A Comparative Analysis of Silicon Phthalocyanine Photosensitizers for in vivo Photodynamic Therapy of RIF-1 Tumors in C3H Mice

Photofrin® photodynamic therapy (PDT) has recently received FDA approval for the palliative treatment of to-tally and partially obstructing esophageal malignancies. However, there is a need for new PDT photosensitizers because Photofrin has a number of undesirable features. The purpose of this study was to evaluate the efficacy of four amine-bearing silicon phthalocyanines—Pc4, Pc10, Pc12 and Pc18—as potential PDT photosensitizers. Equimolar concentrations of these Pc were found to be highly effective at causing the regression of RIF-1 tumors trans-planted to C3H/HeN mice. The amount of Pc4 necessary to cause an equivalent amount of tumor regression in this model system was substantially less than the amount of Photofrin. The cutaneous phototoxicity of the silicon Pc photosensitizer was assessed by the utilization of the murine ear-swelling model. When C3H mice were exposed to 167 J/cm² of polychromatic visible light from a UVB-filtered solar simulator, which emitted UV radiation and visible light above 320 nm, the Pc produced little, if any, cutaneous photosensitivity. These results indicate that Pc4, Pc10, Pc12 and Pc18 are at least as effective as Photofrin in PDT protocols, while at the same time addressing many of the drawbacks of Photofrin.     

The immunosuppressive effects of phthalocyanine photodynamic therapy in mice are mediated by CD4+ and CD8+ T cells and can be adoptively transferred to naive recipients

Photodynamic therapy (PDT) is a promising treatment modality for malignant tumors but it is also immunosuppressive which may reduce its therapeutic efficacy. The purpose of our study was to elucidate the role of CD4+ and CD8+ T cells in PDT immunosuppression. Using silicon phthalocyanine 4 (Pc4) as photosensitizer, nontumor-bearing CD4 knockout (CD4-/-) mice and their wild type (WT) counterparts were subjected to Pc4-PDT in a manner identical to that used for tumor regression (1 cm spot size, 0.5 mg kg(-1) Pc4, 110 J cm(-2) light) to assess the effect of Pc4-PDT on cell-mediated immunity. There was a decrease in immunosuppression in CD4-/- mice compared with WT mice. We next examined the role of CD8+ T cells in Pc4-PDT-induced immunosuppression using CD8-/- mice following the same treatment regimen used for CD4-/- mice. Similar to CD4-/- mice, CD8-/- mice exhibited less immunosuppression than WT mice. Pc4-PDT-induced immunosuppression could be adoptively transferred with spleen cells from Pc4-PDT treated donor mice to syngenic naive recipients (P < 0.05) and was mediated primarily by T cells, although macrophages were also found to play a role. Procedures that limit PDT-induced immunosuppression but do not affect PDT-induced regression of tumors may prove superior to PDT alone in promoting long-term antitumor responses.     

Lysosomal signaling enhances mitochondria-mediated photodynamic therapy in A431 cancer cells: role of iron

In photodynamic therapy (PDT), light activates a photosensitizer added to a tissue, resulting in singlet oxygen formation and cell death. The photosensitizer phthalocyanine 4 (Pc 4) localizes primarily to mitochondrial membranes in cancer cells, resulting in mitochondria-mediated cell death. The aim of this study was to determine how lysosomes contribute to PDT-induced cell killing by mitochondria-targeted photosensitizers such as Pc 4. We monitored cell killing of A431 cells after Pc 4-PDT in the presence and absence of bafilomycin, an inhibitor of the vacuolar proton pump of lysosomes and endosomes. Bafilomycin was not toxic by itself, but greatly enhanced Pc 4-PDT-induced cell killing. To investigate whether iron loading of lysosomes affects bafilomycin-induced killing, cells were incubated with ammonium ferric citrate (30 μM) for 30 h prior to PDT. Ammonium ferric citrate enhanced Pc 4 plus bafilomycin-induced cell killing without having toxicity by itself. Iron chelators (desferrioxamine and starch-desferrioxamine) and the inhibitor of the mitochondrial calcium (and ferrous iron) uniporter, Ru360, protected against Pc 4 plus bafilomycin toxicity. These results support the conclusion that chelatable iron stored in the lysosomes enhances the efficacy of bafilomycin-mediated PDT and that lysosomal disruption augments PDT with Pc 4.     

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.     

