Hyperoxygenation enhances the tumor cell killing of photofrin-mediated photodynamic therapy

Tumor hypoxia, either preexisting or as a result of oxygen depletion during photodynamic therapy (PDT) light irradiation, can significantly reduce the effectiveness of PDT-induced cell killing. To overcome tumor hypoxia and improve tumor cell killing, we propose using supplemental hyperoxygenation during Photofrin-PDT. The mechanism for the tumor cure enhancement of the hyperoxygenation-PDT combination is investigated using an in vivo-in vitro technique. A hypoxic tumor model was established by implanting mammary adenocarcinoma in the hind legs of mice. Light irradiation (200 J/cm2 at either 75 or 150 mW/cm2), under various oxygen supplemental conditions (room air, carbogen, 100% normobaric or hyperbaric oxygen), was delivered to animals that received 12.5 mg/kg Photofrin 24 h before light irradiation. Tumors were harvested at various time points after PDT and grown in vitro for colony formation analysis. Treated tumors were also analyzed histologically. The results show that when PDT is combined with hyperoxygenation, the hypoxic condition could be improved and the cell killing rate at various time points after PDT could be significantly enhanced over that without hyperoxygenation, suggesting an enhanced direct and indirect cell killing associated with high-concentration oxygen breathing. This study further confirms our earlier observation that when a PDT treatment is combined with hyperoxygenation it can be more effective in controlling hypoxic tumors.     

In vitro toxicity testing of zinc tetrasulfophthalocyanines in fibroblast and keratinocyte cells for the treatment of melanoma cancer by photodynamic therapy

A series of water-soluble tetrasulfonated metallophthalocyanines (MPcs) dyes have been studied to be used as a drug or photosensitizer (PS) in photodynamic therapy (PDT) for the treatment of cancers. During PDT the PS is administrated intravenously or topically to the patient before laser light at an appropriate wavelength is applied to the cancerous area to activate the PS. The activated PS will react with oxygen typically present in the cancerous tissue to generate reactive oxygen species for the destruction of the cancerous tissue. This in vitro study aimed at investigating the cytotoxic effects of different concentrations of zinc tetrasulfophthalocyanines (ZnTSPc) activated with a diode laser (λ = 672 nm) on melanoma, keratinocyte and fibroblast cells. To perform this study 3 × 10? cells/ml were seeded in 24-well plates and allowed to attach overnight, after which cells were treated with different concentrations of ZnTSPc. After 2h, cells were irradiated with a constant light dose of 4.5J/cm2. Post-irradiated cells were incubated for 24 h before cell viability was measured using the CellTiter-Blue Viability Assay. Data indicated high concentrations of ZnTSPc (60-100 μg/ml) in its inactive state are cytotoxic to the melanoma cancer cells. Also, results showed that photoactivated ZnTSPc (50 μg/ml) was able to reduce the cell viability of melanoma, fibroblast and keratinocyte cells to 61%, 81% and 83% respectively. At this photosensitizing concentration the efficacy the treatment light dose of 4.5J/cm2 against other light doses of 2.5J/cm2, 7.5J/cm2 and 10J/cm2 on the different cell lines were analyzed. ZnTSPc at a concentration of 50 μg/ml activated with a light dose of 4.5J/cm2 was the most efficient for the killing of melanoma cancer cells with reduced killing effects on healthy normal skin cells in comparison to the other treatment light doses. Melanoma cancer cells after PDT with a photosensitizing concentration of 50μg/ml and a treatment light dose of 4.5J/cm2 showed certain apoptosis characteristics such as chromatin condensation and fragmentation of the nucleus. This concludes that low concentrations of ZnTSPc activated with the appropriate light dose can be used to induce cell death in melanoma cells with the occurrence of minimal damage to surrounding healthy tissue.     

Potentiation of photodynamic therapy with hypericin by mitomycin C in the radiation-induced fibrosarcoma-1 mouse tumor model

Hypericin, a polycyclic quinone obtained from plants of the genus Hypericum, has been shown to be a promising photosensitizer. We investigated the combination of hypericin-photodynamic therapy (PDT) and a bioreductive drug mitomycin C (MMC) in the present study. The radiation-induced fibrosarcoma-1 tumors were exposed to laser light (120 J/cm2 at 595 nm) 24 h after an intravenous injection of hypericin (1 mg/kg). Hypericin-PDT alone significantly decreased tumor perfusion and oxygen tension as demonstrated by India ink staining technique and OxyLite pO2 measurement, respectively. The in vivo-in vitro cell-survival assay revealed about 60% direct tumor cell killing immediately after PDT. No significant delayed tumor cell death was observed after PDT, which suggests that vascular damage does not contribute significantly to the overall tumor cell death. Injection of a 2.5 mg/kg dose of MMC 20 min before light application significantly decreased tumor cell survival and delayed tumor growth compared with PDT or MMC alone. No greater skin reaction was observed after the combination of MMC and PDT than after PDT alone. Our study demonstrates that combining hypericin-PDT with MMC can be effective in enhancing tumor response with little side effect.     

