Vertical ordering sensitivity of solid supported DPPC membrane to alamethicin and the related loss of cell viability.

BACKGROUND: Experimental studies of antimicrobial peptides interacting with lipid membranes recently attracted growing interest due to their numerous biomedical applications. However, the influence of such peptides on the structural organisation of lipid membranes in connection with the actual cell response still remains an elusive issue. METHODS: X-ray diffraction was employed on detecting the sensitivity of the periodical spacing of dipalmitoyl-phosphatidyl-choline stacked as solid-supported bilayers to the presence of varying amounts of the peptide alamethicin in a wide range of peptide-to-lipid molar ratios. These results were then correlated with the effects of alamethicin on biological membranes in vitro as observed by optical microscopy and microculture tetrazolium assay on the tumour cells HeLa to provide a comprehensive and quantitative analysis of these effects, based on a dose-response relationship. RESULTS: The experiments allowed correlating the periodical spacing and the peptide-to-lipid molar ratio on alamethicin-dipalmitoyl-phosphatidyl-choline samples. Two different trends of periodical spacing vs. peptide-to-lipid molar ratio clearly appeared at low and high hydration levels, showing intriguing non-linear profiles. Unexpected correspondences were observed between the peptide-to-lipid molar ratio range where the changes in dipalmitoyl-phosphatidyl-choline structure occur and the alamethicin doses which alter the viability and the plasma membrane morphology of HeLa. CONCLUSIONS: Alamethicin might induce either mechanical or phase changes on dipalmitoyl-phosphatidyl-choline bilayers. Such easily accessible ordering information was well-calibrated to predict the alamethicin doses necessary to trigger cell death through plasma membrane alterations. GENERAL SIGNIFICANCE: This benchmark combined study may be valuable to predict bioeffects of several antimicrobial peptides of biomedical relevance.

Cationic liposomes formulated with DMPC and a gemini surfactant traverse the cell membrane without causing a significant bio-damage.

Cationic liposomes have been intensively studied both in basic and applied research because of their promising potential as non-viral molecular vehicles. This work was aimed to gain more information on the interactions between the plasmamembrane and liposomes formed by a natural phospholipid and a cationic surfactant of the gemini family. The present work was conducted with the synergistic use of diverse experimental approaches: electro-rotation measurements, atomic force microscopy, ζ-potential measurements, laser scanning confocal microscopy and biomolecular/cellular techniques. Electro-rotation measurements pointed out that the interaction of cationic liposomes with the cell membrane alters significantly its dielectric and geometric parameters. This alteration, being accompanied by significant changes of the membrane surface roughness as measured by atomic force microscopy, suggests that the interaction with the liposomes causes locally substantial modifications to the structure and morphology of the cell membrane. However, the results of electrophoretic mobility (ζ-potential) experiments show that upon the interaction the electric charge exposed on the cell surface does not vary significantly, pointing out that the simple adhesion on the cell surface of the cationic liposomes or their fusion with the membrane is to be ruled out. As a matter of fact, confocal microscopy images directly demonstrated the penetration of the liposomes inside the cell and their diffusion within the cytoplasm. Electro-rotation experiments performed in the presence of endocytosis inhibitors suggest that the internalization is mediated by, at least, one specific pathway. Noteworthy, the liposome uptake by the cell does not cause a significant biological damage.

Interactions of DMPC and DMPC/gemini liposomes with the cell membrane investigated by electrorotation.

The electrorotation technique was utilized to investigate the interactions between a mouse fibroblast cell line and zwitterionic liposomes formed by a natural phospholipid or cationic liposomes formulated with the same phospholipid and a cationic gemini surfactant. The application of this technique allowed an accurate characterization of the passive dielectric behavior of the plasma membrane by the determination of its specific capacitance and conductance. Changes of these parameters, upon interaction with the liposomes, are related to variations in the structure and or in the transport properties of the membrane. Cells were exposed to both types of liposomes for 1 or 4h. Electrorotation data show a dramatic reduction of the dielectric parameters of the plasma membrane after one hour treatment. After 4h of treatment the effects are still observed only in the case of the cationic liposomes. Surprisingly, these same treatments did not cause a relevant biological damage as assessed by standard viability tests. A detailed discussion to rationalize this phenomenon is presented.

