Influence of Dimethyl Sulfoxide on the Low-Temperature Behavior of

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B: Biomaterials and Membranes

The Influence of Dimethyl Sulfoxide on the Low-Temperature Behavior of Cholesterol-Loaded Palmitoyl-oleyl-phosphatidylcholine Membranes Beatrice Gironi, Marco Paolantoni, Assunta Morresi, Paolo Foggi, and Paola Sassi J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b02333 • Publication Date (Web): 30 May 2018 Downloaded from http://pubs.acs.org on May 30, 2018

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The Journal of Physical Chemistry

The Influence of Dimethyl Sulfoxide on the LowTemperature Behavior of Cholesterol-Loaded Palmitoyl-oleyl-phosphatidylcholine Membranes

Beatrice Gironi, a Marco Paolantoni, a Assunta Morresi, a Paolo Foggi a,b,c,d and Paola Sassi a,e*

a

Dipartimento di Chimica, Biologia e Biotecnologie, Università di Perugia, Via Elce di Sotto 8,

06123 Perugia, Italy b

European Laboratory for Non Linear Spectroscopy (LENS), Università di Firenze, via Nello

Carrara 1, 50019 Sesto Fiorentino, Florence, Italy c

CNR-INO, Via Nello Carrara 1, 50019 Sesto Fiorentino, Florence, Italy

d

CNR-ICCOM, Via Madonna del Piano 10, 50019 Sesto Fiorentino, Florence, Italy

e

Centro di Eccellenza sui Materiali Innovativi Nanostrutturati (CEMIN), Università di Perugia, Via

Elce di Sotto 8, 06123 Perugia, Italy *

[email protected]

1 ACS Paragon Plus Environment

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ABSTRACT: The properties of lipid membranes at low temperature are important for a number of biomedical and biotechnological applications and the success of these applications depends on understanding the effects of temperature changes on intermolecular lipid-lipid and lipid-water interactions. Here we use FTIR spectroscopy to study lipid suspensions in water/dimethyl sulfoxide (DMSO) solutions in the -60 to 30 °C range. DMSO is a cryo-preservative agent of cellular systems, and its action is largely related to its interaction with the lipid membrane, especially in the low temperature regime. In the present work we analyze the effects of solvent composition on the structural and thermotropic properties of cholesterol (chol)-loaded liposomes of palmitoyloleylphosphatidylcholine (POPC) since POPC/chol liposomes are suitable models of the plasmatic membrane. To this extent, we compare the properties of lipid vesicles suspended in water and water/DMSO solution at 0.10 DMSO mole fraction and we observe that the gel phase of the membrane has an increased thermal stability on DMSO addition. We estimate that the amount of unfrozen water at T= -60 °C is much reduced by the presence of DMSO, both in the gel and the liquid ordered phase of the membrane. Interestingly, we also evidence a reduced hydration of the lipid heads in the presence of DMSO when the vesicles are dispersed in a liquid solution whereas the addition of DMSO does not alter the hydration state of phosphate and carbonyl groups in the frozen state of the membrane.


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1. INTRODUCTION Biomembranes are highly functional sites of living systems and are central to many fundamental processes, such as the transport of materials, recognition, adhesion and signaling1,2. The numerous specific functions occurring in these membranes are reflected in the complex composition of the lipid bilayer and the large variety of interactions among different molecular species. These interactions define the stability of the different membrane phases such as the ordered gel phase (L), the disordered liquid phase (L) and the ordered liquid phase (LO): the structure of this latter is someway intermediate between the formers and is related to the presence of cholesterol3. Together with sphingomyelins (SM) and phosphatidylcholines, cholesterol (chol) is the main constituent of the outer leaflet of the animal cell membranes. The interaction between cholesterol and phospholipids, creating ordered domains in monolayer and bilayer membranes, is of particular interest for the functions and viability of biological systems4. In fact, micro- and nanometer sized lipid domains migrating inside the bilayer have been proposed as the stage structures for specific biological functions3,5-7. These domains (lipid rafts) are supposed to be enriched in cholesterol that makes them more ordered, thicker and, thus, suitable binding sites for certain membrane proteins. The high levels of cholesterol usually suggest the presence of an LO structure in these rafts4-6, despite the fact that the phase state is difficult to be recognized in cells and sometimes also in model membranes7. The existence of LO domains inside the membrane seems to be related to a selective preference of cholesterol for saturated lipids; in saturated lipids like dipalmitoylphosphatidylcholine (DPPC) and SM the phase diagrams indicate a liquid ordered-liquid disordered (LO - L) coexistence when cholesterol is present

