Supramolecular assembly of a thermoresponsive steroidal surfactant with an oppositely charged thermoresponsive block copolymer.

Supramolecular rearrangements are crucial in determining the response of stimuli sensitive soft matter systems such as those formed by mixtures of oppositely charged amphiphiles. Here mixtures of this kind were prepared by mixing the cationic block copolymer pAMPTMA30-b-pNIPAAM120 and an anionic surfactant obtained by the modification of the bile salt sodium cholate. As pure components, the two compounds presented a thermoresponsive self-assembly at around 30-35 °C; a micelle formation in the case of the copolymer and a transition from fibers to tubes in the case of the bile salt derivative. When both were present in the same solution they associated into mixed aggregates that showed complex thermoresponsive features. At room temperature, the core of the aggregate was comprised of a supramolecular twisted ribbon of the bile salt derivative. The block copolymers were anchored on the surface of this ribbon through electrostatic interactions between their charged blocks and the oppositely charged heads of the bile salt molecules. The whole structure was stabilized by a corona of the uncharged blocks that protruded into the surrounding solvent. By increasing the temperature to 30-34 °C the mixed aggregates transformed into rods with smooth edges that associated into bundles and clusters, which in turn induced clouding of the solution. Circular dichroism allowed us to follow progressive rearrangements of the supramolecular organization within the complex, occurring in the range of temperature of 20-70 °C.

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.

Cluster phases of decorated micellar solutions with macrocyclic ligands.

An aqueous self-assembled micellar system (sodium dodecyl sulfate, SDS, decorated with various adhesive sites, cryptand Kryptofix 222 and crown ether 18-Crown-6 molecules) has been investigated by dynamic light scattering (DLS) and small angle x-ray scattering (SAXS) to have insights into the micellar structure, the micellar interactions, and the aggregation properties of the system. DLS demonstrates the existence of populations of aggregates in the submicrometer/micrometer range, while the Guinier analysis of the SAXS curves helps in detailing objects smaller than 30 nm. The aggregates of micelles are here named cluster phases of micelles (CPMs). Considering that SDS micelles in water do not aggregate at low concentration, it is shown that macrocyclic ligands induce the SDS micelle aggregation as a function of the concentration (i.e., investigated ligand/SDS molar ratios are 5.0, 1.5, 1.0, and 0.5) and hydrophobicity of the adhesive sites. The sizes and the percentages of the micelles and the CPMs have been monitored to test the stability and reversibility of the system. DLS results clearly show that the aggregation processes of the decorated micelles are reproducible at time intervals of the order of 1 month, while the stability may not be entirely maintained after a year. As an issue of particular relevance, the higher the ligand/surfactant molar ratio, the larger are the CPMs induced. The K222 ligand results in being more effective in promoting the micellar aggregation than 18C6 as a consequence of the different hydrophobicity.

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.

Deviations from a simple Debye relaxation in aqueous solutions of differently flexible polyions induced by polymer concentration.

The electrical conductivity of aqueous solutions of differently flexible polymers has been measured in the frequency range from 1 MHz to 2 GHz. This note evidences how the shape parameter alpha of the conductivity relaxation associated with the orientational polarization of the aqueous phase depends on the polymer concentration and, moreover, reflects the different concentration regimes of the polymer solution (dilute, semidilute entangled and nonentangled, and concentrated regimes). The relevance of this dependence, as far as the microscopic environment of water molecules is concerned, is briefly discussed.

Polyion-induced cluster formation in different colloidal polyparticle aqueous suspensions.

The formation of aggregates in polyion-induced charged colloidal particles in aqueous suspension is characterized, under appropriate conditions, by two complementary effects, known as re-entrant condensation and charge inversion, which are considered as proof for the existence of a cluster phase. In this paper, we extend our previous investigation to a set of aqueous colloidal particle suspensions, such as polystyrene spheres, colloidal gold particles, and polylactic acid particles. These systems are characterized by the evolution of the average size of the aggregates and their surface electrical charge (charge inversion) by means of dynamic light-scattering measurements and laser Doppler electrophoretic techniques. The results, together with the previous ones concerning liposome particles, support the notion of a common behavior of this group of complex colloids characterized by short-ranged attractive interactions. The study provides some insights into these structures, which are potentially useful in biotechnological applications, such as multicompartmental carriers in nonviral drug delivery.

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.

Dielectric properties of differently flexible polyions: a scaling approach.

The dielectric relaxations associated with counterion polarization along some typical polyion lengths have been measured in an extended frequency range (from 10 kHz to 2 GHz) for four different polyelectrolyte solutions, differing for the polyion molecular weight and the backbone stiffness. Here, we deal with the so-called intermediate dielectric relaxation, falling between the polarization process concerning the whole polyion chain and the polarization process associated with the field-induced re-orientation of the water molecule dipoles. These observed intermediate relaxations have been characterized by means of two parameters, i.e., the dielectric strength Deltaepsilon and the relaxation frequency nu0, and their dependence on the polyion concentration has been described according to the scaling model of a polyelectrolyte solution. These dependencies follow the expected exponents of the scaling laws, both for the dilute and semidilute (unentangled and entangled) regimes. The different concentration regimes evidenced from dielectric relaxation measurements are in very good agreement with the ones determined by means of the zero-shear viscosity measurements. Our results confirm that the intermediate dielectric relaxation in polyelectrolyte solutions should be attributed to counterion fluctuations along some segments (e.g. the subunits of the Mandel model) of the polyion chain, independently of its overall stiffness. This counterion polarization effect is rather confined to the local structure of the polyion chain instead of the whole chain and it is largely independent of the polyion conformation.

