Showing papers by "Lina Bezdetnaya published in 2008"

Quantum dots in axillary lymph node mapping: Biodistribution study in healthy mice

Abstract: Background Breast cancer is the first cause of cancer death among women and its incidence doubled in the last two decades. Several approaches for the treatment of these cancers have been developed. The axillary lymph node dissection (ALND) leads to numerous morbidity complications and is now advantageously replaced by the dissection and the biopsy of the sentinel lymph node. Although this approach has strong advantages, it has its own limitations which are manipulation of radioactive products and possible anaphylactic reactions to the dye. As recently proposed, these limitations could in principle be by-passed if semiconductor nanoparticles (quantum dots or QDs) were used as fluorescent contrast agents for the in vivo imaging of SLN. QDs are fluorescent nanoparticles with unique optical properties like strong resistance to photobleaching, size dependent emission wavelength, large molar extinction coefficient, and good quantum yield.

Photodynamic therapy with intratumoral administration of Lipid-Based mTHPC in a model of breast cancer recurrence

Abstract: The weak intratumoral fluorescence at early time points could be explained by concentration quenching within the liposomes as evidenced from fluorescence polarization studies. Progressive mTHPC redistribution from liposomes and its further incorporation into tumor tissue resulted in fluorescence build-up over time with a maximum at 24 hours post-injection. This correlates perfectly with the best therapeutic effect at this time point. The absence of total cure can be attributed to inhomogeneous photosensitizer distribution. mTHPC is reabsorbed into the blood stream but the total administered amount is much reduced as opposed to systemic administration so that repeated PDT sessions might be favorable in terms of side effects and tumor response.

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

Abstract: 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).

Analysis of differential PDT effect in rat bladder tumor models according to concentrations of intravesical hexyl-aminolevulinate

Abstract: The hexylester of 5-aminolevulinic acid (HAL) is a very efficient precursor of the photosensitizer protoporphyrin IX (PpIX) for photodynamic therapy (PDT). Our previous study, performed in rat orthotopic bladder tumors, indicated an opposite effect of HAL/PpIX-PDT according to HAL concentration. The present study investigated possible reasons for this differential effect considering the impact of extracted amounts of PpIX in normal and tumor bearing bladders along with PpIX distribution in distinctive histopathological layers. High performance liquid chromatography (HPLC) analysis of tumor and normal bladder tissues after 8 mM and 16 mM HAL instillation showed that PpIX was the main porphyrin species. The PpIX production in tumor bladders instilled with 8 mM HAL was significantly higher than after 16 mM HAL. Fluorescence confocal microscopy demonstrated a punctuate bright fluorescence pattern in tumor zones of bladders instilled with 8 mM HAL, whereas a more diffuse cytoplasmatic fluorescence distribution was observed after 16 mM HAL instillation. Immunofluorescence staining together with transmission electron microscopy showed severe mitochondrial damage in tumor zones of bladders treated with 8 mM HAL/PpIX PDT, with intact mitochondria in tumor zones of bladders treated with 16 mM HAL/PpIX PDT. We conclude that the differential response to HAL/PpIX PDT in function of HAL concentrations could be attributed to diminished PpIX synthesis and differential intracellular localisation of PpIX. Mitochondria were shown to be the critical photodamaged sites of HAL/PpIX PDT and as such tissue sensitivity to treatment can be estimated through investigation of intracellular PpIX distribution.

