Experimental evidence and physiological significance of the ascorbate passive diffusion through the lipid bilayer.

The diverse range of functions performed by ascorbate in many metabolic processes requires its effective redistribution between various aqueous body compartments. It is believed that this hydrophilic molecule needs protein transporters for crossing the biological membrane barriers. Any effective model reflecting the ascorbate distribution within the body requires bi-directional fluxes, but only the ascorbate transporters facilitating its intake by cells have been identified to date. The cellular efflux of this molecule still lacks proper mechanistic explanation, nevertheless data suggesting possible passive ascorbate transport recently appeared. In the paper, we provide experimental evidences that ascorbate associates efficiently with the lipid bilayer interface and slowly crosses its hydrophobic core. The measured logPmembrane/water and membrane permeability coefficient equal to 3 and 10-7 - 10-8 cm/s, respectively. The ascorbate passive diffusion across the lipid bilayer provides the missing element needed for the construction of a consistent physiological model describing the ascorbate local homeostasis. The model was effectively used for the construction of the mechanistic description of the processes, which facilitate the ascorbate homeostasis in the brain.

Effects of electroformation protocol parameters on quality of homogeneous GUV populations.

Giant unilamellar vesicles (GUVs) have become one of extensively studied biological bilayer models especially when investigating topological and mechanical properties of cell membranes. They are also used to visualize membrane-related phenomena. However, the method of preparation and the effects of parameters of preparation on the vesicular structure are extensively varied. Therefore, it is important to understand how the process of formation of GUVs influences the outcome population, as it can influence the outcome of the experiment that is planned. Therefore, in this study, we investigated the effects of protocol parameters of electroformation on properties of homogeneous population of POPC GUVs. The parameters investigated in this study are duration of electroformation, usage of electrodes and frequency of applied AC field and its voltage. The properties investigated, which can be used to describe GUV populations are average diameter of vesicle, the amount of lipid molecules in population, and structure of vesicles. According to our results, prolonged time (greater than 4 h) does not influence outcome; however, parameters of applied electrical field (voltage and frequency) did significantly influence the properties of obtained POPC GUV populations.

Alterations of biomechanics in cancer and normal cells induced by doxorubicin.

Mechanical properties of biological structures play an important role in regulating cellular activities and are critical for understanding metabolic processes in cancerous cells and the effects of drugs. For some cancers, such as acute myeloid leukaemia, chemotherapy is one of preferential methods. However, due to the lack of selectivity to cancer cells, cytostatic agents cause toxicity to normal tissues. Here, we study the effect of doxorubicin (DOX) on the mechanical properties of DNA molecules, leukemic blast cells and erythrocytes, using optical tweezers. In addition, we controlled the subcellular distribution of the drug by confocal microscopy. Our results indicated that doxorubicin affects mechanical properties of cellular structures. In all cases the drug reduced mechanical strength of examined objects. For the leukemic cells the drug subcellular distribution was predominantly nuclear with some particulate cytoplasmic fluorescence. In erythrocytes, doxorubicin showed fluorescence mainly in cytoplasm and plasma membrane. The lowering of blast cells stiffness may be due to the interaction of doxorubicin with nuclear structures, especially with nucleic acids, as our studies with DNA confirmed. In addition, it is known that DOX inhibits the polymerization of actin and thus cytoskeletal modification may also be important in reducing of cell mechanical strength. In the case of erythrocytes - the non-nucleated cells, a significant effect on the decrease of cell stiffness, besides the cytoskeleton, may have the interaction of the drug with the cell membrane. Experiments with model phospholipid membranes confirmed that observed increase in cell elasticity originates, among other things, from the drug incorporation in the lipid membrane itself. The lowering of mechanical strength of leukemic cells may have an significant impact on the effectiveness of chemotherapy. However, the fact that doxorubicin interacts not only with proliferating cancer cells, but also with the health ones may explains the high toxicity of the drug at the therapeutic concentrations. Our observations also suggest that chemotherapy with doxorubicin may decrease the risk of vascular complications in acute leukemia, due to increasing the cell elasticity.

Changes in lipid membrane mechanics induced by di- and tri-phenyltins.

Organotin compounds, being biologically active, affect a variety of cellular functions due to their ability to accumulate in and penetrate biological membranes. These compounds influence the distribution of electrostatic charges, alter organization, disrupt molecular dynamics and change mechanical properties of biological membranes. It was found that the membrane/water partition coefficient equals 4, a value significantly higher than octanol/water partition coefficient. In addition, the effect of di- and tri-phenyltin chlorides on the mechanics of model lipid membranes was measured for the first time. It has been determined that phenyltins affect the global model lipid bilayer properties by reducing the membrane expansion modulus, when measured using micromanipulation technique, and elevating the bending rigidity coefficient of the lipid bilayer, as determined with the flickering noise spectroscopy. In addition, the elevated water permeability shows that phenyltins also cause the local defects formation in the lipid bilayer, i.e. lipid pores. These data shows that phenyltins may interfere indirectly with variety cellular processes by altering non-specifically the entire cellular membrane system. Accordingly, when phenyltins are added to macrophages in culture, they inflict massive alterations of cell morphology and interfere with membrane-associated processes, as visualized using fluorescence labelling of selected subcellular compartments.

