Pharmacokinetics of Tetra (m-hydroxyphenyl)chlorin in Human Plasma and Individualized Light Dosimetry in Photodynamic Therapy

The pharmacokinetics of the photosensitizer 5,10,15,20-tetra( m -hydroxyphenyl) chlorin(mTHPC) was investigated in the plasma of 20 patients by absorption and fluorescence spectroscopy. The temporal behavior was characterized by a rapid decrease in concentration during the first minutes after intravenous injection of 0.15 mg/kg mTHPC. A minimum concentration in the plasma was reached after about 45 min. The drug concentration then increased again, attaining a maximum after about 10 h, after which it decreased again with a halflife of about 30 h. Irradiation tests in the oral cavity at different time intervals after the injection revealed that the tissue re-action was only partially correlated with the mTHPC plasma level. The tissue response was stronger at later drug-light intervals (1–4 days) than during the first hours after injection even though the mTHPC plasma concentration was higher at the shorter times. Relative mTHPC concentrations were also measured in the mucosae of the oral cavity, the esophagus and the bronchi of 27 patients by light-induced fluorescence spectroscopy using an optical fiber-based spectrometer. These measurements were performed prior to photodynamic therapy (PDT), 4 days after injection of the photosensitizer. Highly significant linear correlations were found between the relative mTHPC concentrations in the mucosae of these three organs. Likewise, the plasma levels of mTHPC measured just before PDT were significantly correlated with the mTHPC concentrations in the three types of mucosae mentioned above. These results indicate that mTHPC plasma levels measured just before PDT can be used for PDT light dosimetry.     

In vitro investigation on the pH dependence of the absorption and fluorescence properties of the photosensitizer mTHPC

Fluorescence excitation efficiency is of great importance for photodynamic diagnosis. Because usually a difference in the interstitial pH between normal and tumor tissue occurs, it is necessary to assess the impact of pH on the fluorescence emission intensity of the photosensitizer meta-tetrahydroxyphenylchlorin (mTHPC) in this context. The results obtained by in vitro fluorescence measurements clearly indicate that pH values below 6 lead to a significant decrease in the fluorescence intensity. In the physiological range of pH 6.5-7.2, however, no pH dependence was found. Besides the decrease in the fluorescence intensity of mTHPC for pH < 6, changes in the spectral shape of the absorption were found. These changes can be utilized for "dual-wavelength ratio imaging," using mTHPC as a pH-sensitive indicator with the excitation pair 405 nm/436 nm in the range of pH 3.5-6.     

In vivo mTHPC photobleaching in normal rat skin exhibits unique irradiance-dependent features

We report measurements performed on the normal skin of rats in vivo, which provide information on the photobleaching kinetics and mechanisms of the photosensitizer meso-tetrahydroxyphenyl chlorin (mTHPC). Loss of mTHPC fluorescence was monitored using in vivo fluorescence spectroscopy during photodynamic therapy (PDT) performed using 650 nm laser irradiation. The bleaching was evaluated for irradiances of 5, 20 and 50 mW cm(-2). Two distinct phases of mTHPC photobleaching were observed. In the first phase there was no obvious irradiance dependence in the loss of fluorescence vs fluence. The second phase was initiated by an irradiance-dependent discontinuity in the slope of the bleaching curve, after which the photobleaching rates showed an irradiance dependence consistent with an oxygen-dependent reaction process. To investigate the unusual shape of the in vivo bleaching curves, we measured the PDT-induced changes in O2 concentrations in mTHPC-sensitized spheroids irradiated with 2, 5 and 20 mW cm(-2) of 650 nm light. The oxygen concentration data indicated no unusual features within the range of fluences where the discontinuities in fluorescence were observed during in vivo spectroscopy. The fluorescence from the in vivo bleaching experiments thus reports a phenomenon that is not reported by measurements of the photochemical oxygen consumption in the spheroids.     

