Total Light Dose Research Articles

Effect of Photodynamic Therapy with Methylene Blue onAcanthamoebaIn Vitro

To evaluate the disinfectant effect of methylene blue (MB)-mediated photodynamic therapy (PDT) on a pathogenic strain of Acanthamoeba. Acanthamoeba castellanii (ATCC 50370) used in this study were treated under one of four experimental conditions: light irradiation and incubation in MB (L+M+), light irradiation and incubation in physiologic solution (L+M-), incubation in MB only (L-M+), and incubation in physiologic solution (L-M-). M+ trophozoites were incubated in either 0.25 or 0.5 mM MB for 10 minutes. L+ organisms were irradiated for 30 minutes following incubation in solution. A halogen lamp (660 ± 10 nm) with a maximum output of 6 mW/cm(2) was used as the PDT light source. After treatment, antiacanthamoeba activity was evaluated by checking the respiratory activity of the amoeba with 5-cyano-2,3-tetrazolium chloride (CTC) staining. We also determined whether the effect of PDT with MB had been retained or augmented when it was performed in combination with conventional antiamoebic agents. MB-PDT suppressed the respiratory activity of trophozoites in an MB-concentration-dependent manner at total light doses of 10.8 J/cm(2). The respiratory activity of each group as a percentage of that of L-M- is as follows: L+M+ 11.6% (0.5 mM), 60.9% (0.25 mM); L-M+ 116.5% (0.5 mM), 105.5% (0.25 mM); L+M- 107.6%; and L-M- 106.3%. (L+M+ versus L-M- P < 0.05). MB-PDT had a synergistic effect when used in combination with polyhexamethylene biguanide (PHMB) or amphotericin B, but not with voriconazole. MB-PDT is effective against Acanthamoeba in vitro and has synergistic effects with PHMB and amphotericin B.

Incomplete efficacy of 5‐aminolevulinic acid (5 ALA) photodynamic therapy in the treatment of widespread extramammary Paget's disease

Photodynamic therapy (PDT) using 5-aminolevulinic acid (5-ALA) is an effective treatment for several conditions such as Bowen's disease, subsets of basal cell carcinomas and actinic keratosis. Surgical resection is the first-choice therapy for extramammary Paget's disease (EMPD), but extensive resection is highly invasive and recurrences are frequent. We report two cases of genital EMPD treated by PDT with partial efficacy. The first patient, a 78-year-old male, suffered from pubic and scrotal Paget's disease for 6 years despite numerous treatments. The second patient, a 78-year-old female, had vulvar involvement for 2 years that was resistant to multiple treatments. The disease was recurrent and chronic with important pruritus and significant impact on the quality of life. Methyl 5-aminolevulinate was applied for 3 h, and irradiation was applied with red light (630 nm) using a total light dose of 37 J/cm(2) for a period of 10 min. The patients were treated every 2 to 4 weeks for a total of at least three treatments. Both patients experienced a partial transient reduction in their symptoms. One patient had a partial transient remission (< 50% reduction of the involved surface), whereas in the other patient, PDT failed to reduce the surface area of the lesions.

Morphological and histological changes of glioma cells immediately after 5-aminolevulinic acid mediated photodynamic therapy

<title/>Objective: Photodynamic therapy (PDT) using 5-aminolevulinic acid (5-ALA) is a new therapeutic modality for malignant glioma. This study is designed to evaluate acute morphological and histological changes of glioma cells after 5-ALA mediated PDT using fluorescence microscopy and three-dimensional spheroid models.Methods: Monolayer cultures and spheroids (small and large sizes) of C6 (rat glioma cell line) and U251MG (human glioma cell line) cells were co-incubated for 4 hours with 5-ALA (100 μg/ml) and irradiated using a diode laser (635±5 nm, 5-100 mW/cm2, and total light dose 2·5-50 J/cm2). Growth kinetics of the spheroids were followed for 7 days after PDT and morphological changes were assessed by phase contrast microscopy. In addition to hematoxylin and eosin and the terminal deoxynucleotidyl transferase-mediated dUTP-FITC nick end-labeling, various fluorescent dyes including calcein AM, ethidium homodimer-1 (EthD-1), annexin V-FITC, propidium iodide and Hoechst 33342, were used to clarify the mechanism of cell death after PDT.Results: Although PDT spheroids of both cell lines showed growth inhibition during the first few days, resumption of spheroid growth on day 3 after irradiation was observed in most spheroids. Hematoxylin and eosin staining showed cells with condensed chromatin within the superficial layer of the PDT spheroids. In the PDT groups, monolayer cells in the irradiated area were negative for calcein AM and clearly positive for EthD-1. In C6 PDT spheroids, the outer layer of the spheroid was negative for calcein AM and positive for EthD-1. In monolayer cells, cells in the irradiation area in PDT groups were positive for propidium iodide and negative for Hoechst 33342.Conclusion: Light and fluorescence microscopic findings suggested necrotic cell death as an anti-tumor effect in the acute phase after PDT. These findings were observed in both C6 and U251MG monolayer culture cells and spheroids. Although various total light doses resulted in significant differences in the growth rate of spheroids after PDT, resumption of spheroid growth occurred in most spheroids.

