PDT-induced Damage Research Articles

Inhibition of the HIF-1 Survival Pathway as a Strategy to Augment Photodynamic Therapy Efficacy.

Photodynamic therapy (PDT) is a non-to-minimally invasive treatment modality that utilizes photoactivatable drugs called photosensitizers to disrupt tumors with locally photoproduced reactive oxygen species (ROS). Photosensitizer activation by light results in hyperoxidative stress and subsequent tumor cell death, vascular shutdown and hypoxia, and an antitumor immune response. However, sublethally afflicted tumor cells initiate several survival mechanisms that account for decreased PDT efficacy. The hypoxia inducible factor 1 (HIF-1) pathway is one of the most effective cell survival pathways that contributes to cell recovery from PDT-induced damage. Several hundred target genes of the HIF-1 heterodimeric complex collectively mediate processes that are involved in tumor cell survival directly and indirectly (e.g., vascularization, glucose metabolism, proliferation, and metastasis). The broad spectrum of biological ramifications culminating from the activation of HIF-1 target genes reflects the importance of HIF-1 in the context of therapeutic recalcitrance. This chapter elaborates on the involvement of HIF-1 in cancer biology, the hypoxic response mechanisms, and the role of HIF-1 in PDT. An overview of inhibitors that either directly or indirectly impede HIF-1-mediated survival signaling is provided. The inhibitors may be used as pharmacological adjuvants in combination with PDT to augment therapeutic efficacy.

Role of Bcl-2 Family Proteins in Photodynamic Therapy Mediated Cell Survival and Regulation.

Photodynamic therapy (PDT) is a treatment modality that involves three components: combination of a photosensitizer, light and molecular oxygen that leads to localized formation of reactive oxygen species (ROS). The ROS generated from this promising therapeutic modality can be lethal to the cell and leads to consequential destruction of tumor cells. However, sometimes the ROS trigger a stress response survival mechanism that helps the cells to cope with PDT-induced damage, resulting in resistance to the treatment. One preferred mechanism of cell death induced by PDT is apoptosis, and B-cell lymphoma 2 (Bcl-2) family proteins have been described as a major determinant of life or death decision of the death pathways. Apoptosis is a cellular self-destruction mechanism to remove old cells through the biological event of tissue homeostasis. The Bcl-2 family proteins act as a critical mediator of a life–death decision of cells in maintaining tissue homeostasis. There are several reports that show cancer cells developing resistance due to the increased interaction of the pro-survival Bcl-2 family proteins. However, the key mechanisms leading to apoptosis evasion and drug resistance have not been adequately understood. Therefore, it is critical to understand the mechanisms of PDT resistance, as well as the Bcl-2 family proteins, to give more insight into the treatment outcomes. In this review, we describe the role of Bcl-2 gene family proteins’ interaction in response to disease progression and PDT-induced resistance mechanisms.

Lipophilicity of Bacteriochlorin-Based Photosensitizers as a Determinant for PDT Optimization through the Modulation of the Inflammatory Mediators.

Photodynamic therapy (PDT) augments the host antitumor immune response, but the role of the PDT effect on the tumor microenvironment in dependence on the type of photosensitizer and/or therapeutic protocols has not been clearly elucidated. We employed three bacteriochlorins (F2BOH, F2BMet and Cl2BHep) of different polarity that absorb near-infrared light (NIR) and generated a large amount of reactive oxygen species (ROS) to compare the PDT efficacy after various drug-to-light intervals: 15 min. (V-PDT), 3h (E-PDT) and 72h (C-PDT). We also performed the analysis of the molecular mechanisms of PDT crucial for the generation of the long-lasting antitumor immune response. PDT-induced damage affected the integrity of the host tissue and developed acute (protocol-dependent) local inflammation, which in turn led to the infiltration of neutrophils and macrophages. In order to further confirm this hypothesis, a number of proteins in the plasma of PDT-treated mice were identified. Among a wide range of cytokines (IL-6, IL-10, IL-13, IL-15, TNF-α, GM-CSF), chemokines (KC, MCP-1, MIP1α, MIP1β, MIP2) and growth factors (VEGF) released after PDT, an important role was assigned to IL-6. PDT protocols optimized for studied bacteriochlorins led to a significant increase in the survival rate of BALB/c mice bearing CT26 tumors, but each photosensitizer (PS) was more or less potent, depending on the applied DLI (15 min, 3 h or 72 h). Hydrophilic (F2BOH) and amphiphilic (F2BMet) PSs were equally effective in V-PDT (>80 cure rate). F2BMet was the most efficient in E-PDT (DLI = 3h), leading to a cure of 65 % of the animals. Finally, the most powerful PS in the C-PDT (DLI = 72 h) regimen turned out to be the most hydrophobic compound (Cl2BHep), allowing 100 % of treated animals to be cured at a light dose of only 45 J/cm2.

