Relationship between mTHPC fluorescence photobleaching and cell viability during in vitro photodynamic treatment of DP16 cells

An implicit dosimetric model has been proposed in which biological damage caused by photodynamic therapy (PDT) is monitored through the decrease in sensitizer fluorescence during treatment. To investigate this, in vitro experiments were performed in which DP16 cells were incubated in meta-tetra(hydroxyphenyl)chlorin (mTHPC) and then irradiated with 514 nm light. Photosensitizer concentration, fluence rate and oxygenation were independently controlled and monitored during the treatment. Fluorescence of mTHPC was continuously monitored via a charge-coupled device-coupled spectrometer during treatment and, at selected fluence levels, cell viability was determined using a trypan blue exclusion assay. The relationship of cell viability to normalized fluorescence was obtained for the different treatment conditions. The relationship was independent of cell medium oxygenation, treatment fluence rate and sensitizer incubation concentration except at a high mTHPC concentration (4 microg/mL). This relationship suggests that fluorescence bleaching may be used to predict mTHPC PDT damage in vitro.     

Effects of fluence rate on cell survival and photobleaching in meta-tetra-(hydroxyphenyl)chlorin-photosensitized Colo 26 multicell tumor spheroids

We report the influence of fluence rate on the photobleaching and cell survival in Colo 26 multicell spheroids photosensitized by meta-tetra-(hydroxyphenyl)chlorin (mTHPC). Photosensitizer degradation and therapeutic efficacy increased dramatically and progressively when the fluence rate was reduced over the range from 90 to 5 mW cm-2. These experimental results were compared to a mathematical model of photobleaching based on self-sensitized singlet oxygen reactions with the photosensitizer ground state. This model incorporates photophysical parameters obtained from microelectrode measurements of oxygen depletion at the surface of mTHPC-sensitized spheroids and was refined by including the inhomogeneous distribution of mTHPC in spheroids and oxygen depletion in the bulk medium. Since the model is consistent with the experimental data we conclude that the fluence rate dependence of the cell survival and of mTHPC photobleaching is due to photochemical oxygen consumption and a predominantly singlet oxygen-mediated mechanism of mTHPC photobleaching. The threshold dose of reacting singlet oxygen was calculated to be 7.9 +/- 2.2 mM in this system.     

Calculation of singlet oxygen dose using explicit and implicit dose metrics during benzoporphyrin derivative monoacid ring A (BPD-MA)-PDT in vitro and correlation with MLL cell survival

Photodynamic therapy (PDT) oxygen consumption, clonogenic cell survival, fluorescence photobleaching and photoproduct formation were investigated during benzoporphyrin derivative monoacid (BPD-MA)-PDT of MAT-LyLu cells in vitro. Cells were incubated with BPD-MA concentrations of 0.1, 0.5 or 2.5 μg mL(-1) for 2 h and then treated with 405 nm light under oxygenated and hypoxic conditions. Fluorescence spectra were acquired during treatment, and photobleaching and photoproduct generation were quantified using singular value decomposition of the spectra. Cell survival was measured at set times during the treatment using a colony-forming assay. The amount of oxygen consumed by PDT per photon absorbed decreased with BPD-MA intracellular concentration. Survival was correlated with the total amount of oxygen consumed by PDT per unit volume, which is assumed to be equivalent to the amount of singlet oxygen that reacted. A photobleaching-based singlet oxygen dose metric was also found to predict survival independent of intracellular BPD-MA concentration. The BPD-MA photoproduct was bleached during the treatment. Two singlet oxygen dose metrics based on photoproduct kinetics could not be correlated with cell survival over the full range of intracellular BPD-MA concentrations used.     

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.     

