Infrared Nanospectroscopy Reveals DNA Structural Modifications upon Immobilization onto Clay Nanotubes.

The growing demand for innovative means in biomedical, therapeutic and diagnostic sciences has led to the development of nanomedicine. In this context, naturally occurring tubular nanostructures composed of rolled sheets of alumino-silicates, known as halloysite nanotubes, have found wide application. Halloysite nanotubes indeed have surface properties that favor the selective loading of biomolecules. Here, we present the first, to our knowledge, structural study of DNA-decorated halloysite nanotubes, carried out with nanometric spatially-resolved infrared spectroscopy. Single nanotube absorption measurements indicate a partial covering of halloysite by DNA molecules, which show significant structural modifications taking place upon loading. The present study highlights the constraints for the use of nanostructured clays as DNA carriers and demonstrates the power of super-resolved infrared spectroscopy as an effective and versatile tool for the evaluation of immobilization processes in the context of drug delivery and gene transfer.

Experimental Evidence of Single-Stranded DNA Adsorption on Multiwalled Carbon Nanotubes.

Noncovalent DNA functionalization is one of the most used routes for the easy dispersion of carbon nanotubes (CNTs) yielding DNA-CNTs complexes with promising applications. Definition of the structure of adsorbed DNA is crucial, but the organization of polymer at the carbon interface is far from being understood. In comparison to single-walled nanotubes, not much effort has been devoted to assessing the structure of the adsorbed DNA on multiwalled carbon nanotubes (MWCNTs), where their metallic nature, large size, and polydispersity represent serious obstacles for both experimental and theoretical studies. As a contribution to fill this lack in these aspects, we investigated DNA-MWCNT complexes by dielectric spectroscopy (DS) which is sensitive to even small changes in the charge distribution at charged interfaces and was largely employed in studying the electric and conformational properties of polyelectrolytes, such as DNA, in aqueous solutions and at interfaces. The dielectric relaxation in the MHz range is the signature of DNA adsorption on CNTs and sheds light on its conformational properties. A detailed analysis of the conductivity of the DNA-MWCNT suspensions unequivocally proves that DNA is adsorbed in a single-stranded conformation while excess DNA reassociates without interfering with the stability of the complexes.

Deoxycholic acid and l-Phenylalanine enrich their hydrogel properties when combined in a zwitterionic derivative.

HYPOTHESIS: Sodium Deoxycholate (NaDC) and Phenylalanine (Phe) are important biological hydrogelators. NaDC hydrogels form by lowering the pH or by increasing the ionic strength. Phe gels form from saturated solution by thermal induction and slow kinetics. The resulting gels hold great potential in medicine and biology as drug carriers and models for fundamental self-assembly in pathological conditions. Based on this background it was hypothesized that a Phe substituted NaDC could provide a molecule with expanded gelling ability, merging those of the precursors. EXPERIMENTS: We coupled both building blocks in a zwitterionic derivative bearing a Phe residue at the C3 carbon of NaDC. The specific zwitterionic structure, the concurrent use of Ca2+ ions for the carboxyl group coordination and the pH control generate conditions for the formation of hydrogels. The hydrogels were analyzed by combining UV and circular dichroism spectroscopies, rheology, small angle X-ray scattering and atomic force microscopy. FINDINGS: Hydrogel appearance occurs in conditions that are uncovered in the case of the pure Phe and NaDC: self-standing gels form instantaneously at room temperature, in the 10-12 pH range and down to concentration of 0.17 wt%. Both thixotropic and shake resistant gels can form depending on the derivative concentration. The gels show an uncommon thermal stability in the scanned range of 20-60 °C. The reported system concurrently enriches the hydrogelation properties of two relevant building blocks. We anticipate some potential applications of such gels in materials science where coordination of metal ions can be exploited for templating inorganic nanostructures.

Shear Rheology Control of Wrinkles and Patterns in Graphene Oxide Films.

Drying graphene oxide (GO) films are subject to extensive wrinkling, which largely affects their final properties. Wrinkles were shown to be suitable in biotechnological applications; however, they negatively affect the electronic properties of the films. Here, we report on wrinkle tuning and patterning of GO films under stress-controlled conditions during drying. GO flakes assemble at an air-solvent interface; the assembly forms a skin at the surface and may bend due to volume shrinkage while drying. We applied a modification of evaporative lithography to spatially define the evaporative stress field. Wrinkle alignment is achieved over cm2 areas. The wavelength (i.e., wrinkle spacing) is controlled in the µm range by the film thickness and GO concentration. Furthermore, we propose the use of nanoparticles to control capillary forces to suppress wrinkling. An example of a controlled pattern is given to elucidate the potential of the technique. The results are discussed in terms of classical elasticity theory. Wrinkling is the result of bending of the wet solid skin layer assembled on a highly elastic GO dispersion. Wavelength selection is the result of energy minimization between the bending of the skin and the elastic deformation of the GO supporting dispersion. The results strongly suggest the possibility to tune wrinkles and patterns by simple physicochemical routes.

Interactions and effects of BSA-functionalized single-walled carbon nanotubes on different cell lines.

Functionalized carbon nanotubes (CNTs) have shown great promise in several biomedical contexts, spanning from drug delivery to tissue regeneration. Thanks to their unique size-related properties, single-walled CNTs (SWCNTs) are particularly interesting in these fields. However, their use in nanomedicine requires a clear demonstration of their safety in terms of tissue damage, toxicity and pro-inflammatory response. Thus, a better understanding of the cytotoxicity mechanisms, the cellular interactions and the effects that these materials have on cell survival and on biological membranes is an important first step for an appropriate assessment of their biocompatibility. In this study we show how bovine serum albumin (BSA) is able to generate homogeneous and stable dispersions of SWCNTs (BSA-CNTs), suggesting their possible use in the biomedical field. On the other hand, this study wishes to shed more light on the impact and the interactions of protein-stabilized SWCNTs with two different cell types exploiting multidisciplinary techniques. We show that BSA-CNTs are efficiently taken up by cells. We also attempt to describe the effect that the interaction with cells has on the dielectric characteristics of the plasma membrane and ion flux using electrorotation. We then focus on the BSA-CNTs' acute toxicity using different cellular models. The novel aspect of this work is the evaluation of the membrane alterations that have been poorly investigated to date.

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.

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.

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.

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.

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.

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.