Photodynamic effects of chlorine e6 on bioencapsulated tumour spheroids

Abstract: Introduction Since Sutherland developed the multicellular tumor spheroid (MTS) model to mimic the 3D-structure of small size solid tumors (R. Sutherland et al., 1970), MTS have been found to be useful in several aspects of tumor biology, including studies in the field of radiation biology and photodynamic therapy (PDT). Cellular organization of MTS allows to imitate in vivo small size tumors much better than 2D in vitro models (G. Hamilton, 1998). MTS were demonstrated to represent quite realistically the 3D growth and organization of solid tumors, and consequently to simulate well the cell-cell interactions and microenvironmental conditions found in tumor tissue. This similarities to a tumor xenograft let us apply MTS as a more rapid and valid in vitro model for anticancer drug screening compared to a monolayer culture. MTS could be formed from monolayer tumor cells grown by various in vitro classical methods, such as liquid-overlay, spinner flask and gyratory rotation systems. At the same time all classical methods are time consuming and can not provide the production of MTS with narrow spheroid size distribution within a range of 300 – 900 µm. More over, some tumor cells cannot form spheroids in suspension. The method proposed by Markvicheva et al. (Markvicheva et al., 2003) for microencapsulated MTS production provides several advantages over all classical techniques, such as generation of significant spheroid quantities, production of MTS of desired sizes, generation of MTS based on tumor and non-tumor cells which normally can't form aggregates in suspension culture. The objective of this research was to estimate the response of a novel in vitro model based on encapsulated MTS to PDT. Chlorine e6 was chosen as a model photosensitizer. Materials and Methods Chemicals: Sodium alginate (medium viscosity), EDTA and CaCl2 were from Sigma. All solutions for cell immobilization were prepared using 0.9% NaCl. Oligochitosan (MM 3500 Da, DD 98 %) was kindly provided by Prof. A.Bartkowiak (Poland). Chlorine e6 (Ce6) was supplied by Porphyrin Products (Logan, UT, USA). Ce6 stock solution (2 mM) was prepared in dimethyl sulfoxide (DMSO) and stored at -20oC. Before being added into the cell cultures, Ce6 was further diluted in the culture medium. All solutions for cell immobilization were prepared using 0.9% NaCl. Cells and cell cultivation media: In our study MCF-7 human adenocarcinoma cell line was used. The cells were cultured as a monolayer in DMEM medium supplemented with 10 mg/l human insulin, 10 % fetal calf serum (FCS) BioClot at 370C in a 5 % CO2 humidified atmosphere and were reseeded into fresh medium every 2-3 days. Bioencapsulation of tumor cells in microcapsules: Cell precipitate (107 cells), obtained by trypsinization of monolayer culture was mixed with 2 ml of a sterilized sodium alginate solution, and the mixture was extruded using an electrostatic bead generator into 0.5 % CaCl2 by peristaltic pump. The obtained hydrogel microbeads were incubated with 0.2 % oligochitosan solution for 10 min, in order to form alginate-oligochitosan membrane on microbeads surface. Then microbeads were washed 3 times with physiological saline. In order to get hollow microcapsules, the microbeads were incubated in 50 mM EDTA solution for 10 min and they were again washed and transferred into cultivation medium. Empty microcapsules were prepared as mentioned above. Cultivation of bioencapsulated cells to generate MTS: The resulted alginate-oligochitosan microcapsules with MCF-7 cells were cultivated in RPMI medium supplemented with 10 % FBS in 150 cm2 (Corning Inc.) flasks at 370C in 5 % CO2 for 2-4 weeks until MTS were formed. Ce6 non-specific adsorption on microcapsule surface: Microcapsules were incubated in Ce6 solution in the darkness at various final Ce6 concentrations for 24 h. Then supernatants and microcapsules were washed in the physiological solution, and were analysed using a computer-controlled luminescence spectrofluorimetre (Perkin-Elmer LS50B). The excitation wavelength was 410 nm, and spectra were collected at emission wavelength ranged between 600 – 800 nm. Ce6 solution in the same conditions but without microcapsules was used as a control. Ce6 cytotoxicity: Encapsulated MTS (100 µl) were incubated with Ce6 (0 - 34 nM) in 24-well plates for 24h. Each well contained 0.5 ml of RPMI medium supplemented with 2 % FBS. Cell viability was measured using MTT-assay. MCF-7 cells growing in monolayer were used as control. The cytotoxicity was expressed in the form of the viability using the following formula: Viability (%) = (Viable cells concentration in experiment / Viable cells concentration in control) x 100. Every experiment was repeated three times. Ce6 photoxicity: Encapsulated MTS (100 µl) were