Effects of oxyethylated glycerol cryoprotectants on phase transitions of DPPC model membranes

Aim. To determine the effect of the oxyethylated glycerol cryoprotectants (OEGn) with polymerization degrees n = 5, 25, 30 on the phase states and phase transitions of dipalmitoylphosphatidylcholine (DPPC)-based model membranes. Methods. Differential scanning calorimetry. Results. Model lipid membra...

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Дата:	2015
Автори:	Kasian, N.A., Krasnikova, A.O., Vashchenko, O.V., Lisetski, L.N., Zinchenko, A.V., Kompaniets, A.M., Ratushna, M.V.
Формат:	Стаття
Мова:	English
Опубліковано:	Інститут молекулярної біології і генетики НАН України 2015
Назва видання:	Вiopolymers and Cell
Теми:	Bioorganic Chemistry
Онлайн доступ:	https://nasplib.isofts.kiev.ua/handle/123456789/152446
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Цитувати:	Effects of oxyethylated glycerol cryoprotectants on phase transitions of DPPC model membranes / N.A. Kasian, A.O. Krasnikova, O.V. Vashchenko, L.N. Lisetski, A.V. Zinchenko, A.M. Kompaniets, M.V. Ratushna // Вiopolymers and Cell. — 2015. — Т. 31, № 2. — С. 146-153. — Бібліогр.: 31 назв. — англ.

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spelling	nasplib_isofts_kiev_ua-123456789-1524462025-02-09T21:36:04Z Effects of oxyethylated glycerol cryoprotectants on phase transitions of DPPC model membranes Вплив кріопротекторів групи оксиетильованих похідних гліцерину на фазові переходи модельних мембран на основі ДПФХ Влияние криопротекторов группы оскиэтилированных производных глицерина на фазовые переходы модельных мембран на основе ДПФХ Kasian, N.A. Krasnikova, A.O. Vashchenko, O.V. Lisetski, L.N. Zinchenko, A.V. Kompaniets, A.M. Ratushna, M.V. Bioorganic Chemistry Aim. To determine the effect of the oxyethylated glycerol cryoprotectants (OEGn) with polymerization degrees n = 5, 25, 30 on the phase states and phase transitions of dipalmitoylphosphatidylcholine (DPPC)-based model membranes. Methods. Differential scanning calorimetry. Results. Model lipid membranes on water/OEGn and water/glycerol subphases with varying cryoprotectant concentrations from 0 to ~ 100 % w/w were studied. A significant raise in the pre-transition and main phase transition temperatures with increasing OEGn concentration was noted whereas the membrane melting peak persist to 100 % w/w OEGn. A sharp increase in the melting enthalpy was observed for OEGn = 5. Conclusions. The solvating ability of the subphase in DPPC membranes decreases in the order water > glycerol > OEGn = 5 > OEGn = 25 > OEGn = 30, which correlates with the relative number of groups effectively contributing to the solvation process. Мета. Встановлення впливу кріопротекторів групи оксиетильованих похідних гліцерину (ОЕГn) зі ступенями полімеризації n = 5, 25 и 30 на фазові стани та фазові переходи модельних ліпідних мембран на основі ДПФХ. Методи. Диференціальна скануюча калориметрія. Результати. Досліджено модельні ліпідні мембрани на субфазі вода/ОЕГn та вода/гліцерин із варіюванням концентрації кріопротектору від 0 до ~100 мас. %. Зі збільшенням концентрації ОЕГn суттєво зростають температури передпереходу та плавлення модельної мембрани, при цьому пік плавлення зберігається до 100 мас. % ОЕГn. Для ОЕГn=5 віднайдено значне зростання ентальпії плавлення мембрани. Висновки. Сольватуюча здатність