The Korean Society For Biotechnology And Bioengineering

Current Issues

Korean Society for Biotechnology and Bioengineering Journal - Vol. 32 , No. 2

[ Research Paper ]
Korean Society for Biotechnology and Bioengineering Journal - Vol. 32, No. 2, pp.103-107
ISSN: 1225-7117 (Print) 2288-8268 (Online)
Print publication date 30 Jun 2017
Received 17 Mar 2017 Revised 04 May 2017 Accepted 10 May 2017
DOI: https://doi.org/10.7841/ksbbj.2017.32.2.103

Effect of Trehalose on Biological Membranes with Respect to Phase of the Membranes
Jin-Won Park*

Correspondence to : *Department of Chemical and Biomolecular Engineering, College of Energy and Biotechnology, Seoul National University of Science and Technology, Seoul 139-743, Korea Tel: +82-2-970-6605, Fax: +82-2-977-8317 e-mail: jwpark@seoultech.ac.kr


© 2017 The Korean Society for Biotechnology and Bioengineering
Funding Information ▼

Abstract

The effect of the trehalose incorporation on the biological membranes was investigated with respect to the phase of the membranes using the fluorescence intensity change. Spherical phospholipid bilayers, vesicles, were prepared only with the variation in the phase of each layer via a double emulsion technique. In the aqueous inside of the vesicles, 8-Aminonaphthalene- 1,3,6-trisulfonic acid disodium salt(ANTS) was encapsulated. As a quencher, p-Xylene-bis(N-pyridinium bromide)(DPX) was included in the buffer where the vesicles were dispersed. The fluorescence scale was calibrated with the fluorescence of ANTS vesicles in p-Xylene-bis(N-pyridinium bromide)(DPX)-included-buffer taken as 100% fluorescence and the mixture of ANTS and DPX in the buffer as 0% fluorescence. Trehalose injection into the vesicle solution led the distortion of the membrane. It was found that the distortion was related to the phase of each layer the vesicle up on the ratio of trehalose to lipid. In the identical measurements at glucose, the behavior of the distortion was completely different from that of trehalose. These results seem to depend on the stability of the vesicles, due to the osmotic and volumetric effects on the headgroup packing disruption.


Keywords: Trehalose, Vesicles, Lipid layer phase

1. INTRODUCTION

Sugars have been widely found in living organisms [1]. Soluble sugars have unique functions for liposomes, intact membranes, and whole cells against desiccation and fusion caused by freezing or freeze-drying [2-4]. Therefore, studies on the effects of saccharides on a cell membrane, such as on its permeability and stability, are very important for understanding the functions, such as protection and preservation [2,5]. Especially, trehalose has been studied as a disaccharide which indicates the most effective protection against water stress among saccharides [6]. Furthermore, it has been proposed that trehalose induced the structure preservation against freezing or drying in the systems [7]. Consequently, it is suggested that trehalose avoided the transition between gel and liquid crystalline phase during rehydration and inhibited the leakage from the system inside due to packing defect [8].

Since lipid bilayers, as biomimetic membranes, provide a barrier between extracellular and intracellular compartments of a biological cell, the layers have been useful to investigate the various biochemical processes such as cell fusion, exocytosis, and endocytosis. The structural changes of the membranes, caused by the injection of the external reagents, were greatly studied using the release of the encapsulated aqueous contents [9]. Two types of assays are complementary, because the layer fusion is the concomitant mixing of bilayers and aqueous contents. The lipid mixing assay provides the information at the same time for the structural change of the membranes with and without the incorporation of the reagents. Additionally, it must be excluded that the contents merely leakage across the membrane without the change. Therefore, the aqueous-space assay is essential to confirm the change caused by the incorporation [10,11].

The stabilizing effect of trehalose on lipid layers is well documented, but not well understood [12,13]. To gain insight into how this disaccharide interacts with bilayers, monolayers are commonly used to study lipid surfaces in contact with trehalose solution [14-16]. The applicability of these results to cell membranes relies on the equivalence of the two systems in their response to trehalose. Although the headgroup/water interfaces in the two systems may be similar to make monolayers effective bilayer surrogates, a bilayer cannot simply be considered to consist of two independent monolayers [17,18]. In this work, we thus investigated the systematic effect of trehalose on the layer with respect to phase asymmetry on vesicle rupture, including the comparison to that of glucose.


