Лаборатория изучает взаимосвязь между структурой и функцией липидов – основного компонента клеточной мембраны. В результате исследований сотрудники получают липидные конъюгаты с биологически активными молекулами (в том числе с лекарствами), поверхностно активные вещества, липиды, несущие репортерные группировки (липидные зонды – уникальные инструменты для исследователей) и др.

Разрабатываются эффективные схемы синтеза флуоресцентных и фотоаффинных липидных зондов, спроектированных с учетом свойств липидных мембран. В настоящее время создаются передовые системы доставки лекарств для наномедицины.

Лаборатория оснащена всем необходимым для анализа (ВЭЖХ и ГЖХ) и химического синтеза липидов (включая редкие установки для окисления синглетным кислородом и озонирования).

В области синтеза, выделения и очистки, изучения, а главное – применения полученных липидных субстанций Лаборатория сотрудничает с другими лабораториями ИБХ РАН, а также с Московским технологическим университетом (МИТХТ), МГУ, РНИМУ им. Н.И. Пирогова, Саратовским и Нижегородским университетами, Институтом эпидемиологии и микробиологии им. Н.Ф. Гамалеи, Институтом глазных болезней им. Гельмгольца, Российским онкологическим научным центром им. Н.Н. Блохина РАМН, Университетом Умео (Швеция), Институтом Хормеля (США), Льежским университетом (Бельгия) и др.

Лаборатория химии липидов была создана в 1963 году, всего через четыре года после основания ИБХ. Возглавил новую Лабораторию Лев Давыдович Бергельсон. Ему удалось провести колоссальную работу по систематизации способов выделения липидов из природных источников, изучению структуры и развитию химического синтеза липидов и их аналогов, а также серьезно продвинуться в области исследования строения и функций биологических мембран, роли липидов в различных патологиях. В 1991 году Лабораторию возглавил Юлиан Георгиевич Молотковский, с тех пор особое развитие получила тематика флуоресцентных липидных зондов. С 2009 года лабораторией руководит Елена Львовна Водовозова, и сейчас основное направление исследований – фармацевтические разработки.

Накопленный за 50 лет опыт – фактически созданная за эти годы научная школа – позволяет сотрудникам лаборатории находить неординарные решения в области липидной химии и успешно справляться со сложными задачами.

• Создание наноразмерных систем направленной доставки лекарств на основе липосом, липофильных пролекарств и липофильных гликоконъюгатов (молекулярных адресов) (рис. 1, 2). Показано, что адресные лекарственные липосомы по противоопухолевому эффекту значительно превосходят исходные лекарства и липосомы, не оснащенные углеводным лигандом (рис. 3).
• Синтез новых флуоресцентных зондов липидной природы, изучение с их помощью строения и функций мембран и закономерностей переноса энергии возбуждения между флуорофорами (рис. 4 и 5).

• Осуществлен синтез природных ненасыщенных жирных кислот на основе стереохимии реакции Виттинга.
• Обнаружены явления дедифференцировки липидного состава органелл раковых клеток (Л.Д. Бергельсон и Э.В. Дятловицкая).
• Исследовано распространение и роль нового класса диольных липидов (Л.Д. Бергельсон и В.А. Вавер).
• Синтезировано и доказано строение бактериальных липоаминокислот, а также проведен синтез ненасыщенного фосфатидилинозита (Л.Д. Бергельсон и Ю.Г. Молотковский).
• Изучены топологии мембран с помощью спектроскопии ЯМР (Л.Д. Бергельсон и Л.И. Барсуков – в сотрудничестве с В.Ф. Быстровым).
• Обнаружены и доказаны строения нового класса бактериальных орнитин-содержащих липидов (Л.Д. Бергельсон и С.Г. Батраков).
• Синтезирована и применена в биологических исследованиях большая гамма флуоресцентных и фотоаффинных липидных зондов (Л.Д. Бергельсон, Ю.Г. Молотковский, Е.Л. Водовозова, И.И. Михалев, И.А. Болдырев).
• Разработана адресная липосомальная доставка в опухоли липид-модифицированных противораковых агентов (Ю.Г. Молотковский, Е.Л. Водовозова, Г.П. Гаенко ― в сотрудничестве с Н.В. Бовиным).
• Показано, что разработанные липосомальные препараты гемосовместимы (Н.Р. Кузнецова, Е.Л. Водовозова – в сотрудничестве с Льежским университетом и Центром наномедицины в Будапеште).
• Показано in vivo (на модели карциномы легких Льюис), что противоопухолевый эффект 100-нм липосом, сконструированных на основе природных фосфолипидов и липофильного пролекарства мелфалана и несущих тетрасахаридный лиганд селектинов сиалил-Льюис Х (SiaLeX), обусловлен специфическим антивасклярным эффектом в ткани опухоли (Е.Л. Водовозова, Н.Р. Кузнецова – в сотрудничестве с Н.В. Бовиным и РОНЦ РАМН).
• Показано (на модели эндотелиальных клеток пупочной вены человека), что SiaLeX-липосомы, нагруженные липофильным пролекарством мелфалана, селективно доставляют лекарство в клетки, активированные фактором некроза опухоли альфа (TNF-α) (А.С. Алексеева, Е.Л. Водовозова – в сотрудничестве с Институтом эпидемиологии и микробиологии).
• Исследованы функции вновь открытого церамид-1-фосфат-переносящего белка (C1P), широко распространенного в клетках тканей млекопитающих с применением набора флуоресцентномеченых фосфо- и гликолипидов. Показано, что C1P модифицирует активность фосфолипазы А2 и тем опосредует биосинтез эйкозаноидов (Ю.Г. Молотковский в сотрудничестве с Институтом Хормеля и др.).

Рис. 2. Электронные микрофотографии реплик с поверхностей скола замороженных дисперсий: А, а — липосом c МТХ-DG;Б, б — липосом c Mlph-DG.

Рис. 3. Динамика выживания мышей BLRB с перевитой аденокарциномой молочной железы в различных экспериментальных группах (n=10). Мыши получили в/в инъекции на 3-й и 7-й день после перевивки опухоли. Группы: 1 — мерфалан (сарколизин); 2 — пустые липосомы; 3 — липосомы + пролекарство; 4 — липосомы + пролекарство + конъюгат SiaLe^X; 5 — липосомы + конъюгат SiaLe^X; контроль —  физ. раствор.

Рис. 4. Набор флуоресцентных зондов для исследования свойств мембран на разном расстоянии от поверхности мембраны. Графики на заднем плане — профили параметра порядка набора зондов в бислоях разного состава.

Рис. 5. Структура ганглиозидного зонда BODIPY–FL–C3–GM1 — флуоресцентного маркера рафтов.

Текущие гранты:

РНФ № 14-15-00128 ««Ворота» гематоэнцефалического барьера: механизмы регуляции, их зависимость от состояния организма и возраста, способы коррекции с помощью супрамолекулярных транспортных систем» Соисполнитель-рук. Е.Л. Водовозова, головная организация – Саратовский государственный университет

РФФИ № 15-04-07415-а «Исследование липид-белковых взаимодействий в биологических мембранах и в клетках с помощью флуоресцентных липидных зондов» Рук. Ю.Г. Молотковский.

РФФИ № 16-04-01585-а «Изучение состояния противоопухолевых липосом, несущих в бислое липофильное пролекарство метотрексата, в плазме крови человека » Рук. Е.Л. Водовозова.

РФФИ № 15-33-20523 мол_а_вед «Новые флуоресцентные индикаторы активности и субстратной специфичности различных типов фосфолипазы А2"» Рук. И.А. Болдырев.

РФФИ № 16-34-01237 мол_а «Изучение механизмов проникновения в клетки и внутриклеточного транспорта липосомальных форм липофильного пролекарства метотрексата» Рук. А.С. Алексеева

Избранные публикации

1. Tretiakova D., Onishchenko N., Boldyrev I., Mikhalyov I., Tuzikov A., Bovin N., Evtushenko E., Vodovozova E. (2018). Influence of stabilizing components on the integrity of antitumor liposomes loaded with lipophilic prodrug in the bilayer. COLSUB 166, 45–53 [+]

   Previously, we proposed a liposomal formulation of melphalan (Mlph)—a chemotherapeutic alkylating agent—incorporated in a fluid lipid bilayer in the form of dioleoylglyceride ester. In this work, we compared the stabilizing effect of different amphiphiles included in the Mlph-liposomes, such as phosphatidylinositol (PI), ganglioside GM₁, a conjugate of N-carboxymethyl-modified oligoglycine with dioleoylphosphatidylethanolamine (acidic lipopeptide), and polyethylene glycol (2000 Da) conjugated with dipalmitoylphosphatidylethanolamine (PEG-lipid), upon incubation in human serum. Mean hydrodynamic diameter values (86–90 nm) were similar among different liposome samples, while zeta potential values considerably varied. The formulations were incubated in human serum at 37 °C for different time intervals up to 24 h. Liposome integrity was evaluated by changes in fluorescence upon leakage of calcein or disruption of Förster resonance energy transfer between donor and acceptor fluorescent lipid probes in the bilayer. The best stabilization of liposomes was achieved upon the addition of ganglioside GM₁ or the acidic lipopeptide. Inclusion of 10 mol% PI improved liposome stability only for the first 4 h of incubation. Pegylated liposomal formulations of melphalan lipophilic prodrug with fluid phase bilayer were the least stable, which is probably due to the propensity of the PEG-lipid to exit liposome membranes. Cholesterol-containing bilayers of liquid ordered phase, supplemented with sufficient amounts of the PEG-lipid, showed good stability in serum.

   ID:2061
2. Alekseeva A.S., Tretiakova D.S., Chernikov V.P., Utkin Y.N., Molotkovsky J.G., Vodovozova E.L., Boldyrev I.A. (2017). Heterodimeric V. nikolskii phospholipases A2 induce aggregation of the lipid bilayer. Toxicon 133, 169–179 [+]

   We report that the action of the heterodimeric phospholipases A2 (PLA2s) from Vipera nikolskii, which comprises enzymatically active basic subunit and inactive acidic PLA2 homologue, on the lipid bilayer results in the aggregation and stacking of bilayers. These processes are demonstrated using two independent methods (fluorescence spectroscopy and electron microscopy). Aggregation of bilayers is possible because both subunits of the V. nikolskii heterodimer contain a membrane-binding site (also known as IBS). Thus, when the two IBSs bind to the membrane, the heterodimer acts as a connecting agent. Heterodimers induce aggregation of negatively charged bilayers composed of phosphatidylglycerol and do not induce aggregation of neutral bilayers composed of phosphatidylcholine.