Vacata- and divacataporphyrin: New photosensitizers for application in photodynamic therapy-an in vitro study

The photodynamic therapy is a well-known method of treatment of both malignant tumors and non-tumor lesions in human patients. In the present study, we aimed at evaluating the in vitro efficacy of the new photosensitizing agents, vacataporphyrin (VP), and divacataporphyrin (DVP). The effectiveness of VP and DVP was compared to well-known photosensitizers, that is, hematoporphyrin derivative (HPD) and chlorin e6 (Ce6) in identical in vitro conditions. The experiment was performed on a well-established breast cancer cell line, MCF-7 and compared to HCV 29T11-2-D1 cell line. Cells were incubated in standard conditions and they were exposed to different concentrations of VP, DVP, HPD, and Ce6, that is, 180, 90, 45, 22.5, and 10 µg/ml. After incubation with photosensitizers, the cells were washed, medium was exchanged and the cells were subjected to irradiation at the proper wavelengths, light intensity (100 mW/sq cm), and total light doses 4.5 and 9 J/sq cm. Our results showed that the VP and DVP are potent photosensitizers and the photocytotoxic effect after the incubation with DVP was much better than that of VP. The cytotoxic effects of VP and DVP were less intensive than these of HPD and Ce6. VP and DVP also accumulated well in the tumor cells. Our results also indicated that the VP and DVP effectiveness on MCF-7 cells was photosensitizer dose and light dose dependent. The overall properties revealed by both new porphyrins and particularly a possibility for excitation at a higher wavelength and thus a deeper tissue penetration, make them promising candidates for further in vivo experiments.

Tissue Uptake Study and Photodynamic Therapy of Melanoma‐Bearing Mice with a Nontoxic, Effective Chlorin

Chlorins have intense red absorptions and high tumor affinities that make them interesting candidates for photodynamic therapy (PDT) of cancer. This paper reports cytotoxicity, phototoxicity, in vitro cellular uptake, and in vivo biodistribution and PDT efficacy of a synthetic chlorin derivative (TCPCSO₃H) towards Cloudman melanoma cells (S91). No cytotoxic effects were observed in vitro at concentrations up to 20 μm, and no toxicity was observed in vivo in DBA mice with doses up to 2 mg kg⁻¹. Pharmacokinetics and biodistribution of TCPCSO₃H were evaluated in vivo in DBA mice bearing S91 tumors. TCPCSO₃H demonstrated preferential accumulation in S91 mouse melanoma, with tumor-to-normal tissue ratios of 5 and 11 for muscle and skin, respectively, 24 h after intravenous injection of 2 mg kg⁻¹. Photodynamic therapy performed under these conditions with 70 mW cm⁻² diode laser irradiation at 655 nm for 25 min (total light dose=105 J cm⁻²) resulted in scab formation, followed by temporary or permanent (>60 days) tumor remission. According to the Kaplan-Meier analysis, the median survival time of the control group was 9 days, whereas that of the treated group was 38 days.