N-acetylcysteine protects against chorioretinal damage induced by photodynamic therapy for experimental choroidal neovascularization in a rat model

We explored the protective effects of N-acetylcysteine (NAC) on chorioretinal damage induced by photodynamic therapy (PDT) in an experimental rat model of choroidal neovascularization (CNV). Experimental CNV was induced by an argon laser in 24 Brown Norway rats 7 days prior to PDT. Commencing 1 day after CNV induction, 0.5 mL of NAC was orally administered daily to the NAC + group (12 rats), and 0.5 mL of normal saline to the NAC- group (12 rats). Diode laser treatment was delivered for 42 s (total energy, 25 J/cm2) to the left eye prior to verteporfin infusion (PDT-) and to the right eye 15-20 min after such infusion (PDT+). Fluorescein angiography was performed just prior to PDT and enucleation to evaluate fluorescein leakage and CNV closure. We compared the CNV thickness, PDT-induced apoptosis [evaluated via terminal dUTP nick-end labeling (TUNEL)], fluorescein angiographic data, and extents of immunohistofluorescent staining for cleaved caspase-3 and superoxide dismutase (SOD) between the two groups. Fourteen days after diode laser treatment, the CNV closure rate was significantly higher in the PDT-treated than the control group. However, the CNV closure rates did not differ significantly between the NAC- and NAC + groups. The TUNEL activity (a measure of PDT-induced apoptosis) of retinal cells was higher in the NAC-/PDT + than the NAC+/PDT + group at 1, 3, 7, and 14 days. The cleaved caspase-3 and SOD levels were higher in the NAC-/PDT + than the NAC+/PDT + group at 3 and 7 days. PDT triggers oxygen radical-induced injury to, and apoptosis in, the retina. NAC may reduce PDT-induced damage to the retina without compromising the therapeutic efficacy of CNV.

Galactodendritic phthalocyanine targets carbohydrate-binding proteins enhancing photodynamic therapy.

Photosensitizers (PSs) are of crucial importance in the effectiveness of photodynamic therapy (PDT) for cancer. Due to their high reactive oxygen species production and strong absorption in the wavelength range between 650 and 850 nm, where tissue light penetration is rather high, phthalocyanines (Pcs) have been studied as PSs of excellence. In this work, we report the evaluation of a phthalocyanine surrounded by a carbohydrate shell of sixteen galactose units distributed in a dendritic manner (PcGal16) as a new and efficient third generation PSs for PDT against two bladder cancer cell lines, HT-1376 and UM-UC-3. Here, we define the role of galacto-dendritic units in promoting the uptake of a Pc through interaction with GLUT1 and galectin-1. The photoactivation of PcGal16 induces cell death by generating oxidative stress. Although PDT with PcGal16 induces an increase on the activity of antioxidant enzymes immediately after PDT, bladder cancer cells are unable to recover from the PDT-induced damage effects for at least 72 h after treatment. PcGal16 co-localization with galectin-1 and GLUT1 and/or generation of oxidative stress after PcGal16 photoactivation induces changes in the levels of these proteins. Knockdown of galectin-1 and GLUT1, via small interfering RNA (siRNA), in bladder cancer cells decreases intracellular uptake and phototoxicity of PcGal16. The results reported herein show PcGal16 as a promising therapeutic agent for the treatment of bladder cancer, which is the fifth most common type of cancer with the highest rate of recurrence of any cancer.

Optimal Photosensitizers for Photodynamic Therapy of Infections Should Kill Bacteria but Spare Neutrophils

Photodynamic therapy (PDT) for localized microbial infections exerts its therapeutic effect both by direct bacterial killing and also by the bactericidal effects of host neutrophils stimulated by PDT. Therefore, PDT-induced damage to neutrophils must be minimized, while direct photoinactivation of bacteria is maintained to maximize the therapeutic efficacy of antimicrobial PDT in vivo. However, there has been no study in which the cytocidal effect of PDT on neutrophils was investigated. In this study, the cytocidal effects of PDT on neutrophils were evaluated using different antimicrobial photosensitizers to find suitable candidate photosensitizers for antimicrobial PDT. PDT on murine peripheral-blood neutrophils was performed in vitro using each photosensitizer at a concentration that exerted a maximum bactericidal effect on methicillin-resistant Staphylococcus aureus, and morphological alteration and viability of neutrophils were studied. Most neutrophils were viable (>80%) after PDT using toluidine blue-O (TB) or methylene blue (MB), while neutrophils showed morphological change and their viabilities were decreased (<70%) after PDT using other photosensitizers (erythrosine B, rose bengal, crystal violet, Photofrin, new methylene blue and Laserphyrin). These results suggest that PDT using TB or MB can preserve host neutrophils while exerting a significant therapeutic effect on in vivo localized microbial infection.

