Photodynamic therapy associating Photogem (R) and blue LED on L929 and MDPC-23 cell culture

To evaluate the cytotoxicity of PDT (photodynamic therapy) with Photogem (R) associated to blue LED (light-emitting diode) on L929 and MDPC-23 cell cultures, 30000 cells/cm(2) were seeded in 24-well plates for 48 h, incubated with Photogem (R) (10, 25 or 50 mg/l) and irradiated with an LED source (460 +/- 3 nm; 22 mW/cm(2)) at two energy densities (25.5 or 37.5 J/cm(2)). Cell metabolism was evaluated by the MTT (methyltetrazolium) assay (Dunnet`s post hoc tests) and cell morphology by SEM (scanning electron microscopy). Flow cytometry analysed the type of PDT-induced cell death as well and estimated intracellular production of ROS (reactive oxygen species). There was a statistically significant decrease of mitochondrial activity (90% to 97%) for all Photogem (R) concentrations associated to blue LED, regardless of irradiation time. It was also demonstrated that the mitochondrial activity was not recovered after 12 or 24 h, characterizing irreversible cell damage. PDT-treated cells presented an altered morphology with ill-defined limits. In both cell lines, there was a predominance of necrotic cell death and the presence of Photogem (R) or irradiation increased the intracellular levels of ROS. PDT caused severe toxic effects in normal cell culture, characterized by the reduction of the mitochondrial activity, morphological alterations and induction of necrotic cell death.

Effectiveness of Photogem (R) activated by LED on the decontamination of artificial carious bovine dentin

The aim of this study was the evaluation of the effectiveness of photodynamic therapy on the decontamination of artificially induced carious bovine dentin, using Photoge(R) as the photosensitizer agent and an LED device as a light source. Dentin samples obtained from bovine incisors were immersed in sterile broth supplemented by Lactobacillus acidophillus 10(8) colony formation units (CFU) and Streptococcus mutans 10 8 CFU. Different concentrations of photosensitizer, PA = 1 mg/ml, PB = 2 mg/ml, and PC = 3 mg/ml, and two fluences, D = 24 J/cm(2) and D = 48 J/cm(2), were investigated. After CFU counting per milligram of carious dentin and statistical analysis, we observed that the photodynamic therapy (PDT) parameters used were effective for bacterial reduction in the in vitro model under study. The best result was achieved with the application of Photoge(R) at 2 mg/ml and photoactivated under 24 J/cm(2) showing a survival factor of 0.14. At higher photosensitizer concentrations, a higher dark toxicity was observed. We propose a simple mathematical expression for the determination of PDT parameters of photosensitizer concentration and light fluence for different survival factor values. Since LED devices are simpler and cheaper compared to laser systems, it would be interesting to verify their efficacy as a light source in photodynamic therapy for the decontamination of carious dentin.

Photodynamic therapy associating Photogem (R) and blue LED on L929 and MDPC-23 cell culture

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Toxicity of photodynamic therapy with LED associated to Photogem (R): An in vivo study

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Toxicity of photodynamic therapy with LED associated to Photogem (R): An in vivo study

The aims of this study were to evaluate the effects of PhotogemA (R)-mediated photosensitization on rat palatal mucosa and the biodistribution of the photosensitizer in this tissue. A solution of PhotogemA (R) (500 or 1000 mg/l) was applied to the palatal mucosa for 30 min and the exposure time to blue LED (460 nm) was 20 min (144 J/cm(2)). At 0, 1, 3, and 7 days, palatal mucosa was photographed for macroscopic analysis. After killing, the palate was removed for microscopic analysis. Thermal mapping evaluated temperature change in the tissue during irradiation. All experimental groups revealed intact mucosa in the macroscopic analysis. Tissue alterations were observed microscopically for only four out of 80 animals subjected to PDT. Fluorescence emitted by PhotogemA (R) was identified and was limited to the epithelial layer. A temperature increase from 35 to 41A degrees C was recorded. PhotogemA (R)- mediated PDT was not toxic to the rat palatal mucosa.