Neither elevated nor reduced CO 2 affects the photophysiological performance of the marine Antarctic diatom Chaetoceros brevis

Enhanced or reduced pCO 2 (partial pressure of CO 2) may affect the photosynthetic performance of marine microalgae since changes in pCO 2 can influence the activity of carbon concentrating mechanisms, modulate cellular RuBisCO levels or alter carbon uptake efficiency. In the present study we compared the photophysiology of the Antarctic diatom Chaetoceros brevis at two pCO 2 extremes: 750 ppmv (2× ambient) and 190 ppmv (0.5× ambient) CO 2. Cultures were acclimated to four irradiance regimes: two regimes simulating deep or shallow vertical mixing, and two regimes mimicking limiting and saturating stable water column conditions. Then, growth rate, pigmentation, RuBisCO large subunit expression, RuBisCO activity, photosynthesis vs irradiance curves, effective quantum yield of PSII (F v/F m), and POC were measured. The four irradiance regimes induced a suite of photophysiological responses, ranging from low light acclimation to efficient photoprotection. Growth was reduced under the low constant and the deep mixing regime, compared to the shallow mixing and the stable saturating regime. Low stable irradiance resulted in higher light harvesting pigment concentrations, lower RuBisCO activity and a lower light saturation point (E k) compared to the other irradiance regimes. Highest RuBisCO activity as well as P max levels was measured in the shallow mixing regime, which received the highest total daily light dose. Photoprotection by xanthophyll cycling was observed under all irradiance regimes except the low stable irradiance regime, and xanthophyll cycle pool sizes were higher under the dynamic irradiance regimes. For the fluctuating irradiance regimes, F v/F m was hardly affected by previous excess irradiance exposure, suggesting minimal PSII damage. No significant differences between the two pCO 2 levels were found, with respect to growth, pigment content and composition, photosynthesis, photoprotection and RuBisCO activity, for all four irradiance regimes. Thus, within the range tested, pCO 2 does not significantly affect the photophysiological performance of C. brevis.

Photodynamic Antimicrobial Chemotherapy in Aquaculture: Photoinactivation Studies of Vibrio fischeri

BackgroundPhotodynamic antimicrobial chemotherapy (PACT) combines light, a light-absorbing molecule that initiates a photochemical or photophysical reaction, and oxygen. The combined action of these three components originates reactive oxygen species that lead to microorganisms' destruction. The aim was to evaluate the efficiency of PACT on Vibrio fischeri: 1) with buffer solution, varying temperature, pH, salinity and oxygen concentration values; 2) with aquaculture water, to reproduce photoinactivation (PI) conditions in situ.Methodology/Principal FindingsTo monitor the PI kinetics, the bioluminescence of V. fischeri was measured during the experiments. A tricationic meso-substituted porphyrin (Tri-Py+-Me-PF) was used as photosensitizer (5 µM in the studies with buffer solution and 10–50 µM in the studies with aquaculture water); artificial white light (4 mW cm−2) and solar irradiation (40 mW cm−2) were used as light sources; and the bacterial concentration used for all experiments was ≈107 CFU mL−1 (corresponding to a bioluminescence level of 105 relative light units - RLU). The variations in pH (6.5–8.5), temperature (10–25°C), salinity (20–40 g L−1) and oxygen concentration did not significantly affect the PI of V. fischeri, once in all tested conditions the bioluminescent signal decreased to the detection limit of the method (≈7 log reduction). The assays using aquaculture water showed that the efficiency of the process is affected by the suspended matter. Total PI of V. fischeri in aquaculture water was achieved under solar light in the presence of 20 µM of Tri-Py+-Me-PF.Conclusions/SignificanceIf PACT is to be used in environmental applications, the matrix containing target microbial communities should be previously characterized in order to establish an efficient protocol having into account the photosensitizer concentration, the light source and the total light dose delivered. The possibility of using solar light in PACT to treat aquaculture water makes this technology cost-effective and attractive.

