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.
Recent major discoveries in membrane biophysics hold the key to a modern understanding of the origin of life on Earth. Membrane bilayer vesicles have been shown to provide a multifaceted ...microenvironment in which protometabolic reactions could have developed. Cell-membrane-like aggregates of amphiphilic molecules capable of retaining encapsulated oligonucleotides have been successfully created in the laboratory. Sophisticated laboratory studies on the origin of life now show that elongation of the DNA primer takes place inside fatty acid vesicles when activated nucleotide nutrients are added to the external medium. These studies demonstrate that cell-like vesicles can be sufficiently permeable to allow for the intake of charged molecules such as activated nucleotides, which can then take part in copying templates in the protocell interior. In this Review we summarize recent experiments in this area and describe a possible scenario for the origin of primitive cells, with an emphasis on the elongation of encapsulated nucleotides.
A molecular‐diversity‐oriented approach for the preparation of well‐defined polycationic amphiphilic cyclodextrins (paCDs) as gene‐delivery systems is reported. The synthetic strategy takes advantage ...of the differential reactivity of primary versus secondary hydroxyl groups on the CD torus to regioselectively decorate each rim with cationic elements and lipophilic tails, respectively. Both the charge density and the hydrophobic–hydrophilic balance can be finely tuned in a highly symmetrical architecture that is reminiscent of both cationic lipids and cationic polymers, the two most prominent types of nonviral gene vectors. The monodisperse nature of paCDs and the modularity of the synthetic scheme are particularly well suited for structure–activity relationship studies. Gel electrophoresis revealed that paCDs self‐assemble in the presence of plasmid DNA (pDNA) to provide homogeneous, stable nanoparticles (CDplexes) of 70–150 nm that fully protect pDNA from the environment. The transfection efficiency of the resulting CDplexes has been investigated in vitro on BNL‐CL2 and COS‐7 cell lines in the absence and presence of serum and found to be intimately dependent on architectural features. Facial amphiphilicity and the presence of a cluster of cationic and hydrogen‐bonding centers for cooperative and reversible complexation of the polyanionic DNA chain is crucial to attain high transgene expression levels with very low toxicity profiles. Further enhancement of gene expression, eventually overcoming that of polyplexes from commercial polyethyleneimine (PEI) polymers (22 kDa), is achieved by building up space‐oriented dendritic polycationic constructs.
DNA delivery: Segregated, space‐oriented polycationic and lipophilic domains have been installed onto a β‐cyclodextrin (βCD) scaffold by using a molecular‐diversity‐oriented strategy. The resulting polycationic amphiphilic CDs (paCDs) efficiently compact and deliver plasmid DNA (pDNA) into cells (see graphic), thus promoting transgene expression, in a topology‐dependent manner.
► 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.
Prodrugs of HIV protease inhibitors Vierling, Pierre; Greiner, Jacques
Current pharmaceutical design,
08/2003, Letnik:
9, Številka:
22
Journal Article
Recenzirano
Despite the efficiency of the present polytherapies against AIDS, HIV replication continues indicating difficulties in drug adherence, drug-drug interactions, resistance issues, and the existence of ...reservoirs or sanctuaries for the virus. Moreover, most of the current FDA-approved HIV protease inhibitors (PIs) display disadvantageous physicochemical and pharmacological properties such as low water solubility, low oral bioavailability and/or low level of penetration into the HIV sanctuaries resulting from their in vivo binding to the plasma proteins and to the Multi-Drug-Resistant P-glycoprotein, their rapid metabolization and inactivation by the liver cytochrome P450 enzymes. To overcome these suboptimal pharmacokinetics, high daily doses must be ingested, which complicate patient adherence to the prescribed regimen and contribute to the appearance of serious long-term metabolic complications and to the decrease of the viral treatment outcome. Another attractive alternative aimed at improving the safety, pharmacokinetics, and therapeutic potency of the current PIs is to modify these PIs into pharmacologically inactive prodrugs which are converted in vivo into their parent active drug. The present review is dedicated to the different prodrug approaches, including the "lipophilic", "hydrophilic", "active transport" and "double-drug" prodrug strategies, which have been applied more particularly to the current HIV PIs used in clinic. Among the strategies explored up to now, the most successful one was the "hydrophilic" prodrug approach which has led to the discovery of fosamprenavir, a phosphate ester prodrug of amprenavir, which has reached phase III clinical trials. This success gives strong support for the search of PI prodrugs as a therapeutic alternative in addition to the development of new and well-tolerated PIs.
Monodisperse facial amphiphiles consisting of a β-cyclodextrin (βCD) platform exposing a multivalent display of cationic groups at the primary rim and bearing hydrophobic chains at the secondary ...oxygens have been prepared by implementing two very robust “click” methodologies, namely cuprous cation-catalyzed azide–alkyne cycloaddition (CuAAC) and thiourea-forming reaction. Most interestingly, the use of solid-supported Cu(I) catalysts was found to be very well suited for multiple CuAAC while facilitating purification of the C 7-symmetric macromolecular triazole adducts. The strategy is compatible with molecular diversity-oriented approaches, which has been exploited to generate a small library of click polycationic amphiphilic CDs (paCDs) for assessing the influence of structural modifications in the ability to complex, compact, and protect pDNA and the efficiency of the resulting paCD:pDNA nanocomplexes (CDplexes) to deliver DNA into cells and promote transfection. The results indicate that fine-tuning the hydrophilic/hydrophobic balance is critical to achieve optimal self-assembling properties and stability of the resulting CDplexes in saline- and serum-containing media. Triazole-type paCDs were, in general, less efficient in promoting gene transfection than thiourea-type derivatives. Nevertheless, the current body of results support that the “dual click” approach implying sequential CuAAC and thiourea-forming reactions represents a versatile strategy to optimize the gene delivery capabilities of cyclodextrin-based facial amphiphiles.
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.
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.
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