Showing papers by "Lina Bezdetnaya published in 2011"

Redistribution of meta-tetra(hydroxyphenyl)chlorin (m-THPC) from conventional and PEGylated liposomes to biological substrates

Abstract: We used the phenomenon of previously described photoinduced fluorescence quenching and fluorescence polarization to evaluate the transfer of meta-tetra(hydroxyphenyl)chlorin (m-THPC) from commercial high-drug load liposomes to plasma proteins and model membranes. Fluorescence quenching of m-THPC in liposomes by iodide indicates that part of m-THPC in PEGylated liposomes is localized in the PEG shell, while the rest is bound to the lipid bilayer. It was shown that the two molecule pools in the commercial PEGylated liposomal formulation Fospeg® condition the characteristics of the m-THPC release kinetics. A substantial percentage of m-THPC from Fospeg® is released much faster than from the conventional liposomal formulation Foslip®. Using the technique of resonance light scattering, it was shown that partial m-THPC aggregation is present in liposomes with very high drug loads, higher in PEGylated liposomes compared to conventional ones.

mTHPC-based photodynamic therapy induction of autophagy and apoptosis in cultured cells in relation to mitochondria and endoplasmic reticulum stress

Abstract: Photodynamic therapy (PDT), an approved anticancer treatment, is reported as a potent inducer of programmed cell death (PCD) by both apoptosis and autophagy The present study investigated the kinetics of both autophagy and caspase activation in MCF-7 cells submitted to mTHPC-PDT upon condition of treatment promoting ER accumulation of mTHPC Fluence-dependent immediate cytochrome c (cyt C) release followed by caspase-9 and -7 activation at 1 h post-PDT evidenced a mitochondrial oxidative stress triggered by high light doses leading to >90% of cell death ER oxidative stress was monitored by the induction of the glucose-related protein chaperone GRP78 From 6 h post-PDT, GRP78 induction was accompanied by the conversion of LC3-I into LC3-II, the hallmark of autophagosome formation The formation of acid vesicles evidenced by fluorescence microscopy was obvious from 22 h post-PDT Twenty-four hours post-PDT, cyt C release decreased and caspase-9 cleavage disappeared, while the expression of cleaved caspase-7 remained significant At the same time, the profiles of GRP78, cleaved caspase-7 and LC3-II expression were similar irrespective of light doses In contrast to an inhibitor of caspase activation Z-VAD-FMK, the use of autophagy inhibitor, Wortmannin, impaired cytotoxicity along with an increase in caspase-7 activation These results demonstrate a valuable contribution of autophagy to cell death in mTHPC-photosensitized MCF-7 cells

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

Abstract: 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.

Evaluation of combined matrix‐assisted laser desorption/ionization time‐of‐flight and matrix‐assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry experiments for peptide mass fingerprinting analysis

Abstract: Peptide Mass Fingerprinting (PMF) is still of significant interest in proteomics because it allows a large number of complex samples to be rapidly screened and characterized. The main part of post-translational modifications is generally preserved. In some specific cases, PMF suffers from ambiguous or unsuccessful identification. In order to improve its reliability, a combined approach using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICRMS) was evaluated. The study was carried out on bovine serum albumin (BSA) digest. The influence of several important parameters (the matrix, the sample preparation method, the amount of the analyte) on the MOWSE score and the protein sequence coverage were evaluated to allow the identification of specific effects. A careful investigation of the sequence coverage obtained by each kind of experiment ensured the detection of specific peptides for each experimental condition. Results highlighted that DHB-FTICRMS and DHB- or CHCA-TOFMS are the most suited combinations of experimental conditions to achieve PMF analysis. The association (convolution) of the data obtained by each of these techniques ensured a significant increase in the MOWSE score and the protein sequence coverage. Copyright © 2011 John Wiley & Sons, Ltd.

Improvement of fluorescence diagnosis of bladder cancer with ALA dendrimers

Abstract: Fluorescence guided cystoscopy with 5-aminolevulinic acid (5-ALA) or its hexyl derivative (h-ALA) is used in bladder cancer diagnosis. However, the lack of specificity and the fast photobleaching of the induced sensitizer protoporphyrin IX (PpIX) impair the complete resection of the tumor during cystoscopy. To overcome these drawbacks, we investigated the possibility to use dendrimers bearing a high ALA payload (18 molecules). We investigated the in vitro hydrolysis of ALA and the PpIX synthesis rate with ALA equimolar concentrations of h-ALA and 18-ALA. We further instilled prodrugs intravesically in rats bearing orthotopic bladder tumors and analyzed the PpIX distribution on frozen sections by fluorescence microscopy. A slow mono-exponential hydrolysis of the dendrimers was observed, compared with the rather fast release of ALA from h-ALA. Using dendrimers, the porphyrin levels observed to 24 h didn't significantly change, thus limiting PpIX synthesis to levels observed using h-ALA. The incomplete hydrolysis of ALA from dendrimer resulted in sustained PpIX synthesis in cells up to 24 h after exposure to the prodrug. Steric hindrance to esterase access would be a rate-determining factor in sequential release of ALA residues from the dendrimer. In vivo, the best tumor vs. muscle specificity of 18-ALA dendrimers was obtained after 1h instillation following 4h resting time with a tumor to healthy urothelium rate of 3/1 and a tumor to muscle ratio of 3.5/1. The continuous synthesis of PpIX has ensured a sustained ratio about 2.5/1 to 24 h after the end of instillation of prodrug. By comparison with h-ALA or smaller dendrimers with 6-ALA residues, 18-ALA dendrimers presented an increased tumor vs. urothelium ratio. Because of their size, 18-ALA dendrimers are less prone to enter a very cohesive structure. We have confirmed by the detection of esters ALA, and PpIX in serum from rats following intravesical administration that 18-ALA dendrimers were less taken up by the systemic circulation, contrary to h-ALA or 6-ALA. Dendrimers will more easily penetrate the tumor, explaining the high tumor to muscle ratio and the deeper penetration as compared to h-ALA. The prolonged and sustained PpIX synthesis and the improved selectivity are primary indicators that ALA dendrimers could possibly overcome drawbacks of ALA/h-ALA fluorescence guided cystoscopy.