субфази, що оцінена по зниженню температури плавлення сухого ДПФХ, знижується у низці вода > гліцерин > ОЕГn=5 ОЕГn=25 ОЕГn=30, що корелює зі зменшенням питомої кількості груп, що ефективно беруть участь у сольватації. Цель. Установление влияния криопротекторов группы оксиэтилированных производных глицерина (ОЭГn) со степенями полимеризации n = 5, 25 и 30 на фазовые состояния и фазовые переходы модельных липидных мембран на основе ДПФХ. Методы. Дифференциальная сканирующая калориметрия. Результаты. Исследованы модельные липидные мембраны на субфазе вода/ОЭГn и вода/глицерин с варьированием концентрации криопротектора от 0 до ~100 масс. %. С увеличением концетрации ОЭГn существенно возрастают температуры предперехода и плавления модельной мембраны, при этом пик плавления сохраняется вплоть до 100 масс. % ОЭГn. Для ОЭГn=5 обнаружено значительное возрастание энтальпии плавления мембраны. Выводы. Сольватирующая способность субфазы, оцененная по снижению температуры плавления сухого ДПФХ, снижается в ряду вода > глицерин > ОЭГn=5 > ОЭГn=25 > ОЭГn=30, что коррелирует с уменьшением удельного количества эффективно участвующих в сольватации групп. 2015 Article Effects of oxyethylated glycerol cryoprotectants on phase transitions of DPPC model membranes / N.A. Kasian, A.O. Krasnikova, O.V. Vashchenko, L.N. Lisetski, A.V. Zinchenko, A.M. Kompaniets, M.V. Ratushna // Вiopolymers and Cell. — 2015. — Т. 31, № 2. — С. 146-153. — Бібліогр.: 31 назв. — англ. 0233-7657 DOI: http://biopolymers.org.ua/doi/bc.0008DA https://nasplib.isofts.kiev.ua/handle/123456789/152446 577.352:544.015.4:547.426 en Вiopolymers and Cell application/pdf Інститут молекулярної біології і генетики НАН України
institution	Digital Library of Periodicals of National Academy of Sciences of Ukraine
collection	DSpace DC
language	English
topic	Bioorganic Chemistry Bioorganic Chemistry
spellingShingle	Bioorganic Chemistry Bioorganic Chemistry Kasian, N.A. Krasnikova, A.O. Vashchenko, O.V. Lisetski, L.N. Zinchenko, A.V. Kompaniets, A.M. Ratushna, M.V. Effects of oxyethylated glycerol cryoprotectants on phase transitions of DPPC model membranes Вiopolymers and Cell
description	Aim. To determine the effect of the oxyethylated glycerol cryoprotectants (OEGn) with polymerization degrees n = 5, 25, 30 on the phase states and phase transitions of dipalmitoylphosphatidylcholine (DPPC)-based model membranes. Methods. Differential scanning calorimetry. Results. Model lipid membranes on water/OEGn and water/glycerol subphases with varying cryoprotectant concentrations from 0 to ~ 100 % w/w were studied. A significant raise in the pre-transition and main phase transition temperatures with increasing OEGn concentration was noted whereas the membrane melting peak persist to 100 % w/w OEGn. A sharp increase in the melting enthalpy was observed for OEGn = 5. Conclusions. The solvating ability of the subphase in DPPC membranes decreases in the order water > glycerol > OEGn = 5 > OEGn = 25 > OEGn = 30, which correlates with the relative number of groups effectively contributing to the solvation process.
format	Article
author	Kasian, N.A. Krasnikova, A.O. Vashchenko, O.V. Lisetski, L.N. Zinchenko, A.V. Kompaniets, A.M. Ratushna, M.V.
author_facet	Kasian, N.A. Krasnikova, A.O. Vashchenko, O.V. Lisetski, L.N. Zinchenko, A.V. Kompaniets, A.M. Ratushna, M.V.
author_sort	Kasian, N.A.