Phthalocyanine 4 (Pc 4) photodynamic therapy of human OVCAR-3 tumor xenografts

Photodynamic therapy (PDT) is a cancer treatment modality utilizing a photosensitizer, light and oxygen. Photodynamic therapy with Photofrin has been approved by the U.S. Food and Drug Administration for treatment of advanced esophageal and early lung cancer. Because of certain drawbacks associated with the use of Photofrin, there is a need to identify new photosensitizers for human use. The photosensitizer Pc 4 (HOSiPc-OSi[CH3]2[CH2]3N[CH3]2) has yielded promising PDT effects in many in vitro and in vivo systems. The aim of this study was to assess the usefulness of Pc 4 as a PDT photosensitizer for a human tumor grown as a xenograft in athymic nude mice. The ovarian epithelial carcinoma (OVCAR-3) was heterotransplanted subcutaneously in athymic nude mice. Sixty mice bearing OVCAR-3 tumors (approximately 80-130 mm3) were divided into six groups of 10 animals each, three for controls and three for treatment. The Pc 4 was given by tail vein injection, and 48 h later a 1 cm area encompassing the tumor was irradiated with light from a diode laser coupled to a fiberoptic terminating in a microlens (lambda = 672 nm, 150 J/cm2, 150 mW/cm2). Tumors of control animals receiving no treatment, light alone or Pc 4 alone continued to grow. Of animals receiving 0.4 mg/kg Pc 4 and light, one (10%) had a complete response and was cured (no regrowth up to 90 days post-PDT), while all others (90%) had a partial response and were delayed in regrowth. Of animals receiving 0.6 mg/kg Pc 4 and light, eight (80%) had a complete response, and two of these were cured. Of animals receiving 1.0 mg/kg Pc 4 and light, six (60%) had a complete response, and two of these were cured. In additional experiments, tumors from animals treated with Pc 4 (1 mg/kg) and light were removed 15, 30, 60 and 180 min post-PDT, and from these tumors DNA and protein were extracted. Agarose gel electrophoresis revealed the presence of apoptotic DNA fragmentation as early as 15 min post-PDT. Western blotting showed the cleavage of the 116 kDa native poly(ADP-ribose) polymerase (PARP) into fragments of approximately 90 kDa, another indication of apoptosis, and the presence of p21/WAF1/CIP1 (p21) in all PDT-treated tumors. These changes did not occur in control tumors. Pc 4 appears to be an effective photosensitizer for PDT of human tumors grown as xenografts in nude mice. Early apoptosis, as revealed by PARP cleavage, DNA fragmentation and p21 overexpression, may be responsible for the excellent Pc 4-PDT response. Clinical trials of Pc 4-PDT are warranted.     

APOPTOSIS DURING PHOTODYNAMIC THERAPY-INDUCED ABLATION OF RIF-1 TUMORS IN C3H MICE: ELECTRON MICROSCOPIC, HISTOPATHOLOGIC AND BIOCHEMICAL EVIDENCE

Abstract Very little is known about the applicability of the metabolic and biochemical events observed in cell culture systems to in vivo tumor shrinkage following photodynamic therapy (PDT). The purpose of this study was to assess whether PDT induces apoptosis during tumor ablation in vivo . We treated radiation-induced fibrosarcoma (RIF-1) tumors grown in C3H/HeN mice with PDT employing three photosensitizers, Photofrin-II, chloroaluminum phthalocyanine tetrasulfonate, or Pc IV (a promising phthalocyanine developed in this laboratory). Each photosensitizer was injected intraperitoneally and 24 h later the tumors were irradiated with an appropriate wavelength of red light using an argon-pumped dye laser. During the course of tumor shrinkage, the tumors were removed at 1, 2, 4 and 10 h post-PDT for DNA fragmentation, histopathologic, and electron microscopic studies. Markers of apoptosis, viz . the ladder of nucleosome-size DNA fragments, increased apoptotic bodies, and condensation of chromatin material around the periphery of the nucleus, were evident in tumor tissue even 1 h post-PDT; the extent of these changes increased during the later stages of tumor ablation. No changes were observed in tumors given photosensitizer alone or irradiation alone. Our data suggest that the damage produced by in vivo PDT may activate endonucleolysis and chromatin condensation, and that apoptosis is an early event in tumor shrinkage following PDT.     

