Franco Tardani

Interactions between single-walled carbon nanotubes and lysozyme.

Dispersions of single-walled and non-associated carbon nanotubes in aqueous lysozyme solution were investigated by analyzing the stabilizing effect of both protein concentration and pH. It was inferred that the medium pH, which significantly modifies the protein net charge and (presumably) conformation, modulates the mutual interactions with carbon nanotubes. At fixed pH, in addition, the formation of protein/nanotube complexes scales with increasing lysozyme concentration. Electrophoretic mobility, dielectric relaxation and circular dichroism were used to determine the above features. According to circular dichroism, lysozyme adsorbed onto nanotubes could essentially retain its native conformation, but the significant amount of free protein does not allow drawing definitive conclusions on this regard. The state of charge and charge distribution around nanotubes was inferred by combining electrophoretic mobility and dielectric relaxation methods. The former gives information on changes in the surface charge density of the complexes, the latter on modifications in the electrical double layer thickness around them. Such results are complementary each other and univocally indicate that some LYS molecules take part to binding. Above a critical protein/nanotube mass ratio, depletion phenomena were observed. They counteract the stabilization mechanism, with subsequent nanotube/nanotube aggregation and phase separation. Protein-based depletion phenomena are similar to formerly reported effects, observed in aqueous surfactant systems containing carbon nanotubes.

Delivery of RNA and Its Intracellular Translation into Protein Mediated by SDS-CTAB Vesicles: Potential Use in Nanobiotechnology

Catanionic vesicles are supramolecular aggregates spontaneously forming in water by electrostatic attraction between two surfactants mixed in nonstoichiometric ratios. The outer surface charges allow adsorption to the biomembrane by electrostatic interactions. The lipoplex thus obtained penetrates the cell by endocytosis or membrane fusion. We examined the possible cytotoxic effects and evaluated the transfection efficiency of one vesicle type as compared to known commercial carriers. We show that the individual components of two different vesicles types, CTAB (cetyltrimethylammonium bromide) and DDAB (didodecyldimethylammonium bromide) are detrimental for cell survival. We also assayed the cytotoxicity of SDS-DDAB vesicles and showed dose and time dependency, with the DDAB component being per se extremely cytotoxic. The transfection efficiency of exogenous RNA mediated by SDS-CTAB increases if vesicles assemble in the presence of the reporter RNA; finally, freezing abrogates the transfection ability. The results of our experimental strategy suggest that catanionic vesicles may be adopted in gene therapy and control of antiproliferative diseases.

Lysozyme binds onto functionalized carbon nanotubes

Single walled carbon nanotubes have singular physicochemical properties making them attractive in a wide range of applications. Studies on carbon nanotubes and biological macromolecules exist in literature. However, ad hoc investigations are helpful to better understand the interaction mechanisms. We report on a system consisting of single walled carbon nanotubes and lysozyme. The phenomenology of nanotube-protein interactions and its effects on protein conformation were determined. We investigated the formation of oxidized nanotube-lysozyme conjugates, by studying the effect of both protein concentration and pH. Electrophoretic mobility, dielectric spectroscopy and dynamic light scattering were used to determine the interaction pathways, monitoring the surface charge density and the size of the complexes. The results allowed identifying the conditions of surface saturation at different pH values. The secondary structure of nanotube-adsorbed protein was controlled by circular dichroism; it was observed that it substantially retains its native conformation. Interestingly, we found that the interactions among oxidized nanotubes and lysozyme molecules are mainly of electrostatic nature and easily tunable by varying the pH of the solutions.

Effects of single-walled carbon nanotubes on lysozyme gelation.

The possibility to disperse carbon nanotubes in biocompatible matrices has got substantial interest from the scientific community. Along this research line, the inclusion of single walled carbon nanotubes in lysozyme-based hydrogels was investigated. Experiments were performed at different nanotube/lysozyme weight ratios. Carbon nanotubes were dispersed in protein solutions, in conditions suitable for thermal gelation. The state of the dispersions was determined before and after thermal treatment. Rheology, dynamic light scattering and different microscopies investigated the effect that carbon nanotubes exert on gelation. The gelation kinetics and changes in gelation temperature were determined. The effect of carbon and lysozyme content on the gel properties was, therefore, determined. At fixed lysozyme content, moderate amounts of carbon nanotubes do not disturb the properties of hydrogel composites. At moderately high volume fractions in carbon nanotubes, the gels become continuous in both lysozyme and nanotubes. This is because percolating networks are presumably formed. Support to the above statements comes by rheology.

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.

Encapsulating carbon nanotubes in aqueous ds-DNA anisotropic phases: shear orientation and rheological properties

Carbon nanotubes reinforce polymer composites providing nanotube-based nanohybrids with potentially outstanding properties. The dispersion quality, however, influences the performances of the resulting materials. Therefore, new preparation procedures and efficient dispersion strategies are needed. A new method encapsulating single-walled carbon nanotubes in a nematic phase of double stranded DNA–water–NaCl is reported here. The procedure relies on osmotic compression and on its role in compacting DNA–nanotube composites. An anionic polymer (sodium dextransulfate) was added to the above dispersions and segregative phase separation was induced. DNA–nanotube composites were concentrated and phase-separated from the coexisting polymer solution. In this way, high concentrations of carbon nanotubes can be incorporated in the DNA-rich phase, inducing a transition from liquid- to solid-like behavior. The resulting nematic fluids are homogeneous and orient when shear stresses are applied. The kinetics of re-alignment was determined by rheological and spectroscopic methods. The effect of the nanotubes on the resulting behavior was accounted for. A slowing down of DNA motion observed in such composite matrices suggests interactions with nanotubes.

Titration of DNA/Carbon Nanotube Complexes with Double-Chained Oppositely Charged Surfactants

1/1 dispersions of ss-DNA/CNT complexes in mass ratios were investigated in a mixture with didodecyldimethylammonium bromide, DDAB. Depending on the amounts of the surface-active agent and of the complexes, solutions, precipitates, or re-dissolution occur. DDAB titrates the phosphate groups on the outer surface of the complex and controls the phase sequence in these systems. The combination of different experimental methods determined the phases that occur therein. The results are based on optical absorbance, Dynamic Light Scattering, ionic conductivity, ζ-potential, optical microscopy and AFM. From the above findings a (pseudo)-binary phase diagram is attained. The system has strong similarities with polymer-surfactant mixtures. In fact, its properties conform to cases in which interactions between rigid rod-like polyelectrolytes and oppositely charged species take place. The peculiarities of double-chained DDAB in the process imply significant differences with respect to the behavior of single chain surfactants. In fact, DDAB associates into vesicular entities, when the homologous single chain species forms small micellar aggregates.

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.