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pH and salt-induced reversible aggregation of nonionic synthetic glycolipid vesicles
Colloids and Surfaces A: Physicochemical and Engineering Aspects 207 (2002) 215– 221 www.elsevier.com/locate/colsurfa
pH and salt-induced reversible aggregation of nonionic synthetic glycolipid vesicles Li-Qiang Zheng a,*, Ling-ling Shui a, Qiang Shen a, Gan-Zuo Li a, Teruhiko Baba b, Hiroyuki Minamikawa b, Masakatsu Hato b a
Key Laboratory of Colloid and Interface Chemistry of State Education Ministry, Shandong Uni6ersity, Jinan 250100, China Surface Engineering Laboratory, Department of Polymer Physics, National Institute of Materials and Chemical Research, 1 -1 Higashi, Tsukuba, Ibaraki 305 -8565, Japan
b
Received 20 July 2001; accepted 5 March 2002
Abstract Salt and pH can induce reversible aggregation of vesicles composed of a nonionic synthetic glycolipid, 1,3-di-o-phytanyl-2-o-(b-maltotriosyl) glycerol. The aggregation of the vesicles appears reversible with respect to a change in the pH value of the medium as seen from the reversible change in the turbidity. It was also found that in an acidic region (pH 4–6), the size of the aggregated vesicles are well above 1000 nm, an indication of vesicle aggregation. But in an alkaline region (pH 8–10), the sizes are 110–130 nm, close to their original size of 100– 110 nm, which strongly suggests the reversible disaggregation and also confirms the lack of vesicle fusion. The n-potentials of vesicles are measured in the presence of NaCl with the pH changes of the vesicle suspension. It is seen that the n-potential of vesicles changes with the pH value. The surface charges of the Mal3(Phyt)2 vesicles arise from two independent mechanisms; one is excess ‘adsorption’ of OH− ions at the vesicle– water interfaces and the other is dissociation of hydroxyl groups in a high pH region (pH \11). The changes of the surface charges are thought to be the major factor which induces the aggregation and disaggregation of this nonionic glycolipid vesicle. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Glycolipid; Vesicle; Aggregation; Disaggregation; n-Potential; Surface charge
1. Introduction Most bilayer lipids found in biological systems have two hydrocarbon chains attached to a polar group, which is usually a glycerol derivative. In plants and bacteria, the dominant membrane * Corresponding author E-mail address: [email protected] (L.-Q. Zheng).
lipids are the glycolipids, with monosugar or disugar attached to the glycerol. Lipid bilayers are the basic building units of biological membranes. A planar membrane can curve and generate a reticulum structure or curve cylindrically into microtubules. Abundant biological processes involve transformations between such bilayer structures. Glycolipids are one of the major components of cell membranes and believed to be involved in
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variety of physiological events [1] such as molecular recognition at cell surfaces [2– 5]. It is believed that glycolipids are progressively gaining importance in biology and technology [1,2,6– 9]. But, to our knowledge, very few studies on glycolipid vesicle aggregation have been made so far. Webb, Menikh, Fragata and coworkers have recently investigated a salt-induced aggregation of vesicles of thylakoid galactolipid digalactosyl diglyceride (DGDG) and monogalactosyl diglyceride (MGDG) [10 – 13]. Webb and coworkers found that salt can induce the reversible aggregation of DGDG vesicles and this phenomena was explained in terms of salt-induced change in hydration of the bilayer surfaces that leads to a reduction of hydration repulsion [10]. Fragata and his coworkers later proposed a similar mechanism in which the initial step in DGDG aggregation is an ion-induced decrease in interfacial polarity [13]. In this paper, We have investigated the stability of the vesicles composed of a nonionic glycolipid in the presence of salt and with the changes of pH. It was found that pH and salt can induce the reversible aggregation– disaggregation of the nonionic glycolipid vesicles we have investigated. The mechanism of this process was been researched by means of the n-potential measurement. To our knowledge, the aggregation behaviour of a nonionic synthesized glycolipid vesicles has not been studied before.
2. Materials and methods
2.1. Materials Synthetic glycolipid, 1,3-di-o-phytanyl-2-o-(bmaltotriosyl) glycerol, Mal3(Phyt)2 (Fig. 1) are the
Fig. 1. Chemical structure of the synthetic glycolipid 1,3-di-ophytanyl-2-o-(b-maltotriosyl) glycerol (Mal3(Phyt)2).
same materials described in the previous paper [14,15]. The purity of this glycolipid has been confirmed to be better than 96% by thin-layer chromatography, 1H-NMR and elemental analysis. The glycolipid was stored as a stock solution in CHCl3/CH3OH (2:1 by volume) at − 20 °C. CHCl3 and CH3OH were purchased from Dojindo. They were of a spectroscopic reagent grade and were used without further purification. Water was prepurified with a house made purification system (RO membrane, ion-exchange column and 0.22 mm filter) and further purified with an Elga UHQ unit just before experiments.