5,8-10

. On the

contrary, for unsaturated lipids as palmitoyl-oleylphosphatidylcholine (POPC) the presence of LO structures

3,11

, as well as of a single disordered phase at all cholesterol contents up to 40% and

temperatures >25 °C 12, were proposed. In this scenario of bilayer structures and phase domains, hydration plays a fundamental role since natural membranes are fully hydrated and hydration water serves to the structure and function 3 ACS Paragon Plus Environment


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of cells so much to be in fact a part of the system itself 13. In artificial membranes, a sharp increase of the L to L transition (melting) temperature is observed when the number of water molecules per lipid molecule is less than about ten14. This value corresponds to the number of water molecules directly bounded to lipid headgroups (nW’) and it is larger in the liquid than the gel phase of the membrane14,15. What is called the hydration number of the phospholipid molecule (nW) is not necessarily limited to nW’; in fact, the hydration water represents the interface between the lipid unit and the bulk-like water, structurally and dynamically different from the latter 13,14. Its extent correlates with the area per lipid head group, i.e. the surface offered by the lipid molecule to the solvent that is in turn depending on the packing (phase) of hydrophobic chains 14. The hydration water does not freeze and maintains a certain motional freedom even at very low temperatures 14,16. The properties of the membrane at low temperatures are important for a number of biomedical and biotechnological applications like freeze-drying, cryosurgery and cryopreservation

17-19

. The success of these

applications depends on understanding the effects of temperature changes on intermolecular lipidlipid and lipid-water interactions. However, it is not always easy to analyze these systems in the subzero regime. Fortunately, spectroscopy techniques allow to probe both water and membrane even in the frozen state, leading to the possibility of studying the change of interactions in a wide temperature range 20. Here we address the molecular-scale interactions between cholesterol and POPC in water –DMSO solutions, paying particular attention to the interplay between cholesterol and DMSO in determining the hydration and structural properties of related membranes. POPC bilayers are employed as model membranes as this lipid has a low melting temperature3,11, it is present in natural membranes 21, and is frequently used in biophysical studies 1-3; despite the numerous investigations performed on POPC membranes, a few experimental data are actually available for the frozen state of the system. Moreover, the present study concerns the role of DMSO in the solvating medium owing to its relevance as permeability enhancer of cellular systems22,23 but, most of all, as cryo-protective agent 20-25

, and both these functions are related to the interaction sulfoxide/water, and/or 4 ACS Paragon Plus Environment

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sulfoxide/membrane. In this work the effects induced by DMSO on the L, L and LO lipid phases are investigated at different temperatures and lipid composition. To this extent, we used Fourier Transform InfraRed (FTIR) spectroscopy to monitor the hydrophobic, hydrophilic and interface regions of the bilayer as a function of temperature, in the -60 to 30 °C range. In addition, we analyzed the water libration-bending combination band at about 2200 cm-1, to evaluate the amount of nonfrozen water in the presence of lipid vesicles and to give a complete description of the hydration state of the membrane in the low temperature regime.

2. EXPERIMENTAL 2.1 Preparation of Liposomes: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and cholesterol (chol) powders were purchased from Avanti Polar Lipids, and used without further purification. DMSO (purity>99%) was purchased from Sigma-Aldrich. Different amounts of chol were added to POPC to obtain concentrations corresponding to 0.00, 0.15, 0.30 and 0.50 mole fractions (xchol) respect to the total lipid content. The chloroform solution of the lipid mixture was allowed to evaporate in a fume hood. The solution was then placed in a desiccator where pressure was reduced by a vacuum pump overnight. The end result of this evaporation was a lipid film. Hydration of this lipid film was accomplished by adding water, or water/DMSO solution at 0.10 DMSO mole fraction (xDMSO), to achieve a POPC concentration of 100 mg ml-1; besides, the hydration process was conducted at a temperature above the main phase transition of the lipid mixture. The hydrated lipid suspension underwent 3 freeze/thaw cycles by alternately placing the sample vial in liquid nitrogen and in warm water. This procedure yields stacked multilamellar vesicles; after preparation, the vesicles were left in a refrigerator at 4 °C for one day before performing FTIR measurements.



