Sensitizers For Photodynamic Therapy Research Articles

A study of the stability of tri(glucosyloxyphenyl)chlorin, a sensitizer for photodynamic therapy, in human colon tumoural cells: a liquid chromatography and MALDI-TOF mass spectrometry analysis

Graphic Asymmetrical glycoconjugated tetrapyrrolic macrocycles are under study as efficient sensitizers for photodynamic therapy (PDT). In this context, tri( meta - O -β-glucopyranosyloxyphenyl)chlorin [TPC( m - O -Glu) 3 ] 2a / 3a was found to be four times more photoactive in vitro than Foscan ® . In a further study of this interesting glycoconjugate, its metabolism by cellular glycosidases in HT29 cells has to be explored. Cellular extracts of HT29 cells incubated with TPC( m - O -Glu) 3 (24 h, 6 μM) were analyzed by MALDI-TOF mass spectrometry and high performance liquid chromatography (HPLC). In MALDI-TOF mass spectra, the presence of compounds distinct from TPC( m - O -Glu) 3 ( m / z 1151) were observed at m / z 989, 827 and 665 corresponding to the loss of one, two or three glucose units (162 u) and were be ascribed to TPC( m -OH)( m - O -Glu) 2 2 / 3b , b ′, b ″, TPC( m -OH) 2 ( m - O -Glu) 2 / 3c , c ′, c ″ and TPC( m -OH) 3 isomers 2d / 3d , respectively. The porphyrins resulting from chlorin oxidation TPP( m - O -Glu) 3 4a , TPP( m -OH)( m - O -Glu) 2 4b , b ″, TPP( m -OH) 2 ( m - O -Glu) 4c , c ″ and TPP( m -OH) 3 4d were also observed. The HPLC profile ( λ anal. =420 nm) showed eight peaks consistent with mass spectra. The kinetics of deglucosylation was studied from HPLC profiles between 1 and 48 h incubation. The concentration of triglucoconjugated and diglucoconjugated molecules was maximum around 3 and 8 h incubation, respectively, whereas, totally deglucosylated species appeared only after incubation for more than 10 h. The fully deglycosylated porphyrin TPP( m -OH) 3 is the final metabolite, being observed at a concentration 15 times higher than that of the remaining TPC( m - O -Glu) 3 2a / 3a . Compared to the photobiological activity of the parent molecule [TPC( m - O -Glu) 3 ], a three times higher TPP( m -OH) 3 concentration was necessary to observe a similar in vitro photoactivity.

Efficiency of singlet oxygen generation of aminosquarylium cyanines

Several symmetrical N-methylamino- and N,N-diethylaminosquarylium cyanine dyes bearing benzothiazole, benzoselenazole and quinoline nuclei, displaying strong absorption within the “phototherapeutic window” (600–1000nm), were investigated for the efficiency of singlet oxygen production, aiming their potential sensitising ability for photodynamic therapy (PDT). The assessment was performed determining the corresponding quantum yields of singlet oxygen generation, measuring the luminescence decay of the dyes in the near infrared. By combining the exhibited absorption and the obtained quantum yields, some of the dyes can be regarded as potential candidates as sensitizers for PDT.

Photodynamic effect of novel chlorin e6 derivatives on a single nerve cell

Chlorin e 6 and its derivatives are promising sensitizers for photodynamic therapy (PDT). In order to compare the photodynamic effects of 8 novel derivatives of chlorin e 6 and to explore some mechanisms of their effects at the cellular level, we studied PDT-induced changes in bioelectric activity of crayfish mechanoreceptor neuron that was used as a sensitive experimental model. Neurons were insensitive to red laser irradiation (632.8 nm; 0.3 W/cm 2) or to photosensitizers alone, but changed firing rate and died under the photodynamic effect of nanomolar concentrations of sensitizers. The dynamics of neuron responses depended on photosensitizer type and concentration. The dependence of neuron lifetime on photosensitizer concentration allowed comparing efficiencies of different photosensitizers. Radachlorin was the most potent photosensitizer comparable with mTHPC. High photodynamic efficiency of some chlorin e 6 derivatives was related to weak dependence of neuron lifetime on sensitizer concentration, indicating to the initiation of 2–3 secondary processes such as free radical membrane damage by one absorbed photon. Photodynamic efficiency of sensitizers depended on amphiphilicity influencing their intracellular localization.

Ultrafast studies of the excited-state dynamics of copper and nickel phthalocyanine tetrasulfonates: potential sensitizers for the two-photon photodynamic therapy of tumors.

