Preparation and characterization of metallomicelles of Ru(II). Cytotoxic activity and use as vector.

The use of nanovectors in several medicinal treatments has reached a great importance in the last decade. Some drugs need to be protected to increase their lifetimes in the blood flow, to avoid degradation, to be delivered into target cells or to decrease their side effects. The goal of this work was to design and prepare nanovectors formed by novel surfactants derived from the [Ru(bpy)3]2+ complex. These amphiphilic molecules are assembled to form metallomicelles which can act as pharmaceutical agents and, at the same time, as nanovectors for several drugs. TEM images showed a structural transition from spherical to elongated micelles when the surfactant concentration increased. Fluorescence microscopy confirmed the internalization of these metallomicelles into diverse cell lines and cytotoxicity assays demonstrated specificity for some human cancer cells. The encapsulation of various antibiotics was carried out as well as a thorough study about the DNA condensation by the metallomicelles. To the best of our knowledge, applications of these metallomicelles have not been shown in the literature yet.

Cooperative interaction between metallosurfactants, derived from the [Ru(2,2'-bpy)3](2+) complex, and DNA.

With the idea of improving and advancing the design and preparation of new reagents based on cationic surfactants for gene therapy, two luminescent metallosurfactants derived from the [Ru(2,2'-bpy)3](2+) complex were synthesized. Their interaction with DNA and the effect they exert on the conformation of the polynucleotide were studied by using different techniques. The equilibrium binding constants, Kb, of the two surfactants to DNA were obtained at different molar ratios X=[surfactant]/[DNA]. The observed sigmoidal dependence of Kb on X confirms the cooperative character of the binding. After the addition of a determined surfactant concentration, the condensation of the polymer was observed. The amount of surfactant needed to produce this conformational change is lower for the double stranded surfactant than for the single chain surfactant due to a stronger hydrophobic interaction. The addition of α-cyclodextrin molecules to the metallosurfactant/DNA solutions results in polynucleotide decompaction, which confirms the importance of the hydrophobic interactions in the condensation of the polynucleotide. Results also show the importance of choosing both a proper system to study and the most seeming measuring technique to use. It is demonstrated that, in some cases, the use of several techniques is desirable to obtain reliable and accurate results.

Binding of DNA by a dinitro-diester calix[4]arene: denaturation and condensation of DNA.

A study of a dinitro-diester calix[4]arene (5,17-(3-nitrobenzylideneamino)-11,23-di-tert-butyl-25,27-diethoxycarbonyl methyleneoxy-26,28-dihydroxycalix[4]arene) interaction with calf-thymus DNA was carried out using several techniques. The measurements were done at various molar ratios X=[calixarene]/[DNA]. Results show diverse changes in the DNA conformation depending on the X value. Thus, at low macrocycle concentrations, the calixarene binds to the polynucleotide. This interaction, mainly in groove mode, weakens the hydrogen bonds between base pairs of the helix inducing denaturation of the double strands, as well as condensation of the macromolecule, from an extended coil state to a globular state. An opposite effect is observed at X molar ratios higher than 0.07. The de-condensation of DNA happens, that is, the transition from a compact state to a more extended conformation, probably due to the stacking of calixarene molecules in the solution. Results also show the importance of making a proper choice of the system under consideration.

Conformational changes of DNA in the presence of 12-s-12 gemini surfactants (s=2 and 10). Role of the spacer's length in the interaction surfactant-polynucleotide.

A multifaceted study on the interaction of calf-thymus DNA with two different cationic gemini surfactants alkanediyl-α-ω-bis(dodecyldimethyl-amonium)bromide, 12-s-12,2Br(-) (with s=2, G2, and 10, G10) was carried out. The measurements were done at different molar ratios X=[surfactant]/[DNA]. Results show two different conformational changes in DNA: a first compaction of the polynucleotide corresponding to a partial conformational (not total) change of DNA from an extended coil state to a globular state that happens at the lower molar ratio X. A second change corresponds to a breaking of the partial condensation, that is, the transition from the compacted state to a new more extended conformation (for the higher X values) different to the initial extension. According to circular dichroism spectra and dynamic light scattering measurements, this new state of DNA seems to be similar to a ψ-phase. Measurements confirm that interactions involved in the compaction are different to those previously obtained for the analog surfactant CTAB. X values at which the conformational changes happen depend on the length of the spacer in the surfactant along with the charge of the polar heads.