Photodynamic Therapy of Human Glioma (U87) in the Nude Rat

Abstract— We measured the response of normal brain and the human U87 glioma implanted in the brain of rats (n = 65) to photodynamic therapy (PDT) using Photofrin as the sensitizer. Normal brain and U87 tumor implanted within brain of athymic (nude) rats were subjected to PDT (12.5 mg/kg of Photofrin) at increasing optical energy doses (35 J/cm², 140 J/cm², 280 J/cm²) of 632 nm light. Photofrin concentration in tumor, brain adjacent to tumor and normal brain were measured in a separate population of rats. Twenty-four hours after PDT, the brains were removed, sectioned, stained with hematoxylin and eosin (H&E), and the volumes of the PDT-induced lesion measured. Photofrin concentration in tumor greatly exceeded that of normal brain and brain adjacent to tumor (>20×). Both normal brain and U87 tumor exhibited superficial tissue damage with PDT at 35 J/cm². However, both normal and tumor-implanted brain exhibited tissue damage with increasing optical dose. A heterogeneous pattern of pannecrosis along with a uniform volume of pannecrosis was detected in the tumor. In contrast, normal brain exhibited a uniform sharply demarcated volume of necrosis. Our data indicate that the U87 human brain tumor model and the normal brain in the athymic rat are sensitive to PDT and Photofrin with an optical dose-dependent response to treatment.     

Magnetic Resonance Imaging Evaluation of Photodynamic Therapy-Induced Hemorrhagic Necrosis in the Murine M1 Tumor Model

Abstract— Proton magnetic resonance imaging (MRI) and histological methods were used to evaluate photodynamic therapy (PDT)-induced hemorrhagic necrosis in the murine Ml tumor within 72 h of treatment of male DBA/2 mice. The effects of three photosensitizing drugs were investigated: Photofrin (n = 4), Zn (II) phthalocyanine (n = 7) and benzoporphyrin derivative monoacid ring A (n = 11). As noted in previous studies of PDT using MRI, MRI makes possible serial, noninvasive, in vivo observation of tissue response to PDT. Our serial study of MRI and histological data confirms that tumors responded in the same way to PDT treatment using the three photosensitizing drugs: vascular damage followed by hemorrhagic necrosis. Most importantly and unlike previous MRI studies of PDT, we used a very high field magnet that enhanced the effect of magnetic susceptibility on image signal when blood is processed by the body after PDT-induced hemorrhagic necrosis. This last finding demonstrates the utility of high field magnets and the importance of localized, serial experiments in future magnetic resonance studies of PDT.     

PHOTODYNAMIC THERAPY OF CHEMICALLY- AND ULTRAVIOLET B RADIATION-INDUCED MURINE SKIN PAPILLOMAS BY CHLOROALUMINUM PHTHALOCYANINE TETRASULFONATE

Photodynamic therapy (PDT) of cancer combines irradiation of tumors with visible light following selective uptake of the photosensitizer by the tumor cells. PhotofrinR-II (Pf-II) is the only photosensitizer which is in clinical use in PDT, whereas chloroaluminum phthalocyanine tetrasulfonate (AlPcTS) has also shown promise in preclinical studies. In most such studies, the effectiveness of the photosensitizers has been assessed in implanted tumor model systems rather than in model systems where tumors are allowed to grow in their own connective tissue matrix. In this study the pharmacokinetics, tumor ablation capability and cutaneous photosensitization response of AlPcTS have been assessed in mice bearing chemically- and ultraviolet B radiation (UVB)-induced benign skin papillomas. When tumor-bearing animals were injected intraperitoneally with AlPcTS (5 mg/kg body wt), maximum tumor:normal skin ratio of 2.4 was observed at 48 h, at which time the mice were irradiated within the absorption spectrum of the photosensitizer. In tumor ablation studies with SENCAR mice bearing chemically-induced skin tumors, AlPcTS resulted in greater than 80% ablation in tumor volume at 20 days post-irradiation. In cutaneous photosensitization response, AlPcTS produced only transient effects (no effect after 24 h) in SENCAR mice. Pharmacokinetics data, tumor ablation effects and cutaneous photosensitization response of AlPcTS were comparable in SKH-1 hairless mice bearing UVB-induced skin tumors. Our data indicate that AlPcTS produces significant photodynamic effects towards the ablation of murine skin tumors, and that it does not produce prolonged cutaneous photosensitivity.     