In order to evaluate the potential of copper and nickel phthalocyanine tetrasulfonates as sensitizers for two-photon photodynamic therapy, we conducted kinetic femtosecond measurements of transient absorption and bleaching of their excited state dynamics in aqueous solution. Samples were pumped with 620 nm and 310 nm laser light, which allowed us to study relaxation processes from both the first and second singlet (or doublet for the copper phthalocyanine) excited states. A second excitation from the first excited triplet state, approximately 685 and 105 ps after the first excitation for copper and nickel phthalocyanine tetrasulfonate respectively, was the most efficient way to bring the molecules to an upper triplet state. Presumably this highest triplet state can inflict molecular damage on adjacent biomolecules int eh absence of oxygen, resulting in the desired cytotoxic cellular response. Transient absorption spectra at different fixed delays indicate that optimum efficiency would require that the second photon has a wavelength of approximately 750 nm.

The Cellular Uptake of Sensitizers Bound to Cyclodextrin Carriers

Photodynamic therapy of cancer uses the interaction of sensitizers and light to destroy cancer cells. In this study we tested the cellular uptake of meso-tetrakis(4-sulfonatophenyl)porphine (TPPS4) and its complex PdTPPS4 in the presence or absence of 2-hydroxypropyl-cyclodextrins (hpCDs) on G361 human melanoma cells. Self-fluorescence in G361 cells were measured by Perkin-Elmer LS50B luminometer equipped with well plate reader accessory. Morphological changes in cells have been evaluated using inversion fluorescent microscope Olympus IX 70 and image analysis. The uptake of the sensitizer PdTPPS4 at the given time interval from 1 to 48 hours is markedly higher than the uptake of TPPS4. The highest uptake was found for sensitizer PdTPPS4 in combination with hpbetaCD. TPPS4 and PdTPPS4 especially in the supramolecular complex with nontoxic cyclodextrin carriers represent efficient sensitizers for photodynamic therapy in vitro on G361 cells.

Photodynamic Effects of Radachlorin® on Cervical Cancer Cells

Photodynamic therapy (PDT) is a novel treatment modality, which produces local tissue necrosis with laser light following the prior administration of a photosensitizing agent. Radachlorin has recently been shown to be a promising PDT sensitizer. In order to elucidate the antitumor effects of PDT using Radachlorin on cervical cancer, growth inhibition studies on a HPV-associated tumor cell line, TC-1 cells in vitro and animals with an established TC-1 tumor in vivo were determined. TC-1 tumor cells were exposed to various concentrations of Radachlorin and PDT, with irradiation of 12.5 or 25 J/cm(2) at an irradiance of 20 mW/cm(2) using a Won-PDT D662 laser at 662 nm in vitro. C57BL/6 mice with TC-1 tumor were injected with Radachlorin via different routes and treated with PDT in vivo. A growth suppression study was then used to evaluate the effects at various time points after PDT. The results showed that irradiation of TC-1 tumor cells in the presence of Radachlorin induced significant cell growth inhibition. Animals with established TC-1 tumors exhibited significantly smaller tumor sizes over time when treated with Radachlorin and irradiation. PDT after the application of Radachlorin appears to be effective against TC-1 tumors both in vitro and in vivo.

Spectral properties of stilbazolium merocyanines – potential sensitizers in photodynamic therapy and diagnosis: Part I. Merocyanines in model systems

Spectral and photochemical properties of seven stilbazolium merocyanines of different chain length and side groups attached, dissolved in ethanol or methanol solutions as well as in polyvinyl alcohol water solutions and films, were investigated. The absorption, fluorescence emission and fluorescence excitation spectra as well as steady state photoacoustic and laser induced optoacoustic spectroscopy signals were measured.Triplet states are, because of their slow decay time, usually very effective in photochemical reaction; therefore dyes with efficient triplet states generation are suitable for destruction of illuminated diseased cells. The laser induced optoacoustic measurements were used to evaluate the efficiency of generation and decay times of dye triplet states. The quenching of the dye triplet state by oxygen was also studied.The results show that properties of merocyanines and their interactions with macromolecules depend strongly on the type of side groups attached. The dyes with NO2 groups attached exhibit low yield of fluorescence therefore they could be useful rather in photodynamic therapy (PDT) than for diagnostic purposes. Some of the merocyanines investigated exhibit high yields of triplet state generation and, therefore, should be effective in photodynamic reactions.Further investigations will be carried out with the same dyes on healthy and diseased cells in order to show whether it is possible, on the grounds of spectral and photochemical properties of dye molecules in simple model systems, to predict the efficiencies of the dye incorporation into cells and the yield of photodynamic reactions.