   ID:1797
3. Alekseeva A.S., Moiseeva E.V., Onishchenko N.R., Boldyrev I.A., Singin A.S., Budko A.P., Shprakh Z.S., Molotkovsky J.G., Vodovozova E.L. (2017). Liposomal formulation of a methotrexate lipophilic prodrug: assessment in tumor cells and mouse T-cell leukemic lymphoma. Int J Nanomedicine 12, 3735–3749 [+]

   In a previous study, a formulation of methotrexate (MTX) incorporated in the lipid bilayer of 100-nm liposomes in the form of diglyceride ester (MTX-DG, lipophilic prodrug) was developed. In this study, first, the interactions of MTX-DG liposomes with various human and mouse tumor cell lines were studied using fluorescence techniques. The liposomes composed of egg phosphatidylcholine (PC)/yeast phosphatidylinositol/MTX-DG, 8:1:1 by mol, were labeled with fluorescent analogs of PC and MTX-DG. Carcinoma cells accumulated 5 times more MTX-DG liposomes than the empty liposomes. Studies on inhibitors of liposome uptake and processing by cells demonstrated that the formulation used multiple mechanisms to deliver the prodrug inside the cell. According to the data from the present study, undamaged liposomes fuse with the cell membrane only 1.5-2 hours after binding to the cell surface, and then, the components of liposomal bilayer enter the cell separately. The study on the time course of plasma concentration in mice showed that the area under the curve of MTX generated upon intravenous injection of MTX-DG liposomes exceeded that of intact MTX 2.5-fold. These data suggested the advantage of using liposomal formulation to treat systemic manifestation of hematological malignancies. Indeed, the administration of MTX-DG liposomes to recipient mice bearing T-cell leukemic lymphoma using a dose-sparing regimen resulted in lower toxicity and retarded lymphoma growth rate as compared with MTX.

   ID:1796
4. Shenkarev Z.O., Melnikova D.N., Finkina E.I., Sukhanov S.V., Boldyrev I.A., Gizatullina A.K., Mineev K.S., Arseniev A.S., Ovchinnikova T.V. (2017). Ligand Binding Properties of the Lentil Lipid Transfer Protein: Molecular Insight into the Possible Mechanism of Lipid Uptake. Biochemistry 56 (12), 1785–1796 [+]

   The lentil lipid transfer protein, designated as Lc-LTP2, was isolated from Lens culinaris seeds. The protein belongs to the LTP1 subfamily and consists of 93 amino acid residues. Its spatial structure includes four α-helices (H1-H4) and a long C-terminal tail. Here, we report the ligand binding properties of Lc-LTP2. The fluorescent 2-p-toluidinonaphthalene-6-sulfonate binding assay revealed that the affinity of Lc-LTP2 for saturated and unsaturated fatty acids was enhanced with a decrease in acyl-chain length. Measurements of boundary potential in planar lipid bilayers and calcein dye leakage in vesicular systems revealed preferential interaction of Lc-LTP2 with the negatively charged membranes. Lc-LTP2 more efficiently transferred anionic dimyristoylphosphatidylglycerol (DMPG) than zwitterionic dimyristoylphosphatidylcholine. Nuclear magnetic resonance experiments confirmed the higher affinity of Lc-LTP2 for anionic lipids and those with smaller volumes of hydrophobic chains. The acyl chains of the bound lysopalmitoylphosphatidylglycerol (LPPG), DMPG, or dihexanoylphosphatidylcholine molecules occupied the internal hydrophobic cavity, while their headgroups protruded into the aqueous environment between helices H1 and H3. The spatial structure and backbone dynamics of the Lc-LTP2-LPPG complex were determined. The internal cavity was expanded from ∼600 to ∼1000 Å(3) upon the ligand binding. Another entrance into the internal cavity, restricted by the H2-H3 interhelical loop and C-terminal tail, appeared to be responsible for the attachment of Lc-LTP2 to the membrane or micelle surface and probably played an important role in the lipid uptake determining the ligand specificity. Our results confirmed the previous assumption regarding the membrane-mediated antimicrobial action of Lc-LTP2 and afforded molecular insight into its biological role in the plant.

   ID:1779
5. Третьякова Д.С., Онищенко Н.Р., Вострова А.Г., Водовозова Е.Л. (2017). Взаимодействия противоопухолевых липосом, несущих липофильные пролекарства в бислое, с белками плазмы крови. Биоорг. хим. 43 (6), 661–673 [+]

   Взаимодействия с белками плазмы крови 100-нанометровых липосом из яичного фосфатидилхолина и фосфатидилинозита пекарских дрожжей, несущих в бислое диглицеридные сложноэфирные конъюгаты мелфалана (Mlph-L) и метотрексата (MTX–L), исследованы с помощью иммуноблоттинга. Ранее в тестах на гемосовместимость нами показано, что липосомы не влияют на основные форменные элементы крови. Однако MTX–L активировали систему комплемента (СК), в отличие от Mlph-L. В настоящей работе показано, что замена фосфатидилинозита в качестве стабилизирующего липида Mlph-L на конъюгат с полиэтиленгликолем или введение в бислой адресного углеводного липофильного конъюгата незначительно влияет на взаимодействия липосом с важнейшими компонентами СК и аполипопротеинами, а также на их общую адсорбционную способность. Mlph-L не вызывали фрагментации белка С3 – центрального компонента СК, в отличие от МТХ–L, что подтверждает реактогенность последних. Анализ связывания МТХ–L с белком С3 и его фрагментами, а также регуляторными белками СК и иммуноглобулинами позволяет сделать вывод об активации СК по механизму альтернативного пути. Ранее нами показано, что при уменьшении концентрации пролекарства в бислое до уровня, соответствующего режиму низкодозовой терапии метотрексатом, активации СК не происходит.

   ID:2062
6. Chupin V.V., Boldyrev I.A. (2017). 3-{4-[(E)-{4-[(E)-Phenyldiazenyl]phenyl}diazenyl]phenoxy}propane-1,2-diol. Molbank 2017 (1), M932 [+]

   Title compound was designed to be a black quencher of pyrene fluorescence. It was made amphiphilic to serve as a membrane-bound probe. The synthesis is a two-step procedure. The first step is a Mitsunobu reaction of [{(phenyldiazenyl)phenyl}diazenyl]phenol with 1,2-O-isopropylideneglycerol. The second step is the cleavage of the isopropylidene protecting group. The title compound has the extinction coefficient 59,000 at λmax = 380 nm. The Forster distance between the title compound and the pyrene was found to be 37.8 Å.

   ID:1705
7. Zhai X., Gao Y.G., Mishra S.K., Simanshu D.K., Boldyrev I.A., Benson L.M., Bergen H.R. 3rd, Malinina L., Mundy J., Molotkovsky J.G., Patel D.J., Brown R.E. (2017). Phosphatidylserine Stimulates Ceramide 1-Phosphate (C1P) Intermembrane Transfer by C1P Transfer Proteins. J. Biol. Chem. 292 (6), 2531–2541 [+]

   Genetic models for studying localized cell suicide that halt the spread of pathogen infection and immune response activation in plants include Arabidopsis accelerated-cell-death 11 mutant (acd11). In this mutant, sphingolipid homeostasis is disrupted via depletion of ACD11, a lipid transfer protein that is specific for ceramide 1-phosphate (C1P) and phyto-C1P. The C1P binding site in ACD11 and in human ceramide-1-phosphate transfer protein (CPTP) is surrounded by cationic residues. Here, we investigated the functional regulation of ACD11 and CPTP by anionic phosphoglycerides and found that 1-palmitoyl-2-oleoyl-phosphatidic acid or 1-palmitoyl-2-oleoyl-phosphatidylglycerol (≤15 mol %) in C1P source vesicles depressed C1P intermembrane transfer. By contrast, replacement with 1-palmitoyl-2-oleoyl-phosphatidylserine stimulated C1P transfer by ACD11 and CPTP. Notably, "soluble" phosphatidylserine (dihexanoyl-phosphatidylserine) failed to stimulate C1P transfer. Also, none of the anionic phosphoglycerides affected transfer action by human glycolipid lipid transfer protein (GLTP), which is glycolipid-specific and has few cationic residues near its glycolipid binding site. These findings provide the first evidence for a potential phosphoglyceride headgroup-specific regulatory interaction site(s) existing on the surface of any GLTP-fold and delineate new differences between GLTP superfamily members that are specific for C1P versus glycolipid.

   ID:1703
8. Koukalová A., Amaro M., Aydogan G., Gröbner G., Williamson P.T.F., Mikhalyov I., Hof M., Sachl R. (2017). Lipid Driven Nanodomains in Giant Lipid Vesicles are Fluid and Disordered. Sci Rep 7 (1), 5460 [+]
   It is a fundamental question in cell biology and biophysics whether sphingomyelin (SM)- and cholesterol (Chol)- driven nanodomains exist in living cells and in model membranes. Biophysical studies on model membranes revealed SM and Chol driven micrometer-sized liquid-ordered domains. Although the existence of such microdomains has not been proven for the plasma membrane, such lipid mixtures
   have been often used as a model system for ‘rafts’. On the other hand, recent super resolution and single molecule results indicate that the plasma membrane might organize into nanocompartments. However, due to the limited resolution of those techniques their unambiguous characterization is still missing. In this work, a novel combination of Förster resonance energy transfer and Monte Carlo simulations (MC-FRET) identifies directly 10 nm large nanodomains in liquid-disordered model membranes composed of lipid mixtures containing SM and Chol. Combining MC-FRET with solidstate wide-line and high resolution magic angle spinning NMR as well as with fluorescence correlation spectroscopy we demonstrate that these nanodomains containing hundreds of lipid molecules are fluid and disordered. In terms of their size, fluidity, order and lifetime these nanodomains may represent a relevant model system for cellular membranes and are closely related to nanocompartments suggested to exist in cellular membranes.
   ID:1971
9. Алексеева А.С., Третьякова Д.С., Мельникова Д.Н., Молотковский Юл.Г., Болдырев И.А. (2016). Новый флуоресцентный мембранный зонд (2,3;5,6 бисциклогексил)bodipy меченный фосфатидилхолн. Биоорг. хим. 42 (3), 339–344 [+]

   Новый мембранный зонд бисциклогексил BODIPY (BCHB) меченный фосфатидилхолин структурно очень близок в 1,3,5,7 тетраметил BODIPY (TMB) меченному фосфатидилхолину и синтезируется по аналогичной схеме. Системы сопряженных связей BCHB и TMB формально идентичны, однако спектральные характеристики BCHB заметно отличаются, что делают BCHB хорошим акцептором фёрстеровского резонансного переноса (FRET) для TMB. Показано, что FRET пара фосфатидилхолинов на основе BCHB и TMB является перспективным инструментом для исследования мембранных систем, например, межмембранного липидного переноса.
    

