Patrizia Andreozzi

Multi- to unilamellar transitions in catanionic vesicles.

Sodium dodecylsulfate (SDS) and cetyltrimethylammonium bromide (CTAB) dispersed in aqueous solution form catanionic vesicles. Depending on composition, such vesicles show different net charge, stability, and interaction capability, indicative of the strong impact that catanionic systems may have in gene therapy and drug delivery technologies. To reveal the interplay among composition, net charge, sensitivity to temperature changes, vesicle size, and inner structure, a series of experiments on catanionic vesicles prepared at different SDS/CTAB mole ratios was performed. Dynamic light scattering, small-angle X-ray scattering, and zeta-potential experiments allow one to characterize an unexpected critical phenomenon at the nanoscale level. On heating, vesicles increase in size, but at a critical temperature an abrupt vesicle size reduction has been observed, together with a transition from multi- to a unilamellar state. The critical temperature regularly depends on the SDS/CTAB mole ratio. The unilamellar state obtained upon heating is retained for weeks. These phenomena suggest a new way to produce stable unilamellar vesicles with tunable size and charge.

Supra-molecular Association and Polymorphic Behaviour In Systems Containing Bile Acid Salts.

A wide number of supra-molecular association modes are observed in mixtures containing water and bile salts, BS, (with, eventually, other components). Molecular or micellar solutions transform into hydrated solids, fibres, lyotropic liquid crystals and/or gels by raising the concentration, the temperature, adding electrolytes, surfactants, lipids and proteins. Amorphous or ordered phases may be formed accordingly. The forces responsible for this very rich polymorphism presumably arise from the unusual combination of electrostatic, hydrophobic and hydrogen-bond contributions to the system stability, with subsequent control of the supra-molecular organisation modes. The stabilising effect due to hydrogen bonds does not occur in almost all surfactants or lipids and is peculiar to bile acids and salts. Some supra-molecular organisation modes, supposed to be related to malfunctions and dis-metabolic diseases in vivo, are briefly reported and discussed.

Biological activity of SDS-CTAB cat-anionic vesicles in cultured cells and assessment of their cytotoxicity ending in apoptosis

SDS-CTAB cat-anionic vesicles are supramolecular aggregates forming complexes with biopolymers and enter the cells via membrane fusion or endocytosis. Different applicative areas exist: gene therapy, drug delivery and nanotechnology. We previously examined the absorption/release of biopolymers from vesicles in solution. Here we evaluate their cytotoxicity in cultured cells; to this end we characterized the vesicles and analyzed their biological effects at cellular and molecular level. At low concentration these vesicles have scarce consequences on normal cell growth; at higher dosage they activate apoptotic death processes, due to membrane damage. In conclusion, the use of these particles in nano-biotechnology represents an actual possibility.

Catanionic vesicles formed with arginine-based surfactants and 1,2-dipalmitoyl-sn-glycero-3-phosphate monosodium salt.

We report on mixing an anionic diacyl phospholipid (1,2-dipalmitoyl-sn-glycero-3-phosphate monosodium salt, DPPA) with either monoacyl and diacyl arginine-based surfactants. These mixtures are part of the rich family of pseudo-triple-chain and pseudo-tetra-chain catanionic mixtures, respectively. Vesicle size and zeta-potential were measured at several mixing ratios. Additional information on counterion binding, vesicle size, and integrity was obtained from ion selective electrode and Cryo-TEM measurements. Addition of positively charged surfactants to DPPA results in an increase of vesicle size. However, zeta-potential shows different trends, depending on whether water or acid media are used as solvent. In the latter, zeta-potential values progressively approach 0 upon addition of amino acid based surfactants. In water, surprisingly, zeta-potential values become more negative. The results are discussed in terms of modifications in counterion binding and vesicle size.

Electrostatic Interactions between a Protein and Oppositely Charged Micelles

Micellar solutions made of a fully fluorinated surfactant, LiPFN, form water-soluble complexes with lysozyme in a wide concentration range. Such complexes are stabilized by electrostatic and, very presumably, double-layer interactions. The mixtures were investigated by combining electrophoretic mobility, DLS, and dielectric relaxation methods. The former gives information on the surface charge density of protein-micelle complexes and indicates that the resulting adducts retain a negative charge (i.e., charge neutralization is incomplete). The double-layer thickness of proteins, micelles, and protein-micelle complexes is also connected to the dielectric relaxation frequency. Changes in particle size (inferred by DLS), charge density, and double-layer thickness are closely interrelated to each other. A model was developed to quantify such properties.