Interaction between like-charged polyelectrolyte-colloid complexes in electrolyte solutions: a Monte Carlo simulation study in the Debye-Hückel approximation.

We study the effective interaction between differently charged polyelectrolyte-colloid complexes in electrolyte solutions via Monte Carlo simulations. These complexes are formed when short and flexible polyelectrolyte chains adsorb onto oppositely charged colloidal spheres, dispersed in an electrolyte solution. In our simulations the bending energy between adjacent monomers is small compared to the electrostatic energy, and the chains, once adsorbed, do not exchange with the solution, although they rearrange on the particles surface to accommodate further adsorbing chains or due to the electrostatic interaction with neighbor complexes. Rather unexpectedly, when two interacting particles approach each other, the rearrangement of the surface charge distribution invariably produces antiparallel dipolar doublets that invert their orientation at the isoelectric point. These findings clearly rule out a contribution of dipole-dipole interactions to the observed attractive interaction between the complexes, pointing out that such suspensions cannot be considered dipolar fluids. On varying the ionic strength of the electrolyte, we find that a screening length kappa(-1), short compared with the size of the colloidal particles, is required in order to observe the attraction between like-charged complexes due to the nonuniform distribution of the electric charge on their surface ("patch attraction"). On the other hand, by changing the polyelectrolyte/particle charge ratio xi(s), the interaction between like-charged polyelectrolyte-decorated particles, at short separations, evolves from purely repulsive to strongly attractive. Hence, the effective interaction between the complexes is characterized by a potential barrier, whose height depends on the net charge and on the nonuniformity of their surface charge distribution.

Kinetic arrest in polyion-induced inhomogeneously charged colloidal particle aggregation.

Polymer chains adsorbed onto oppositely charged colloidal particles can significantly modify the particle-particle interactions. For sufficient amounts of added polymers, the original electrostatic repulsion can even turn into an effective attraction and relatively large aggregates can form. The attractive interaction contribution between two particles arises from the correlated adsorption of polyions at the oppositely charged particle surfaces, resulting in a non-homogeneous surface charge distribution. Here, we investigate the aggregation kinetics of polyion-induced colloidal complexes through Monte Carlo simulation, in which the effect of charge anisotropy is taken into account by a DLVO-like inter-particle potential, as recently proposed by Velegol and Thwar (Langmuir 17, 7687 (2001)). The results reveal that the aggregation process slows down due to the progressive increase of the potential barrier height upon clustering. Within this framework, the experimentally observed cluster phases in polyelectrolyte-liposome solutions can be interpreted as a kinetic arrested state.

Counterion condensation of differently flexible polyelectrolytes in aqueous solutions in the dilute and semidilute regime.

The low-frequency limit of the electrical conductivity (dc conductivity) of differently flexible polyions in aqueous solutions has been measured over an extended polyion concentration range, covering both the dilute and semidilute (entangled and unentangled) regime, up to the concentrated regime. The data have been analyzed taking into account the different flexibility of the polymer chains according to the scaling theory of polyion solutions, in the case of flexible polyions, and according to the Manning model, in the case of rigid polyions. In both cases, the fraction f of free counterions, released into the aqueous phase from the ionizable polyion groups, has been evaluated and its dependence on the polyion concentration determined. Our results show that the counterion condensation follows at least three different regimes in dependence on the polyion concentration. The fraction f of free counterions remains constant only in the semidilute regime (a region that we have named the Manning regime), while there is a marked dependence on the polyion concentration both in the dilute and in the concentrated regime. These results are briefly discussed in the light of the scaling theory of polyelectrolyte aqueous solutions.

Polyelectrolyte-induced aggregation of liposomes: a new cluster phase with interesting applications.