   ID:1498
10. German S.V., Navolokin N.A., Kuznetsova N.R., Zuev V.V., Inozemtseva O.A., Aniskov A.A., Volkova E.K., Bucharskaya A.B., Maslyakova G.N., Fakhrullin R.F., Terentyuk G.S., Vodovozova E.L., Gorin D.A. (2015). Liposomes loaded with hydrophilic magnetite nanoparticles: Preparation and application as contrast agents for magnetic resonance imaging. Colloids Surf B Biointerfaces 135, 109–15 [+]

    Magnetic fluid-loaded liposomes (MFLs) were fabricated using magnetite nanoparticles (MNPs) and natural phospholipids via the thin film hydration method followed by extrusion. The size distribution and composition of MFLs were studied using dynamic light scattering and spectrophotometry. The effective ranges of magnetite concentration in MNPs hydrosol and MFLs for contrasting at both T2 and T1 relaxation were determined. On T2 weighted images, the MFLs effectively increased the contrast if compared with MNPs hydrosol, while on T1 weighted images, MNPs hydrosol contrasting was more efficient than that of MFLs. In vivo magnetic resonance imaging (MRI) contrasting properties of MFLs and their effects on tumor and normal tissues morphology, were investigated in rats with transplanted renal cell carcinoma upon intratumoral administration of MFLs. No significant morphological changes in rat internal organs upon intratumoral injection of MFLs were detected, suggesting that the liposomes are relatively safe and can be used as the potential contrasting agents for MRI.

    ID:1421
11. Malinina L., Simanshu D.K., Zhai X., Samygina V.R., Kamlekar R., Kenoth R., OchoaLizarralde B., Malakhova M.L., Molotkovsky J.G., Patel D.J., Brown R.E. (2015). Sphingolipid transfer proteins defined by the GLTP-fold. Q. Rev. Biophys. 48 (3), 281–322 [+]

    Glycolipid transfer proteins (GLTPs) originally were identified as small (~24 kDa), soluble, amphitropic proteins that specifically accelerate the intermembrane transfer of glycolipids. GLTPs and related homologs now are known to adopt a unique, helically dominated, two-layer 'sandwich' architecture defined as the GLTP-fold that provides the structural underpinning for the eukaryotic GLTP superfamily. Recent advances now provide exquisite insights into structural features responsible for lipid headgroup selectivity as well as the adaptability of the hydrophobic compartment for accommodating hydrocarbon chains of differing length and unsaturation. A new understanding of the structural versatility and evolutionary premium placed on the GLTP motif has emerged. Human GLTP-motifs have evolved to function not only as glucosylceramide binding/transferring domains for phosphoinositol 4-phosphate adaptor protein-2 during glycosphingolipid biosynthesis but also as selective binding/transfer proteins for ceramide-1-phosphate. The latter, known as ceramide-1-phosphate transfer protein, recently has been shown to form GLTP-fold while critically regulating Group-IV cytoplasmic phospholipase A2 activity and pro-inflammatory eicosanoid production.

    ID:1480
12. Alekseeva A., Kapkaeva M., Shcheglovitova O., Boldyrev I., Pazynina G., Bovin N., Vodovozova E. (2015). Interactions of antitumour Sialyl Lewis X liposomes with vascular endothelial cells. Biochim. Biophys. Acta 1848 (5), 1099–1110 [+]

    Recently, we showed that tetrasaccharide selectin ligand SiaLe(X) provided targeted delivery of liposomes loaded in the bilayer with melphalan lipophilic prodrug to tumour endothelium followed by severe injury of tumour vessels in a Lewis lung carcinoma model. Here, we study the impact of SiaLe(X) ligand on the interactions of liposomes with human umbilical vein endothelial cells (HUVEC) using flow cytometry, spectrofluorimetry and confocal microscopy. Liposomes composed of egg phosphatidylcholine/yeast phosphatidylinositol/1,2-dioleoyl glycerol ester of melphalan, 8:1:1, by mol, and varying percentages of lipophilic SiaLe(X) conjugate were labelled with BODIPY-phosphatidylcholine. The increase in SiaLe(X) content in liposomes led to a proportional increase in their uptake by cytokine-activated cells as opposed to non-activated HUVEC: for 10% SiaLe(X) liposomes, binding avidity and overall accumulation increased 14- and 6-fold, respectively. The early stages of intracellular traffic of targeted liposomes in the activated cells were monitored by co-localisation with the trackers of organelles. Endocytosis of SiaLe(X) liposomes occurred mostly via clathrin-independent pathways, which does not contradict the available literature data on E-selectin localisation in the plasma membrane. Using dual fluorescence labelling, with rhodamine-labelled phospholipid and calcein encapsulated at self-quenching concentrations, we found that SiaLe(X) liposomes undergo rapid (within minutes) internalisation by activated HUVEC accompanied by the disruption of liposomes; non-activated cells consumed a negligible dose of liposomes during at least 1.5h. Our data evidence the selective effect of SiaLe(X) formulations on activated endothelial cells and indicate their potential for intracellular delivery of melphalan lipophilic prodrug.

    ID:1242
13. Privalova A.M., Uglanova S.V., Kuznetsova N.R., Klyachko N.L., Golovin Yu.I., Korenkov V.V., Vodovozova E.L., Markvicheva E.A. (2015). Microencapsulated Multicellular Tumor Spheroids as a Tool to Test Novel Anticancer Nanosized Drug Delivery Systems In Vitro. J. Nanosci. Nanotechnol. 15 (7), 4806–4814 [+]

    In the study, MCF-7 human breast adenocarcinoma cells were used to study cytotoxicity of novel anticancer nanosized formulations, such as docetaxel-loaded nanoemulsion and liposomal formulation of a lipophilic methotrexate (MTX) prodrug. In Vitro study of cytotoxicity was carried out in 2 models, namely using 3D In Vitro model based on multicellular tumor spheroids (MTS) and 2D monolayer culture. MTS were generated by tumor cell cultivation within alginate-oligochitosanmicro-capsules. In the case of the monolayer culture, cell viability was found to be 25, 18 and 12% for the samples containing nanoemulsion at concentrations 20, 300 and 1000 nM of docetaxel, respectively, after 48 hs incubation. For MTS these values were higher, namely 33, 23 and 18%, respectively. Cytotoxicity of liposomal MTX prodrug-based formulation with final concentration of 1, 2, 10, 50, 100 and 1000 nM in both models was also studied. MTX liposomal formulation demonstrated lower cytotoxicity on MTS compared to intact MTX. Moreover, MTS were also more resistant to both liposomal formulation and intact MTX than the monolayer culture. Thus, at 1000 nM MTX in the liposomal form, cell viability in MTS was 1.4-fold higher than that in the monolayer culture. MTS could be proposed as a promising tool to test novel anticancer nanosized formulations In Vitro.

    ID:1142
14. Malakhov M.V., Dubinnyi M.A., Vlasova N.V., Zgoda V.G., Efremov R.G., Boldyrev I.A. (2014). End-group differentiating ozonolysis of furocoumarins. RSC Advances 4 (106), 61277–61280 [+]

    Ozonolysis of furocoumarins followed by reductive work-up yields not only common symmetrical dialdehydes, but also o-formylumbelliferones with moderate-to-high yields. Simultaneous formation of both products accounts for the transformation of carbonyl oxides – products of primary ozonide ring opening.

    ID:1097
15. Alekseeva A.S., Korotaeva A.A., Samoilova E.V., Volynsky P.E., Vodovozova E.L., Boldyrev I.A. (2014). Secretory phospholipase A2 activity in blood serum: The challenge to sense. Biochem. Biophys. Res. Commun. 454 (1), 178–182 [+]

    Excess levels of secretory phospholipase A2 (sPLA2) is known to contribute to several inflammatory diseases including vascular inflammation correlating with coronary events in coronary artery disease. Thus a method to monitor sPLA2 activity in blood serum is urgently needed. Such method is still a challenge since existing fluorescent probes do not allow to monitor sPLA2 activity directly in blood serum. Here we analyze and overcome barriers in sPLA2 sensing methodology and report a fluorescent probe and a kinetic model of its hydrolysis by sPLA2. New probe is designed with a fluorophore and a quencher not interfering binding to the enzyme. At the same time phospholipid matrix bearing the probe promotes efficient initial quenching of the fluorophore. Kinetic model of probe hydrolysis takes into account signal change due to the side processes. The probe and the kinetic model applied together prove the concept that the activity of sPLA can be measured directly in blood serum.

    ID:1124
16. Vlasenko Yu.V., Alekseeva A.S., Vodovozova E.L. (2014). Synthesis of a Fluorescent Analogue of Methotrexate Lipophilic Prodrug. Russ. J. Bioorgan. Chem. 40 (1), 114–117 [+]
    A fluorescent analogue of the lipophilic prodrug of antitumor agent methotrexate has been synthesized. The conjugate consists of a residue of rac 1 [13 (Me 4 BODIPY 8)tridecanoyl] 2 oleoylglycerol connected to methotrexate by an ester bond via β Ala N carbonylmethylene linker (Me 4 BODIPY 8 stands for 4,4 difluoro 1,3,5,7 tetramethyl 4 bora 3a,4a diaza s indacene 8 yl). The probe is designed for incor poration in the membrane of the liposomal vehicle to study a mechanism of interaction with tumor cells and intracellular traffic.
    ID:1001
17. Sachl R., Amaro M., Aydogan G., Koukalová A., Mikhalyov I.I., Boldyrev I.A., Humpolíčková J., Hof M. (2014). On multivalent receptor activity of GM1 in cholesterol containing membranes. Biochim. Biophys. Acta , [+]

    Gangliosides located at the outer leaflet of plasma membrane are molecules that either participate in recognizing of exogenous ligand molecules or exhibit their own receptor activity, which are both essential phenomena for cell communication and signaling as well as for virus and toxin entry. Regulatory mechanisms of lipid-mediated recognition are primarily subjected to the physical status of the membrane in close vicinity of the receptor. Concerning the multivalent receptor activity of the ganglioside GM1, several regulatory strategies dealing with GM1 clustering and cholesterol involvement have been proposed. So far however, merely the isolated issues were addressed and no interplay between them investigated. In this work, several advanced fluorescence techniques such as Z-scan fluorescence correlation spectroscopy, Förster resonance energy transfer combined with Monte Carlo simulations, and a newly developed fluorescence antibunching assay were employed to give a more complex portrait of clustering and cholesterol involvement in multivalent ligand recognition of GM1. Our results indicate that membrane properties have an impact on a fraction of GM1 molecules that is not available for the ligand binding. While at low GM1 densities (~1 %) it is the cholesterol that turns GM1 headgroups invisible, at higher GM1 level (~4 %) it is purely the local density of GM1 molecules that inhibits the recognition. At medium GM1 content, cooperation of the two phenomena occurs. This article is part of a Special Issue entitled: Nanoscale membrane orgainisation and signalling.