Photodynamic therapy for malignant mesothelioma: preclinical studies for optimization of treatment protocols

Effective photodynamic therapy (PDT) depends on the optimization of factors such as drug dose, drug-light interval, fluence rate and total light dose (or fluence). In addition sufficient oxygen has to be present for the photochemical reaction to occur. Oxygen deficits may arise during PDT if the photochemical reaction consumes oxygen more rapidly than it can be replenished, and this could limit the efficacy of PDT. In this study we investigated the influence of the drug-light interval, illumination-fluence rate and total fluence on PDT efficacy for the photosensitizer meta-tetrahydroxyphenylchlorin (mTHPC). The effect of increasing the oxygenation status of tumors during PDT was also investigated. PDT response was assessed from tumor-growth delay and from cures for human malignant mesothelioma xenografts grown in nude mice. Tumor-bearing mice were injected intravenously with 0.15 or 0.3 mg.kg-1 mTHPC, and after intervals of 24-120 h, the subcutaneous tumors were illuminated with laser light (652 nm) at fluence rates of 20, 100 or 200 mW.cm-2. Tumor response was strongly dependent on the drug-light interval. Illumination at 24 h after photosensitization was always significantly more effective than illumination at 72 or 120 h. For a drug-light interval of 24 h the tumor response increased with total fluence, but for longer drug-light intervals even high total fluences failed to produce a significant delay in tumor regrowth. No fluence-rate dependence of PDT response was demonstrated in these studies. Nicotinamide injection and carbogen breathing significantly increased tumor oxygenation and increased the tumor response for PDT schedules with illumination at 24 h after photosensitizer injection.     

Simple Photodynamic Therapy Dose Models Fail to Predict the Survival of MLL Cells After HPPH–PDT In Vitro

Fluorescence photobleaching, photodynamic therapy (PDT) oxygen consumption and clonogenic cell survival were investigated during 2-(1-hexyloxethyl)-2-devinyl pyropheophoribde-a (HPPH) PDT of MAT-LyLu cells in vitro . Cells were incubated with HPPH concentrations of 0.24, 1.2, 3.6 or 12 μ m for 4 h and then treated with 650 nm light under oxygenated and hypoxic conditions. Fluorescence spectra were acquired during treatment and photobleaching was quantified using singular value decomposition of the spectra. Cell survival was measured at set times during the treatment using a colony forming assay. Intracellular fluorescence lifetime measurements were also performed at each incubation concentration. The photobleaching kinetics did not follow first- or second-order kinetics and the fluorescence lifetime was similar for all intracellular concentrations. As the intracellular concentration of drug was increased, the amount of singlet oxygen and the absorbed quanta per cell required to achieve the same cell kill increased. Singlet oxygen dose was calculated using one- and two-compartment models of HPPH intracellular distribution. It was found that a two-compartment model, in which a PDT-sensitive binding site saturates at low concentrations, accounts for the observed photobleaching, oxygen consumption and cell survival.     

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.     

A Comparison of Dose Metrics to Predict Local Tumor Control for Photofrin‐mediated Photodynamic Therapy

This preclinical study examines light fluence, photodynamic therapy (PDT) dose and “apparent reacted singlet oxygen,” [¹O₂]_rx, to predict local control rate (LCR) for Photofrin‐mediated PDT of radiation‐induced fibrosarcoma (RIF) tumors. Mice bearing RIF tumors were treated with in‐air fluences (50–250 J cm^?2) and in‐air fluence rates (50–150 mW cm^?2) at Photofrin dosages of 5 and 15 mg kg^?1 and a drug‐light interval of 24 h using a 630‐nm, 1‐cm‐diameter collimated laser. A macroscopic model was used to calculate [¹O₂]_rx and PDT dose based on in vivo explicit dosimetry of the drug concentration, light fluence and tissue optical properties. PDT dose and [¹O₂]_rx were defined as a temporal integral of drug concentration and fluence rate, and singlet oxygen concentration consumed divided by the singlet oxygen lifetime, respectively. LCR was stratified for different dose metrics for 74 mice (66 + 8 control). Complete tumor control at 14 days was observed for [¹O₂]_rx ≥ 1.1 mm or PDT dose ≥1200 μm J cm^?2 but cannot be predicted with fluence alone. LCR increases with increasing [¹O₂]_rx and PDT dose but is not well correlated with fluence. Comparing dosimetric quantities, [¹O₂]_rx outperformed both PDT dose and fluence in predicting tumor response and correlating with LCR.     