Different charged colloidal particles have been shown to be able to self-assemble, when mixed in an aqueous solvent with oppositely charged linear polyelectrolytes, forming long-lived finite-size mesoscopic aggregates. On increasing the polyelectrolyte content, with the progressive reduction of the net charge of the primary polyelectrolyte-decorated particles, larger and larger clusters are observed. Close to the isoelectric point, where the charge of the adsorbed polyelectrolytes neutralizes the original charge of the particles' surface, the aggregates reach their maximum size, while beyond this point any further increase of the polyelectrolyte-particle charge ratio causes the formation of aggregates whose size is progressively reduced. This re-entrant condensation behavior is accompanied by a significant overcharging. Overcharging, or charge inversion, occurs when more polyelectrolyte chains adsorb on a particle than are needed to neutralize its original charge so that, eventually, the sign of the net charge of the polymer-decorated particle is inverted. The stability of the finite-size long-lived clusters that this aggregation process yields results from a fine balance between long-range repulsive and short-range attractive interactions, both of electrostatic nature. For the latter, besides the ubiquitous dispersion forces, whose supply becomes relevant only at high ionic strength, the main contribution appears due to the non-uniform correlated distribution of the charge on the surface of the polyelectrolyte-decorated particles ('charge-patch' attraction). The interesting phenomenology shown by these system has a high potential for biotechnological applications, particularly when the primary colloidal particles are bio-compatible lipid vesicles. Possible applications of these systems as multi-compartment vectors for the simultaneous intra-cellular delivery of different pharmacologically active substances will be briefly discussed.

Long-term physical evolution of an elastomeric ultrasound contrast microbubble.

HYPOTHESIS: One of the main assets of crosslinked polymer-shelled microbubbles (MBs) as ultrasound-active theranostic agents is the robustness of the shells, combined with the chemical versatility in modifying the surface with ligands and/or drugs. Despite the long shelf-life, subtle modifications occur in the MB shells involving shifts in acoustic, mechanical and structural properties. EXPERIMENTS: We carried out a long-term morphological and acoustic evolution analysis on elastomeric polyvinyl-alcohol (PVA)-shelled MBs, a novel platform accomplishing good acoustic and surface performances in one agent. Confocal laser scanning microscopy, acoustic spectroscopy and AFM nanomechanics were integrated to understand the mechanism of PVA MBs ageing. The changes in the MB acoustic properties were framed in terms of shell thickness and viscoelasticity using a linearised oscillation theory, and compared to MB morphology and to nanomechanical analysis. FINDINGS: We enlightened a novel, intriguing ageing time evolution of the PVA MBs with double behaviour with respect to a crossover time of ∼50 days. Before, significant changes occur in MB stiffness and shell thickness, mainly due to a massive release of entangled PVA chains. Then, the MB resonance frequency increases together with shell thickening and softening. Our benchmark study is of general interest for emerging viscoelastomeric bubbles towards personalised medicine.

Infrared spectra of phosphatidylethanolamine-cardiolipin binary system.

We report on the infrared spectra of binary di-palmitoyl-phosphatidyl-ethanolamine-cardiolipin (DPPE 1-x CLPx) monolayers and multilamellar vesicles as a function of CLP molar fraction x and temperature T. These data, which clearly show the presence of, at least, two kind of lipid domains with different thermodynamic stability and ordering of the lipid acyl chains, are consistent with similar domains observed in Langmuir-Blodgett films of the same binary system. Infrared results suggest the presence of lateral phase separation phenomena both in the bilayers and in the monolayers build up with this binary lipid mixture. These results further support the hypothesis that, within these structures, DPPE 1-x CLPx molecules, for given values of x, are organized in a superlattice as shown by thermodynamic and AFM measurements.

Influence of temperature on microdomain organization of mixed cationic-zwitterionic lipidic monolayers at the air-water interface.