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2.2 Fourier Transform Infrared Spectroscopy (FTIR): Infrared absorption measurements were carried out with a BRUKER Tensor27 spectrometer, at 2 cm-1 resolution, in the 5000-900 cm-1 wavenumber range. Spectra analysis was carried out using the OPUS 6.5 Bruker Optics software. For FTIR measurements 10 L of lipid suspension was sandwiched between two CaF2 windows. A variable temperature FTIR600 InfraRed Stage (Linkam Scientific Instruments, Tadworth, Surrey, UK) was used in the -60 to 30 °C range. The temperature dependence of the FTIR spectra was studied by cooling the sample from 20 °C to -60 °C at a rate of approximately 1 °C min-1. The sample was thermalized at -60 °C for 30 minutes and subsequently heated at a rate of 1 °C/min, while acquiring FTIR spectra every 60 s. 2.3 Manipulation and Treatment of Spectroscopic Data. The hydration state of the membrane was monitored on the antisymmetric stretching mode (PO) of phosphate units at 1200-1250 cm-1 20,2527

; interface hydration was monitored on the position of the carbonyl stretching band (CO) at about

1740 cm-1 (see Figure1) 27. As a probe of membrane fluidity we analysed the CH2 symmetric stretching band ( νCH2 ) at approximately 2850 cm-1, as previously described 25,26. The band position of νCH2 signal was obtained by the “Peak piking” routine of Opus 6.5 software (BrukerOptics), using the “standard mode” evaluation (x-coordinate of relative maximum). To obtain the  parameter, the first derivative of νCH2 data plotted as a function of temperature was determined by using the “Differentiate” routine of OriginPro 8.0 software of OriginLab Corporation, without smoothing. The melting curves of ice for pure water, water/DMSO solution at xDMSO=0.10, and lipid suspensions at xDMSO=0.00 and xDMSO=0.10, were obtained by evaluation of the integrated intensity (2100-2650 cm-1 range) of the libration-bending combination band (LB) in the -60 °C to 30 °C temperature range. In fact, this band is a very sensitive marker of the water aggregation state 28-30 as evidenced in Figure 1. To obtain the melting curves of ice, all the spectra were normalized to the LB 6 ACS Paragon Plus Environment

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integrated intensity at 10 °C to refer to the same amount of liquid water in the different samples. In the 2100-2650 cm-1 region, the contribution of liquid water to the integrated intensity of the combination band at low temperatures was considered negligible. In case of pure water at T= -60 °C, the total water volume is frozen since the temperature is below the homogeneous nucleation temperature (i.e. the lowest temperature at which liquid water can exist) Thn= -42 °C 31. For lipid suspensions, we evaluated the fraction of unfrozen water from the relative decrease of intensity of LB at -60 °C respect to the pure solvent; this fraction fUW was then multiplied by the number RWl of water molecules per lipid molecule used to prepare the lipid suspension, thus obtaining the number nW of unfrozen water molecules per lipid molecule: nW = fUW  RWl.

T= -60 °C T= 30 °C

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


 CH2

CO

LB

PO

0.00

3000

2500

2000

1500

Wavenumber / cm

-1

1000

Figure 1. FTIR spectra of POPC aqueous suspension at -60 °C and 30 °C.



3. RESULTS AND DISCUSSION Membrane lipids can exist in different kinds of organized structures depending on the type of lipid molecule, degree of hydration, and other variables such as temperature and pressure 1,3,14,32. The IR active vibrations of lipids are very sensitive to the conformation and packing of molecules in the bilayer and both their band position and intensity are often used to follow the gel to liquid phase transition 27. 2854

(a) XDMSO=0,00

2853

CH2 / cm

-1

POPC + XCHOL=0,00 POPC + XCHOL=0,15 POPC + XCHOL=0,30

2852

POPC + XCHOL=0,50 2851

2850

-60

-40

-20

0 20 Temperature / °C

2854

(b) 2853

XDMSO=0,10 POPC + XCHOL= 0,00

-1

POPC + XCHOL= 0,15

CH2 / cm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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2852

POPC + XCHOL= 0,30 POPC + XCHOL= 0,50

2851

2850

-60

-40

-20

0

Temperature / °C

20

Figure 2. Melting curves of POPC/chol suspensions in water (a) and water/DMSO solution (b).


