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


Introduction
Most of biologically relevant substances can modify the structure and functioning of biomembranes, in particular, by their interaction with lipid bilayers [1][2][3].Cryoprotectants, which are used for suppression of the ice formation in cells upon freezing, could also be expected to have a marked membranotropic activity, both directly and by water-mediated mechanisms.At a high concentration of cryoprotectants, signifi cant effects on the structure and barrier properties of biomembranes were noted, as well as inhibition of the membrane protein activity [4][5][6].Therefore, clarifi cation of the mechanisms of cryoprotectant action at the cellular and sub-cellular levels is an important problem of modern biophysics.A standard approach in studies of the membranotropic action is the use of model lipid membranes, in particular, 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» cryoprotectants like glycerol and dimethylsulfoxide (DMSO).It is generally accepted that DMSO tends to be localized 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 increase of penetrability, up to eventual disintegration of the lipid bilayer [7][8][9].As a membranotropic agent (MTA), DMSO substantially increases the melting (main phase transition) temperature T m of the lipid membranes based on hydrated dipalmitoylphosphatidylcholine (DPPC) [10].
As compared with DMSO, hydrophilic properties of glycerol are more pronounced, with its partition coeffi 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 activity 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 liposomes 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 without 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][16][17][18][19]. Thus, trehalose decreases the melting temperature T m of dry DPPC even stronger than water, also increasing the enthalpy of this phase transition [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 result, many undesirable processes commonly noted at the transition from liquid crystalline to gel phase are suppressed, e.g. the phase separation of lipids or aggregation of membrane proteins [16][17][18][19].
An important aspect of the membranotropic action of many cryoprotectants (methanol, ethanol, ethylene glycol, glycerol) is the formation of the interdigitated gel phase L βI , when hydrocarbon «tails» of lipids penetrate (fully or partially) into the adja-cent layer [12,[20][21][22].The liquid crystalline L a -phase is preserved, whereas the intermediate phase P β' disappears [12,21,22].Along with the X-ray structural data, the formation of the interdigitated phase is refl ected also in the increased membrane melting enthalpy [22], intensity changes in the Raman bands of methylene groups [23], etc.
Thus, most of the cryoprotectants show the pronounced membranotropic action due to the direct interactions with the polar region of the lipid membrane and strong interaction with water [6,24,25].Therefore, further studies of the membranotropic action of cryoprotectants and clarifi cation of its mechanism 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 oxyethylation of glycerol a homologous series of compounds could be obtained with very similar properties but different molecular mass.Such substancesoxyethylated glycerol derivatives with different degree of polymerization n (OEG n ) [26][27][28][29] -to our knowledge, have never been studied from the viewpoint of their membranotropic effect.In our work, we studied the phase transitions of DPPC model membranes using water/OEG n (n = 5, 25 and 30) mixtures as a subphase.In the subphases, water was gradually replaced by the cryoprotectant in continuous series of the water/OEG n ratios.For comparison, control experiments in the identical conditions were carried out for DPPC membranes with water/glycerol mixtures as a subphase (as noted above, the membranotropic properties of glycerol could be considered as suffi ciently well studied).

Materials
Oxyethylated glycerol cryoprotectants with polymerizatiom degrees 5, 25 and 30 (OEG n=5 , OEG n=25, OEG n=30 ) were synthesized at Barva chemical plant (Ukraine) by the technology developed at the Institute for Problems of Cryobiology and Cryomedicine [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 о C and intensive stirring [30].Comparison of the pre-transition (T p ) and main phase transition (T m ) temperatures with the literature data [30,31] was used for control of the model membrane quality.Binary DPPC+water systems with water content from 0 % to 97 % w/w , as well as DPPC multilamellar dispersions on the water/OEG n and water/glycerol subphase (ternary systems DPPC+[water/OEG n ] and DPPC+[water/glycerol]) were obtained in the similar 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 parameters of the phase transitions were determined using the original Mettler DSC 1 STAR e software.The experimental error for T m value was ±0.1 °C, for ∆H m value -±1.5 J/g.

