-sulfofenyl-porfin (TPPS4) a fotosenzibilizátor -aminolevulovákyselina (ALA) s následným ozářením zdrojemviditelného světla vlnové délky 600-800 nm v dávkách120-200 J/cm2.Šlo o 16 os
The group of 18 patients (11 males and 7 females) treated by photodynamic therapy (PDT) at theDepartment of Dermatology of the Faculty Hospital in Hradec Králové during the period 1999-2003was followed. Photosensitizers meso-tetra-para-sulphophenyl-porphin (TPPS4) and -aminolevulicacid (ALA) followed by irradiation by a source of visible light with the wave length of 600-800 nm indoses of 120-200
- Keywords
- TPPS4,
- MeSH
- Carcinoma, Basal Cell therapy MeSH
- Bowen's Disease therapy MeSH
- Radiation Dosage MeSH
- Adult MeSH
- Photochemotherapy methods instrumentation MeSH
- Photosensitizing Agents administration & dosage adverse effects therapeutic use MeSH
- Aminolevulinic Acid administration & dosage adverse effects therapeutic use MeSH
- Middle Aged MeSH
- Humans MeSH
- Aged MeSH
- Treatment Outcome MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Review MeSH
- Comparative Study MeSH
V práci je souhrnně charakterizováno dermatologické využití fotodynamické diagnostiky a terapie.Je zde popsán princip, provedení, fotosenzibilizátory, indikace a také nežádoucí účinky.
The article gives an overview of photodynamic diagnostics and therapy in dermatology. Principles,execution, photosensitizers, indications and side effects are mentioned.
- Keywords
- TPPS4, ALA, metvix krém, levulan-kerastick,
- MeSH
- Photochemotherapy methods adverse effects utilization MeSH
- Photosensitizing Agents administration & dosage chemistry therapeutic use MeSH
- Hematoporphyrins MeSH
- Skin Absorption MeSH
- Skin Diseases diagnosis therapy MeSH
- Aminolevulinic Acid MeSH
- Light MeSH
- Publication type
- Review MeSH
Východisko. Přesné vymezení ložisek některých kožních projevů, např. bazaliómů, je někdy pro terapeutický zákrok obtížné, a není-li odstraněný útvar celý, často dochází k recidivám. V některých případech pacienti odmítají radikální výkony. Pak připadá v úvahu bolestivější kryoterapie, která rovněž nezaručuje destrukci všech chorobných buněk. Proto se v posledních letech rozvíjí nový diagnostický a terapeutický postup. Metody a výsledky. Postižená ložiska jsou ošetřena injekčně nebo mastí (pod tzv. okluzí) obsahující fotosenzibilizátor, který má zvláštní afinitu k rychle se dělícím buňkám. Po uplynutí adekvátní doby se zjišťuje pomocí UV lampy přítomnost fotosenzibilizátoru a následně se ložisko ozáří paprsky o příslušné vlnové délce (620–680 nm). Výsledky terapie sice nejsou patrné v krátké době, ale jejich efekt bývá kosmeticky velice dobrý. Závěry. Fotodynamickou terapii (PDD) a fotodynamickou diagnostiku (PDT) použili autoři k léčbě různých dermatóz (bazaliómy, metastázy maligního melanómu, verrucae vulgares, keratoakantóm, solitární ložiska T lymfomu –mycosis fungoides,m. Bowen, psoriasis vulgaris, palmoplantární pustulóza, keratóma solare) s velmi slušným léčebným i kosmetickým efektem. Výsledky jsou uvedené v přehledné tabulce v textu. Autoři rozhodně netvrdí, že PDT je jedinou a zázračnou terapií použitelnou k léčbě kožních nádorů či jiných chorob. Přesto se však domnívají, že tato metoda může při vhodné aplikaci rozšířit škálu dosavadních terapeutických postupů. Její výhodou je selektivnost, prakticky žádné zatížení pacienta, dobrá snášenlivost, slušný až výborný kosmetický efekt.
Background. Some skin lesions e.g. basal cell carcinomas are sometimes difficult to remove completely and frequent relapses can develop after their imperfect removal. In case the patient refuses to undergo a radical surgical intervention, more painful alternative like cryotherapy comes into consideration as a method of tumour destruction. Not even such a procedure does guarantee complete destruction of all tumour cells. During the last years new diagnostics and therapeutic methods like photodynamic diagnostic and photodynamic therapy have been developed and they became subjects of our interest. Methods and Results. Lesions were treated with photosenzitizer (meso-tetra-para-sulphonato-phenyl-porfine – TPPS4) administered in an injection or in the ointment under occlusion. Six to 24 hours later we checked presence of photosensitizer in the lesions and in positive cases we irradiated the lesions with light of suitable wave length (630 nm). Conclusions. PDD and PDT were used for diagnostics and treatment of different dermatoses (basal cell carcinomas, malignant melanoma metastases, verrucae vulgares, keratoacanthomas, solitary lesions of T lymphoma – mycosis fungoides, m. Bowen, psoriasis vulgaris, pustulosis palmoplantaris, solar keratoma) with very good medical and cosmetic effect. Results are presented in the table. Authors do not consider the PDT to be the only and miraculous method relevant to treatment of all skin tumours or other skin diseases. They are of the opinion that this technique, when properly used, can extend the scale of therapeutic methods. The advantage of PDT is its selectivity, good tolerance and generally good cosmetic effect.