The molecular basis of prostate cancer cell escape from protoporphyrin IX-based photodynamic therapy

Abstract Introduction Photodynamic therapy (PDT) uses the combination of a photosensitizing drug and light to cause selective damage to solid tumors. For early or localized disease, PDT can be a curative therapy with many advantages over available alternatives. For more advanced or disseminated disease, curative therapy is usually not possible with current technologies of light application, nevertheless it can improve quality of life and lengthen survival. More recently, there has been increasing evidence that PDT can also induce systemic anti-tumor immunity, indicating that PDT can become a rational therapeutic option, even if not all tumor cells are primarily eliminated by PDT. One prerequisite to minimize tumor relapse in these settings is to elucidate how tumor cells can cope with the PDT-induced damage. Material and methods Here we have characterized the rescue response of human PC-3 prostate cancer cells exposed in vitro to sublethal PDT after 5-aminolevulinic acid-induced protoporphyrin IX sensitization at the transcriptome level using Affymetrix HG U133 Plus 2.0 oligonucleotide microarrays. Cells were irradiated with laser light at a wavelength of 635 nm adjusted to an irradiance of 100 mW/cm 2 with irradiations of 1.5 and 3 J/cm 2 . Results The early response was characterized by the up-regulation of early response genes like FOS , JUN , EGR1 , ATF3 , DUSP , heat shock protein genes as well as histone genes, therefore resembling the early response of tumor cells to high dose PDT but without signs of irreversible cell damage. Twenty-four hours after PDT the cells still express high level of early response genes but 235 additional probe sets/genes were now significantly up-regulated (≥3 x ) that were not up-regulated 4 h after PDT. The most prominently up-regulated genes belong to gene families encoding the aldo-keto reductases, fibroblast growth factors, and HSP40-related proteins. In terms of a possible anti-tumor immune response it is noteworthy that also a multitude of chemokine and interleukin genes were up-regulated by the tumor cells upon PDT. Most of them are involved in granulocyte attraction and activation and some are also important for angiogenesis. Conclusion In conclusion, the global molecular characterization of the rescue response to PDT of tumor cells indicates that PDT rather favors an anti-tumor immune response than tumor immune escape reactions. Therefore, combining PDT and immunotherapy seem to be an attractive direction for the establishment of novel multimodal tumor therapies.

5.09] The molecular basis of tumor cell escape from protoporphyrin IX-based photodynamic therapy

Objective Photodynamic therapy (PDT) uses a combination of a photosensitizing drug and light to cause selective damage to solid tumors. For early or localized disease, PDT can be a curative therapy with many advantages over available alternatives. For more advanced or disseminated disease curative therapy is usually not possible with current technologies of light application, however it can improve quality of life and lengthen survival. More recently there is increasing evidence that PDT can also induce systemic anti-tumor immunity, indicating that PDT could become a rational therapeutic option, even if not all tumor cells are primarily eliminated by PDT. One prerequisite to minimize tumor relapse in these settings is to elucidate how tumor cells can cope with the PDT-induced damage. Material and methods Here we have characterized the rescue response of human prostate cancer and glioblastoma cells exposed in vitro to sub-lethal PDT after 5-aminolevulinic acid-induced protoporphyrin IX sensitization at the transcriptome level using Affymetrix U133 2.0 oligonucleotide microarrays. Cells were irradiated with laser light at a wavelength of 635 nm adjusted to an irradiance of 100 mW/cm 2 with fluences varying between 0.5 and 3 J/cm 2 . Results The early response was characterized by the up-regulation of early response genes like FOS, JUN, EGR1, ATF3, DUSP, heat-shock protein genes as well as histone and metallothionein genes, therefore resembling the early response of tumor cells to high-dose PDT but without signs of irreversible cell damage. Twenty-four hours after PDT the cells still expressed high level of early response genes but 241 additional probe sets/genes were significantly up-regulated (5 x ) compared to those that were not up-regulated (⩽2 x ) 4 h after PDT. The most prominently up-regulated genes belong to gene families encoding the aldo-keto reductases, fibroblast growth factors and Hsp40-related proteins. In terms of a possible anti-tumor immune response it is noteworthy that also a multitude of chemokines and interleukins were up-regulated by the tumor cells upon PDT. Most of them are involved in granulocyte attraction and activation and some are also important for angiogenesis. Conclusion In conclusion, the global molecular characterization of the rescue response to PDT of tumor cells indicated that PDT rather favors an anti-tumor immune response than tumor immune escape reactions. Therefore combining PDT and immunotherapy seem to be an attractive direction for the establishment of novel multimodal tumor therapies.

