An engineered POSS drug delivery system for copper(II) anticancer metallodrugs in a selective application toward melanoma cells.

In this work, a polyhedral silsesquioxane (POSS) was used as an engineered drug delivery system for two oxindolimine-copper(II) anticancer complexes, [Cu(isaepy)]+ and [Cu(isapn)]+. The interest in hybrid POSS comes from the necessity of developing materials that can act as adjuvants to improve the cytotoxicity of non-soluble metallodrugs. Functionalization of POSS with a triazole ligand (POSS-atzac) permitted the anchorage of such copper complexes, producing hybrid materials with efficient cytotoxic effects. Structural and morphological characterizations of these copper-POSS systems were performed by using different techniques (IR, NMR, thermogravimetric analysis). A combination of continuous-wave (CW) and pulsed EPR (HYSCORE) spectroscopies conducted at the X-band have enabled the complete characterization of the coordination environment of the copper ion in the POSS-atzac matrix. Additionally, the cytotoxic effects of the loaded materials, [Cu(isapn)]@POSS-atzac and [Cu(isaepy)]@POSS-atzac, were assessed toward melanomas (SK-MEL), in comparison to non-tumorigenic cells (fibroblast P4). Evaluation of their nuclease activity or ability to facilitate cleavage of DNA indicated concentrations as low as 0.6 µg mL-1, while complete DNA fragmentation was observed at 25 µg mL-1. By using adequate scavengers, investigations on active intermediates responsible for their cytotoxicity were performed, both in the absence and in the presence of ascorbate as a reducing agent. Based on the observed selective cytotoxicity of these materials toward melanomas, investigations on the reactivity of these complexes and corresponding POSS-materials with melanin, a molecule that contributes to melanoma resistance to chemotherapy, were carried out. Results indicated the main role of the binuclear copper species, formed at the surface of the silica matrix, in the observed reactivity and selectivity of these copper-POSS systems.

A bioinspired nitrone precursor to a stabilized nitroxide radical.

Nitrones derived from natural antioxidants are emerging as highly specific therapeutics against various human diseases, including stroke, neurodegenerative pathologies, and cancer. However, the development of useful pseudo-natural nitrones requires the judicious choice of a secondary metabolite as the precursor. Betalains are nitrogen-containing natural pigments that exhibit marked antioxidant capacity and pharmacological properties and, hence, are ideal candidates for designing multifunctional nitrones. In this work, we describe the semisynthesis and properties of a biocompatible and antioxidant betalain-nitrone called OxiBeet. This bio-based compound is a better radical scavenger than ascorbic acid, gallic acid, and most non-phenolic antioxidants and undergoes concerted proton-coupled electron transfer. The autoxidation of OxiBeet produces a persistent nitroxide radical, which, herein, is studied via electron paramagnetic resonance spectroscopy. In addition, femtosecond transient absorption spectroscopy reveals that excited state formation is not required for the oxidation of OxiBeet. The results are compared with those obtained using betanin, a natural betalain, and pBeet, the imine analog of OxiBeet. The findings of this study will enable the development of antioxidant and spin-trap nitrones based on the novel N-oxide 1,7-diazaheptamethinium scaffold and betalain dyes with enhanced hydrolytic stability in aqueous alkaline media.

Unlike reactivity of mono- and binuclear imine-copper(II) complexes toward melanoma cells via a tyrosinase-dependent mechanism.

The cytotoxicity of a dinuclear imine-copper (II) complex 2, and its analogous mononuclear complex 1, toward different melanoma cells, particularly human SKMEL-05 and SKMEL-147, was investigated. Complex 2, a tyrosinase mimic, showed much higher activity in comparison to complex 1, and its reactivity was verified to be remarkably activated by UVB-light, while the mononuclear compound showed a small or negligible effect. Further, a significant dependence on the melanin content in the tumor cells, both from intrinsic pigmentation or stimulated by irradiation, was observed in the case of complex 2. Similar tests with keratinocytes and melanocytes indicated a much lower sensitivity to both copper (II) complexes, even after exposition to UV light. Clonogenic assays attested that the fractions of melanoma cells survival were much lower under treatment with complex 2 compared to complex 1, both with or without previous irradiation of the cells. The process also involves generation of reactive oxygen species (ROS), as verified by EPR spectroscopy, and by using fluorescence indicators. Autophagic assays indicated a remarkable formation of cytoplasmic vacuoles in melanomas treated with complex 2, while this effect was not observed in similar treatment with complex 1. Monitoring of specific protein LC3 corroborated the simultaneous occurrence of autophagy. A balance interplay between different modes of cell death, apoptosis and autophagy, occurs when melanomas were treated with the dinuclear complex 2, in contrast to the mononuclear complex 1. These results pointed out to different mechanisms of action of such complexes, depending on its nuclearity.

