Photodynamic treatment with fractionated light decreases production of reactive oxygen species and cytotoxicity in vitro via regeneration of glutathione

Photodynamic therapy removes unwanted or harmful cells by overproduction of reactive oxygen species (ROS). Fractionated light delivery in photodynamic therapy may enhance the photodynamic effect in tumor areas with insufficient blood supply by enabling the reoxygenation of the treated area. This study addresses the outcome of fractionated irradiation in an in vitro photodynamic treatment (PDT) system, where deoxygenation can be neglected. Our results show that fractionated irradiation with light/dark intervals of 45/60 s decreases ROS production and cytotoxicity of PDT. This effect can be reversed by addition of 1,3-bis-(2-chlorethyl)-1-nitrosurea (BCNU), an inhibitor of the glutathione reductase. We suggest that the dark intervals during irradiation allow the glutathione reductase to regenerate reduced glutathione (GSH), thereby rendering cells less susceptible to ROS produced by PDT compared with continuous irradiation. Our results could be of particular clinical importance for photodynamic therapy applied to well-oxygenated tumors.     

Effect and Mechanism of a New Photodynamic Therapy with Glycoconjugated Fullerene

When irradiated, fullerene efficiently generates reactive oxygen species (ROS) and is an attractive photosensitizer for photodynamic therapy (PDT). Ideally, photosensitizers for PDT should be water-soluble and tumor-specific. Because cancer cells endocytose glucose more effectively than normal cells, the characteristics of fullerene as a photosensitizer were improved by combining it with glucose. The cytotoxicity of PDT was studied in several cancer cell lines cultured with C₆₀-(Glc)1 (d -glucose residue pendant fullerene) and C₆₀-(6Glc)1 (a maltohexaose residue pendant fullerene) subsequently irradiated with UVA₁. PDT alone induced significant cytotoxicity. In contrast, PDT with the glycoconjugated fullerene exhibited no significant cytotoxicity against normal fibroblasts, indicating that PDT with these compounds targeted cancer cells. To investigate whether the effects of PDT with glycoconjugated fullerene were because of the generation of singlet oxygen (¹O₂), NaN₃ was added to cancer cells during irradiation. NaN₃ extensively blocked PDT-induced apoptosis, suggesting that PDT-induced cell death was a result of the generation of ¹O₂. Finally, to investigate the effect of PDT in vivo, melanoma-bearing mice were injected intratumorally with C₆₀-(Glc)1 and irradiated with UVA₁. PDT with C₆₀-(Glc)1 suppressed tumor growth. These findings indicate that PDT with glycoconjugated fullerene exhibits tumor-specific cytotoxicity both in vivo and in vitro via the induction of ¹O₂.     

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

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

Active oxygen intermediates in the degradation of hematoporphyrin derivative in tumor cells subjected to photodynamic therapy

Hematoporphyrin derivative (HPD), a sensitizer used in photodynamic therapy (PDT) of malignancies, is progressively destroyed during the treatment. Prior studies suggested that upon PDT the photobleaching of HPD in tumor tissues is largely mediated by self-sensitized singlet oxygen. However, little is known about the role of other reactive oxygen species (ROS). The main aim of this work was to clarify the significance of H2O2, superoxide (O2.(-)) and hydroxyl (OH.) radicals in bleaching of HPD in tumor cells subjected to PDT. Experiments were performed on Ehrlich ascites carcinoma (EAC) cells, which were loaded with HPD in PBS and then irradiated with red light at 630 nm in the same buffer. Studies showed that photosensitization of EAC cells by HPD led to the formation of significant amounts of H2O2, O2.(-) and OH., and that these ROS could be involved in the photobleaching of HPD during PDT. In fact, we found that addition of catalase (CAT, a scavenger H2O2), Cu/Zn-superoxide dismutase (Cu/Zn-SOD) and Tiron (scavengers of O2.(-)), Na-benzoate, mannitol and deferoxamine (scavengers of OH.) caused a substantial decrease in the rate of HPD photobleaching in EAC cells. In these cells, the inhibitory effects of Na-benzoate, mannitol and deferoxamine on the photodegradation of HPD correlated well with suppression of the OH. generation, a highly active oxidizer. In EAC cells, the glutathione redox cycle and CAT (scavengers of H2O2) as well as Cu/Zn-SOD was found to suppress the photoinduced degradation of HPD. It was also established that HPD can directly scavenge H2O2 and oxygen free radicals; in a phosphate buffer its second-order rate constants were measured as 5.51+/-0.32 x 10(3)M(-1)s(-1) (for the reaction with O2.(-)), 5.08+/-0.31 x 10(4)M(-1)s(-1) (for H2O2), and 3.44+/-0.08 x 10(10)M(-1)s(-1) (for OH.). Thus, our data suggest that OH. could be one of the main oxidants mediating the photobleaching behavior of HPD in malignancies. Studies showed that photoexcited moieties of HPD can oxidize cell proteins with the formation of protein peroxides (PPO), which currently are regarded as a new form of ROS. Model experiments suggest that PPO could also participate in bleaching of HPD in tumors treated with PDT. It was found that HPD may destroy in tumor cells after cessation of photoirradiation and that this event is largely mediated by the presence of H2O2, a precursor of OH(.).     