Synthesis and comparison of photodynamic activity of alkylheteroatom substituted azaphthalocyanines

Optimal reaction conditions were developed for synthesis of octakis(butylamino), octakis(butylsulfanyl) and octakis(butoxy) azaphthalocyanines (AzaPc’s) with central metal Mg, Zn and metal-free. Their photodynamic activity was measured and compared as a dye-sensitised photooxidation of 1,3-diphenylisobenzofurane (DPBF). Compounds with alkylamino substituent are very poor producers of the singlet oxygen and therefore not suitable as sensitizers for photodynamic therapy (PDT). On the other hand, compounds with alkylsulfanyl and alkoxy substituents possess very good photodynamic activity and are suitable for PDT.

In vitro toxicity testing of supramolecular sensitizers for photodynamic therapy

We report the phototoxicity of meso-tetrakis(4-sulphonatophenyl)porphine (TPPS4) and zinc metallocomplex (ZnTPPS4) sensitizers in the presence or absence of 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) on G361human melanoma cells. Morphological changes in cell cultures have been evaluated using inversion fluorescent microscope and image analysis. Viability of cells was determined by means of molecular probes for fluorescence microscopy (LIVE/DEAD kit- double staining with Calcein AM and Ethidium Homodimer). The quantitative changes of cell viability in relation to sensitizers concentrations and irradiation doses were proved by fluorometric measurement with fluoroscan Ascent. We found that the most effective sensitizer is ZnTPPS4 bound to HP-β-CD, since the IC 50 value was 12.5 g/ml at the dose of light radiation of 10 J/cm 2.

Synthesis of ether- and carbon-linked polycarboranyl porphyrin dimers for cancer therapies

Porphyrin dimers bearing multiple carborane cages for potential use as sensitizers in boron neutron capture therapy (BNCT) and photodynamic therapy (PDT) were synthesized from protoporphyrin dimethyl ester and characterized. Diastereomeric ether-linked dimers bearing four closo carborane cages (40 boron atoms) were found to be unstable to the acidic conditions necessary for conversion into water-soluble salts. In contrast, the carboncarbon-linked dimers bearing six icosahedral carboranes (60 boron atoms) were stable to acid and could be isolated as water-soluble sodium salts. In vitro and in vivo studies of these novel molecules are currently under investigation.

Singlet oxygen generation ability of squarylium cyanine dyes

The quantum yields for singlet oxygen generation of several squarylium cyanine dyes derived from benzothiazole, benzoselenazole and quinoline, displaying absorption within the so-called “phototherapeutic window” (600–1000 nm), were determined, envisioning their potential usefulness for photodynamic therapy (PDT). The determination was performed by a direct method measuring the luminescence decay of the dyes in the near infrared. Considering the absorption and the quantum yields displayed by some of the dyes, these seemed to be potential candidates as sensitizers for PDT.

Applications of photothermic methods in photodynamic therapy investigations

The applications of steady state photoacoustic and time resolved photothermal methods are carried out in our laboratory. Based on these methods, the selection of optimal sensitizers for photodynamic therapy and photodynamic diagnosis of cancer were described. Additionally, in order to establish the fate of absorbed energy, the absorption and fluorescence spectra were measured. All spectra were measure using natural and/or linearly polarized light because of polarized spectroscopy delivers information about the sample structures. Spectral and photochemical properties of selected sensitizers (merocyanines, porphyrines and phthalocyanines) were investigated. All dyes were first investigated in model systems (fluid solutions or rigid matrix) and later incorporated into resting or stimulated cells as well as into cancer cells delivered from cell lines. Stimulated cells could serve as models of malignant tissue and the properties of these cells at various procedures of stimulation were compared. It was shown that steady state photoacoustic, which is less perturbed by scattering than absorption, is very useful in the establishment of the efficiency of sensitizer incorporation into cells whereas a time resolved photothermal method (laser induced optoacoustic spectroscopy) enabled the establishment of a yield of dye triplet states generation. The triplet states are very active in photochemical reactions. Therefore, on the basis of their yield, it is possible to predict the efficiency of light induced lesions of malignant cells.