Hexvix ® mediated rat bladder photodynamic therapy: The impact of fluence rate on normal bladder preservation

Bladder PDT, the first FDA indication, has been abandoned in favour of photodiagnosis with ALA or derivatives. Major drawback of this treatment modality is prolonged and severe irritative bladder symptoms, due to lack of selectivity of the drug and generalized urothelial necrosis. Low fluence rates are known to potentiate therapeutic efficacy due to an oxygen sparing regime. We have evaluated the effect of low fluence rate PDT on therapeutic outcome and side effects in rat tumor bearing bladders. Bladders were instilled with 8 mM hexylaminolevulinate and illuminated with 635 nm light to a total light dose of 20 or 15 J/cm2 at 100 or 15 mW/cm2, 5 rats per group. Fluence rates of 15 mW/cm2 did not provoke enhancement of cardiac and respiratory rhythms as opposed to 100 mW/cm2, neither was hematuria observed post treatment. Macroscopic aspects of bladders at 100 mW/cm2 show thickened bladders with hemorrhagic areas whereas 15 J/cm2 does not alter macroscopic features. Pathology revealed the absence of viable superficial tumors, irrespective of irradiation conditions. Inflammatory volume was calculated to be 96 a.u. for high fluence high light dose, reduced to 18 a.u. when reducing the light dose and 12 a.u. for the low fluence rates, irrespective of the light dose. Normal urothelial is preserved over the total bladder in case of 15 mW/cm2, whereas major portions of the bladder show absence of reduction of cell layers at 100 mW/cm2. Low fluence rate PDT does not hamper therapeutic efficacy and minimizes local side effects in rats.

Drug and light delivery strategies for photodynamic antimicrobial chemotherapy (PACT) of pulmonary pathogens: A pilot study

Pulmonary disease is the main cause of morbidity and mortality in cystic fibrosis (CF) suffers, with multidrug-resistant Pseudomonas aeruginosa and Burkholderia cepacia complex as problematic pathogens in terms of recurrent and unremitting infections. Novel treatment of pulmonary infection is required to improve the prognosis and quality of life for chronically infected patients. Photodynamic antimicrobial chemotherapy (PACT) is a treatment combining exposure to a light reactive drug, with light of a wavelength specific for activation of the drug, in order to induce cell death of bacteria. Previous studies have demonstrated the susceptibility of CF pathogens to PACT in vitro. However, for the treatment to be of clinical use, light and photosensitizer must be able to be delivered successfully to the target tissue. This preliminary study assessed the potential for delivery of 635 nm light and methylene blue to the lung using an ex vivo and in vitro lung model. Using a fibre-optic light delivery device coupled to a helium-neon laser, up to 11% of the total light dose penetrated through full thickness pulmonary parenchymal tissue, which indicates potential for multiple lobe irradiation in vivo. The mass median aerodynamic diameter (MMAD) of particles generated via methylene blue solution nebulisation was 4.40 μm, which is suitable for targeting the site of infection within the CF lung. The results of this study demonstrate the ability of light and methylene blue to be delivered to the site of infection in the CF lung. PACT remains a viable option for selective killing of CF lung pathogens.

Biodistribution and Photodynamic Efficacy of a Water‐Soluble, Stable, Halogenated Bacteriochlorin against Melanoma

The in vitro phototoxicity of a photostable, synthetic, water-soluble, halogenated bacteriochlorin, 5,10,15,20-tetrakis(2-chloro-5-sulfophenyl)bacteriochlorin (TCPBSO3H), toward mouse melanoma (S91) cells is ∼60-fold higher than that of the analogous porphyrin, and is associated with very weak toxicity in the dark; 90% of S91 cells were killed in response to a light dose of 0.26 J cm(-2) in the presence of [TCPBSO3H]=5 μM. In vivo toxicity toward DBA mice is very low, even at doses of 20 mg kg(-1). In vivo pharmacokinetics and biodistribution of TCPBSO3H were studied in DBA mice with S91 tumors; 24 h after intraperitoneal injection of 10 mg kg(-1), TCPBSO3H demonstrated preferential accumulation in S91 mouse melanoma, with tumor-to-normal tissue ratios of 3 and 5 for muscle and skin, respectively. Photodynamic therapy (PDT) performed under these conditions, with 90 mW cm(-2) diode laser irradiation at λ 750 nm for 20 min (total light dose of 108 J cm(-2)), resulted in tumor regression. Tumor recurrence was observed only approximately two months after treatment, confirming the efficacy of this PDT against melanoma.

