Kinetics of the adhesion of DMPC liposomes on a mercury electrode. Effect of lamellarity, phase composition, size and curvature of liposomes, and presence of the pore forming peptide mastoparan X

Liposomes suspended in aqueous electrolyte solutions can adhere at mercury electrodes. The adhesion is a complex process that starts with the docking and opening and leads to a spreading, finally resulting in the formation of islands of adsorbed lecithin molecules. The adhesion process can be followed by chronoamperometry, and a detailed analysis of the macroscopic and microscopic kinetics can be performed yielding rate constants and activation parameters. By using giant unilamellar liposomes and multilamellar liposomes, the effect of lamellarity and liposome size could be elucidated for liposomes in the liquid crystalline, gel, and superlattice phase states. Below the phase transition temperature, the time constant of opening of the liposomes (i.e., the irreversible binding of the lecithin molecules on the preliminary contact interface liposome|mercury and the therewith associated disintegration of the liposome membrane on that spot) is shown to be strongly size dependent. The activation energy, however, of that process is size independent with the exception of very small liposomes. That size dependence of time constants is a result of the size dependence of the initial contact area. The time constant and the activation energies of the spreading step exhibit a strong size dependence, which could be shown to be due to the size dependence of rate and activation energy of pore formation. Pore formation is necessary to release the solution included in the liposomes. This understanding was corroborated by addition of the pore inducing peptide Mastoparan X to the liposome suspension. The obtained results show that electrochemical studies of liposome adhesion on mercury electrodes can be used as a biomimetic tool to understand the effect of membrane properties on vesicle fusion.     

The lipid composition determines the kinetics of adhesion and spreading of liposomes on mercury electrodes

The dependence of membrane properties on their composition was studied by following the adhesion and spreading of unilamellar and multilamellar liposomes on static mercury electrodes with the help of chronoamperometry. The analysis of the peak-shaped signals allows determining the kinetic parameters of the three-step adhesion-spreading process. The presence of cholesterol in the membrane stabilizes the bilayer in the liquid-crystalline phase, and destabilizes the gel phase. The kinetic parameters also show the effect of superlattice formation in the DMPC-cholesterol system. The detergent triton X-100 is only incorporated in the liquid-crystalline DMPC membranes, and it is expelled to the solution when the membrane is transformed to the gel phase. In the liquid-crystalline membrane, it enhances the adhesion-spreading of liposomes on mercury. The lytic peptides mastoparan X and melittin affect the adhesion-spreading in a similar manner. For the rupture-spreading step, their effect is explained by pore formation. The results obtained with lecithins of different length suggest that the bilayer opening process has much in common with flip-flop translocations. For this process the activation energies were found to be independent of the chain length of the lecithin molecules, while the preexponential factor in the Arrhenius equation decreases drastically for longer chains.     

Influence of the pore size of reversed phase materials on peptide purification processes

The influence of the pore size of a chromatographic reversed phase material on the adsorption equilibria and diffusion of two industrially relevant peptides (i.e. a small synthetic peptide and insulin) has been studied using seven different reversed phase HPLC materials having pore sizes ranging from 90 Å to 300 Å. The stationary phase pore size distribution was obtained by inverse size exclusion measurement (iSEC). The effect of the pore size on the mass transfer properties of the materials was evaluated from Van Deemter experiments. It has been shown that the lumped mass transfer coefficient increases linearly with the average pore size. The Henry coefficient and the impurity selectivity were determined in diluted conditions. The saturation capacity of the main peptides was determined in overloaded conditions using the inverse method (i.e. peak fitting). It was shown that the adsorption equilibria of the peptides on the seven materials is well described by a surface-specific adsorption isotherm. Based on this a lumped kinetic model has been developed to model the elution profile of the two peptides in overloaded conditions and to simulate the purification of the peptide from its crude mixture. It has been found that the separation of insulin from its main impurity (i.e. desamido-insulin) was not affected by the pore size. On the other hand, in the case of the synthetic peptide, it was found that the adsorption of the most significant impurity decreases with the pore size. This decrease is probably due to an increase in silanol activity with decreasing pore size.     