Interaction between monomers of two surfactants derived from the [Ru(2,2'-bpy)(3)](2+) complex and α, ß and γ-cyclodextrins: formation of [2]- and [3]-pseudorotaxanes.

Two new surfactants derived from the tris(2,2'-bipyridine) ruthenium(II) complex, [Ru(2,2'-bpy)(3)](2+), were synthesized and characterized: the double-tailed [Ru(2,2'-bipy)2(4,4'-(C(11)H(23))(2)-2,2'-bipy)](2+) surfactant (RuC11) and the mono-tailed [Ru(2,2'-bipy)(2)(4-(CH(3))-4'-(C(13)H(27))-2,2'-bipy)](2+) surfactant (RuC13). The main characteristic of these species is the presence of an inorganic complex as the polar head of the surfactant with interesting luminescence properties, which were used to study the interaction of these cationic surfactants with α-, ß- and γ-cyclodextrins (CD). The results showed the formation of [2]- and [3]-pseudorotaxanes. The binding constant values as well as the stoichiometry of the complexes formed were obtained; the results were confirmed, from a qualitative point of view, with NMR spectra.

Compaction and decompaction of DNA induced by the cationic surfactant CTAB.

A multifaceted study on the interaction of the cationic surfactant CTAB with calf thymus DNA was carried out by using different techniques. The measurements were done at different molar ratios X = [CTAB]/[DNA]. Results show the conformational change that DNA suffers due to the interaction with surfactant molecules at low molar ratios: the condensation of the polynucleotide, from an extended coil state to a globular state. The effect observed at the higher molar ratios is worth noting: the decondensation of DNA, that is, the transition from a compact state to a more extended conformation. Experimental data obtained confirm that this latter state is not exactly the same as that found in the absence of the surfactant. Attractive interactions between different parts of the molecule by ion correlation effects are the driving force to produce both the compaction and decompaction events. Results also show the importance of choosing both a proper system for the study and the most seeming measuring technique to use. The study demonstrates that, in some cases, the use of several techniques is desirable in obtaining reliable and accurate results.

Photoinduced electron-transfer reactions: a study of the diffusion-controlled and activation-diffusion-controlled processes.

The diffusion-controlled electron transfer rate constants (k(d)) of several quenching reactions of ruthenium complexes [Ru(L)(3)](2+*) (L = bpy, phen, and 4,7-(CH(3))(2)phen) with [Fe(CN)(6)](3-) were experimentally determined at different concentrations of NaNO(3). From these rate constants, the effective values of viscosity coefficients for NaNO(3) solutions were calculated using EMSA (exponential mean spherical approximation) and EF (Eigen-Fuoss) approaches in order to take into account the mean force potential between reactants. The reliability of the effective parameters were checked through calculations of the rate constants of the reaction [IrCl(6)](2-)+ [Ru(bpy)(3)](2+)* in these NaNO(3) solutions. The rate constants of this reaction were also obtained by fluorescence quenching measurements. The agreement between the two sets of data (experimental and predicted) is excellent. The trends of association (k(d)) and dissociation (k(-d)) rate constants for 2+/3-, 2+/2-, and 2+/2+ reactions in NaNO(3) solutions are discussed. The use of effective diffusion coefficients for estimating k(d) and k(-d) allowed us to obtain the intrinsic electron transfer rate constant (k(et)) for the activation-diffusion-controlled process between [Ru(bpy)(3)](2+)* and [Co(NH(3))(5)Cl](2+) complexes from the observed (quenching) rate constant. The trend of electron-transfer rate constant in NaNO(3) for this reaction was rationalized by using the Marcus electron-transfer treatment.