HIGH‐POWER PULSED LIGHT FOR DECONTAMINATION OF CHICKEN FROM FOOD PATHOGENS: A STUDY ON ANTIMICROBIAL EFFICIENCY AND ORGANOLEPTIC PROPERTIES

ABSTRACTThe aim of this study was to evaluate the efficacy of high‐power pulsed light technique to decontaminate the surface of chicken from food pathogens Salmonella enterica serovar Typhimurium and Listeria monocytogenes as well as from total aerobic mesophils in a nonthermal way. Moreover, it was necessary to investigate the organoleptic properties of treated chicken. The spread plate method was used to evaluate antimicrobial efficiency of high‐power pulsed light on S. Typhimurium and L. monocytogenes in vitro and after inoculation on the surface of skinless chicken breast meat. High‐power pulsed light treatment (1,000 pulses, treatment duration 200 s, total ultraviolet light dose 5.4 J/cm2) reduced viability of S. Typhimurium and L. monocytogenes inoculated on the surface of chicken by 2–2.4 log10 (N/N0) colony forming unit (cfu)/mL order. In addition, total aerobic mesophils on the surface of meat were diminished by 2 log10 (N/N0) cfu/mL. All experiments were performed under nonthermal conditions (&lt;42C). Data obtained on investigation of chemical changes in the treated chicken breasts indicated that the intensity of lipid peroxidation in control and treated chicken samples differed in 0.16 mg malondialdehyde per kilogram of chicken meat. Meanwhile, taste panelists testing organoleptic properties of treated chicken did not detect any changes of raw chicken, chicken broth or cooked chicken meat when it was treated under nonthermal conditions in comparison with control. In conclusion, high‐power pulsed light is a fast, effective and environmentally friendly chicken surface decontamination technology in reducing aerobic plate counts as well as inoculated pathogens. It is important to note that no significant changes in meat lipid peroxidation or sensory characteristics were detected in treated chicken under nonthermal conditions.PRACTICAL APPLICATIONSConstructed equipment and obtained data may serve in the future for advanced development of high‐power pulsed light technique which could be used for nonthermal decontamination of different food matrices (fruits, vegetables, eggs shell, fish and meat) and food‐related packaging surfaces, medical devices, processing equipment for the food, medical and pharmaceutical industries.

Rose Bengal Acetate photodynamic therapy-induced autophagy

Photodynamic therapy (PDT), an anticancer therapy requiring the exposure of cells or tissue to a photosensitizing drug followed by irradiation with visible light of the appropriate wavelength, induces cell death by the efficient induction of apoptotic as well as non-apoptotic mechanisms, such as necrosis and autophagy, or a combination of all three mechanisms. However, the exact role of autophagy in photodynamic therapy is still a matter of debate. To understand the role of autophagy in PDT, we investigated the induction of autophagy in HeLa cells photosensitized with Rose Bengal Acetate (RBAc). After incubation with Rose Bengal Acetate (10-5 M), HeLa cells were irradiated for 90 seconds (green LED DPL 305, emitting at 530 +15 nm to obtain 1.6 J/cm2 as the total light dose) and allowed to recover for 72 h. Induction of autophagy and apoptosis were observed with peaks at 8 h and 12 h after irradiation, respectively. Autophagy was detected by biochemical (Western Blotting for the LC3B protein) and morphological criteria (TEM, cytochemistry). In addition, the pan-caspase inhibitor, z-VAD, was unable to completely prevent cell death. The simultaneous onset of apoptosis and autophagy following Rose Bengal Acetate PDT is of remarkable interest in light of the findings that autophagy can result in the class II presentation of antigens and thus, explain why low dose PDT can yield anti-tumor immune responses.

Sewage bacteriophage inactivation by cationic porphyrins: influence of light parameters