Oxygen dependence of two-photon activation of zinc and copper phthalocyanine tetrasulfonate in Jurkat cells

Abstract Photodynamic therapy (PDT), the use of light-activated drugs, is a promising treatment of cancer as well as several nonmalignant conditions. However, the efficacy of one-photon (1-gamma) PDT is limited by hypoxia, which can prevent the production of the cytotoxic singlet oxygen ((1)O(2)) species, leading to tumor resistance to PDT. To solve this problem, we propose an irradiation protocol based on a simultaneous, two-photon (2-gamma) excitation of the photosensitizer (Ps). Excitation of the Ps triplet state leads to an upper excited triplet state T(n) with distinct photochemical properties, which could inflict biologic damage independent of the presence of molecular oxygen. To determine the potential of a 2-gamma excitation process, Jurkat cells were incubated with zinc or copper phthalocyanine tetrasulfonate (ZnPcS(4) or CuPcS(4)). ZnPcS(4) is a potent (1)O(2) generator in 1-gamma PDT, while CuPcS(4) is inactive under these conditions. Jurkat cells incubated with either ZnPcS(4) or CuPcS(4) were exposed to a 670 nm continuous laser (1-gamma PDT), 532 nm pulsed-laser light (2-gamma PDT), or a combination of 532 and 670 nm (2-gamma PDT). The efficacy of ZnPcS(4) to photoinactivate the Jurkat cells decreased as the concentration of oxygen decreased for both the 1-gamma and 2-gamma protocols. In the case of CuPcS(4), cell phototoxicity was measured only following 2-gamma irradiation, and its efficacy also decreased at a lower oxygen concentration. Our results suggest that for CuPcS(4) the T(n) excited state can be populated after 2-gamma irradiation at 532 nm or the combination of 532 and 670 nm light. Dependency of phototoxicity upon aerobic conditions for both 1-gamma and 2-gamma PDT suggests that reactive oxygen species play an important role in 1-gamma and 2-gamma PDT.     

Simultaneous two-photon excitation of photofrin in relation to photodynamic therapy

Photodynamic therapy (PDT), the use of light-activated drugs (photosensitizers), is an emerging treatment modality for tumors as well as various nononcologic conditions. Single-photon (1-gamma) PDT is limited by low specificity of the photosensitizer, leading to damage to healthy tissue adjacent to the diseased target tissue. One solution is to use simultaneous two-photon (2-gamma) excitation with ultrafast pulses of near-IR light. Due to the nonlinear interaction mechanism, 2-gamma excitation with a focused beam is localized in three dimensions, allowing treatment volumes on the order of femtoliters. We propose that this will be valuable in PDT of age-related macular degeneration (AMD), which causes blindness due to abnormal choroidal neovasculature and which is currently treated by 1-gamma PDT. Here, Photofrin has been used as the photosensitizer to demonstrate proof-of-principle of 2-gamma killing of vascular endothelial cells in vitro. The 2-gamma absorption properties of Photofrin were investigated in the 750-900 nm excitation wavelength range. It was shown that 2-gamma excitation dominates over 1-gamma excitation above 800 nm. The 2-gamma absorption spectrum of Photofrin in the 800-900 nm excitation wavelength range was measured. The 2-gamma cross section decreased from about 10 GM (1 GM = 10(-50) cm4 s/photon) at 800 nm to 5 GM at 900 nm. Adherent YPEN-1 endothelial cells were then incubated with Photofrin for 24 h and then treated by PDT at 850 nm where the 1-gamma contribution was negligible. Cell death was monitored with the use of 2-gamma scanning laser microscopy. The light doses required for killing were high (6300 J cm(-2) for approximately 50% killing), but 2-gamma cytotoxicity was unequivocally demonstrated. Although Photofrin is, per se, not a good choice for 2-gamma PDT due to its low 2-gamma cross section, this work provides baseline data to guide the development of novel photosensitizers with much higher 2-gamma cross sections (>100 GM), which will be required for 2-gamma PDT of AMD (and other conditions) to be clinically practical.     

