Layla Pires

Photodynamic Therapy in Pythium insidiosum – An In Vitro Study of the Correlation of Sensitizer Localization and Cell Death

Pythiosis is an infectious disease caused by Pythium insidiosum, a fungus-like organism. Due to the lack of ergosterol on its cell membrane, antibiotic therapy is ineffective. The conventional treatment is surgery, but lesion recurrence is frequent, requiring several resections or limb amputation. Photodynamic therapy uses photo-activation of drugs and has the potential to be an attractive alternative option. The in vitro PDT response on the growing of Pythium insidiosum culture was investigated using three distinct photosensitizers: methylene blue, Photogem, and Photodithazine. The photosensitizer distribution in cell structures and the PDT response for incubation times of 30, 60, and 120 minutes were evaluated. Methylene blue did not penetrate in the pathogens cell and consequently there was no PDT inactivation. Photogem showed heterogenous distribution in the hyphal structure with small concentration inside the cells. Porphyrin-PDT response was heterogenous, death and live cells were observed in the treated culture. After 48 hours, hyphae regrowth was observed. Photodithazine showed more homogenous distribution inside the cell and with the specific intracellular localization dependent on incubation time. Photodithazine first accumulates in intracellular vacuoles, and at incubation times of one hour, it is located at all cell membranes. Higher inhibition of the growing rates was achieved with Photodithazine -PDT, over 98%. Our results showed that the photosensitizers that cross more efficiently the Pythium insidiosum membranes are able to cause extensive damage to the organism under illumination and therefore, are the best options for clinical treatment.

Photodynamic Therapy for Pythiosis

BACKGROUND Pythiosis is a life-threatening disease caused by Pythium insidiosum. Photodynamic therapy (PDT) is an alternative treatment to surgery that uses the interaction of a photosensitizer, light and molecular oxygen to cause cell death. OBJECTIVES To evaluate the effect of PDT on the in vitro growth of P. insidiosum and in an in vivo model of pythiosis. METHODS For in vitro studies, two photosensitizers were evaluated: a haematoporphyrin derivative (Photogem(®)) and a chlorine (Photodithazine(®)). Amphotericin B was also evaluated, and the control group was treated with sterile saline solution. All experiments (PDT, porphyrin, chlorine and light alone, amphotericin B and saline solution) were performed as five replicates. For in vivo studies, six rabbits were inoculated with 20,000 zoospores of P. insidiosum, and an area of 1 cm(3) was treated using the same sensitizers. The PDT irradiation was performed using a laser emitting at 660 nm and a fluence of 200 J/cm(2) . Rabbits were clinically evaluated daily and histopathological analysis was performed 72 h after PDT. RESULTS For in vitro assays, inhibition rates for PDT ranged from 60 to 100% and showed better results in comparison to amphotericin B. For the in vivo assays, after PDT, histological analysis of lesions showed a lack of infection up to 1 cm in depth. CONCLUSIONS AND CLINICAL IMPORTANCE In vitro and in vivo studies showed that PDT was effective in the inactivation of P. insidiosum and may represent a new approach to treating pythiosis.

Optical clearing of melanoma in vivo: characterization by diffuse reflectance spectroscopy and optical coherence tomography

Abstract. Melanoma is the most aggressive type of skin cancer, with significant risk of fatality. Due to its pigmentation, light-based imaging and treatment techniques are limited to near the tumor surface, which is inadequate, for example, to evaluate the microvascular density that is associated with prognosis. White-light diffuse reflectance spectroscopy (DRS) and near-infrared optical coherence tomography (OCT) were used to evaluate the effect of a topically applied optical clearing agent (OCA) in melanoma in vivo and to image the microvascular network. DRS was performed using a contact fiber optic probe in the range from 450 to 650 nm. OCT imaging was performed using a swept-source system at 1310 nm. The OCT image data were processed using speckle variance and depth-encoded algorithms. Diffuse reflectance signals decreased with clearing, dropping by ∼90% after 45 min. OCT was able to image the microvasculature in the pigmented melanoma tissue with good spatial resolution up to a depth of ∼300  μm without the use of OCA; improved contrast resolution was achieved with optical clearing to a depth of ∼750  μm in tumor. These findings are relevant to potential clinical applications in melanoma, such as assessing prognosis and treatment responses. Optical clearing may also facilitate the use of light-based treatments such as photodynamic therapy.

Evaluation of the Photodynamic Therapy effect using a tumor model in Chorioallantoic Membrane with Melanoma cells

Photodynamic Therapy (PDT) is a type of cancer treatment that is based on the interaction of light (with specific wavelength), a photosensitizing agent and molecular oxygen. The photosensitizer (PS) is activated by light and reacts with oxygen resulting in the production of singlet oxygen that is highly reactive and responsible for the cell death. The Chick Chorioallantoic Membrane (CAM) model is a transparent membrane that allows visualization and evaluation of blood vessels and structural changes, where a tumor model was developed. Two induction tumor models were investigated: tumor biopsy or cell culture. It was used a murine melanoma cell B16F10 in culture and a biopsy from a xenograft tumor in hairless mouse. Two PS were tested: Photodithazine® and Photogem®, a chlorine and porphyrin compounds, respectively. Using intravenous administration, the light-drug interval was of 30 minutes, 1 and 3 hours. Illumination was performed at 630 nm and 660 nm, and the vascular and tumor response was monitored and analyzed. The PS distribution was checked with confocal microscopy. This model can be useful to study several parameters of PDT and the effect of this therapy in the cancer treatment since it allows direct visualization of its effects.

Subcellular localization and photodynamic activity of Photodithazine (glucosamine salt of chlorin e6) in murine melanoma B16-F10: an in vitro and in vivo study

Photodynamic therapy (PDT) is already a good option for the clinical treatment of several lesions, including mainly nonmelanoma skin cancers. However, cutaneous melanoma treatment remains a challenge when using PDT. One of the reasons for its reduced efficacy is the high pigmentation of melanoma cells. The object of our study is to evaluate the feasibility of the Photodithazine as a photosensitizer for melanoma. Photodithazine is already used in some malignant tumors with satisfactory results and has significant absorption band around 660 nm where the absorption of melanin is low. In this study, we measured the subcellular localization and photodynamic activity of Photodithazine (PDZ) in murine melanoma B16-F10 cell culture. Additionally, a PDT procedure was applied in an animal melanoma model. This first result demonstrates that Photodithazine is more localized at mitochondria in B16F10 cell culture and the cell viability is reduced to less than 90% using 1 µg/mL (PDZ) and 2 J/cm2. We also noticed a rapid PDZ (less than one hour) accumulation in a murine melanoma model. The treatment of melanoma resulted in 20 % more animal survival after one session of PDT compared with the control group. More studies are required to evaluate the cytotoxic effects of Photodithazine at human melanoma.