title	Effects of oxyethylated glycerol cryoprotectants on phase transitions of DPPC model membranes
title_short	Effects of oxyethylated glycerol cryoprotectants on phase transitions of DPPC model membranes
title_full	Effects of oxyethylated glycerol cryoprotectants on phase transitions of DPPC model membranes
title_fullStr	Effects of oxyethylated glycerol cryoprotectants on phase transitions of DPPC model membranes
title_full_unstemmed	Effects of oxyethylated glycerol cryoprotectants on phase transitions of DPPC model membranes
title_sort	effects of oxyethylated glycerol cryoprotectants on phase transitions of dppc model membranes
publisher	Інститут молекулярної біології і генетики НАН України
publishDate	2015
topic_facet	Bioorganic Chemistry
url	https://nasplib.isofts.kiev.ua/handle/123456789/152446
citation_txt	Effects of oxyethylated glycerol cryoprotectants on phase transitions of DPPC model membranes / N.A. Kasian, A.O. Krasnikova, O.V. Vashchenko, L.N. Lisetski, A.V. Zinchenko, A.M. Kompaniets, M.V. Ratushna // Вiopolymers and Cell. — 2015. — Т. 31, № 2. — С. 146-153. — Бібліогр.: 31 назв. — англ.
series	Вiopolymers and Cell
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fulltext	146 ISSN 0233-7657 Biopolymers and Cell. 2015. Vol. 31. N 2. P. 146–153 doi: http://biopolymers.org.ua/doi/bc.0008DA UDC 577.352:544.015.4:547.426 Effects of oxyethylated glycerol cryoprotectants on phase transitions of DPPC model membranes N. A. Kasian1, A. O. Krasnikova1, O. V. Vashchenko1, L. N. Lisetski1, A. V. Zinchenko2, A. M. Kompaniets2, M. V. Ratushna3 1 Institute for Scintillation Materials, NAS of Ukraine 60, Lenin Ave., Kharkiv, Ukraine, 61001 2 Institute for Problems of Cryobiology and Cryomedicine, NAS of Ukraine 23, Pereyaslovska Str., Kharkiv, Ukraine, 61015 3 Institute of Neurology, Psychiatry and Narcology, NAMS of Ukraine 46, Akademika Pavlova Str., Kharkiv, Ukraine, 61068 kasian@isma.kharkov.ua Aim. To determine the effect of the oxyethylated glycerol cryoprotectants (OEGn) with polymerization degrees n = 5, 25, 30 on the phase states and phase transitions of dipalmitoylphosphatidylcholine (DPPC)- based model membranes. Methods. Differential scanning calorimetry. Results. Model lipid membranes on water/OEGn and water/glycerol subphases with varying cryoprotectant concentrations from 0 to ~ 100 % w/w were studied. A signifi cant raise in the pre-transition and main phase transition temperatures with in- creasing OEGn concentration was noted whereas the membrane melting peak persist to 100 % w/w OEGn. A sharp increase in the melting enthalpy was observed for OEGn = 5. Conclusions. The solvating ability of the subphase in DPPC membranes decreases in the order water > glycerol > OEGn = 5 > OEGn = 25 > OEGn =30, which correlates with the relative number of groups effectively contributing to the solvation process. K e y w o r d s: model lipid membranes, oxyethylated glycerol cryoprotectants, phase transitions, solvation. © 2015 N. A. Kasian et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited Introduction Most of biologically relevant substances can modify the structure and functioning of biomembranes, in particular, by their interaction with lipid bilayers [1–3]. Cryoprotectants, which are used for suppres- sion of the ice formation in cells upon freezing, could also be expected to have a marked membranotropic activity, both directly and by water-mediated mecha- nisms. At a high concentration of cryoprotectants, signifi cant effects on the structure and barrier prop- erties of biomembranes were noted, as well as inhi- bition of the membrane protein activity [4–6]. There- fore, clarifi cation of the mechanisms of cryopro- tectant action at the cellular and sub-cellular levels is an important problem of modern biophysics. A stan- dard approach in studies of the membranotropic ac- tion is the use of model lipid membranes, in particu- lar, of phospholipid water dispersions in the form of liposomes or lamellar structures [1, 2]. Many aspects of the membranotropic action have been extensively studied for «traditional» cryopro- tectants like glycerol and