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.

Assembly kinetics in binary mixtures of strongly attractive colloids.

The early stages of aggregation kinetics in a binary mixture of asymmetric colloids, aggregating irreversibly via biotin-streptavidin bonds, are experimentally and numerically studied. Experiments are performed by DLS methods, and data are analyzed in terms of a Smoluchowski-like coagulation equation. Focus is on the case of small (S) biotin-covered particles interacting with large (L) streptavidin-covered ones. The small particles act as linkers between the large ones. The dependence of S-L and L-L aggregation rate constants on the ratio between concentration of small and large particles is investigated, to detect the concentration at which aggregation of large particles is most effective.

Exploring the pH Sensitivity of Poly(allylamine) Phosphate Supramolecular Nanocarriers for Intracellular siRNA Delivery

Silencing RNA (siRNA) technologies emerge as a promising therapeutic tool for the treatment of multiple diseases. An ideal nanocarrier (NC) for siRNAs should be stable at physiological pH and release siRNAs in acidic endosomal pH, fulfilling siRNA delivery only inside cells. Here, we show a novel application of polyamine phosphate NCs (PANs) based on their capacity to load negatively charged nucleic acids and their pH stability. PANs are fabricated by complexation of phosphate anions from phosphate buffer solution (PB) with the amine groups of poly(allylamine) hydrochloride as carriers for siRNAs. PANs are stable in a narrow pH interval, from 7 to 9, and disassemble at pHs higher than 9 and lower than 6. siRNAs are encapsulated by complexation with poly(allylamine) hydrochloride before or after PAN formation. PANs with encapsulated siRNAs are stable in cell media. Once internalized in cells following endocytic pathways, PANs disassemble at the low endosomal pH and release the siRNAs into the cytoplasm. Confocal laser scanning microscopy (CLSM) images of Rhodamine Green labeled PANs (RG-PANs) with encapsulated Cy3-labeled siRNA in A549 cells show that siRNAs are released from the PANs. Colocalization experiments with labeled endosomes and either labeled siRNAs prove the translocation of siRNAs into the cytosol. As a proof of concept, it is shown that PANs with encapsulated green fluorescence protein (GFP) siRNAs silence GFP in A549 cells expressing this protein. Silencing efficacy was evaluated by flow cytometry, CLSM, and Western blot assays. These results open the way for the use of poly(allylamine) phosphate nanocarriers for the intracellular delivery of genetic materials.

Study of the Impact of Polyanions on the Formation of Lipid Bilayers on Top of Polyelectrolyte Multilayers with Poly(allylamine hydrochloride) as the Top Layer

The impact of polyanions on the formation of lipid bilayers on top of polyelectrolyte multilayers (PEMs) with poly(allylamine hydrochloride) (PAH) as the top layer is studied for the deposition of vesicles of mixed lipid composition, 50:50 molar ratio of zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and negatively charged 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS). PEMs are assembled with polystyrene sulfonate (PSS), poly(acrylic acid) (PAA), and alginic acid sodium salt (Alg) as polyanions. The assembly of the vesicles on the PEMs is followed by means of the quartz crystal microbalance with dissipation. Fluorescence recovery after photobleaching measurements are applied to evaluate bilayer formation. Whereas a bilayer is formed on top of PAH/PSS multilayers, the vesicles are adsorbed on top of PAH/Alg and PAH/PAA multilayers, remaining unruptured or only partially fused. The influence of the surface composition of the PEM and of the bulk properties of the film are analyzed. The phosphate ions present in phosphate-buffered saline (PBS) play a fundamental role in bilayer formation on top of PAH/PSS as they complex with PAH and render the surface potential close to zero. For PAH/PAA and PAH/Alg, PBS renders the surface negative. X-ray photoelectron spectroscopy shows that the dibasic phosphate ions from PBS complex preferentially with PAH in PAH/PAA and PAH/Alg multilayers, whereas monobasic phosphates complex with PAH in PAH/PSS. An explanation for the absence of bilayer formation on PAH/PAA and PAH/Alg is given on the basis of the different affinities of phosphate ions for PAH in combination with the different polyanions.