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In Figure 2a the melting curves of POPC vesicles dissolved in water are presented; they were obtained by plotting the origin of νCH2 band as a function of temperature. In fact, the CH2 symmetric stretching band blue-shifts on increasing the gauche segments in the single acyl chain, or reducing the interactions among the chains: this allows to evidence the main transition of the membrane

27

.

The data of the chol-free sample (blue circles) allow recognizing the temperature domains of the gel and liquid phases for T< -5 °C and T> 3 °C, respectively. In the cholesterol loaded vesicles, the gelto-liquid phase transition is much less cooperative since cholesterol is not homogeneously distributed and only the cholesterol-poor domains can still undertake the transition; on the contrary, the cholesterol-rich regions are characterized by a LO structure that is stable in the entire temperature range. The melting curves in Figure 2b are obtained for the samples at xDMSO=0.10 and indicate an overall up-shift of the melting range of about 5 °C / 8 °C upon addition of DMSO in the solvent medium. In Table 1, the melting temperatures of different samples are listed; they were obtained at the first derivative maximum of melting curves of xchol= 0.00,0.15,0.30 samples (Figure 2).

Table 1. Melting Temperatures of POPC/chol Samples Tm at xDMSO=0.00 xchol (°C) 0.00 -1.5 0.15 0.5 0.30 6.0

Tm at xDMSO=0.10 (°C) 3.0 9.0 11.0

We used the data of Figure 2, to evaluate the parameter (∂νCH2 ⁄∂T)/ νCH2 Due to the relation between νCH2 and the membrane packing 20,25,26,33, we observed that the  parameter shows a temperature behavior that is very similar to the one of the thermal expansion coefficient (∂V⁄∂T)/ Vof lipid systems . These quantities ( and ) have the same physical dimension (°C -1); they both show a maximum variation in the melting range, and are higher in the liquid than the gel phase of the membrane. 9 ACS Paragon Plus Environment


The data of Figure 3a suggest that the expansivity of POPC bilayer at low temperatures does not change on cholesterol addition, even at 0.5 cholesterol mole fraction (xchol=0.50). This means that the thermal expansivity of L and LO phases are almost the same; in fact, in the chol–free sample and at xchol= 0.50 the stable phase of the lipid membrane is L and LO, respectively. At higher temperatures  depends on the amount of cholesterol; at 0.15 chol mole fraction we observe an appreciable increase of expansivity but a further cholesterol addition reduces  to the low value of the LO phase. 0.16 0.14

XCHOL=0.00

0.12

XCHOL=0.15

(a)

(°C-1*103)

XCHOL=0.30 XCHOL=0.50

0.03

0.02

0.01

0.00 0.16

(°C-1*103)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.14

XCHOL=0.00; XDMSO=0.00

0.12


0.10


(b)


0.08 0.03 0.02 0.01 0.00

-60

-50

-40

-30

-20

-10

0

10

20

30

Temperature / °C

Figure 3. (a): Temperature dependence of (𝜕𝜈𝐶𝐻2 ⁄𝜕𝑇)/ 𝜈𝐶𝐻2 for aqueous suspension of POPC/chol samples.(b): comparison between data of aqueous and DMSO solution for xchol=0.00 and xchol=0.50 samples. For POPC/chol systems the thermal expansivity and the partial molar volume of lipids in the LO phase were observed to be significantly lower than the liquid L phase

35

: moreover, a 10

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maximum increase of  value was observed at xchol=0.24, especially in the 2 to 30 °C range 35. Our  data are qualitatively in agreement with these results since they suggest an initial increase and a following decrease of expansivity on cholesterol addition. As displayed in Figure 3b, the temperature dependence of  is slightly altered when the solvent contains DMSO at 0.1 mole fraction: we obtain the same values at lower and higher temperatures but the melting range (when present) is up-shifted of about 4 °C. This result is evidence that, with respect to what is monitored by the hydrophobic part of the bilayer, the effect of temperature is not sensitively altered by the presence of DMSO in the system, regardless of whether it is the phase of the membrane. xchol= 0.0

xDMSO= 0.0 xDMSO= 0.1

xchol= 0.5

xDMSO= 0.0 xDMSO= 0.1

Absorbance

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.000

1700

0.000

T = -60 °C 1720

1740

1760

Wavenumber / cm

-1

T = 30 °C

1700

1720

1740

1760

Wavenumber / cm-1

Figure 4. C=O stretching profile CO for POPC/chol water suspensions at -60 °C (a) and 30 °C (b).