dimethylsulfoxide (DMSO). It is generally accepted that DMSO tends to be local- ized in the membrane close to carbonyl, glycerol and adjacent methylene groups [7]. Depending on the concentration, DMSO can lead to lateral extension of the membrane, a decrease of its thickness and in- crease of penetrability, up to eventual disintegration of the lipid bilayer [7–9]. As a membranotropic agent 147 Effects of oxyethylated glycerols on DPPC membrane (MTA), DMSO substantially increases the melting (main phase transition) temperature Tm of the lipid membranes based on hydrated dipalmitoylphospha- tidylcholine (DPPC) [10]. As compared with DMSO, hydrophilic properties of glycerol are more pronounced, with its partition co- effi cient in the «n-octanol – water» system being equal to 0.005 (~0.25 for DMSO), which explains a weaker penetration of glycerol through the cell membrane [11]. The literature data of the membranotropic activ- ity of glycerol are rather contradictory. According to [6, 12, 13], glycerol is localized in the re gion of polar groups, affecting the thickness of the phospholipid membrane and inter-membrane layer of the solvent. Another opinion is that glycerol does not penetrate to the region of polar groups of the bilayer and does not affect the membrane thickness, and its interaction with water is stronger than that with the lipid polar groups [14]. It has been shown [15] that temperatures and enthalpies of phase transitions of phospholipid li- posomes are not affected substantially when glycerol substitutes for water, and liposomes of basically the same structure can exist with subphases of water, glycerol or ethylene glycol [12]. Such a remarkable property (the ability to replace water in conditions of membrane dehydration with- out effects on the lamellar organization of lipids and their transition from the liquid crystalline to the gel phase) has been also noted for disaccharides and urea [15–19]. Thus, trehalose decreases the melting temperature Tm of dry DPPC even stronger than wa- ter, also increasing the enthalpy of this phase transi- tion [19]. This effect is assumed to have an important biophysical signifi cance. Lowering of the membrane melting temperature in the dehydration conditions preserves its liquid crystalline phase state. As a re- sult, many undesirable processes commonly noted at the transition from liquid crystalline to gel phase are suppressed, e.g. the phase separation of lipids or ag- gregation of membrane proteins [16–19]. An important aspect of the membranotropic ac- tion of many cryoprotectants (methanol, ethanol, ethylene glycol, glycerol) is the formation of the in- terdigitated gel phase LβI, when hydrocarbon «tails» of lipids penetrate (fully or partially) into the adja- cent layer [12, 20–22]. The liquid crystalline La-pha- se is preserved, whereas the intermediate phase Pβ’ disappears [12, 21, 22]. Along with the X-ray struc- tural data, the formation of the interdigitated phase is refl ected also in the increased membrane melting en- thalpy [22], intensity changes in the Raman bands of methylene groups [23], etc. Thus, most of the cryoprotectants show the pro- nounced membranotropic action due to the direct interactions with the polar region of the lipid mem- brane and strong interaction with water [6, 24, 25]. Therefore, further studies of the membranotropic ac- tion of cryoprotectants and clarifi cation of its mech- anism are very important for the development of new cryoprotectant media and within the framework of the general problem of membrane interaction with guest compounds. One of the promising methods for the synthesis of new cryoprotectants is oxyethylation. Thus, by oxy- ethylation of glycerol a homologous series of com- pounds could be obtained with very similar proper- ties but different molecular mass. Such substances – oxyethylated glycerol derivatives with different de- gree of polymerization n (OEGn) [26–29] – to our knowledge, have never been studied from the view- point of their membranotropic effect. In our work, we studied the phase transitions of DPPC model membranes using water/OEGn (n = 5, 