Monitoring ALA‐induced PpIX Photodynamic Therapy in the Rat Esophagus Using Fluorescence and Reflectance Spectroscopy

The presence of phased protoporphyrin IX (PpIX) bleach kinetics has been shown to correlate with esophageal response to 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT) in animal models. Here we confirm the existence of phased PpIX photobleaching by increasing the temporal resolution of the fluorescence measurements using the therapeutic illumination and long wavelength fluorescence detection. Furthermore fluorescence differential pathlength spectroscopy (FDPS) was incorporated to provide information on the effects of PpIX and tissue oxygenation distribution on the PpIX bleach kinetics during illumination. ALA at a dose of 200 mg kg(-1) was orally administered to 15 rats, five rats served as control animals. PDT was performed at an in situ measured fluence rate of 75 mW cm(-2) using a total fluence of 54 J cm(-2). Forty-eight hours after PDT the esophagus was excised and histologically examined for PDT-induced damage. Fluence rate and PpIX photobleaching at 705 nm were monitored during therapeutic illumination with the same isotropic probe. A new method, FDPS, was used for superficial measurement on saturation, blood volume, scattering characteristics and PpIX fluorescence. Results showed two-phased PpIX photobleaching that was not related to a (systematic) change in esophageal oxygenation but was associated with an increase in average blood volume. PpIX fluorescence photobleaching measured using FDPS, in which fluorescence signals are only acquired from the superficial layers of the esophagus, showed lower rates of photobleaching and no distinct phases. No clear correlation between two-phased photobleaching and histologic tissue response was found. This study demonstrates the feasibility of measuring fluence rate, PpIX fluorescence and FDPS during PDT in the esophagus. We conclude that the spatial distribution of PpIX significantly influences the kinetics of photobleaching and that there is a complex interrelationship between the distribution of PpIX and the supply of oxygen to the illuminated tissue volume.

PEG-m-THPC-mediated Photodynamic Effects on Normal Rat Tissues¶

Photodynamic therapy (PDT) of malignancies uses light to activate a photosensitizer preferentially accumulated in cancer cells. The first pegylated photosensitizer, tetrakis-(m-methoxypolyethylene glycol) derivative of 7,8-dihydro-5,10,15,20-tetrakis(3-hydroxyphenyl)-21-23-[H]-porphyrin (PEG-m-THPC), was evaluated in non–tumor-bearing rats. The aim of this study was to assess the photodynamic threshold for damage and its sequelae in normal rat tissue. Thirty-five Fischer rats were sensitized with 3, 9 or 30 mg/kg body weight PEG-m-THPC. Colon, vagina and perineum were irradiated with laser light of 652 nm wavelength and an optical dose of 50, 150 or 450 J/cm fiber length. Temperature in the pelvis was measured during PDT. Three days following PDT the effect on skin, vagina, colon, striated muscle, connective tissue, nerves and blood vessels was assessed by histology. The healing of the above-mentioned tissues was assessed on two rats 3 and 8 weeks after PDT using 9 mg/kg PEG-m-THPC activated with 450 J/cm laser light. No dark toxicity was observed. PDT using 30 mg/kg PEG-m-THPC induced severe necrosis irrespective of the optical dose. Body weight of 9 or 3 mg/kg activated with less than 450 J/cm induced moderate or no damage. No substantial increase in body temperature was seen during PDT. Tissues with severe PDT-induced damage seem to have a good tendency to regenerate. We conclude that within the dose required for tumor treatment PEG-m-THPC is a safe photosensitizer with promising properties. PDT of the colon mucosa below 9 mg/kg PEG-m-THPC and 150 J/cm seems to be safe. All other tissues can be exposed to 9 mg/kg PEG-m-THPC activated with less than 450 J/cm laser light with little side effects.

