Carlotta Pucci

Albumin binding onto synthetic vesicles

Vesicular entities were obtained by mixing didodecyldimethylammonium bromide and sodium dodecylsulfate in non-stoichiometric ratios. The vesicles bear a positive surface charge, due to the cationic species being in excess, and adsorb significant amounts of protein, presumably by electrostatic interactions. We modulated the net charge of bovine serum albumin by pH and observed its binding onto the above vesicles. Binding is controlled by the net charge of vesicles and albumin: it is substantial when albumin has negative charges in excess and is negligible, or non-existent, below its iso-electric point. For pH values >6.0, the binding efficiency increases in proportion to protein charge. Surface coverage changes in proportion to pH when the number of charges neutralized upon binding remains the same. The size of protein–vesicle lipo-plexes was inferred by dynamic light scattering and their charge by ζ-potential. The structure of albumin was evaluated by circular dichroism spectroscopy and estimates of α-helix, β-strand and random coil contents were achieved. Increasing the β-strand and random coil contents subsequent to binding suggests a significant interaction between vesicles and albumin. Attempts to determine the binding efficiency were made by elaborating ζ-potential values. The results were interpreted in terms of a Gibbs adsorption isotherm. Accordingly, it is possible to estimate the binding energy under different pH conditions.

Confining ss-DNA/carbon nanotube complexes in ordered droplets

In 1/1 mass ratio mixtures made of single strand DNA and single-walled carbon nanotubes lyotropic nematic phases are formed. The process is assisted by segregative phase separation procedures. The liquid crystalline order occurring therein was confirmed by optical polarizing microscopy and zero-shear rheology. The resulting nematic droplets were dispersed in protein or cationic surfactant solutions, under appropriate pH and/or ionic strength conditions. The components of the hosting fluid(s) rapidly adsorb onto the droplets, form a permanent peel on their surface, and confine them. The peel resists osmotic gradients and has significant stability. The distribution of the species in the droplet and in the peel was determined by SEM. Data indicate that the peel contains protein or surfactant, depending on the titrant, when the core is rich in DNA and nanotubes. According to electron microscopy, nematic order in the droplets is partly retained.

Catanionic vesicles and DNA complexes: a strategy towards novel gene delivery systems

Catanionic vesicles are appealing vectors in non-viral gene transfection. They possess high kinetic stability and the preparation procedures are easy and cheap. In addition, their size and charge are easily modulated by varying the mole ratio between the components. For these reasons, we investigated the interactions between positively charged catanionic vesicles made of didecyldimethylammonium bromide (DiDAB) and 8-hexadecyl sulfate (8-SHS) with calf thymus DNA. Strongly associating complexes are obtained and their structure depends on DNA content. At low concentration, DNA/vesicles complexes are stable, with features very similar to bare vesicles. In the presence of DNA, multi-lamellar entities are formed; the process is promoted by the aggregation and rearrangement of DNA/vesicle complexes. Surface adsorption onto vesicles increases in proportion to DNA content. In such conditions, ζ-potential abruptly decreases, because of the formation of large clusters in which the vesicular identity is retained. Thereafter, precipitation occurs. The solid obtained accordingly is a lamellar phase with DNA sandwiched between the lamellae. The 1D distance between DNA molecules in the lamellar phase and the precipitate composition depend on the biopolymer content. The double helix of DNA undergoes a reversible compaction process that favors penetration into cells and protects it from nucleases degradation. Finally, addition of the anionic surfactant to the complexes favors DNA release, allowing for a specific signal controlled release.

Binding of a protein or a small polyelectrolyte onto synthetic vesicles.

Catanionic vesicles were prepared by mixing nonstoichiometric amounts of sodium bis(2-ethylhexyl) sulfosuccinate and dioctyldimethylammonium bromide in water. Depending on the concentration and mole ratios between the surfactants, catanionic vesicular aggregates are formed. They have either negative or positive charges in excess and are endowed with significant thermodynamic and kinetic stability. Vesicle characterization was performed by dynamic light scattering and electrophoretic mobility. It was inferred that vesicle size scales in inverse proportion with its surface charge density and diverges as the latter quantity approaches zero and/or the mole ratio equals unity. Therefore, both variables are controlled by the anionic/cationic mole ratio. Small-angle X-ray scattering, in addition, indicates that vesicles are unilamellar. Selected anionic vesicular systems were reacted with poly-L-lysine hydrobromide or lysozyme. Polymer binding continues until complete neutralization of the negatively charged sites on the vesicles surface is attained, as inferred by electrophoretic mobility. Lipoplexes are formed as a result of significant electrostatic interactions between cationic polyelectrolytes and negatively charged vesicles.