The overall adhesion-spreading process of liposomes on a mercury electrode is controlled by a mixed diffusion and reaction kinetics mechanism

Using high-resolution chronoamperometric measurements, with sampling each 1.333 μs, the initial step of the adhesion-spreading of liposomes on a mercury electrode was studied. These measurements allow getting a deeper insight into the first interaction of the liposomes with the mercury electrode, and they show that the overall adhesion-spreading process at different potentials is partially controlled by a fast but weak interaction equilibrium resulting in a mixed diffusion- and reaction-kinetics-controlled mechanism of the overall reaction. The authors dedicate this contribution to Keith Oldham on the occasion of his 80th birthday. Since my (FS) first meeting with Keith Oldham in Alan Bond’s laboratory in Australia in 1987, I had the privilege to get Keith’s unerring advice and have stimulating discussions with him for which I like to cordially thank him.     

Bursting and spreading of liposomes on the surface of a static mercury drop electrode

When liposomes suspended in an aqueous electrolyte solution hit the surface of a static mercury drop electrode (SMDE) they burst and spread. These events lead to capacitive signals and allow counting of liposomes as well as the determination of the number of molecules in each liposome.     

Coarse-grained molecular dynamics studies of the concentration and size dependence of fifth- and seventh-generation PAMAM dendrimers on pore formation in DMPC bilayer

We have performed molecular dynamics (MD) simulations of multiple copies of unacetylated G5 and G7 and acetylated G5 dendrimers in dimyristoylphosphatidylcholine bilayers with explicit water using the coarse-grained model developed by Marrink et al. (J. Phys. Chem. B 2007, 111, 7812) with the inclusion of long-range electrostatics. When initially clustered together near the bilayer, neutral acetylated dendrimers aggregate, whereas cationic unacetylated dendrimers do not aggregate, but separate from each other, similar to the observations from atomic force microscopy by Mecke et al. (Chem. Phys. Lipids 2004, 132, 3). The bilayers interacting with unacetylated dendrimers of higher concentration are significantly deformed and show pore formation on the positively curved portions, while acetylated dendrimers are unable to form pores. Unacetylated G7 dendrimers bring more water molecules into the pores than do unacetylated G5 dendrimers. These results agree qualitatively with experimental results showing that significant cytoplasmic-protein leakage is produced by unacetylated G7 dendrimers at concentrations as low as 10 nM, but only at a much higher concentration of 400 nM for unacetylated G5 dendrimers (Bioconjugate Chem. 2004, 15, 774). This good qualitative agreement indicates that the effect on pore formation of the concentration and size of large nanoparticles can be studied through coarse-grained MD simulations, provided that long-range electrostatic interactions are included.     

Membrane fluidity of tetramyristoyl cardiolipin (TMCL) liposomes studied by chronoamperometric monitoring of their adhesion and spreading at the surface of a mercury electrode

Giant unilamellar liposomes of the synthetic cardiolipin 1′,3′-bis[1,2-dimyristoyl-sn-glycero-3-phospho]-sn-glycerol give chronoamperometric current peaks at a stationary mercury electrode. The signals are due to the adhesion and spreading of the liposomes on the hydrophobic mercury surface. The potential dependence shows a minimum of the peak frequency at the point of zero charge, a large maximum of peak frequency at about ?0.2?V and a second, however, smaller maximum at ?0.8?V. The electrochemical behaviour of the liposomes indicates phase transitions of the cardiolipin which could be also observed in differential scanning calorimetry.     