The thermodynamic behavior of mixed DOTAP-DPPC monolayers at the air-water interface has been investigated in the temperature range from 15 to 45 degrees C, covering the temperature interval where the thermotropic phase transition of DPPC, from solid-like to liquid-like, takes place. Based on the regular solution theory, the miscibility of the two lipids in the mixed monolayer was evaluated in terms of the excess Gibbs free energy of mixing DeltaG(ex), activity coefficients f(1) and f(2) and interaction parameter omega between the two lipids. The mixed DOTAP-DPPC film was found to have positive deviations from ideality at low DOTAP mole fractions, indicating a phase-separated binary mixture. This effect depends on the temperature and is largely conditioned by the structural chain conformation of the DPPC lipid monolayer. The thermodynamic parameters associated to the stability and the miscibility of these two lipids in a monolayer structure have been discussed in the light of the phase diagram of the DOTAP-DPPC aqueous mixtures obtained from differential scanning calorimetry measurements. The correlation between the temperature behavior of DOTAP-DPPC monolayers and their bulk aqueous mixtures has been briefly discussed.

Properties of mixed DOTAP-DPPC bilayer membranes as reported by differential scanning calorimetry and dynamic light scattering measurements.

We have investigated the effect of a cationic lipid [DOTAP] on both the thermotropic phase behavior and the structural organization of aqueous dispersions of dipalmitoyl-phosphatidylcholine [DPPC] by means of high-sensitivity differential scanning calorimetry and dynamic light scattering measurements. We find that the incorporation of increasing quantities of DOTAP progressively reduces the temperature and the enthalpy of the gel-to-liquid crystalline transition. We are further showing that, in mixed DOTAP-DPPC systems, the reduction of the phase transition temperature is accompanied by a reduction of the average size of the structures present in the aqueous mixtures, whatever the DOTAP concentration is. These results, which extend a previous investigation by Campbell et al. (Campbell, R. B.; Balasubramanian, S. V.; Straubinger, R. M.; Biochim. Biosphys. Acta 2001, 27, 1512.) limited to a DOTAP concentration below 20 mol %, confirm that the insertion of cationic head groups in zwitterionic phosphatidylcholine bilayers facilitates the formation of stable, relatively small, unilamellar vesicles. This self-assembling restructuring from an aqueous multilamellar structure toward a liposomal phase is favored by decreasing the phospholipid phase transition temperature and by increasing the temperature of the system. This reduction of the average size and the appearance of a stable liposomal phase is also promoted by a heating and cooling thermal treatment.

Strong repulsive interactions in polyelectrolyte-liposome clusters close to the isoelectric point: a sign of an arrested state.

Charged colloidal particles whose interacting potential is governed by a short-range attraction and a long-range screened electrostatic repulsion contributions form aggregates whose shape, size, and overall charge are sensitively dependent on the balance between attraction and repulsion. In some cases, this class of colloidal systems shows an equilibrium cluster phase, where particles associate and dissociate reversibly into clusters. When the aggregation of the charged particles is induced by adding an oppositely charged polyion, very close to the isoelectric condition, the interaggregate interactions become very strong and a dynamical arrested state seems to occur. We provide some experimental evidences of this structural arrest in a colloid system composed by vesicles built up by a cationic lipid stuck together by an oppositely charged linear polyion, by means of the combined use of static and dynamic light scattering technique complemented by laser Doppler electrophoretic measurements. Our results show that the second virial coefficient, which is related to the potential of mean force between two adjacent aggregates, markedly increases in the vicinity of the isoelectric point. We interpret this increase as a print of strong interparticle interactions, yielding to a dynamical arrested state via cluster growth.

Radiofrequency dielectric loss relaxation in polyion-induced liposome aggregates.