25 and 30) mixtures as a subphase. In the subphases, water was gradually replaced by the cryoprotectant in continu- ous series of the water/OEGn ratios. For comparison, control experiments in the identical conditions were carried out for DPPC membranes with water/glyc- erol mixtures as a subphase (as noted above, the membranotropic properties of glycerol could be con- sidered as suffi ciently well studied). Materials and Methods Materials Oxyethylated glycerol cryoprotectants with polym- erizatiom degrees 5, 25 and 30 (OEGn=5, OEGn=25, OEGn=30) were synthesized at Barva chemical plant (Ukraine) by the technology developed at the Insti- tute for Problems of Cryobiology and Cryomedicine 148 N. A. Kasian, A. O. Krasnikova, O. V. Vashchenko et al. [29], where they were additionally purifi ed. The chemical structure of OEGs is shown in Fig. 1. DPPC (Avanti Polar Lipids, USA) was used for preparation of model membranes in the form of multilamellar dispersions on the water/cryoprotectant subphases. Model lipid membrane preparation DPPC multilamellar dispersions were obtained by lipid hydration (1:3 w/w) and incubation at room temperature for 7-8 days with periodical heating up to ~50 оC and intensive stirring [30]. Comparison of the pre-transition (Tp) and main phase transition (Tm) temperatures with the literature data [30, 31] was used for control of the model membrane quality. Bi- nary DPPC+water systems with water content from 0 % to 97 % w/w , as well as DPPC multilamellar dispersions on the water/OEGn and water/glycerol subphase (ternary systems DPPC+[water/OEGn] and DPPC+[water/glycerol]) were obtained in the simi- lar way. The subphase composition was varied from 100 % water to ~100 % cryoprotectant. Differential scanning calorimetry Thermograms of the systems studied were obtained using a Mettler DSC 1 calorimeter (Mettler Toledo, Switzerland). The samples (approx. 20 mg) were placed into aluminium crucible and sealed. For each sample, no less than four cooling-heating cycles were recorded with scanning rate 2 K/min. The pa- rameters of the phase transitions were determined using the original Mettler DSC 1 STARe software. The experimental error for Tm value was ±0.1 °C, for ∆Hm value – ±1.5 J/g. Results and Discussion Fig. 2 shows the thermograms of model phospho- lipid membranes on the water/OEGn=5 subphase nor- malized with respect to the mass of dry DPPC for different cryoprotectant content in the subphase. The thermograms obtained with OEGn=25, OEGn=30 and glycerol in the same conditions are qualitatively si- milar, though the effect of glycerol upon phase tran- sition peaks of the DPPC membranes is much weak- er. In the DPPC-water system (0 %), two phase tran- sitions are observed in the heating mode: pretransi- Fig. 1. Chemical structure of OEGn, k < n Fig. 2. DSC thermograms of DPPC membranes on water/OEGn=5 subphase in the heating mode for samples with different mass concentration of OEGn=5 in the subphase Fig. 3. Phase diagram of DPPC membranes on water/glycerol subphases with different glycerol content: 1 – melting tem pe ra- ture Tm, 2 – pre-transition temperature Tp. Insert shows the mel- ting enthalpy. Designations of phases are the same as given in the text 149 Effects of oxyethylated glycerols on DPPC membrane tion (Tp = 35.6 C) from the Lβ’ gel phase to the inter- mediate («ripple») phase Pβ’, and the main phase transition or the membrane melting (Tm = 41.8 C) from the Pβ’ phase to the liquid crystalline Lα phase, which is in agreement with the literature data [30, 31]. For all the OEGn used (n = 5, 25, 30), the main transition peak was preserved in all the concentra- tion range up to complete substitution of OEG for water in the DPPC+[water/OEGn] system. A similar picture was reported for replacing water with glyc- erol [12], which is also confi rmed by our data. Based on our DSC thermograms and literature data [12], the phase diagram for DPPC membrane on the water/glycerol subphase can be presented (Fig. 3). The effect of glycerol upon Tp and Tm is rather weak; at glycerol concentration in the subphase above 60 % w/w the pretransition peak