2. MATERIALS AND METHODS

Dipalmitoylphosphatidylcholine (DPPC) Dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidic acid (DPPA), dioleoylphosphatidic acid (DOPA) and trehalose (α-D-glucopyranosyl-α-D-glucopyranoside), ANTS, DPX were purchased from Avanti Polar Lipids (Alabaster, AL) and Sigma Aldrich (St. Louis, MO), respectively. These reagents were used without further purification. The lipids of either DPPA or DOPA were dissolved in 10 mL of tert-butyl methyl ether at 10 mg/mL, followed by adding 100 μL distilled water of 25 mM ANTS, 10 mM Tris-HCl at pH 7.4. Therefore, the micelles with either DPPA or DOPA were prepared by extrusion through the 50 nm pores of 78 mm diameter PTFE membranes above the transition temperature of the desired lipid. Several drops (less than 10 μL) of the micelle solution and tert-butyl methyl ether solution of 10 mg/mL either DPPC or DOPC were continuously added through a 22 gauge needle inserted into the 10 mL aqueous solutions of 90 mM DPX, 10 mM Tris-HCl at pH 7.4, respectively. The final lipid concentration of the aqueous solution was 1 mg/mL. During the addition, the solution was magnetically stirred under the nitrogen stream condition. The vesicle solution was acquired from the supernatant of the solution that underwent through the centrifugation (3700 × g). These procedures are well known as a way to prepare vesicles [19].

The diameter measurements of the micelles and the vesicles were, respectively, conducted using ELS-8000 (Otsuka Electronics Co. Ltd, Osaka, Japan) so that the formation of the vesicles could be confirmed. The diameters of the micelles and the vesicles were 75 ± 10 nm and 80 ± 10 nm. The viscosity and the refractive index of the tert-butyl methyl ether are 0.23 cP and 1.3686, respectively [20]. Besides the measurement of the diameters, no leakage of the ANTS molecules indicated that the structure of each layer was conserved. Otherwise, the fluorescence intensity at 530 nm would be changed tremendously with the addition of several drops of aqueous solutions of 10 mM Tris-HCl at pH 3.0 into the vesicle solution (excitation at 384 nm and emission at 530 nm). The tremendous change in the intensity was observed only with detergent (Tween 20) treatment (Fig. 1). Without the treatment, no change was found after the addition of pH 3 DI water drops. Therefore, the encapsulation was successfully achieved.


Fig. 1. 
Fluorescence intensity change after the addition of pH 3 distilled water drops.

The amount of trehalose or glucose was determined with the desired ratio of trehalose to PC (0, 0.1, 0.3, 0.5, 0.7, and 1.0). The fluorescence intensity was monitored in real time with a Wallac Victor3 multiwell fluorimeter (Perkin-Elmer, Waltham, MA). Because the ANTS has different fluorescence intensities when it is exposed to the DPX, the changes in the intensity of the vesicle solution between with the sugar-solution injection and with buffer-only solution injection means that the structural change in the lipid layers occurs. The fluorescence scale was calibrated with the fluorescence of ANTS vesicles, made with the solid phase of the layers in p-Xylene-bis(N-pyridinium bromide)(DPX)-included-buffer, taken as 100% fluorescence. For 0% of the scale, the well-mixed solution of ANTS and DPX, prepared with the amount used for the vesicle solution, was selected. Therefore, a quantitative observation of the intensity was conducted to investigate the effect of the trehalose on the layer with respect to the phase of the layer, including the comparison to that of the glucose.


3. RESULTS AND DISCUSSION

The phase of the lipid layers was adjusted with the phospholipids whose transition temperature was considered [21]. All of the experiment was performed at room temperature, unless otherwise specified. Dioleoyl lipids were used for the liquid phase since their transition temperature was much lower than room temperature, while dipalmitoyl lipids were for solid at room temperature. Therefore, four types of vesicles were prepared (Fig. 2).


Fig. 2. 
Types of vesicles. Straight line corresponds to solid phase of the lipid layer, and curved line to its liquid phase. (A) Solid phases for both layers. (B) Solid phase for outer layer and liquid phase for inner layer. (C) Liquid phase for outer layer and solid phase for inner layer. (D) Solid phase for both layers.