Influence of lipid composition on the thermotropic behavior and size distribution of mixed cationic liposomes

Cationic liposomes are studied mainly as nonviral nucleic acid delivery systems and to a lesser extent as carriers/adjuvants of vaccines and as low-molecular-weight drug carriers. It is well established that the performance and the biological activity of liposomes in general are strongly related to their physicochemical properties. We investigated the thermotropic behavior and the size distribution of mixed cationic liposomes formulated with different percentages of 1,2 dimyristoyl-sn-glycero-3-phosphatidylcholine and one of four cationic amphiphiles characterized by a pyrrolidinium headgroup with the aim of achieving a better understanding of how the molecular structure of the cationic amphiphile and its mole percentage affect the physicochemical properties of the liposomes. Multilamellar vesicles and large unilamellar vesicles were studied by differential scanning calorimetry and turbidity, respectively, to characterize the thermotropic behavior and lipid phase, whereas dynamic light scattering was used to determine size distribution. This study shows that subtle modifications in the cationic amphiphile's molecular structure and in liposome composition may have dramatic effects on the organization of the liposome bilayer and hence on the morphological and physicochemical features of the liposomes, thus being highly relevant to the biological features investigated previously.     

Effect of silicon dioxide on the formation of the phase composition and pore structure of titanium dioxide with the anatase structure

The formation of the structure of titanium dioxide modified with silicon dioxide, which was introduced as tetraethyl orthosilicate, was studied. It was found that the formation of the nanocrystalline structure of TiO₂ occurred upon the modification of titanium dioxide with silicon dioxide. This nanocrystalline structure of TiO₂ was formed by highly dispersed anatase particles of size 6–10 nm stabilized by silicon oxide layers, which were formed upon the decomposition of tetraethyl orthosilicate. An increase in the modifier concentration resulted in a deceleration of the growth of anatase particles and an increase in the temperature of the phase transition of anatase to rutile. It was found that the anatase phase in the samples containing 5–15 wt % SiO₂ was stable up to 1000°C. The stabilization of highly dispersed anatase particles facilitated the retention of the developed fine-pore structure of xerogels with a pore diameter of 4 nm up to 900°C.     

Effect of nanoscale topography on fibronectin adsorption,focal adhesion size and matrix organisation

Phase separation of PLLA/PS (50/50, w/w) solutions during a spin-casting process gives rise to well-defined nanotopographies of 14, 29 and 45 nm deep pits depending on the concentration of the solution. Their influence on the biological activity of fibronectin (FN) was investigated. FN adsorption was quantified by radiolabelling the protein. The amount of adsorbed FN was higher on the 14 nm deep pit nanotopography than on the other two surfaces. FN distribution between valleys and peaks was investigated by AFM combined with image analysis. FN tends to adsorb preferentially on the valleys of the nanotopography only for the 14 nm system and when adsorbed from solutions of concentration lower than 10 μg/ml. Higher concentration of the FN solution leads to evenly distribution of the protein throughout the surface; moreover, there is no difference in the distribution of the protein between valleys and peaks for the other two systems (29 and 45 nm) irrespective of the concentration of the FN solution. The biological activity of the adsorbed protein layer was assessed by investigating MC3T3 osteoblast-like cells adhesion, FN reorganisation and late matrix formation on the different substrates. Even if initial cell adhesion is excellent for every substrate, the size of the focal adhesion plaques increases as the size of the pits in the nanotopography does. This is correlated to FN reorganisation, which only takes places on the 29 and 45 nm deep pits surfaces, where enhanced late matrix production was also found.     

The role of nutrient presence on the adhesion kinetics of Burkholderia cepacia G4g and ENV435g

The adhesion kinetics of Burkholderia cepacia G4g and ENV435g have been investigated in a radial stagnation point flow (RSPF) system under well-controlled hydrodynamics and solution chemistry. The sensitivity of adhesion behavior to nutrient condition was also examined. Supplementary cell characterization techniques were conducted to evaluate the viability, hydrophobicity, electrophoretic mobility, size, and charge density of cells grown in both nutrient rich Luria broth (LB) and nutrient poor basal salts medium (BSM). Comparable adhesion kinetics were observed for the wild-type (G4g) and mutant (ENV435g) grown in the same medium; however, the attachment efficiency increased with the level of nutrient presence for both cell types by approximately 60%. Nutrient condition altered deposition due to its impact on the surface charge characteristics and size of the cells. Adhesion behavior was consistent with expectations based on classical Derjaguin–Landau–Verwey–Overbeek (DLVO) theory for colloidal interactions, as the adhesion efficiency increased with ionic strength. However, the results also suggest the involvement of non-DLVO type interactions that influence cell adhesion. Systematic experimentation with B. cepacia in the RSPF system demonstrated that the ENV435g mutant is not “adhesion deficient”; rather, adhesion for both the G4g and ENV435g was a function of the nutrient condition and resulting cell surface chemistry.     