    ID:1095
18. Kuznetsova N.R., Vodovozova E.L. (2014). Differential binding of plasma proteins by liposomes loaded with lipophilic prodrugs of methotrexate and melphalan in the bilayer. Biochemistry Mosc. 79 (8), 797–804 [+]

    Immediately upon contact with blood, nanosized drug delivery systems become coated with a so-called protein corona. The quantitative and qualitative composition of the corona defines not only the behavior of the nanocarrier in the circulation but, ultimately, the pharmacokinetics and biodistribution of the encapsulated drug as well. In turn, the composition of the protein corona depends on the surface properties of the nanoparticles, such as size and distribution of charge and functional groups on the particle surface. Liposomes belong to the most bio- and hemocompatible drug delivery systems feasible for intravenous route of administration required in chemotherapy of metastasizing tumors. However, knowledge on the interactions of liposomes of various compositions with blood plasma proteins remains fragmentary. Moreover, all nanosized drug delivery systems are potential targets for the innate immunity system, primarily the complement (C) system, which underlies frequent cases of hypersensitivity reactions. Recently, in a panel of in vitro hemocompatibility tests, we demonstrated that liposomes built of natural phospholipids - egg phosphatidylcholine and phosphatidylinositol from Saccharomyces cerevisiae - and loaded with diglyceride conjugates of anticancer drugs melphalan and methotrexate, did not affect the morphology and numbers of the main blood cell types. While preparations with melphalan prodrug were also inert in coagulation and C activation tests, methotrexate-loaded liposomes caused impaired coagulation and C activation. The aim of this work was to study the interactions of liposomes carrying prodrugs of melphalan and methotrexate with blood plasma proteins in vitro. Data on protein binding capacity of liposomes obtained with classical gel permeation chromatography techniques allowed for prediction of rather rapid elimination of the liposomes from circulation. A number of differences revealed through immunoblotting of the liposome-bound proteins agree with the previously obtained data on C activation. The possible mechanism of C activation by methotrexate-containing liposomes is discussed.

    ID:1141
19. Trusova V.M., Molotkovsky J.G., Kinnunen P.K.J., Gorbenko G.P. (2014). Structural aspects of cytochrome c – cardiolipin interactions: Förster resonance energy transfer study. , 173–223 [+]

    Cytochrome c (cyt c) is a mitochondrial membrane hemoprotein of high physiological importance. Fisrt, cyt c is one of the key elements of respiration chain transferring electrons from cyt c reductase (bc1 complex) to cyt c oxidase. Second, release of cyt c from the intermembrane space of mitochondria into the cytosol triggers the apoptotic pathway. The idea that specific interactions between cyt c and cardiolipin (CL), the main lipid component of mitochondrial membrane, are crucial to the protein biological activities, constantly receives further corroboration from both theoretical and experimental studies. Despite considerable progress achieved in the field of cyt c – CL biophysics, the detailed structural description of protein-lipid complexation is still lacking. In the present study we applied Förster resonance energy transfer (RET) technique to give comprehensive characterization of cyt c binding to the model lipid membranes composed of the mixtures of zwitterionic lipid phosphatidylcholine (PC) with anionic lipids phosphatidylglycerol (PG), phosphatidylserine (PS) or cardiolipin (CL) in different molar ratios. The donor-acceptor pairs were represented by either anthrylvinyl-labeled PC (AV-PC) or anthrylvinyl-labeled CL (AV-CL) incorporated in trace amounts in lipid vesicles, and heme moiety of cyt c. Association of the protein with the lipid bilayers led to the decrease in donor fluorescence reflecting energy transfer from AV fluorophore to heme. The most effective RET was found for CL-containing membranes. This observation has been interpreted in terms of higher affinity of cyt c to CL as compared to other anionic lipids. In order to get understanding of protein specificity to CL, RET was measured as a function of CL content and ionic strength. Monte Carlo analysis of multiple datasets revealed a complex interplay between several processes, namely i) lipid demixing; ii) CL transition into extended conformation; iii) formation of hexagonal phase. The switch between these states was found to be controlled by CL content and salt concentration. These characteristics of cyt c – CL interaction are of great interest not only in the context of regulating cyt c electron transfer and apoptotic propensities, but also from the viewpoint of the protein biogenesis.

    ID:1149
20. Zhytniakivska O., Trusova V., Gorbenko G., Kirilova E., Kalnina I., Kirilov G., Molotkovsky J., Tulkki J., Kinnunen P. (2014). Location of novel benzanthrone dyes in model membranes as revealed by resonance energy transfer. J Fluoresc 24 (3), 899–907 [+]

    Förster resonance energy transfer (FRET) between anthrylvinyl-labeled phosphatidylcholine (AV-PC) as a donor and newly synthesized benzanthrones (referred to here as A8, A6, AM12, AM15 and AM18) as acceptors has been examined to gain insight into molecular level details of the interactions between benzanthrone dyes and model lipid membranes composed of zwitterionic lipid phosphatidylcholine and its mixtures with anionic lipids cardiolipin (CL) and phosphatidylglycerol (PG). FRET data were quantitatively analyzed in terms of the model of energy transfer in two-dimensional systems taking into account the distance dependence of orientation factor. Evidence for A8 location in phospholipid headgroup region has been obtained. Inclusion of CL and PG into PC bilayer has been found to induce substantial relocation of A6, AM12, AM15 and AM18 from hydrophobic membrane core to lipid-water interface.

    ID:1148
21. Kuznetsova N.R., Stepanova E.V., Peretolchina N.M., Khochenkov D.A., Boldyrev I.A., Bovin N.V., Vodovozova E.L. (2014). Targeting liposomes loaded with melphalan prodrug to tumour vasculature via the Sialyl Lewis X selectin ligand. J Drug Target 22 (3), 242–250 [+]

    Earlier we showed that liposome formulation of DL-melphalan lipophilic prodrug bearing tetrasaccharide Sialyl Lewis X (SiaLe(X)) caused prolonged therapeutic effect on mammary cancer in mice. Here, we compare antivascular effect of SiaLe(X)-liposomes loaded with diglyceride ester of melphalan (Mlph) against SiaLe(X)-free formulation in Lewis lung carcinoma model. Methods: Liposomes of egg phosphatidylcholine/yeast phosphatidylinositol/1,2-dioleoyl glycerol (DOG) conjugate of Mlph/±SiaLe(X)-PEG8-15-DOG, 8:1:1:0.2 by mol, were prepared by standard extrusion. After two intravenous injections with Mlph or liposomes under either standard or delayed treatment protocols, vascular-disrupting effects of the preparations were evaluated basing on tumour section histomorphology, lectin perfusion assay and immunohistochemistry (anti-CD31 staining) data. Also, untreated mice were administered with fluorescently-labelled liposomes to assess their distribution in tumour sections with confocal laser scanning microscopy. Results: Two injections of SiaLe(X)-liposomes reproducibly caused severe injuries of tumour vessels. SiaLe(X)-liposomes co-localized with CD31 marker on vascular endothelium while the non-targeted formulation extravasated into tumour. Discussion: Cytotoxic SiaLe(X)-liposomes exhibit superior vascular-disrupting properties compared to non-targeted liposomes, yet the effect starts to transform into gain in tumour growth inhibition only under delayed treatment regimen. Conclusion: SiaLe(X)-ligand provides targeting of cytotoxic liposomes to tumour endothelium and subsequent antivascular effect.

    ID:997
22. Zhai X., Boldyrev I.A., Mizuno N.K., Momsen M.M., Molotkovsky J.G., Brockman H.L., Brown R.E. (2014). Nanoscale Packing Differences in Sphingomyelin and Phosphatidylcholine Revealed by BODIPY Fluorescence in Monolayers: Physiological Implications. Langmuir 30 (11), 3154–64 [+]

    Phosphatidycholines (PC) with two saturated acyl chains (e.g., dipalmitoyl) mimic natural sphingomyelin (SM) by promoting raft formation in model membranes. However, sphingoid-based lipids, such as SM, rather than saturated-chain PCs have been implicated as key components of lipid rafts in biomembranes. These observations raise questions about the physical packing properties of the phase states that can be formed by these two major plasma membrane lipids with identical phosphocholine headgroups. To investigate, we developed a monolayer platform capable of monitoring changes in surface fluorescence by acquiring multiple spectra during measurement of a lipid force-area isotherm. We relied on the concentration-dependent emission changes of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)-labeled PC to detect nanoscale alterations in lipid packing and phase state induced by monolayer lateral compression. The BODIPY-PC probe contained an indacene ring with four symmetrically located methyl (Me) substituents to enhance localization to the lipid hydrocarbon region. Surface fluorescence spectra indicated changes in miscibility even when force-area isotherms showed no deviation from ideal mixing behavior in the surface pressure versus cross-sectional molecular area response. We detected slightly better mixing of Me4-BODIPY-8-PC with the fluid-like, liquid expanded phase of 1-palmitoyl-2-oleoyl-PC compared to N-oleoyl-SM. Remarkably, in the gel-like, liquid condensed phase, Me4-BODIPY-8-PC mixed better with N-palmitoyl-SM than dipalmitoyl-PC, suggesting naturally abundant SMs with saturated acyl chains form gel-like lipid phase(s) with enhanced ability to accommodate deeply embedded components compared to dipalmitoyl-PC gel phase. The findings reveal a fundamental difference in the lateral packing properties of SM and PC that occurs even when their acyl chains match.