2.2. Preparation of glycolipid 6esicles Glycolipid vesicles with uniform size (110 nm in diameter) were prepared by extrusion of frozen– thawed vesicles based on the method described by Mayer et al. [16]. A thin film of Mal3(Phyt)2 was formed on the wall of a test tube by removing CHCl3 in vacuum for at least 24 h. Multilamellar vesicles were obtained by dispersing the dried glycolipid thin film in pure water with vortexing and sonication for 5 min in an USC-1 bath-type sonicator at 65 W output and at room temperature. The resulting vesicles were then frozen in liquid nitrogen and thawed in a water bath at 30 °C. The freeze– thaw cycle was usually repeated five times. The frozen–thawed vesicles were then extruded more than ten times through doubly stacked polycarbonate filters with 0.1 mm pore size (Nucleopore Corp., Pleasanton, CA) by an extruder (Lipex Biomembranes Inc., Vancouver). Polycarbonate filters were extensively washed with pure water beforehand in order to remove amphiphilic polymeric materials. Most of the operations for vesicle preparation were performed in an atmosphere of HEPA filtered air in order to minimize dust contamination. The finally obtained vesicles were judged as unilamellar vesicles by NBD-PE/ dithionite method [17]. Unless otherwise noted, the final concentrations of glycolipid in suspensions were around 0.5 mM for both vesicle aggregation and vesicle size measurements and around 0.1 mM for n-potential measurements, respectively.
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2.3. Turbidity measurements and quasi-elastic light scattering Turbidity readings at 600 nm were obtained on a UVIDEC-320 double beam spectrophotometer using pure water as blanks. Vesicles were diluted to 0.5 mM with pure water just before experiment, then small amounts of concentrated salt solutions were added and A600 were recorded immediately. The pH of suspension was estimated by parallel pH titration measurements in a salt solution on an HM-30S pH meter Vesicle (and aggregated vesicles) diameters were estimated by quasi-elastic light scattering with the ELS-800TS (Photal, Otsuka Electronics). Samples were irradiated by a 5 mV Helium– Neon laser at 632.8 nm and the autocorrelation function used to obtain the mean vesicle diameter as described previously [16].
2.4. n-potential measurement The n-potential of Mal3(Phyt)2 vesicles was measured by an ELS-800TS electrophoretic light scattering apparatus. The cell was made air-tight to prevent the absorption of CO2 into the sample solutions. The concentration of the vesicles was usually adjusted to around 0.1 mM in order to achieve an optimum light scattering intensity. The measurements were usually repeated at least three times and then the averaged values were obtained. Calibration measurements for n-potential were performed with uniform size (0.204 mm in diameter) polystyrene latex beads (Dow Chemical Co.) suspended in a 1.5 mg ml − 1 sodium dodecylsulfate with 10 mM aqueous solution.
3. Results and discussions
3.1. pH-dependent aggregation–disaggregation of Mal3(Phyt)2 6esicles: turbidity and particle sizes Fig. 2 shows values of A600 as functions of pH and NaCl concentrations. When concentrated HCl is added into a vesicle suspension in pure water (A600 = 0.02, pH 5.5– 6.0), the values of A600 increased rapidly at pH 4.5 and reaches a plateau
Fig. 2. Variations of optical density at 600 nm (A600) of Mal3(Phyt)2 vesicle suspensions as functions of pH and NaCl concentrations at 25 °C. (a) 0 mM NaCl, (b) 1 mM NaCl, (c) 5 mM NaCl, (d) 10 mM NaCl, first addition of HCl, (open square), first addition of NaOH (circle), second addition of HCl (triangle), second addition of NaOH (closed square).
value of A600 = 0.45 below pH 4.0 (square in the curve a in Fig. 2). When a concentrated aqueous NaOH was then added to the turbid vesicle suspension (A600 = 0.45, pH 3.5), the values of A600 again started to decrease at around pH 4.5, reverted to the original value of A600 = 0.02 above pH 5.5 and remained practically constant as values of pH further increase (open circle in the curve a). The second titration cycle with HCl and NaOH followed the same curve as that obtained for the first titration cycle. Essentially similar reversible turbidity—pH profiles can be observed with regard to vesicles suspended in an aqueous NaCl. These experiments make clear that there is a threshold pH for the turbidity, which shifts toward higher pH values as NaCl concentration increases (Fig. 2 and Table 1). We obtain an aggregation threshold pH, (pH)th, by extrapolating the rapidly changing portion of the A600 –pH curve to the pH axis.
3.2. Re6ersibility of Mal3(Phyt)2 6esicle aggregation–disaggregation The above results strongly suggest that Mal3(Phyt)2 vesicles show rapid aggregation below (pH)th and disaggregation above (pH)th. In order to confirm this conclusion and examine
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process is reversible and membrane fusion scarcely occurs during this process, except the vesicles in 0 mM NaCl suspensions, ionic strengths were almost constant in the present pH regions. Finally it is noted that the osmotic pressure-induced changes in A600 are not significant even when the ionic strength changed from 0.3 to 12.5 mM (Table 1 and the baseline of the curves in Fig. 2).