The modified fluorescence based vesicle fluctuation spectroscopy technique for determination of lipid bilayer bending properties.

Lipid bilayer is the main constitutive element of biological membrane, which confines intracellular space. The mechanical properties of biological membranes may be characterized by various parameters including membrane stiffness or membrane bending rigidity, which can be measured using flicker noise spectroscopy. The flicker noise spectroscopy exploits the spontaneous thermal undulations of the membrane. The method is based on the quantitative analysis of a series of microscopic images captured during thermal membrane fluctuations. Thus, measured bending rigidity coefficient depends on the image quality as well as the selection of computational tools for image processing and mathematical model used. In this work scanning and spinning disc confocal microscopies were used to visualize fluctuating membranes of giant unilamellar vesicles. The bending rigidity coefficient was calculated for different acquisition modes, using different fluorescent probes and different image processing methods. It was shown that both imaging approaches gave similar bending coefficient values regardless of acquisition time. Using the developed methodology the effect of fluorescent probe type and aqueous phase composition on the value of the membrane bending rigidity coefficient was measured. Specifically it was found that the bending rigidity coefficient of DOPC bilayer in water is smaller than that determined for POPC membrane. It has been found that the POPC and DOPC bending rigidities coefficient in sucrose solution was lower than that in water. Fluorescence imaging makes possible the quantitative analysis of membrane mechanical properties of inhomogeneous membrane.

The Effect of a Fluorophore Photo-Physics on the Lipid Vesicle Diffusion Coefficient Studied by Fluorescence Correlation Spectroscopy.

Fluorescence Correlation Spectroscopy (FCS) is a technique, which allows determination of the diffusion coefficient and concentration of fluorescent objects suspended in the solution. The measured parameter is the fluctuation of the fluorescence signal emitted by diffusing molecules. When 100 nm DOPC vesicles labeled with various fluorescent dyes (Fluorescein-PE, NBD-PE, Atto488 DOPE or ßBodipy FL) were measured, different values of diffusion coefficients have been obtained. These diffusion coefficients were different from the expected values measured using the dynamic light scattering method (DLS). The FCS was initially developed for solutions containing small fluorescent molecules therefore the observed inconsistency may result from the nature of vesicle suspension itself. The duration of the fluorescence signal may depend on the following factors: the exposure time of the labeled object to the excitation beam, the photo-physical properties (e.g., stability) of a fluorophore, the theoretical model used for the calculations of the diffusion coefficient and optical properties of the vesicle suspension. The diffusion coefficients determined for differently labeled liposomes show that its dependence on vesicle size and quantity of fluorescent probed used for labeling was significant demonstrating that the fluorescence properties of the fluorophore itself (bleaching and/or blinking) were critical factors for a correct outcome of FCS experiment. The new, based on combined FCS and DLS measurements, method for the determination of the focal volume prove itself to be useful for the evaluation of a fluorescence dye with respect to its applicability for FCS experiment.

Effect of monovalent anions on water transmembrane transport.

Biological structures consist of lipid bilayers immersed in the aqueous phase. They can be considered as a two-phase system where the two phases are separated (connected) by the transition regions (interphases), which properties are affected by both lipid and aqueous phases. Interphase structure and dynamics might influence properties and/or functioning of the lipid bilayer core, including osmotically induced water flow. It has been shown previously that the osmotically induced water flow depends on the membrane lipid composition, which determines the packing of the membrane core, but the role of the aqueous phase composition has not been studied previously. Ionic composition of an aqueous phase may affect behavior of biological structures. Chaotropic ions interact strongly with a lipid bilayer affecting its molecular packing whereas kosmotropic ions have only limited effect. In the paper result of studies on the dependence of the osmotically induced water flow on the ionic composition of the aqueous phase are presented. The stopped flow technique, with the light scattering as the detection quantity, was adopted, and a new experimental design was developed for the monitoring of the intravesicle salt concentration. Obtained results show that the strength of selected anions interactions with the lipid bilayer does not correlate with their capacity to affect the osmotically induced water flow. Specifically, the measured water permeability coefficient does not depend on the quantity and type of ions present in the aqueous phase.

The effect of lipid oxidation on the water permeability of phospholipids bilayers.

The effect of lipid oxidation on water permeability of phosphatidylcholine membranes was investigated by means of both scattering stopped flow experiments and atomistic molecular dynamics simulations. Formation of water pores followed by a significant enhancement of water permeability was observed. The molecules of oxidized phospholipids facilitate pore formation and subsequently stabilize water in the membrane interior. A wide range of oxidation ratios, from 15 to 100 mol%, was considered. The degree of oxidation was found to strongly influence the time needed for the opening of a pore. In simulations, the oxidation ratio of 75 mol% was found to be a threshold for spontaneous pore formation in the tens of nanosecond timescale, whereas 15 mol% of oxidation led to significant water permeation in the timescale of seconds. Once a pore was formed, the water permeability was found to be virtually independent of the oxidation ratio.