Histological analyses of thermal effect caused by 1.2 W diode laser irradiation at rat periodontal pockets

The aim of this in vivo study was to evaluate the thermal effects caused by 810 nm 1.2 W diode laser irradiation of periodontal tissues. Despite all data available concerning the laser application for periodontal treatment, one of the most relevant challenges is to prevent the harmful tissue heating induced by different clinical protocols. Periodontal pockets were induced at molars in 96 rats. Several irradiation powers under CW mode were investigated: 0, 400, 600, 800, 1000, 1200 mW. The pockets were irradiated using a 300 A mu m frontal illumination fiber. The animals were killed at 4 or 10 days after irradiation. The mandible was surgically removed and histologically processed. The histological sections stained with H/E demonstrated that irradiation parameters up to 1000 mW were thermally safe for the periodontal tissues. The sections stained with Brown & Brenn technique evidenced bacteria in the periodontal tissues. Consequently, the diode laser irradiation as a unique treatment was not capable to eliminate bacteria of the biofilm present in the pockets. According to the methodology used here, it was concluded that the thermal variation promoted by a diode laser can cause damage to periodontal tissues depending on the energy density used. The 1.2 W diode laser irradiation itself does not control the bacteria present in the biofilm of the periodontal pockets without mechanical action. The knowledge of proper high intensity laser parameters and methods of irradiation for periodontal protocols may prevent any undesirable thermal damage to the tissues.

Can efficiency of the photosensitizer be predicted by its photostability in solution?

We have investigated a possible correlation between the photostability and photodynamic efficacy for different photosensitizers; hematoporphyrin derivatives and chlorines. To perform such analysis, we combined the depth of necrosis (d (nec)) measurement, expressed by the light threshold dose and a photodegradation parameter, measured from investigation of photosensitizer degradation in solution. The d (nec) analysis allows us to determine the light threshold dose and compare its value with the existent results in the literature. The use of simple models to understand basic features of Photodynamic Therapy (PDT) may contribute to the solid establishment of dosimetry in PDT, enhancing its use in the clinical management of cancers and others lesions. Using hematoporphyrin derivatives and chlorines photosensitizers we investigated their properties related to the photodegradation in solution and the light threshold dose (D (th)) in rat livers.

Hematoporphyrin-based photodynamic therapy for cutaneous squamous cell carcinoma in cats

Photodynamic therapy (PDT) using a haematoporphyrin derivative (Photogem (R), General Physics Institute and clustes Ltda) as photosensitizer and light emitting diodes (LEDs) as the light source was evaluated in 12 cats with cutaneous squamous cell carcinoma. Lesions were illuminated with LEDs, (300 J/cm for 30 min) 24 h after the administration of the photosensitizer. Clinical responses were classified as complete disappearance of the tumour with total re-epithelialization; partial response (a reduction greater than 50%); and no response (less than 50% reduction). Tumours localized to the pinna treated with one (n = 3) or two (n = 4) applications of PDT yielded no response. Highly invasive tumours of the nose and nasal planum also showed no response, after two treatments (n = 2). A combination of PDT and surgery was performed in three cases. Two cats showed partial response and one complete response with one application of therapy 30 days after nasal surgery. Small and noninfiltrative lesions (n = 3) of the nasal planum showed a PR with one application (n = 2) and a CR with two applications (n = 1). This study shows that PDT using Photogem (R) and LEDs can provide local control of low-grade feline squamous cell carcinoma. The addition of PDT to surgery in more invasive cases may help prevent recurrence.

Phototransformation of hematoporphyrin in aqueous solution: Anomalous behavior at low oxygen concentration

The photoactivation of a photosensitizer is the initial step in photodynamic therapy (PDT) where photochemical reactions result in the production of reactive oxygen species and eventually cell death. In addition to oxidizing biomolecules, some of these photochemical reactions lead to photosensitizer degradation at a rate dependent on the oxygen concentration among other factors. We investigated photodegradation of Photogem A (R) (28 mu M), a hematoporphyrin derivative, at different oxygen concentrations (9.4 to 625.0 mu M) in aqueous solution. The degradation was monitored by fluorescence spectroscopy. The degradation rate (M/s) increases as the oxygen concentration increases when the molar ratio of oxygen to PhotogemA (R) is greater than 1. At lower oxygen concentrations (< 25 mu M) an inversion of this behavior was observed. The data do not fit a simple kinetic model of first-order dependence on oxygen concentration. This inversion of the degradation rate at low oxygen concentration has not previously been demonstrated and highlights the relationship between photosensitizer and oxygen concentrations in determining the photobleaching mechanism(s). The findings demonstrate that current models for photobleaching are insufficient to explain completely the effects at low oxygen concentration.