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

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

Radical induced quartet photosensitizers with high ~1O_2 production for in vivo cancer photodynamic therapy

Singlet oxygen(¹O₂) is a strong oxidant which plays important roles in photodynamic therapy(PDT). The exploitation of photosensitizers with high ¹O₂ production is crucial to improve PDT efficiency. In this study, a radical labeled quartet photosensitizer Cy-DENT is reported with high singlet oxygen quantum yield(Φ_Δ=32.3%) due to a radical enhanced inter-system crossing(ISC) process. After the introduction of 2,2,6,6-tetramethylpiperidinyloxy(TEMPO) radical, quartet state ⁴[R,T] of CyDENT could be formed to give an over 20-fold enhancement of singlet oxygen quantum yield compared to Cy-DEN(without TEMPO radical) under irradiation of near infrared(NIR) light. In addition, the ¹O_(2 )production is well controlled by varying the electron-donating ability of the terminal substituent group. Cy-DENT possesses good cell permeability and is localized in mitochondria. Under the irradiation of 700 nm light, Cy-DENT can produce high levels of ROS to destroy the mitochondria membrane potential and induce cell apoptosis. Through the encapsulation of PEG-SS-PCL micelle, Cy-DENT can be effectively delivered to tumors and suppresses the tumor growth after PDT treatment.     

Antioxidant inhibitors potentiate the cytotoxicity of photodynamic therapy

Photodynamic therapy (PDT) is an increasingly popular anticancer treatment that uses photosensitizer, light and tissue oxygen to generate cytotoxic reactive oxygen species (ROS) within illuminated cells. Acting to counteract ROS-mediated damage are various cellular antioxidant pathways. In this study, we combined PDT with specific antioxidant inhibitors to potentiate PDT cytotoxicity in MCF-7 cancer cells. We used disulphonated aluminium phthalocyanine photosensitizer plus various combinations of the antioxidant inhibitors: diethyl-dithiocarbamate (DDC, a Cu/Zn-SOD inhibitor), 2-methoxyestradiol (2-ME, a Mn-SOD inhibitor), l-buthionine sulfoximine (BSO, a glutathione synthesis inhibitor) and 3-amino-1,2,4-triazole (3-AT, a catalase inhibitor). BSO, singly or in combination with other antioxidant inhibitors, significantly potentiated PDT cytotoxicity, corresponding with increased ROS levels and apoptosis. The greatest potentiation of cell death over PDT alone was seen when cells were preincubated for 24 h with 300 μM BSO plus 10 mM 3-AT (1.62-fold potentiation) or 300 μM BSO plus 1 μM 2-ME (1.52-fold), or with a combination of all four inhibitors (300 μM BSO, 10 mM 3-AT, 1 μM 2-ME and 10 μM DDC: 1.4-fold). As many of these inhibitors have already been clinically tested, this work facilitates future in vivo studies.     

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

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

Differential effects of photofrin, 5-aminolevulinic acid and calphostin C on glioma cells

