Šárka Perutková

Titanium nanostructures for biomedical applications

Titanium and titanium alloys exhibit a unique combination of strength and biocompatibility, which enables their use in medical applications and accounts for their extensive use as implant materials in the last 50 years. Currently, a large amount of research is being carried out in order to determine the optimal surface topography for use in bioapplications, and thus the emphasis is on nanotechnology for biomedical applications. It was recently shown that titanium implants with rough surface topography and free energy increase osteoblast adhesion, maturation and subsequent bone formation. Furthermore, the adhesion of different cell lines to the surface of titanium implants is influenced by the surface characteristics of titanium; namely topography, charge distribution and chemistry. The present review article focuses on the specific nanotopography of titanium, i.e. titanium dioxide (TiO2) nanotubes, using a simple electrochemical anodisation method of the metallic substrate and other processes such as the hydrothermal or sol-gel template. One key advantage of using TiO2 nanotubes in cell interactions is based on the fact that TiO2 nanotube morphology is correlated with cell adhesion, spreading, growth and differentiation of mesenchymal stem cells, which were shown to be maximally induced on smaller diameter nanotubes (15 nm), but hindered on larger diameter (100 nm) tubes, leading to cell death and apoptosis. Research has supported the significance of nanotopography (TiO2 nanotube diameter) in cell adhesion and cell growth, and suggests that the mechanics of focal adhesion formation are similar among different cell types. As such, the present review will focus on perhaps the most spectacular and surprising one-dimensional structures and their unique biomedical applications for increased osseointegration, protein interaction and antibacterial properties.

Mechanics and electrostatics of the interactions between osteoblasts and titanium surface.

Due to oxidation and adsorption of chloride and hydroxyl anions, the surface of titanium (Ti) implants is negatively charged. A possible mechanism of the attractive interaction between the negatively charged Ti surface and the negatively charged osteoblasts is described theoretically. It is shown that adhesion of positively charged proteins with internal charge distribution may give rise to attractive interaction between the Ti surface and the osteoblast membrane. A dynamic model of the osteoblast attachment is presented in order to study the impact of geometrically structured Ti surfaces on the osteoblasts attachment. It is indicated that membrane-bound protein complexes (PCs) may increase the membrane protrusion growth between the osteoblast and the grooves on titanium (Ti) surface and thereby facilitate the adhesion of osteoblasts to the Ti surface. On the other hand, strong local adhesion due to electrostatic forces may locally trap the osteoblast membrane and hinder the further spreading of osteointegration boundary. We suggest that the synergy between these two processes is responsible for successful osteointegration along the titanium surface implant.

Role of phospholipid asymmetry in the stability of inverted hexagonal mesoscopic phases.

The role of phospholipid asymmetry in the transition from the lamellar (L(alpha)) to the inverted hexagonal (H(II)) phase upon the temperature increase was considered. The equilibrium configuration of the system was determined by the minimum of the free energy including the contribution of the isotropic and deviatoric bending and the interstitial energy of phospholipid monolayers. The shape and local interactions of a single lipid molecule were taken into account. The minimization with respect to the configuration of the lipid layers was performed by a numerical solution of the system of the Euler-Lagrange differential equations and by the Monte Carlo simulated annealing method. At high enough temperature, the lipid molecules attain a shape exhibiting higher intrinsic mean and deviatoric curvatures, which fits better into the H(II) phase than into the L(alpha) phase. Furthermore, the orientational ordering of lipid molecules in the curvature field expressed as the deviatoric bending provides a considerable negative contribution to the free energy, which stabilizes the nonlamellar H(II) phase. The nucleation configuration for the L(alpha)-H(II) phase transition is tuned by the isotropic and deviatoric bending energies and the interstitial energy.

Mechanical stability of membrane nanotubular protrusions influenced by attachment of flexible rod-like proteins

It is indicated that nonhomogeneous lateral distribution of membrane attached and flexible rod-like proteins (MRPs) may stabilize nanotubular membrane protrusions. We have shown that curvature induced accumulation of MRPs in the nanotubular membrane protrusion and the corresponding reduction of the membrane free energy are possible if the decrease of the deviatoric free energy of MRPs in the nanotubular protrusions is large enough to overcome the increase of the free energy due to decrease of configurational entropy in the process of lateral sorting of MRPs. The decrease of isotropic curvature energy of MRPs in the region of membrane protrusion is usually not sufficient for substantial MRPs sorting and consequent stabilization of the nanotubular membrane protrusions.

Influence of nanoparticle-membrane electrostatic interactions on membrane fluidity and bending elasticity

The aim of this work is to investigate the effect of electrostatic interactions between the nanoparticles and the membrane lipids on altering the physical properties of the liposomal membrane such as fluidity and bending elasticity. For this purpose, we have used nanoparticles and lipids with different surface charges. Positively charged iron oxide (γ-Fe2O3) nanoparticles, neutral and negatively charged cobalt ferrite (CoFe2O4) nanoparticles were encapsulated in neutral lipid 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine and negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine lipid mixture. Membrane fluidity was assessed through the anisotropy measurements using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene. Though the interaction of both the types of nanoparticles reduced the membrane fluidity, the results were more pronounced in the negatively charged liposomes encapsulated with positively charged iron oxide nanoparticles due to strong electrostatic attractions. X-ray photoelectron spectroscopy results also confirmed the presence of significant quantity of positively charged iron oxide nanoparticles in negatively charged liposomes. Through thermally induced shape fluctuation measurements of the giant liposomes, a considerable reduction in the bending elasticity modulus was observed for cobalt ferrite nanoparticles. The experimental results were supported by the simulation studies using modified Langevin-Poisson-Boltzmann model.

