Cyclodextrin-Based Nanostructure Efficiently Delivers siRNA to Glioblastoma Cells Preferentially via Macropinocytosis.

Small interfering ribonucleic acid (siRNA) has the potential to revolutionize therapeutics since it can knockdown very efficiently the target protein. It is starting to be widely used to interfere with cell infection by HIV. However, naked siRNAs are unable to get into the cell, requiring the use of carriers to protect them from degradation and transporting them across the cell membrane. There is no information about which is the most efficient endocytosis route for high siRNA transfection efficiency. One of the most promising carriers to efficiently deliver siRNA are cyclodextrin derivatives. We have used nanocomplexes composed of siRNA and a ß-cyclodextrin derivative, AMC6, with a very high transfection efficiency to selectively knockdown clathrin heavy chain, caveolin 1, and p21 Activated Kinase 1 to specifically block clathrin-mediated, caveolin-mediated and macropinocytosis endocytic pathways. The main objective was to identify whether there is a preferential endocytic pathway associated with high siRNA transfection efficiency. We have found that macropinocytosis is the preferential entry pathway for the nanoparticle and its associated siRNA cargo. However, blockade of macropinocytosis does not affect AMC6-mediated transfection efficiency, suggesting that macropinocytosis blockade can be functionally compensated by an increase in clathrin- and caveolin-mediated endocytosis.

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.

Trehalose-based Siamese twin amphiphiles with tunable self-assembling, DNA nanocomplexing and gene delivery properties.

An original family of multivalent vectors encompassing gemini and facial amphiphilicity, namely cationic Siamese twin surfactants, has been prepared from the disaccharide trehalose; molecular engineering lets us modulate the self-assembling properties and the topology of the nanocomplexes with plasmid DNA for efficient gene delivery in vitro and in vivo.

Molecular determinants for cyclo-oligosaccharide-based nanoparticle-mediated effective siRNA transfection.

AIM: To study the structural requirements that a cyclooligosaccharide-based nanoparticle must fulfill to be an efficient siRNA transfection vector. MATERIALS & METHODS: siRNA protection from degradation by RNAses, transfection efficiency and the thermodynamic parameters of the nanoparticle/siRNA interactions were studied on pairs of amphiphilic molecules using biochemical techniques and molecular dynamics. RESULTS: The lower the siRNA solvent accessible surface area in the presence of the nanoparticle, higher the protection from RNAse-mediated degradation in the corresponding nanocomplex; a moderate nanoparticle/siRNA binding energy value further facilitates reversible complexation and binding to the target cellular mRNA. CONCLUSION: The use, in advance, of these parameters will provide a useful indication of the potential of a molecular nanoparticle as siRNA transfecting vector.

Trehalose- and glucose-derived glycoamphiphiles: small-molecule and nanoparticle Toll-like receptor 4 (TLR4) modulators.

An increasing number of pathologies have been linked to Toll-like receptor 4 (TLR4) activation and signaling, therefore new hit and lead compounds targeting this receptor activation process are urgently needed. We report on the synthesis and biological properties of glycolipids based on glucose and trehalose scaffolds which potently inhibit TLR4 activation and signaling in vitro and in vivo. Structure-activity relationship studies on these compounds indicate that the presence of fatty ester chains in the molecule is a primary prerequisite for biological activity and point to facial amphiphilicity as a preferred architecture for TLR4 antagonism. The cationic glycolipids here presented can be considered as new lead compounds for the development of drugs targeting TLR4 activation and signaling in infectious, inflammatory, and autoimmune diseases. Interestingly, the biological activity of the best drug candidate was retained after adsorption at the surface of colloidal gold nanoparticles, broadening the options for clinical development.