Monodisperse amphiphilic oligoethyleneimine (OEI)−β-cyclodextrin (βCD) 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 βCD 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.
Abstract 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.
Experiments of magnetolysis, i.e., destruction of cells induced with magnetic particles (MPs) submitted to the application of a magnetic field, were conducted on HepG2 cancer cells. We herein ...demonstrate the usefulness of combining anisotropic MPs with an alternative magnetic field in magnetolysis. Thus, the application of an alternative magnetic field of low frequency (a few Hertz) in the presence of anisotropic, submicronic particles allowed the destruction of cancer cells “in vitro”. We also show that a constant magnetic field is far less efficient than an oscillating one. Moreover, we demonstrate that, at equal particle volume, it is much more efficient to utilize spindle shaped particles rather than spherical ones. In order to get deeper insight into the mechanism of magnetolysis experiments, we performed a study by AFM, which strongly supports that the magnetic field induces the formation of clusters of particles becoming then large enough todamage cell membranes.
•Magnetic force was applied on cancer cells through magnetic particles.•The penetration depth was predicted, both for spherical and ellipsoidal particles.•Alternative force was shown to damage the cells contrary to static force.•The effect of indentation of magnetic particles was compared to the one of AFM tips.•The damage was attributed to the formation of clusters of particles.
► A general model to obtain the viscoelastic properties of a cell using AFM is proposed. ► It is exact for any time dependent displacement of the cantilever head. ► Its application to cancer cell ...reveals a short time relaxation not observed before. ► A power law model does not properly describe the creep relaxation function. ► A cut-off frequency appears in the shear modulus.
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.
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.
A collection of homologous monodisperse facial amphiphiles consisting of an α-, β- or γ-cyclodextrin (α, β 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 βCD 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.
The incorporation of carbohydrate functional elements in the architecture of polycationic amphiphilic cyclodextrins (paCDs) provides glycosylated paCDs (pGaCDs) that form transfectious nanocomplexes ...(glycoCDplexes) with pDNA. In this study, we aimed at elucidating the internalization mechanisms at play and their incidence in transfection efficiency for glycoCDplexes formulated with 6-amino-6-deoxy-β-
d
-glucopyranosyl-appended pGaCDs in comparison with mannosylated and non-glycosylated congeners. Preliminary data showed a relatively high uptake of the 6-aminoglucosylated nanocomplexes by BNL-CL2 hepatocytes that correlated with a strong affinity towards the galactose-specific peanut agglutinin (PNA) lectin, suggesting that the galactose-binding asialoglycoprotein receptor at the surface of hepatocytes might be involved in glycoCDplex internalization. Transfection kinetics, internalization rates and protein expression data in BNL-CL2 ASGPR-expressing cells and COS-7 ASGPR-devoid epithelial cells in the absence and presence of different inhibitors of clathrin-dependent (chlorpromazine), caveolae-dependent (genistein) and macropinocytosis (amiloride) endocytic routes evidenced significant differences in cell uptake pathways and fate of glycoCDplexes as compared with CDplexes. Most importantly, such differences were dependent on the cell type and on the carbohydrate coating moiety. Clathrin-mediated uptake in BNLCL-2 cells is particularly favored for the 6-amino-6-deoxyglucose CDplexes, supporting the interplay of specific recognition phenomena. Competitive uptake and transfection experiments conducted in the presence of asialofetuin or of a polyclonal ASGPR-antibody, as well as siRNA-mediated ASGPR-specific gene knockdown, supported the involvement of ASGPR, firmly demonstrating the dual role of the 6-amino-6-deoxyglucose motif as DNA and lectin receptor ligand. The results reinforce the use of carbohydrates in glycoCDplexes to modulate cellular uptake and transfection capabilities in a cell-dependent manner.
β-Cyclodextrin-based glycoCDplexes are internalized through several redundant pathways whose relative prevalence depends on the coating sugar and on the cell line.
A quantitative method for the reduction of disulfides, which uses a totally recyclable solid phase supported reducing agent, is reported.
d,
l-α-Lipoic acid was quantitatively condensed on a highly ...stable 100% PEG Aminomethyl-ChemMatrix® resin that can swell in aqueous media as well as in organic solvents. Lipoic residue, subsequently reduced to its dihydrolipoyl form, was utilized as a reducing agent for highly valuable disulfide compounds.
Precise control over the architecture of gene carriers is instrumental to manipulate gene delivery efficiency. Combining cationic centers and carbohydrate motifs into monodisperse architectures has ...been proposed as a suitable strategy to impart nucleic acid condensation abilities while preserving biocompatibility. Herein, we have assessed the influence of the arrangement and orientation of cationic elements on the self-assembling and gene transfer capabilities of polycationic glycoamphiphilic cyclodextrins (pGaCDs). For such purposes, a series of cyclodextrin multiconjugates bearing aminoglucoside motifs at their primary rim and hexanoyl chains at the secondary positions were synthesized. In the presence of pDNA, pGaCDs self-assemble into nanoaggregates that promote cellular uptake and gene expression in COS-7 cells with efficiencies that are intimately associated with the arrangement of amino functionalities imposed by the aminoglucoside antennae onto the cyclodextrin-scaffolded cluster. Although transfection efficiencies were lower than those observed for polyethyleneimine (PEI)-based polyplexes and previously-reported polycationic amphiphilic cyclodextrins (paCDs), the results reported herein illustrate (i) the dramatic influence that subtle architectural modifications exert on the supramolecular organization of pGaCDs and (ii) the virtues of monodisperse systems for tailoring gene transfer capabilities.
Precise control over the architecture of gene carriers is instrumental to manipulate gene delivery efficiency. Combining cationic centers and carbohydrate motifs into monodisperse architectures has ...been proposed as a suitable strategy to impart nucleic acid condensation abilities while preserving biocompatibility. Herein, we have assessed the influence of the arrangement and orientation of cationic elements on the self-assembling and gene transfer capabilities of polycationic glycoamphiphilic cyclodextrins (pGaCDs). For such purposes, a series of cyclodextrin multiconjugates bearing aminoglucoside motifs at their primary rim and hexanoyl chains at the secondary positions were synthesized. In the presence of pDNA, pGaCDs self-assemble into nanoaggregates that promote cellular uptake and gene expression in COS-7 cells with efficiencies that are intimately associated with the arrangement of amino functionalities imposed by the aminoglucoside antennae onto the cyclodextrin-scaffolded cluster. Although transfection efficiencies were lower than those observed for polyethyleneimine (PEI)-based polyplexes and previously-reported polycationic amphiphilic cyclodextrins (paCDs), the results reported herein illustrate (i) the dramatic influence that subtle architectural modifications exert on the supramolecular organization of pGaCDs and (ii) the virtues of monodisperse systems for tailoring gene transfer capabilities.
The self-assembling and gene transfer capabilities of monodisperse amphiphilic aminoglucoside-cyclodextrin conjugates depend on the amino disposition at the glycationic head.
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