RESISTANCE TO PHOTODYNAMIC THERAPY IN RADIATION INDUCED FIBROSARCOMA-1 and CHINESE HAMSTER OVARY-MULTI-DRUG RESISTANT CELLS in vitro

A degree of resistance to photodynamic therapy (PDT) has been induced in radiation-induced fibrosarcoma-1 (RIF-1) tumor cells by repeated photodynamic treatment with Photofrin (4 or 18 h incubation) in vitro to the 0.1-1% survival level, followed by regrowth from single surviving colonies. The resistance is shown as increased cell survival in the strain designated RIF-8A, compared to the wild-type RIF-1 cells, when exposed to increasing Photofrin concentration for 18 h incubation and fixed light exposure. No difference was found between RIF-1 and RIF-8A in the uptake of Photofrin per unit cell volume at 18 h incubation. Resistance to PDT was also observed in Chinese hamster ovary-multi-drug resistant (CHO-MDR) cells compared to the wild-type CHO cells, possibly associated with decreased cellular concentration of Photofrin in the former. By contrast, the PDT-resistant RIF-8A cells did not show any cross-resistance to Adriamycin, nor was there any significant drug concentration difference between RIF-1 and RIF-8A. These findings suggest that different mechanisms are responsible for PDT-induced resistance and multi-drug resistance.     

Porphyrin and Nonporphyrin Photosensitizers in Oncology: Preclinical and Clinical Advances in Photodynamic Therapy

Photodynamic therapy (PDT) is now a well-recognized modality for the treatment of cancer. While PDT has developed progressively over the last century, great advances have been observed in the field in recent years. The concept of dual selectivity of PDT agents is now widely accepted due to the relative specificity and selectivity of PDT along with the absence of harmful side effects often encountered with chemotherapy or radiotherapy. Traditionally, porphyrin-based photosensitizers have dominated the PDT field but these first generation photosensitizers have several disadvantages, with poor light absorption and cutaneous photosensitivity being the predominant side effects. As a result, the requirement for new photosensitizers, including second generation porphyrins and porphyrin derivatives as well as third generation photosensitizers has arisen, with the aim of alleviating the problems encountered with first generation porphyrins and improving the efficacy of PDT. The investigation of nonporphyrin photosensitizers for the development of novel PDT agents has been considerably less extensive than porphyrin-based compounds; however, structural modification of nonporphyrin photosensitizers has allowed for manipulation of the photochemotherapeutic properties. The aim of this review is to provide an insight into PDT photosensitizers clinically approved for application in oncology, as well as those which show significant potential in ongoing preclinical studies.     

CHLORIN AND PORPHYRIN DERIVATIVES AS POTENTIAL PHOTOSENSITIZERS IN PHOTODYNAMIC THERAPY

In order to find a photosensitizer with better optical properties and pharmacokinetics than Photofrin II, a series of new photosensitizers related to methyl pheophorbide-a and chlorin-e6 were synthesized. These compounds absorb at substantially longer wavelengths (lambda max 660 nm) than does Photofrin II (630 nm) and show promise for use in photodynamic therapy. Among the porphyrins, we observed that long carbon chain ether derivatives are better photosensitizers than their ester analogs. These sensitizers were tested for in vivo photosensitizing activity vis-a-vis Photofrin II, using the standard screening system of DBA/2 mice bearing transplanted SMT/F tumors. Most of these photosensitizers were found to have better tumoricidal photosensitizing activity than Photofrin II and demonstrated more rapid attenuation of normal tissue photosensitivity with time after administration vis-a-vis Photofrin II.     

Induction of systemic neutrophil response in mice by photodynamic therapy of solid tumors.

Photodynamic therapy (PDT) of solid tumors elicits a strong, acute inflammatory response characterized by a rapid and massive infiltration of activated neutrophils into the tumor. The present study investigated the impact of PDT on the systemic and local (treatment site) kinetics of neutrophil trafficking and activity in mouse SCCVII and EMT6 tumor models. Differential leukocyte counts in the peripheral blood of treated mice revealed a pronounced neutrophilia developing rapidly after Photofrin porfimer sodium (Photofrin)- or tetra(m-tetrahydroxyphenyl)chlorin (mTHPC)-based PDT. Significant neutrophilia was also observed upon PDT treatment of normal dorsal skin but not on the footpad of tumor-free mice. The changes in circulating neutrophil numbers were accompanied by an efflux of these cells from the bone marrow. An increased proportion of cells with high L-selectin (CD62L antigen) expression was found among bone-marrow-residing neutrophils 6-24 h after PDT, and in neutrophils in the peripheral circulation and treated tumors 24 h after therapy. Complement inhibition completely prevented the development of PDT-induced neutrophilia. The results of the present study demonstrate that treatment of solid tumors by PDT induces a strong and protracted increase in systemic neutrophil numbers mediated by complement activation. This reaction reflects rapid and massive mobilization and activation of neutrophils for the destruction of PDT-treated tumor tissue.     