Tumor selectivity at short times following systemic administration of a liposomal temoporfin formulation in a murine tumor model

Meso-tetra(hydroxyphenyl)chlorin (mTHPC) (INN: Temoporfin) is one of the most potent photodynamically active substances in clinical use. Treatment protocols for Temoporfin-mediated photodynamic therapy often rely on drug-light intervals of several days in order for the photosensitizer to accumulate within the target tissue, though tumor selectivity is limited. Here, the mTHPC localization was studied at 2-8 h following systemic administration of a liposomal Temoporfin formulation (0.15 mg kg(-1) b.w.) in HT29 human colon adenocarcinoma in NMRI nu/nu mice. Photosensitizer distribution within tumor and internal organs was investigated by means of high performance liquid chromatography following chemical extraction, as well as in situ fluorescence imaging and point-monitoring fluorescence spectroscopy. For tumor tissue, the Temoporfin concentrations at 4 h (0.16+/-0.024 ng mg(-1)) and 8 h (0.18+/-0.064 ng mg(-1)) were significantly higher than at 2 h (0.08+/-0.026 ng mg(-1)). The average tumor-to-muscle and the tumor-to-skin selectivity were 6.6 and 2, respectively, and did not vary significantly with time after photosensitizer injection. In plasma, the Temoporfin concentration was low (0.07+/-0.07 ng mg(-1)) and showed no significant variation with time. Our results indicate a rapid biodistribution and clearance from the bloodstream. Within the same type of organ, data from both fluorescence methods generally exhibited a significant correlation with the extraction results.     

Comparison of photosensitizer (AIPcS2) quantification techniques: in situ fluorescence microsampling versus tissue chemical extraction

A noninvasive in situ fluorescence-based method for the quantification of the photosensitizer chloroaluminum disulfonated phthalocyanine was compared to the highly accurate but nonreal time ex vivo spectrofluorometry method. Our in vivo fluorescence technique is designed to allow real-time assessment of photosensitizer in tumor and normal tissues and therefore temporally optimal light delivery. Laser-induced fluorescence was used to measure photosensitizer concentration from multiple microscopic regions of tissue. Ex vivo chemical extraction was used to quantify photosensitizer concentration in the same volume of tissue. The amount of photosensitizer in the vascular and/or parenchymal compartments of skeletal muscle and liver was determined by quantifying fluorescent signal in vivo, ex vivo and after blood removal. Confocal microscopy was used to spatially document photosensitizer localization 30 min and 24 h after delivery. While a linear correlation can exist between the fluorescence intensity measured by our fiber-optic bundle system and actual tissue concentration, temporal changes to this calibration line exist as the photosensitizer changes its partitioning fraction between the blood (vasculature) and the tissue parenchyma. In situ photosensitizer fluorescence microsampling (dosimetry) systems can be performed in real time and linearly correlated to actual tissue concentration with minimal intertissue variance. Tissue-specific differences may require temporal alterations in the calibration.     

Photobleaching of hypericin bound to human serum albumin,cultured adenocarcinoma cells and nude mice skin

Hypericin is a promising photosensitizer for photodynamic therapy (PDT) characterized by a high yield of singlet oxygen. Photobleaching of hypericin has been studied by means of absorption and fluorescence spectroscopy in different biological systems: in human serum albumin solution, in cultured human adenocarcinoma WiDr cells and in the skin of nude mice. Prolonged exposure to light (up to 95 min, 100 mW/cm2) of wavelength around 596 nm induced fluence-dependent photobleaching of hypericin in all studied systems. The photobleaching was not oxygen dependent, and singlet oxygen probably played no significant role. Emission bands in the spectral regions 420-560 nm and above 600 nm characterize the photoproducts formed. An emission band at 615-635 nm was observed after irradiation of cells incubated with hypericin or of mouse skin in vivo but not in albumin solution. The excitation spectrum of these products resembled that of hypericin. Hypericin appears to be more photostable than most sensitizers used in PDT, including mTHPC and Photofrin.     

mTHPC-mediated photodynamic diagnosis of malignant brain tumors.

