Lina Bezdetnaya

Correlation between in vivo pharmacokinetics, intratumoral distribution and photodynamic efficiency of liposomal mTHPC

Foslip is a recently designed third generation photosensitiser based on unilamellar dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol (DPPC/DPPG) liposomal formulations of meta-tetra(hydroxyphenyl)chlorine (mTHPC). The present study investigates Foslip behaviour and its photodynamic efficiency in EMT6 xenografted nude mice at different times following i.v. administration of 0.3 mg kg(-1) mTHPC in a Foslip formulation. Plasma pharmacokinetics and biodistribution were studied by high performance liquid chromatography and were described by a three compartments analysis with half-lifes of 0.13, 4.31 and 35.7 h. The highest tumour to muscle ratios were observed at 6 and 15 h post-administration. Intratumoral distribution was carried out using two photon excitation confocal microscopy. Progressive efflux from the vascular compartment was noted in favour of tumour parenchyma, which was almost completed at 15 h. The best tumour response was obtained for a drug-light interval of 6 h, interval for which mTHPC was present in both endothelial and parenchyma cells. Tumour and plasma concentrations however were far below their maximal values. Based on these observations, we assume that the presence of mTHPC in both vasculature and tumour cells is required for optimal PDT efficacy.

Fluorescence imaging of Foscan® and Foslip in the plasma membrane and in whole cells

A fluorescence microscope equipped with a condenser for total internal reflection (TIR) illumination was combined with a pulsed laser diode and a time-gated image intensifying camera for fluorescence lifetime measurements of single cells. In particular, fluorescence patterns, decay kinetics, and lifetime images of the lipophilic photosensitizers Foscan and Foslip were studied in whole cells as well as in close vicinity to their plasma membranes. Fluorescence lifetimes of both photosensitizers in cultivated HeLa cells decreased from about 8 ns at an incubation time of 3 h to about 5 ns at an incubation time of 24 h. This seems to result from an increase in aggregation (or self-quenching) of the photosensitizers during incubation. Selective measurements within or in close proximity to the plasma membrane indicate that Foscan and Foslip are taken up by the cells in a similar way, but may be located in different cellular sites after an incubation time of 24 h. A combination of TIR and fluorescence lifetime imaging microscopy (FLIM), described for the first time, appears to be promising for understanding some key mechanisms of photodynamic therapy (PDT).

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.

Fluence rate effects in meta-tetra(hydroxyphenyl)chlorin (mTHPC) photosensitized HT29 multicell tumor spheroids: comparative study with Colo26 spheroids

The present study evaluates the impact of the fluence rate of red light irradiation ((lambda) = 650 nm) on photodynamic efficacy in mTHPC-sensitized HT29 and Colo26 spheroids. Photocytotoxicity, computed as product of the cell yield and plating efficiency, increased progressively when the fluence rate was reduced from 90 to 30 mW cm-2 in both types of spheroids. The observed photocytotoxicity was mainly due to an immediate rather than a delayed cell death for HT29 spheroids. An opposite effect was observed for Colo26 spheroids. The plausible explanations could be the difference in penetration of mTHPC into the spheroids as well as their heterogeneous structure.

Effect of antioxidants on PDT treatment of cultured tumor cells

Lipid peroxidation (LP) is involved in cell damage induced by photodynamic treatment (PDT) sensitized by some lipophylic porphyrins. We investigated an effect of lipophylic antioxidant (alpha) -tocopherol and its water-soluble analog, trolox, on meta-tetra(hydroxyphenyl)chlorin (mTHPC) sensitized PDT (413 nm) of cultured human colon adenocarcinoma cells (HT29). Cell survival was measured by the 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide conversion to farmazan (MTT assay). Both antioxidants in concentrations lower than 0.1 mM did not affect photokilling of HT29 cells. These data might suggest that LP is not of crucial importance in cell damage photosensitized by mTHPC. One mM (alpha) -tocopherol or trolox decreased cell survival by ca. 15 and 13% respectively. Both antioxidants increased PDT- induced damage of HT29. Potentiation was evident as the decrease in the initial shoulder part of fluence dependence curve. We propose that antioxidants at height, pro-oxidant concentrations can potentiate PDT induced killing of tumor cells.

Increase in reactive oxygen species production and phagocytic activity of polymorphonuclear neutrophils stimulated by preirradiated hematoporphyrin-derivative solution

We have studied the influence of preirradiated by visible light hematoporphyrin derivative (HpD) solution in PBS on the production of reactive oxygen species (ROS) and phagocytosis of latex particles by rat peritoneal polymorphonuclear neutrophils (PMN), and also on the delayed type hypersensitivity reaction (DTH) to sheep red blood cells in mice. The release of ROS and phagocytic activity were observed by means of registration of the luminol- enhanced chemiluminescence (ChL) in the absence and the in the presence of latex particles. Non-irradiated HpD did not influence neither spontaneous ChL response, nor latex- activated. HpD preirradiated by 135 J/m2 did not affect spontaneous, but increased latex-activated ChL response by 20 percent. This fact indicates an increase in PMN phagocytic activity under the treatment with preirradiated HpD. Increase in preirradiation fluence up to 8.1 kJ/m2 resulted in significant enhancement of spontaneous ChL and inhibition of latex-activated ChL response of PMN. Results of spectroscopic analysis showed negligible decease in HpD Soret band after preirradiation of HpD by the highest fluences used in this study. We could not detect any significant photoproduct formation by differential absorption spectroscopy. Earlier, we have propose the photoinduced aggragation as one of the possible mechanisms of photodegradation of aqueous porphyrin solutions. In all probability, the increase in ROS production by PMN, treated with preirradiated HpD can be attributed to the phagocytosis of aggregates formed. It is possible that ROS can influence directly the DTH-effector cells leading to the observed decrease in DTH reaction level.

Quantum yields of photodegradation of protoporphyrin IX in solution: luminescent, spectrophotometric, and biological testing of photobleaching

In the present study, the biological testing of the photodegradation of protoporphyrin IX (PP IX) is proposed. The method implies preirradiation of PP IX ((lambda) < 415 nm) in buffer solution (with or without serum) with the following adding to cells and irradiation of this mixture ((lambda) < 415 nm). The increase in survival with increase in preirradiation fluence was used for estimation of rate and quantum yield of PP IX photobleaching in solutions. The quantum yield estimated by the proposed method was compared with quantum yields of photodegradation of PP IX by decay in absorbance and decay in fluorescence. The highest quantum yield of photodegradation of PP IX was obtained by the test of cells photoinactivation, the lowest by the decay monitored by absorbance. The different values of quantum yield of photodegradation are discussed in the view of photobleaching of monomeric forms which are much more photolabile than aggregates and of photoproduct formation. Biological testing of photodegradation of monomeric species of PP IX seems to be the most specific.