Characteristics of the immunosuppression induced by cutaneous photodynamic therapy: persistence, antigen specificity and cell type involved

Relatively little is known about the immunosuppression induced in mice which have received cutaneous photodynamic therapy (PDT). Consequently, experiments were undertaken using mice which received dorsal PDT using Photofrin as the photosensitizer in an attempt to characterize the overall nature of the immunosuppression. Photoirradiation of mice at various times after injection indicated there was no correlation between photosensitivity and immunosuppression. The suppression was found to be adoptively transferable and antigen specific suggesting the generation of suppressor cells. Selective cell depletions prior to adoptive transfer indicated a CD4+ T cell to be responsible for the immunosuppression. Interestingly, using allogeneic spleen cells, no effect on the delayed type hypersensitivity (DTH) response was found. The results indicate that the suppression induced by cutaneous PDT, with the exception of the lack of DTH suppression, is similar to that induced by UVB irradiation but unlike that reported using laser PDT of the peritoneal cavity. This suggests that not only the type of photoirradiation but also the site of photoirradiation might determine the character of the induced immunosuppression.     

Photodynamic Therapy of 9L Gliosarcoma with Liposome-Delivered Photofrin

Abstract— The effect of Photofrin encapsulated in a liposome delivery vehicle for photodynamic therapy (PDT) of the 9L gliosarcoma and normal rat brain was tested. We hypothesized that the liposome vehicle enhances therapeutic efficacy, possibly by increasing tumor tissue concentration of Photofrin. Male Fisher rats bearing a 9L gliosarcoma were treated 16 days after intracerebral tumor implantation with either Photofrin in dextrose (n = 5) or Photofrin in liposome (n = 6). Nontumor-bearing animals were treated with Photofrin delivered either in dextrose (n = 4) or liposome (n = 4) vehicle. Tissue concentrations of Photofrin delivered either in dextrose (n = 4) or liposome (n = 4) vehicle were measured in tumor, brain adjacent to tumor and in normal brain tissue. Photofrin was administered (intraperitoneally) at a dose of 12.5 mg/kg and PDT (17 J/cm₂ of 632 nm light at 100 mW/cm₂) was performed 24 h after Photofrin administration. Brains were removed 24 h after PDT and stained with hematoxylin and eosin for analysis of cellular damage. The PDT using Photofrin in the liposome vehicle caused significantly more damage to the tumor ( P < 0.001) than did PDT with Photofrin in dextrose. The PDT of tumor with Photofrin delivered in liposomes caused a 22% volume of cellular necrosis, while PDT of tumor with Photofrin delivered in dextrose caused only scattered cellular damage. Photofrin concentration in tumors was significantly higher ( P = 0.021) using liposome (33.8 ± 18.9 μg/g) compared to dextrose delivery (5.5 ± 1.5 μg/g). Normal brain was affected similarly in both groups, with only scattered cellular necrosis. Our data suggest that the liposome vehicle enhances the therapeutic efficacy of PDT treatment of 9L tumors.     

Quantitative analysis of Pc 4 localization in mouse lymphoma (LY-R) cells via double-label confocal fluorescence microscopy

Photodynamic therapy (PDT) is a novel cancer therapy that uses light-activated drugs (photosensitizers) to destroy tumor tissue. Reactive oxygen species produced during PDT are thought to cause the destruction of tumor tissue. However, the precise mechanism of PDT is not completely understood. To provide insight into the in vitro mechanisms of PDT, we studied the subcellular localization of the photosensitizer HOSiPcOSi(CH3)2-(CH2)3N(CH3)2 (Pc 4) in mouse lymphoma (LY-R) cells using double-label confocal fluorescence microscopy. This technique allowed us to observe the relative distributions of Pc 4 and an organelle-specific dye within the same cell via two, spectrally distinct, fluorescence images. To quantify the localization of Pc 4 within different organelles, linear correlation coefficients from the fluorescence data of Pc 4 and the organelle-specific dyes were calculated. Using this measurement, the subcellular spatial distributions of Pc 4 could be successfully monitored over an 18 h period. At early times (0-1 h) after introduction of Pc 4 to LY-R cells, the dye was found in the mitochondria, lysosomes and Golgi apparatus, as well as other cytoplasmic membranes, but not in the plasma membrane or the nucleus. Over the next 2 h, there was some loss of Pc 4 from the lysosomes as shown by the correlation coefficients. After an additional incubation period of 2 h Pc 4 slowly increased its accumulation in the lysosomes. The highest correlation coefficient (0.65) was for Pc 4 and BODIPY-FL C5 ceramide, which targets the Golgi apparatus, and also binds to other cytoplasmic membranes. The correlation coefficient was also high (0.60) for Pc 4 and a mitochondria-targeting dye (Mitotracker Green FM). Both of these correlation coefficients were higher than that for Pc 4 with the lysosome-targeting dye (Lysotracker Green DND-26). The results suggest that Pc 4 binds preferentially and strongly to mitochondria and Golgi complexes.     