A combination of techniques to evaluate photodynamic efficiency of photosensitizers

Photodynamic therapy, used mainly for cancer treatment and microorganisms inaction, is based on production of reactive oxygen species by light irradiation of a sensitizer. Hematoporphyrin derivatives as Photofrin (R) (PF) Photogem (R) (PG) and Photosan (R) (PF), and chlorin-c6-derivatives as Photodithazine (R)(PZ), have suitable sensitizing properties. The present study provides a way to make a fast previous evaluation of photosensitizers efficacy by a combination of techniques: a) use of brovine serum albumin and uric acid as chemical dosimeters; b) photo-hemolysis of red blood cells used as a cell membrane interaction model, and c) octanol/phosphate buffer partition to assess the relative lipophilicity of the compounds. The results suggest the photodynamic efficient rankings PZ > PG >= PF > PS. These results agree with the cytotoxicity of the photosensitizers as well as to chromatographic separation of the HpDs, both performed in our group, showing that the more lipophilic is the dye, the more acute is the damage to the RBC membrane and the oxidation of indol, which is immersed in the hydrophobic region of albumin.

Analysis of the combined effect of lasers of different wavelengths for PDT outcome using 600, 630, and 660 nm

We investigated the effects of photodynamic therapy (PDT) outcome when combining three laser systems that produce light in three different wavelengths (600, 630, and 660 nm). Cooperative as well as independent effects can be observed. We compared the results of the combined wavelengths of light with the effect of single laser for the excitation of the photosensitizer. In the current experiment, the used photosensitizer was Photogem (R) (1.5 mg/kg). Combining two wavelengths for PDT, their cumulative dose and different penetrability may change the overall effect of the fluence of light, which can be effective for increasing the depth of necrosis. This evaluation was performed by comparing the depth and specific aspect of necrosis obtained by using single and dual wavelengths for irradiation of healthy liver of male Wistar rats. We used 15 animals and divided them in five groups of three animals. First, Photogem (R) was administered; follow by measurement of the fluorescence spectrum of the liver before PDT to confirm the level of accumulation of photosensitizer in the tissue. After that, an area of 1 cm(2) of the liver was illuminated using different laser combinations. Qualitative analysis of the necrosis was carried out through histological and morphological study. [GRAPHICS] (a) - microscopic images of rat liver cells, (b) - superficial necrosis caused by PDT using dual-wavelength illumination, (c) - neutrophilic infiltration around the vessel inside the necrosis, and (d) - neutrophilic infiltration around the vessel between necrosis and live tissue (C) 2011 by Astro Ltd. Published exclusively by WILEY-VCH Verlag GmbH & Co. KGaA

Photostability of different chlorine photosensitizers

In this paper, we report the photodegradation of three different chlorine photosensitizers (Photoditazine (R), Radachlorin (R), and Foscan (R)). The photosensitizer degradation was analyzed by changes in the fluorescence spectrum during illumination. The rate of fluorescence variation was normalized to the solution absorption and the photon energy resulting in the determination of the necessary number of photons to be absorbed to induce photosensitizer photodegradation. The parameter for rate of the molecules decay, the photon fluence rate and optical properties of the solution allow us to determine the photosensitizer stability in solution during illumination. The results show that the order of susceptibility for photodegradation rate is: Radachlorin (R) < Photoditazine (R) < Foscan (R). This difference in the photodegradation rate for Foscan can be explained by the high proportion of aggregates in solution that inhibit the photo-oxidative process that impede the singlet oxygen formation. We hypothesize that there is a correlation between photodegradation rate and photodynamic efficacy witch is governed by the singlet oxygen formation responsible for the most relevant reaction of the cell death photodynamic induction. Then its is important to know the photostability of different types of drugs since the photodegradation rate, the photodegradation as well as the photodynamic efficacy are strong correlated to the oxygen concentration in the tissue.