Photodynamic therapy in combination with intravitreal triamcinolone for myopic choroidal neovascularization

Editor, Photodynamic therapy (PDT) using verteporfin has been shown to be an effective treatment of subfoveal choroidal neovascularization (CNV) due to age-related macular degeneration (AMD) and pathologic myopia. However, recent studies have suggested that PDT can induce collateral damage, especially to the choriocapillary layer (CCL) and retinal pigment epithelium (RPE), and that it paradoxically has angiogenic properties itself (Ruiz-Moreno & Montero 2003; Schmidt-Erfurth et al. 2003; Gelisken et al. 2004; Postelmans et al. 2004). In pathologic myopia, CCL and RPE may show progressive atrophy even without the presence of CNV. Therefore, especially in PDT of CNV due to pathologic myopia, the number of PDT sessions should be as few as possible. Intravitreal injections of triamcinolone acetonide (IVTA) have been shown to have angiostatic and antiexudative effects (Gillies et al. 2003; Jonas et al. 2003), and preliminary studies in AMD patients suggest that the combination of PDT and IVTA may reduce the number of PDT sessions necessary to occlude the neovascular complex and may be beneficial for patients who do not respond to PDT in the first sessions (Spaide et al. 2003; Rechtman et al. 2004). Theoretically, a combination of PDT and IVTA may be particularly advantageous in the treatment of myopic CNV, but this has not yet been reported. We report on a 35-year-old man with an anisometropic myopia (right eye − 4.5 D, left eye −17.0 D), who experienced the onset of metamorphopsia and a loss of visual acuity (VA) in the left eye from 0.7 to 0.3 due to subfoveal classic CNV (greatest linear dimension 600 µm). Visual acuity in the right eye was 1.0. He received PDT using verteporfin, which led to a rapid reduction of metamorphopsia and a slight increase in VA to 0.4. About 4 weeks later metamorphopsia recurred and VA dropped again. Fluorescein angiography showed reperfusion and leakage of the CNV. Altogether the subject received five PDT sessions at 10–12-week intervals with early postoperative temporary occlusion of the CNV, reduced metamorphopsia and VA increase, which were then followed by reperfusion of the CNV, increasing metamorphopsia and decreasing VA. During follow-up, ophthalmoscopy and fluorescein angiography revealed an increasing atrophy of the RPE and CCL, corresponding in size and shape to the PDT treatment spots. Visual acuity decreased to 0.08. At last, after giving informed consent, the patient received a sixth PDT session (at which point the greatest linear dimension of the CNV was 1800 µm), which was combined with an intravitreal injection of about 20 mg triamcinolone acetonide. The intravitreal injection was performed 2 weeks prior to the PDT; the injection technique has been described previously (Jonas et al. 2003). Over the 8 months since this last intervention, VA has stabilized at 0.125 in the absence of metamorphospia. Fluorescein angiography has shown staining of the lesion and a surrounding RPE window defect. Ophthalmoscopically and in optical coherence tomography no subretinal fluid has been detected. The lens shows signs of early posterior subcapsular cataract development. At present, PDT is the therapy of choice for subfoveal classic CNV due to myopia. However, its biological effects (Schmidt-Erfurth et al. 2003) may induce a lesion reperfusion and growth, and additional damage to the RPE and CCL may occur with increasing numbers of PDT sessions. Highly myopic patients are especially susceptible to this PDT-induced damage, which may override the beneficial effects of the treatment in either the short or the longterm. The present case report suggests that a combination therapy of PDT plus IVTA may be beneficial in myopic patients who do not respond to PDT alone. Furthermore, it suggests that the side-effects of PDT (in terms of the temporary increase in the expression of angiogenic growth factors and collateral damage to the RPE) may be reduced, and as a corollary, the number of re-treatments may be decreased by combining PDT with intravitreal triamcinolone acetonide. The possible benefits of a combination therapy may outweigh the risks of IVTA. Further studies are warranted.

Enhancing the therapeutic responsiveness of photodynamic therapy with the antiangiogenic agents SU5416 and SU6668 in murine nasopharyngeal carcinoma models