whether or not vesicle (membrane) fusion occurs under acidic pH conditions, we have measured the average size and polydispersity of suspended particles during the pH titration cycles in varied NaCl concentrations. The results are summarized in Table 1. The average size of Mal3(Phyt)2 vesicles just after the preparation is about 11095 nm. The polydispersity, which is defined as a measure of the width of the particle size distribution curves obtained from the cumulant analysis, is in all cases well below 0.1, corresponding to a narrow size distribution. In the lower pH region below (pH)th, the average size and polydispersity again exhibit values very close to those of original 110nm size vesicles. Practically identical change in the size and polydispersity of vesicles in the lower and higher pH regions can be observed during the second titration cycle. These results indicate that the pH-dependent aggregation– disaggregation
3.3. Salt-induced the aggregation of Mal3(Phyt)2 6esicle A study of cation-induced aggregation of Mal3(Phyt)2 vesicle has been made by measuring the turbidity of vesicle suspensions. It is found that turbidity changes during the titration of Mal3(Phyt)2 vesicles with various chloride salts shown in Fig. 3. The observed sequence of cation effect on vesicle aggregation is:
Table 1 Effects of NaCl concentration on the aggregation threshold pH and the vesicle aggregation reversibility observed from average diameter and polydipersity of vesicles at 25 °C Aggregation threshold pH
pHa
0.0
4.7 (0.02 mM)
Pure water 3.6 9.1 3.2 11.2
1.0
5.7
5.0
10.0
[NaCl] (mM)
a b
Inonic strengthb (mM)
Average diameter (nM)
Polydispersity
0.0 0.25 0.26 0.89 2.5
111.6 \1500 112.8 \1500 118.8
0.03 0.95 0.03 0.30 0.07
Saline water 3.5 9.1 3.7 11.3
1.0 1.35 1.37 1.55 3.45
107.3 \1500 107.8 \1500 110.5
0.02 0.89 0.03 1.08 0.09
6.2
Saline water 3.5 9.1 3.8 11.2
5.0 5.32 5.33 5.48 7.18
107.3 \1500 108.8 \1500 111.9
0.02 0.62 0.03 0.61 0.08
7.2
Saline water 3.5 9.4 3.6 9.1
10.0 10.35 10.37 10.56 12.46
105.8 \1500 117.2 \1500 118.7
0.04 0.40 0.07 1.30 0.07
Given by the alternate addition (2 cycles) of HCl and NaOH to a vesicle suspension. Actual ionic strength at the aggregation threshold pH.
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properties of distearoyl derivatives of MGDG and DGDG. These authors interpreted their data as indicating that hydrated ions penetrate into the region between adjacent headgroups and cause monolayer expansion. Since these investigators did not report a difference between the efficacies of Na+ and Ca2 + , it is likely that they investigated a different phenomena from that reported here. A direct analogy between these results and ours is, of course, difficult because of the differences in structures of the glycolipids. Clearly, however, glycolipid–ion interactions are extensive. Fig. 3. Variations of optical density at 600 nm (A600) during the sequential addition of chloride salts to 100 nm Mal3(Phyt)2 vesicles dispersed in water. (e) Ca2 + , (a) Cs+, (b) K+, (c) Li+, (d) Na+. Data show the means of three or four replicates from a representative experiment. The measurements were done at neutral pH.
Ca2 + \Cs+ \K+ $Li+ \Na+ For the aggregation of glycolipid vesicles, the most effective cations are those with small hydrated radii or large crystal radii (Ca2 + , Cs+ and K+), while the least effective are those with large hydrated radii or small crystal radii (Na+ and Li+) [18]. The above result fully corresponds with the previous conclusion [18]. It is thought that interactions between approaching membranes in aqueous solutions are dominated by attractive Van der Waals forces, repulsive electrostatic forces, and repulsive hydrostatic forces [19]. Obviously, there is no electrostatic repulsive forces in nonionic glycolipid bilayers, so the major repulsive force preventing Mal3(Phyt)2 vesicle aggregation is the hydration force. Supporting evidence for such a view was obtained by Johnston et al. [20] who observed glycolipid–ion interactions in cerebroside monolayers and concluded that ion-induced changes in water structure could explain observed effects of salts on gluco- and galacto-cerebroside monolayer expansion. Wieslander et al. [21] reported effects of CaCl2 and MgCl2 on the degree of hydration of Acholeplasma laidlawii diglucosyldiacylgycerol as detected by 2H-NMR. Tomoaia-Cotisel et al. [22] have observed effects of salts on the monolayer
3.4. The surface charge of Mal3(Phyt)2 6esicles Fig. 4 shows the n-potential of Mal3(Phyt)2 vesicles as functions of pH and NaCl concentrations. It is seen that the n-potentials of Mal3(Phyt)2 vesicles strongly depend on the values of both pH and ionic strength of the aqueous phase; negative values of the n-potential initially rise as pH increases, and then go through a maximum at pH$10. Finally, they decrease as pH further increases. In the aqueous 10 mM NaCl, a maximum of the n-potential decreases in the pH 6–11 region. Above pH 12, n-potential is approximately constant at around − 40 mV irrespective of NaCl concentrations because the ionic strength is now mainly determined by NaOH rather than by NaCl concentrations. In an acidic region below pH 4, estimation of precise values of
Fig. 4. n-potentials of Mal3(Phyt)2 vesicles as functions of pH and NaCl concentration at 25 °C.