The DODAB–AOT–water system: vesicle formation and interactions with salts or synthetic polyelectrolytes

The phase behavior of the dioctyldimethylammonium bromide (DODAB)–sodium bis(2-ethylhexyl)sulfosuccinate (AOT)–water system was determined. Studies dealt with dilute concentration regimes, at an overall surfactant concentration lower than 30.0 mmol kg−1, and in the cationic rich side. The system was investigated by combining visual inspection with optical microscopy, fluorescence microscopy, dynamic light scattering, small-angle X-ray scattering, and electrophoretic mobility. At high [DODAB]/[AOT] mole ratios, stable and unilamellar catanionic vesicles are observed in a narrow region of the phase diagram. Vesicles are characterized by nearly constant hydrodynamic diameters, in the 300–400 nm size range, and positive ζ-potentials (about 40–50 mV). A further increase of AOT content induces the onset of a biphasic region, where vesicles are in equilibrium with a lamellar phase. In the above concentration regimes, addition of sodium bromide destabilizes vesicles and favors the formation of a lamellar phase. Under the same conditions, conversely, a synthetic polyelectrolyte, sodium polyacrylate, does not alter the phase behavior. It induces the growth of particle size and decreases the ζ-potential. The complexes formed by catanionic vesicles and sodium polyacrylate aggregate and form clusters, and readily re-dissolve when more polymer is added.

Size and charge modulation of surfactant-based vesicles.

Nonstoichimetric mixtures of two oppositely charged surfactants, such as sodium dodecylsulfate and hexadecyltrimethylammonium bromide or tetradecyltrimethylammonium bromide and tetraethylammonium perfluorooctanesulfonate, a fluorinated species, form vesicles in dilute concentration regimes of the corresponding phase diagrams. Vesicles size and charge density are tuned by changing the mole ratio between oppositely charged species, at fixed overall surfactant content. They are also modulated by adding neutral electrolytes, or raising T. In the investigated regions, mixtures made of sodium dodecylsulfate/hexadecyltrimethylammonium bromide show ideality of mixing, the other non ideality and phase separation. The formation of unilamellar vesicles occurs in the sodium dodecylsulfate/hexadecyltrimethylammonium bromide mixture, but not in the other. DLS, viscosity, and electrophoretic mobility quantified the above effects. Surface charge density, surface tension, elasticity, and osmotic pressure concur to the stability of unilamellar vesicles and a balance between the above contributions is demonstrated. The results are relevant for practical applications of vesicles as carriers in biomedicine.

Ion distribution around synthetic vesicles of the cat-anionic type.

Aqueous alkyltrimethylammonium bromides, or dialkyldimethylammonium ones, were mixed with sodium alkyl sulfates and dialkanesulfonates. Depending on the overall surfactant concentration, charge and/or mole ratios, cat-anionic vesicles were formed by mixing nonstoichiometric amounts of oppositely charged species. The resulting vesicles are thermodynamically and kinetically stable. ζ-potential and dynamic light scattering characterized the systems. As a rule, cat-anionic vesicles have sizes in the 10(2)-10(3) nm range and bear significant amounts of surface charges. At fixed surfactant concentration, the vesicle surface charge density scales with mole ratios and tends to zero as the latter approach unity. Conversely, the hydrodynamic radius diverges when the cationic/anionic mole ratio is close to 1. The double-layer thickness and surface charge density are controlled by mole ratios and addition of NaBr, which plays a role in vesicle stability. The salt screens the surface charge density and modulates both vesicle size and double-layer thickness. Slightly higher concentrations of NaBr induce the transition from vesicles toward lamellar phases. The electrokinetic properties of cat-anionic dispersions were analyzed by dielectric relaxation experiments. The measured properties are sensitive to vesicle size distributions. In fact, the relaxation frequency shifts in proportion to vesicle polydispersity. Model calculations proposed on that purpose supported the experimental findings.