Aggregation susceptibility on phototransformation of hematoporphyrin derivatives

Photosensitizers used in PDT suffer degradation by light. In this work, photobleaching of Photogem((R)) (PG), Photofrin((R)) (PF), and Photosan((R)) (PS), hematoporphyrin derivatives, were induced by light in the presence or absence of 1% Triton X-100. The degradation efficiency in the absence of 1% Triton X-100 follows the sequence: Pf > PF > Ps, which means that PF presented a greater degradation than PF and PS. Forever, in the presence of the surfactant the degradation efficiency is different: PF congruent to PS > PF. Besides aggregation susceptibility, studies in cell culture (tumor and non tumor cells) and in animals (depth of necrosis) were performed, trying to correlate the stability of these photosensitizers with their photodynamic effect. The results suggest that PF presents higher light induced photo-cytotoxicity than PF and PS for both types of cells. For the depth of necrosis studies, more aggregated photosensitizer showed a longer time to accumulate in liver (30 min for PG, 120 h for PF and 720 h for PS). The, to establish an ideal dosimetry in PDT, one must consider the intrinsic physical chemistry characteristics of the photosensitizer as well as their ability to undergo photobleaching.

Fungicidal effect of photodynamic therapy against fluconazole-resistant Candida albicans and Candida glabrata

P>Although photodynamic therapy (PDT) has shown great promise for the inactivation of Candida species, its effectiveness against azole-resistant pathogens remains poorly documented. This in vitro study describes the association of Photogem (R) (Photogem, Moscow, Russia) with LED (light emitting diode) light for the photoinactivation of fluconazole-resistant (FR) and American Type Culture Collection (ATCC) strains of Candida albicans and Candida glabrata. Suspensions of each Candida strain were treated with five Photogem (R) concentrations and exposed to four LED light fluences (14, 24, 34 or 50 min of illumination). After incubation (48 h at 37 degrees C), colonies were counted (CFU ml-1). Single-species biofilms were generated on cellulose membrane filters, treated with 25.0 mg l-1 of Photogem (R) and illuminated at 37.5 J cm-2. The biofilms were then disrupted and the viable yeast cells present were determined. Planktonic suspensions of FR strains were effectively killed after PDT. It was observed that the fungicidal effect of PDT was strain-dependent. Significant decreases in biofilm viability were observed for three strains of C. albicans and for two strains of C. glabrata. The results of this investigation demonstrated that although PDT was effective against Candida species, fluconazole-resistant strains showed reduced sensitivity to PDT. Moreover, single-species biofilms were less susceptible to PDT than their planktonic counterparts.

Non-homogeneous liver distribution of photosensitizer and its consequence for photodynamic therapy outcome

Background: Photodynamic therapy is mainly used for treatment of malignant lesions, and is based on selective location of a photosensitizer in the tumor tissue, followed by light at wavelengths matching the photosensitizer absorption spectrum. In molecular oxygen presence, reactive oxygen species are generated, inducing cells to die. One of the limitations of photodynamic therapy is the variability of photosensitizer concentration observed in systemically photosensitized tissues, mainly due to differences of the tissue architecture, cell lines, and pharmacokinetics. This study aim was to demonstrate the spatial distribution of a hematoporphyrin derivative, Photogem(R), in the healthy liver tissue of Wistar rats via fluorescence spectroscopy, and to understand its implications on photodynamic response. Methods: Fifteen male Wistar rats were intravenously photosensitized with 1.5 mg/kg body weight of Photogem(R). Laser-induced fluorescence spectroscopy at 532nm-excitation was performed on ex vivo liver slices. The influence of photosensitizer surface distribution detected by fluorescence and the induced depth of necrosis were investigated in five animals. Results: Photosensitizer distribution on rat liver showed to be greatly non-homogeneous. This may affect photodynamic therapy response as shown in the results of depth of necrosis. Conclusions: As a consequence of these results, this study suggests that photosensitizer surface spatial distribution should be taken into account in photodynamic therapy dosimetry, as this will help to better predict clinical results. (C) 2010 Elsevier B.V. All rights reserved.