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Maria-Minodora Iordache, Mihaela-Georgeta Moisescu, Eugenia Kovács, Tudor Savopol
Liposomes, Gentamicin, Microwaves, Fluorescence, Generalized polarization, Phase transition
Liposomes are largely used today as drug carriers. Their behavior in various chemical and physical environments should be well known in view of a rational design of vesicles of appropriate formulations. We investigated the liposomal membrane behavior under different physical and chemical conditions such as exposure to antibiotics, radiofrequecy fields and thermal changes. Measuring the Generalized Polarization of laurdan-labeled liposomes in the presence of the charged aminoglycoside gentamicin, under exposure to 2.45 GHz and under cyclic variation of temperature, the phase transition behavior of the phospholipids was characterized.
Daniela S. Iancu, C.B. Iancu, Mihaela G. Moisescu, T. Savopol, Eugenia Kovacs
Reactive oxygen species, 2’,7’-Dichlorodihydrofluorescein acetate, Microvolumetric fluorospectrometer.
Detecting reactive oxygen species (ROS) in living cells is a difficult task because of their short lifetime, presence of cellular antioxidants, risk of cell death and unwanted transformations of the detector substance. Fluorescence-based techniques are currently most widely used, with high sensitivity and easy processing of samples but the classical instruments need a high amount of samples and consumables. We optimized and validated a microvolumetric fluorescence-based method for ROS detection in living cells, using 2’,7’-dichlorodihydrofluorescein acetate (H2DCFDA) and a microvolumetric fluorospectrometer. In our experimental conditions we determined that a measurement volume of 3 µL containing between 125 and 500 cells was sufficient for a reliable measurement of oxidized 2’,7’-dichlorofluorescein fluorescence. The method was applied on H2O2 and catalase treated MEF k41 cells. The catalase preloaded cells presented a lower fluorescent signal under H2O2 stress. Our results show that this microvolumetric method is suitable for oxidative stress specific detection even when compounds able to discriminate the various molecular categories of ROS are added. The measurements could be performed with high accuracy, demonstrating that the microvolumetric method provides a sensitive, fast and cost-effective means for ROS detection in living cells.
I Roateşi, Tudor Savopol, Mihaela G. Moisescu, Eugenia Kovacs
Liposome delivery, Active targeting, Functionalized liposomes, Apob100, Endocytosis
Liposomes can be modified and functionalized with different ligands to control their biological properties, such as longevity, targeting ability, and intracellular penetration, in a desired fashion (for example in photodynamic therapy). The aim of this study was to obtain functionalized liposomes with apolipoprotein ApoB100 ligand to achieve active intracellular targeted delivery via LDL-receptor, by naturally occurring endocytotic pathway. Human malignant melanoma A375 cells were used as model cells for in vitro evaluation of cellular endocytosis efficiency. The functionalized ApoB100-liposomes were incubated with cultured cells. Incubation periods of 2 and 4 hours were used to obtain ligand-receptor cellular endocytosis and fluorescence expression was evaluated. The functionalized liposomes displayed remarkably higher intracellular transfection efficiency comparatively to simple liposomal suspension. Liposomes functionalized with ApoB100 protein could function as excellent active targeting ligands and could be promising vectors for active targeted photosensitizer delivery in photodynamic therapy.
Nicolescu, Alina; Dolenko, Brion; Bezabeh, Tedros; Stefan, Lorena-Ivona; Ciurtin, Coziana; Kovacs, Eugenia; Smith, Ian C. R.; Simionescu, Bogdan C.; Deleanu, Calin
NMR spectroscopy; Urinary metabolites; diabetes statistical classification chemometry
A NMR dataset with non-buffered urine samples consisting of 73 controls and 94 type II diabetes was subject to an in-house statistical classifier. A model was developed based on two glucose-free regions of the spectrum and those maximally discriminatory subregions selected most often by the algorithm were noted. The final classifier achieved 83.0% sensitivity and 83.6% specificity, with 83.2% overall accuracy. There were five spectral subregions selected by the algorithm as most relevant for discrimination. The protocol works well with non-buffered samples and has the potential for an automated clinical diagnosis of diabetes.
