A novel fluorescent thrombin binding aptamer (TBA), conjugated with the environmentally sensitive dansyl probe at the 3'-end and a β-cyclodextrin residue at the 5'-end, has been efficiently synthesized exploiting Cu(I)-catalyzed azide-alkyne cycloaddition procedures. Its conformation and stability in solution have been studied by an integrated approach, combining in-depth NMR, CD, fluorescence, and DSC studies. ITC measurements have allowed us to analyze in detail its interaction with human thrombin. All the collected data show that this bis-conjugated aptamer fully retains its G-quadruplex formation ability and thrombin recognition properties, with the terminal appendages only marginally interfering with the conformational behavior of TBA. Folding of this modified aptamer into the chairlike, antiparallel G-quadruplex structure, promoted by K(+) and/or thrombin binding, typical of TBA, is associated with a net fluorescence enhancement, due to encapsulation of dansyl, attached at the 3'-end, into the apolar cavity of the β-cyclodextrin at the 5'-end. Overall, the structural characterization of this novel, bis-conjugated TBA fully demonstrates its potential as a diagnostic tool for thrombin recognition, also providing a useful basis for the design of suitable aptamer-based devices for theranostic applications, allowing simultaneously both detection and inhibition or modulation of the thrombin activity.
A Raman-assisted crystallographic study on the adduct between AziRu, a Ru(III) complex with high antiproliferative activity, and RNase A is presented. The protein structure is not perturbed significantly by the Ru label. The metal coordinates to ND atoms of His105 or of His119 imidazole rings, losing all of its original ligands but retaining octahedral, although distorted, coordination geometry. The AziRu binding inactivates the enzyme, suggesting that its antitumor action can be exerted by a mechanism of competitive inhibition.
An efficient drug delivery strategy is presented for novel anticancer amphiphilic ruthenium anionic complexes, based on the formation of stable nanoparticles with the cationic lipid 1,2-dioleyl-3-trimethylammoniumpropane chloride (DOTAP). This strategy is aimed at ensuring high ruthenium content within the formulation, long half-life in physiological media, and enhanced cell uptake. An in-depth microstructural characterization of the aggregates obtained mixing the ruthenium complex and the phospholipid carrier at 50/50 molar ratio is realized by combining a variety of techniques, including dynamic light scattering (DLS), small angle neutron scattering (SANS), neutron reflectivity (NR), electron paramagnetic resonance (EPR), and zeta potential measurements. The in vitro bioactivity profile of the Ru-loaded nanoparticles is investigated on human and non-human cancer cell lines, showing IC(50) values in the low μM range against MCF-7 and WiDr cells, that is, proving to be 10-20-fold more active than AziRu, a previously synthesized NAMI-A analog, used for control. Fluorescence microscopy studies demonstrate that the amphiphilic Ru-complex/DOTAP formulations, added with rhodamine-B, are efficiently and rapidly incorporated in human MCF-7 breast adenocarcinoma cells. The intracellular fate of the amphiphilic Ru-complexes was investigated in the same in vitro model by means of an ad hoc designed fluorescently tagged analog, which exhibited a marked tendency to accumulate within or in proximity of the nuclei.
The binding properties of AziRu, a ruthenium(III) complex with high antiproliferative activity, toward a hen egg white lysozyme have been investigated by X-ray crystallography and Raman microscopy. The data provide clear evidence on the mechanism of AziRu-protein adduct formation and of ligand exchange in the crystal state.
Nucleic acid-based aptamers can be selected from combinatorial libraries of synthetic oligonucleotides to bind, with affinity and specificity similar to antibodies, a wide range of biomedically relevant targets. Compared to protein therapeutics, aptamers exhibit significant advantages in terms of size, non-immunogenicity and wide synthetic accessibility. Various chemical modifications have been introduced in the natural oligonucleotide backbone of aptamers in order to increase their half-life, as well as their pharmacological properties. Very effective alternative approaches, devised in order to improve both the aptamer activity and stability, were based on the design of polyvalent aptamers, able to establish multivalent interactions with the target: thus, multiple copies of an aptamer can be assembled on the same molecular- or nanomaterial-based scaffold. In the present review, the thrombin binding aptamers (TBAs) are analyzed as a model system to study multiple-aptamer constructs aimed at improving their anticoagulation activity in terms of binding to the target and stability to enzymatic degradation. Indeed - even if the large number of chemically modified TBAs investigated in the last 20 years has led to encouraging results - a significant progress has been obtained only recently with bivalent or engineered dendritic TBA aptamers, or assemblies of TBAs on nanoparticles and DNA nanostructures. Furthermore, the modulation of the aptamers activity by means of tailored drug-active reversal agents, especially in the field of anticoagulant aptamers, as well as the reversibility of the TBA activity through the use of antidotes, such as porphyrins, complementary oligonucleotides or of external stimuli, are discussed.