The fluorescence intensity change from the trehalose injection is listed for each condition in Table 1. The change was different at each phase of the lipid layer. At the solid phase of the outer layer, the intensity was tremendously decreased up to around 0.5 disaccharide molecules per lipid. The change of the intensity at the solid phase of the outer layer seems consistent with the previous researches. It has been suggested that excess sugars were observed for disaccharide of solute:lipid ratios more than 0.5:1 and excluded from the hydration layer near phospholipid headgroups [22]. It has been proposed that the increase in the sugar:lipid ratio led to an increase in the lamellar repeat spacing caused by the trehalose incorporation [23]. The increase was not proportional with the sugar composition. The mechanical property of the layer also showed a similar behavior for the ratios [24].

Table 1. 
Fluorescence intensity for each phase of the vesicle layer after the injection of trehalose and glucose into the vesicle solution
Outer-Layer Solid Phase
Inner-Layer Solid Phase Inner-Layer Liquid Phase
Ratio of Trehalose to Lipid Ratio of Trehalose to Lipid
Fluorescence Intensity (%) 0 0.1 0.3 0.5 0.7 1.0 0 0.1 0.3 0.5 0.7 1.0
100 90 70 50 50 50 70 60 45 35 35 35
Outer-Layer Liquid Phase
Inner-Layer Solid Phase Inner-Layer Liquid Phase
Ratio of Trehalose to Lipid Ratio of Trehalose to Lipid
Fluorescence Intensity (%) 0 0.1 0.3 0.5 0.7 1.0 0 0.1 0.3 0.5 0.7 1.0
70 65 55 45 40 35 30 30 30 30 30 30
Outer-Layer Solid Phase
Inner-Layer Solid Phase Inner-Layer Liquid Phase
Ratio of Glucose to Lipid Ratio of Glucose to Lipid
Fluorescence Intensity (%) 0 0.2 0.6 1.0 1.4 2.0 0 0.2 0.6 1.0 1.4 2.0
100 100 100 100 100 100 70 70 70 70 70 70
Outer-Layer Liquid Phase
Inner-Layer Solid Phase Inner-Layer Liquid Phase
Ratio of Glucose to Lipid Ratio of Glucose to Lipid
Fluorescence Intensity (%) 0 0.2 0.6 1.0 1.4 2.0 0 0.2 0.6 1.0 1.4 2.0
70 60 50 40 35 30 30 25 20 15 10 10

However, at the liquid phase of the outer layer, the intensity behavior was different upon the phase of the inner layer. At both liquid phases of the bilayer, little change was observed. For further investigation, the ANTS-encapsulted-vesicles of both liquid phase layers were monitored in DPX solution with trehalose and without trehalose. The intensity with trehalose was higher than that without trehalose (Fig. 3). This result appears to mean that the trehalose enabled the membrane to be more stable for the liquid phase of the membrane. The previous investigations were supportive for this interpretation [25-27]. The change of the intensity was determined mostly by the phase of the outer layer due to the direction of the exposure to the trehalose, but the phase of the inner layer seem non negligible. The more trehalose molecules were on the layer at the liquid phase of the outer layer with the solid phase of the inner layer, the less stable was the lipid layer. At the liquid phase of the outer layer, the intensity was decreased gradually at the solid phase of the inner layer with the increase in the ratio of the trehalose to the lipid. Moreover, at the solid phase of the outer layer, the intensity was relatively less decreased at the liquid phase of the inner layer than that at the solid phase of the inner layer. From this decrease, it may be claimed that the effect induced by the trehalose incorporation on each layer was interfered by the mismatch between the tail groups of each layer. The dependency between each layer was consistent with the results published before [28].


Fig. 3. 
Fluorescence intensity of both liquid phase layers. (——) Vesicles prepared with the buffer including 1.0 ratio of trehalose to lipid. (- - -) Vesicles prepared with the buffer non-including trehalose.

For the comparison to the effect of the trehalose, the glucose was utilized. Since the trehalose is a dimer of glucose, the identical measurements were repeated at the glucose twice of the trehalose concentration. The intensity behaviors with the glucose were completely different from those with the trehalose. Only a slight decrease of the intensity at even 2:1 ratio of glucose: lipid was observed at the solid phase of the outer layer. However, at the liquid phase of the outer layer, the intensity was gradually decreased with the increase in the ratio of the glucose per the lipid. The results were supported from the previous results of the surface pressure-area isotherm of that the isotherm was little changed only at the solid phase of the layer with the glucose [29]. The fluorescence intensity of 20% below indicated that most of the vesicles lost their initial structure [30]. Therefore, the low intensities at both liquid phases of the layers with 1.0 ratio of the glucose to the lipid may be caused by not only the permeability across the layers but also the loss of the vesicles. All of these results described above seem attributable to the osmotic and volumetric effects on the headgroup packing disruption. This interpretation is supported by the molecular dynamic simulation in which the hydrogen bonding by the trehalose for the liquid phase of the biological membranes promoted the order of the lipid tails [31].