Design, syntheses, characterization, and cytotoxicity studies of novel heterobinuclear oxindolimine copper(II)-platinum(II) complexes.

Herein, the design and syntheses of two new mononuclear oxindolimine-copper(II) (1 and 2) and corresponding heterobinuclear oxindolimine Cu(II)Pt(II) complexes (3 and 4), are described. All the isolated complexes were characterized by spectroscopic techniques (UV/Vis, IR, EPR), in addition to elemental analysis and mass spectrometry. Cyclic voltammetry (CV) measurements showed that in all cases, one-electron quasi-reversible waves were observed, and ascribed to the formation of corresponding copper(I) complexes. Additionally, waves related to oxindolimine ligand reduction was verified, and confirmed using analogous oxindolimine-Zn(II) complexes. The Pt(IV/II) reduction, and corresponding oxidation, for complexes 3 and 4 occurred at very close values to those observed for cisplatin. By complementary fluorescence studies, it was shown that glutathione (GSH) cannot reduce any of these complexes, under the experimental conditions (room temperature, phosphate buffer 50mM, pH7.4), using an excess of 20-fold [GSH]. All these complexes showed characteristic EPR spectral profile, with parameters values gǁ>g⊥ suggesting an axially distorted environment around the copper(II) center. Interactions with calf thymus-DNA, monitored by circular dichroism (CD), indicated different effects modulated by the ligands. Finally, the cytotoxicity of each complex was tested toward different tumor cells, in comparison to cisplatin, and low values of IC50 in the range 0.6 to 4.0µM were obtained, after 24 or 48h incubation at 37°C. The obtained results indicate that such complexes can be promising alternative antitumor agents.

Inhibition of cyclin-dependent kinase CDK1 by oxindolimine ligands and corresponding copper and zinc complexes.

Oxindolimine-copper(II) and zinc(II) complexes that previously have shown to induce apoptosis, with DNA and mitochondria as main targets, exhibit here significant inhibition of kinase CDK1/cyclin B protein. Copper species are more active than the corresponding zinc, and the free ligand shows to be less active, indicating a major influence of coordination in the process, and a further modulation by the coordinated ligand. Molecular docking and classical molecular dynamics provide a better understanding of the effectiveness and kinase inhibition mechanism by these compounds, showing that the metal complex provides a stronger interaction than the free ligand with the ATP-binding site. The metal ion introduces charge in the oxindole species, giving it a more rigid conformation that then becomes more effective in its interactions with the protein active site. Analogous experiments resulted in no significant effect regarding phosphatase inhibition. These results can explain the cytotoxicity of these metal complexes towards different tumor cells, in addition to its capability of binding to DNA, and decreasing membrane potential of mitochondria.

Oxindole-Schiff base copper(II) complexes interactions with human serum albumin: spectroscopic, oxidative damage, and computational studies.

