Magdalena Przybylo

The cellular internalization of liposome encapsulated protoporphyrin IX by HeLa cells.

The proper lipid composition of liposomes designed to carry drugs determines their surface properties ensuring their accumulation within selected tissue. The electrostatic potential and surface topology of liposomes affect the internalization by single cells. The high-resolution imaging of cancer cells and the distribution of protoporphyrin-loaded liposomes within the cytoplasm and its dependence on the liposome surface properties are presented. In the paper, HeLa cells were used to investigate the uptake of porphyrin-loaded liposomes and liposomes alone by means of confocal and differential interference contrast microscopies. The effect of liposomes surface electrostatic potential and surface topology on their intracellular distribution was evaluated. The time evolution of the intracellular distribution of liposomes labelled with Rhodamine-PE was examined on HeLa cells. These studies allow for the identification of the liposome lipid composition so the efficient delivery of the active substance to cancer cells will be achieved. The obtained results showed that neutral PC-liposomes are the most efficiently internalized by HeLa cells. Moreover, results showed that properties of liposomes affect not only the internalization efficiency of the photosensitizer but also its distribution within the cells, as revealed by colocalization measurements.

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 alteration of lipid bilayer dynamics by phloretin and 6-ketocholestanol.

Lipid bilayer properties are quantified with a variety of arbitrary selected parameters such as molecular packing and dynamics, electrostatic potentials or permeability. In the paper we determined the effect of phloretin and 6-ketocholestanol (dipole potential modifying agents) on the membrane hydration and efficiency of the trans-membrane water flow. The dynamics of water molecules within the lipid bilayer interface was evaluated using solvent relaxation method, whereas the osmotically induced trans-membrane water flux was estimated with the stopped-flow method using the liposome shrinkage kinetics. The presence of phloretin or 6-ketocholestanol resulted in a change of both, the interfacial hydration level and osmotically driven water fluxes. Specifically, the presence of 6-ketocholestanol reduced the amount and mobility of water in the membrane interface. It also slows the osmotically induced water flow. The interfacial hydration change caused by phloretin was much smaller and the effect on osmotically induced water flow was opposite to that of 6-ketocholestanol.

Improved method to evaluate the ability of compounds to destabilize the cellular plasma membrane.

In the paper, we present an improved method for evaluation of a compound ability to destabilize erythrocyte plasma membrane. The proposed method is based on the continuous monitoring of the light scattered by erythrocytes exposed to osmotic pressure differences. The kinetics of hemolysis depends on the plasma membrane mechanics and the extent of the osmotic stress. Generally, the osmotic pressure difference of approximately 150 mOsm is taken for measurements, as a result of the equal volume mixing with the physiological salt solutions. In this approach the hemolytic process completion is not established which may result in poor quality and reproducibility of the experimental data. In consequence, inaccurate parameters of the kinetic are determined due to the low quality fitting to the, widely used, single exponential model. In the paper we propose a new experimental protocol allowing to determine the extended set of parameters for kinetics of hemolysis. Namely, the method of the minimal osmotic pressure difference determination is proposed which ensures the completeness of the hemolytic process. This step allows improving the quality and exactness of the calculated parameters. The developed methodology was tested on two qualitatively different, biologically relevant, experiments; evaluation of the peptide effect on the plasma membrane properties and differentiating between human and rabbit erythrocytes.

Molecular crowding has no effect on the dilution thermodynamics of the biologically relevant cation mixtures

The ionic composition of intracellular space is rigorously maintained in the expense of high-energy expenditure. It has been recently postulated that the cytoplasmic ionic composition is optimized so the energy cost of the fluctuations of calcium ion concentration is minimized. Specifically, thermodynamic arguments have been produced to show that the presence of potassium ions at concentrations higher than 100 mM reduce extend of the energy dissipation required for the dilution of calcium cations. No such effect has been measured when sodium ions were present in the solution or when the other divalent cation magnesium was diluted. The experimental observation has been interpreted as the indication of the formation of ionic clusters composed of calcium, chloride and potassium. In order to test the possibility that such clusters may be preserved in biological space, the thermodynamics of ionic mixtures dilution in solutions containing albumins and model lipid bilayers have been measured. Obtained thermograms clearly demonstrate that the energetics of calcium/potassium mixture is qualitatively different from calcium/sodium mixture indicating that the presence of the biologically relevant quantities of proteins and membrane hydrophilic surfaces do not interfere with the properties of the intracellular aqueous phase.

Molecular machines-a new dimension of biological sciences

Abstract Biological systems are characterized by directional and precisely controlled flow of matter and information along with the maintenance of their structural patterns. This is possible thanks to sequential transformations of information, energy and structure carried out by molecular machines. The new perception of biological systems, including their mechanical aspects, requires the implementation of tools and approaches previously developed for engineering sciences. In this review paper, a biological system is presented in a new perspective as an ensemble of coordinated molecular devices functioning in the limited space confined by the biological membrane. The working of a molecular machine is presented using the example of F0F1 ATPase, and the general conditions necessary for the coordination of a large number of functional units are described.

Analysis of metal surfaces coated with europium-doped titanium dioxide by laser induced breakdown spectroscopy.

The surface passivation with titanium sol-gel coatings is a frequently used technique to control the adsorption of selected biological macromolecules and to reduce the exposure of the bulk material to biological matter. Due to the increasing number of new coating-preparation methods and new gel compositions with various types of additives, the quality and homogeneity determination of the surface covering is a critical factor affecting performance of any implanted material. While coating thickness is easy to determine, the homogeneity of the surface distribution of coating materials requires more elaborate methodologies. In the paper, the laser induced breakdown spectroscopy (LIBS) based method, capable to quantitate the homogeneity and uniformity of the europium in titanium dioxide sol-gel coatings on stainless steel surfaces prepared with two different procedures: spin-coating and dip-coating, is presented. The emission intensity of titanium has been used to determine the coating thickness whereas the relative values of europium and titanium emission intensities provide data on the coating homogeneity. The obtained results show that the spin-coating technique provides better surface coverage with titanium dioxide. However, when the surface coating compositions were compared the dip-coating technique was more reliable.

Chapter three - A Multi Time-Scale Approach of the Lipid Bilayer Dynamics

Abstract The lipid bilayer is a supramolecular aggregate with complex dynamics. Independent and collective motions of lipid molecules span a wide range of timescales. In order to understand the lipid bilayer functioning, the correlation between various motions needs to be understood. Molecules with low molecular weight, when placed in the lipid bilayer, may alter membrane properties depending on their location. The effect of two membrane active molecules, ethanol and lidocaine, on the lipid bilayer was investigated in three different timescales by means of dedicated fluorescence techniques. The interfacial water dynamics was measured using “solvent relaxation” technique at picosecond timescale. The lateral lipid molecule mobility along the lipid bilayer surface was determined with fluorescence correlation spectroscopy at millisecond timescale, and collective lipid processes, such as spontaneous lipid pore formation, were monitored with fluorescence stopped-flow technique at the timescale of seconds and longer. When the lipid bilayer was exposed to the ethanol molecules, changes at all probed timescales have been detected, whereas in the presence of lidocaine, only the alterations in interfacial water mobility and spontaneous lipid pore formation were observed. The introduction of lidocaine left the lateral lipid mobility unaffected. These results indicate that there might happen loose correlations between processes occurring in different timescales within the same lipid bilayer.