The invasive nature of malignant gliomas makes treatment by surgery alone extremely difficult. However, the preferential accumulation of photosensitisers in neoplastic tissues suggests photodynamic therapy (PDT) may be useful as an adjuvant therapy following tumour resection. In this study, the potential use of three different photosensitisers, namely Photofrin, 5-aminolevulinic acid (5-ALA) and calphostin C in the treatment of glioma was investigated. The uptake, cytotoxicity on U87 and GBM6840 glioma cell lines were determined by flow cytometry and MTT assay respectively. Their effect on glioma cell invasiveness was evaluated by (1) measuring the levels of matrix degradation enzymes matrix metalloproteinase (MMP)-2 and -9 using gelatin zymography, and (2) Matrigel invasion assay. The results showed that uptake of calphostin C reached saturation within 2 h, while Photofrin and 5-ALA induced protoporphyrin IX (PpIX) levels elevated steadily up to 24 h. Photocytotoxic effect on the two glioma cell lines was similar with LD50 at optimal uptake: 1 microg/mL Photofrin at 1.5 J/cm(2); 1 mM 5-ALA at 2 J/cm(2) and 100 nM calphostin C at 2 J/cm(2). The inhibition in cell proliferation after Photofrin treatment was similar for both cell lines, which correlated to more cells being arrested in the G0/G1 phase of the cell cycle (P<0.01). By contrast, U87 was more sensitive to calphostin C whereas GBM6840 was more susceptible to 5-ALA treatment. The ability of both cell lines to migrate through the Matrigel artificial basement membrane was significantly reduced after PDT (P<0.001). This might be due to a decreased production in MMP-2 and MMP-9, together with the reduction of adhesion molecule expression. Photofrin was most superior in inhibiting cell invasion and calphostin C was least effective in reducing adhesion molecule expression. Taken together, PDT could be useful in the treatment of gliomas but the choice of photosensitisers must be taken into consideration.     

DNA binding and photocleavage in vitro by new dirhodium(II) dppz complexes: correlation to cytotoxicity and photocytotoxicity

Two new dirhodium(II) complexes possessing the intercalating dppz ligand (dppz = dipyrido[3,2-a:2',3'-c]phenazine), cis-[Rh(2)(mu-O(2)CCH(3))(2)(dppz)(eta(1)-O(2)CCH(3))(CH(3)OH)](+) (1) and cis-[Rh(2)(mu-O(2)CCH(3))(2)(dppz)(2)](2+) (2), were synthesized and characterized as potential agents for photochemotherapy. Various techniques show that 1 binds to DNA through intercalation, although some aggregation of the complex on the DNA surface is also present. In contrast, 2 does not intercalate between the DNA bases; however, strong hypochromic behavior is observed in the presence of DNA, which can be attributed to intermolecular pi-stacking of 2 enhanced by the polyanion. The apparent DNA binding constants determined using optical titrations are compared to those from dialysis experiments. Both complexes photocleave pUC18 plasmid in vitro under irradiation with visible light (lambda(irr) >or= 395 nm, 15 min), resulting in the nicked, circular form. Greater photocleavage is observed for 1 relative to 2, which may be due to the ability of 1 to intercalate between the DNA bases. The cytotoxicity toward human skin cells (Hs-27) measured as the concentration at which 50% cell death is recorded, LC(50), was found to be 135 +/- 8 microM for 2 in the dark (30 min), which is significantly lower than those of 1 (LC(50) = 27 +/- 2 microM) and Rh(2)(O(2)CCH(3))(4) (LC(50) = 15 +/- 2 microM). Irradiation of cell cultures containing 1 and Rh(2)(O(2)CCH(3))(4) with visible light (400-700 nm, 30 min) has little effect on their cytotoxicity, with LC(50) values of 21 +/- 3 and 13 +/- 2 microM, respectively. Interestingly, a 3.4-fold increase in the toxicity of 2 is observed when the cell cultures are irradiated (400-700 nm, 30 min), resulting in LC(50) = 39 +/- 1 microM. The greater toxicity of 1 compared to 2 in the dark may be related to the ability of the former compound to intercalate between the DNA bases. The lower cytotoxicity of 2, together with its significantly greater photocytotoxicity, makes this complex a potential agent for photodynamic therapy (PDT). These results suggest that intercalation or strong DNA binding may not be a desirable property of a potential PDT agent.     

Porphyrin-fullerene C60 dyads with high ability to form photoinduced charge-separated state as novel sensitizers for photodynamic therapy