Photodynamic therapy (PDT) is a promising therapeutic modality using a tumor localizing photosensitizer and light to destroy tumor cells. A major limitation of PDT is tumor recurrence, which is partly due to neovascularization. The objective of the present study was to determine whether combination therapy with PDT and antiangiogenic agents (i.e. SU5416 and SU6668) would be more effective in controlling tumor recurrence in a mouse model of human CNE2 poorly differentiated nasopharyngeal carcinoma compared with PDT or antiangiogenic agents administered alone. Athymic mice bearing CNE2 tumor xenografts received daily i.p. injections of 20 mg/kg SU5416 or 100 mg/kg SU6668 for 28 consecutive days either alone or following a single hypericin-PDT treatment. Significant inhibition of CNE2 tumor growth was observed in all treatment groups. Differences in 4x tumor growth time, the number of mice with 4x tumor growth, tumor growth inhibition as well as the percent of mice surviving were not statistically significant among individual treatment groups. However, the number of mice with 4x tumor growth observed in SU6668 monotherapy and combined PDT and SU6668 treatment groups was significantly less than that in the control group (P<0.05 and 0.01, respectively). Moreover, compared with the control group, only the combined PDT and SU6668 treatment significantly extended survival of tumor-bearing host mice (P<0.05). The semiquantitative RT-PCR results showed that the expression of HIF-1alpha, VEGF, COX-2 and bFGF were increased in PDT-treated tumor samples collected 24 h post-PDT, suggesting that PDT-induced damage to tumor microvasculature and the resultant hypoxia upregulate the expression of certain proangiogenic factors. The effectiveness of PDT can be enhanced by antiangiogenic treatment with the synthetic RTK inhibitors. Of the two synthetic RTK inhibitors tested, SU6668 was more effective than SU5416 in enhancing tumor responsiveness to PDT.

DNA damages after SIM01 photodynamic treatment

Photodynamic therapy (PDT) is a new approach to cancer treatment for a variety of malignant tumors. A new photosensitizer, 2,3-dihydro-5,15-di(3,5-dihydroxyphenyl)porphyrin (SIM01), had been evaluated for its genotoxic effects on glioma cells (C6). Comet assay had been used to evaluate the potential genotoxic effect induced by SIM01 on the C6 cells. When SIM01 had been shown to be a powerful sensitizer no DNA strand break was detected in the absence of light. SIM01 localized in cytoplasm but not in the nucleus of the tumors cells, which supported the finding of undetectable DNA damage under darkness and low photodynamic dose. Cell exposure to 20Jcm(-2) after an incubation time of 2h with 0, 0.25, 0.5, 2 or 4μgmL(-1) induced less than 25% of cell death but significant Tail Moment changes. If DNA damage intensity increased according to SIM01 doses under light exposure, importance of repair seemed to increase proportionally to PDT-induced damage. Positive controls consisted of doxorubicin-treated C6 cells this mutagen being known to induce genetic damage. Whatever the conditions used SIM01 appeared to be less deleterious than doxorubicin. As the comet assay can not give us the certitude that no mutation, photoadducts or oxidative damage had been developed under light exposure this point will have to be verified with another mutagenicity assay. SIM01 appears to be safe from a mutagenic point of view something of importance as tumors of small volume in patients with a long lifespan are at first indicated for PDT.

Monitoring PDT-induced damage using spectrally resolved reflectance imaging of tissue oxygenation

Photodynamic therapy (PDT) with chlorin e 6 (Chl) was monitored in vivo using vital microscopy and Fourier transform spectral imaging (FT-SI). Mammary C26 colon carcinoma, implanted intradermally in a mouse, was irradiated at 650 nm with various radiant exposures, 3 h after administration of 5 mg/kg Chl. The photodynamic response (PDR) in the skin flap with tumor was expressed as microcirculation disturbances (thrombi formation, multiple embolizations, arteriolar occlusion and venous stasis) and, dependent on the radiant exposure, was transient or permanent. These biological manifestations were accompanied by a change in hemoglobin (Hb)/oxyhemoglobin (HbO 2) absorption spectra obtained in vivo by FT-SI. False-color mapping of hemoglobin oxygen saturation (OS) visualized the alteration of tissue oxygenation. The results demonstrate, for the first time, that FT-SI can serve as a sensitive non-invasive tool for OS monitoring of PDT effects.

Foscan®-based photodynamic treatment in vivo: correlation between efficacy and Foscan accumulation in tumor, plasma and leukocytes

The tumoricidal effect of Foscan-mediated photodynamic therapy may involve both vessel and tumor cell destruction. The relevant importance of each mechanism seems to be defined by the time interval between photosensitizer administration and illumination (drug-light interval, DLI). Short drug-light intervals favor vascular damage due to the preferential photosensitizer accumulation in the tumor vasculature, whereas long drug-light intervals trigger direct tumor cell damage due to the dye localization in the tumor. The purpose of this study was to investigate the influence of tumor, plasma and leukocyte concentrations of Foscan at different times after photosensitizer delivery on PDT response. Both pharmacokinetic and tumor-response studies were carried out in nude mice bearing s.c. Colo26 tumors. One to 96 h after i.v. injection of 0.5 mg/kg Foscan, animals were exposed to 10 J/cm(2) 652-nm light delivered at 30 mW/cm(2). Mean tumor regrowth time was determined for each schedule of treatment and correlated to Foscan distribution in the compartments of interest at the time of illumination. PDT efficacy was greatest for irradiations performed at 6 and 12 h post Foscan injection and limited at 96 h. Unlike tumor and plasma Foscan concentrations, photosensitizer accumulation in leukocytes exhibited a good correlation with PDT efficacy. The results suggest that leukocytes could play an important role in the mechanism of PDT-induced vascular damage either by being one of the main effector compartments or by better reflecting Foscan accumulation in endothelial cells compared to plasma. The prevalence of indirect damage was highlighted by the fact that PDT efficacy was not modified by the use of a higher fluence rate of irradiation (160 mW/cm(2)), which depleted intratumor oxygen and did not restrain PDT-induced cell toxicity.

