Efficient Delivery of MicroRNA and AntimiRNA Molecules Using an Argininocalix[4]arene Macrocycle.

MicroRNAs (miRNAs) are short non-coding RNA molecules acting as gene regulators by repressing translation or by inducing degradation of the target RNA transcripts. Altered expression of miRNAs may be involved in the pathogenesis of many severe human diseases, opening new avenues in the field of therapeutic strategies, i.e., miRNA targeting or miRNA mimicking. In this context, the efficient and non-toxic delivery of premiRNA and antimiRNA molecules might be of great interest. The aim of the present paper is to determine whether an argininocalix[4]arene is able to efficiently deliver miRNA, premiRNA, and antimiRNA molecules to target cells, preserving their biological activity. This study points out that (1) the toxicity of argininocalix[4]arene 1 is low, and it can be proposed for long-term treatment of target cells, being that this feature is a pre-requisite for the development of therapeutic protocols; (2) the delivery of premiRNA and antimiRNA molecules is efficient, being higher when compared with reference gold standards available; and (3) the biological activity of the premiRNAs and antimiRNAs is maintained. This was demonstrated using the argininocalix[4]arene 1 in miRNA therapeutic approaches performed on three well-described experimental model systems: (1) the induction of apoptosis by antimiR-221 in glioma U251 cells; (2) the induction of apoptosis by premiR-124 in U251 cells; and (3) the inhibition of pro-inflammatory IL-8 and IL-6 genes in cystic fibrosis IB3-1 cells. Our results demonstrate that the argininocalix[4]arene 1 should be considered a very useful delivery system for efficient transfer to target cells of both premiRNA and antimiRNA molecules, preserving their biological activity.

Cyclodextrin- and calixarene-based polycationic amphiphiles as gene delivery systems: a structure-activity relationship study.

Multi-head/multi-tail facial amphiphiles built on cyclodextrin (CD) and calixarene (CA) scaffolds are paradigmatic examples of monodisperse gene delivery systems. The possibility to precisely control the architectural features at the molecular level offers unprecedented opportunities for conducting structure-activity relationship studies. A major requirement for those channels is the design of a sufficiently diverse ensemble of compounds for parallel evaluation of their capabilities to condense DNA into transfection nanoparticles where the gene material is protected from the environment. Here we have undertaken the preparation of an oriented library of ß-cyclodextrin (ßCD) and calix[4]arene (CA4) vectors with facial amphiphilic character designed to ascertain the effect of the cationic head nature (aminothiourea-, arginine- or guanidine-type groups) and the macrocyclic platform on the abilities to complex plasmid DNA (pDNA) and in the efficiency of the resulting nanocomplexes to transfect cells in vitro. The hydrophobic domain, formed by hexanoyl or hexyl chains, remains constant in each series, matching the overall structure found to be optimal in previous studies. DLS, TEM and AFM data support that all the compounds self-assemble in the presence of pDNA through a process that involves initially electrostatic interactions followed by formation of ßCD or CA4 bilayers between the oligonucleotide filaments. Spherical transfectious nanoparticles that are monomolecular in DNA are thus obtained. Evaluation in epithelial COS-7 and human rhabdomyosarcoma RD-4 cells evidenced the importance of having primary amino groups in the vector to warrant high levels of transfection, probably because of their buffering capacity. The results indicate that the optimal cationic head depends on the macrocyclic core, aminothiourea groups being preferred in the ßCD series and arginine groups in the CA4 series. Whereas the transfection efficiency relationships remain essentially unchanged within each series, irrespective of the cell type, the optimal platform (ßD or CA4) strongly depends on the cell type. The results illustrate the potential of monodisperse vector prototypes and diversity-oriented strategies on identifying the optimal candidates for gene therapy applications.

Glycoligand-targeted core-shell nanospheres with tunable drug release profiles from calixarene-cyclodextrin heterodimers.

Stable core-shell nanospheres self-assemble in water from heterodimers combining a hydrophobic calix[4]arene moiety and a hydrophilic ß-cyclodextrin head; their potential to encapsulate and provide sustained release of the anticancer drug docetaxel and undergo surface post-modification with glycoligands targeting the macrophage mannose receptor is discussed.

Completing the folate biosynthesis pathway in Plasmodium falciparum: p-aminobenzoate is produced by a highly divergent promiscuous aminodeoxychorismate lyase.

Enzymes that produce or recycle folates are the targets of widely used antimalarial drugs. Despite the interest in the folate metabolism of Plasmodium falciparum, the molecular identification of ADCL (aminodeoxychorismate lyase), which synthesizes the p-aminobenzoate moiety of folate, remained unresolved. In the present study, we demonstrate that the plasmodial gene PF14_0557 encodes a functional ADCL and report a characterization of the recombinant enzyme.

Arginine clustering on calix[4]arene macrocycles for improved cell penetration and DNA delivery.

Cell-penetrating peptides are widely used as molecular transporters for the internalization inside cells of various cargo, including proteins and nucleic acids. A special role is played by arginine-rich peptides and oligoarginines covalently linked or simply mixed with the cargo. Here we report cell-penetrating agents in which arginine units are clustered on a macrocyclic scaffold. Instead of using long peptides, four single arginine units were covalently attached to either the upper or lower rim of a calix[4]arene, kept in the cone conformation building a 'parallel' cyclic array. These new macrocyclic carriers show high efficiency in DNA delivery and transfection in a variety of cell lines.