Molecular umbrella-amphotericin B conjugates.

A tetrawalled and an octawalled molecular umbrella conjugate of amphotericin B (AmB) have been synthesized. Both conjugates exhibit high water solubility, a low tendency to aggregate, negligible hemolytic activity at 100 µM, and an ability to release a derivative of AmB under reducing conditions that exhibits high antifungal activity. Whereas the larger, octawalled conjugate shows slight adsorption to liposomal membranes and an ability to cross them by passive transport, the tetrawalled analogue shows significant adsorption and much lower bilayer transport activity. The potential of molecular umbrella-AmB conjugates as therapeutic agents is briefly discussed.

In vivo characterization of dynein-driven nanovectors using Drosophila oocytes.

Molecular motors transport various cargoes including vesicles, proteins and mRNAs, to distinct intracellular compartments. A significant challenge in the field of nanotechnology is to improve drug nuclear delivery by engineering nanocarriers transported by cytoskeletal motors. However, suitable in vivo models to assay transport and delivery efficiency remain very limited. Here, we develop a fast and genetically tractable assay to test the efficiency and dynamics of fluospheres (FS) using microinjection into Drosophila oocytes coupled with time-lapse microscopy. We designed dynein motor driven FS using a collection of dynein light chain 8 (LC8) peptide binding motifs as molecular linkers and characterized in real time the efficiency of the FS movement according to its linker's sequence. Results show that the conserved LC8 binding motif allows fast perinuclear nanoparticle's accumulation in a microtubule and dynein dependent mechanism. These data reveal the Drosophila oocyte as a new valuable tool for the design of motor driven nanovectors.

Amphiphilic oligoethyleneimine-ß-cyclodextrin "click" clusters for enhanced DNA delivery.

Monodisperse amphiphilic oligoethyleneimine (OEI)-ß-cyclodextrin (ßCD) clusters have been prepared, and their potential as gene delivery systems has been evaluated in comparison with a nonamphiphilic congener. The general prototype incorporates tetraethyleneimine segments linked to the primary rim of ßCD through either triazolyl or thioureidocysteaminyl connectors. Transfection efficiency data for the corresponding CD:pDNA nanocomplexes (CDplexes) in BNL-CL2 murine hepatocytes evidenced the strong beneficial effect of facial amphiphilicity.

A general approach for the microrheology of cancer cells by atomic force microscopy.

The determination of the viscoelastic properties of cells by atomic force microscopy (AFM) is mainly realized by looking at the relaxation of the force when a constant position of the AFM head is maintained or at the evolution of the indentation when a constant force is maintained. In both cases the analysis rests on the hypothesis that the motion of the probe before the relaxation step is realized in a time which is much smaller than the characteristic relaxation time of the material. In this paper we carry out a more general analysis of the probe motion which contains both the indentation and relaxation steps, allowing a better determination of the rheological parameters. This analysis contains a correction of the Hertz model for large indentation and also the correction due to the finite thickness of the biological material; it can be applied to determine the parameters representing any kind of linear viscoelastic model. This approach is then used to model the rheological behavior of one kind of cancer cell called Hep-G2. For this kind of cell, a power law model does not well describe the low and high frequency modulus contrary to a generalized Maxwell model.

Polycationic amphiphilic cyclodextrins as gene vectors: effect of the macrocyclic ring size on the DNA complexing and delivery properties.

A collection of homologous monodisperse facial amphiphiles consisting of an α-, ß- or γ-cyclodextrin (α, ß or γCD) platform exposing a multivalent display of cationic groups at the primary rim and bearing hexanoyl chains at the secondary hydroxyls have been prepared to assess the influence of the cyclooligosaccharide core size in their ability to complex, compact and protect pDNA and in the efficiency of the resulting nanocondensates (CDplexes) to deliver DNA into cells and promote transfection in the presence of serum. All the polycationic amphiphilic CDs (paCDs) were able to self-assemble in the presence of the plasmid and produce transfectious nanoparticles at nitrogen/phosphorous ratios ≥5. CDplexes obtained from ßCD derivatives generally exhibited higher transfection capabilities, which can be ascribed to their ability to form inclusion complexes with cholesterol, thereby enhancing biological membrane permeability. The presence of thiourea moieties as well as increasing the number of primary amino groups then favour cooperative complexation of the polyphosphate chain, enhancing the stability of the complex and improving transfection. In the α and γCD series, however, only the presence of tertiary amino groups in the cationic clusters translates into a significant improvement of the transfection efficiency, probably by activating endosome escape by the proton sponge mechanism. This set of results illustrates the potential of this strategy for the rational design and optimisation of nonviral gene vectors.

Mannosyl-coated nanocomplexes from amphiphilic cyclodextrins and pDNA for site-specific gene delivery.

Fully homogeneous facial amphiphiles consisting in a cyclodextrin (CD) platform onto which a polycationic cluster and a multi-tail hydrophobic moiety have been installed (polycationic amphiphilic CDs; paCDs) self-organized in the presence of plasmid DNA to form nanometric complexes (CDplexes) which exhibit broad-range transfection capabilities. We hypothesized that biorecognizable moieties located at the hydrophilic rim in the CD scaffold would be exposed at the surface of the corresponding nanoparticles after DNA-promoted aggregation, endowing the system with molecular recognition abilities towards cell receptors. This concept has been demonstrated by developing an efficient synthetic strategy for the preparation of multivalent polycationic glyco-amphiphilic CDs (pGaCDs). Self-assembled nanoparticles obtained from mannosylated pGaCDs and pDNA (average hydrodynamic diameter 80 nm) have been shown to be specifically recognized by mannose-specific lectins, including concanavalin A (Con A) and the human macrophage mannose receptor (MMR). Further macrophage adhesion studies indicated that unspecific binding, probably due to electrostatic interactions with negatively charged cell membrane components, can also operate. The relative specific versus non-specific internalization is dependent on the pGaCD:pDNA proportion, being optimal at a protonable nitrogen/phosphate (N/P) ratio of 5. The resulting GlycoCDplexes were shown to specifically mediate transfection in Raw 264.7 (murine macrophage) cells expressing the mannose-fucose receptor in vitro. FACS experiments confirmed that transfection using these nanoparticles is mannose-dependent, supporting the potential of the approach towards vectorized gene delivery.