Synthesis, and in vitro and in vivo evaluation of a diphenylchlorin sensitizer for photodynamic therapy

This paper reports the synthesis of a new diphenylchlorin photosensitizer, 2,3-dihydro-5,15-di(3,5-dihydroxyphenyl)porphyrin (SIM01). The photodynamic properties, cell uptake and localization of SIM01 were compared with those of structurally related meso-tetra(hydroxyphenyl)chlorin (m-THPC). In vitro studies were conducted on rat glioma cells (C6) and human adenocarcinoma (HT-29), and in vivo studies on human colon adenocarcinoma cells (HT-29) and human prostate adenocarcinoma cells (PC3). Both dyes showed an absorption maximum at around 650 nm, with a molar extinction coefficient of 13 017 M −1 cm −1 for SIM01 and 22 718 M −1 cm −1 for m-THPC. Their capacity to generate singlet oxygen was identical, but differences in partition coefficients indicated that SIM01 was slightly more hydrophilic. In vitro, SIM01 was slightly more phototoxic than m-THPC for C6 cells (4.8 vs. 6.8 μg ml −1). However, phototoxicities were nearly identical for HT29 cells (0.45 μg ml −1 for 5 h incubation followed by 300 mW, 20 J cm −2). Pharmacokinetics in vivo in mice, as determined by fibre spectrofluorimetry, showed that the SIM01 fluorescence signal in the tumor was maximal between 6 and 12 h after injection, as compared to 72 h for m-THPC. With a 2 mg kg −1 dye dose and laser irradiation at 300 J cm −2 (650 nm, 300 mW), the optimal PDT response occurred when the interval between injection and irradiation was 6 h for SIM01 and 24 h for m-THPC. For SIM01 with 5 mg kg −1 injection, the optimal PDT response occurred with a 12 h delay and with the same irradiation parameters as described above, in this case the tumor response showing 40% growth. Considering the tumor volume doubling time, the value was 6.5 days in the control group and increased to 13.5 days with SIM01. Thus, SIM01 may be a powerful sensitizer characterized by strong in vitro and in vivo phototoxicity and faster tissue uptake and elimination than m-THPC.

癌光線力学療法用増感剤としてのノン-ペリフェラル位アルキル基置換アルキルベンゾポルフィラジン異性体の励起三重項寿命

Synthesis of zinc non-peripheral substituted alkylbenzopyridoporphyrazines and separation of their regio isomers have been reported. The triplet state lifetime of zinc non-peripheral substituted alkylbenzopyridoporphyrazines in polymer film was measured by laser flash photolysis in order to estimate an ability to use the sensitizers for photodynamic therapy of cancer. The lowest symmetric isomer of zinc non-peripheral substituted alkylbenzopyridoporphyrazines showed the longest triplet state lifetime.

Association between the photodynamic loss of Bcl-2 and the sensitivity to apoptosis caused by phthalocyanine photodynamic therapy.

We have reported that photodynamic therapy (PDT) using the photosensitizer phthalocyanine (Pc) 4 and red light damages the antiapoptotic protein Bcl-2. Recently, using transient transfection of Bcl-2 deletion mutants, we identified the membrane anchorage domains of Bcl-2 as necessary to form the photosensitive target. However, it is not clear how Bcl-2 photodamage sensitizes cells to Pc 4-PDT-induced apoptosis, whether overall cell killing is also sensitized or how up-regulation of Bcl-2 in tumors might make them more or less responsive to Pc 4-PDT. In this study we report on MCF-7c3 cells (human breast cancer cells expressing stably transfected procaspase-3) overexpressing wild-type Bcl-2 or certain deletion mutants in either a transient or a stable mode. By flow cytometric analysis of transiently transfected cells, we found that wild-type Bcl-2, Bcl-2delta33-54 and Bcl-2delta37-63 (each of which can be photodamaged) protected cells from apoptosis caused by Pc 4-PDT. In contrast, Bcl-2delta210-239, which lacks the C-terminal transmembrane domain and cannot be photodamaged, afforded no protection. We then evaluated the PDT sensitivity of transfected cell lines stably overexpressing high levels of wild-type Bcl-2 or one of the Bcl-2 mutants. Overexpression of wild-type Bcl-2, Bcl-2delta33-54 or Bcl-2delta37-63 resulted in relative resistance of cells to Pc 4-PDT, as assessed by morphological apoptosis or loss of clonogenicity. Furthermore, overexpression of Bcl-2 also inhibited the activation-associated conformational change of the proapoptotic protein Bax, and higher doses of Pc 4 and light were required to activate Bax in cells expressing high levels of Bcl-2. Many advanced cancer cells have elevated amounts of Bcl-2. Our results show that increasing the dose of Pc 4-PDT can overcome the resistance afforded by either Bcl-2 or the two mutants. PDT regimens that photodamage Bcl-2 lead to activation of Bax, induction of apoptosis and elimination of the otherwise resistant tumor cells.