Photophysical parameters, photosensitizer retention and tissue optical properties completely account for the higher photodynamic efficacy of meso-tetra-hydroxyphenyl-chlorin vs Photofrin

Meso-tetra-hydroxyphenyl-chlorin (mTHPC) is one of the most potent photosensitizers currently available for clinical photodynamic therapy (PDT). However the reason or reasons for its high photodynamic efficacy remain(s) unresolved. To investigate the PDT efficacy of mTHPC vs Photofrin we use the knowledge of photophysical parameters extracted from the analysis of oxygen electrode measurements in spheroids to compute and compare their respective singlet oxygen (1O2) dose depositions. The electrode measurements indirectly report the bleaching kinetics of mTHPC and indicate that its photobleaching mechanism is consistent with 1O2-mediated reactions. mTHPC's photodegradation via 1O2 reactions is confirmed by a more direct evaluation of the spatially resolved fluorescence in confocal sections of intact spheroids during irradiation. The PDT efficacy comparisons establish that mTHPC's enhanced potency may be accounted for completely on the basis of its ability to sequester tightly in cells and its photophysical properties, in particular its higher extinction coefficient at a redshifted wavelength. We extend the efficacy comparison to include the influence of hemoglobin absorption of PDT treatment light and show that incorporating the influence of wavelength-dependent light attenuation in tissue further contributes to significantly higher efficacy for mTHPC- vs Photofrin-PDT.     

Reactive Oxygen Species Explicit Dosimetry for Photofrin-mediated Pleural Photodynamic Therapy

Explicit dosimetry of treatment light fluence and implicit dosimetry of photosensitizer photobleaching are commonly used methods to guide dose delivery during clinical PDT. Tissue oxygen, however, is not routinely monitored intraoperatively even though it is one of the three major components of treatment. Quantitative information about in vivo tissue oxygenation during PDT is desirable, because it enables reactive oxygen species explicit dosimetry (ROSED) for prediction of treatment outcome based on PDT-induced changes in tumor oxygen level. Here, we demonstrate ROSED in a clinical setting, Photofrin-mediated pleural photodynamic therapy, by utilizing tumor blood flow information measured by diffuse correlation spectroscopy (DCS). A DCS contact probe was sutured to the pleural cavity wall after surgical resection of pleural mesothelioma tumor to monitor tissue blood flow (blood flow index) during intraoperative PDT treatment. Isotropic detectors were used to measure treatment light fluence and photosensitizer concentration. Blood-flow-derived tumor oxygen concentration, estimated by applying a preclinically determined conversion factor of 1.5 × 10⁹ μMs cm⁻² to the blood flow index, was used in the ROSED model to calculate the total reacted reactive oxygen species [ROS]rx. Seven patients and 12 different pleural sites were assessed and large inter- and intrapatient heterogeneities in [ROS]rx were observed although an identical light dose of 60 J cm⁻² was prescribed to all patients.     

The influence of oxygen depletion and photosensitizer triplet-state dynamics during photodynamic therapy on accurate singlet oxygen luminescence monitoring and analysis of treatment dose response

