INFLUENCE OF QUERCETIN/IRON (II) COMPLEX ON PHASE TRANSITIONS OF PHOSPHATIDYLCHOLINE OR PHOSPHATIDYLETHANOLAMINE CONTAINING LIPOSOMES

Elena A. Yagolnik ^a, Yury S. Tarahovsky ^b,c,*, Svetlana M. Kuznetsova^b, Eugeny N. Muzafarov ^a,d, Yuri A. Kim ^e

^aTula State University, Tula, 300000, Russia

^bInstitute of Theoretical and Experimental Biophysics, RAS, Pushchino, Moscow Region, 142290, Russia

^cResearch-Educational Center "Bionanophysics," Moscow Institute of Physics and Technology, 141700, Dolgoprudniy, Russia

^dInstitute of Basic Biological Problems, RAS, Pushchino, Moscow Region, 142290, Russia

^eInstitute of Cell Biophysics, RAS, Pushchino, Moscow Region, 142290, Russia

ABSTRACT

We studied influence of quercetin/iron (II) complexes on phase transitions of the phospholipid bilayer of liposomes. Differential scanning calorimetry (DSC) of liposomes prepared from dimiristoyl-phosphatidylcholine (DMPC) and palmitoyl-oleoyl-phosphatidylethanolamine (POPE) demonstrated that quercetin/iron complexes cannot interact with liposomes because, as it was detected by photon-correlation spectroscopy, they produce nano- and micro-particles insoluble in water. However if quercetin was added to liposomes followed by iron, the quercetin/iron complex interacted with the phospholipid bilayer and influenced lipid phase transitions. The most pronounced changes were observed in the transition from the bilayer to the hexagonal H_II phase of POPE liposomes treated by quercetin followed by iron. DSC detected a considerable increase in the transition width and a shift of the transition maximum towards higher temperatures. The revealed influence of flavonoid/iron complexes on lipid phase transitions should be considered in designing liposomal vehicles for flavonoids and drug delivery.

Flavonoids attract attention because they can influence the human health. These natural compounds are regarded not only as potent antioxidants, but also as important biological regulators essential for prevention of various diseases including cardiovascular, autoimmune, inflammatory, endocrine, neurodegenerative, and cancer [1]. Because of hydrophobicity they are preferably interact with biological membranes [2]. Here we studied the influence of quercetin on two most important phospholipids of cell membranes phosphatidylcholine and phosphatidylethanolamine. Phosphatidylcholine tends to produce stable bilayer in a wide range of temperatures. Phosphatidylethanolamine is able to produce bilayer structures only in the vicinity of the lipid melting point known as the main phase transition (T_m). At higher temperature phosphatidylethanolamine is subjected to a transition from the bilayer to the hexagonal H_II phase. The transition temperature depends the on hydrocarbon chains composition [3]. Phosphatidylethanolamine isolated from mammalian cells may produce hexagonal H_II phase at a physiological temperature. It is supposed that in a living cell the ratio of lipids producing bilayer and nonbilayer structures is homeostatically regulated to optimize conditions for membrane proteins functioning. A disbalance of lipid phase properties could be a reason of numerous diseases [4].

The ability of flavonoid-iron complexes to interact with the bilayer was demonstrated by DSC. As follows from DSC thermograms, iron cations in concentrations 10^-6 M, which is close to the iron concentration in human blood, had a little effect on the DMPC melting (Fig.1), while quercetin-iron complex influenced the lipid melting weaker than free quercetin. The effect could be observed only when quercetin was added to liposomes first and about 30 minutes later the iron cations were added. In the cases when the compounds were added in the reverse order, or when the preliminary prepared mixture of quercetin and iron were added to liposomes we did not see any influence of the complex on the lipid melting.

A similar change of T_m transition was observed after sequential additions of quercetin and 30 min later of iron to POPE liposomes (Fig.2). The influence of flavonoid-iron complexes on T_h transition of POPE at 69°C was much stronger than

Fig. 1 and 2. Influence of quercetin and quercetin/iron complexes on DSC thermogram of: (1) – DMPC liposomes . Concentration of lipid was 2,95·10^-4 М (0,2 mg/ml), quercetin – 4·10^-5 М, FeSO₄ – 4·10^-6 M; (2) – POPE liposomes. Concentration of lipid was 1,95·10^-3 M (2 mg/ml), quercetin – 4·10^-4 М, FeSO₄ – 4·10^-5 M. The thermograms presented here are of untreated liposomes (a); liposomes treated by quercetin (b); treated by iron (c); treated by quercetin followed by iron added 30 min later (d); treated by preliminary produced quercetin/iron complex (e). For DSC we used DASM-4 (IBP RAS, Pushchino, Russia).

Fig. 3. Photon-correlation spectroscopy analyses of particle size present in mixture of quercetin and FeSO₄ in concentration used for DSC. The particle size distribution was analised on N4 PLUS Submicron Particle Size Analyzer (Beckman Coulter, USA).

that on T_m transition described above. In the presence of quercetin-iron complexes the maximum of the transition was shifted by about 5 degrees to higher temperatures while the width of the transition increased and the height decreased. These changes were more pronounced after the sequential addition of quercetin and iron. Addition of preliminarily prepared complexes of quercetin and iron had much lower effect on the lipid transition. This effect could be explained by low solubility of quercetin-iron complex in water and formation of insoluble particles of 10 – 15 nm and of 1 – 5 μm detected by photon-correlation spectroscopy (Fig.3).

Phosphatidylcholine and phosphatidylethanolamine are often used for preparation of liposomes applicable for drugs and genes delivery. The delivery efficiency depends on the phase transitions of lipids [5]. The ability of flavonoid-metal complexes to influence the phase behavior of phosphatidylethanolamine could be especially important in liposomes containing this lipid, for example those used in gene therapy [6]. Liposomes loaded with flavonoids may considerably increase the flavonoids concentration in blood and facilitate their bioavailability [7]. Lipophilic complexes of flavonoids with iron could be useful for development of liposomal vehicles for drug and gene delivery.

Acknowledgment

We thank CCU MIPT and REC "Nanotechnology" of MIPT for the equipment and chemicals used in this work. Financial support from the ONEXIM group and the Skolkovo Foundation and the Ministry of Education and Science of the Russian Federation, agreement 14.B37.21.1515 is gratefully acknowledged.

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