C60 Fullerene Clusters Stabilize the Biologically Inactive Form of Topotecan.

There is a range of experimental proofs that biologically relevant compounds change their activity in the presence of C60 fullerene clusters in aqueous solution, which most frequently act as a nanoplatform for drug delivery. Inspired by this evidence, we made an effort to investigate the interaction of fullerene clusters with the antibiotic topotecan (TPT). This study proceeded in three steps, namely, UV/vis titration to confirm complexation and in vitro assays on proliferating and nonproliferating cells to elucidate the role of C60 fullerene in the putative change in TPT activity. Surprisingly, although the nonproliferating cell assay is consistent with the titration data and confirms complex formation, it contradicted the results of the proliferating cell assay. The latter showed that the mixture of TPT and fullerene affects the cells in the same way as pure TPT, as if there were no fullerenes in solution at all, whereas the action of TPT was expected to be enhanced. We explained this contradiction by the specific stabilization of the biologically inactive carboxylate form of the antibiotic adsorbed in the alkaline shell of large fullerene clusters, which leads to neutralization of the drug delivery function and almost zero net biological effect of the antibiotic in vitro. The practical outcome of the work is that fullerene clusters can be used for the selective delivery of pH-sensitive drug forms.

Interceptor potential of C60 fullerene aqueous solution: a comparative analysis using the example of the antitumor antibiotic mitoxantrone.

We performed a qualitative and quantitative analysis of intermolecular interactions in aqueous solution between the antitumor antibiotic mitoxantrone and C60 fullerene in comparison with interactions between the antibiotic and well-known aromatic molecules such as caffeine and flavin mononucleotide, commonly referred to as interceptor molecules. For these purposes, we obtained equilibrium hetero-association constants of these interactions using a UV/Vis titration experiment. Special attention was paid to the interaction of C60 fullerene with mitoxantrone, which has been quantified for the first time. Based on the theory of interceptor-protector action and using a set of measured equilibrium constants we managed to estimate the relative biological effect of these mixtures in a model living system, taking human buccal epithelium cells as an example. We demonstrated that C60 fullerene is able to restore the functional activity of the buccal epithelium cell nucleus after exposure to mitoxantrone, which makes it possible to use C60 fullerene as regulator of medico-biological activity of the antibiotic.

Hetero-association models of non-covalent molecular complexation.

The present review discusses the current state-of-the-art in building models enabling the description of non-covalent equilibrium complexation of different types of molecules in solution, which results in the formation of supramolecular structures different in length and composition (hetero-association or supramolecular multicomponent co-polymerisation). The description is focused on standard physical and chemical quantities such as experimental observables and equilibrium parameters of interaction (equilibrium constants and concentrations). The major partial cases of the hetero-association models, such as finite and indefinite isodesmic and cooperative complexations, and Benesi-Hildebrand and Langmuir adsorption models are considered. Future challenges in the development of the hetero-association models are provided.

Study of the properties of doxorubicin-resistant cells affected by acute leucosis.

The stiffness of cell membrane was found to be one of the factors determining resistance of a cell in vitro to antibiotic doxorubicin action. Membranes of surviving cells are negatively charged (-35 - -30 mV) and have high values of stiffness (2.2-5.1 µÐ Ð°) at the doxorubicin concentrations in the medium of 1-500 µg/ml. If the drug concentration and exposure time are being increased, only cells with 'soft' membrane (0.25-1 µÐ Ð°) and positive surface potential (15-29 mV) survive. The data obtained have important prognostic value in studying drug resistance of tumour blood cells and can be used as objective markers of efficiency of the antitumor therapy.

Determination of the equilibrium constant of C60 fullerene binding with drug molecules.

We report a new analytical method that allows the determination of the magnitude of the equilibrium constant of complexation, Kh, of small molecules to C60 fullerene in aqueous solution. The developed method is based on the up-scaled model of C60 fullerene-ligand complexation and contains the full set of equations needed to fit titration datasets arising from different experimental methods (UV-Vis spectroscopy, 1H NMR spectroscopy, diffusion ordered NMR spectroscopy, DLS). The up-scaled model takes into consideration the specificity of C60 fullerene aggregation in aqueous solution and allows the highly dispersed nature of C60 fullerene cluster distribution to be accounted for. It also takes into consideration the complexity of fullerene-ligand dynamic equilibrium in solution, formed by various types of self- and hetero-complexes. These features make the suggested method superior to standard Langmuir-type analysis, the approach used to date for obtaining quantitative information on ligand binding with different nanoparticles.