Radical tumor resection is the basis for the prolonged survival of patients suffering from malignant brain tumors such as glioblastoma multiforme. We have carried out a phase-II study involving 22 patients with malignant brain tumors to assess the feasibility and the effectiveness of the combination of intraoperative photodynamic diagnosis and fluorescence-guided resection (FGR) mediated by the second-generation photosensitizer meta-tetrahydroxyphenylchlorin (mTHPC). In addition, intraoperative photodynamic therapy (PDT) was performed. Several commercially available fluorescence diagnostic systems were investigated for their applicability in clinical practice. We have adapted and optimized a diagnostic system that includes a surgical microscope, an excitation light source (filtered to 370-440 nm), a video camera detection system and a spectrometer for clear identification of the mTHPC fluorescence emission at 652 nm. Especially in regions of faint fluorescence, it turned out to be essential to maximize the spectral information by optimizing and matching the spectral properties of all components, such as excitation source, camera and color filters. To sum up, on the basis of 138 tissue samples derived from 22 tumor specimens, we have been able to achieve a sensitivity of 87.9% and a specificity of 95.7%. This study demonstrates that mTHPC-mediated intraoperative FGR followed by PDT is a highly promising concept in improving the radicality of tumor resection combined with a therapeutic approach.     

Temporally and spatially heterogeneous distribution of mTHPC in a murine tumor observed by two-color confocal fluorescence imaging and spectroscopy in a whole-mount model

Efficient intratumor delivery of anticancer drugs and photosensitizers is an important factor in the success of chemotherapy and photodynamic therapy, respectively. Unfortunately, their adequate and uniform intratumor distribution is impeded by several physiological barriers and by binding to tissue components. Measurement of gross tumor drug accumulation is a routine method of investigating the uptake and clearance of chemotherapy agents and photosensitizers but tells little about their extravascular spatial distribution. We use whole-mount two-color confocal fluorescence imaging and imaging spectroscopy of unprocessed excised murine tumor fragments to investigate the intratumor distribution of the photosensitizer meso-tetrahydroxyphenyl chlorin (mTHPC) as a function of distance from blood vessels perfused with 0.2 mum diameter fluorescent microspheres. Significant mismatches between drug and perfused vasculature are caused by heterogeneities in tumor blood supply. We describe complex microscopic mTHPC gradients that reverse dramatically relative to the perfused vasculature with time after injection. This imaging technique can be applied to screen the dynamic intratumor distribution of other fluorescent photosensitizers and anticancer drugs.     

Photodynamic effects of mTHPC on human colon adenocarcinoma cells: photocytotoxicity,subcellular localization and apoptosis

The photodynamic properties of meta-tetra(hydroxyphenyl)chlorin (mTHPC), a promising second-generation photosensitizer, were investigated using a human colon adenocarcinoma cell line (Colo 201 cells). The study on photocytotoxicity using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay showed that mTHPC was an effective photosensitizer on Colo 201 cells. The photocytotoxicity of mTHPC showed both drug and light dose-dependent characteristics. To reach LD50, namely, the dose at which 50% of the cells were killed, only 0.45+/-0.15 microg/mL of mTHPC and 3 J/cm2 of light dose were required. The presence of 10% fetal calf serum in culture medium significantly decreased the incorporation of mTHPC into cells and resulted in the reduction of photodynamic efficacy. Using confocal laser scanning microscopy, mTHPC was first shown to localize in lysosomes rather than in mitochondria. Furthermore, nuclear stainings demonstrated that photodynamic therapy with mTHPC induced apoptosis in Colo 201 cells.     

Photodynamic Mechanisms induced by a Combination of Hypericin and a Chlorin Based‐Photosensitizer in Head and Neck Squamous Cell Carcinoma Cells