apparently disappears, and the Tm peak enthalpy increases by ~5 J/g, which can be a marker for formation of the interdigitated gel phase LβI [20]; this is confi rmed by the X-ray structural data [12]. The phase transition temperatures Tp and Tm for systems DPPC+[water/OEGn=5] (a), DPPC+[water/ OEGn=25] and DPPC+[water/OEGn=30] (b) are pre- sented in Fig. 4. With higher OEGn concentrations, the phase transition temperatures increased. This ef- fect is more marked for Tp than for Tm, which sug- gests the predominant interaction of the cryopro- tectants with the polar region of the membranes. When the OEGn fraction in the subphase exceeds 20 %, w/w the pre-transition peak is getting overlapped with the stronger melting peak. The melting peak be- comes smeared at the OEGn content above 30 %, re- fl ecting weaker cooperativity of the main phase tran- sition; however, it persists on the thermograms up to ~100 % of OEGn in the subphase. Two regions can be singled out on the Tm vs. OEGn concentration de- pendence. Up to 40 % of the cryoprotectant, Tm grows slowly, and above 40 % the Tm increase becomes rather noticeable, especially for OEGn=25 and OEGn=30. The maximum increase in Tm is by ~25 C at ~100 % OEGn=30. It can be assumed that the OEGn cryopro- tectants, similarly to glycerol, lead to interdigitation of the lipid membrane. However, the melting enthal- py (ΔHm) data support this assumption only for the DPPC+[water/OEGn=5] system: ΔHm increase is obser- ved when the cryoprotectant content in the subphase increased from 20 to 40 % (Insert, Fig. 4, A). For DPPC+[water/OEGn=25] and DPPC+[water/OEGn=30], no clear jumps of ΔHm on the concentration depen- dence are observed. Further clarifi cation on eventual induction of the interdigitated phases by OEGn cryo- protectants could probably be obtained by the X-ray structural analysis. Let us consider in more detail the effects of cryo- protectants on the melting temperature of the phos- Fig. 4. Temperatures of melting Tm (1) and pre-transition Tp (2) in systems DPPC+[water/OEGn=5] (A), DPPC+[water/OEGn=25] and DPPC+[water/OEGn=30] (B) as function of OEGn content in the subphase. Insert shows the values of melting enthalpy ΔHm A B 150 N. A. Kasian, A. O. Krasnikova, O. V. Vashchenko et al. pholipid membranes. In Fig. 5 one can see Tm as a function of water concentration for ternary systems (DPPC+[water/OEGn=5], DPPC+[water/OEGn=25], DPPC+ [water/OEGn=30] and DPPC+[water/glycerol]). For com- parison, the data for the binary system DPPC+water (without cryoprotectant) are also presented. From the data of Fig. 5, it can be concluded that the effects of cryoprotectants on the melting tem- perature of lipid membranes can be considered in two aspects: 1) an increase in Tm when water is re- placed by the cryoprotectant, and 2) lowering Tm due to solvation of the dry lipid by the cryoprotectant. Let us consider this in more details. It is known that in the binary system DPPC+water the value of Tm for the fully hydrated DPPC is practi- cally constant [18, 30]. With continuous dehydrati- on, when the water content becomes less than ~30 % (which corresponds to ~18 water molecules per one molecule of DPPC), Tm noticeably increased, reach- ing ~110 C for dry DPPC [18] (Fig. 5). This can be explained by stronger Van-der-Waals interactions between the hydrocarbon «tails» due to a decrease in the cross-section area of polar lipid «heads» [19]. In the ternary systems, when water is replaced by cryo- protectant, the melting temperature increases, as- suming the values between Tm of hydrated DPPC and Tm of dry DPPC (Fig. 6, A). The Tm increase is rather small when the OEGn concentration is still below ~40 % w/w. One can assume that in this concentra- tion region there is enough water in the system for hydration of both the lipid and OEGn. When the cry- oprotectant content is further increased, the available water is not suffi cient for hydration of the polar re- gion of the lipid membrane and the OEGn molecules, and solvation of the membrane by the cryoprotectant molecules begins to play an important role. Thus, with further dehydration