    ID:1012
23. Simanshu D.K., Zhai X., Munch D., Hofius D., Markham J.E., Bielawski J., Bielawska A., Malinina L., Molotkovsky J.G., Mundy J.W., Patel D.J., Brown R.E. (2014). Arabidopsis accelerated cell death 11, ACD11, is a ceramide-1-phosphate transfer protein and intermediary regulator of phytoceramide levels. Cell Rep 6 (2), 388–99 [+]

    The accelerated cell death 11 (acd11) mutant of Arabidopsis provides a genetic model for studying immune response activation and localized cellular suicide that halt pathogen spread during infection in plants. Here, we elucidate ACD11 structure and function and show that acd11 disruption dramatically alters the in vivo balance of sphingolipid mediators that regulate eukaryotic-programmed cell death. In acd11 mutants, normally low ceramide-1-phosphate (C1P) levels become elevated, but the relatively abundant cell death inducer phytoceramide rises acutely. ACD11 exhibits selective intermembrane transfer of C1P and phyto-C1P. Crystal structures establish C1P binding via a surface-localized, phosphate headgroup recognition center connected to an interior hydrophobic pocket that adaptively ensheaths lipid chains via a cleft-like gating mechanism. Point mutation mapping confirms functional involvement of binding site residues. A π helix (π bulge) near the lipid binding cleft distinguishes apo-ACD11 from other GLTP folds. The global two-layer, α-helically dominated, "sandwich" topology displaying C1P-selective binding identifies ACD11 as the plant prototype of a GLTP fold subfamily.

    ID:1147
24. Chugunov A.O., Volynsky P.E., Krylov N.A., Boldyrev I.A., Efremov R.G. (2014). Liquid but Durable: Molecular Dynamics Simulations Explain the Unique Properties of Archaeal-Like Membranes. Sci Rep 4, 7462 [+]

    Археи, прежде известные как архебактерии, в основном являются экстремофилами: их среда обитания — это высокие температура, давление, соленость и кислотность. Возможно, «особый путь» архей был определен необычными свойствами их мембран, существенно отличающихся по составу от «обычных» фосфолипидов у бактерий и эукариот. В Лаборатории моделирования биомолекулярных систем ИБХ РАН провели компьютерное исследование архейных мембран, объяснив взаимосвязь между химической структурой липидов и физическими свойствами мембран. Статья опубликована в журнале Scientific Reports. По ее материалам написан пресс-релиз: «Прочные, но гибкие: молекулярная динамика объясняет уникальность биомембран архей».

    ID:1110
25. Ivanova E.A., Maslov M.A., Kabilova T.O., Puchkov P.A., Alekseeva A.S., Boldyrev I.A., Vlassov V.V., Serebrennikova G.A., Morozova N.G., Zenkova M.A. (2013). Structure-transfection activity relationships in a series of novel cationic lipids with heterocyclic head-groups. Org. Biomol. Chem. 11, 7164–7178 [+]

    Cationic liposomes are promising candidates for the delivery of various therapeutic nucleic acids. Here, we report a convenient synthesis of carbamate-type cationic lipids with various hydrophobic domains (tetradecanol, dialkylglycerol, cholesterol) and positively charged head-groups (pyridinium, N-methylimidazolium, N-methylmorpholinium) and data on the structure-transfection activity relationships. It was found that single-chain lipids possess high surface activity, which correlates with high cytotoxicity due to their ability to disrupt the cellular membrane by combined hydrophobic and electrostatic interactions. Liposomes containing these lipids also display high cytotoxicity with respect to all cell lines. Irrespective of chemical structures, all cationic lipids form liposomes with similar sizes and surface potentials. The characteristics of complexes composed of cationic liposomes and nucleic acids depend mostly on the type of nucleic acid and P/N ratios. In the case of oligodeoxyribonucleotide delivery, the transfection activity depends on the type of cationic head-group regardless of the type of hydrophobic domain: all types of cationic liposomes mediate efficient oligonucleotide transfer into 80-90% of the eukaryotic cells, and liposomes based on lipids with N-methylmorpholinium cationic head-group display the highest transfection activity. In the case of plasmid DNA and siRNA, the type of hydrophobic domain determines the transfection activity: liposomes composed of cholesterol-based lipids were the most efficient in DNA transfer, while liposomes containing glycerol-based lipids exhibited reasonable activity in siRNA delivery under serum-free conditions.

    ID:873
26. Zhai X., Momsen W.E., Malakhov D.A., Boldyrev I.A., Momsen M.M., Molotkovsky J.G., Brockman H.L., Brown R.E. (2013). GLTP-fold interaction with planar phosphatidylcholine surfaces is synergistically stimulated by phosphatidic acid and phosphatidylethanolamine. J. Lipid Res. 54 (4), 1103–13 [+]

    Among amphitropic proteins, human glycolipid transfer protein (GLTP) forms a structurally-unique fold that translocates on/off membranes to specifically transfer glycolipids. Phosphatidylcholine (PC) bilayers with curvature-induced packing stress stimulate much faster glycolipid intervesicular transfer than nonstressed PC bilayers raising questions about planar cytosol-facing biomembranes being viable sites for GLTP interaction. Herein, GLTP-mediated desorption kinetics of fluorescent glycolipid (tetramethyl-boron dipyrromethene (BODIPY)-label) from lipid monolayers are assessed using a novel microfluidics-based surface balance that monitors lipid lateral packing while simultaneously acquiring surface fluorescence data. At biomembrane-like packing (30-35 mN/m), GLTP uptake of BODIPY-glycolipid from POPC monolayers was nearly nonexistent but could be induced by reducing surface pressure to mirror packing in curvature-stressed bilayers. In contrast, 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) matrices supported robust BODIPY-glycolipid uptake by GLTP at both high and low surface pressures. Unexpectedly, negatively-charged cytosol-facing lipids, i.e., phosphatidic acid and phosphatidylserine, also supported BODIPY-glycolipid uptake by GLTP at high surface pressure. Remarkably, including both 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (5 mol%) and POPE (15 mol%) in POPC synergistically activated GLTP at high surface pressure. Our study shows that matrix lipid headgroup composition, rather than molecular packing per se, is a key regulator of GLTP-fold function while demonstrating the novel capabilities of the microfluidics-based film balance for investigating protein-membrane interfacial interactions.

    ID:853
27. Samygina V.R., OchoaLizarralde B., Popov A.N., CaboBilbao A., GonideCerio F., Molotkovsky J.G., Patel D.J., Brown R.E., Malinina L. (2013). Structural insights into lipid-dependent reversible dimerization of human GLTP. Acta Crystallogr. D Biol. Crystallogr. 69 (Pt 4), 603–16 [+]

    Human glycolipid transfer protein (hsGLTP) forms the prototypical GLTP fold and is characterized by a broad transfer selectivity for glycosphingolipids (GSLs). The GLTP mutation D48V near the `portal entrance' of the glycolipid binding site has recently been shown to enhance selectivity for sulfatides (SFs) containing a long acyl chain. Here, nine novel crystal structures of hsGLTP and the SF-selective mutant complexed with short-acyl-chain monoSF and diSF in different crystal forms are reported in order to elucidate the potential functional roles of lipid-mediated homodimerization. In all crystal forms, the hsGLTP-SF complexes displayed homodimeric structures supported by similarly organized intermolecular interactions. The dimerization interface always involved the lipid sphingosine chain, the protein C-terminus (C-end) and α-helices 6 and 2, but the D48V mutant displayed a `locked' dimer conformation compared with the hinge-like flexibility of wild-type dimers. Differences in contact angles, areas and residues at the dimer interfaces in the `flexible' and `locked' dimers revealed a potentially important role of the dimeric structure in the C-end conformation of hsGLTP and in the precise positioning of the key residue of the glycolipid recognition centre, His140. ΔY207 and ΔC-end deletion mutants, in which the C-end is shifted or truncated, showed an almost complete loss of transfer activity. The new structural insights suggest that ligand-dependent reversible dimerization plays a role in the function of human GLTP.

    ID:1000
28. Kuznetsova N.R., Svirshchevskaya E.V., Skripnik I.V., Zarudnaya E.N., Benke A.N., Gaenko G.P., Molotkovskiĭ Yu.G., Vodovozova E.L. (2013). Interaction of liposomes bearing a lipophilic doxorubicin prodrug with tumor cells. Biochemistry (Moscow) Supplement Series A: Membrane and Cell Biology 7 (1), 12–20 [+]

    When used as nanosized carriers, liposomes enable targeted delivery and decrease systemic toxicity of antitumor agents significantly. However, slow unloading of liposomes inside cells diminishes the treatment efficiency. The problem could be overcome by the adoption of lipophilic prodrugs tailored for incorporation into lipid bilayer of liposomes. We prepared liposomes of egg yolk phosphatidylcholine and yeast phosphatidylinositol bearing a diglyceride conjugate of an antitumor antibiotic doxorubicin (a lipophilic prodrug, DOX-DG) in the membrane to study how these formulations interact with tumor cells. We also prepared liposomes of rigid bilayer-forming lipids, such as a mixture of dipalmitoylphosphatidylcholine and cholesterol, bearing DOX in the inner water volume, both pegylated (with polyethylene glycol (PEG) chains exposed to water phase) and non-pegylated. Efficiency of binding of free and liposomal doxorubicin with tumor cells was evaluated in vitro using spectrofluorimetry of cell extracts and flow cytometry. Intracellular traffic of the formulations was investigated by confocal microscopy; co-localization of DOX fluorescence with organelle trackers was estimated. All liposomal formulations of DOX were shown to distribute to organelles retarding its transport to nucleus. Intracellular distribution of liposomal DOX depended on liposome structure and pegylation. We conclude that the most probable mechanism of the lipophilic prodrug penetration into a cell is liposome-mediated endosomal pathway.

    ID:996
29. Krasnov V.P., Korolyova M.A., Vodovozova E.L. (2013). Nano-sized melphalan and sarcolysine drug delivery systems: synthesis and prospects of application. Russian Chemical Reviews 82 (8), 783–814 [+]

    The results of experimental studies concerned with the development of nano-sized drug delivery systems for the antitumour drugs sarcolysine and melphalan are generalized. The structures and biological activities of nanocarriers in comparison with unmodified drugs are discussed. Particular attention is given to the liposomes containing lipid derivatives of sarcolysine and melphalan in the lipid bilayer.

    ID:1002
30. Gorbenko G., Trusova V., Sood R., Molotkovsky J., Kinnunen P. (2012). The effect of lysozyme amyloid fibrils on cytochrome c-lipid interactions. Chem. Phys. Lipids 165 (7), 769–76 [+]

    Protein polymerization into ordered fibrillar structures (amyloid fibrils) is currently associated with a range of pathological conditions. Recent studies clearly indicate that amyloid cytotoxicity is provoked by a continuum of cross-β-sheet aggregates including mature fibrils. In view of the possible diversity of cytotoxicity mechanisms, the present study addressed the question of whether protein conversion into amyloid fibrils can modify its competitive membrane adsorption behavior. Using a combination of resonance energy transfer, dynamic light scattering and fluorescence quenching techniques, the competitive binding of either monomeric or polymerized lysozyme, and cytochrome c to the model lipid membranes composed of phosphatidylcholine mixtures with varying proportions of phosphatidylglycerol, phosphatidylserine or cardiolipin has been studied. The ability of fibrillar lysozyme to induce dissociation of cytochrome c from the membrane binding sites proved to be markedly stronger than that of its monomeric counterpart, with desorption process displaying cooperativity features upon increasing the charge of lipid bilayer. The decreased efficiency of tryptophan fluorescence quenching by acrylamide and short-wavelength shift of emission maximum observed upon membrane binding of lysozyme fibrils were rationalized in terms of fluorophore transfer into interfacial bilayer region. It is hypothesized that electrostatic interactions play predominant role in determining the lipid-associating and competitive abilities of fibrillar lysozyme.