The results from the present study with each phase of the lipid layer appear uniquely consistent with the response that the biological functions are preserved with respect to the transition temperature of the layer. Since the functions can be performed at the liquid phases of both layers, the liquefaction of the layers, led by the trehalose incorporation, may relate to the functions. From the present and previous studies, it is inferred that the trehalose induces the disruption effect on the layer, which cholesterol does.


4. CONCLUSION

In this study, the fluorescence intensity of vesicles was monitored at each phase of the layer. The intensity at the solid phase of the outer layer decreased proportionally to the increase in the trehalose concentration up to 0.5 of trehalose to lipid. In the identical measurements at the solid phase of the outer layer with glucose, just a slight change in the intensity was observed with the increase in the glucose composition from 0% glucose even up to even 2:1 ratio of glucose:lipid. At the liquid phase, the relative change of the intensity was opposite each other of the sugars compared to the solid phase. These results seem attributed to the osmotic and volumetric effect on the headgroup packing disruption. The present study may provide a platform to control biological functions related to cellular processes. Therefore, it would be interesting to investigate the saccharide effect on the behavior of the agent-triggered cells.


Acknowledgments

This study was supported by the Research Program funded by the Seoul National University of Science and Technology. We thank all of the members of Department of Chemical and Biomolecular Engineering at the Seoul National University of Science and Technology for help and valuable discussions. We thank Prof. Heongkyu Ju and Mr. Jisu Kim at the Gachon University for valuable help.