of the system, Tm increases, not reaching, however, the values noted for the dry DPPC because of solvation of the polar region of the DPPC membrane by the OEGn molecules (Fig. 6, A). Accounting for [16–19], it can be concluded that the lowering of Tm of the dehydrated lipid due to its solvation by the cryoprotectant is of an obvious bio- physical signifi cance. One may assume that this val- ue refl ects the solvating ability of the subphase with Fig. 5. Melting temperature Tm of DPPC membranes as function of water concentration in subphases water/glycerol (1), water/ OEGn=5 (2), water/OEGn=25 (3), water/OEGn=30 (4) and water (5) Fig. 6. Melting temperature of DPPC membranes on different subphases as function of the number of polar centers N (A) and relative number of polar centers N/M (B) in the solvating molecule: 1 – water, 2 – glycerol, 3 – OEGn=5, 4 – OEGn=25, 5 – OEGn=30 A B 151 Effects of oxyethylated glycerols on DPPC membrane respect to the lipid membrane. The obtained experi- mental data show that at a higher polymerization de- gree n of the cryoprotectant the decrease in Tm be- comes less pronounced, i.e., the solvation ability of OEGn decreases with the increase in n. Taking water as the reference, the solvation ability of the subphase decreases in the series: water > glycerol > OEGn=5 > OEGn=25 > OEGn=30. In this series the number of polar groups N per one molecule increases (Fig. 6, A), but the number of po- lar groups normalized with respect to the molecular mass M (N/M) decreases (Fig. 6, B). In the OEGn molecules two kinds of polar centers can be distin- guished that can interact with the polar region of the membrane (and with water) – hydroxyl groups (–OH) and ethoxy groups (–CH2–CH2–O–). With a higher n, the relative number of ethoxy groups is larger, and that of hydroxyl groups is smaller. It should be noted that only hydroxyl groups are present in water and glycerol molecules; the solvating abilities of water and glycerol (estimated by Tm depression of dry DPPC) are close to one another and are much higher as compared with OEGn. In other words, the hydrox- yl groups are more effi cient as solvating agents for DPPC membrane than the ethoxy groups. This statement can be supported by following specu- lations. In zwitterionic DPPC molecules there are two charged centers, namely, negatively charged oxygen atoms in phosphate groups and positively charged ni- trogen in choline groups. Unlike negative charge of oxygens, the nitrogen positive charge is screened by three methyl groups. So, the membrane solvation pro- cess is essentially determined by interaction between negatively charged DPPC phosphate groups and atoms with suffi cient positive charge from the subphase mol- ecules. This positive charge is located on hydrogen of the hydroxyl groups, which can explain their higher solvating ability as compared with ethoxy groups. Returning to Fig. 6, B, the non-linear plot describ- ing the solvating ability of subphase is a multi-pa- rameter function, depending on both quantity and quality of polar centers in subphase molecules. In- deed, for OEGn ( points 3 to 5 in Fig. 6, B) the Tm values dramatically increase, whereas the relative number of polar centers is slightly diminishing. Additionally, with a higher molecular mass of the cryoprotectant the probability of effi cient interaction of all its polar groups with the polar groups of the lipid molecules decreases due to steric factors. Thus, the decrease in the solvating ability of the sub- phase for DPPC membranes is in the following or- der: water > glycerol > OEGn = 5 > OEGn = 25 > OEGn=30. This can be explained by the lowering of the relative number of hydroxyl groups that effectively contrib- ute to the solvation process. Conclusions For the fi rst time, the DPPC model membranes on the water/oxyethylated glycerols (OEGn) subphase with varying OEGn concentrations from 0 to ~ 100 % were studied by DSC method. It has been shown that the OEGn cryoprotectants substitute for water, with a substantial increase in the pre-transition and melting temperatures of the model membranes. The melting peak