A.V. Yudintsev¹, V.M. Trusova¹, G.P. Gorbenko¹, T. Deligeorgiev²,

A. Vasilev², N. Gadjev²

¹V.N. Karazin Kharkov National University, 4 Svobody Sq., Kharkov, 61077

²Department of Applied Organic Chemistry, Faculty of Chemistry, University of Sofia, Bulgaria

Lanthanide effect on structural state of model membranes

Liposomes, spherical self-closed structures formed by one or several concentric lipid bilayers currently represent a major interest for drug delivery. While their lipidic bilayer help solubilizing hydrophobic compounds, their internal aqueous center provides a way of encapsulating hydrophilic drugs. Liposome-based delivery systems are particularly attractive due to a number of advantages, such as biocompatibility, complete biodegradability, low toxicity, ability to carry both hydrophilic and lipophilic payloads and protect them from chemical degradation and transformation, increased therapeutic index of a drug, improved pharmacokinetic and pharmacodynamic profiles compared to free drugs, reduced side effects, etc. Development of liposomal carriers is heavily based on the evaluation of membrane-partitioning and bilayer-modifying properties of the drug. This is important not only for achieving maximum payload without compromising liposome stability, but also for prediction of therapeutic and toxic effects of a certain compound, because membrane interactions may prove critical for drug absorption, distribution, metabolism and elimination in an organism. Of particular importance is the development of liposomal formulations of new classes of antineoplastic drugs with alternative mode of cytotoxic action and nonoverlapping mechanisms of drug resistance. One of such classes is represented by lanthanide coordination complexes which have been reported to possess high cytotoxic potential.

In the present work we concentrated our efforts on the pre-formulation studies of the two synthesized Eu(III) coordination complexes referred to here as LC1 and LC2. More specifically, our goal was twofold: i) to characterize membrane partition properties of LC, and ii) to clarify the effects of these compounds on physicochemical properties and structural state of the phosphatidylcholine (PC) model lipid membranes.

a) b)

Fig. 1. Chemical structure of Eu(III) coordination complexes: a) LC1, b) LC2

LC1 and LC2 are asymmetric Eu(III) coordination complexes with diverse O-containing chelate ligands which are thought to serve at least two main functions – bind tightly Eu(III), providing the rigidity of the whole-molecule structure, and shield lanthanide ion from quenching and destabilizing effects of water. These compounds are characterized by broad absorption spectrum in the range 240-320 nm with the peak at 266 nm. Association of LC1 and LC2 with PC membranes was followed by the absorbance increase, without any shift of maximum position. The observed dependencies of the absorbance changes on lipid concentration were analyzed to quantify the drug redistribution between aqueous and lipid phases in terms of partition coefficient. Partition coefficients for LC1 and LC2 were found to be ca. 1.4×10⁵ and 6.7×10³, respectively. Allowing for zwitterionic nature of PC molecules and the fact that lanthanide complexes under study are highly hydrophobic compounds bearing no charge, it seems reasonable to consider partitioning process as being driven primarily by hydrophobic effect. It is tempting to suppose that different lipophilicity of the examined drugs originates from the differences in their structures. To explore the drug effect on liposome structural properties we employed pH-indicator dye bromothymol blue (BTB) and fluorescent membrane probe pyrene.

At physiological pH there exists an equilibrium between protonated and deprotonated BTB forms. This dye responds to the changes in environmental conditions by the shifts of its protolytic and partition equlibria. The distribution of different dye forms between aqueous and lipid phases is determined by the properties of liposomal membranes and manifests itself in the changes of BTB absorbance. Membrane association of different BTB species can be quantitatively described in terms of the partition coefficients. As have been observed at low ionic strength partition coefficient of the protonated dye form, is several orders of magnitude larger than of the deprotonated ones, i.e. the extent of membrane association of the deprotonated form is negligibly small. Due to high hydrophobicity of BTB species the dye binding to membranes is driven mainly by non-ionic interactions. Incorporation of Eu(III) complexes into PC bilayer gives rise to increase of partition coefficients of protonated BTB species, suggesting that these agents can perturb membrane structure, presumably through generation of structural defects and altering the conformation of PC headgroups. These findings were further corroborated by pyrene excimerization studies.

Emission spectrum of pyrene monomers is featured by a well-defined vibronic structure with five major vibronic bands between 370 and 400 nm. Relative intensities of vibronic transitions exhibit clear dependence on solvent polarity. The variations of the intensity ratio of the third to the first vibronic bands (R_III) upon varying drug-to-lipid molar ratio were found to lie mostly within experimental accuracy. This implies that polarity of pyrene microenvironment remains virtually unchanged on lanthanide incorporation into lipid bilayer, i.e. the drugs do not affect the probe distribution across a lipid bilayer. In contrast to the transverse pyrene location, lateral distribution of the probe undergoes changes upon the drug addition. This is evidenced by the observed increase of another parameter recovered from the pyrene spectra – excimer-to-monomer fluorescence intensity ratio, which reflects the rate of probe lateral diffusion within membrane plane. This parameter was found to increase in the presence of Eu complexes suggesting decreased degree of lipid packing. Importantly, the magnitude of all the observed bilayer-modifying effects appeared to be rather small, the property, which, in combination with appreciable lipophilicity of the investigated compounds is favorable for the development of liposome-based carriers of these potential anticancer drugs.