Photodynamic therapy has been used to inactivate microorganisms through the use of targeted photosensitizers. Although the photoinactivation of microorganisms has already been studied under different conditions, a systematic evaluation of irradiation characteristics is still limited. The goal of this study was to test how the light dose, fluence rate and irradiation source affect the viral photoinactivation of a T4-like sewage bacteriophage. The experiments were carried out using white PAR light delivered by fluorescent PAR lamps (40 W m(-2)), sun light (600 W m(-2)) and an halogen lamp (40-1690 W m(-2)). Phage suspensions and two cationic photosensitizers (Tetra-Py(+)-Me, Tri-Py(+)-Me-PF) at concentrations of 0.5, 1.0 and 5.0 microM were used. The results showed that the efficacy of the bacteriophage photoinactivation is correlated not only with the sensitizer and its concentration but also with the light source, energy dose and fluence rate applied. Both photosensitizers at 5.0 microM were able to inactivate the T4-like phage to the limit of detection for each light source and fluence rate. However, depending of the light parameters, different irradiation times are required. The efficiency of photoinactivation is dependent on the spectral emission distribution of the light sources used. Considering the same light source and a fixed light dose applied at different fluence rates, phage inactivation was significantly higher when low fluence rates were used. In this way, the light source, fluence rate and total light dose play an important role in the effectiveness of the antimicrobial photodynamic therapy and should always be considered when establishing an optimal antimicrobial protocol.

P69 Does the fluence rate influence clinical outcome and pain experienced by the patients with actinic keratosis treated with PDT?

Background Topical 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT) has been established in recent years as an effective treatment for non-melanoma skin cancers, including actinic keratosis (AKs). Available literature data suggest that low fluence rates are preferable in terms of efficacy and tolerability for treating patients suffering from disseminated AKs. Aims To identify factors contributing to pain induction during PDT, and to detect the optimal fluence rate, in conjunction with the clinical outcome. Methods Fifty adults, with an overall of 150 histologically confirmed lesions of AKs were recruited in the study. Inclusion criteria were the presence of at least 3 discrete AKs, located on face or scalp, and age over 18. Treatment protocol for each lesion included two therapeutic sessions of topical PDT, one week apart. Each lesion was randomly allocated to 25, 50 or 75 mW/cm2. The total light dose was equal to 75 J/cm2 and was maintained the same during all treatment sessions. A 20% 5-aminolevulinic acid (ALA) cream was applied over the lesion, and after 4 h of light-impenetrable occlusion dressing, the lesion was illuminated with red light (570–670 nm) by a non-coherent light source (Waldmann PDT 1200, Waldmann-Medizin-Technik, Villingen-Schwenningen, Germany). Pain experienced by patients during illumination was recorded using a visual analogue scale (VAS) graded from 0 (no pain) to 10 (unbearable pain). Follow-up visits were held at months 3, 6 and 12 after treatment. Clinical outcome was defined as complete response (CR; complete absence of any clinical sign indicative of AKs) or as non-complete response (non-CR; remaining clinical signs indicative of AKs). Results Mean VAS score did not significantly differ between the groups of 25 and 50 mW/cm2. However, mean VAS was significantly higher at the group of 75 mW/cm2. With respect to the clinical outcome and the rate of recurrences during the first year of follow-up, no differences were observed. Conclusion According to our observations a fluence rate of 50 mW/cm2 is equally effective and better tolerated by patients treated with topical 5-ALA PDT for AKs.

Photosensitization-based inactivation of food pathogen Listeria monocytogenes in vitro and on the surface of packaging material

The study was focused on the susceptibility of Listeria monocytogenes ATCL3C 7644 cells and biofilms to non-thermal antimicrobial treatment – photosensitization in vitro and after adhesion to the surface of packaging material. L. monocytogenes was incubated with 5-aminolevulinic acid (ALA) (7.5 mM) for 0–2 h and illuminated with visible light. The LED-based light source used for the illumination emitted light λ = 400 nm with energy density 20 mW/cm 2. The illumination time varied 0–20 min, and a total light dose reached 0–24 J/cm 2. The obtained data indicate that L. monocytogenes produces endogenous porphyrins after incubation with 7.5 mM ALA. Subsequent illumination of cells remarkably inactivates (4 log) them in vitro. Photosensitization diminished population of Listeria cells adhered onto the packaging material by 3.7 log and inactivated bacterial biofilms by 3.1 log. It was shown that antimicrobial efficiency of photosensitization depended on the illumination time, incubation with ALA time as well as on the used ALA concentration. In conclusion, cells and biofilms of L. monocytogenes ATCL3C 7644 can be effectively inactivated by ALA-based photosensitization in the solution as well as adhered onto the surface of packaging material. Obtained data support the idea, that photosensitization as non-thermal and effective antimicrobial treatment has potential to develop into environmentally safe, surface decontamination technique.