Binding of Ru(NH3)5pz2+ to 4-sulfocalix[4]arene sodium salt. Effects of the host-guest interaction on electron transfer processes.

The reactions [Ru(NH3)5pz]2++S2O8(2-) and [Ru(NH3)5pz]2++[Co(C2O4)3]3- have been studied in solutions of 4-sulfocalix[4]arene sodium salt. Results show a binding of the ruthenium complex to the calixarene with a 2:1 stoichiometry; that is, a ruthenium molecule binds to two calixarene molecules. This stoichiometry changes when NaCl is added to the medium. Thus, a mixture of 1:1 and 2:1 adducts is found in the presence of 0.1 mol dm(-3) NaCl and only 1:1 adducts when the salt concentration is increased up to 0.3 mol dm(-3). Results show that the binding of the ruthenium complex to the calixarene is due to electrostatic and nonelectrostatic interactions. Kinetic data are interpreted by using the pseudophase model and taking into account the stoichiometry of the ruthenium binding to calixarene. The presence of a supporting electrolyte in the medium produces ion pair formation which exerts an influence on the kinetic rate constants.

Cooperative and noncooperative binding of *Ru(bpy)3(2+) to DNA and SB4.5G dendrimers.

The process *Ru(bpy)(3)(2+) + S(2)O(8)(2-) in two different reaction media, the SB4.5G dendrimer and DNA solutions, was studied. In both media, the receptors have anionic characteristics. This fact will produce a binding of the ruthenium complex to the two receptors by attractive electrostatic interactions. On the contrary, the peroxodisulfate ions will be preferentially located in the aqueous solution due to electrostatic repulsions with the receptors. Despite the similarities of the receptors, some differences are observed in these two reaction media. These differences arise from the fact that the binding of the *Ru(bpy)(3)(2+) complex to DNA shows a negative cooperativity, whereas the binding to the dendrimer is noncooperative in character. The anticooperative character of the binding that happens in DNA solutions becomes noncooperative when an electrolyte, NaNO(3), is added to the medium. This is related to a condensation of the salt's counterions on the surface of the DNA which produces a decrease of the equilibrium constant corresponding to the binding of the complex to the receptor. Therefore, it is shown that the ionic strength of the reaction medium exerts a great influence on the cooperative nature of the ligand/receptor binding. This also explains the different behavior observed in DNA and dendrimer solutions.

Formation of a rotaxane from the end-capping process of a pseudorotaxane. Effects of the solvent.

The effects that the solvent exerts on the end-capping process of a pseudorotaxane formed by the [Ru(NH 3) 5(4,4'-bpy)]2+ complex and beta-cyclodextrin were studied. In this process the 4,4'-bpy ligand acts as rigid axle and the cyclodextrin as ring or macrocycle. The stopper used was the [Fe(CN) 5 H2O]3(-) complex. The solvents used were mixtures of ethyleneglycol-water and tert-butyl alcohol-water. Results showed similar, although strange, behavior in both media studied. Thus, a decrease of the observed rate constant was obtained when the concentration of cyclodextrin increases for all the media studied. However, at fixed cyclodextrin concentrations, an increase of k obs was obtained when small quantities of the cosolvent were added to the medium and, further, a decrease of k obs for the higher quantities of the organic solvents. This strange behavior could be explained by taking into account electrostatic and specific solvent (solvent-solvent and solvent-solute) effects.

Rigidity and/or flexibility of calixarenes. effect of the p-sulfonatocalix[n]arenes (n = 4, 6, and 8) on the electron transfer process [Ru(NH3)5pz]2+ + Co(C2O4)3(3-).