References
1. Crowe, J. H., and L. M. Crowe, (1992), Water and life, p5-7, Springer, Berlin, Germany.
2. Crowe, J. H., L. M. Crowe, and S. A. Jackson, (1983), Preservation of structural and functional activity in lyophilized sarcoplasmic reticulum, Arch. Biochem. Biophys, 220, p477-484.
3. Crowe, L. M., J. H. Crowe, A. Rudolph, C. Womersley, and L. Appel, (1985), Preservation of freeze- dries liposomes by trehalose, Arch. Biochem. Biophys, 242, p240-247.
4. Leslie, S. B., S. A. Teter, L. M. Crowe, and J. H. Crowe, (1994), Trehalose lowers membrane phase transitions in dry yeast cells, Biochim. Biophys. Acta, 1192, p7-13.
5. Carpenter, J. F., L. M. Crowe, and J. H. Crowe, (1987), Stabilization of dry phospholipid bilayers and proteins by sugars, Biochim. Biophys. Acta, 923, p109-115.
6. Lerbret, A., P. Bordat, F. Affouard, A. Hédoux, Y. Guinet, and M. Descamps, (2007), How do trehalose, maltose, and sucrose influence some structural and dynamical properties of lysozyme? Insight from molecular dynamics simulations, J. Phys. Chem. B, 111, p9410-9420.
7. Crowe, L. M., and J. H. Crowe, (1995), Liposomes, New Systems and New Trends in Their Applications, p89-110, Editions de Santé, Paris, France.
8. Lambruschini, C., A. Relini, A. Ridi, L. Cordone, and A. Gliozzi, (2000), Trehalose interacts with phospholipid polar heads in Langmuir monolayers, Langmuir, 16, p5467-5470.
9. Wilschut, J., and D. Papahadjopoulos, (1979), Ca2+-induced fusion of phospholipid vesicles monitored by mixing of aqueous contents, Nature, 281, p690-692.
10. Ellens, H., J. Bentz, and F. C. Szoka, (1984), pH-induced destabilization of phosphatidylethanolamine-containing liposomes: role of bilayer contact, Biochemistry, 27, p1532-1538.
11. Bentz, J., N. Düzgüneş, and S. Nir, (1985), Temperature dependence of divalent cation induced fusion of phosphatidylserine liposomes: Evaluation of the kinetic rate constants, Biochemistry, 24, p1064-1072.
12. Wolkers, W. F., N. J. Walker, Y. Tamari, F. Tablin, and J. H. Crowe, (2003), Towards a clinical application of freeze-dried human platelets, Cell Preservation Technology, 1, p175-188.
13. Karger, S., (1992), Biological Product Freeze-Drying and Formulation, p156-169, In J. C. May, and F. Brown (eds), Developments in Biological Standardization, 74, Publishers, Basel, Switzerland.
14. Crowe, J. H., M. A. Whittam, D. Chapman, and L. M. Crowe, (1984), Interactions of phospholipid monolayers with carbohydrates, Biochim. Biophys. Acta, 769, p151-159.
15. Diaz, S., F. Lairión, J. Arroyo, A. C. Biondi de Lopez, and E. A. Disalvo, (2001), Contribution of phosphate groups to the dipole potential of dimyristoylphosphatidycholine membranes, Langmuir, 17, p852-855.
16. Luzardo M del, C., F. Amalfa, A. M. Nũnez, S. Diaz, A. C. Biondi de Lopez, and E. A. Disalvo, (2000), Effect of trehalose and sucrose on the hydration and dipole potential of lipid bilayers, Biophys. J. , 78, p2452-2458.
17. Nagle, J. F., (1986), Theory of lipid monolayer and bilayer chain-melting phase transitions, Faraday Discuss. Chem. Soc, 81, p151-162.
18. Nagle, J. F., and S. Tristram-Nagle, (2000), Structure of lipid bilayers, Biochim. Biphys. Acta, 1469, p159-195.
19. New, R. R. C., (1990), Liposomes: a practical approach, p20-41, Academic Press, New York, USA.
20. Lide, D. R., (2005), CRC handbook of chemistry and physics: A ready-reference book of chemical and physical data, 85th ed., p220, CRC Press, Boca Raton, USA.
21. Park, J.-W., and D. J. Ahn, (2008), Temperature effect on nanometer-scale physical properties of mixed phospholipid monolayers, Colloids Surf. B: Biointerfaces, 62, p157-161.
22. Westh, P., (2008), Glucose, sucrose and trehalose are partially excluded from the interface of hydrated DMPC bilayers, Phys. Chem. Chem. Phys, 10, p4110-4112.
23. Lennéa., T., C. J. Garveyb, K. L. Kosterc, and G. Bryant, (2010), Kinetics of the lamellar gel-fluid transition in phosphatidylcholine membranes in the presence of sugars, Chem. Phys. Lipids, 163, p236-242.
24. Hur, J., and J.-W. Park, (2015), Trehalose-induced variation in mechanical properties of vesicles in aqueous solution, J. Membrane Biol, 248, p1121-1125.
25. Kapla, J., J. Wohlert, B. Stevensson, O. Engström, G. Widmalm, A. Maliniak, (2013), Molecular dynamics simulations of membranesugar interactions, J. Phys. Chem. B, 117, p6667-6673.
26. Kent, B., T. Hunt, T. A. Darwich, T. Hauß, C. J. Garvey, and G. Bryant, (2014), Localization of trehalose in partially hydrated DOPC bilayers: insights into cryoprotective mechanisms, J. R. Soc. Interface, 11, p20140069.
27. Brüning, B.-A., S. Prévost, R. Stehle, R. Steitz, P. Falus, B. Farago, and T. Hellweg, (2014), Bilayer undulation dynamics in unilamellar phospholipid vesicles: Effect of temperature, cholesterol and trehalose, Biochim. et Biophys. Acta, 1838, p2412-2419.
28. Park, J.-W., (2010), First-leaflet phase effect on properties of phospholipid bilayer formed through vesicle adsorption on LB monolayer, J. Membr. Biol, 237, p107-114.
29. Nakata, S., T. Shiota, N. Kumazawa, and M. Denda, (2012), Interaction between a monosaccharide and a phospholipid molecular layer, Colloids Surf. B: Physicochem. Eng. Aspects, 405, p14-18.
30. Park, J.-W, (2014), Composition effect of the outer layer on the vesicle fusion catalyzed by Phospholipase D, Bull. Korean Chem. Soc, 35, p3509-3513.
31. Pereira, C., and P. H. Hünenberger, (2006), Interaction of the Sugars Trehalose, Maltose and Glucose with a Phospholipid Bilayer: A Comparative Molecular Dynamics Study, J. Phys. Chem. B, 110, p15572-15581.