It is known that cholesterol can interact with the carbonyl functionality and align with the hydrophobic tails of the phospholipid molecule; besides, it has a preferential interaction with saturated rather than monounsaturated lipids 1-4,36,37. We observed the signature of the POPC/chol interaction in the spectral profile of POPC carbonyl stretching band CO at about 1740 cm-1.In Figure 11 ACS Paragon Plus Environment


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4 the CO profiles for the aqueous suspensions of POPC/chol vesicles at low and high temperatures are shown. At T= -60 °C the band has a maximum at about 1735 cm-1 and a shoulder appears at about 1720 cm-1: this shoulder is related to the H-bonding interactions between POPC carbonyls and water, and between carbonyls and chol hydroxyls. In the presence of DMSO the band is unchanged both for the chol-free and the xchol=0.50 sample, thus: the interactions on the interface region of the frozen membrane are not modified by changing the solvent composition. However, a blue-shift of the band is observed when the vesicles are dissolved in the liquid solvent, as shown in Figure 4b; this suggests a reduced hydration caused by DMSO especially for the chol-free POPC sample. Since the hydration properties have a fundamental impact on the structure and function of lipid membranes, we used the integrated intensity of the combination band at 2100-2650 cm-1, to monitor the water solid-to-liquid transition; in fact, this band shows a sharp decrease of intensity and a red-shift of the maximum at the transition temperature (TICE) due to the different extinction coefficient of libration and bending combination band for liquid and solid H2O 28. In Figure 5 the melting curves of pure water and water/DMSO solution at xDMSO=0.10 are shown as black squares and circles, respectively. Pure water data show that a strong decrease of LB molar extinction coefficient LB is observed at the solid to liquid transition. Actually, a temperature dependence of LB is also detected for both the solid (T 0°C) absorption at 2100-2650 cm-1: this is due to the strong sensitivity of LB to reveal the weakening of H-bonding interactions on increasing temperature within the ice or liquid water domain

29,38

. At low

temperatures, the intensity of the band is proportional to ice concentration: the strong difference between pure water and water/DMSO solution shows that DMSO addition strongly reduces the amount of frozen water and shifts the melting temperature from 0 °C to -17 °C

39

. In Figure 5 the

intensity of the water combination band for lipid suspensions is also shown: in the presence of lipid vesicles we observe the same TICE values of the relative solvent. Whether not changing TICE, the presence of liposomes causes a reduction of ice content at low temperatures. In fact, the water 12 ACS Paragon Plus Environment

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surrounding the hydrophilic surface of the membrane does not freeze at tens of degrees below the equilibrium freezing temperature, even in the presence of ice crystals14-16; this fraction of water is not simply referred to the molecules H-bonded to phospholipid polar heads but to hydration layers extending a few molecular diameters 40. The reduction of ice content we observed in the POPC aqueous suspension at T< TICE is 7 % ca. respect to the pure solvent ( fUW = 0.07 ), and the difference between pure water and lipid suspension is maintained in the 0 °C to -60 °C range (see black and blue squares of Figure 5). Considering the mole ratio between water and POPC ( RWl  410:1 ) , we find that 30 water molecules per POPC molecule ( nW = 30 ) do not freeze. This number is close to the hydration number nW=31 found by Kučerka et al. for fully hydrated POPC in the liquid state, and is much larger than what they indicate as the number of water molecules directly interacting with POPC head groups ( nW’=9 )

15

. Actually, the estimate of nW depends on the sensitivity of the technique to reveal any

difference respect to bulk water induced by the solute 41. Any comparison between results of different measurements cannot be done precisely but can only provide evidence of similarities; however, we can compare values obtained with the same procedure.



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Figure 5. Integrated intensity (see experimental section) of libration-bending combination band of water in pure solvents (black squares and black circles), in lipid aqueous suspensions (blue and redcrossed squares) and lipid suspensions in water/DMSO solution (blue and red-crossed circles).