The photodynamic activities of a porphyrin-C60 dyad (P-C60) and its metal complex with Zn(II) (ZnP-C60) were compared with 5-(4-acetamidophenyl)-10,15,20-tris(4-methoxyphenyl)porphyrin (P), both in homogeneous medium-bearing photooxidizable substrates and in vitro on the Hep-2-human-larynx-carcinoma cell line. This study represents the first evaluation of dyads, with a high capacity to form a photoinduced charge-separated state, to act as agents to inactivate cells by photodynamic therapy (PDT). Absorption and fluorescence spectroscopic studies were performed in toluene and N,N-dimethylformamide (DMF). The emission of the porphyrin moiety in the dyads is strongly quenched by the attached fullerene C60 moiety. The singlet molecular oxygen, O2(1delta(g)), productions (phi(delta)) were determined using 9,10-dimethylanthracene (DMA). The values of phi(delta) were strongly dependent on the solvent's polarity. Comparable phi(delta) values were found for dyads and P in toluene, while O2(1delta(g)) production was significantly diminished for the dyads in DMF. In more polar solvent, the stabilization of charge-transfer state takes place, decreasing the efficiency of porphyrin triplet-state formation. Also, both dyads photosensitize the decomposition of L-tryptophan in DMF. In biological medium, no dark cytotoxicity was observed using sensitizer concentrations < or = 1 microM and 24 h of incubation. The uptake of sensitizers into Hep-2 was studied using 1 microM of sensitizer and different times of incubation. Under these conditions, a value of approximately 1.5 nmol/10(6) cells was found between 4 and 24 h of incubation. The cell survival after irradiation of the cells with visible light was dependent upon light-exposure level. A higher photocytotoxic effect was observed for P-C60, which inactivates 80% of cells after 15 min of irradiation. Moreover, both dyads keep a high photoactivity even under argon atmosphere. Thus, depending on the microenvironment where the sensitizer is localized, these compounds could produce biological photodamage through either an O2(1delta(g))-mediated photoreaction process or a free-radicals mechanism under low oxygen concentration. These results show that molecular dyads, which can form a photoinduced charge-separated state, are a promising model for phototherapeutic agents, with potential applications in cell inactivation by PDT.     

Analysis of pulmonary microvasculature changes after photodynamic therapy delivered to distant sites

Photodynamic therapy (PDT) can exert local damage by direct tumor cytotoxicity, by disruption of the microvasculature or by a combination of these effects. Although systemic effects after PDT of small tissue areas (< 1% total body surface area) are unlikely, treatment of larger areas may result in an accumulated effect leading to toxicity. Several investigators have described animal death after high dose PDT to tumors on the hind limb of animals and hypothesized that a toxic shock syndrome caused by vasoactive agents released after PDT is responsible. Because one of the most vulnerable organs to toxic shock injury is the lung, we studied the systemic effects of local PDT to this organ by intravital microscopy using a pulmonary window chamber. The PDT treatment conditions (25 mg/kg Photofrin, 24 h, 150 J/cm2 630 nm, maximum area 6.28 cm2) were chosen that produce systemic toxicity and lethality in rats. Adhesion of leukocytes in the lung was monitored in vivo using anti-CD-13-labeled microspheres. The progression of pulmonary edema was assessed by monitoring the leakage of rhodamine-labeled albumin and by wet-to-dry lung weight ratios. Although an increased leukocyte adherence was observed and a significant number of animals died after the extensive PDT treatment, no biologically significant lung edema could be demonstrated. These data indicate that lung edema and acute respiratory distress syndrome is not the cause of death in these animals and that the toxicity is related to other mechanisms including circulatory shock after extensive muscle damage.     

PHOTODYNAMIC THERAPY OF A MURINE TUMOR FOLLOWING SENSITISATION WITH CHLORO ALUMINUM SULFONATED PHTHALOCYANINE

Abstract Syngeneic mice bearing a colorectal carcinoma (Colo 26) growing subcutaneously in the flank region received photodynamic therapy (PDT) when the tumor was 7-12 mm diameter. Light (emission at 675 nm from an argon ion pumped dye laser, laser energy 100 J, power 50 mW) was delivered to the tumor 24-28 h after the i.v. injection of a single dose of chloro aluminum sulfonated phthalocyanine (ClAlSPc) at 5 mg kg¹ body weight. Control tumor-bearing animals received (1) phosphate buffered saline (PBS) but not PDT, (2) ClAlSPc but not PDT, or (3) PBS injection plus PDT. Five days later PDT tumors of the ClAlSPc + PDT group were markedly reduced in size (mean weight 0.075 ± 0.027 g) as compared with those from control groups 1 (0.408 ± 0.167 g), 2 (0.475 ± 0.143 g) or 3 (0.376 ± 0.153 g). Histological examination revealed that ClAlSPc + PDT induced severe necrosis and cytotoxicity of neoplastic cells with viable tumor limited to a small peripheral margin. Animals in the ClAlSPc + PDT group in a repeat experiment survived significantly longer than animals in the three control groups suggesting that ClAlSPc may be a useful photosensitiser for PDT of cancer.     