    ID:999
31. Kuznetsova N.R., Sevrin C., Lespineux D., Bovin N.V., Vodovozova E.L., Mészáros T., Szebeni J., Grandfils C. (2011). Hemocompatibility of liposomes loaded with lipophilic prodrugs of methotrexate and melphalan in the lipid bilayer. Journal of controlled release : official journal of the Controlled Release Society , [+]

    A panel of in vitro tests intended for evaluation of the nano-sized drug delivery systems' compliance with human blood was applied to liposomal formulations of anticancer lipophilic prodrugs incorporated into the lipid bilayer. Liposomes on the basis of natural phosphatidylcholine (PC) and phosphatidylinositol (PI), 8:1 (mol) were loaded with 10mol% of either methotrexate or melphalan 1,2-dioleoylglyceride esters (MTX-DOG and Mlph-DOG respectively) and either decorated with 2mol% of sialyl Lewis X/A (SiaLe(X/A)) tetrasaccharide ligand or not. Hemolysis rate, red blood cells and platelets integrity and size distribution, complement (C) activation, and coagulation cascade functioning were analyzed upon the material incubation with whole blood. Both formulations were negatively charged with the zeta potential value being higher in the case of MTX-DOG liposomes, which also were larger than Mlph-DOG liposomes and more prone to aggregation. Accordingly, in hemocompatibility tests Mlph-DOG liposomes did not provoke any undesirable effects, while MTX-DOG liposomes induced significant C activation and abnormal coagulation times in a concentration-dependent manner. Reactivity of the liposome surface was not affected by the presence of SiaLe(X/A) or PI. Decrease in liposome loading with MTX-DOG from 10 to 2.5% resulted in lower surface charge density, smaller liposome size and considerably reduced impact on C activation and coagulation cascades.

    ID:644
32. Semenova A.A., Chugunov A.O., Dubovskii P.V., Chupin V.V., Volynsky P.E., Boldyrev I.A. (2011). The role of chain rigidity in lipid self-association: Comparative study of dihexanoyl- and disorbyl-phosphatidylcholines. Chem. Phys. Lipids 165, 382–386 [+]

    In the course of structure-function investigations of lipids a phosphatidylcholine molecule with short and rigid tails, di-2,4-hexadienoylphosphatidylcholine (DiSorbPC), was synthesized and studied in comparison with its saturated analog, dihexanoylphosphatidylcholine (DHPC). Conjugated double bonds in the acyl chains in DiSorbPC reduce considerably the number of possible conformers of the lipid within an aggregate. This leads to impaired packing of unsaturated acyl chains and thus, to a surprisingly high (115Å(2)) area per molecule for DiSorbPC at the air-water interface and failure to form micelles of regular size and shape. Details on DiSorbPC aggregation and packing provided by a set of experimental techniques combined with molecular dynamics simulations are presented.

    ID:669
33. Alekseeva A.S., Maslov M.A., Antipova N.V., Boldyrev I.A. (2011). Comparison of two lipid/DNA complexes of equal composition and different morphology. Colloids Surf B Biointerfaces 88 (1), 512–6 [+]

    Two types of complexes were prepared from a cationic cholesterol derivative, dioleoylphos-phatidylcholine and DNA. Depending on the preparation procedure complexes were either dense snarls of lipid covered DNA (type A) or multilayer liposomes with DNA between layers (type B). The transfection efficiency of the snarl-shaped complexes was low but positive. The transfection efficiency of the liposome-shaped complexes was zero, while DNA release upon their interaction with anionic liposomes was 1.7 times higher. The differences in transfection efficacy and DNA release could not be ascribed to the difference in resistance of complexes to decomposition upon interaction with anionic liposomes or intracellular environment since the lipid composition of complexes is the same. Instead the complexes in which lipoplex phase is more continuous (type A) should require more anionic lipids or more time within a cell for complete decomposition. Prolonged life time should lead to the higher probability of DNA expression.

    ID:524
34. Moiseeva E.V., Kuznetsova N.R., Svirshchevskaya E.V., Bovin N.V., Sitnikov N.S., Shavyrin A.S., Beletskaya I.P., Combes S., Fedorov A.Y.u., Vodovozova E.L. (2011). Liposome formulations of combretastatin A4 and its 4-arylcoumarin analogue prodrugs: The antitumor effect in the mouse model of breast cancer. Biochemistry (Moscow) Supplement Series B: Biomedical Chemistry Biochemistry (Moscow) Supplement Series B: Biomedical Chemistry 5 (3), 276–283 [+]

    The antimitotic agent combretastatin A-4 (CA-4) has been recently proposed as an antivascular agent for anticancer therapy. In order to reduce systemic toxicity by means of administration in liposome formulations, new lipophilic prodrugs, oleic derivatives of CA-4 and its 4-arylcoumarin analogue (CA4-Ole and ArC-Ole, respectively), have been synthesized in this study. Liposomes with mean diameter of 100 nm prepared on the basis of egg phosphatidylcholine and baker’s yeast phosphatidylinositol quantitatively included up to 15 mol% of CA4-Ole, or 7 mol% of ArC-Ole. To achieve targeting to neovascular endothelium prodrug bearing liposomes decorated with the tetrasaccharide selectin ligand Sialyl Lewis X (SiaLeX) have been also prepared. The antitumor activity was studied in vivo using the model of slow-growing mouse breast cancer. Under the dose used (22 mg/kg) and the administration protocol (four injections, one per a week, starting from the appearance of palpable tumors) cytostatic CA-4 did not reveal any anticancer effect; moreover, it even stimulated tumor growth. The liposome formulations of CA4-Ole did not demonstrate such stimulation. However, to achieve a pronounced antitumor effect, the number of injections of liposomes should be apparently increased. The cytotoxic activity of a novel antimitotic agent ArC was one order of magnitude lower in the human breast carcinoma cell culture in vitro. Nevertheless, in vivo in the mouse model of breast cancer the antitumor effect of this compound corresponded to the double equivalent dose of CA-4. The results demonstrate perspectives of SiaLe^X-liposomes loaded with ArC-Ole: the preparation partially inhibited tumor growth already after the second injection. Thus, subsequent optimization of doses and regimens of administration both for ArC and liposomal ArC-Ole formulations are needed.

    ID:671
35. Mikhalyov I., Samsonov A. (2011). Lipid raft detecting in membranes of live erythrocytes. Biochim. Biophys. Acta 1808 (7), 1930–9 [+]

    The fluorescent probe N-(BODIPY(®)-FL-propionyl)-neuraminosyl-GM(1) (BODIPY-GM(1)) was used to detect lipid rafts in living red blood cells (RBCs) membranes. The probe was detected with fluorescence video microscopy and was found to be uniformly distributed along plasma membrane at room temperature (23°C). At 4°C some probe clearly phase-separated to yield detectable bright spots that were smaller than spatial resolution. As measured by spectrofluorometry, in addition to a major fluorescence peak caused by emissions from monomers, the probe exhibited a red-shifted peak that is characteristic of a BODIPY fluorophore at high local concentrations, indicating that some probe had clustered. Red-shifted fluorescence was the greatest at 4°C, intermediate at 23°C, and the smallest at 37°C. Treating the RBCs with methyl-β-cyclodextrin to remove cholesterol eliminated the red-shifted peak. This strongly indicates that the presence of cholesterol was essential for phase separation of the probe. Fluorometry experiments indicate that rafts exist at 23°C and at 37°C, even though the membrane appears to be uniform at the resolution of microscope. The distinct GM(1) patches distributed over entire membrane of the erythrocytes were observed at both 23°C and at 37°C in RBCs stained with Alexa FL 647 cholera toxin subunit B conjugate (CTB-A647 ). Based on both fluorometry and fluorescence microscopy, some rafts clearly exist at 37°C.

    ID:673
36. Sachl R., Mikhalyov I., Gretskaya N., Olżyńska A., Hof M., Johansson L.B. (2011). Distribution of BODIPY-labelled phosphatidylethanolamines in lipid bilayers exhibiting different curvatures. Phys Chem Chem Phys 13 (24), 11694–701 [+]

    In this paper we have investigated the behaviour of newly synthesised mono-palmitoyl- and dipalmitoyl-phosphatidylethanolamine probes (abbreviated as mPE and dPE, respectively) labelled in the polar headgroup region by either the FL-BODIPY or the 564/570-BODIPY fluorophore and solubilised in lipid systems that exhibit different curvatures. Because of the bulky BODIPY-groups, the monoacyl-form derivatives have a conic-like shape, whereas that for the diacyl derivatives is rather cylindrical. A careful analysis of time-resolved resonance energy transfer experiments by means of analytical models as well as Monte Carlo simulations shows that the mPE derivatives have a comparable affinity to highly curved bilayer regions (torroidal pores formed by magainin-2 in lipid bilayers, or the rims of discoid bicelles) and to planar bilayer regions (i.e. the flat region of lipid bilayers and bicelles). Furthermore, the monoacyl-probes are as compared to the diacyl-probes effectively closer to each other in a lipid bilayer, while none of these probes seems to be randomly distributed. Self-aggregation is most efficiently induced by the larger aromatic 564/570-BODIPY chromophore, but it is suppressed when using the diacyl instead of the monoacyl-form, and/or by attaching BODIPY-groups to the acyl-chain.

    ID:672
37. Vodovozova E.L., Pazynina G.V., Bovin N.V. (2011). Synthesis of diglyceride conjugate of selectin ligand SiaLeX as a vector for targeting of drug-loaded liposomes. Mendeleev Communications 21 (2), 69–71 [+]

    A conjugate of tetrasaccharide Sialyl Lewis X [SiaLe^X, Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ] 3-aminopropyl glycoside and rac-1,2-dioleoyl-3-carboxymethylene[poly(8–15)oxyethylene]oxyacetylamidopropionylglycerol amenable for the incorporation in lipid bilayer of drug-loaded liposomes to achieve targeting in tumors and inflammation foci was obtained by the formation of carboxamide bond.