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.

Effect of temperature on the reentrant condensation in polyelectrolyte-liposome complexation.

Interactions of oppositely charged macroions in aqueous solution give rise to intriguing aggregation phenomena, resulting in finite-size, long-lived clusters, characterized by a quite narrow size distribution. Particularly, the adsorption of highly charged linear polyelectrolytes on oppositely charged colloidal particles is strongly correlated and some short-range order arises from competing electrostatic interactions between like-charged polymer chains (repulsion) and between polymer chains and particle surface (attraction). In these systems, in an interval of concentrations around the isoelectric point, relatively large clusters of polyelectrolyte-decorated particles form. However, the mechanisms that drive the aggregation and stabilize, at the different polymer/particle ratios, a well-defined size of the aggregates are not completely understood. Nor is clear the role that the correlated polyion adsorption plays in the aggregation, although the importance of "patchy interactions" has been stressed as the possible source of attractive interaction term between colloidal particles. Different models have been proposed to explain the formation of the observed cluster phase. However, a central question still remains unanswered, i.e., whether the clusters are true equilibrium or metastable aggregates. To elucidate this point, in this work, we have investigated the effect of the temperature on the cluster formation. We employed liposomes built up by DOTAP lipids interacting with a simple anionic polyion, polyacrylate sodium salt, over an extended concentration range below and above the isoelectric condition. Our results show that the aggregation process can be described by a thermally activated mechanism.

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.

Liposome-induced DNA compaction and reentrant condensation investigated by dielectric relaxation spectroscopy and dynamic light scattering techniques.

Interaction of DNA with oppositely charged objects, such as multivalent ions, cationic surfactants, cationic liposomes, basic proteins, and alcohols, up to nano- or mesoscopic particles, gives rise to a very interesting and fascinating phenomenology, where the shape, size, and stability of the resulting aggregates depend on a delicate balance between different driving forces, mainly of electrostatic origin. We have studied the cationic liposome-DNA complexes during the whole complexation process, below, close to, and above the isoelectric condition, where the number of cationic lipids equals the number of phosphate groups on the DNA chain. We took advantage of the combined use of dynamic light scattering, laser Doppler electrophoretic mobility, and radio-wave dielectric relaxation measurements in order to characterize both the structural parameters (hydrodynamic radius) and the electrical parameters (charge and counterion concentration) of the resulting structures. These structures are fundamentally of two types, clusters of liposomes stuck together by DNA chains (cluster phase in low-density colloidal suspension) and coexisting DNA coils and DNA globules, according to the procedure through which interactions occur (liposomes in excess DNA solution or DNA in excess liposome suspension).

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.

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.

Polyion-induced liposomal vesicle aggregation: a radiowave dielectric relaxation study.

The radiowave dielectric properties of aqueous heterogeneous systems during the complexation of charged polyions and oppositely charged liposomal particles have been measured in a wide frequency range, between 100 Hz and 2 GHz. The formation of a polyion-liposome complex driven by the correlated polyion adsorption at the particle surface implies two concomitant effects referred to as reentrant condensation and charge inversion. Both of them are governed by electrostatic interactions and there is now strong evidence, based on experiments and simulations, that counterion release is the driving force of the aggregation process. From this point of view, dielectric technique may offer a suitable tool in the investigation of the structural properties of these aggregates. In spite of the fact that interaction of polyions with oppositely charged surfaces was extensively experimentally investigated, there are no papers concerning the dielectric properties during the polyion-induced aggregation. To get an insight into this important topic, the authors present here an extensive set of radiowave dielectric measurements of liposomal vesicle aqueous suspensions where the liposome aggregation was induced by an oppositely charged polyion. The aggregation was followed from the beginning, when most of the isolated liposomes predominate, up to the formation of polyion-coated liposomes of inverted charge, crossing the isoelectric condition, where large, almost neutral, aggregates appear. The authors describe the observed dielectric dispersions as due to counterion polarization in the adjacency of the liposome and liposome aggregate surface, primarily governed by the zeta potential, according to the standard electrokinetic model.

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.

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.

Dielectric scaling in polyelectrolyte solutions with different solvent quality in the dilute concentration regime.

In this note, we present a set of radiowave dielectric spectroscopy measurements of two dilute, differently-charged polyelectrolyte solutions, under different solvent conditions. We have found that both the dielectric strength, Delta epsilon, and the relaxation time, tau(ion), of the dielectric relaxation process associated with the counterion polarization along a length scale of the order of the correlation length obey the scaling laws with the polyion concentration, according to the Ito model. This is verified with good accuracy independently of the quality of the solvent, which has been varied from poor to good solvent conditions. This finding supports evidence to the fact that, in dilute solutions, the counterion polarization is independent of the polyion concentration, in spite of what occurs at the semi-dilute concentrations.