To date, singlet oxygen ((1)O(2)) luminescence (SOL) detection was predictive of photodynamic therapy (PDT) treatment responses both in vitro and in vivo, but accurate quantification is challenging. In particular, the early and strongest part of the time-resolved signal (500-2000ns) is difficult to separate from confounding sources of luminescence and system noise, and so is normally gated out. However, the signal dynamics change with oxygen depletion during PDT, so that this time gating biases the (1)O(2) measurements. Here, the impact of gating was investigated in detail, determining the rate constants from SOL and direct pO(2) measurements during meso-tetra(hydroxyphenyl)chlorin (mTHPC)-mediated PDT of cells in vitro under well-controlled conditions. With these data as input, numerical simulations were used to examine PDT and SOL dynamics, and the influence of various time gates on cumulative SOL signals. It is shown that gating can underestimate the SOL at early treatment time points by ～40% and underestimate the cumulative SOL signal by 20-25%, representing significant errors. In vitro studies with both mTHPC and aminolevulinic acid-photosensitizer protoporphyrin IX demonstrate that rigorous analysis of SOL signal kinetics is then crucial in order to use SOL as an accurate and quantitative PDT dose metric.     

Advanced Photosensitizer Activation Strategies for Smarter Photodynamic Therapy Beacons

Photodynamic therapy (PDT) is a clinical treatment in which a light‐absorbing drug called a photosensitizer (PS) is combined with light and molecular oxygen to generate cytotoxic singlet oxygen. PDT provides additional tissue selectivity compared to conventional chemotherapy as singlet oxygen is generated only in areas in which PS accumulates and that are simultaneously illuminated by a light source with sufficient irradiance and dose. Early PDT beacons built on this concept by adding an analyte‐responsive element that simultaneously turns on PDT and fluorescence, providing both an additional layer of selectivity and real‐time feedback of the PS′s activation state. More recent PDT beacons have expanded this idea, with new methods now available for sensing analytes, generating singlet oxygen, and reporting treatment status. In this Minireview, we consider developments in advanced activation strategies implemented in therapeutic and theranostic beacons.     

Fluorescence Photobleaching of ALA-induced Protoporphyrin IX during Photodynamic Therapy of Normal Hairless Mouse Skin: The Effect of Light Dose and Irradiance and the Resulting Biological Effect

The photobleaching of 5-aminolaevulinic acid (ALA)-induced protoporphyrin IX (PpIX) was investigated during superficial photodynamic therapy (PDT) in normal skin of the SKH HRt hairless mouse. The effects of light dose and fluence rate on the dynamics and magnitude of photobleaching and on the corresponding PDT-induced dam-age were examined. The results show that the PDT damage cannot be predicted by the total light dose. Photo-bleaching was monitored over a wide range of initial PpIX fluorescence intensities. The rate of PpIX photo-bleaching is not a simple function of fluence rate but is dependent on the initial concentration of sensitizer. Also, at high fluence rates (50–150 mW/cm², 514 nm) oxygen depletion is shown to have a significant effect. The rate of photobleaching with respect to light dose and the corresponding PDT damage both increase with decreasing fluence rate. We therefore suggest that the definition of a bleaching dose as the light dose that causes a 1/e reduction in fluorescence signal is insufficient to describe the dynamics of photobleaching and PDT-induced dam-age. We have detected the formation of PpIX photoproducts during the initial period of irradiation that were themselves subsequently photobleached. In the absence of oxygen, PpIX and its photoproducts are not photo-bleached. We present a method of calculating a therapeutic dose delivered during superficial PDT that demonstrates a strong correlation with PDT damage.     

In Vivo Fluence Rate Effect in Photodynamic Therapy of Early Cancers with Tetra(m-hydroxyphenyl)chlorin

Abstract— Several parameters affect clinical trials in photodynamic therapy and influence the therapeutic outcome. Beside drug dose, light dose, drug-light interval and other variables, the fluence rate is a parameter that can influence the therapeutic results. In this study we have evaluated the fluence rate effect with a second-generation photosensitizer, tetra( m -hydroxyphenyl)chlorin (mTHPC) using a 7,12-dimethylbenz(a)anthracene induced early squamous cell carcinoma of the Syrian hamster cheek pouch as a tumor model. Following injection of 0.5 mg/kg of mTHPC, irradiation tests were performed at two drug-light intervals, 4 and 8 days. Wavelength and light dose were adapted from those applied routinely in clinical trials. Irradiations at 652 nm were carried out with fluences ranging from 8 to 20 J/cm² delivered at fluence rates of 15 and 150 mW/cm². Similar tests were also performed at 514 nm with a fluence of 80 J/cm² delivered at fluence rates ranging from 25 to 125 mW/cm². At both wavelengths and drug-light intervals for a given fluence, the higher fluence rates resulted in less tissue damage in tumor and healthy mucosae. However, the lower fluence rates yielded slightly less therapeutic selectivity. This study confirms that the fluence rate is of major importance in clinical PDT.     