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n-potential becomes difficult on account of the aggregation of Mal3(Phyt)2 vesicles. Nevertheless we have estimated n-potentials of Mal3(Phyt)2 vesicles using highly diluted vesicles (0.03– 0.01 mM lipid) to minimize the effects of large aggregates. It is found that as pH decreases below 4, the decrease in n-potential seems to level off; the n-potentials are about −39 3 mV at pH 3.6 and 09 3 mV at pH 1.6. The n-potential measurements have demonstrated that the Mal3(Phyt)2 vesicle– water interfaces are negatively charged. Though it has been commonly recognized that virtually all solid– liquid (and vapor– liquid) interfaces acquire charges by dissociation or adsorption of ionic constituents of the systems [23], the present observations again raise the question, what is the molecular origin of the interfacial charges at the sugar– water interface? In this context, it is worthwhile to resolve analogous negative charging phenomena observed for nonpolar oil – water and gas bubble– water interfaces both in the presence and in the absence of nonionic surfactants. The common interpretation of the phenomena is that the charges arise from an excess adsorption of hydroxyl ions onto the interface concerned. This hypothesis is mainly based on the reduction of interfacial charges as the solution pH is lowered. This is also the case for the present system. Marinova et al. [24] have critically examined different hypotheses about the origin of interfacial charges of nonpolar oil– water interfaces and concluded that hydroxyl ions were released by the dissociation– association equilibrium of water adsorb at oil– water interface in support for the OH− adsorption hypothesis. Taking account of the pH dependence of the interfacial charges of Mal3(Phyt)2 vesicles, we here assume that interfacial charges of the Mal3(Phyt)2 arise from two independent mechanisms; one is excess adsorption of OH− ions at the vesicle– water interfaces and the other is dissociation of hydroxyl groups of the maltotriose headgroup in a high pH regime (\ pH 11). That is because dissociation of hydroxyl group of sugar groups occurs in high pH conditions: a value of pKa of hydroxyl groups in amylose is estimated as about 13 [25]. Thus the main contribution of the surface
charge comes from the adsorption of OH− group in the system.
4. Conclusions The main conclusions are summarized as follows, 1. Large unilamellar vesicles made from a nonionic synthetic glycolipid, 1,3-di-o-phytanyl-2o-(b-maltotriosyl) glycerol showed the pH-dependent aggregation– disaggregation process; the vesicle aggregation occurs in the lower pH region and the vesicle disaggregation occurs in the higher pH region. This process is almost reversible and the aggregation threshold pH is dependent on NaCl concentration. 2. Large unilamellar vesicles of Mal3(Phyt)2 aggregate strongly in the presence of physiologically relevant levels of aqueous salt solutions.The mechanism in which vesicle aggregation occurs is probably related to the degree of hydration of the bilayer surface and the charging of the vesicle surface. 3. The negative surface charge on apparently nonionic glycolipid vesicle was observed by using the electrophoretic mobility measurements. The value of n-potential was dependent on pH and NaCl concentration. Charge reversal point of vesicle can not be evidently observed between pH 1.6 and 13, which means that proton little adsorb on the vesicle surface in contrast to hydroxyl ion. 4. The adsorption of hydroxyl ions and dissociation of hydroxyl groups of the saccharide moiety of a glycolipid molecule are considered to be the most probable causes of the charging of the vesicle surface.
Acknowledgements We wish to thank to the Natural Sciences Fund Foundation of China (Grant No. 29973023, China) and the New Energy and Technology Development Organization (NEDO, Japan) for financial support.
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References [1] (a) W. Curatolo, Biochim. Biophys. Acta 906 (1987) 111; (b) W. Curatolo, Biochim. Biophys. Acta 906 (1987) 137 references therein. [2] S. Hakomori, Pure Appl. Chem. 63 (1991) 473. [3] L. Eggens, B.A. Fenderson, T. Toyokuni, B. Dean, M.R. Stroud, S. Hakomori, J. Biol. Chem. 264 (1989) 9476. [4] N. Kojima, Trends Glycosci. Glycotechnol. 4 (1992) 491. [5] K. Kates, in: M. Kates (Ed.), Handbook of Lipid Research 6, Glycolipids, Phosphoglycolipids, and Sulfoglycolipids, Plenum Press, New York, London, 1990, pp. 1–122. [6] H.-J. Hinz, L. Six, K.-P. Ruess, M. Lieflander, Biochemistry 24 (1985) 806. [7] R. Koynova, M. Caffrey, Chem. Phys. Lipids 69 (1994) 181 references therein. [8] W. Von Rybinski, Curr. Opin. Coll. Int. Sci. 1 (1996) 587 references therein. [9] F. Nilsson, O. Soderman, I. Johansson, Langmuir 12 (1996) 902. [10] M.S. Webb, C.P.S. Tilcock, B.R. Green, Biochim. Biophys. Acta 938 (1988) 323. [11] M.S. Webb, B.R. Green, Biochim. Biophys. Acta 1030 (1990) 231. [12] A. Menikh, M. Fragata, Eur. Biophys. J. 22 (1993) 249.