was clearly observed up to ~100 % OEGn. With OEGn substituting for water, the melting tempera- tures of DPPC membranes were lower as compared with dry DPPC. The cryoprotectant concentration ran ge was determined (20–40 % w/w) where the melting enthalpy of the DPPC membrane on the wa- ter/OEGn=5 subphase sharply increased. The solvat- ing ability of the subphase with respect to DPPC membranes decreases in the following order water > glycerol > OEGn=5 > OEGn=25 > OEGn=30, which cor- relates with the relative number of the groups effec- tively contributing to the solvation process. REFERENCES 1. Pignatello R, Musumeci T, Basile L, Carbone C, Puglisi G. Biomembrane models and drug-biomembrane interaction stud- ies: Involvement in drug design and development. J Pharm Bioallied Sci. 2011;3(1):4–14. 2. Peetla C, Stine A, Labhasetwar V. Biophysical interactions with model lipid membranes: applications in drug discovery and drug delivery. Mol Pharm. 2009;6(5):1264–76. 3. Seydel JK, Wiese M. Drug-membrane interactions: analysis, drug distribution, modeling. Wiley-VCH Verlag GmbH, Wein- heim, 2002, 349 p 4. Nardid O. Study of low-molecular effect of cryoprotectants on mitochondria respiratory chain by spin probe EPR. Prob- 152 N. A. Kasian, A. O. Krasnikova, O. V. 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Phys Rev A. 1989; 40(5):2712–30. 153 Effects of oxyethylated glycerols on DPPC membrane Влияние криопротекторов группы оскиэтилированных производных глицерина на фазовые переходы модельных мембран на основе ДПФХ Н. А. Касян, А. О. Красникова, О. В. Ващенко, Л. Н. Лисецкий, А. В. Зинченко, А. М. Компаниец, М. В. Ратушная Цель. Установление влияния криопротекторов группы окси- этилированных производных глицерина (ОЭГn) со степенями полимеризации n = 5, 25 и 30 на фазовые состояния и фазовые переходы модельных липидных мембран на основе ДПФХ. Методы. Дифференциальная сканирующая калориметрия. Ре- зультаты. Исследованы модельные липидные мембраны на субфазе вода/ОЭГn и вода/глицерин с варьированием концен- трации криопротектора от 0 до ~100 масс. %. С увеличени- ем концетрации ОЭГn существенно возрастают температуры предперехода и плавления модельной мембраны, при этом пик плавления сохраняется вплоть до 100 масс. % ОЭГn. Для ОЭГn=5 обнаружено значительное возрастание энтальпии плавления мембраны. Выводы. Сольватирующая способность субфа- зы, оцененная по снижению температуры плавления сухого ДПФХ, снижается в ряду вода > глицерин > ОЭГn=5 > ОЭГn=25 > > ОЭГn=30, что коррелирует с уменьшением удельного коли- чества эффективно участвующих в сольватации групп. Ключевые слова: модельне липидные мембраны, ок- сиэтилированные производные глицерина, фазовые перехо- ды, сольватация Вплив кріопротекторів групи оксиетильованих похідних гліцерину на фазові переходи модельних мембран на основі ДПФХ Н. О. Касян, А. О. Краснікова, О. В. Ващенко, Л. М. Лисецький, О. В. Зінченко, А. М. Компанієць, М. В. Ратушна Мета. Встановлення впливу кріопротекторів групи оксие- тильованих похідних гліцерину (ОЕГn) зі ступенями полі- меризації n = 5, 25 и 30 на фазові стани та фазові переходи модельних ліпідних мембран на основі ДПФХ. Методи. Диференціальна скануюча калориметрія. Результати. До- сліджено модельні ліпідні мембрани на субфазі вода/ОЕГn та вода/гліцерин із варіюванням концентрації кріопротек- тору від 0 до ~100 мас. %. Зі збільшенням концентрації ОЕГn суттєво зростають температури передпереходу та плавлен- ня модельної мембрани, при цьому пік плавлення зберіга- ється до 100 мас. % ОЕГn. Для ОЕГn=5 віднайдено значне зростання ентальпії плавлення мембрани. Висновки. Сольватуюча здатність субфази, що оцінена по зниженню температури плавлення сухого ДПФХ, знижується у низці вода > гліцерин > ОЕГn=5 > ОЕГn=25 > ОЕГn=30, що корелює зі зменшенням питомої кількості груп, що ефективно беруть участь у сольватації. Ключов і слова: модельні ліпідні мембрани, оксиетильо- вані похідні гліцерину, фазові переходи, сольватація. Received 26.11.2014

Effects of oxyethylated glycerol cryoprotectants on phase transitions of DPPC model membranes

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