Facile synthesis of N, N′-dimethylated N-confused porphyrins

N-Confused tetraarylporphyrins react with CH 3I in the presence of Na 2CO 3 to give, in high yield, N, N′-dimethylated N-confused porphyrin salts, which are mixtures of structural isomers. The structures of the major isomers were determined by X-ray diffraction and NMR spectroscopic analyses. These N, N′-dimethylated N-confused tetraarylporphyrin salts generate singlet oxygen when irradiated at long wavelengths in the visible region, and therefore, are potential sensitizers for photodynamic therapy.

Singlet oxygen generation by 9-acetoxy-2,7,12,17-tetrakis-(β-methoxyethyl)-porphycene (ATMPn) in solution

Despite the approval of Photofrin® in various countries, chemically pure sensitizers for photodynamic therapy (PDT) are still needed. Based on numerous experiments in vitro, singlet oxygen is considered to play the major role in PDT. Therefore, the generation of singlet oxygen by the promising, hydrophobic photosensitizer 9-acetoxy-2,7,12,17-tetrakis-(β-methoxyethyl)-porphycene (ATMPn) in deuterated ethanol (EtOD) has been investigated in detail by time-resolved near-infrared spectroscopy. Using a new, highly sensitive infrared photomultiplier the complete time dependence of singlet oxygen luminescence was measured at 1270 nm. The respective relaxation rates and rate constants of the involved ATMPn triplet T1 state and oxygen 1Δg state were determined, in particular at different oxygen concentrations. Therefore, the oxygen concentration dependence of the proportion PT of the ATMPn triplet T1 state population quenched by oxygen, which is proportional to the singlet oxygen quantum yield ΦΔ, could be calculated. The result shows that PT decreased significantly with decreasing oxygen concentration. The completely different oxygen concentrations in vitro and in vivo lead therefore to a different quantum yield of singlet oxygen generation in both situations. Thus, one has to be careful when transferring results from in vitro experiments to photodynamic therapy in vivo, in particular regarding the role of singlet oxygen (type-II reaction) as compared to the type-I reaction.

Inverted porphyrins and expanded porphyrins: An overview

Porphyrins and metallopophyrins have attracted the attention of chemists for the past 100 years or more owing to their widespread involvement in biology. More recently, synthetic porphyrins and porphyrin-like macrocycles have attracted the attention of researchers due to their diverse applications as sensitizers for photodynamic therapy, MRI contrasting agents, and complexing agents for larger metal ions and also for their anion binding abilities. The number of π-electrons in the porphyrin ring can be increased either by increasing the numberof conjugated double bonds between the pyrrole rings or by increasing the number of heterocyclic rings. Thus, 22π sapphyrins, 26π rubyrins, 30π heptaphyrins, 34π octaphyrins and higher cyclic polypyrrole analogues containing 40π, 48π, 64π, 80π and 96π systems have recently been reported in the literature. These macrocycles show rich structural diversity where normal and different kinds of inverted structures have been identified. In this review, an attempt has been made to collect the literature of the inverted porphyrins and expanded porphyrins reported until December 2001. Since themeso aryl expanded porphyrins have tendency to form both inverted and non-inverted structures more emphasis has been given tomeso aryl expanded porphyrins.

New sulfonamide and sulfonic ester porphyrins as sensitizers for photodynamic therapy

New sulfonamide and sulfonic ester porphyrins were prepared and their in vitro characteristics as sensitizers for photodynamic therapy were assessed. Fluorescence spectra were characterised by two main peaks, located between 650-680 nm and 705-740 nm, respectively. Some of these new porphyrins (three out of nine) showed good photodynamic properties in the in vitro assays. In the absence of light, these porphyrins are not toxic. With 50 J.cm−2 illumination light (514 nm) they induced mortality in 50% of HT29 cells with 2 to 4.5 μg/ml. Comparison of in vitro phototoxic efficacy of these three compounds with other photosensitizers already described confirms interest in their phototoxic properties.

Indocyanine green as a prospective sensitizer for photodynamic therapy of melanomas.

Spectroscopic, photochemical and biological properties of indocyanine green (ICG) are presented. Light over 800 nm is effectively absorbed by ICG. This property as well as photochemical behaviour of ICG make it a very suitable dye for photodynamic treatment of melanoma cells. Cytotoxicity of ICG itself and the effect of photodynamic therapy (PDT) were evaluated by following the growth of human (SKMEL 188) and mouse (S91) melanoma cells. The surviving fraction of the cells irradiated (lambda(ex) = 830 nm) vs non-irradiated, treated with the same dose of ICG, is significantly decreased (5- to 10-fold). These results show that ICG is a very promising dye for photodynamic therapy of melanomas.