732 ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2018, Vol. 44, No. 6, pp. 732–739. © Pleiades Publishing, Ltd., 2018. Behavior of Doxorubicin Lipophilic Conjugates in Liposomal Lipid Bilayers 1 A. S. Alekseeva a , A. O. Chugunov a, b , P. E. Volynsky a , N. R. Onishchenko a , J. G. Molotkovsky a , R. G. Efremov a, b, c , I. A. Boldyrev a, d , and E. L. Vodovozova a, 2 a Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997 Russia b Higher School of Economics, Moscow, 101000 Russia c Moscow Institute of Physics and Technology, Dolgoprudnyi, 141700 Russia d National University of Science and Technology MISiS, Moscow, 119049 Russia Received April 27, 2018; in final form, June 4, 2018 AbstractPreparation of liposomal formulations containing water-soluble drugs in the form of lipophilic prodrugs in their lipid bilayer is of considerable interest. Previously, we synthesized doxorubicin dioleoyl glyceride and oleoyl conjugates intended for incorporation into fluid-phase liposomal bilayers. In this work, we studied the behavior of lipid conjugates in bilayers prepared from palmitoyl oleoyl phosphatidylcholine and dimyristoyl phosphatidylcholine using methods of fluorescence spectroscopy and molecular modeling. The conjugates were shown to have limited mobility in lipid bilayers, which can be explained by the formation of hydrogen bonds between the doxorubicin aglycone and the lipid phosphate groups. In the liposome mem- brane, lipophilic conjugates also tend to form clusters through interaction of doxorubicin moieties. Oleoyl chains stretch in parallel to the acyl residues of phospholipids. Due to the formation of a larger number of hydrogen bonds, the oleoyl conjugates interacted with the bilayer more effectively than the dioleoyl glyceride counterparts. These properties of doxorubicin conjugates can affect both the possibility of their incorporation into the lipid bilayer (from the therapeutic effect point of view) and intracellular release of the antibiotic drug by means of enzymolysis. Keywords: doxorubicin, fluorescence anisotropy, lipid bilayer, lipophilic prodrugs, liposomes, molecular dynamics DOI: 10.1134/S1068162019010023 INTRODUCTION The use of liposomes as drug delivery systems is an accepted approach to increase the efficacy of treat- ment and improve the quality of life of cancer patients [1]. Liposome-based drugs occupy the leading posi- tion in the world market of nanomedical products for systemic use. A known example of the effective antitu- mor therapy with a liposomal drug is Doxil®/Cae- lyx® (also a generic drug Lipodox® since 2013), which is composed of pegylated (i.e., covered with polyethyleneglycol chains to avoid the capture by reticuloendothelial system cells) 100-nm liposomes bearing an encapsulated antibiotic doxorubicin [2]. Lipid bilayers of these drugs are built of saturated phospholipids and cholesterol forming tight “con- densed” membranes resistant to premature damage in the blood stream. In the manufacturing process of these drugs, the method of effective encapsulation of water soluble substances into the inner volume known as “remote loading,” or active loading, is used: the drug diffuses through the lipid bilayer against the salt concentration gradient [3]. In the liposome, the drug, as a rule, accumulates in the form of a precipitant and forms a crystalline phase, which supports an increase in the capacity of the nanosized carrier (molar drug- to-lipid ratio reaches 0.2–0.25). It is difficult to receive a therapeutic dose of the active ingredient upon passive encapsulation of the drug solution into liposomes, since the inner water volume of nanosized carriers is too small. On the other hand, the method of active loading can only be used for amphiphilic weak acids or bases including anthracycline antibiotics, partic- ularly, doxorubicin. Along with the virtues, Doxil® liposomes have some shortcomings, which include hindered internal- ization by different tumor cells, as well as slow DOX intracellular release [4, 5]. Therefore, the search for Abbreviations: DOX, doxorubicin; DOX-DG, DOX dioleoyl glyceride conjugate; DOX-Ole, oleoyl conjugate; DMPC, 1,2-dimiristoyl-sn-glycerophosphocholine; MD, molecular dynamics; PBS, phosphate-buffered saline; POPC, 1-palmitoyl- 2-oleoyl-sn-glycerophosphocholine. 1 The manuscript is based on the report at the conference “Lipids of the XXI century. First quarter,” October 22–23, 2018, Moscow. 2 Corresponding author: phone: +7 (495) 330-6610; e-mail: elvod@lipids.ibch.ru.