The reaction [Ru(NH3)5pz]2+ + Co(C2O4)33- has been studied in aqueous solutions of p-sulfonatocalix[n]arene (n = 4, 6, and 8). The results are interpreted by using the pseudophase model. Results show that the rigidity and/or flexibility of the assembled rings have a great effect on the thermodynamics of inclusion of the guest into the host and, therefore, on the kinetics of the electron transfer processes that take place in these media. The obtained results are discussed from the viewpoint of two types of interactions: electrostatic and nonelectrostatic. From surface potential measurements, the guest-host interactions have been demonstrated to be mainly due to nonelectrostatic interactions, although the species are charged. So, the nonelectrostatic contribution to the equilibrium constant in all the calixarenes studied is 1 order of magnitude higher than the electrostatic one (Knel = 144 and 884 mol-1 dm3 for p-sulfonatocalix[n]arene (n = 4 and 6, respectively) and Kel approximately 10 mol-1 dm3). Electrostatic interactions also affect the kinetic results.

Salt and solvent effects on the kinetics and thermodynamics of the inclusion of the ruthenium complex [Ru(NH3)5(4,4'-bpy)]2+ in beta-cyclodextrin.

The influences of solvents (in water-cosolvent mixtures) and salts on the kinetics and thermodynamics of the inclusion of [Ru(NH3)5(4,4'-bpy)]2+ in beta-cyclodextrin (beta-CD) have been studied. Solvent effects on the kinetics can be described as a consequence of the competition of the cosolvent for the beta-CD cavity. The salt effects on the kinetics depend on the ion pairing of the anions with the [Ru(NH3)5(4,4'-bpy)]2+ complex. On the other hand, the solvent effects on the equilibrium constant depend on the stabilization of the 4,4'-bipyridine ligand in the water-cosolvent mixture relative to water. Finally, salt effects on the equilibrium constant are interpreted as a consequence of ion pairing between the anion of the salt and the inclusion complex.

Salt and solvent effects on the kinetics of the oxidation of the excited state of the [Ru(bpy)3]2+ complex by S2O8(2-).

The title reaction was studied in different reaction media: aqueous salt solutions (NaNO3) and water-cosolvent (methanol) mixtures. The observed rate constants, k(obs), show normal behavior in the solutions containing the electrolyte, that is, a negative salt effect. However, the solvent effect is abnormal, because a decrease of the rate constant is observed when the dielectric constant of the reaction medium decreases. These effects (the normal and the abnormal) can be explained using the Marcus-Hush treatment for electron transfer reactions. To apply this treatment, the true, unimolecular, electron-transfer rate constants, k(et), have been obtained from k(obs) after calculation of the rate constants corresponding to the formation of the encounter complex from the separate reactants, k(D), and the dissociation of this complex, k(-D). This calculation has been carried out using an exponential mean spherical approach (EMSA).

Method for the evaluation of the reorganization energy of electron transfer reactions produced under restricted geometry conditions.

The kinetics of the electron transfer reactions between S(2)O(8)(2-) and the complexes (Ru(NH(3))(5)L)(2+) (L = pyridine, pyrazine, and 4-cyanopyridine) have been studied in micellar (SDS) solutions. A method for the evaluation of the reorganization energy of these reactions, based on the comparison of their rate constants, is proposed. From the results obtained, we concluded that the observed rate constants go through a minimum for the surfactant concentration in which the reorganization energy goes through a maximum. The method can be applied to any kind of restricted geometry conditions.

Strength and character of peptide/anion interactions.

The binding free energy of complex [Co(C(2)O(4))(3)](3-) to three peptides H-Lys-Gly-Lys-Gly-Lys-Gly-Lys-NH(2) (P-1), H-(Lys-Gly-Lys-Gly-Lys-Gly-Lys)(2)-NH(2) (P-2), H-(Lys-Gly-Lys-Gly-Lys-Gly-Lys)(3)-NH(2) (P-3) and to the monomers (amino acids) forming the peptides has been obtained using the kinetics of the electron-transfer reaction between [Ru(NH(3))(5)py](2+) and [Co(C(2)O(4))(3)](3-) as the probe. The polymerization of the monomers increases the negative free energy of binding and changes its character, noncooperative for the monomers and anticooperative for the peptides. This increase in the negative free energy represents a driving force for the polymerization process. The magnitude of the gain in negative free energy, as a consequence of the anticooperative character of the binding of the cobalt complex to the peptide, depends on the ratio of [complex]/[monomers].