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[13] M. Fragata, A. Menikh, S. Robert, J. Phys. Chem. 97 (1993) 13920. [14] Hato, M., Minamikawa, H., Tamada, K., Baba, T., Tanabe, Y., Adv. Coll. Int. Sci., in press. [15] H. Minamikawa, M. Hato, Langmuir 16 (1998) 4503. [16] L.D. Mayer, M.J. Hope, P.R. Cullis, Biochim. Biophys. Acta 858 (1986) 161. [17] H.J. Gruber, H. Schindler, Biochim. Biophys. Acta 1189 (1994) 212. [18] M.S. Webb, C.P.S. Tilcock, B.R. Green, Biochim. Biophys. Acta 938 (1988) 323. [19] J.N. Israelachvilli, in: V. Degiorgio, M. Corti (Eds.), Physics of Amphiphiles: Micelles, Vesicles and Microemulsions, North-Holland, Amsterdam, 1985, pp. 24 – 58. [20] D.S. Johnston, E. Coppard, D. Chapman, Biochim. Biophys. Acta 815 (1985) 325. [21] A. Wieslander, J. Ulmius, G. Lindblom, K. Fontell, Biochim. Biophys. Acta 512 (1978) 241. [22] M. Tomoaia-Cotisel, J. Zsako, E. Chifu, P.J. Quinn, Chem. Phys. Lipids 34 (1983) 55. [23] D.F. Evans, H. Wennerstrom, The Colloidal Domain, VCH Publishers, Inc, New York, 1994. [24] K.G. Marinova, R.G. Alargova, N.D. Denkov, O.D. Velev, D.N. Petsev, I.B. Ivanov, R.P. Borwankar, Langmuir 12 (1996) 2045. [25] H.L. Doppert, A.J. Staverman, J. Polym. Sci. A-1 (4) (1966) 2367 – 2372.
pH and salt-induced reversible aggregation of nonionic synthetic glycolipid vesicles Li-Qiang Zheng a,*, Ling-ling Shui a, Qiang Shen a, Gan-Zuo Li a, Teruhiko Baba b, Hiroyuki Minamikawa b, Masakatsu Hato b a
Key Laboratory of Colloid and Interface Chemistry of State Education Ministry, Shandong Uni6ersity, Jinan 250100, China Surface Engineering Laboratory, Department of Polymer Physics, National Institute of Materials and Chemical Research, 1 -1 Higashi, Tsukuba, Ibaraki 305 -8565, Japan
b
Received 20 July 2001; accepted 5 March 2002
Abstract Salt and pH can induce reversible aggregation of vesicles composed of a nonionic synthetic glycolipid, 1,3-di-o-phytanyl-2-o-(b-maltotriosyl) glycerol. The aggregation of the vesicles appears reversible with respect to a change in the pH value of the medium as seen from the reversible change in the turbidity. It was also found that in an acidic region (pH 4–6), the size of the aggregated vesicles are well above 1000 nm, an indication of vesicle aggregation. But in an alkaline region (pH 8–10), the sizes are 110–130 nm, close to their original size of 100– 110 nm, which strongly suggests the reversible disaggregation and also confirms the lack of vesicle fusion. The n-potentials of vesicles are measured in the presence of NaCl with the pH changes of the vesicle suspension. It is seen that the n-potential of vesicles changes with the pH value. The surface charges of the Mal3(Phyt)2 vesicles arise from two independent mechanisms; one is excess ‘adsorption’ of OH− ions at the vesicle– water interfaces and the other is dissociation of hydroxyl groups in a high pH region (pH \11). The changes of the surface charges are thought to be the major factor which induces the aggregation and disaggregation of this nonionic glycolipid vesicle. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Glycolipid; Vesicle; Aggregation; Disaggregation; n-Potential; Surface charge
1. Introduction Most bilayer lipids found in biological systems have two hydrocarbon chains attached to a polar group, which is usually a glycerol derivative. In plants and bacteria, the dominant membrane * Corresponding author E-mail address: [email protected] (L.-Q. Zheng).
lipids are the glycolipids, with monosugar or disugar attached to the glycerol. Lipid bilayers are the basic building units of biological membranes. A planar membrane can curve and generate a reticulum structure or curve cylindrically into microtubules. Abundant biological processes involve transformations between such bilayer structures. Glycolipids are one of the major components of cell membranes and believed to be involved in
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L.-Q. Zheng et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 207 (2002) 215–221
variety of physiological events [1] such as molecular recognition at cell surfaces [2– 5]. It is believed that glycolipids are progressively gaining importance in biology and technology [1,2,6– 9]. But, to our knowledge, very few studies on glycolipid vesicle aggregation have been made so far. Webb, Menikh, Fragata and coworkers have recently investigated a salt-induced aggregation of vesicles of thylakoid galactolipid digalactosyl diglyceride (DGDG) and monogalactosyl diglyceride (MGDG) [10 – 13]. Webb and coworkers found that salt can induce the reversible aggregation of DGDG vesicles and this phenomena was explained in terms of salt-induced change in hydration of the bilayer surfaces that leads to a reduction of hydration repulsion [10]. Fragata and his coworkers later proposed a similar mechanism in which the initial step in DGDG aggregation is an ion-induced decrease in interfacial polarity [13]. In this paper, We have investigated the stability of the vesicles composed of a nonionic glycolipid in the presence of salt and with the changes of pH. It was found that pH and salt can induce the reversible aggregation– disaggregation of the nonionic glycolipid vesicles we have investigated. The mechanism of this process was been researched by means of the n-potential measurement. To our knowledge, the aggregation behaviour of a nonionic synthesized glycolipid vesicles has not been studied before.