Hidden entropic contribution in the thermodynamics of molecular complexation.

It has become an axiom that the thermodynamic analysis of non-covalent molecular complexation is intrinsically model-dependent, i.e. the set of implicitly or explicitly introduced assumptions may strongly affect the thermodynamic parameters. This review deals with the entropy change accompanying molecular complexation, which results in the formation of ordered structures. In simple terms the key question may be formulated as follows: 'If the molecular complexation leads to the formation of supramolecular structures, then, the system entropy changes due to ordering. So, how is this factor accounted for in standard models of molecular complexation and does it have any effect on the magnitude of measured thermodynamic parameters of complexation?' The structure of the review is made as a collection of cases in which the hidden entropy change has been recognized among the most influential factors.

C60 fullerene affects elastic properties and osmoregulation reactions of human lymphocytes.

The influence of aqueous solution of pristine C60 fullerene (C60FAS) on functional activity of lymphocytes from a healthy person was studied for the first time. By means of atomic force microscopy, it was found that C60FAS in a concentration of 0.1 mg/ml increases the stiffness of the lymphocyte membrane by 41% (p < 0.05) and lowers the functional activity of the plasmalemma surface, thereby constraining the use of its membrane material in physiological reactions using a hypotonic model in vitro. However, a cell retains the ability to regulate its volume and demonstrates relative resistance to hypo-osmotic stress. The resistance of lymphocytes in hypo-osmotic medium is facilitated by activation of the nucleus by C60 fullerene particles, which regulates the implementation of two consistent phases of an increase and decrease of cell volume, thereby retaining cell viability. All these indicate the impact of C60 fullerene on the cellular nucleus.

Shape-independent model (SHIM) approach for studying aggregation by NMR diffusometry.

NMR diffusometry has been gaining wide popularity in various areas of applied chemistry for investigating diffusion and complexation processes in solid and aqueous phases. To date, the application of this method to study aggregation phenomena proceeding beyond the dimer stage of assembly has been restricted by the need for a priori knowledge of the aggregates' shape, commonly difficult to know in practice. We describe here a comprehensive analysis of aggregation parameter-dependency on the type and shape selected for modeling assembly processes, and report for the first time a shape-independent model (designated the SHIM approach), which may be used as an alternative in cases when information on aggregates' shapes is unavailable. The model can be used for determining equilibrium aggregation parameters from self-diffusion NMR data including equilibrium self-association constant and changes in enthalpy, ΔH, and entropy, ΔS.

Quantitative correlation of the in vitro biological effect with parameters of molecular complexation in mutagen-interceptor systems.

According to the theory of interceptor-protector action a quantitative link between the physico-chemical parameters of molecular complexation and in vitro biological effect in aromatic drug-interceptor systems must exist. In the present communication such link between relative change in mutagenicity of IQ-type aromatic mutagens on addition of aromatic interceptor molecules with equilibrium hetero-association constants of mutagen-interceptor complexation has been found using the published in vitro data in bacteria cell systems.

Interceptor effect of C60 fullerene on the in vitro action of aromatic drug molecules.

C60 fullerenes are spherical molecules composed purely of carbon atoms. They inspire a particularly strong scientific interest because of their specific physico-chemical properties and potential medical and nanotechnological applications. In this work we are focusing on studying the influence of the pristine C60 fullerene on biological activity of some aromatic drug molecules in human buccal epithelial cells. Assessment of the heterochromatin structure in the cell nucleus as well as the barrier function of the cell membrane was performed. The methods of cell microelectrophoresis and atomic force microscopy were also applied. A concentration-dependent restoration of the functional activity of the cellular nucleus after exposure to DNA-binding drugs (doxorubicin, proflavine and ethidium bromide) has been observed in human buccal epithelial cells upon addition of C60 fullerene at a concentration of ~10(-5 )M. The results were shown to follow the framework of interceptor/protector action theory, assuming that non-covalent complexation between C60 fullerene and the drugs (i.e., hetero-association) is the major process responsible for the observed biological effects. An independent confirmation of this hypothesis was obtained via investigation of the cellular response of buccal epithelium to the coadministration of the aromatic drugs and caffeine, and it is based on the well-established role of hetero-association in drug-caffeine systems. The results indicate that C60 fullerene may reverse the effects caused by the aromatic drugs, thereby pointing out the potential possibility of the use of aromatic drugs in combination with C60 fullerene for regulation of their medico-biological action.

