Polyelectrolyte microcapsules with entrapped multicellular tumor spheroids as a novel tool to study the effects of photodynamic therapy.

In the current study, semi-permeable alginate-oligochitosan microcapsules for multicellular tumor spheroids (MTS) generation were elaborated and tested, to estimate a response of the microencapsulated MTS (MMTS) to photodynamic therapy (PDT). The microcapsules (mean diameter 600 µm) with entrapped human breast adenocarcinoma MCF-7 cells were obtained using an electrostatic bead generator, and MMTS were generated by in vitro long-term cell cultivation. The formed MMTS were incubated in Chlorin e6 photosensitizer solution and then irradiated using 650-nm laser light. The cell viability was measured by MTT-assay in 24 h after irradiation, and histological analysis was performed. The proposed MTS-based model was found to be more resistant to the PDT than the two-dimensional monolayer cell culture model. Thus, MMTS could be considered as a promising three-dimesional in vitro model to estimate the doses of drugs or parameters for PDT in vitro before carrying out preclinical tests.

Effect of irradiation fluence rate on the efficacy of photodynamic therapy and tumor oxygenation in meta-tetra (hydroxyphenyl) chlorin (mTHPC)-sensitized HT29 xenografts in nude mice.

We present direct experimental evidence of the fluence-rate-dependent, radiation-induced variations in intratumor oxygen partial pressure (pO(2)) in HT29 human colon adenocarcinoma xenografts subjected to meta-tetra(hydroxyphenyl)chlorin (mTHPC)-based photodynamic therapy (PDT). The data establish a correlation between tumor oxygenation and treatment outcome. Tumor-bearing mice were injected with 0.3 mg/kg photosensitizer and subjected 72 h later to a 12 J/cm(2) red light dose administered at fluence rates of 5, 30, 90 and 160 mW/cm(2). A significant decrease in mean and median pO(2) was registered at approximately half of the total radiation fluence was delivered in tumors treated at rates of 160 and 90 mW/cm(2). Conversely, with the two lower fluence rates, intratumor pO(2) was maintained at levels comparable to those measured before illumination. Tumor oxygenation values registered shortly after every treatment protocol were at least equal to baseline levels, thus excluding the possibility of significant acute vessel damage during illumination. The tumor regrowth profile correlated with the pO(2) values monitored during irradiation. Tumors treated with fluence rates of 5 and 30 mW/cm(2) exhibited significantly longer tumor quadrupling times than those treated at 160 and 90 mW/cm(2). Improved tumor destruction could be expected by reducing the rate and the extent of oxygen depletion during meta-tetra(hydroxyphenyl)chlorin photodynamic therapy using low fluence rates.

Modulation of delayed type hypersensitivity in mice treated with photoproducts of various photosensitizers used in photodynamic therapy.

Photodynamic therapy is frequently accompanied by the induction of immunosuppression. The photochemical mechanisms behind the induction of this immunosuppression are not clear. The purpose of this study was to evaluate the potential of photoproducts of merocyanine 540 (MC540), protoporphyrin IX (PPIX) and hematoporphyrin derivative (HpD) to cause modulation (suppression/activation) of the T cell immune response in vivo. The approach that we have adopted is the pre-irradiation of a photosensitizer solution with the subsequent application of the products of photosensitizer photodegradation in animals. In this approach the photochemical mechanisms of type I and II are not involved in the photosensitized modification of biological targets in vivo. Using the model of delayed type hypersensitivity (DTH) reaction to sheep red blood cells in mice, we have demonstrated that the photoproducts of three essentially different photosensitizers affect T-cell immunity. The HpD photoproducts had a suppressive effect on the DTH, while products of PPIX photodegradation enhanced the DTH nearly twice. Pre-irradiated MC540 strongly modulated the DTH response, i.e. the DTH was enhanced at low doses and inhibited at higher doses. Our results strongly indicate that at least part of the photodynamic therapy-induced immunomodulation may occur via the photobleaching of photosensitizers accompanied by the generation of photoproducts, which can affect T cell immunity.