    ID:670
38. Konshina A.G., Boldyrev I.A., Utkin Y.N., Omelkov A.V., Efremov R.G. (2011). Snake cytotoxins bind to membranes via interactions with phosphatidylserine head groups of lipids. PLoS ONE 6 (4), e19064 [+]

    The major representatives of Elapidae snake venom, cytotoxins (CTs), share similar three-fingered fold and exert diverse range of biological activities against various cell types. CT-induced cell death starts from the membrane recognition process, whose molecular details remain unclear. It is known, however, that the presence of anionic lipids in cell membranes is one of the important factors determining CT-membrane binding. In this work, we therefore investigated specific interactions between one of the most abundant of such lipids, phosphatidylserine (PS), and CT 4 of Naja kaouthia using a combined, experimental and modeling, approach. It was shown that incorporation of PS into zwitterionic liposomes greatly increased the membrane-damaging activity of CT 4 measured by the release of the liposome-entrapped calcein fluorescent dye. The CT-induced leakage rate depends on the PS concentration with a maximum at approximately 20% PS. Interestingly, the effects observed for PS were much more pronounced than those measured for another anionic lipid, sulfatide. To delineate the potential PS binding sites on CT 4 and estimate their relative affinities, a series of computer simulations was performed for the systems containing the head group of PS and different spatial models of CT 4 in aqueous solution and in an implicit membrane. This was done using an original hybrid computational protocol implementing docking, Monte Carlo and molecular dynamics simulations. As a result, at least three putative PS-binding sites with different affinities to PS molecule were delineated. Being located in different parts of the CT molecule, these anion-binding sites can potentially facilitate and modulate the multi-step process of the toxin insertion into lipid bilayers. This feature together with the diverse binding affinities of the sites to a wide variety of anionic targets on the membrane surface appears to be functionally meaningful and may adjust CT action against different types of cells.

    ID:525
39. Tuuf J., Kjellberg M.A., Molotkovsky J.G., Hanada K., Mattjus P. (2011). The intermembrane ceramide transport catalyzed by CERT is sensitive to the lipid environment. Biochim. Biophys. Acta 1808 (1), 229–35 [+]

    The in vitro activity of the ceramide transporter, CERT has been studied using a fluorescence assay. CERT is responsible for the in vivo non-vesicular trafficking of ceramide between the endoplasmic reticulum and Golgi. In this study we have examined how the membrane environment surrounding the ceramide substrate, the membrane packing density and the membrane charge, are affecting the ceramide transfer activity. To examine this we have used an anthrylvinyl-labeled ceramide analogue. We found that if ceramide is in a tightly packed environment such as in sphingomyelin or dipalmitoylphosphatidylcholine containing membranes, the CERT transfer activity is markedly reduced. Ceramide in fluid membranes on the other hand are available for CERT mediated transfer. CERT also favors membranes that contain phosphatidylinositol 4-monophospate, due to its binding capacity of the pleckstrin homology domain towards phosphatidylinositol 4-monophospate. From this study we conclude that the membrane matrix surrounding ceramide, that is ceramide miscibility, is largely affecting the transfer activity of CERT.

    ID:675
40. Kamlekar R.K., Gao Y., Kenoth R., Molotkovsky J.G., Prendergast F.G., Malinina L., Patel D.J., Wessels W.S., Venyaminov S.Y., Brown R.E. (2010). Human GLTP: Three distinct functions for the three tryptophans in a novel peripheral amphitropic fold. Biophys. J. 99 (8), 2626–35 [+]

    Human glycolipid transfer protein (GLTP) serves as the GLTP-fold prototype, a novel, to our knowledge, peripheral amphitropic fold and structurally unique lipid binding motif that defines the GLTP superfamily. Despite conservation of all three intrinsic Trps in vertebrate GLTPs, the Trp functional role(s) remains unclear. Herein, the issue is addressed using circular dichroism and fluorescence spectroscopy along with an atypical Trp point mutation strategy. Far-ultraviolet and near-ultraviolet circular dichroism spectroscopic analyses showed that W96F-W142Y-GLTP and W96Y-GLTP retain their native conformation and stability, whereas W85Y-W96F-GLTP is slightly altered, in agreement with relative glycolipid transfer activities of >90%, ∼85%, and ∼45%, respectively. In silico three-dimensional modeling and acrylamide quenching of Trp fluorescence supported a nativelike folding conformation. With the Trp⁹⁶-less mutants, changes in emission intensity, wavelength maximum, lifetime, and time-resolved anisotropy decay induced by phosphoglyceride membranes lacking or containing glycolipid and by excitation at different wavelengths along the absorption-spectrum red edge indicated differing functions for W142 and W85. The data suggest that W142 acts as a shallow-penetration anchor during docking with membrane interfaces, whereas the buried W85 indole helps maintain proper folding and possibly regulates membrane-induced transitioning to a glycolipid-acquiring conformation. The findings illustrate remarkable versatility for Trp, providing three distinct intramolecular functions in the novel amphitropic GLTP fold.

    ID:677
41. Boldyrev I.A., Molotkovskiĭ J.G. (2010). New 4,4-Difluoro-3a,4a-Diaza-s-Indacene (BODIPY)-Labeled Sphingolipids for Membrane Studies. Russ. J. Bioorgan. Chem. 36 (4), 508–511 [+]

    The synthesis of a series of new fluorescently labeled sphingolipids containing a 4,4-difluoro-
    1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-yl (Me4-BODIPY-8) group at the ω-position of a
    fatty acyl residue is described. The obtained probes were used in studies of biological and model membrane
    systems.

    ID:337
42. Sachl R., Boldyrev I., Johansson L.B.A. (2010). Localisation of BODIPY-labelled phosphatidylcholines in lipid bilayers. Phys. Chem. Chem. Phys. 12, 6027–6034 [+]

    A series of sn-2 acyl-labelled phosphatidyl-cholines (PC), bearing 4,4-difluoro-1-3-5-7-tetra-methyl-4-bora-3a,4a-diaza-s-indacene-8-yl (Me₄-BODIPY) at the end of the C_n-acyl chains were solubilised in unilamellar vesicles and studied with respect to the order and location of the Me₄-BODIPY (denoted: B) group. The obtained results are based on time-resolved electronic energy transfer from donors (2-(9-anthroyloxy)-stearic acid) localised in the lipid–water interface to acceptors BnPC (n = 3, 5, 7, 9, 11, 13, 15), as well as the energy migration among the Me₄-BODIPY groups of BnPC:s. The donor–acceptor and the donor–donor experiments strongly suggest that the Me₄-BODIPY group in BnPC tends to loop back close to the lipid–water interface. The Me₄-BODIPY groups, residing in the two bilayer leaflets, are located at approximately the same depth, and transversally separated by ca. 27 Å for all n-values. Close to the interface, the optimal transversal distribution widens somewhat with increasing length of the sn-2 acyl chain. The obtained order parameter profile of the BnPC:s is also compatible with such a location.

    ID:326
43. Mikhalyov I., Gretskaya N., Johansson L.B. (2009). Fluorescent BODIPY-labelled GM1 gangliosides designed for exploring lipid membrane properties and specific membrane-target interactions. Chem. Phys. Lipids 159 (1), 38–44 [+]

    New fluorophore-labelled G(M1) gangliosides have been synthesised and spectroscopically characterised. Spectroscopically different BODIPY groups were covalently linked, specifically to either the polar or the hydrophobic part of the ganglioside molecule. The absorption and fluorescence spectroscopic properties are reported for 564/571-BODIPY- and 581/591-BODIPY-labelled G(M1). Each of the different BODIPY groups is highly fluorescent and depolarisation experiments provide molecular information about the spatial distribution in lipid bilayers, as well as order and dynamics. From experiments performed on two spectroscopically different BODIPY:s, specific interactions can be revealed by monitoring the rate/efficiency of donor-acceptor electronic energy transfer. Systems of particular interest for applying these probes are e.g. mixtures of lipids, and peptides/proteins interacting with lipid membranes.

    ID:218
44. Kuznetsova N., Kandyba A., Vostrov I., Kadykov V., Gaenko G., Molotkovsky J., Vodovozova E. (2009). Liposomes loaded with lipophilic prodrugs of methotrexate and melphalan as convenient drug delivery vehicles. J. Drug. Deliv. Sci. Techn. 19, 51–59 [+]

    Liposomal formulations prepared by extrusion from natural phospholipids and 1,2-dioleoylglycerol conjugates of methotrexate and melphalan (egg phosphatidylcholine–phosphatidylinositol–prodrug, 8:1:1, by mol.) were characterized by size, composition and stability. Both prodrugs were shown to incorporate completely into unilamellar liposomes with the mean size below 100 nm and form stable dispersions containing the drug concentrations relevant for systemic injections in animals. For long-term storage, the dispersions can be  subjected to deep freezing (- 196°C) and stored at - 70°C; before usage, they should be defrosted and treated shortly in an ultrasonic bath. According to the example of methotrexate conjugate, stability of prodrug ester bond in liposomal formulation towards hydrolysis by human plasma esterases during 24-h incubation were established. Also, liposomes bearing methotrexate conjugate were shown to overcome resistance of human leukemia cells related to impaired transport of initial drug across the membrane.

    ID:106
45. Boldyrev I.A., Gaenko G.P., Moiseeva E.V., Deligeorgiev T., Kaloianova S., Lesev N., Vasilev A., Molotkovskiĭ Iu.G. (2009). [1,10-phenantroline europium complexes: their inclusion in liposomes and cytotoxicity]. Bioorg. Khim. 37 (3), 408–13 [+]

    For a series of 1,10-phenantroline tris-beta-diketonate europium complexes (EuC), cytotoxic activity on the HBL-100 human breast carcinoma cells was determined. Liposomal preparation of the most active EuC, V12, was also tested for cytotoxicity. Testing of this preparation in vivo on starting lethal murine model of T cell leukemic lymphoma ASF-LL showed that the inclusion of V12 in liposomes did not increase its antitumour activity in a local mode of administration.