In DMSO samples, the liquid solution at xDMSO=0.10 is stable at T> -17 °C; at T -17 °C a fraction of water progressively crystallizes by decreasing the temperature. At T< -17 °C POPC membranes further reduce the fraction of ice respect to the pure solvent but the fact that TICE doesn’t change suggests that most of liquid water at low temperatures is still hydrating DMSO and just a small fraction is retained by lipid membranes. Actually, in the pure water/DMSO solution at T= -60 °C, the fraction of ice is about 57% of total water and coexists with a xDMSO=0.21 liquid solution 42. At this temperature a relative decrease of 7% ice content is observed in the presence of POPC vesicles, as evaluated from our data of LB intensity shown in Figure 5 (blue respect to black circles); this means that a further 17 waters per POPC molecule are retained in the liquid phase of the sample. This estimate considers that in lipid suspension DMSO hydration does not change, and only the additional 14 ACS Paragon Plus Environment

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water fraction is hydrating the lipid membrane. The decrease of lipid hydration number nW from 30 of pure water suspension to 17 of DMSO sample could justify the slight increase of POPC melting temperature. In fact, an hydration number nW=17 is still well above the number of waters tightly bounded to polar heads 15, and only when dehydration affects these water molecules the increase of melting temperature is much larger 14,25. The integrated intensity of water combination band evaluated for the xchol= 0.50 sample at xDMSO=0.00 and xDMSO=0.10 are compared with data obtained for chol-free samples (see red symbols in comparison to blue symbols of Figure 5). In both cases a sensitive decrease of ice formation is observed respect to POPC membranes (16% and 17% at xDMSO=0.00 and xDMSO=0.10, respectively), thus suggesting a relative increase of hydration water related to the presence of cholesterol; however, in these samples the water/lipid mole ratio is much lower (205:1). We evaluated that 34 and 21 waters per lipid molecule (considering both POPC and chol) are retained on the surface of the lipid bilayer in the absence and presence of DMSO, respectively. Actually, for the xchol=0.50 sample the two lipids are not equally solvent-exposed since cholesterol is more inserted into the bilayer 36,43 thus the POPC hydration is likely to exceed the 34 (or 21) water molecules here evaluated. In Table 2 nW values obtained for the frozen state of the different lipid samples are reported.

Table 2. Lipid Hydration Numbers at T= -60 °C POPC (L phase) xDMSO =0.00 30 xDMSO =0.10 17

POPC/chol xchol=0.50 (L phase) 34 21

Interestingly this estimate suggests that, despite the slight increase of non-frozen water when passing from the gel to the liquid ordered state of the membrane, the effect of DMSO addition on lipid hydration is roughly the same: a 40% below the value of water suspension. To gain more insights on the hydration properties of polar heads, an analysis of the phosphate IR bands was accomplished. Actually, the PO band at about 1250 cm-1 is a sensitive marker of water15 ACS Paragon Plus Environment


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phosphate contacts showing a red-shift at about 1230 cm-1 for H-bonded groups 25-27. In fact, for the same system (see for example the pink curves of Figure 6a and Figure 6b) the PO band shifts at higher wavenumbers on increasing temperature. In Figure 6a, the comparison between PO spectral profiles in the presence (pink and red curves) and absence (cyan and blue curves) of DMSO, shows that the hydration of phosphate units is not altered by the different solvent composition at T= -60 °C, and this holds true for both L and LO phases of the membrane. Figure 6b shows that the band has a blue shift on DMSO addition when the L or LO phase of the membrane are stable at 30 °C. The effect of DMSO addition is the same for PO and CO bands. This result is different from what we recently observed for DPPC vesicles where the phosphate units of the gel state resulted to be less hydrated in the presence of DMSO, both in the L and L phases

26

. The polar surface of DPPC and POPC lipid

bilayers is perfectly the same. The fundamental difference between the gel state of DPPC and POPC is the thermal stability since the saturated lipid has a melting temperature much greater (Tm =41 °C) than the monounsaturated one (Tm = -2 °C). In Figure 7a the PO bands for the L phase of DPPC and POPC membranes in the absence of DMSO are shown: the comparison between curves suggests that the exposure of phosphate groups to the solvent is very similar, despite the great temperature difference. The intensity of the 1250 cm-1 shoulder of non-hydrated phosphate groups is lower for DPPC than POPC on DMSO addition ( see Figure 7b): this means that the hydration degree of the saturated lipid is lower even if the amount of liquid water in the system is much higher. According to what observed for DPPC 26 and POPC vesicles we deduce that DMSO reduces the extension of the hydration layer at sub-zero temperatures, but decreases the number of water molecules directly bounded to phosphate groups only when the vesicles are dispersed in a liquid solvent.