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.     

Modeling of a type II photofrin-mediated photodynamic therapy process in a heterogeneous tissue phantom

We present a quantitative framework to model a Type II photodynamic therapy (PDT) process in the time domain in which a set of rate equations are solved to describe molecular reactions. Calculation of steady-state light distributions using a Monte Carlo method in a heterogeneous tissue phantom model demonstrates that the photon density differs significantly in a superficial tumor of only 3 mm thickness. The time dependences of the photosensitizer, oxygen and intracellular unoxidized receptor concentrations were obtained and monotonic decreases in the concentrations of the ground-state photosensitizer and receptor were observed. By defining respective decay times, we quantitatively studied the effects of photon density, drug dose and oxygen concentration on photobleaching and cytotoxicity of a photofrin-mediated PDT process. Comparison of the dependences of the receptor decay time on photon density and drug dose at different concentrations of oxygen clearly shows an oxygen threshold under which the receptor concentration remains constant or PDT exhibits no cytotoxicity. Furthermore, the dependence of the photosensitizer and receptor decay times on the drug dose and photon density suggests the possibility of PDT improvement by maximizing cytotoxicity in a tumor with optimized light and drug doses. We also discuss the utility of this model toward the understanding of clinical PDT treatment of chest wall recurrence of breast carcinoma.     

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.     

Strategies for enhanced photodynamic therapy effects

Photodynamic therapy (PDT) is a treatment modality for the selective destruction of cancerous and nonneoplastic pathologies that involves the simultaneous presence of light, oxygen and a light-activatable chemical called a photosensitizer (PS) to achieve a cytotoxic effect. The photophysics and mechanisms of cell killing by PDT have been extensively studied in recent years, and PDT has received regulatory approval for the treatment of a number of diseases worldwide. As the application of this treatment modality expands with regard to both anatomical sites and disease stages, it will be important to develop strategies for enhancing PDT outcomes. This article focuses on two broad approaches for PDT enhancement: (1) mechanism-based combination treatments in which PDT and a second modality can be designed to either increase the susceptibility of tumor cells to PDT or nullify the treatment outcome-mitigating molecular responses triggered by PDT of tumors, and (2) the more recent approaches of PS targeting, either by specific cellular function-sensitive linkages or via conjugation to macromolecules.     

Improvement of tumor response by manipulation of tumor oxygenation during photodynamic therapy

Photodynamic therapy (PDT) requires molecular oxygen during light irradiation to generate reactive oxygen species. Tumor hypoxia, either preexisting or induced by PDT, can severely hamper the effectiveness of PDT. Lowering the light irradiation dose rate or fractionating a light dose may improve cell kill of PDT-induced hypoxic cells but will have no effect on preexisting hypoxic cells. In this study hyperoxygenation technique was used during PDT to overcome hypoxia. C3H mice with transplanted mammary carcinoma tumors were injected with 12.5 mg/kg Photofrin and irradiated with 630 nm laser light 24 h later. Tumor oxygenation was manipulated by subjecting the animals to 3 atp (atmospheric pressure) hyperbaric oxygen or normobaric oxygen during PDT light irradiation. The results show a significant improvement in tumor response when PDT was delivered during hyperoxygenation. With hyperoxygenation up to 80% of treated tumors showed no regrowth after 60 days. In comparison, when animals breathed room air, only 20% of treated tumors did not regrow. To explore the effect of hyperoxygenation on tumor oxygenation, tumor partial oxygen pressure was measured with microelectrodes positioned in preexisting hypoxic regions before and during the PDT. The results show that hyperoxygenation may oxygenate preexisting hypoxic cells and compensate for oxygen depletion induced by PDT light irradiation. In conclusion, hyperoxygenation may provide effective ways to improve PDT efficiency by oxygenating both preexisting and treatment-induced cell hypoxia.     

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.     

Nonlinear dynamics of intracellular methylene blue during light activation of cell cultures