X-ray photoelectron spectroscopy and magnetic studies on the effect of pore size, wall thickness, and wall composition on superparamagnetic cobaltocene mesoporous Nb, Ta, and Ti composites

Recent results in our group demonstrated that mixed oxidation state mesoporous niobium oxide cobaltocene composites display superparamagnetism at certain composition ratios. This was the first report of superparamagnetism in nanoscale molecular ensembles. A series of mesoporous niobium oxide materials were synthesized in order to understand the role of pore size and thickness of the walls in the mesostructure on the magnetic properties. Mesoporous Ti oxide and Ta oxide composites were also synthesized in order to investigate the effect of changing the wall composition on the magnetic properties of this new series of materials. All samples were characterized by X-ray diffraction, nitrogen adsorption, ultraviolet spectroscopy, X-ray photoelectron spectroscopy, scanning electron microscopy, and superconducting quantum interference device magnetometry. The results of this study showed that variation of wall thickness or pore size in the Nb system had little effect on the properties and that superparamagnetism most likely arises from mixed oxidation state cobaltocene grains residing in the individual pores and not from the free electrons in the mesostructure or much larger domains. The Langevin function was applied to the isothermal magnetic data from the Nb composites and gave mean superparamagnetic particle sizes of ca. 14 nm in each system. The Co(II) to Co(III) ratios in these materials were approximately 1:1. The Ti and Ta materials showed no sign of superparamagnetism and only very low levels of neutral cobaltocene in the pores. This suggests that a critical amount of cobaltocene is required to bring about superparamagnetic behavior.     

A DSC study of paeonol-encapsulated liposomes, comparison the effect of cholesterol and stigmasterol on the thermotropic phase behavior of liposomes

Thermotropic phase behaviors of paeonol-encapsulated liposomes containing stigmasterol or cholesterol have been investigated by differential scanning calorimetry. We compared the thermotropic phase behavior of pure dipalmitoylphosphatidylcholine (DPPC) liposomes, sterol/DPPC liposomes, and paeonol/sterol/DPPC liposomes increasing the ratio of paeonol to sterol from 0 to 1, by analyzing the calorimetric parameters of main phase transition of liposomes including phase transition temperature (onset temperature and peak temperature) and phase transition cooperativity. The results showed that paeonol could incorporate into the hydrophobic region of DPPC, thus, decrease phase transition temperature of DPPC. Though stigmasterol interacts with DPPC less favorably than cholesterol, thermotropic phase behavior of paeonol/cholesterol/DPPC liposomes and that of paeonol/stigmasterol/DPPC liposomes are very similar. A phase separation occurred when the molar ratio of paeonol to sterol reached 1:1 in paeonol-encapsulated liposomes, where a paeonol-rich domain coexisted with a sterol-rich domain. The packing order of acyl chains of DPPC in sterol-rich domain is a little higher than that in paeonol-rich domain.     

Effect of urea on the phase composition and strength of phosphogypsum grains

The effect of urea addition on the composition and strength of grains produced by pelletization of wet phosphogypsum and its subsequent drying was studied experimentally.     