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T= -60 °C

T= 30 °C

xchol= 0.0 water xDMSO = 0.1 xchol= 0.5

Absorbance

water xDMSO = 0.1

Absorbance

0.000

0.000

1150

1200

1250

Wavenumber / cm

1150

-1

1200

1250

Wavenumber / cm

-1

Figure 6. PO band for POPC suspensions of chol-free and xchol=0.50 samples at T= -60 °C and T=30 °C xDMSO= 0.0

xDMSO= 0.1

POPC T= -60 °C DPPC T= 25 °C

POPC T= -60 °C DPPC T= 25 °C

Absorbance

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.000

(a) 1150

(b)

0.000

1200

1250

Wavenumber / cm

-1

1150

1200

1250

Wavenumber / cm

-1



Page 18 of 25

Figure 7. PO band for the L phase of POPC and DPPC membranes in the absence (a) and presence (b) of DMSO.

4. CONCLUSIONS The present work elucidates the role played by the solvent composition in defining the hydration properties of cholesterol loaded POPC membranes in the -60 °C to 30 °C temperature range, and particularly in the sub-zero regime. DMSO at 0.10 mole fraction was added to aqueous suspension of liposomes: our results show that the thermal expansivity of both the L, L and LO phases of the membrane are almost unaffected by the presence of the co-solvent. The thermal expansivity is not altered by the presence of DMSO because its molecules are not dispersed in the bilayer: they rather act at the level of polar heads and alters the hydration degree of the membrane. In fact, when lipid vesicles are dispersed in the liquid solvent, the presence of DMSO reduces the hydration of both phosphate and carbonyl groups; while in the frozen state of the membrane the H-bonding interactions on both interface and polar regions of the bilayer result to be unchanged respect to xDMSO=0.00 system. At low temperatures, we evaluated the fraction of non-frozen water to assess the aqueous layer surrounding the hydrophilic surface as a separation from the bulk solvent. When the vesicles are in the frozen state, we observed that, even if the number of water molecules directly bonded to phosphate groups is not changing, the sulfoxide has a strong impact since it causes a 40% lowering the value of unfrozen water molecules. Actually, as observed by Wolfe et al.

40

, the estimate of an

amount of unfrozen water much larger than bound water could have a kinetic origin. Anyway, under the experimental conditions we used, the co-solvent alters the hydration of phosphate and carbonyl groups when the membrane is dispersed in the liquid solvent, but is irrelevant in the frozen state. The thermal treatment we applied is similar to the one usually adopted in cryo-preservation protocols of cells

44,45

, where DMSO is added to cellular samples at room temperature and then a treatment at

controlled freezing rate is applied. Our data suggest that, if the reduced hydration of polar heads at 18 ACS Paragon Plus Environment

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30 °C is due to the proximity of DMSO, then the sulfoxide is expelled from the surface of the lipid bilayer on freezing the system. This preferential exclusion of DMSO is probably driven by its strong interaction with water 46; this interaction reduces any contact of DMSO with lipid head groups but also keep water in the sulfoxide hydration shell thus reducing the extent of lipid hydration water. This could explain the peculiar cryo-protective action of DMSO 47.

Corresponding author: Paola Sassi Dipartimento di Chimica, Biologia e Biotecnologie, Università di Perugia, Via Elce di Sotto 8, 06123 Perugia, Italy e-mail: [email protected] Acknowledgements The authors are grateful to Prof. John Crowe (Univ. California-Davis) and Prof. Willem F. Wolkers (Leibniz Univ. Hannover) for the fruitful discussion and valuable suggestions. Authors acknowledge financial support from the italian Centro Nazionale Trapianti by the project “Studio di cellule per uso clinico umano, con particolare riferimento a modelli cellulari (liposomi) e linee cellulari in interazione con crioconservanti e con materiali biocompatibili”; and the University of Perugia by the project “Fondo d’Ateneo per la ricerca di base 2014 - Sintesi e caratterizzazione spettroscopica di nanoibridi per la diagnosi e il trattamento del glioblastoma “.



Page 20 of 25

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TOC GRAPHIC

liquid

frozen -60

-40

-20

0

20

Temperature / °C


Influence of Dimethyl Sulfoxide on the Low-Temperature Behavior of

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