2. Materials and methods
2.1. Materials Synthetic glycolipid, 1,3-di-o-phytanyl-2-o-(bmaltotriosyl) glycerol, Mal3(Phyt)2 (Fig. 1) are the
Fig. 1. Chemical structure of the synthetic glycolipid 1,3-di-ophytanyl-2-o-(b-maltotriosyl) glycerol (Mal3(Phyt)2).
same materials described in the previous paper [14,15]. The purity of this glycolipid has been confirmed to be better than 96% by thin-layer chromatography, 1H-NMR and elemental analysis. The glycolipid was stored as a stock solution in CHCl3/CH3OH (2:1 by volume) at − 20 °C. CHCl3 and CH3OH were purchased from Dojindo. They were of a spectroscopic reagent grade and were used without further purification. Water was prepurified with a house made purification system (RO membrane, ion-exchange column and 0.22 mm filter) and further purified with an Elga UHQ unit just before experiments.
2.2. Preparation of glycolipid 6esicles Glycolipid vesicles with uniform size (110 nm in diameter) were prepared by extrusion of frozen– thawed vesicles based on the method described by Mayer et al. [16]. A thin film of Mal3(Phyt)2 was formed on the wall of a test tube by removing CHCl3 in vacuum for at least 24 h. Multilamellar vesicles were obtained by dispersing the dried glycolipid thin film in pure water with vortexing and sonication for 5 min in an USC-1 bath-type sonicator at 65 W output and at room temperature. The resulting vesicles were then frozen in liquid nitrogen and thawed in a water bath at 30 °C. The freeze– thaw cycle was usually repeated five times. The frozen–thawed vesicles were then extruded more than ten times through doubly stacked polycarbonate filters with 0.1 mm pore size (Nucleopore Corp., Pleasanton, CA) by an extruder (Lipex Biomembranes Inc., Vancouver). Polycarbonate filters were extensively washed with pure water beforehand in order to remove amphiphilic polymeric materials. Most of the operations for vesicle preparation were performed in an atmosphere of HEPA filtered air in order to minimize dust contamination. The finally obtained vesicles were judged as unilamellar vesicles by NBD-PE/ dithionite method [17]. Unless otherwise noted, the final concentrations of glycolipid in suspensions were around 0.5 mM for both vesicle aggregation and vesicle size measurements and around 0.1 mM for n-potential measurements, respectively.
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2.3. Turbidity measurements and quasi-elastic light scattering Turbidity readings at 600 nm were obtained on a UVIDEC-320 double beam spectrophotometer using pure water as blanks. Vesicles were diluted to 0.5 mM with pure water just before experiment, then small amounts of concentrated salt solutions were added and A600 were recorded immediately. The pH of suspension was estimated by parallel pH titration measurements in a salt solution on an HM-30S pH meter Vesicle (and aggregated vesicles) diameters were estimated by quasi-elastic light scattering with the ELS-800TS (Photal, Otsuka Electronics). Samples were irradiated by a 5 mV Helium– Neon laser at 632.8 nm and the autocorrelation function used to obtain the mean vesicle diameter as described previously [16].
2.4. n-potential measurement The n-potential of Mal3(Phyt)2 vesicles was measured by an ELS-800TS electrophoretic light scattering apparatus. The cell was made air-tight to prevent the absorption of CO2 into the sample solutions. The concentration of the vesicles was usually adjusted to around 0.1 mM in order to achieve an optimum light scattering intensity. The measurements were usually repeated at least three times and then the averaged values were obtained. Calibration measurements for n-potential were performed with uniform size (0.204 mm in diameter) polystyrene latex beads (Dow Chemical Co.) suspended in a 1.5 mg ml − 1 sodium dodecylsulfate with 10 mM aqueous solution.
3. Results and discussions
3.1. pH-dependent aggregation–disaggregation of Mal3(Phyt)2 6esicles: turbidity and particle sizes Fig. 2 shows values of A600 as functions of pH and NaCl concentrations. When concentrated HCl is added into a vesicle suspension in pure water (A600 = 0.02, pH 5.5– 6.0), the values of A600 increased rapidly at pH 4.5 and reaches a plateau
Fig. 2. Variations of optical density at 600 nm (A600) of Mal3(Phyt)2 vesicle suspensions as functions of pH and NaCl concentrations at 25 °C. (a) 0 mM NaCl, (b) 1 mM NaCl, (c) 5 mM NaCl, (d) 10 mM NaCl, first addition of HCl, (open square), first addition of NaOH (circle), second addition of HCl (triangle), second addition of NaOH (closed square).
value of A600 = 0.45 below pH 4.0 (square in the curve a in Fig. 2). When a concentrated aqueous NaOH was then added to the turbid vesicle suspension (A600 = 0.45, pH 3.5), the values of A600 again started to decrease at around pH 4.5, reverted to the original value of A600 = 0.02 above pH 5.5 and remained practically constant as values of pH further increase (open circle in the curve a). The second titration cycle with HCl and NaOH followed the same curve as that obtained for the first titration cycle. Essentially similar reversible turbidity—pH profiles can be observed with regard to vesicles suspended in an aqueous NaCl. These experiments make clear that there is a threshold pH for the turbidity, which shifts toward higher pH values as NaCl concentration increases (Fig. 2 and Table 1). We obtain an aggregation threshold pH, (pH)th, by extrapolating the rapidly changing portion of the A600 –pH curve to the pH axis.