The aim of this study was to elucidate photodynamic therapy (PDT) effects mediated by hypericin and a liposomal meso‐tetrahydroxyphenyl chlorin (mTHPC) derivative, with focus on their 1:1 mixture, on head and neck squamous cell carcinoma cell lines. Absorption, excitation and photobleaching were monitored using fluorescence spectrometry, showing the same spectral patterns for the mixture as measured for single photosensitizers. In the mixture mTHPC showed a prolonged photo‐stability. Singlet oxygen yield for light‐activated mTHPC was Φ_Δ = 0.66, for hypericin Φ_Δ = 0.25 and for the mixture Φ_Δ = ~0.4. A linear increase of singlet oxygen yield for mTHPC and the mixture was found, whereas hypericin achieved saturation after 35 min. Reactive oxygen species fluorescence was only visible after hypericin and mixture‐induced PDT. Cell viability was also more affected with these two treatment options under the selected conditions. Examination of death pathways showed that hypericin‐mediated cell death was apoptotic, with mTHPC necrotic and the 1:1 mixture showed features of both. Changes in gene expression after PDT indicated strong up‐regulation of selected heat‐shock proteins. The application of photosensitizer mixtures with the features of reduced dark toxicity and combined apoptotic and necrotic cell death may be beneficial in clinical PDT. This will be the focus of our future investigations.     

Time-dependent biodistribution of tetra(m-hydroxyphenyl)chlorin and benzoporphyrin derivative monoacid ring A in the hamster model: comparative fluorescence microscopy study

The pharmacokinetics of the photosensitizer used play a key role in the understanding of the mechanism of photodynamic therapy-induced damage. Fluorescence microscopy was used to compare time-dependent biodistribution of tetra(m-hydroxyphenyl)chlorin (mTHPC) and benzoporphyrin derivative monoacid ring A (BPD-MA) in different hamster tissues, including an early, chemically induced, squamous cell carcinoma. Following injection of 0.5 mg/kg body weight of mTHPC and 2.0 mg/kg BPD-MA, groups of three animals were sacrificed at different time points and a series of fluorescence micrographs from different excised organs were analyzed. The highest fluorescence intensities of mTHPC were observed at 96 h for squamous epithelia and skin and at 48 h for smooth muscle. There is no real peak of BPD-MA fluorescence between 30 min and 3 h in the basal epithelial layers, fibroconnective tissue, muscles or blood vessels. At 4 h after injection, the fluorescence level of BPD-MA decreased and at 24 h it had returned to background level in all observed tissues. The significantly faster clearance of BPD-MA is the principal advantage as compared to mTHPC. However, similar localization patterns in different tissues with essentially vascular affinity represent a possible disadvantage for treating early malignancies with BPD-MA as compared to mTHPC, which is mainly localized in various epithelia. For both photosensitizers no significant selectivity between early squamous cell carcinoma and healthy mucosae is seen. Pharmacokinetic studies of different photosensitizers in an appropriate animal model are essential for selecting new-generation photosensitizers with the most favorable localization for photodynamic therapy of early malignancies in hollow organs.     

Pharmacokinetics and pharmacodynamics of tetra(m-hydroxyphenyl)chlorin in the hamster cheek pouch tumor model: comparison with clinical measurements