Study of the Photodynamic Effect on the A549 Cell Line by Atomic Force Microscopy and the Influence of Green Tea Extract on the Production of Reactive Oxygen Species

We studied the morphology of the A549 cell line (human lung carcinoma cells) before and after photodynamic therapy (PDT) by atomic force microscopy. PDT was induced by an efficient light-emitting diode source with total light dose of 15 J cm(-2) in the presence of the sensitizer zinc-5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrine. In the presence of molecular oxygen, light activation of the photosensitizer, which accumulates in cancer cells, leads to the local production of reactive oxygen species (ROS). This is one of several reasons leading to cell death, and in some cases we could observe signs of apoptosis. We detected the kinetics of ROS production to be dependent on the presence of green tea extract.

Inactivation of food pathogenBacillus cereusby photosensitizationin vitroand on the surface of packaging material

The study was focused on the possibility to inactivate food pathogen Bacillus cereus by 5-aminolevulinic acid (ALA) - based photosensitization in vitro and after adhesion on the surface of packaging material. Bacillus cereus was incubated with ALA (3-7.5 mmol l(-1)) for 5-60 min in different environment (PBS, packaging material and wheat grains) and afterwards illuminated with visible light. The light source used for illumination emitted light at lambda = 400 nm with energy density at the position of the cells, 20 mW cm(-2). The illumination time varied from 0 to 20 min, and subsequently a total energy dose was between 0 and 24 J cm(-2). The obtained results indicate that B. cereus after the incubation with 3-7.5 mmol l(-1) ALA produces suitable amounts of endogenous photosensitizers. Following illumination, micro-organism inactivated even by 6.3 log. The inactivation of B. cereus after adhesion on the surface of food packaging by photosensitization reached 4 log. It is important to note that spores of B. cereus were susceptible to this treatment as well; 3.7-log inactivation in vitro and 2.7-log inactivation on the surface of packaging material were achieved at certain experimental conditions. Vegetative cells and spores of Gram-positive food pathogen B. cereus were effectively inactivated by ALA-based photosensitization in vitro. Moreover, the significant inactivation of B. cereus adhered on the surface of packaging material was observed. It was shown that photosensitization-based inactivation of B. cereus depended on the total light dose (illumination time) as well as on the amount of endogenous porphyrins (initial ALA concentration, time of incubation with ALA). Our previous data, as well as the one obtained in this study, support the idea that photosensitization with its high selectivity, antimicrobial efficiency and nonthermal nature could serve in the future for the development of completely safe, nonthermal surface decontamination and food preservation techniques.

Charge effect on the photoinactivation of Gram-negative and Gram-positive bacteria by cationic meso-substituted porphyrins

BackgroundIn recent times photodynamic antimicrobial therapy has been used to efficiently destroy Gram (+) and Gram (-) bacteria using cationic porphyrins as photosensitizers. There is an increasing interest in this approach, namely in the search of photosensitizers with adequate structural features for an efficient photoinactivation process. In this study we propose to compare the efficiency of seven cationic porphyrins differing in meso-substituent groups, charge number and charge distribution, on the photodynamic inactivation of a Gram (+) bacterium (Enterococcus faecalis) and of a Gram (-) bacterium (Escherichia coli). The present study complements our previous work on the search for photosensitizers that might be considered good candidates for the photoinactivation of a large spectrum of environmental microorganisms.ResultsBacterial suspension (107 CFU mL-1) treated with different photosensitizers concentrations (0.5, 1.0 and 5.0 μM) were exposed to white light (40 W m-2) for a total light dose of 64.8 J cm-2. The most effective photosensitizers against both bacterial strains were the Tri-Py+-Me-PF and Tri-Py+-Me-CO2Me at 5.0 μM with a light fluence of 64.8 J cm-2, leading to > 7.0 log (> 99,999%) of photoinactivation. The tetracationic porphyrin also proved to be a good photosensitizer against both bacterial strains. Both di-cationic and the monocationic porphyrins were the least effective ones.ConclusionThe number of positive charges, the charge distribution in the porphyrins' structure and the meso-substituent groups seem to have different effects on the photoinactivation of both bacteria. As the Tri-Py+-Me-PF porphyrin provides the highest log reduction using lower light doses, this photosensitizer can efficiently photoinactivate a large spectrum of environmental bacteria. The complete inactivation of both bacterial strains with low light fluence (40 W m-2) means that the photodynamic approach can be applied to wastewater treatment under natural light conditions which makes this technology cheap and feasible in terms of the light source.