Necrosis-like death with plasma membrane damage against cervical cancer cells by photodynamic therapy.

In order to elucidate the antitumor effect of photodynamic therapy (PDT) using the photosensitizing agent hematoporphyrin derivative (Photogem) and a diode laser, we evaluated the cell death of uterine cancer cell lines (CaSki, HT3, HeLa, and SKOV-3) and mice transplanted with TC-1 lung cancer cells. Morphological changes, MTT assay, flow cytometry, cytotoxicity, and tumor growth-inhibition study were evaluated at various time intervals after PDT. The results showed that the survival rates of each cell line decreased with time and dose-response after performing PDT. Also, PDT-induced damage of cancer cells was almost entirely confined to necrosis of the tumor cells in the early time courses. The irradiation of CaSki cells in the presence of Photogem induced plasma membrane disruption and cell shrinkage, indicating the plasma membrane as the main target for Photogem. In the experiment in vivo, the time courses of Photogem with irradiation showed significantly longer survival and a significantly smaller tumor size compared to those in the untreated control groups, and resorption of the tumor after PDT treatment was observed. Collectively, our results indicated that Photogem possesses tumor-specific affinity, and necrosis-like death with plasma membrane damage was postulated to be the principal mechanism of the antitumor effect of PDT using Photogem.

Photogem Induces Necrosis in Various Uterine Cervical Cancer Cell Lines by PDT

In order to elucidate the antitumor effect of photodynamic therapy (PDT), using a derivative of the photosensitizing agent hematoporphyrin (Photogem) and a diode laser, the cell death of uterine cancer cell lines (CaSki, HT3, HeLa, and SKOV-3), and mice transplanted with TC-1 lung cancer cells, were evaluated. The morphological changes, MTT assay, flow cytometry, cytotoxicity and tumor growth inhibition study were evaluated at various time intervals after the PDT. The results showed that the survival rates of each cell line decreased with time and dose response after performing the PDT. Also, the PDT-induced damage of cancer cells was almost entirely confined to necrosis of the tumor cells in the early time courses. The irradiation of CaSki cells in the presence of Photogem induced plasma membrane disruption and cell shrinkage, indicating the plasma membrane as the main target for Photogem. In the in vivo experiment, significantly longer survival and a significantly smaller tumor size were seen over the time courses of the Photogem with irradiation compared to the untreated control groups; resorption of the tumor was also observed after the PDT treatment. Collectively, our results indicated that Photogem possesses anti-tumor effects, and necrosis-like death, with plasma membrane damage, was postulated to be the principal mechanism of the antitumor effect of the PDT using Photogem.

Topical 5-aminolevulinic acid-photodynamic therapy of hairless mouse skin using two-fold illumination schemes: PpIX fluorescence kinetics, photobleaching and biological effect.

Light fractionation with dark periods of the order of hours has been shown to considerably increase the efficacy of 5-aminolevulinic acid-photodynamic therapy (ALA-PDT). Recent investigations have suggested that this increase may be due to the resynthesis of protoporphyrin IX (PpIX) during the dark period following the first illumination that is then utilized in the second light fraction. We have investigated the kinetics of PpIX fluorescence and PDT-induced damage during PDT in the normal skin of the SKH1 HR hairless mouse. A single illumination (514 nm), with light fluences of 5, 10 and 50 J cm-2 was performed 4 h after the application of 20% ALA, to determine the effect of PDT on the synthesis of PpIX. Results show that the kinetics of PpIX fluorescence after illumination are dependent on the fluence delivered; the resynthesis of PpIX is progressively inhibited following fluences above 10 J cm-2. In order to determine the influence of the PpIX fluorescence intensity at the time of the second illumination on the visual skin damage, 5 + 95 and 50 + 50 J cm-2 (when significantly less PpIX fluorescence is present before the second illumination), were delivered with a dark interval of 2 h between light fractions. Each scheme was compared to illumination with 100 J cm-2 in a single fraction delivered 4 or 6 h after the application of ALA. As we have shown previously greater skin damage results when an equal light fluence is delivered in two fractions. However, significantly more damage results when 5 J cm-2 is delivered in the first light fraction. Also, delivering 5 J cm-2 at 5 mW cm-2 + 95 J cm-2 at 50 mW cm-2 results in a reduction in visual skin damage from that obtained with 5 + 95 J cm-2 at 50 mW cm-2. A similar reduction in damage is observed if 5 + 45 J cm-2 are delivered at 50 mW cm-2. PpIX photoproducts are formed during illumination and subsequently photobleached. PpIX photoproducts do not dissipate in the 2 h dark interval between illuminations.