    ID:639
46. Vodovozova E.L., Pazynina G.V., Tuzikov A.B., Grechishnikova I.V., Molotkovsky J.G. (2009). Synthesis of photoreactive inorganic probes--instruments for studying membrane lectins. Bioorg. Khim. 30 (2), 174–81 [+]

    A method for the synthesis of photoaffinity neoglycolipid probes with a highly efficient carbene-generating diazocyclopentadien-2-ylcarbonyl (Dcp) label, which can be radioiodinated under standard oxidation conditions, was developed. The probes are intended for incorporation into the lipid bilayer. They are lipophilic glycoconjugates on the basis of an amphiphilic aglycone built up from a diacylglycerol and a polyethylene glycol spacer (with a polymerization degree of 9-16) bearing the Dcp label at the terminal unit. The location of the label in the aglycone provides the possibility of one-step preparation of a wide range of probes using various carbohydrate synthons. We have synthesized photoaffinity neoglycoconjugates containing the oligosaccharides: sialyl LewisX tetrasaccharide and A trisaccharide, which is specific to some tumor cells. A probe containing an inactive pentaol (aminodeoxyglucitol) was also synthesized to detect nonspecific binding. The Dcp label is bound to the probe molecule by ester bond; its lability under alkaline conditions facilitates the analysis of cross-linked products after photoaffinity labeling. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 2; see also http://www.maik.ru.

    ID:242
47. Boldyrev I.A., Zhai X., Momsen M.M., Brockman H.L., Brown R.E., Molotkovsky J.G. (2007). New BODIPY lipid probes for fluorescence studies of membranes. J. Lipid Res. 48 (7), 1518–32 [+]

    Many fluorescent lipid probes tend to loop back to the membrane interface when attached to a lipid acyl chain rather than embedding deeply into the bilayer. To achieve maximum embedding of BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorophore into the bilayer apolar region, a series of sn-2 acyl-labeled phosphatidylcholines was synthesized bearing 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-yl (Me(4)-BODIPY-8) at the end of C(3)-, C(5)-, C(7)-, or C(9)-acyl. A strategy was used of symmetrically dispersing the methyl groups at BODIPY ring positions 1, 3, 5, and 7 to decrease fluorophore polarity. Iodide quenching of the phosphatidylcholine probes in bilayer vesicles confirmed that the Me(4)-BODIPY-8 fluorophore was embedded in the bilayer. Parallax analysis of Me(4)-BODIPY-8 fluorescence quenching by phosphatidylcholines containing iodide at different positions along the sn-2 acyl chain indicated that the penetration depth of Me(4)-BODIPY-8 into the bilayer was determined by the length of the linking acyl chain. Evaluation using monolayers showed minimal perturbation of <10 mol% probe in fluid-phase and cholesterol-enriched phosphatidylcholine. Spectral characterization in monolayers and bilayers confirmed the retention of many features of other BODIPY derivatives (i.e., absorption and emission wavelength maxima near 498 nm and approximately 506-515 nm) but also showed the absence of the 620-630 nm peak associated with BODIPY dimer fluorescence and the presence of a 570 nm emission shoulder at high Me(4)-BODIPY-8 surface concentrations. We conclude that the new probes should have versatile utility in membrane studies, especially when precise location of the reporter group is needed.

    ID:214
48. Boldyrev I.A., Molotkovskiĭ I.G. (2006). [A synthesis and properties of new 4,4-difluoro-3a,4a-diaza-s-indacene (BODIPY))-labeled lipids]. Bioorg. Khim. 32 (1), 87–92 [+]

    A series of fluorescently labeled fatty acids of various chain lengths with 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-yl (Me4-BODIPY-8) residue in the omega-position were synthesized. These acids were used to prepare new fluorescently labeled phosphatidylcholines, sphingomyelin, and galactosyl ceramide. Taking into account the symmetry of the Me4-BODIPY-8-fluorophore, one can presume that, in most bilayer membrane systems, this fluorophore is would be embedded into the bilayer.

    ID:331
49. Boldyrev I.A., Molotkovskiĭ Yu.G. (2005). A synthesis of new rigid fluorescent bichromophoric probes for studying mechanisms of donor-donor energy migration. Bioorg. Khim. 31 (3), 331–4 [+]

    Three new fluorescent probes were synthesized for improving the method of studying donor-donor energy migration (DDEM). Each probe has two identical fluorescent 7-diethylaminocoumarin-3-carbonyl groups attached to a rigid bisteroid dodecacyclic spacer through additional inserts. In two probes, the inserts are beta-Ala and L-Ser residues, which provide for a different nearest environment of the fluorophores. The third probe has identical beta-Ala inserts. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 3; see also http://www.maik.ru.

    ID:328
50. Boldyrev I.A., Molotkovskiĭ Yu.G. (2004). Fluorescent properties of 9-anthracenecarboxamides. Bioorg. Khim. 30 (6), 649–55 [+]

    A number of new 9-anthracenecarboxamides are synthesized in order to create new fluorescent probes for studying biological systems. The parameters of their fluorescence in organic solvents of various polarities are investigated, and possible mechanisms of internal quenching of fluorescence of these compounds are discussed. One of the compounds, 4-ethoxycarbonylphenylamide of 9-anthracenecarboxylic acid, is shown to be a promising basis for the development of a new fluorescent probe. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 6; see also http://www.maik.ru.

    ID:329
51. Vodovozova E.L., Tsibizova E.V., Molotkovsky J.G. (2001). One-step iodination of the diazocyclopentadien-2-ylcarbonyl group—a new and convenient preparation of effective radiolabelled photoaffinity probes. J. Chem. Soc., Perkin Trans. 1  (200), 2221–2228 [+]

    A detailed study devoted to direct iodination of the photoactivatable diazocyclopentadien-2-ylcarbonyl (Dcp) group is presented. The iodination does not influence the high carbene reactivity of the Dcp-generated carbene. It was shown that the Dcp substituent forms 4-mono-, 5-mono- and 4,5-diiododerivatives upon iodination under oxidative conditions (76, 20 and 4%, respectively, when DcpOMe 2 is iodinated). Photolysis of the individual products of iodination in cyclohexane resulted in rather high insertion into non-activated CH bonds, without noticeable loss of iodine. Syntheses of new phospholipid and ganglioside membrane probes are also described which incorporate the Dcp function via a labile ester bond. A [125I]-Dcp-phosphatidylcholine probe exhibiting high specific radioactivity (∼500 Ci mmol
    1) was easily prepared at yields of 90% (on the starting Na125I), by using peracetic acid as an oxidant.
    Furthermore, it was successfully used for photolabelling of the integral protein hemagglutinin in a well-characterised influenza virus model. In summary, the Dcp group is efficient for labelling a wide variety of molecules, and as such, it provides a new tool for exploring a diverse range of biological systems.

    ID:182
52. Vodovozova E.L., Moiseeva E.V., Grechko G.K., Gayenko G.P., Nifant'ev N.E., Bovin N.V., Molotkovsky J.G. (2000). Antitumour activity of cytotoxic liposomes equipped with selectin ligand SiaLe(X), in a mouse mammary adenocarcinoma model. Eur. J. Cancer 36 (7), 942–9 [+]

    The overexpression of lectins by malignant cells compared with normal ones can be used for the targeting of drug-loaded liposomes to tumours with the help of specific carbohydrate ligands (vectors). Recently we have shown that liposomes bearing specific lipid-anchored glycoconjugates on a polymeric matrix bind in vitro to human malignant cells more effectively and, being loaded with a lipophilic prodrug of merphalan, reveal higher cytotoxic activity compared with unvectored liposomes. In this study, carbohydrate-equipped cytotoxic liposomes were tested in vivo in a mouse breast cancer model, BLRB-Rb (8.17)1Iem strain with a high incidence of spontaneous mammary adenocarcinoma (SMA). Firstly, a cell line of the SMA was established which was then used to determine the specificity of the tumour cell lectins. After screening of the lectin specificity of a number of fluorescent carbohydrate probes, SiaLe(X) was shown to be the ligand with the most affinity, and a lipophilic vector bearing this saccharide was synthesised. Then different liposomal formulations of the synthetic merphalan lipid derivative and SiaLe(X) vector were prepared and applied in the treatment of mice with grafted adenocarcinomas. The results of the tumorigenesis data show that the therapeutic efficacy of merphalan increases sharply after its insertion as a lipophilic prodrug into the membrane of SiaLe(X)-vectored liposomes.

    ID:109
53. Vodovozova E.L., Gayenko G.P., Razinkov V.I., Korchagina E.Y., Bovin N.V., Molotkovsky J.G. (1998). Saccharide-assisted delivery of cytotoxic liposomes to human malignant cells. Biochem. Mol. Biol. Int. 44 (3), 543–53 [+]

    The overexpression of lectins by malignant cells was applied for in vitro targeting of liposomes equipped with a saccharide vector and loaded in the lipid phase with a lipid derivative of anticancer agent sarcolysine. The lectin specificity of human leukemia HL-60 and human lung adenocarcinoma ACL cells was revealed by tests with fluorescein-labeled sugar probes. With the help of fluorescent lipid dye it was shown that active saccharide ligands increased the level of the vectored liposome binding to malignant cells by 50-80% as compared to liposomes without vector or with inactive one. The degree of liposome/cell membrane fusion was monitored fluorometrically and was shown to be complete and independent of the vectors. The targeted drug-loaded liposomes had the cytotoxic activity 2-4 times higher as compared to the vector-free ones.

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54. Batrakov S.G., Bergelson L.D. (1978). Lipids of the Streptomycettes. Structural investigation and biological interrelation. Review. Chem. Phys. Lipids 21 (1-2), 1–29 [+]

    During a systematic investigation of lipids of Streptomycetes a series of compounds of biochemical and microbiological interest have been isolated and characterized. These include several menaquinones, glycosyl diglycerides (glucuronosyl and isoladobinosym diglycerides), two ornithino lipids and a diol phospholipid. Some of these lipids were not known previously as constituents of streptomycete cells although they have been encountered elsewhere; others have proved to be novel lipids. The results of structural studies of these lipids are reviewed and some of their possible biological functions are discussed.

    ID:222
55. Molotkovsky J.G., Bergelson L.D. (1973). Synthesis of unsaturated mixed acid phosphatidylinositol of natural configuration. A new procedure for resolving racemic alcohols.  (11), 135–147 ID:216
56. Bergelson L.D., Dyatlovitskaya E.V., Torkhovskaya T.I., Sorokina I.B., Gorkova N.P. (1968). Dedifferentiation of phospholipid composition in subcellular particles of cancer cells. FEBS Lett. 2 (2), 87–90 [+]

    In this pioneer work, the phenomenon of phospholipid dedifferentiation (composition leveling) in the tumor cell organelles was described for the first time.

    ID:225
57. Bergelson L.D., Vaver V.A., Prokazova N.V., Ushakov A.N., Popkova G.A. (1966). Diol lipids. Biochim. Biophys. Acta 116 (3), 511–20 [+]

    In the first review on the recently discovered diol lipids, the data on their structures and distribution in natural sources are summarized, and a hypothesis of their metabolic role is expressed.

    ID:224

Водовозова Елена Львовна

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