CD and EPR were used to characterize interactions of oxindole-Schiff base copper(II) complexes with human serum albumin (HSA). These imine ligands form very stable complexes with copper, and can efficiently compete for this metal ion towards the specific N-terminal binding site of the protein, consisting of the amino acid sequence Asp-Ala-His. Relative stability constants for the corresponding complexes were estimated from CD data, using the protein as competitive ligand, with values of log K(CuL) in the range 15.7-18.1, very close to that of [Cu(HSA)] itself, with log K(CuHSA) 16.2. Some of the complexes are also able to interfere in the alpha-helix structure of the protein, while others seem not to affect it. EPR spectra corroborate those results, indicating at least two different metal species in solution, depending on the imine ligand. Oxidative damage to the protein after incubation with these copper(II) complexes, particularly in the presence of hydrogen peroxide, was monitored by carbonyl groups formation, and was observed to be more severe when conformational features of the protein were modified. Complementary EPR spin-trapping data indicated significant formation of hydroxyl and carbon centered radicals, consistent with an oxidative mechanism. Theoretical calculations at density functional theory (DFT) level were employed to evaluate Cu(II)-L binding energies, L-->Cu(II) donation, and Cu(II)-->L back-donation, by considering the Schiff bases and the N-terminal site of HSA as ligands. These results complement previous studies on cytotoxicity, nuclease and pro-apoptotic properties of this kind of copper(II) complexes, providing additional information about their possibilities of transport and disposition in blood plasma.

Double-strand DNA cleavage induced by oxindole-Schiff base copper(II) complexes with potential antitumor activity.

Some oxindole-Schiff base copper(II) complexes have already shown potential antitumor activity towards different cells, inducing apoptosis in a process modulated by the ligand, and having nuclei and mitochondria as main targets. Here, three novel copper(II) complexes with analogous ligands were isolated and characterized by spectroscopic techniques, having their reactivity compared to the so far most active complex in this class. Cytotoxicity experiments carried out toward human neuroblastoma SH-SY5Y cells confirmed its pro-apoptosis property. DNA cleavage studies were then performed in the presence of these complexes, in order to verify the influence of ligand structural features in its nuclease activity. All of them were able to cause double-strand DNA scissions, giving rise to nicked circular Form II and linear Form III species, in the presence of hydrogen peroxide. Additionally, DNA Form II was also detected in the absence of peroxide when the most active complex, [Cu(isaepy)2]2+ 1, was used. In an effort to better elucidate their interactions with DNA, solutions of the different complexes titrated with DNA had their absorption spectra monitored. An absorbance hyperchromism observed at 260 nm pointed to the intercalation of these complexes into the DNA structure. Further, investigations of 2-deoxy-d-ribose (DR) oxidation catalyzed by each of those complexes, using 2-thiobarbituric acid reactive species (TBARS) method, and detection of reactive oxygen species (ROS) formation by spin-trapping EPR, suggested that their mechanism of action in performing efficiently DNA cleavage occurs preferentially, but not only by oxidative pathways.

Investigations of different carbohydrate anomers in copper(II) complexes with D-glucose, D-fructose, and D-galactose by Raman and EPR spectroscopy.

With the aim of verifying different carbohydrate anomers coordinated to copper(II) ions, some copper(II) complexes with D-glucose (Glc), D-fructose (Fru), and D-galactose (Gal) were prepared and investigated by spectroscopic techniques. Their compositions were verified by elemental, ICP-AES and thermal analyses, in addition to conductivity measurements. The compounds isolated were consistent with the formula Na2[Cu2(carbohydrate)3].8H2O and Na[Cu2(carbohydrate)3].6H2O for the aldoses Glc and Gal, respectively, and Na2[Cu3(carbohydrate)4].8H2O in the case of the ketose, Fru. EPR spectra of these solids showed a rhombic environment around the metal center and suggested the presence of different anomers of the carbohydrates in each case. By Raman spectroscopy, it was possible to verify the predominance of the beta anomer of d-glucose in the corresponding copper complex, while in the free ligand the alpha anomer is predominant. In the case of the analogous complex with d-galactose, the spectrum of the complex shows bands of both anomers (alpha and beta) in approximately the same relative intensities as those observed in the isolated free ligand spectrum. On the other hand, for the complex with d-fructose a mixture of both furanose (five-membered ring) and pyranose (six-membered ring) structures was detected with prevalence of the furanose structure. Based on variations in the relative intensities of characteristic Raman bands, the binding site for copper in the fructose ligand was identified as most likely the 1-CH2OH and the anomeric 1-OH, while in beta-D-glucose it is presumably the anomeric 1-OH and the O-5 atom. These results indicated that EPR and Raman spectroscopy are suitable supporting techniques for the characterization of carbohydrate anomers coordinated to paramagnetic ions.