Pilot Study on Light Dosimetry Variables for Photodynamic Therapy of Barrett's Esophagus with High-Grade Dysplasia

Photodynamic therapy (PDT) is used to treat Barrett's esophagus with high-grade dysplasia and mucosal carcinoma. Outcomes are variable with some patients having persistent disease, whereas others develop strictures. The aims of this study were (a) to compare porfimer sodium tissue uptake, light dose, and esophageal thickness with clinical outcomes and (b) to determine the selectivity of porfimer sodium uptake in diseased and normal epithelium. Forty-eight hours after porfimer sodium infusion, patients underwent mucosal biopsy for quantification of the porfimer sodium. Laser light was delivered at 48 hours and again 24 or 48 hours later. Porfimer sodium was extracted from the biopsy samples and quantified using fluorescence spectroscopy. The enhanced photodynamic dose was determined as [porfimer sodium content * light dose/esophageal thickness]. PDT efficacy was determined 6 to 8 weeks later based on persistence or complete ablation of dysplasia or carcinoma. Mean porfimer sodium content of 6.2 mg/kg (range, 2.6-11.2 mg/kg) and mean total light dose of 278 J/cm (range, 225-360 J/cm) resulted in a complete treatment. Mean porfimer sodium tissue content of 3.9 mg/kg (range, 2.1-8.1 mg/kg) and mean total light dose of 268 J/cm (range, 250-350 J/cm) resulted in an incomplete treatment. The total esophageal thickness (range, 1.7-6.0 mm) and enhanced photodynamic dose were correlated with treatment outcome. Esophageal thickness is the strongest predictor of treatment outcome. The porfimer sodium content of Barrett's and normal tissue is not significantly different. "Photodynamic dose" for esophageal PDT should incorporate the esophageal thickness.

Acute Morphological and Histological Changes of Glioma Cells after 5-Aminolevulinic Acid Mediated Photodynamic Therapy

Objective Photodynamic therapy (PDT) using 5-aminolevulinic acid (5-ALA) is a new therapeutic modality for malignant glioma. This study is designed to evaluate acute morphological and histological changes of glioma cells after 5-ALA mediated PDT using fluorescence microscopy and three dimensional spheroid models.Methods Monolayer cultures and spheroids (small and large sizes) of C6 (rat glioma cell line) and U251MG (human glioma cell line) cells were co-incubated for 4 hours with 5-ALA (100μg/ml) and irradiated using a diode laser (635±5 nm, 5-100 mW/cm2, total light dose 2.5-50 J/cm2). Growth kinetics of the spheroids were followed for 7 days after PDT and morphological changes were assessed by phase-contrast microscopy. In addition to hematoxylin-eosin (H.E.) and the terminal deoxynucleotidyl transferase-mediated dUTP-FITC nick end-labeling, various fluorescent dyes including Calcein AM, Ethidium homodimer (ethD-1), Annexin V-FITC, Propidium iodide (PI) and Hoechst33342, were used to clarify the mechanism of cell death after PDT.Results Although PDT spheroids of both cell lines showed growth inhibition during the first few days, resumption of spheroid growth on day 3 after irradiation was observed in most spheroids. H.E staining showed cells with condensed chromatin within the superficial layer of the PDT spheroids. In the PDT groups, monolayer cells in the irradiated area were negative for Calcein AM and clearly positive for EthD-1. In C6 PDT spheroids, the outer layer of the spheroid was negative for Calcein AM and positive for EthD-1. In monolayer cells, cells in the irradiation area in PDT groups were positive for PI and negative for Hoechst33342. Conclusion Light and fluorescence microscopic findings suggested necrotic cell death as an anti-tumor effect in the acute phase after PDT. These findings were observed in both C6 and U-251MG monolayer culture cell and spheroids. Although various total light doses resulted in significant differences in the growth rate of spheroids after PDT, resumption of spheroid growth occurred in most spheroids.