Methylene blue (MB+) is a well-known dye in medicine and has been discussed as an easily applicable drug for topical treatment in photodynamic therapy (PDT). Methylene blue can potentially be used as a redox indicator to detect the important redox reactions that are induced during PDT. The kinetics of this process was analyzed on a subcellular level with confocal laser scanning microscopy. BKEz-7 endothelial cells were incubated 4 h with 1 microM MB+. The fluorescence dynamics of MB+ during irradiation with 633 nm light was observed with subcellular resolution. Images were acquired at 0.5 s intervals (frame rate 1 image/0.5 s). Fluorescence was observed in the red channel of the laser scanning microscope. Synchronously, the phase-contrast image was visualized with the green channel. Morphological changes could therefore be correlated with the dynamics of MB+. In addition, the light-dose-dependent phototoxicity at 633 nm irradiation was determined by viable cell counting. After an induction period (phase I), fast fluorescent spikes could be observed in the whole cytoplasm, which decayed with a time constant of about 20 s (phase II), followed by a period of nearly constant fluorescence intensity (phase III) and exponential photobleaching (phase IV). Phase II exhibits highly nonlinear kinetics, which is hypothesized to correlate probably with a nonlinear quantal production of reactive oxygen species (ROS). Morphological cell changes were not observed during phase II. During phase III, a pycnotic cell nucleus developed. From the determination of viable cells we can conclude that a light dose applied within phase II was only sublethal in correlation with morphological observations. Overproduction of ROS leading finally to cell killing during phases III and IV is discussed.     

STRESS PROTEIN EXPRESSION IN MURINE TUMOR CELLS FOLLOWING PHOTODYNAMIC THERAPY WITH BENZOPORPHYRIN DERIVATIVE

Abstract— Photodynamic therapy (PDT) has been proven as a method of tumor eradication and is currently being used clinically to treat a wide variety of malignancies. Although it is understood that the interaction of light and sensitizer results in the production of potentially damaging oxygen species, the mechanism by which tumors are destroyed has yet to be defined fully. Using a new porphyrin sensitizer, benzoporphyrin derivative(BPD), we examined protein expression in murine tumor cells following treatment as an indication of molecular changes to target tissue concurrent with PDT-mediated damage. In order to assess the relevance of the results obtained using an in vitro PDT model, metabolic labeling of proteins synthesized subsequent to PDT was performed both in tumor cells grown and treated in tissue culture dishes and in cells explanted from PDT-treated solid tumors. We observed that the oxidative stress associated with PDT-resulted in the induction of a number or proteins corresponding to a set of heat-shock or stress proteins, and that the pattern of expression was similar when tumor cells were treated in vitro and in vivo . These results support the use of in vitro models in the dissection of the molecular erects of PDT and provide the foundation for future experiments that will examine the role of the immune system in tumor eradication by PDT.     

Porphyrin-bile acid conjugates: from saccharide recognition in the solution to the selective cancer cell fluorescence detection

This paper describes the preparation and use of conjugates of porphyrins and bile acids as ligands to bind to tumor expressed saccharides. Bile acid-porphyrin conjugates were tested for recognition of saccharides that are typically present on malignant tumor cells. Fluorescence microscopy, in vitro PDT cell killing, and PDT of subcutaneous 4T1 mouse tumors is reported. High selectivity for saccharide cancer markers and cancer cells was observed. This in vivo and in vitro study demonstrated high potential use for these compounds in targeted photodynamic therapy.     

Chemiluminescence as a PDT light source for microbial control

The photodynamic therapy (PDT) is a combination of using a photosensitizer agent, light and oxygen that can cause oxidative cellular damage. This technique is applied in several cases, including for microbial control. The most extensively studied light sources for this purpose are lasers and LED-based systems. Few studies treat alternative light sources based PDT. Sources which present flexibility, portability and economic advantages are of great interest. In this study, we evaluated the in vitro feasibility for the use of chemiluminescence as a PDT light source to induce Staphylococcus aureus reduction. The Photogem? concentration varied from 0 to 75 μg/ml and the illumination time varied from 60 min to 240 min.The long exposure time was necessary due to the low irradiance achieved with chemiluminescence reaction at μW/cm2 level. The results demonstrated an effective microbial reduction of around 98% for the highest photosensitizer concentration and light dose. These data suggest the potential use of chemiluminescence as a light source for PDT microbial control, with advantages in terms of flexibility, when compared with conventional sources.     

IMINIUM SALT OF COPPER BENZOCHLORIN (CDS1), A NOVEL PHOTOSENSITIZER FOR PHOTODYNAMIC THERAPY: MECHANISM OF CELL KILLING

The mechanism of cell killing by CDS1, an iminium salt of octaethylbenzochlorin with copper in the aromatic ring, in combination with light from a noncoherent light source was investigated. Using a standard clonogenic assay and the AY-27 FANFT turnor line. photoactivation of CDS1 was shown to be cytotoxic. The photodynamic cell killing ability of CDS1 required the presence of molecular oxygen. The reactive species generated by light activation of CDS1 were effectively quenched by N.N' -diphenyl- p -phenylenediamine. Additionally, the photodynamic effect of CDS1 was not cnhanced by dcuterium oxide. To characterize the reactive oxygen species generated by the photoactivation of CDS1 the well-characterized erythrocyte ghost model was used. Superoxide dismutase and catalase were potcnt inhibitors of CDS1-induced lipid peroxidation of erythrocyte membranes. Sodium azide only partially inhibited lipid peroxidation. These findings differed from the known singlet oxygen generator, tin (II) etiopurpurin dichloride (SnET₂). Sodium azide was a potent inhibitor of SnET₂-induced lipid peroxidation, whereas superoxide dismutase and catalase were totally ineffective. Based on these results, we conclude that CDS1 requires the presence of molecular oxygen for cell killing to occur but appcars to act primarily through a non-singlet oxygen mechanism.     