Results and Discussion
Fig. 2 shows the thermograms of model phospholipid membranes on the water/OEG n=5 subphase normalized with respect to the mass of dry DPPC for different cryoprotectant content in the subphase.The thermograms obtained with OEG n=25 , OEG n=30 and glycerol in the same conditions are qualitatively similar, though the effect of glycerol upon phase transition peaks of the DPPC membranes is much weaker.In the DPPC-water system (0 %), two phase transitions are observed in the heating mode: pretransi- tion (T p = 35.6 C) from the L β' gel phase to the intermediate («ripple») phase P β' , and the main phase transition or the membrane melting (T m = 41.8 C) from the P β' phase to the liquid crystalline L α phase, which is in agreement with the literature data [30,31].For all the OEG n used (n = 5, 25, 30), the main transition peak was preserved in all the concentration range up to complete substitution of OEG for water in the DPPC+[water/OEG n ] system.A similar picture was reported for replacing water with glycerol [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 T p and T m is rather weak; at glycerol concentration in the subphase above 60 % w/w the pretransition peak apparently disappears, and the T m peak enthalpy increases by ~5 J/g, which can be a marker for formation of the interdigitated gel phase L βI [20]; this is confi rmed by the X-ray structural data [12].
The phase transition temperatures T p and T m for systems DPPC+[water/OEG n=5 ] (a), DPPC+[water/ OEG n=25 ] and DPPC+[water/OEG n=30 ] (b) are presented in Fig. 4. With higher OEG n concentrations, the phase transition temperatures increased.This effect is more marked for T p than for T m , which suggests the predominant interaction of the cryoprotectants with the polar region of the membranes.When the OEG n fraction in the subphase exceeds 20 %, w/w the pre-transition peak is getting overlapped with the stronger melting peak.The melting peak becomes smeared at the OEG n content above 30 %, refl ecting weaker cooperativity of the main phase transition; however, it persists on the thermograms up to ~100 % of OEG n in the subphase.Two regions can be singled out on the T m vs. OEG n concentration dependence.Up to 40 % of the cryoprotectant, T m grows slowly, and above 40 % the T m increase becomes rather noticeable, especially for OEG n=25 and OEG n=30 .The maximum increase in T m is by ~25 C at ~100 % OEG n=30 .It can be assumed that the OEG n cryoprotectants, similarly to glycerol, lead to interdigitation of the lipid membrane.However, the melting enthalpy (ΔH m ) data support this assumption only for the DPPC+[water/OEG n=5 ] system: ΔH m increase is observed when the cryoprotectant content in the subphase increased from 20 to 40 % (Insert, Fig. 4, A).For DPPC+[water/OEG n=25 ] and DPPC+[water/OEG n=30 ], no clear jumps of ΔH m on the concentration dependence are observed.Further clarifi cation on eventual induction of the interdigitated phases by OEG n cryoprotectants could probably be obtained by the X-ray structural analysis.
Let us consider in more detail the effects of cryoprotectants on the melting temperature of the phos- From the data of Fig. 5, it can be concluded that the effects of cryoprotectants on the melting temperature of lipid membranes can be considered in two aspects: 1) an increase in T m when water is replaced by the cryoprotectant, and 2) lowering T m 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 T m for the fully hydrated DPPC is practically constant [18,30].With continuous dehydration, when the water content becomes less than ~30 % (which corresponds to ~18 water molecules per one molecule of DPPC), T m noticeably increased, reaching ~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 cryoprotectant, the melting temperature increases, assuming the values between T m of hydrated DPPC and T m of dry DPPC (Fig. 6, A).The T m increase is rather small when the OEG n concentration is still below ~40 % w/w.One can assume that in this concentration region there is enough water in the system for hydration of both the lipid and OEG n .When the cryoprotectant content is further increased, the available water is not suffi cient for hydration of the polar region of the lipid membrane and the OEG n molecules, and solvation of the membrane by the cryoprotectant molecules begins to play an important role.Thus, with further dehydration of the system, T m increases, not reaching, however, the values noted for the dry DPPC because of solvation of the polar region of the DPPC membrane by the OEG n molecules (Fig. 6, A).
Accounting for [16][17][18][19], it can be concluded that the lowering of T m of the dehydrated lipid due to its solvation by the cryoprotectant is of an obvious biophysical signifi cance.One may assume that this value refl ects the solvating ability of the subphase with respect to the lipid membrane.The obtained experimental data show that at a higher polymerization degree n of the cryoprotectant the decrease in T m becomes less pronounced, i.e., the solvation ability of OEG n decreases with the increase in n.Taking water as the reference, the solvation ability of the subphase decreases in the series: water > glycerol > OEG n=5 > OEG n=25 > OEG n=30 .
In this series the number of polar groups N per one molecule increases (Fig. 6, A), but the number of polar groups normalized with respect to the molecular mass M (N/M) decreases (Fig. 6, B).In the OEG n molecules two kinds of polar centers can be distinguished that can interact with the polar region of the membrane (and with water) -hydroxyl groups (-OH) and ethoxy groups (-CH 2 -CH 2 -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 T m depression of dry DPPC) are close to one another and are much higher as compared with OEG n .In other words, the hydroxyl groups are more effi cient as solvating agents for DPPC membrane than the ethoxy groups.
This statement can be supported by following speculations.In zwitterionic DPPC molecules there are two charged centers, namely, negatively charged oxygen atoms in phosphate groups and positively charged nitrogen in choline groups.Unlike negative charge of oxygens, the nitrogen positive charge is screened by three methyl groups.So, the membrane solvation process is essentially determined by interaction between negatively charged DPPC phosphate groups and atoms with suffi cient positive charge from the subphase molecules.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 describing the solvating ability of subphase is a multi-parameter function, depending on both quantity and quality of polar centers in subphase molecules.Indeed, for OEG n ( 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 subphase for DPPC membranes is in the following order: water > glycerol > OEG n = 5 > OEG n = 25 > OEG n=30 .This can be explained by the lowering of the relative number of hydroxyl groups that effectively contribute to the solvation process.

Conclusions
For the fi rst time, the DPPC model membranes on the water/oxyethylated glycerols (OEG n ) subphase with varying OEG n concentrations from 0 to ~ 100 % were studied by DSC method.It has been shown that the OEG n 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 % OEG n .With OEG n substituting for water, the melting temperatures 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 water/OEG n=5 subphase sharply increased.The solvating ability of the subphase with respect to DPPC membranes decreases in the following order water > glycerol > OEG n=5 > OEG n=25 > OEG n=30 , which correlates with the relative number of the groups effectively contributing to the solvation process.