The interaction of PVP complexes of gossypol and its derivatives with an artificial membrane lipid matrix

CELLULAR & MOLECULAR BIOLOGY LETTERS http://www.cmbl.org.pl Received: 20 March 2009 Final form accepted: 18 November 2009 Published online: 20 November 2009 Volume 15 (2010) pp 98-117 DOI: 10.2478/s11658-009-0037-x © 2009 by the University of Wrocław, Poland Research article THE INTERACTION OF PVP COMPLEXES OF GOSSYPOL AND ITS DERIVATIVES WITH AN ARTIFICIAL MEMBRANE LIPID MATRIX MAKSIM IONOV1,2,*, ILNORA TUKFATULLINA1, BAKHTIYAR SALAKHUTDINOV1, NINA BARAM1, MARIA BRYSZEWSKA2 and TAKHIR ARIPOV1 1 A.S. Sadykov Institute of Bioorganic Chemistry, Academy of Sciences of Uzbekistan, 83, st, Kh. Abdullaev, Tashkent, 100125, Uzbekistan, 2Department of General Biophysics, University of Łódź, Banacha 12/16, 90-237 Łódź, Poland Abstract: In this paper, we present the results of a study on the membraneactive properties of gossypol, its derivatives and their polyvinylpyrrolidone complexes as assessed by differential scanning calorimetry and by the fluorescent probe method. The latter revealed the change in polarization of the incident radiation caused by the action of the polyphenol on the artificial membrane lipid matrix. Key words: Gossypol and its derivatives, Lipid membranes, Differential scanning calorimetry, Membrane fluidity INTRODUCTION Gossypol is a binaphthyl dialdehyde polyphenol that is extracted from cotton seeds (Fig. 1A). It is widely known for its antifertility activity [1], but it has other properties, including antiviral, anti-inflammatory and antiphrastic activities [2-5]. Chemical modifications of gossypol have been shown to produce various compounds with interesting and important pharmacological properties. Megosin, * Author for correspondence. e-mail: , tel./fax +48426354474 Abbreviations used: AOA – antioxidant activity; DMPC – dimyristoylphosphatidylcholine; DPH – 1,6-diphenyl-1,3,5-hexatriene; DPPC – dipalmitoyl-phosphatidylcholine; DSC – differential scanning calorimetry; PC – phosphatidylcholine; PE – phosphatidylethanolamine; PVP – N-polyvinylpyrrolidone (8000 MM). CELLULAR & MOLECULAR BIOLOGY LETTERS 99 batriden, gosolidon and other gossypol derivatives have been used in the treatment of various diseases [6-8]. Fig. 1. The molecular structures of gossypol in its aldehyde form (A), MGS-1 (B), megosin (C), and rometin, which is a complex of megosin with PVP (D). Gossypol itself is toxic to humans and other animals, especially in high concentrations. It can cause diarrhoea, hypokalaemia, weakness, oedema, breathlessness, neuritis and paralysis [9]. Chemical modifications can decrease its toxicity. However, gossypol and its derivatives are water-insoluble, which hinders their use in many medical applications. The biological availability of medical preparations depends to a great extent on their water solubility. One way of increasing the solubility of an insoluble or poorly soluble drug molecule is to associate it with a polymer that is cheap, water soluble, and chemically and biologically inert. Such polymers can transport the biologically active molecules in the body and deliver them to the cells, and can also control the durability of their action [10]. In our opinion, differential scanning calorimetry (DSC) is an effective method for distinguishing between these alternatives. As stated in the Materials and Methods section, one way in which the lipid multilamellar dispersions were prepared was by mixing the polyphenol or its PVP complex with lipids, then subjecting the dispersion to DSC. This enabled us to compare the effects of the investigated compounds on the lipid melting curves and to estimate the perturbation of the lipid bilayer structure. 100 Vol. 15. No. 1. 2010 CELL. MOL. BIOL. LETT. In this study, we synthesized complexes between polyphenols and polyvinylpyrrolidone (PVP). PVP is chemically inert at physiological pH. Making complexes by enveloping gossypol or one of its derivatives in the PVP chain conferred water solubility on the polyphenols. PVP molecules have no intrinsic AOA, which enables us to propose the following alternative ways in which AOA may take effect in the preparations studied: (a) the complex penetrates into the whole of the hydrophobic region of the lipid bilayer; (b) the complex exerts AOA only on the surface of the lipid matrix without penetrating into the bilayer; (c) the complex interacts with the polar part of the lipid matrix, then dissociates to release the hydrophobic polyphenol molecules, which penetrate into the hydrophobic region of the lipid, whereupon the AOA takes effect on lipid peroxidation. The antioxidant properties of gossypol and its derivatives become prominent when they interact with either artificial lipid or biological membranes [11-13, 15]. Since they are water-insoluble, their antioxidant activity is presumably conferred onto the hydrophobic region of the membrane lipid matrix. However, it has been shown that water-soluble complexes of PVP with these same compounds also have antioxidant activity in membranes, which begs the questions: since they are parts of a water-soluble complex, how do these polyphenol molecules function as antioxidants in biological membranes, and at what depth in the lipid matrix do the antioxidant reactions take place? MATERIALS AND METHODS Chemicals The lipids dimiristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine (PC), phosphatidylethanolamine (PE) and the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) were from Sigma. Gossypol (2,2’-[1,1’,6,6’,7,7’-hexahydrooxy-3,3’-dimethyl-5,5’-diazopropyl-8,8’diformyl]-dinaphthalene; Fig. 1A) and its derivatives megosin (bis2,2’{[(7,7’,8,8’-tetrahydro-1,1’,6,6’-tetrahydroxy-5,5’-diazopropyl-3,3’-dimethyl7,7’-dioxo)-2,2’-binaphthyl]-8,8’-methyleniminoethane}sodium salt; Fig. 1C) and MGS-1 (bisdimethylaminoethyliminogossypol; Fig. 1B), and their supramolecular complexes with N-polyvinylpyrrolidone (8000 MM), pogosin, rometin (Fig. 1D) and MGS-2, were synthesized in the Polyphenol Laboratory in the Institute of Bioorganic Chemistry of the Uzbekistan Academy of Sciences (Fig. 1). The complexes formed by incorporating gossypol and its derivatives into PVP polymers were stabilized by hydrogen bonds between the functional groups of the interacting molecules [27, 28]. The active polyphenol compounds constituted 9-10% of the complexes, i.e. the ratio of the number of PVP molecules to gossypol or derivative molecules in the complexes was approximately 10:1. CELLULAR & MOLECULAR BIOLOGY LETTERS 101 Preparation of multilamellar lipid dispersions Multilamellar lipid dispersions were prepared from either DPPC or DMPC (300 μM) in 10 mM Tris-HCl buffer, pH 7.5, using the method of multilamellar dispersion preparation described in [14]. Samples for differential scanning calorimetry (DSC) were prepared in one of the following ways. In the first method, samples of the lipids in organic solvents were mixed with ethanolic solutions of gossypol, megosin or MGS1, or their PVP complexes. The (...truncated)