Evaluation of the "DSPM" model on a titania membrane: measurements of charged and uncharged solute retention, electrokinetic charge, pore size, and water permeability

The DSPM (Donnan steric partitioning pore model) was evaluated in the case of a titania membrane with "nanofiltration properties" by measuring the electrokinetic charge, pore size, and water permeability of the membrane, along with charged and uncharged solute retention. The zeta potential values (zeta) were determined from measurements of the electrophoretic mobility (EM) of titania powder forming the filtering layer of the membrane. Zeta potential values were converted into membrane volume charge (X) by assuming two limiting cases: a constant surface charge (sigma(s)(cst)) and a constant surface potential (psi(s)(cst)). The mean pore radius and thickness/porosity ratio of the membrane were determined by permporometry and from water permeability measurements, respectively. Retention measurements were carried out as a function of the permeate volume flux for both neutral solutes (polyethylene glycol PEG of different size) and salts (KCl, MgSO4, K2SO4, and MgCl2) at various pH values. Ionic retentions showed minimum values near the IEP of the membrane. Retention data were analyzed using the DSPM. Very good agreement was found between the pore radius calculated by the model and that determined by permporometry. X values calculated from fitting retention data using the DSPM were also in satisfactorily agreement with X values calculated from EM measurements assuming a constant surface potential for a large pH range. Furthermore, the DSPM leads to X values (X(DSPM)) between those calculated from EM (X(EM)) using the two limiting bounds. In other words, X(DSPM) was higher than X(EM) assuming psi(s)(cst) at pH values far from the isoelectric point (IEP) and lower than X(EM) assuming sigma(s)(cst). These results show that the DSPM is in qualitative agreement with the charge regulation theory (increase of the pore surface potential and decrease of the pore surface charge density with decreasing the pore size). On the other hand, the thickness/porosity ratio of the membrane calculated from solute retention data differed significantly from that determined from water permeability measurements. Moreover, a single value of Deltax/Ak could not be determined from PEG and salt retention data. This means that the Deltax/Ak parameter loses its physical meaning and includes physical phenomena which are not taken into account by the DSPM. Nevertheless, the model satisfactorily predicted the limiting retention, as this is not influenced by the Deltax/Ak parameter.     

Effect of the structural disorder of T-shaped triazolyl-based coordination network compounds on the pore size and stability

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Effects of vacuum annealing on the particle size and phase composition of nanocrystalline tungsten carbide powders

The effects of the vacuum annealing temperature (400–1400°C) on the phase and chemical composition, particle size, and microstresses of the nanocrystalline powders of tungsten carbide WC with 20–60 nm particles were studied by X-ray diffraction and electron microscopy. Vacuum annealing of WC nano-powders at T _ann ≤ 1400°C was accompanied by decarbonization, resulting from the interaction of carbon with the oxygen impurity. Changes in the chemical composition of the nanocrystalline powder of tungsten carbide led to changes in its phase composition. The annealing was accompanied by growth of powder particles due to the aggregation of nanoparticles and by a decrease of microstresses.     

The influence of the pore size, the foaming temperature and the viscosity of the continuous phase on the properties of foams produced by membrane foaming

Membrane foaming is a new method of foaming. To enlarge the knowledge about the influencing factors and to know how to vary the structure of the resulting foam, different factors were evaluated. A whey protein solution with 10% protein was foamed as a model solution by means of a tubular cross-flow filtration membrane. The pore size of the membrane was varied. The smaller the pore size, the smaller the bubbles produced. As a result, the foam firmness increases and less drainage was observed when smaller pore sizes were applied.

An important factor is that the added amount of gas must be stabilised as completely as possible in the foam. In order to achieve this, both the process and the product parameters were varied. Raising the foaming temperature increased the quantity of stabilised gas. The whey proteins then diffuse faster to the bubble surfaces and stabilise these by unfolding and networking reactions to prevent the coalescence of the bubbles.

The product parameter viscosity was found to influence the foaming result in such a way that up to a viscosity of 40 mPa s the incorporated gas bubbles are stabilised by the higher viscosity. At viscosities higher than 40 mPa s it is difficult to incorporate in the bubbles, and the foam structure becomes coarser due to increased coalescence at the pores of the membrane. The foam stability is enhanced with higher viscosities.