Modeling of oxygen transport and cell killing in type-II photodynamic therapy

Photodynamic therapy (PDT) provides an effective option for treatment of tumors and other diseases in superficial tissues and attracts attention for in vitro study with cells. In this study, we present a significantly improved model of in vitro cell killing through Type-II PDT for simulation of the molecular interactions and cell killing in time domain in the presence of oxygen transport within a spherical cell. The self-consistency of the approach is examined by determination of conditions for obtaining positive definitive solutions of molecular concentrations. Decay constants of photosensitizers and unoxidized receptors are extracted as the key indices of molecular kinetics with different oxygen diffusion constants and permeability at the cell membrane. By coupling the molecular kinetics to cell killing, we develop a modeling method of PDT cytotoxicity caused by singlet oxygen and obtain the cell survival ratio as a function of light fluence or initial photosensitizer concentration with different photon density or irradiance of incident light and other parameters of oxygen transport. The results show that the present model of Type-II PDT yields a powerful tool to quantitate various events underlying PDT at the molecular and cellular levels and to interpret experimental results of in vitro cell studies.     

Effect of photosensitizer dose on fluence rate responses to photodynamic therapy

Photodynamic therapy (PDT) regimens that conserve tumor oxygenation are typically more efficacious, but require longer treatment times. This makes them clinically unfavorable. In this report, the inverse pairing of fluence rate and photosensitizer dose is investigated as a means of controlling oxygen depletion and benefiting therapeutic response to PDT under conditions of constant treatment time. Studies were performed for Photofrin-PDT of radiation-induced fibrosarcoma tumors over fluence rate and drug dose ranges of 25-225 mW cm(-2) and 2.5-10 mg kg(-1), respectively, for 30 min of treatment. Tumor response was similar among all inverse regimens tested, and, in general, tumor hemoglobin oxygen saturation (SO2) was well conserved during PDT, although the highest fluence rate regimen (225 mWx2.5 mg) did lead to a modest but significant reduction in SO2. Regardless, significant direct tumor cell kill (>1 log) was detected during 225 mWx2.5 mg PDT, and minimal normal tissue toxicity was found. PDT effect on tumor oxygenation was highly associated with tumor response at 225 mWx2.5 mg, as well as in all other regimens tested. These data suggest that high fluence rate PDT can be carried out under oxygen-conserving, efficacious conditions at low photosensitizer dose. Clinical confirmation and application of these results will be possible through use of minimally invasive oxygen and photosensitizer monitoring technologies, which are currently under development.     

Photodynamic actinometry using microspheres: concept, development and responsivity