The pharmacokinetics (PK) of the photosensitizer tetra(m-hydroxyphenyl)chlorin (mTHPC) was measured by optical fiber-based light-induced fluorescence spectroscopy (LIFS) in the normal and tumoral cheek pouch mucosa of 29 Golden Syrian hamsters with chemically induced squamous cell carcinoma. Similar measurements were carried out on the normal oral cavity mucosa of five patients up to 30 days after injection. The drug doses were between 0.15 and 0.3 mg per kg of body weight (mg/kg), and the mTHPC fluorescence in the tissue was excited at 420 nm. The PK in both human and hamster exhibited similar behavior although the PK in the hamster mucosa was slightly delayed in comparison with that of its human counterpart. The mTHPC fluorescence signal of the hamster mucosa was smaller than that of the human mucosa by a factor of about 3 for the same injected drug dose. A linear correlation was found between the fluorescence signal and the mTHPC dose in the range from 0.075 to 0.5 mg/kg at times between 8 and 96 h after injection. No significant selectivity in mTHPC fluorescence between the tumoral and normal mucosa of the hamsters was found at any of the applied conditions. The sensitivity of the normal and tumoral hamster cheek pouch mucosa to mTHPC photodynamic therapy as a function of the light dose was determined by light irradiation at 650 nm and 150 mW/cm2, 4 days after the injection of a drug dose of 0.15 mg/kg. These results were compared with irradiations of the normal oral and normal and tumoral bronchial mucosa of 37 patients under the same conditions. The reaction to PDT of both types of human mucosae was considerably stronger than that of the hamster cheek pouch mucosa. The sensitivity to PDT became comparable between hamster and human mucosa when the drug dose for the hamster was increased to 0.5 mg/kg. A significant therapeutic selectivity between the normal and neoplastic hamster cheek pouch was observed. Less selectivity was found following irradiations of normal mucosa and early carcinomas in the human bronchi. The pharmacodynamic behavior of mTHPC was determined by test irradiations of the normal mucosa of hamsters and patients between 6 h and 8 days after injection of 0.5 and 0.15 mg/kg in the hamsters and the patients, respectively. The normal hamster cheek pouch showed a maximum response to irradiation 6 h after injection and then decreased continuously to no observable reaction at 8 days after injection. The reaction of the normal human oral mucosa, however, showed an increasing sensitivity to the applied light between 6 h and 4 days after mTHPC injection and then decreased again at 8 days. The hamster model with the chemically induced early squamous cell cancer in the cheek pouch thus showed some similarity to the early squamous cell cancer of the human oral mucosa considering the PK. However, a quantitative difference in fluorescence signal for identical mTHPC doses as well as a significant difference in pharmacodynamic behavior were also observed. The suitability of this animal model for the optimization of PDT parameters in the clinic is therefore limited. Hence great care must be taken in screening new dyes for PDT of early squamous cell cancer of the upper aerodigestive tract based upon observables in the hamster cheek pouch model.     

Biodistribution and bioactivity of tetra-pegylated meta-tetra(hydroxyphenyl)chlorin compared to native meta-tetra(hydroxyphenyl)chlorin in a rat liver tumor model

It has been proposed that the construction of a photosensitizer-polymer conjugate would lead to an increased selective retention of the drug in tumor tissue resulting in an enhancement of selective tumor destruction by light in photodynamic therapy. In this study the kinetics of a tetra-pegylated derivative of meta-tetra(hydroxyphenyl)chlorin (mTHPC-PEG) were compared with those of native meta-tetra(hydroxyphenyl)chlorin (mTHPC) in a rat liver tumor model. In addition, the time course of bioactivity of both drugs was studied in normal liver tissue. Pegylation of mTHPC resulted in a two-fold increase in the plasma half-life time, a five-fold decrease in liver uptake and an increase in the tumor selectivity at early time intervals after drug administration. However, although mTHPC concentrations in liver decrease rapidly with time, mTHPC-PEG liver concentrations increased as a function of time. This led to a loss of tumor selectivity at all but the earliest time points, whereas with mTHPC tumor selectivity increased with time. For both drugs the time course of bioactivity in the liver parallels drug concentration levels with extensive necrosis after irradiation of mTHPC-PEG-sensitized liver tissue up to drug-light intervals of 120 h. It is concluded that on balance mTHPC-PEG does not appear to show any benefits over native mTHPC for the treatment of liver tumors, as normal liver tissue accumulates the compound. However, pegylation is a potentially promising strategy with an increase in tumor selectivity and reduced liver uptake if accumulation in the liver can be prevented.     

Unusual Photoinduced Response of mTHPC Liposomal Formulation (Foslip)

Liposomal formulations of meso-tetra(hydroxyphenyl)chlorin (mTHPC) have already been proposed with the aim to optimize photodynamic therapy. Spectral modifications of these compounds upon irradiation have not yet been investigated. The objective of this study was to evaluate photobleaching properties of mTHPC encapsulated into dipalmitoylphosphatidylcholine (DPPC) liposomes, Foslip. Fluorescence measurements in DPPC liposomes with different DPPC:mTHPC ratios demonstrated a dramatic decrease in fluorescence anisotropy with increasing local mTHPC concentration, thus suggesting strong interactions between mTHPC molecules in lipid bulk medium. Exposure of Foslip suspensions to small light doses (<50 mJ/cm²) resulted in a substantial drop in fluorescence, which, however, was restored after addition to the sample of a non-ionic surfactant Triton X-100. We attributed this behavior to photoinduced fluorescence quenching. This effect depended strongly on the molar DPPC:mTHPC ratio and was revealed only for high local mTHPC concentrations. The results were interpreted supposing energy migration between closely located mTHPC molecules with its subsequent dissipation by the molecules of photoproduct acting as excitation energy traps. We further assessed the effect of photoinduced quenching in plasma protein solution. Relatively slow kinetics of photoinduced Foslip response during incubation in the presence of proteins was attributed to mTHPC redistribution from liposomal formulations to proteins. Therefore, changes in mTHPC distribution pattern in biological systems would be consistent with changes in photoinduced quenching and would provide valuable information on mTHPC interactions with a biological environment.     