3.2. Re6ersibility of Mal3(Phyt)2 6esicle aggregation–disaggregation The above results strongly suggest that Mal3(Phyt)2 vesicles show rapid aggregation below (pH)th and disaggregation above (pH)th. In order to confirm this conclusion and examine
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process is reversible and membrane fusion scarcely occurs during this process, except the vesicles in 0 mM NaCl suspensions, ionic strengths were almost constant in the present pH regions. Finally it is noted that the osmotic pressure-induced changes in A600 are not significant even when the ionic strength changed from 0.3 to 12.5 mM (Table 1 and the baseline of the curves in Fig. 2).
whether or not vesicle (membrane) fusion occurs under acidic pH conditions, we have measured the average size and polydispersity of suspended particles during the pH titration cycles in varied NaCl concentrations. The results are summarized in Table 1. The average size of Mal3(Phyt)2 vesicles just after the preparation is about 11095 nm. The polydispersity, which is defined as a measure of the width of the particle size distribution curves obtained from the cumulant analysis, is in all cases well below 0.1, corresponding to a narrow size distribution. In the lower pH region below (pH)th, the average size and polydispersity again exhibit values very close to those of original 110nm size vesicles. Practically identical change in the size and polydispersity of vesicles in the lower and higher pH regions can be observed during the second titration cycle. These results indicate that the pH-dependent aggregation– disaggregation
3.3. Salt-induced the aggregation of Mal3(Phyt)2 6esicle A study of cation-induced aggregation of Mal3(Phyt)2 vesicle has been made by measuring the turbidity of vesicle suspensions. It is found that turbidity changes during the titration of Mal3(Phyt)2 vesicles with various chloride salts shown in Fig. 3. The observed sequence of cation effect on vesicle aggregation is:
Table 1 Effects of NaCl concentration on the aggregation threshold pH and the vesicle aggregation reversibility observed from average diameter and polydipersity of vesicles at 25 °C Aggregation threshold pH
pHa
0.0
4.7 (0.02 mM)
Pure water 3.6 9.1 3.2 11.2
1.0
5.7
5.0
10.0
[NaCl] (mM)
a b
Inonic strengthb (mM)
Average diameter (nM)
Polydispersity
0.0 0.25 0.26 0.89 2.5
111.6 \1500 112.8 \1500 118.8
0.03 0.95 0.03 0.30 0.07
Saline water 3.5 9.1 3.7 11.3
1.0 1.35 1.37 1.55 3.45
107.3 \1500 107.8 \1500 110.5
0.02 0.89 0.03 1.08 0.09
6.2
Saline water 3.5 9.1 3.8 11.2
5.0 5.32 5.33 5.48 7.18
107.3 \1500 108.8 \1500 111.9
0.02 0.62 0.03 0.61 0.08
7.2
Saline water 3.5 9.4 3.6 9.1
10.0 10.35 10.37 10.56 12.46
105.8 \1500 117.2 \1500 118.7
0.04 0.40 0.07 1.30 0.07
Given by the alternate addition (2 cycles) of HCl and NaOH to a vesicle suspension. Actual ionic strength at the aggregation threshold pH.
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properties of distearoyl derivatives of MGDG and DGDG. These authors interpreted their data as indicating that hydrated ions penetrate into the region between adjacent headgroups and cause monolayer expansion. Since these investigators did not report a difference between the efficacies of Na+ and Ca2 + , it is likely that they investigated a different phenomena from that reported here. A direct analogy between these results and ours is, of course, difficult because of the differences in structures of the glycolipids. Clearly, however, glycolipid–ion interactions are extensive. Fig. 3. Variations of optical density at 600 nm (A600) during the sequential addition of chloride salts to 100 nm Mal3(Phyt)2 vesicles dispersed in water. (e) Ca2 + , (a) Cs+, (b) K+, (c) Li+, (d) Na+. Data show the means of three or four replicates from a representative experiment. The measurements were done at neutral pH.