Fluorescence techniques for determination of the membrane potentials in high throughput screening.

The characterization of small molecules requires identification and evaluation of several predictive parameters, when selecting compounds for pharmacological applications and/or determining their toxicity. A number of them are correlated with the compound interaction with biological membranes and/or capacity to cross them. The knowledge of the extent of adsorption, partition coefficient and permeability along with the compound ability to alter membrane properties are critical for such studies. Lipid bilayers are frequently used as the adequate experimental models of a biological membrane despite their simple structure and a limited number of components. A significant number of the biologically relevant lipid bilayer properties are related to its electrostatics. Three electrostatic potentials were defined for the lipid bilayer; the intrinsic or induced surface electrostatic potential, the dipole potential and the membrane potential. Each of them was measured with dedicated methodologies. The complex measurement protocols and technically demanding instrumentation made the development of efficient HTS approaches for complete characterization of membrane electrostatics practically impossible. However, the rapid development of fluorescence techniques accompanied by rapid growth in diversity and number of dedicated fluorescent probes enabled characterization of lipid bilayer electrostatics in a moderately simple manner. Technically advanced, compact and automated workstations, capable of measuring practically all fluorescence parameters, are now available. Therefore, the proper selection of fluorescent probes with measuring procedures can be designed to evaluate drug candidates in context of their ability to alter membrane electrostatics. In the paper we present a critical review of available fluorescence methods, useful for the membrane electrostatics evaluation and discuss the feasibility of their adaptation to HTS procedures. The significance of the presented methodology is even greater considering the rapid growth of advanced drug formulations, where electrostatics is an important parameter for production processes and pharmacokinetics of the product. Finally, the potential of the membrane electrostatics to emerge as a viable pharmacological target is indicated and fluorescence techniques capable to evaluate this potential are presented.

A method to evaluate a propidium iodide association with oligonucleotides and oligonucleotide-cationic lipid interactions.

Complex molecular ensembles are frequently considered an element of new pharmacological formulations. This is especially evident in the therapies based on genetic information. In order to obtain an effective drug, it is necessary to associate a nucleic acid molecule with the components to ensure the desired aggregate structure and properties. To evaluate the progress of the supromolecular aggregate formation a range of methodologies and techniques are needed to test the quality and uniformity of the formulations. In this paper we propose a procedure which measures the association of a small molecule with nucleic acid using propidium iodide and oligonucleotides as an example. To measure propidium iodide binding constant the oligonucleotide was covalently labeled with fluorescein and then the changes in fluorescence resonance energy transfer (FRET) were determined and handled according to the acceptor-donor titration methodology. The calculated binding constants were in a good agreement with the values published previously. The developed method was then used to evaluate the extent of an oligonucleotide association with the lipid aggregates. It was found that two populations of oligonucleotides are present in all lipid samples studied. The fraction of oligonucleotides associated with liposomes rises with the increase of a cationic lipid content, reaching the constant value when the fraction of cationic lipid exceeded 20 mol%. Energy transfer data combined with these obtained in quenching experiments show that the orientation of the oligonucleotide associated with a lipid bilayer depends on the amount of surface charge.

Application of liposome based sensors in high-throughput screening systems.

High throughout screening is an approach based on the concept which assumes that when sufficiently large library of compounds are tested, the chance of discovering a new active compound is increased. In order to meet this expectation, proper testing criteria need to be devised. Those criteria should be related to the fate of a compound in the organism to have any predictive power. Not long ago, the main criteria were based exclusively on parameters defined by the maximum activity (QSAR). In this system the activity criteria have not been included therefore the compound ability to reach the target is not accounted for. Considering that, the construction of yet another set of parameters has been initiated (QSPR). The parameters are in fact semi-empirical numbers which need to be tested on real, physical models. Whereas the activity tests are straightforward, the pharmacokinetic ones are difficult and controversial. One such parameter describes the critical property of an active compound, namely its ability to cross biological membranes. This review describes new concepts in the determination of the permeability coefficient with the help of methods which are based on liposome biosensors. Two methods using fluorescence probes incorporated in the lipid bilayer of liposome are described in detail and compared to other currently available techniques.

A fluorescence method for determining transport of charged compounds across lipid bilayer.

There is a constant need for simple, economical and time-efficient methods which allow evaluating a compound's ability to penetrate the biological membrane, one of the key parameters needed to characterize biologically active compounds. In the paper we propose a new method of permeability determination. Instead of detecting the compound's concentration directly, we employ an approach in which the membrane interface is labeled with a fluorescein lipid probe; the probe is sensitive to the presence of charged compounds. The fluorescence intensity changes of the dye permanently attached to both sides of a model lipid bilayer are measured. Specifically, the time course of the fluorescence intensity changes following a rapid induction of a non-equilibrium state of the sample allows the evaluation of the membrane permeability for the compound. The method was validated by the determination of the phenyltin compound's transport through the model phosphatidylcholine unilamellar liposome bilayer.