Interaction between Dipolar Lipid Headgroups and Charged Nanoparticles Mediated by Water Dipoles and Ions

In this work, a theoretical model describing the interaction between a positively or negatively charged nanoparticle and neutral zwitterionic lipid bilayers is presented. It is shown that in the close vicinity of the positively charged nanoparticle, the zwitterionic lipid headgroups are less extended in the direction perpendicular to the membrane surface, while in the vicinity of the negatively charged nanoparticle, the headgroups are more extended. This result coincides with the calculated increase in the osmotic pressure between the zwitterionic lipid surface and positively charged nanoparticle and the decrease of osmotic pressure between the zwitterionic lipid surface and the negatively charged nanoparticle. Our theoretical predictions agree well with the experimentally determined fluidity of a lipid bilayer membrane in contact with positively or negatively charged nanoparticles. The prospective significance of the present work is mainly to contribute to better understanding of the interactions of charged nanoparticles with a zwitterionic lipid bilayer, which may be important in the efficient design of the lipid/nanoparticle nanostructures (like liposomes with encapsulated nanoparticles), which have diverse biomedical applications, including targeted therapy (drug delivery) and imaging of cancer cells.

Monovalent Ions and Water Dipoles in Contact with Dipolar Zwitterionic Lipid Headgroups-Theory and MD Simulations

The lipid bilayer is a basic building block of biological membranes and can be pictured as a barrier separating two compartments filled with electrolyte solution. Artificial planar lipid bilayers are therefore commonly used as model systems to study the physical and electrical properties of the cell membranes in contact with electrolyte solution. Among them the glycerol-based polar phospholipids which have dipolar, but electrically neutral head groups, are most frequently used in formation of artificial lipid bilayers. In this work the electrical properties of the lipid layer composed of zwitterionic lipids with non-zero dipole moments are studied theoretically. In the model, the zwitterionic lipid bilayer is assumed to be in contact with aqueous solution of monovalent salt ions. The orientational ordering of water, resulting in spatial variation of permittivity, is explicitly taken into account. It is shown that due to saturation effect in orientational ordering of water dipoles the relative permittivity in the zwitterionic headgroup region is decreased, while the corresponding electric potential becomes strongly negative. Some of the predictions of the presented mean-field theoretical consideration are critically evaluated using the results of molecular dynamics (MD) simulation.

Echinophilic proteins stomatin, sorcin, and synexin locate outside gangliosideM1 (GM1) patches in the erythrocyte membrane.

The detergent (Triton X-100, 4°C)-resistant membrane (DRM)-associated membrane proteins stomatin, sorcin, and synexin (anexin VII) exposed on the cytoplasmic side of membrane were investigated for their lateral distribution in relation to induced ganglioside(M1) (GM1) raft patches in flat (discocytic) and curved (echinocytic) human erythrocyte membrane. In discocytes, no accumulation of stomatin, sorcin, and synexin in cholera toxin subunit B (CTB) plus anti-CTB-induced GM1 patches was detected by fluorescence microscopy. In echinocytes, stomatin, sorcin, and synexin showed a similar curvature-dependent lateral distribution as GM1 patches by accumulating to spiculae induced by ionophore A23187 plus calcium. Stomatin was partly and synexin and sorcin were fully recruited to the spiculae. However, the DRM-associated proteins only partially co-localized with GM1 and were frequently distributed into different spiculae than GM1. The study indicates that stomatin, sorcin, and synexin are echinophilic membrane components that mainly locate outside GM1 rafts in the human erythrocyte membrane. Echinophilicity is suggested to contribute to the DRM association of a membrane component in general.

Vesiculation of biological membrane driven by curvature induced frustrations in membrane orientational ordering

1Condensed Matter Physics Department, Jožef Stefan Institute, Ljubljana, Slovenia; 2Laboratory of Biophysics, Faculty of Electrical Engineering, University of Ljubljana, Ljubljana, Slovenia; 3Department of Complex Systems and Chemical Processing of Information, Institute of Physical Chemistry, Polish Academy of Sciences, Warsaw, Poland; 4Laboratory of Clinical Biophysics, Faculty of Health Studies, University of Ljubljana, Ljubljana, Slovenia; 5Laboratory of Clinical Biophysics, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia; 6Department of Physics, Faculty of Natural Sciences and Mathematics, University of Maribor, Maribor, Slovenia

Interaction between equally charged membrane surfaces mediated by positively and negatively charged macro-ions.

In biological systems, charged membrane surfaces are surrounded by charged molecules such as electrolyte ions and proteins. Our recent experiments in the systems of giant phospholipid vesicles indicated that some of the blood plasma proteins (macro-ions) may promote adhesion between equally charged membrane surfaces. In this work, theory was put forward to describe an IgG antibody-mediated attractive interaction between negatively charged membrane surfaces which was observed in experiments on giant phospholipid vesicles with cardiolipin-containing membranes. The attractive interactions between negatively charged membrane surfaces in the presence of negatively and positively charged spherical macro-ions are explained using functional density theory and Monte Carlo simulations. Both, the rigorous solution of the variational problem within the functional density theory and the Monte Carlo simulations show that spatial and orientational ordering of macro-ions may give rise to an attractive interaction between negatively charged membrane surfaces. It is also shown that the distinctive spatial distribution of the charge within the macro-ions (proteins) is essential in this process.