incubated with Ce6 (8.4 nM) in 24-well plates for 24h. Then MTS were washed with PBS 3 times, and 0.5 ml of RPMI was added in each well. The cells were irradiated by 650 nm diode laser (Coherent, France). Light energy densities were 0.5 – 70 J cm-2 at power density 30 mW cm-2. The cell viability was measured using MTT-assay in 24 h after irradiation. To study the structure of MTS before and after irradiation at various Ce6 concentrations, a set of MTS samples was selected. The samples were fixed in a 2 % (w/v) formaldehyde solution and embedded to the paraffin to prepare thin sectioned on slides. Results and Discussion Cell encapsulation was carried out using a special device, namely an electrostatic bead generator. The best calcium-alginate microbeads with a narrow bead size distribution (within the range of 300 – 600 µm, a mean size 340 ± 40 µm) were prepared using the voltage of 7.8 kV (Tabl.1). At the same time alginate-oligochitosan microcapsules mean diameter was much bigger (608 ± 50 µm) that that one of microbeads (Fig. 1). In order to understand the Ce6 non-specific adsorption on microcapsule surface, microcapsules were incubated with Ce6 solution for 24 h. The non-specific Ce6 sorption by empty microcapsules was 30 nmol per 1 ml of microcapsule slurry. Therefore the Ce6 sorption on the microcapsule surface could be ignored, and there was no necessity to remove the microcapsule membrane at photodynamic treatment of encapsulated MTS. To get MTS model, tumor cells (MCF-7) were microencapsulated and cultivated in 150 ml T-flasks in a 5 % CO2 atmosphere at 37 oC for 2 – 4 weeks. The cell proliferation has been easily observed by light microscope (Leitz, Germany). The cells grew in aggregates which have been increasing in their sizes with the cultivation time. The cell concentration in obtained encapsulated MTS was 5 x 106 cells/ml slurry. The cytotoxicity of Ce6 was estimated as an inhibition rate in cell viability. The cytotoxicity increased with increasing photosensitizer concentration in both encapsulated MTS and monolayer culture, but the viability of encapsulated MTS was higher than that one in monolayer culture (Fig. 2). Results obtained for MCF-7 monolayer were in agreement with data previously reported (JL. Merlin et al., 2003). Maximal non-toxic concentrations were 8.4 µM and 1.7 µM for encapsulated spheroids and for monolayer culture, respectively. These results revealed that the difference between monolayer culture and MTS was rather remarkable even for these previous experiments. These two concentrations were chosen for the next experiments. Parameter Value Voltage (Electrostatic bead generator) 7.8 kV Flow rate(peristaltic pump) 0.5 ml/min Tube diameter(peristaltic pump) 1.3 mm Needle diameter 0.3 mm Sodium alginate solution concentration 1.3 % (w/v) Table 1: Optimized conditions for microcapsule preparation technique using an electrostatic bead generator. Figure 1: Microcapsule size distribution in RPMI medium (a mean diameter is 608±50 µm; a membrane thickness is 70 ± 5 µm). Figure 2: Cell viability in monolayer model (▲) and MTS model (▄) after incubation with Ce6 for 24 h. Figure 3: Cell viability in monolayer model (▲) and MTS model (▄) 24h after PDT at light energy densities 1-70 J cm-2 As can be seen in Fig. 3, phototoxicity increased with light energy density enhance both for spheroids and monolayer culture. However, the cell viability of the encapsulated MTS was higher than that one of monolayer culture, in spite of the fifth-fold Ce6 concentration taken for MTS. For instance, a percentage of viable cells in MTS was tree times bigger compared to monolayer culture at light energy density 10 J cm-2. Conclusions Biocompatible polyelectrolyte microcapsules were used to generate MCF-7 cell based encapsulated MTS. Our results demonstrated that the proposed MTS model was much more resistant to the photodynamic treatment than monolayer model. We concluded that the encapsulated MTS model could mimic small size solid tumors more precisely, than commonly used classical monolayer model. Acknowledgments The authors are greatful to FEBS for support of D. Zaytseva-Zotova with FEBS Collaborative Experimental Scholarship for Central & Eastern Europe. References 1. R. Sutherland et al. (1970) Growth of nodular carcinomas in rodents compared with multi-cell spheroids in tissue culture. Growth 34 271-272 2. G. Hamilton. (1998) Multicellular tumor spheroids as an in vitro model. CancerLett 131 29 3. E. Markvicheva et al. (2003) Encapsulated multicellular tumor spheroids as a novel in vitro model to study small size tumors. ChemInd 57 585-588 4. JL. Merlin et al. (2003) The multidrug resistance modulator SDZ-PSC 833 potentiates the photodynamic activity of chlorine e6 independently on P-glycoprotein in multidrug resistant human breast adenocarcinoma cells. Int J Oncol 22 (4) 733-739