Ca2 + \Cs+ \K+ $Li+ \Na+ For the aggregation of glycolipid vesicles, the most effective cations are those with small hydrated radii or large crystal radii (Ca2 + , Cs+ and K+), while the least effective are those with large hydrated radii or small crystal radii (Na+ and Li+) [18]. The above result fully corresponds with the previous conclusion [18]. It is thought that interactions between approaching membranes in aqueous solutions are dominated by attractive Van der Waals forces, repulsive electrostatic forces, and repulsive hydrostatic forces [19]. Obviously, there is no electrostatic repulsive forces in nonionic glycolipid bilayers, so the major repulsive force preventing Mal3(Phyt)2 vesicle aggregation is the hydration force. Supporting evidence for such a view was obtained by Johnston et al. [20] who observed glycolipid–ion interactions in cerebroside monolayers and concluded that ion-induced changes in water structure could explain observed effects of salts on gluco- and galacto-cerebroside monolayer expansion. Wieslander et al. [21] reported effects of CaCl2 and MgCl2 on the degree of hydration of Acholeplasma laidlawii diglucosyldiacylgycerol as detected by 2H-NMR. Tomoaia-Cotisel et al. [22] have observed effects of salts on the monolayer
3.4. The surface charge of Mal3(Phyt)2 6esicles Fig. 4 shows the n-potential of Mal3(Phyt)2 vesicles as functions of pH and NaCl concentrations. It is seen that the n-potentials of Mal3(Phyt)2 vesicles strongly depend on the values of both pH and ionic strength of the aqueous phase; negative values of the n-potential initially rise as pH increases, and then go through a maximum at pH$10. Finally, they decrease as pH further increases. In the aqueous 10 mM NaCl, a maximum of the n-potential decreases in the pH 6–11 region. Above pH 12, n-potential is approximately constant at around − 40 mV irrespective of NaCl concentrations because the ionic strength is now mainly determined by NaOH rather than by NaCl concentrations. In an acidic region below pH 4, estimation of precise values of
Fig. 4. n-potentials of Mal3(Phyt)2 vesicles as functions of pH and NaCl concentration at 25 °C.
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n-potential becomes difficult on account of the aggregation of Mal3(Phyt)2 vesicles. Nevertheless we have estimated n-potentials of Mal3(Phyt)2 vesicles using highly diluted vesicles (0.03– 0.01 mM lipid) to minimize the effects of large aggregates. It is found that as pH decreases below 4, the decrease in n-potential seems to level off; the n-potentials are about −39 3 mV at pH 3.6 and 09 3 mV at pH 1.6. The n-potential measurements have demonstrated that the Mal3(Phyt)2 vesicle– water interfaces are negatively charged. Though it has been commonly recognized that virtually all solid– liquid (and vapor– liquid) interfaces acquire charges by dissociation or adsorption of ionic constituents of the systems [23], the present observations again raise the question, what is the molecular origin of the interfacial charges at the sugar– water interface? In this context, it is worthwhile to resolve analogous negative charging phenomena observed for nonpolar oil – water and gas bubble– water interfaces both in the presence and in the absence of nonionic surfactants. The common interpretation of the phenomena is that the charges arise from an excess adsorption of hydroxyl ions onto the interface concerned. This hypothesis is mainly based on the reduction of interfacial charges as the solution pH is lowered. This is also the case for the present system. Marinova et al. [24] have critically examined different hypotheses about the origin of interfacial charges of nonpolar oil– water interfaces and concluded that hydroxyl ions were released by the dissociation– association equilibrium of water adsorb at oil– water interface in support for the OH− adsorption hypothesis. Taking account of the pH dependence of the interfacial charges of Mal3(Phyt)2 vesicles, we here assume that interfacial charges of the Mal3(Phyt)2 arise from two independent mechanisms; one is excess adsorption of OH− ions at the vesicle– water interfaces and the other is dissociation of hydroxyl groups of the maltotriose headgroup in a high pH regime (\ pH 11). That is because dissociation of hydroxyl group of sugar groups occurs in high pH conditions: a value of pKa of hydroxyl groups in amylose is estimated as about 13 [25]. Thus the main contribution of the surface
charge comes from the adsorption of OH− group in the system.
4. Conclusions The main conclusions are summarized as follows, 1. Large unilamellar vesicles made from a nonionic synthetic glycolipid, 1,3-di-o-phytanyl-2o-(b-maltotriosyl) glycerol showed the pH-dependent aggregation– disaggregation process; the vesicle aggregation occurs in the lower pH region and the vesicle disaggregation occurs in the higher pH region. This process is almost reversible and the aggregation threshold pH is dependent on NaCl concentration. 2. Large unilamellar vesicles of Mal3(Phyt)2 aggregate strongly in the presence of physiologically relevant levels of aqueous salt solutions.The mechanism in which vesicle aggregation occurs is probably related to the degree of hydration of the bilayer surface and the charging of the vesicle surface. 3. The negative surface charge on apparently nonionic glycolipid vesicle was observed by using the electrophoretic mobility measurements. The value of n-potential was dependent on pH and NaCl concentration. Charge reversal point of vesicle can not be evidently observed between pH 1.6 and 13, which means that proton little adsorb on the vesicle surface in contrast to hydroxyl ion. 4. The adsorption of hydroxyl ions and dissociation of hydroxyl groups of the saccharide moiety of a glycolipid molecule are considered to be the most probable causes of the charging of the vesicle surface.
Acknowledgements We wish to thank to the Natural Sciences Fund Foundation of China (Grant No. 29973023, China) and the New Energy and Technology Development Organization (NEDO, Japan) for financial support.
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