Palla-Papavlu, Alexandra; Paraico, Iurie; Shaw-Stewart, James; Dincă, Valentina; Savopol, Tudor; Eugenia; Lippert, Thomas; Wokaun, Alexander; Dinescu, Maria
Microarrays, Polymer, Biomolecules, Nanosecond, Parameters, Films
The numerous properties of liposomes, i.e., nontoxicity, biodegradability, and their ability to encapsulate different biological active substances in aqueous and lipid phase, make them perfect models of biomembranes. Liposomes made up of phospholipids may be used to study new applications such as cell targeting or, under specific experimental conditions, may be applied in micro and nano-sized biosensors.
This study demonstrates the capability of direct laser printing of liposomes in micron-scale patterns for the realization of biosensors or drug delivery systems.
The transfer experiments were carried out onto ordinary glass substrates, and optical microscopy images reveal that well-defined patterns without splashes can be obtained for a narrow range of laser transfer fluences using 193 nm irradiation and an intermediate triazene polymer. The triazene polymer with different thicknesses was used as sacrificial layer with the purpose of protecting the liposome solution from direct laser irradiation. It was found that the thickness of the sacrificial layer should exceed 150 nm to obtain clean, debris-free patterns. Moreover, the integrity of the liposomes after laser transfer was maintained as demonstrated through fluorescence microscopy. Raman spectroscopy data suggest that the chemical composition of the liposomes does not change for transfer fluences in the range of 40 to 60 mJ/cm(2).
Palla-Papavlu, Alexandra; Paraico, Iurie; Shaw-Stewart, James; Dinca, Valentina; Savopol, Tudor; Kovacs, Eugenia; Lippert, Thomas; Wokaun, Alexander; Dinescu, Maria
Journal:Applied physics a-materials science & processing
Year: 2011
DOI: 10.1007/s00339-010-6114-1
Microarrays; Polymer; Biomolecules; Nanosecond; Parameters; Films
The numerous properties of liposomes, i.e., nontoxicity, biodegradability, and their ability to encapsulate different biological active substances in aqueous and lipid phase, make them perfect models of biomembranes. Liposomes made up of phospholipids may be used to study new applications such as cell targeting or, under specific experimental conditions, may be applied in micro and nano-sized biosensors.
This study demonstrates the capability of direct laser printing of liposomes in micron-scale patterns for the realization of biosensors or drug delivery systems.
The transfer experiments were carried out onto ordinary glass substrates, and optical microscopy images reveal that well-defined patterns without splashes can be obtained for a narrow range of laser transfer fluences using 193 nm irradiation and an intermediate triazene polymer. The triazene polymer with different thicknesses was used as sacrificial layer with the purpose of protecting the liposome solution from direct laser irradiation. It was found that the thickness of the sacrificial layer should exceed 150 nm to obtain clean, debris-free patterns. Moreover, the integrity of the liposomes after laser transfer was maintained as demonstrated through fluorescence microscopy. Raman spectroscopy data suggest that the chemical composition of the liposomes does not change for transfer fluences in the range of 40 to 60 mJ/cm(2).
Following these results, one can envision that liposome patterns obtained by LIFT can be ultimately applied for in vitro and in vivo studies.
Kenaan, Mohamad; Moisescu, Mihaela G.; Savopol, Tudor; Martin, Diana; Arnaud-Cormos, Delia; Leveque, Philippe
Journal: International Journal of Microwave and Wireless Technologies
Year: 2011
Microwave exposure; FDTDG; Generalized polarization; Fluorescence measurements
An in vitro system for 2.45 GHz microwave (MW) exposure with real-time fluorescence measurements is proposed. This system is specifically designed for the measurement of those biophysical parameters of living cells or membrane models which can be quantified by spectrofluorometric methods (e. g. membrane generalized polarization (GP), membrane fluidity, membrane potential, etc.). The novelty of the system consists in the possibility to perform fluorescence measurements on the biological samples simultaneously with their exposure to MW. The MW applicator is an open ended coaxial antenna which is dipped into a cuvette. The distribution of electromagnetic field and specific absorption rate (SAR) in the cuvette are provided from a rigorous electromagnetic numerical analysis performed with a finite difference-time domain (FDTD) based tool. With this system, fluorescence measurements were used to calculate the membrane GP values of giant unilamellar vesicle suspensions that were acquired during exposure to a 1.2 W incident power. For this power, the SAR distribution and mean SAR value for the whole volume were calculated based on temperature measurements made at different positions inside the cuvette.