An Assay for the Quantitation of Photofrin in Tissues and Fluids

A method for determining the concentration of Photofrin in tissues and biological fluids was developed. The procedure is based on the dissolution of biological material with Solvable a commercially available tissue so-lubilizer, followed by porphyrin-specific fluorescence detection and measurement. It was found necessary to use a quadratic standard curve for the estimation of unknown Photofrin concentrations. While this method is limited to compounds that are stable in strong base, it has the advantages of being sensitive, rapid and low cost .     

Localization of tetra(m-hydroxyphenyl)chlorin (Foscan) in human healthy tissues and squamous cell carcinomas of the upper aero-digestive tract, the esophagus and the bronchi: a fluorescence microscopy study

To date, little is known about precise time-dependent distribution and histological localization of tetra(m-hydroxyphenyl)chlorin (mTHPC) in human healthy tissues and squamous cell malignancies in the upper aero-digestive tract. A fluorescence microscopy study was performed on 50 healthy tissue biopsies and on 13 tumors (graded from Tis to T1 SCC) from 30 patients. Tissue samples were taken between 4 h and 11 days following injection of 0.15 mg/kg mTHPC. A fairly comparable distribution pattern in various tissues was observed over time in different patients. Vascular localization of mTHPC fluorescence predominates at a short delay, whereas the dye is essentially located in the tumoral and healthy mucosa after longer delays. A much lower uptake and retention of mTHPC fluorescence was noted in striated muscle and cartilage as compared to neoplastic lesions. No significant selectivity was found between healthy and tumoral mucosa. The obtained data are important to confirm drug-light interval that have been selected for effective PDT for early SCC malignancies while minimizing the risks of over- or under-treatment. The low fluorescence level in striated muscle provides the opportunity to develop interstitial PDT as a treatment modality for invasive SCC of unfavorable locations in the oral cavity or pharynx, such as the base of the tongue.     

Determination of the enantiomeric composition of phenylalanine samples by chemometric analysis of the fluorescence spectra of cyclodextrin guest-host complexes

A novel strategy for determining the enantiomeric composition of phenylalanine samples that combines ordinary fluorescence spectroscopy, guest-host cyclodextrin chemistry, and multivariate regression modeling is investigated. Partial-least-squares regression (PLS-1) models were developed from fluorescence spectral data obtained with a series of samples containing cyclodextrin guest-host complexes of phenylalanine with different known enantiomeric compositions. The regression models were subsequently validated by determining the enantiomeric composition of a set of independently prepared phenylalanine samples. The ability of the models to correctly predict the enantiomeric compositions of future samples was evaluated in terms of the root-mean-square percent relative error (RMS%RE). The RMS%RE in the mol fraction of D-phenylalanine ranged from 1.3% to 3.0% when beta-cyclodextrin was used as the host molecule for different guest-host concentrations. The RMS%RE in the mol fraction of D-phenylalanine obtained in a similar validation study conducted with gamma-cyclodextrin ranged between 1.8% and 4.0% for different guest-host concentrations. Compared with previous studies done in absorption, fluorescence data were found to be more sensitive and the spectral differences observed as a function of enantiomeric composition were more uniformly spaced, making regression modeling more reliable. As a result, good regression models could be made at lower concentrations than were possible previously when absorption measurements were used.     