The Relationship of Phthalocyanine 4 (Pc 4) Concentrations Measured Noninvasively to Outcome of Pc 4 Photodynamic Therapy in Mice

The ability to noninvasively measure photosensitizer concentration at target tissues will allow optimization of photodynamic therapy (PDT) and could improve outcome. In this study, we evaluated whether preirradiation tumor phthalocyanine 4 (Pc 4) concentrations, measured noninvasively by the optical pharmacokinetic system (OPS), correlated with tumor response to PDT. Mice bearing human breast cancer xenografts were treated with 2 mg kg⁻¹ Pc 4 iv only, laser irradiation (150 J cm⁻²) only, Pc 4 followed by fractionated irradiation or Pc 4 followed by continuous irradiation. Laser irradiation treatment was initiated when the tumor to skin ratio of Pc 4 concentration reached a maximum of 2.1 at 48 h after administration. Pc 4 concentrations in tumor, as well as in Intralipid in vitro , decreased monoexponentially with laser fluence. Pc 4-PDT resulted in significant tumor regression, and tumor response was similar in the groups receiving either fractionated or continuous irradiation treatment after Pc 4. Tumor growth delay following Pc 4-PDT correlated with OPS-measured tumor Pc 4 concentrations at 24 h prior to PDT ( R ² = 0.86). In excised tumors, OPS-measured Pc 4 concentrations were similar to the HPLC-measured concentrations. Thus, OPS measurements of photosensitizer concentrations can be used to assist in the scheduling of Pc 4-PDT.     

Immunosuppressive Effects of Silicon Phthalocyanine Photodynamic Therapy

The purpose of this study was to determine if silicon phthalocyanine 4 (Pc 4), a second-generation photosensitizer being evaluated for the photodynamic therapy (PDT) of solid tumors, was immunosuppressive. Mice treated with Pc 4 PDT 3 days before dinitrofluorobenzene sensitization showed significant suppression of their cell-mediated immune response when compared to mice that were not exposed to PDT. The response was dose dependent, required both Pc 4 and light and occurred at a skin site remote from that exposed to the laser. The immunosuppression could not be reversed by in vivo pre-treatment of mice with antibodies to tumor necrosis factor-alpha or interleukin-10. These results provide evidence that induction of cell-mediated immunity is suppressed after Pc 4 PDT. Strategies that prevent PDT-mediated immunosuppression may therefore enhance the efficacy of this therapeutic modality.     

A Requirement for Bid for Induction of Apoptosis by Photodynamic Therapy with a Lysosome- but Not a Mitochondrion-targeted Photosensitizer

Photodynamic therapy (PDT) with lysosome-targeted photosensitizers induces the intrinsic pathway of apoptosis via the cleavage and activation of the BH3-only protein Bid by proteolytic enzymes released from photodisrupted lysosomes. To investigate the role of Bid in apoptosis induction and the role of damaged lysosomes on cell killing by lysosome-targeted PDT, we compared the responses of wild type and Bid-knock-out murine embryonic fibroblasts toward a mitochondrion/endoplasmic reticulum-binding photosensitizer, Pc 4, and a lysosome-targeted sensitizer, Pc 181. Whereas apoptosis and overall cell killing were induced equally well by Pc 4-PDT in both cell lines, Bid^−/− cells were relatively resistant to induction of apoptosis and to overall killing following PDT with Pc 181, particularly at low PDT doses. Thus, Bid is critical for the induction of apoptosis caused by PDT with the lysosome-specific sensitizers, but dispensable for PDT targeted to other membranes.