PEG-m-THPC-mediated photodynamic effects on normal rat tissues.

Photodynamic therapy (PDT) of malignancies uses light to activate a photosensitizer preferentially accumulated in cancer cells. The first pegylated photosensitizer, tetrakis-(m-methoxypolyethylene glycol) derivative of 7,8-dihydro-5,10,15,20-tetrakis(3-hydroxyphenyl)-21-23-[H]-porphyrin (PEG-m-THPC), was evaluated in non-tumor-bearing rats. The aim of this study was to assess the photodynamic threshold for damage and its sequelae in normal rat tissue. Thirty-five Fischer rats were sensitized with 3, 9 or 30 mg/kg body weight PEG-m-THPC. Colon, vagina and perineum were irradiated with laser light of 652 nm wavelength and an optical dose of 50, 150 or 450 J/cm fiber length. Temperature in the pelvis was measured during PDT. Three days following PDT the effect on skin, vagina, colon, striated muscle, connective tissue, nerves and blood vessels was assessed by histology. The healing of the above-mentioned tissues was assessed on two rats 3 and 8 weeks after PDT using 9 mg/kg PEG-m-THPC activated with 450 J/cm laser light. No dark toxicity was observed. PDT using 30 mg/kg PEG-m-THPC induced severe necrosis irrespective of the optical dose. Body weight of 9 or 3 mg/kg activated with less than 450 J/cm induced moderate or no damage. No substantial increase in body temperature was seen during PDT. Tissues with severe PDT-induced damage seem to have a good tendency to regenerate. We conclude that within the dose required for tumor treatment PEG-m-THPC is a safe photosensitizer with promising properties. PDT of the colon mucosa below 9 mg/kg PEG-m-THPC and 150 J/cm seems to be safe. All other tissues can be exposed to 9 mg/kg PEG-m-THPC activated with less than 450 J/cm laser light with little side effects.

Photodynamic Effectiveness and Vasoconstriction in Hairless Mouse Skin after Topical 5‐Aminolevulinic Acid and Single‐ or Two‐fold Illumination

Several options were investigated to increase the efficacy of photodynamic therapy (PDT) using protoporphyrin IX (PpIX) induced by topically applied 5-aminolevulinic acid (ALA). Hairless mice with normal skin or UVB-light-induced skin changes were used as a model. In the first part of the study animals were illuminated immediately (t = 4) or 6 h (t = 10, PpIX fluorescence maximum) after the end of a 4 h ALA application. A total incident light fluence of 100 J/cm2 (514.5 nm) was delivered at a fluence rate of 100 or 50 mW/cm2. The PDT-induced damage to normal skin was more severe after treatment at t = 10 than at t = 4. Illumination at 50 mW/cm2 caused significantly more visible damage than the same light fluence given at 100 mW/cm2. For UVB-illuminated skin, different intervals or fluence rates made no significant difference in the severity of damage, although some qualitative differences occurred. In situ fluence rate measurements during PDT indicated vasoconstriction almost immediately after the start of the illumination. A fluorescein exclusion assay after PDT demonstrated vasoconstriction that was more pronounced in UVB-treated skin than in normal skin. The second part of the study examined the effect of two illuminations. The first illumination bleaches the PpIX fluorescence. At the start of the second illumination, new PpIX had been formed. Light of 514.5 nm was delivered at 100 mW/cm2 to a total incident light fluence of 200 J/cm2 at t = 4 (single illumination) or 100 J/cm2 at t = 4 plus 100 J/cm2 at t = 10. There was no visual difference in skin damage between 100 and 200 J/cm2 single illumination. Two-fold illumination (100 + 100 J/cm2) caused significantly more skin damage, indicating a potentially successful option for increasing the efficacy of topical ALA-PDT.