In this note, we present a set of dielectric loss relaxation measurements of aqueous charged liposome suspensions during the whole aggregation process induced by oppositely charged adsorbing polyions. The system experiences two concomitant effects known as "reentrant condensation" and "charge inversion," resulting in the formation of liposome aggregates whose average size reaches a maximum in the vicinity of the electroneutrality condition, accompanied to a progressive reduction of their overall electrical charge. Far from the neutrality, from both sides, polyion-coated liposomes exist with a charge of opposite sign. The dielectric loss relaxation in these complex aggregating systems has never been measured so far and we report here, for the first time, the dielectric loss behavior of liposomes built up by a cationic lipid and stuck together by poly(acrylate), which is a flexible oppositely charged polyion. The data are analyzed in the framework of standard electrokinetic model theory. The evolution of the aggregation process as a function of the polyion content is mainly characterized by a counterion polarization effect, governed by the surface charge density of the aggregates and hence by the zeta-potential.

Differential effects on membrane permeability and viability of human keratinocyte cells undergoing very low intensity megasonic fields.

Among different therapeutic applications of Ultrasound (US), transient membrane sonoporation (SP) - a temporary, non-lethal porosity, mechanically induced in cell membranes through US exposure - represents a compelling opportunity towards an efficient and safe drug delivery. Nevertheless, progresses in this field have been limited by an insufficient understanding of the potential cytotoxic effects of US related to the failure of the cellular repair and to the possible activation of inflammatory pathway. In this framework we studied the in vitro effects of very low-intensity US on a human keratinocyte cell line, which represents an ideal model system of skin protective barrier cells which are the first to be involved during medical US treatments. Bioeffects linked to US application at 1 MHz varying the exposure parameters were investigated by fluorescence microscopy and fluorescence activated cell sorting. Our results indicate that keratinocytes undergoing low US doses can uptake drug model molecules with size and efficiency which depend on exposure parameters. According to sub-cavitation SP models, we have identified the range of doses triggering transient membrane SP, actually with negligible biological damage. By increasing US doses we observed a reduced cells viability and an inflammatory gene overexpression enlightening novel healthy relevant strategies.

Charge patch attraction and reentrant condensation in DNA-liposome complexes.

We investigated the formation of complexes between cationic liposomes built up by DOTAP and three linear anionic polyions, with different charge density and flexibility, such as a single-stranded ssDNA, a double-stranded dsDNA and the polyacrylate sodium salt [NaPAA] of three different molecular weights. Our aim is to gain further insight into the formation mechanism of polyion-liposome aggregates of different sizes (lipoplexes), by comparing the behavior of DNA with a model polyelectrolyte, such as NaPAA, with approximately the same charge density but with a higher flexibility. We employed dynamic light scattering (DLS) and transmission electron microscopy (TEM) measurements, in order to explore both the hydrodynamic and structural properties of the aggregates resulting from polyion-liposome interaction and to present a comprehensive picture of the complexation process. The phenomenology can be summarized in a charge ratio-dependent scenario, where the main feature is the formation of large equilibrium clusters due to the aggregation of intact polyion-coated vesicles. At increasing polyion-liposome ratio, the size of the clusters continuously increases, reaching a maximum at a well-defined value of this ratio, and then decreases ("reentrant" condensation). The aggregation mechanism and the role of the polyion charge density in the complex formation are discussed in the light of the recent theories on the correlated adsorption of polyelectrolytes at charged interfaces. Within this framework, the phenomena of charge inversion and the reentrant condensation, peaked at the isoelectric point, finds a simple explanation.

Counterion release in overcharging of polyion-liposome complexes.

We present a set of electrical conductivity measurements of a mesoscopic equilibrium cluster phase in the aggregation process of charged particles induced by oppositely charged polyions. These measurements supply strong experimental evidence that correlated adsorption of polyions is driven by the counterion release. This phenomenon, known to occur in DNA-liposome mixtures in lamellar phase, i.e., when liposomes fuse together to form a sandwichlike structure encompassing DNA chains, was not previously observed in aqueous suspension of clusters of intact liposomes stuck together by polyions to form reversible aggregates. A simple statistical model of the lateral correlation of polyions at the particle surface justifies quantitatively the observed behavior of the counterion release, as shown by electrical conductivity measurements.

Conductometric evidence for intact polyion-induced liposome clusters.

