Only a few examples of monodisperse molecular entities that can compact exogenous nucleic acids into nanocomplexes, protect the cargo from the biological environment, facilitate cell internalization, and promote safe transfection have been reported up to date. Although these species open new venues for fundamental studies on the structural requirements that govern the intervening processes and their application in nonviral gene-vector design, the synthesis of these moieties generally requires a relatively sophisticated chemistry, which hampers further development in gene therapy. Herein, we report an original strategy for the reversible complexation and delivery of DNA based on the supramolecular preorganization of a β-cyclodextrin-scaffolded polycationic cluster facilitated by bisadamantane guests. The resulting gemini-type, dual-cluster supramolecules can then undergo DNA-templated self-assembly at neutral pH value by bridging parallel DNA oligonucleotide fragments. This hierarchical DNA condensation mechanism affords transfectious nanoparticles with buffering capabilities, thus facilitating endosomal escape following cell internalization. Protonation also destabilizes the supramolecular dimers and consequently the whole supramolecular edifice, thus assisting DNA release. Our advanced hypotheses are supported by isothermal titration calorimetry, NMR and circular dichroism spectroscopic analysis, gel electrophoresis, dynamic light scattering, TEM, molecular mechanics, molecular dynamics, and transfection studies conducted in vitro and in vivo.
The convergent preparation of Janus molecular nanoparticles by thiourea-"clicking" of α,α'-trehalose halves has been implemented; the strategy allows access to macrocyclic derivatives with seggregated cationic and lipophilic domains that in the presence of DNA undergo pH-dependent self-assembly into lamellar superstructures, as established by electrochemical, structural (SAXS), microscopical (TEM) and computational techniques, that mediate transfection in vitro and in vivo.
The topology of β-cyclodextrin can be molded, from toroidal to ovoid basket-shaped, by the installation of an o- or m-xylylene moiety connecting two consecutive d-glucopyranosyl units through the secondary O-2(I) and O-3(II) positions. This strategy can be exploited advantageously to precast the cavity for preferential inclusion of globular or planar guests as well as to privilege dimeric or monomeric species in water solution.
Two soluble cyanine/β-cyclodextrin derivatives have been synthesized under simultaneous ultrasound/microwave irradiation. UV-Vis, steady-state, time-resolved fluorescence and circular dichroism spectroscopies were used to evaluate their photophysical properties, as well as to study their complexation with the anticancer drug doxorubicin. Titration experiments were performed by monitoring corrected emission intensity. The analysis of fluorescence data provided stability constants for doxorubicin complexes with cyanine/β-cyclodextrins which are 4 orders of magnitude greater than those reported for its complexation with native β-cyclodextrin and one order greater than its association with DNA. The complexation has also been studied using Molecular Mechanics and Molecular Dynamics simulations. Both electrostatic and van de Waals binding energy contributions are important to system stabilization. The potential use of these systems as carriers has been evaluated via in vitro experiments on HeLa cells and by monitoring cell entrance via confocal laser scanning microscopy.
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