Use of Cat-Anionic Vesicles as Molecular Vectors for Gene Transfer into Target Cells

We report on the possibilities to use cat-anionic vesicles as active vectors for transfection technologies. Cat-anionic aggregates are non-stoichiometric mixtures made of oppositely charged surfactants, or lipids. Depending on the relative amounts of two such components, bi- or multi-layered vesicles may be formed. The former ones adsorb bio-polymers on their outer surface, but bi-layers may also contain large amounts of lipophilic species in their interior. The transfection fate depends on the vesicle ability and efficiency in adsorption, which leads, eventually, to fusion with cell membranes. The intra-membrane uptake mechanisms differ significantly, and depend on whether the outer, inner, or bi-layer distribution of the species is considered. The first possibility involves a direct exchange of (supra) molecular entities between fluid surfaces in contact; the second and third, conversely, imply membrane fusion and subsequent transport of material within the cell matrix. A realistic combination of the above possibilities can be envisaged, and would ensure a long term action of the transferred (transfected) formulations. Examples taken from recent literature suggest that a realistic possibility is offered to high-yield molecular penetration: this becomes useful in gene transfer and molecular transfection technologies. Some technological aspects inherent to the above formulations are briefly outlined. The overall effect of penetration across the cell membrane of exogenous material in terms of biocompatibility is quite a formidable task to face with and shall be described in detail.

Formation and Properties of Gels Based on Lipo-plexes

Aqueous systems containing sodium taurodeoxycholate and, eventually, soybean lecithin were investigated. Depending on the relative amounts of two such species, molecular, micellar, vesicular, liquid crystalline, and solid phases were formed. In the presence of bovine serum albumin, micellar and vesicular systems form lipo-plexes. The latter self-organize into gels, depending on composition and thermal treatments. According to scanning electron microscopy, vesicle-based gels obtained from lipo-plexes form sponge-like entities, whereas micelle-based ones self-arrange in fibrous organizations. Gels are characterized by a significant viscoelasticity in a wide temperature and frequency range. Rheological data were interpreted by assuming strict relations between the system response and the self-organization of the lipo-plexes into gels. It was inferred that differences in the gel properties depend on the different self-assembly modes of the aggregates formed by the mentioned lipo-plexes. Use of the above systems in biomedical applications, mostly in the preparation of matrices requiring the use of smart and biocompatible gels, is suggested.

Binding of Protein-Functionalized Entities onto Synthetic Vesicles

Mono-disperse silica nano-particles with pending functional acid groups lying on their surface were reacted with coupling agents and, then, with lysozyme, to get proteinfunctionalized entities. The synthetic procedure reported therein gives tiny amounts of protein functionalized sites; surface coverage by the protein is, thus, moderate. The amount of covalently bound lysozyme was estimated from UV-vis methods and resulted to be about molecules per nano-particle. Electro-phoretic mobility experiments indicate the occurrence of significant variations in surface charge density of functionalized nanoparticles compared to the original ones and ensure a significant binding efficiency onto reconstructed, or synthetic, vesicles. Protein-functionalized nano-particles form clusters and are readily re-dispersed by application of shear methods. Thereafter, they remain in disperse form for long times. According to DLS, protein-functionalized nano-particles interact with either cationic or cat-anionic synthetic vesicles. Care was made to ensure that nano-particles and vesicles have comparable sizes. The above procedure ensures to determine the fate of the reactive pathways by DLS. At room temperature and moderate ionic strength, the binding of protein-functionalized entities onto the aforementioned vesicles is completed in about one hour. The nano-particle vesicle complexes precipitate as fine powders, or form large floating objects, depending on vesicle size, relative concentrations of proteinfunctionalized particles and their net charge (which is related to the pH of the dispersing medium). The binding efficiency for the above processes is controlled by the overlapping of repulsive and attractive interactions between particles and vesicles. The kinetic pathways relative to the interactions between vesicles and nano-particles were investigated, and significant differences were met in the two cases. Some technological implications of the above systems are preliminarily discussed. For instance, it is stated that interactions between nano-particles and vesicles mimic those occurring between cells and solid particles, or viral vectors, located in the medium surrounding vesicles.