Doaga, I. Octavian.; Savopol, Tudor; Neagu, Monica ; Neagu, A ; Kovács, Eugenia
Cell seeding, Adsorption, Collagen scaffold, Fibroblasts, Tissue engineering, Optical density
The process of cell seeding oil biocompatible scaffolds has a major impact oil the morphological evolution of an engineered tissue because it involves all the key factors of tissue formation: cells, matrix, and their mutual interactions. In order to characterize the efficiency of cell seeding techniques, mainly static parameters are used such as cell density, cell distribution, and cell viability. Here, we present an experimental model that incorporates an optical density meter providing real-time information oil the cell seeding velocity, a relevant dynamic parameter of cell-matrix interaction. Our setup may be adapted to fit various Cell seeding protocols. A modified fluorimetric cuvette is used as bioreactor culture flask. The optical density of the magnetically stirred cell suspension is recorded by a digital optoelectronic device. We performed calibration experiments in order to prove that, in our experimental conditions, optical density depends linearly on the number of cells in the unit volume Of Suspension. Control studies showed that, during the time course of a typical experiment (up to 10 h), the cells (murine 3T3 fibroblasts) neither aggregated nor adhered significantly to the walls of the cuvette. Hence, our Setup yields the number of cells attached to the scaffold as a function of time. In order to analyze the experimental seeding curves, we built a kinetic model based on Langmuir’s adsorption theory, which was extended to include a preliminary step of integrin function recovery. We illustrate the proposed approach by two sets of experiments that involved trypsin-EDTA or only EDTA treatment (no trypsin) used to detach the cells from the culture flasks. The data indicate that in both cases cell-matrix adhesion has a sequential, two-step dynamics, but kinetic parameters and attachment site availability depend on the experimental protocol.
Gordeliy VI, Labahn J, Moukhametzianov R, Efremov R, Granzin J, Schlesinger R, Büldt G, Savopol T, Scheidig AJ, Klare JP, Engelhard M
Structural-Changes, Serine Chemotaxis, Photocycle, Receptor, Protein, Diffraction, Resolution, Membrane, Insights, Domain
Microbial rhodopsins, which constitute a family of seven-helix membrane proteins with retinal as a prosthetic group, are distributed throughout the Bacteria, Archaea and Eukaryota(1-3). This family of photoactive proteins uses a common structural design for two distinct functions: light-driven ion transport and phototaxis. The sensors activate a signal transduction chain similar to that of the two-component system of eubacterial chemotaxis(4). The link between the photoreceptor and the following cytoplasmic signal cascade is formed by a transducer molecule that binds tightly and specifically 5 to its cognate receptor by means of two transmembrane helices (TM1 and TM2). It is thought that light excitation of sensory rhodopsin II from Natronobacterium pharaonis (SRII) in complex with its transducer (HtrII) induces an outward movement of its helix F (ref. 6), which in turn triggers a rotation of TM2 (ref. 7). It is unclear how this TM2 transition is converted into a cellular signal. Here we present the X-ray structure of the complex between N. pharaonis SRII and the receptor-binding domain of HtrII at 1.94 Angstrom resolution, which provides an atomic picture of the first signal transduction step. Our results provide evidence for a common mechanism for this process in phototaxis and chemotaxis.
Sajin, Gheorghe; Kovacs, Eugenia; Moraru-Pologea, Roxana; Savopol, Tudor; Sajin, Maria
Biological cells; Effects of electromagnetic radiations; Membrane
The effects of low-level microwaves (2.45 GHz) on the membrane of human erythrocytes were studied measuring the hemoglobin loss and osmotic resistance of erythrocytes exposed to different power densities (0.025-10.0 mW/cm(2)) at different irradiation times. A significant increase of the hemoglobin loss by exposed erythrocytes as well as a strong dependence of the rate of the increase of hemoglobin loss on the initial level of spontaneous hemolysis were observed, It was found that at low power densities (0.84 and 1.36 mW/cm(2)), the hemolysis degree increases quasi-linearily with the exposure time, while at higher density (5 mW/cm(2)), this tendency is reversed after first 10 h of irradiation, It appears like long-term irradiation exerts a protective effect against spontaneous hemolysis caused by blood ageing. The osmotic fragility test performed on samples exposed to 5 mW/cm2 at different irradiation times showed that the osmotic resistance of exposed erythrocytes increases in time, reaching a maximum at the end of irradiation (60 h), while the osmotic resistance of the controls is constant.
Have any questions? We are always open to talk about our projects, creative opportunities and how we can help you.