Standardless multicomponent qNMR analysis of compounds with overlapped resonances based on the combination of ICA and PULCON

A fast and reliable nuclear magnetic resonance (NMR) method for quantitative analysis of targeted compounds with overlapped signals in complex mixtures has been established. The method is based on the combination of chemometric treatment for spectra deconvolution and the PULCON principle (pulse length based concentration determination) for quantification. Independent component analysis (ICA) (mutual information least dependent component analysis (MILCA) algorithm) was applied for spectra deconvolution in up to six component mixtures with known composition. The resolved matrices (independent components, ICs and ICA scores) were used for identification of analytes, calculating their relative concentrations and absolute integral intensity of selected resonances. The absolute analyte concentrations in multicomponent mixtures and authentic samples were then calculated using the PULCON principle. Instead of conventional application of absolute integral intensity in case of undisturbed signals, the multiplication of resolved IC absolute integral and its relative concentration in the mixture for each component was used. Correction factors that are required for quantification and are unique for each analyte were also estimated. The proposed method was applied for analysis of up to five components in lemon and orange juice samples with recoveries between 90% and 111%. The total duration of analysis is approximately 45 min including measurements, spectra decomposition and quantification. The results demonstrated that the proposed method is a promising tool for rapid simultaneous quantification of up to six components in case of spectral overlap and the absence of reference materials. Copyright © 2015 John Wiley & Sons, Ltd.     

The binding characteristics and intracellular localization of temoporfin (mTHPC) in myeloid leukemia cells: phototoxicity and mitochondrial damage

The state of aggregation of the photosensitizer meso-tetrahydroxyphenylchlorin (mTHPC) in both cell free and intracellular environment was elucidated by comparing its absorption and excitation spectra. In methanol, mTHPC existed as monomers and strongly fluoresced. In aqueous solutions such as phosphate-buffered saline (PBS), mTHPC formed nonfluorescent aggregates. Some portion of mTHPC monomerized in the presence of 10% fetal calf serum PBS. In murine myeloid leukemia M1 and WEHI-3B (JCS) cells, cytoplasmic mTHPC were monomeric. By using organelle-specific fluorescent probes, it was found that mTHPC localized preferentially at the mitochondria and the perinuclear region. Photodynamic treatment of mTHPC-sensitized leukemia cells caused rapid appearance of the apoptogenic protein cytochrome c in the cytosol. Results from flow cytometric analysis showed that the release of cytochrome c was especially pronounced in JCS cells, and well correlated with the extent of apoptotic cell death as reported earlier. Electron microscopy revealed the loss of integrity of the mitochondrial membrane and the appearance of chromatin condensation as early as 1 h after light irradiation. We conclude that rapid release of cytochrome c from photodamaged mitochondria is responsible for the mTHPC-induced apoptosis in the myeloid leukemia JCS and M1 cells.     

Development of liposomes containing ethanol for skin delivery of temoporfin: Characterization and in vitro penetration studies

The aim of this study was to develop ethanol-containing (3.3–20%, w/v) liposomes loaded with temoporfin (mTHPC), which presents a highly hydrophobic photosensitizer with low percutaneous penetration, and to investigate their skin penetration enhancing effect. Characterization parameters of liposomes were measured by photon correlation spectroscopy, lamellarity was analyzed by cryo-electron microscopy and mTHPC-content in formulations was determined spectrofotometrically. In order to assess the stability of mTHPC–liposomes at 4 and 23 °C, at predetermined time intervals characterization parameters and mTHPC-content were measured. The in vitro skin penetration of mTHPC was investigated using human abdominal skin mounted in Franz cells. The results indicated that mTHPC–liposomes were of a small particle size, small polydispersity index, negative surface charge, unilamellar or oligolamellar, and of a spherical or oval shape. All liposomes were stable during 12 months’ storage at 4 °C. Increasing the amount of ethanol in mTHPC–liposomes the skin deposition of mTHPC increased also. Liposomes without ethanol delivered the lowest amount of mTHPC into the skin, while liposomes containing 20% ethanol showed the highest penetration enhancement. In conclusion, mTHPC–liposomes containing 20% ethanol could be a promising tool for delivering temoporfin to the skin, which would be beneficial for the photodynamic therapy of cutaneous malignant or non-malignant diseases.