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.     

Scanning electrochemical microscopy (SECM) studies of oxygen transfer across phospholipid monolayers under surface pressure control: comparison of monolayers at air/water and oil/water interfaces

Scanning electrochemical microscopy has been used in combination with a specially designed Langmuir trough to compare the kinetics of oxygen transfer across an L-alpha-phosphatidylethanolamine, distearoyl monolayer spread at three different interfaces: air/water, air/water in contact with an oil lens, and oil/water. The monolayer is shown to reduce the kinetics of interfacial transport, and rate constants for the transport of oxygen across each interface, at different surface pressures, have been evaluated. The results obtained for each interface are compared, and the implications for studies of passive diffusion across cell membranes are discussed.     

Miconazole induces fungistasis and increases killing of Candida albicans subjected to photodynamic therapy

Cutaneous and mucocutaneous Candida infections are considered to be important targets for antimicrobial photodynamic therapy (PDT). Clinical application of antimicrobial PDT will require strategies that enhance microbial killing while minimizing damage to host tissue. Increasing the sensitivity of infectious agents to PDT will help achieve this goal. Our previous studies demonstrated that raising the level of oxidative stress in Candida by interfering with fungal respiration increased the efficiency of PDT. Therefore, we sought to identify compounds in clinical use that would augment the oxidative stress caused by PDT by contributing to reactive oxygen species (ROS) formation themselves. Based on the ability of the antifungal miconazole to induce ROS in Candida, we tested several azole antifungals for their ability to augment PDT in vitro. Although miconazole and ketoconazole both stimulated ROS production in Candida albicans, only miconazole enhanced the killing of C. albicans and induced prolonged fungistasis in organisms that survived PDT using the porphyrin TMP-1363 and the phenothiazine methylene blue as photosensitizers. The data suggest that miconazole could be used to increase the efficacy of PDT against C. albicans, and its mechanism of action is likely to be multifactorial.     

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.     

Monitoring photodynamic therapy of localized infections by bioluminescence imaging of genetically engineered bacteria

The increasing occurrence of multi-antibiotic resistant microbes has led to the search for alternative methods of killing pathogens and treating infections. Photodynamic therapy (PDT) uses the combination of non-toxic dyes and harmless visible light to produce reactive oxygen species that can kill mammalian and microbial cells. Although the photodynamic inactivation of bacteria has been known for over a hundred years, its use to treat infections has not been much developed. This may be partly due to the difficulty of monitoring the effectiveness of PDT in animal models of infection. In order to facilitate this monitoring process, we have developed a procedure that uses bioluminescent genetically engineered bacteria and a light sensitive imaging system to allow real-time visualization of infections. When these bacteria are treated with PDT in vitro, the loss of luminescence parallels the loss of colony-forming ability. We have developed several models of infections in wounds and soft-tissue abscesses in mice that can be followed by bioluminescence imaging. The size and intensity of the infection can be sequentially monitored in a non-invasive fashion in individual mice in real-time. When photosensitizers are introduced into the infected tissue followed by illumination with red light, a light-dose dependent loss of luminescence is seen. If the bacterium is invasive, the loss of luminescence correlates with increased survival of the mice, whilst animals in control groups die of sepsis within five days. Healing of the PDT treated wounds is not impaired and may actually be improved. This approach can allow many animal models of localized infections to be accurately monitored for efficacy of treatment by PDT.     

A Bifunctional Photosensitizer for Enhanced Fractional Photodynamic Therapy: Singlet Oxygen Generation in the Presence and Absence of Light

The photosensitized generation of singlet oxygen within tumor tissues during photodynamic therapy (PDT) is self‐limiting, as the already low oxygen concentrations within tumors is further diminished during the process. In certain applications, to minimize photoinduced hypoxia the light is introduced intermittently (fractional PDT) to allow time for the replenishment of cellular oxygen. This condition extends the time required for effective therapy. Herein, we demonstrated that a photosensitizer with an additional 2‐pyridone module for trapping singlet oxygen would be useful in fractional PDT. Thus, in the light cycle, the endoperoxide of 2‐pyridone is generated along with singlet oxygen. In the dark cycle, the endoperoxide undergoes thermal cycloreversion to produce singlet oxygen, regenerating the 2‐pyridone module. As a result, the photodynamic process can continue in the dark as well as in the light cycles. Cell‐culture studies validated this working principle in vitro.