Theoretical description of metabolism using queueing theory.

A theoretical description of the process of metabolism has been developed on the basis of the Pachinko model (see Nicholson and Wilson in Nat Rev Drug Discov 2:668-676, 2003) and the queueing theory. The suggested approach relies on the probabilistic nature of the metabolic events and the Poisson distribution of the incoming flow of substrate molecules. The main focus of the work is an output flow of metabolites or the effectiveness of metabolism process. Two simplest models have been analyzed: short- and long-living complexes of the source molecules with a metabolizing point (Hole) without queuing. It has been concluded that the approach based on queueing theory enables a very broad range of metabolic events to be described theoretically from a single probabilistic point of view.

Evidence of entropically driven C60 fullerene aggregation in aqueous solution.

In the present work, we report the first experimental evidence of entropically driven C60 fullerene aggregation in aqueous solution, occurring with nearly zero enthalpy change.

Complexation of C60 fullerene with aromatic drugs.

The contributions of various physical factors to the energetics of complexation of aromatic drug molecules with C(60) fullerene are investigated in terms of the calculated magnitudes of equilibrium complexation constants and the components of the net Gibbs free energy. Models of complexation are developed taking into account the polydisperse nature of fullerene solutions in terms of the continuous or discrete (fractal) aggregation of C(60) molecules. Analysis of the energetics has shown that stabilization of the ligand-fullerene complexes in aqueous solution is mainly determined by intermolecular van der Waals interactions and, to lesser extent, by hydrophobic interactions. The results provide a physicochemical basis for a potentially new biotechnological application of fullerenes as modulators of biological activity of aromatic drugs.

On the reliability of quantitation of biological effect in drug-interceptor-DNA systems.

Relative insensitivity of theoretical estimation of biological effect in drug-interceptor-DNA systems is found with respect to variation of parameters of quasiphysiological conditions. The "inertness" of the biological response, in part, justifies the use of parameters of intermolecular interaction, derived from independent physicochemical experiments, in estimation of relative biological effect in the theory of interceptor/protector action.

C60 fullerene aggregation in aqueous solution.

In the present work we develop a novel approach for quantification of the energetics of C60 fullerene aggregation in aqueous media in terms of equilibrium aggregation constant KF. In particular, it is shown that the experimental determination of the magnitude of KF is possible only within the framework of the 'up-scaled aggregation model', considering the C60 fullerene water solution as a solution of fullerene clusters. Using dynamic light scattering (DLS) data we report the value, K(F) = 56,000 M(-1), which is in good agreement with existing theoretical estimates and the results of energetic analyses. It is suggested that the proposed 'up-scaled model' may be used in any instances of non-specific aggregation resulting in formation of large spherical particles. The measurement of the translational diffusion coefficient and the dimensions of the light scattering particles using a DLS approach with respect to C60 fullerene aggregates is found to contain significant systematic errors originating from the interaction effect that is well-known for micellar solutions. As a result, corrections to the equations associated with DLS data are proposed.

Energetics of ligand binding to the DNA minor groove.

In the present work the decomposition of the total Gibbs free energy of ligand-DNA binding onto various physical terms was accomplished for the group of nine DNA minor groove binders (MGB ligands) differing in both structure and charge state. The decomposition protocol includes the analysis of the most complete set of physical factors known to contribute to the complexation process, viz. the net change in the number of degrees of freedom (translational, rotational, vibrations of the chemical bonds and vibrations of the ligand as a whole within the binding site), the conformational entropy, van der Waals, electrostatic and hydrophobic interactions, the polyelectrolyte contribution and the net effect of changes in the number of hydrogen bonds. All of these processes are further decomposed into the interaction with the solvent and the interaction of the ligand with DNA. The principal outcome of the decomposition is the possibility of performing a comparative analysis of the energetic contribution of various physical terms and provide an answer to the question concerning what physical factors stabilize or destabilize the complexes of MGB ligands with DNA.