In this note, we present a set of electrical conductivity measurements of polyion-induced liposome aggregate aqueous suspensions that supports evidence for the existence of a cluster phase in low-density colloidal systems. Heavily NaCl-loaded liposomes, dispersed in a low-conductivity aqueous solution, are forced by electrostatic interactions with oppositely charged polyions to build up into individual aggregates, where the single vesicles maintain their integrity and, upon an external force, are able to release their ionic content. The conductivity data, within the effective medium approximation theory for heterogeneous systems, are in agreement with the picture of a suspension built up by clusters of vesicles which are able to preserve their content from the external medium. This finding opens new possibilities in multicompartment drug delivery techniques.

Group I metabotropic glutamate receptors: implications for brain diseases.

Glutamate is the major excitatory neurotransmitter in the brain and plays a unique role in a variety of central nervous system (CNS) functions. The discovery of the metabotropic receptors (mGluRs), a family of G-protein coupled receptors than can be activated by glutamate, has led to an impressive number of studies in recent years aimed at understanding their biochemical, physiological and pharmacological characteristics. The eight mGluRs now known are divided into three groups according to their sequence homology, signal transduction mechanisms, and agonist selectivity. Group I mGluRs include mGluR1 and mGluR5, which are linked to the activation of phospholipase C; Groups II and III include all others and are negatively coupled to adenylyl cyclases. The availability in recent years of agents selective for Group I mGluRs has made possible the study of the physiological roles of these receptors in the CNS. In addition to mediating glutamatergic neurotransmission, Group I mGluRs can modulate other neurotransmitter receptors, including GABA and the ionotropic glutamate receptors. Group I mGluRs are involved in many CNS functions and may participate in a variety of disorders such as pain, epilepsy, ischemia, and chronic neurodegenerative diseases. This class of receptor may provide important pharmacological therapeutic targets and elucidating its functions will be relevant to develop new treatments for neurological and psychiatric disorders in which glutamatergic neurotransmission is abnormally regulated. In this review anatomical, physiological and pharmacological results are presented with a special emphasis on the role of Group I mGluRs in functional and pathological processes.

Folate-based single cell screening using surface enhanced Raman microimaging.

Recent progress in nanotechnology and its application to biomedical settings have generated great advantages in dealing with early cancer diagnosis. The identification of the specific properties of cancer cells, such as the expression of particular plasma membrane molecular receptors, has become crucial in revealing the presence and in assessing the stage of development of the disease. Here we report a single cell screening approach based on Surface Enhanced Raman Scattering (SERS) microimaging. We fabricated a SERS-labelled nanovector based on the biofunctionalization of gold nanoparticles with folic acid. After treating the cells with the nanovector, we were able to distinguish three different cell populations from different cell lines (cancer HeLa and PC-3, and normal HaCaT lines), suitably chosen for their different expressions of folate binding proteins. The nanovector, indeed, binds much more efficiently on cancer cell lines than on normal ones, resulting in a higher SERS signal measured on cancer cells. These results pave the way for applications in single cell diagnostics and, potentially, in theranostics.

Frequency domain electrical conductivity measurements of the passive electrical properties of human lymphocytes.

An extensive set of electrical conductivity measurements of human lymphocyte suspensions has been carried out in the frequency range from 1 kHz to 100 MHz, where the surface polarization due to the Maxwell-Wagner effect occurs. The data have been analyzed according to well-established heterogeneous system theories and the passive electrical parameters of both the cytoplasmic and nuclear membranes have been obtained. Moreover, a further analysis to take into account the roughness of the membrane surface on the basis of a fractal model yields new estimates for the membrane conductivity and the membrane permittivity, independently of the surface architecture of the cell. These findings are confirmed by measurements carried out at higher frequencies, in the range from 1 MHz to 1 GHz, on lymphocytes dispersed in both hyperosmotic and hypoosmotic media, that influence the surface complexity of the membrane due to the microvillous protrusions. The surface roughness of the cell is described by a macroscopic parameter (the fractal dimension) whose variations are associated to the progressive swelling of the cell, as the osmolality of the solution is changed.