Photodynamic therapy (PDT) relies on three main ingredients, oxygen, light and photoactivating compounds, although the PDT response is definitively contingent on the site and level of reactive oxygen species (ROS) generation. This study describes the development of a novel, fluorescent-based actinometer microsphere system as a means of discerning spatially resolved dosimetry of total fluence and ROS production. Providing a high resolution, localized, in situ measurement of fluence and ROS generation is critical for developing in vivo PDT protocols. Alginate-poly-L-lysine-alginate microspheres were produced using ionotropic gelation of sodium alginate droplets, ranging from 80 to 200 microm in diameter, incorporating two dyes, ADS680WS (ADS) and Rhodophyta-phycoerythrin (RPE), attached to the spheres' inside and outside layers, respectively. To test the responsivity and dynamic range of RPE for ROS detection, the production of ROS was initiated either chemically using increasing concentrations of potassium perchromate or photochemically using aluminum tetrasulphonated phthalocyanine. The generation of singlet oxygen was confirmed by phosphorescence at 1270 nm. The resulting photodegradation and decrease in fluorescence of RPE was found to correlate with increased perchromate or PDT treatment fluence, respectively. This effect was independent of pH (6.5-8) and could be inhibited using sodium azide. RPE was not susceptible to photobleaching with light alone (670 nm; 150 Jcm(-2)). ADS, which absorbs light between 600 and 750 nm, showed a direct correlation between radiant exposure (670 nm; 0-100 Jcm(-2)) and diminished fluorescence. Photobleaching was independent of irradiance (10-40 mW cm(-2)). We propose that actinometer microspheres may provide a means for obtaining high spatial resolution information regarding delivered PDT dose within model systems during investigational PDT development and dosimetric information for clinical extracorporeal PDT as in the case of ex vivo bone marrow purging.     

Effect of meta-tetra(hydroxyphenyl)chlorin (mTHPC)-mediated photodynamic therapy on sensitive and multidrug-resistant human breast cancer cells

Meta-tetra(hydroxyphenyl)chlorin (mTHPC) is in clinical trials for the photodynamic therapy (PDT) of localized-stage cancer. The PDT susceptibility of cells expressing multidrug resistance (MDR) phenotype is an attractive possibility to overcome the resistance to cytotoxic drugs observed during cancer chemotherapy. The accumulation, photocytotoxicity and intracellular localization of mTHPC were examined using the doxorubicin selected MCF-7/DXR human breast cancer cells, expressing P-glycoprotein (P-gp), and the wild-type parental cell line, MCF-7. No significant difference in mTHPC accumulation was observed between the two cell lines up to 3 h contact. The photodynamic activity of mTHPC, measured 24 h after irradiation with red laser light (lambda=650 nm), was significantly greater in MCF-7/DXR as compared to MCF-7 cells. A light dose of 2.5 J cm(-2) inducing 50% of cytotoxicity in MCF-7, resulted in 85% cytotoxicity in MCF-7/DXR. The presence of P-gp inhibitors SDZ-PSC-833 and cyclosporin A did not modify the mTHPC-induced cytotoxicity. The difference in intracellular mTHPC distribution pattern between two cell lines may contribute to different photocytotoxicity. Our results indicate that mTHPC mediated PDT could be useful in killing cells expressing MDR phenotype.     

Activation of heat shock protein 70 promoter with meso-tetrahydroxyphenyl chlorin photodynamic therapy reported by green fluorescent protein in vitro and in vivo

Cellular responses to photodynamic therapy (PDT) include induction of heat shock proteins (HSP). We examined meso-tetrahydroxyphenyl chlorin (mTHPC) PDT-mediated HSP activation in EMT6 cells stably transfected with a plasmid containing the gene for green fluorescent protein (GFP) driven by an hsp70 promoter. mTHPC incubation induced concentration-dependent GFP expression. Irradiation of cells exposed to a sensitizer concentration that induced a slight increase in GFP and no loss of cell viability resulted in fluence-dependent GFP accumulation. In response to drug only and to PDT, GFP levels increased to a maximum of four- to five-fold above control levels with increasing drug or fluence and then decreased at higher doses. A trypan blue-exclusion assay confirmed that decreased GFP levels in both cases were due to a loss of cell viability. For initial evaluation in vivo, HSP70/ GFP-transfected EMT6 tumors were grown in BALB/c mice and subjected to mTHPC-PDT with a fluence of 1 J/cm2. Six hours after PDT, GFP fluorescence was imaged in these tumors through the intact skin in vivo. These results indicate that sublethal doses of mTHPC-PDT stimulate GFP expression under the control of an hsp70 promoter and illustrate the potential of noninvasively monitoring reporter protein fluorescence as a measure of molecular response to PDT.     

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.