Profiles of equilibrium constants for self-association of aromatic molecules.

Analysis of the noncovalent, noncooperative self-association of identical aromatic molecules assumes that the equilibrium self-association constants are either independent of the number of molecules (the EK-model) or change progressively with increasing aggregation (the AK-model). The dependence of the self-association constant on the number of molecules in the aggregate (i.e., the profile of the equilibrium constant) was empirically derived in the AK-model but, in order to provide some physical understanding of the profile, it is proposed that the sources for attenuation of the equilibrium constant are the loss of translational and rotational degrees of freedom, the ordering of molecules in the aggregates and the electrostatic contribution (for charged units). Expressions are derived for the profiles of the equilibrium constants for both neutral and charged molecules. Although the EK-model has been widely used in the analysis of experimental data, it is shown in this work that the derived equilibrium constant, K(EK), depends on the concentration range used and hence, on the experimental method employed. The relationship has also been demonstrated between the equilibrium constant K(EK) and the real dimerization constant, K(D), which shows that the value of K(EK) is always lower than K(D).

The theory of interceptor-protector action of DNA binding drugs.

The review discusses the theory of interceptor-protector action (the IPA theory) as the new self-consistent biophysical theory establishing a quantitative interrelation between parameters measured in independent physico-chemical experiment and in vitro biological experiment for the class of DNA binding drugs. The elements of the theory provide complete algorithm of analysis, which may potentially be applied to any system of DNA targeting aromatic drugs. Such analytical schemes, apart from extension of current scientific knowledge, are important in the context of rational drug design for managing drug's response by changing the physico-chemical parameters of molecular complexation.

Interaction of pseudoephedrine and azithromycin with losartan: Spectroscopic, dissolution and permeation studies.

This study aims at investigating the potential effect of selected cationic drugs (azithromycin (AZN) and pseudoephedrine sulfate (PSD) on the dissolution profile and intestinal permeation of losartan potassium (LOS) that might occur due to ion pair salt formation. DSC, FT-IR and 1H NMR indicated the formation of ion pair salts between LOS and each of AZN and PSD. Based on NMR chemical shifts calculations, utilizing specialized software, the most likely structures of the salt were proposed and revealed interesting structural features. The obtained ion pair products were shown to have lower aqueous solubilities (water and phosphate buffer pH 6.8) and higher apparent partition coefficient values compared to the parent compound. Neither of the cations affected the dissolution of LOS tablet (Cozaar® 100 mg) in the studied media (HCl pH 1.2 and phosphate buffer pH 6.8). Interestingly, AZN significantly increased the dissolution of LOS in phosphate buffer pH 4.5 (f2 = 33), and an explanation based on distinguished association pattern between AZN and LOS (CH/π) was offered. Employing permeation test across Caco-2 cells monolayer, the apparent permeability coefficient (Papp) of LOS increased significantly (from 0.9 × 10-5 cm/s to 1.8 × 10-5 cm/s) in the presence of the selected cations. Therefore, while the employed cationic drugs were not shown to form ion pair salts under the in-vitro dissolution conditions, they may still participate in significant in-vivo interaction with LOS.

Effect of Nicotinamide on the Photolysis of Riboflavin in Aqueous Solution.

The photolysis of riboflavin (RF) in aqueous solution in the presence of nicotinamide (NA) by visible light has been studied in the pH range 1.0-12.0 and the various photoproducts have been identified as known compounds. RF has been determined in degraded solutions by a specific multicomponent spectrometric method in the presence of its photoproducts and NA. The second-order rate constants (k 2) for the bimolecular interaction of RF and NA range from 0.54 (pH 1.0) to 9.66 M(-1) min(-1) (pH 12.0). The log k 2-pH profile for the photolysis reaction follows a sigmoid curve showing a gradual increase in the rate of pH due to a change in the ionization behavior of the molecule. The lower rate in the acid region is probably due to protonation of the molecule since the cationic form of RF is less susceptible to photolysis than the neutral form. Similarly, a slowing of the rate in the alkaline region is due to anion formation of the molecule. NA is involved as an electron acceptor during the sequence of reactions and thus enhances the rate of photolysis of RF. Absorption and fluorescence measurements did not provide evidence for the complex formation between the two compounds under the present conditions.