A highly functionalized aminoacyl nucleolipid based on uridine is here proposed as a novel cationic surfactant. To achieve this, a straightforward, high yielding and versatile protocol has been devised, in principle providing synthetic access to a variety of different, related analogs. Self-aggregation properties of this nucleolipid were determined by using a combined approach, including surface tension, conductivity and DLS measurements. Above the critical micellar concentration of 4 × 10(-5) mol kg(-1), large supramolecular assemblies with a counterion condensation degree of 0.25 were observed. The bioactivity profile of this new compound was investigated on cancer and non cancer cell lines.
A novel phosphoramidite derivative of cholesterol, with an ether-linked hexaethylene glycol (HEG) spacer arm, has been obtained through simple and reproducible solid phase modified oligonucleotide synthesis manipulations. This building block and the known phosphoramidite derivative of 3b-(2-hydroxyethoxy)cholesterol have been exploited in standard oligonucleotide synthesis protocols for the preparation of 5'- conjugates of the G-quadruplex-forming ⁵'TGGGAG³' oligomer, known as the Hotoda's sequence, to produce new potential anti-HIV agents.
In this work, carbohydrate-based systems designed as artificial ion transporters have been surveyed. Despite the large structural diversity and ease of manipulations of carbohydrates, in principle endowed with a variety of desirable properties for ionophoric activity, only few examples of sugar-containing compounds have been reported in the literature for these purposes. The most remarkable example is the family of modified β-cyclodextrins, resulting in active cation and/or anion transporters when long, flexible n-alkyl or oligo-ethylene or butylene glycol chains are appended at the lower rim of the macrocycle. Interesting features have been also found in amphiphilic CyPLOS (Cyclic Phosphate-Linked Oligosaccharide) dimers, that is macrocycles with two phenyl-β-D-glucopyranoside residues, 4,6-linked through phosphodiester bonds, derivatized with tetraethylene glycol tentacles. A wider repertoire of available carbohydrate-based scaffolds is expected to largely stimulate the discovery of novel, efficient artificial ionophores, of great interest for both technological and biomedical applications.
A new organometallic ruthenium complex, named AziRu, along with three amphiphilic nucleoside-based ruthenium complexes, ToThyRu, HoThyRu and DoHuRu, incorporating AziRu in their skeleton, have been synthesized, stabilized in POPC phospholipid formulations and studied for their antineoplastic activity. Self-aggregation behavior of these complexes was investigated, showing that the three synthesized AziRu derivatives able to form liposomes and, under specific conditions, elongated micelles. The formulations prepared in POPC proved to be stable for months and showed high in vitro antiproliferative activity. The here described results open new scenarios in the design of innovative transition metal-based supramolecular systems for anticancer drugs vectorization.
A novel ruthenium complex, linked to a cholesterol-containing nucleolipid (named ToThyCholRu), stabilized by lipid aggregates for antineoplastic therapy is presented. In order to retard the degradation kinetics typically observed for several ruthenium-based antineoplastic agents, ToThyCholRu is incorporated into a liposome bilayer formed by POPC. The resulting nanoaggregates contain up to 15% in moles of the ruthenium complex, and are shown to be stable for several weeks. The liposomes host the ruthenium-nucleolipid complex with the metal ion surrounded by POPC lipid headgroups and the steroid moiety inserted in the more external acyl chain region. These ruthenium-containing liposomes are more effective in inhibiting the growth of cancer cells than a model NAMI-A-like ruthenium complex, prepared for a direct evaluation of their anti-proliferative activity. These results introduce new perspectives in the design of innovative transition-metal-based supramolecular systems for anticancer drug vectorization.