Photodynamic actinometry using microspheres: concept, development and responsivity

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

Tumor Oxygenation Changes Post-Photodynamic Therapy

Abstract— Tumor oxygenation after a photodynamic therapy (PDT) treatment is a critical factor for understanding the post-treatment metabolic pathway of the tumor. It also provides important information for designing combination therapy of PDT and other oxygen-dependent anticancer modalities. In this study, mammary carcinoma in flank and hind leg of C3H mice were subjected to PDT at either subcurative or curative level (12.5 mg/kg Photofrin; 200 or 600 J/cm², respectively). The before and post-PDT tumor oxygenation was measured with an oxygen-sensitive microelectrode. The data revealed that tumor oxygenation at the time of PDT has a profound effect on posttreatment tumor oxygenation, which may largely be due to an interplay between direct PDT cytotoxicity and PDT damage to the tumor microvasculature. Transient reoxygenation occurred after PDT, which may provide a window for improved combination therapy for other oxygen-dependent modalities.     

Effect of photosensitizer dose on fluence rate responses to photodynamic therapy

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

Combination of surgical resection and photodynamic therapy of 9L gliosarcoma in the nude rat

The objective of the present study was to investigate the treatment of 9L gliosarcoma brain tumor in the rat with the combination of surgical resection and photodynamic therapy (PDT). Nude rats with intracranial 7-day-old 9L gliomas were randomly subjected to no treatment, PDT alone (Photofrin: 2 mg kg(-1), optical: 80 J cm(-2)), surgical resection alone or resection combined with 2 mg kg(-1) Photofrin-mediated PDT at an optical dose of 80 J cm(-2). All animals were sacrificed 14 days after tumor implantation. Hematoxylin-and-eosin and immunohistochemical stainings were performed to assess the tumor volume and the expression of vascular endothelial growth factor (VEGF) in the brain adjacent to the tumor (BAT) as well as the tumor cell apoptosis and proliferation. Our data show that both surgical resection alone and PDT alone significantly decreased tumor volume, but furthermore, surgical resection combined with PDT significantly reduced the tumor volume and reduced local tumor infiltration compared to either surgical resection or PDT treatment alone. PDT treatment with or without resection increased tumor apoptosis, but resection alone did not alter the tumor cell apoptosis compared with a nontreatment control group. Both surgical resection alone and PDT alone induced a significant increase in VEGF expression in the BAT; however intraoperative PDT did not further increase VEGF expression, compared with surgery alone or PDT alone. No significant differences were found in tumor cell proliferation as indicated by Ki67 immunoreactivity among the four groups. Our results suggest that PDT enhances the efficacy of surgical resection in the management of malignant gliomas without increasing VEGF expression in the BAT.     

Direct near-infrared luminescence detection of singlet oxygen generated by photodynamic therapy in cells in vitro and tissues in vivo

Singlet oxygen (1O2) is believed to be the major cytotoxic agent involved in photodynamic therapy (PDT). Measurement of 1O2 near-infrared (NIR) luminescence at 1270 nm in biological environments is confounded by the strongly reduced 1O2 lifetime and probably has never been achieved. We present evidence that this is now possible, using a new NIR-sensitive photomultiplier tube. Time-resolved 1O2 luminescence measurements were made in various solutions of aluminum tetrasulphonated phthalocyanine (AlS4Pc) and Photofrin. Measurements were also performed on suspensions of leukemia cells incubated with AlS4Pc, and a true intracellular component of the 1O2 signal was clearly identified. Time-resolved analysis showed a strongly reduced 1O2 lifetime and an increased photosensitizer triplet-state lifetime in the intracellular component. In vivo measurements were performed on normal skin and liver of Wistar rats sensitized with 50 mg/kg AlS4Pc. In each case, a small but statistically significant spectral peak was observed at 1270 nm. The 1O2 lifetime based on photon count rate measurements at 1270 nm was 0.03-0.18 micros, consistent with published upper limits. We believe that these are the first direct observations of PDT-generated intracellular and in vivo 102. The detector technology provides a new tool for PDT research and possibly clinical use.