Aggregation Behavior of Hexadecane Emulsions Induced by Egg Yolk

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Aggregation Behavior of Hexadecane Emulsions Induced by Egg Yolk PC Vesicles Bo Yang,† Hideo Matsumura,‡ Hideo Kise,† and Kunio Furusawa*,§ Institute of Material Science, University of Tsukuba, Tsukuba, Ibaraki 305, Japan, Electrotechnical Laboratory, AIST, MITI, Tsukuba, Ibaraki 305, Japan, and Department of Chemistry, University of Tsukuba, Tsukuba, Ibaraki 305, Japan Received September 8, 1999. In Final Form: December 15, 1999 We studied the aggregation behavior of hexadecane droplets after adding phosphatidylcholine (PC) vesicles to the suspension medium by dynamic light scattering electrophoresis, leakage test of fluorescence, and surface tension measurements. The variable factors in these experiments were (i) the concentration of LaCl3 into the bulk, (ii) the concentrations of the PC vesicles, and (iii) the size of the PC vesicles. The experimental results indicated that during the initial stage of adsorption, PC vesicles hold well-integrated into the aggregates and then they disintegrate gradually into a monolayerlike structure, although the latter process is a relatively slow one. It is concluded that the initial aggregation of emulsion droplets after adding PC vesicles is due to the interparticle bridging induced by the adsorbed PC vesicles.

Introduction Phospholipid vesicles have been extensively applied in various fields of cell biology and medicine both as a model for cell membrane systems and as a pharmaceutical carrier. The interactions between vesicles and various materials, such as vesicle-vesicle,1-3 vesicle-proteins,4,5 vesicle-mica surface,6 and vesicle-polymer microspheres,7 are an important subject in these fields, from both a fundamental and an applied point of view. For example, in their use as a drug delivery system, the stability of vesicles has become a very important problem when the vesicles encounter the various materials in vivo, such as ions, proteins, and oily materials. Many studies on the stability of vesicles against these materials have been conducted. However, the study on the interaction between vesicle and oily material has not been well understood. Oily materials usually exist in vivo as an emulsion (oil in water). So, the studies of interaction between vesicles and oil/water interface are required to clarify the stability of vesicles on the emulsion surface. In our previous reports,8,9 as a basic research, the interactions between phosphatidylcholine (PC) vesicles or mixed vesicles containing PC and phosphatidylserine (PS) with flat oil/water interfaces were extensively investigated by measuring the interfacial tension after adding lipid dispersions into the bulk electrolyte solution. We have shown that the adsorption * To correspondence should be addressed. Tel. and Fax: 81 298 53 4426. E-mail: [email protected]. † Institute of Material Science, University of Tsukuba. ‡ AIST, MITI. § Department of Chemistry, University of Tsukuba. (1) Petsev, D. N. Langmuir 1999, 15, 1096. (2) Ssrvazyan, A. P. Annu. Rev. Biophys. Biophys. Chem. 1991, 20, 321. (3) Carmona-Ribeiro, A. M. J. Phys. Chem. 1993, 97, 11843. (4) Giehl, A.; Lemm, T.; Bartelsen, O.; Sandhoff, K.; Blume, A. Eur. J. Biochem. 1999, 261, 650. (5) Tsunegawa, W.; Sano, H.; Sohma, H.; McCormack, F. X.; Voelker, D. R.; Kuroki, Y. Biochim. Biophys. Acta 1998, 1387, 433. (6) Horn, R. G. Biochim. Biophys. Acta 1984, 778, 224. (7) Carmona-Ribeiro, A. M.; Herrington, T. M. J. Colloid Interface Sci. 1993, 156, 19. (8) Yang, B.; Matsumura, H.; Furusawa, K. J. Colloids Surf. A 1999, 148, 191. (9) Yang, B.; Matsumura, H.; Furusawa, K. J. Colloids Surf. B 1999, 14, 161.

behavior of the vesicles on the flat surface depends strongly on the van der Waals and electrostatic forces between them. In this work, the adsorption behavior of PC vesicles on emulsion droplets (O/W) has been studied by investigating the aggregation behavior of emulsion droplets after adding the PC vesicles using colloid chemical techniques, such as electrophoresis, light scattering, surface tension, and fluorescence spectroscopy. Experimental Section Materials. Egg yolk phosphatidylcholine (PC) (Sigma, product number 7318) was purchased from Sigma Chemical Co. Ltd. (U.S.A.). To evaluate the PC molar concentration, the molecular weight of PC was assumed to be 780 on average. Inorganic chemicals (NaCl, MgCl2, LaCl3) and oil (hexadecane) were of analytical reagent grade and were supplied by Wako Pure Chemical Industry (Japan). The fluorophore, 8-Aminonaphthalene-1,3,6-trisulfonic disodium salt (ANTS), and the quencher, p-xylene-bis-pyridinium bromide (DPX), were obtained from Molecular Probes Inc. (U.S.A.). The water used in all the experiments was purified by the Nanopure system and redistilled (Pyrex model still-1, Iwaki Glass Co. Ltd., Japan). Preparation of Vesicles. Unilamellar PC vesicles were prepared by the extrusion method according to Hope et al.10 Briefly, 30 mg of egg yolk PC was dissolved in CHCl3, and it was evaporated in a round-bottomed flask by rotating it and redissolved in diethyl ether. After the ether was evaporated, a lipid film was formed. Water (10 mL) was added to the flask and the mixture was sonicated for 30 s at 150 W. As a result, a multilamellar vesicle (MLV) suspension was obtained. After 5 freeze/thaw cycles, the lipid dispersion was extruded five times through two stacked polycarbonate filters (Nuclepore, Costar, Cambridge, MA) with different pore size (100, 200, 300 nm). The mean diameter of the resultant vesicles was determined by a light-scattering apparatus (Otsuka Elect. ELS-800). In this paper, three types of PC vesicles with different sizes (100 ( 10, 200 ( 10, 300 ( 10 nm) were used. The concentration of PC lipid was analyzed by the Bartlett method.11 Preparation of O/W Emulsions. Hexadecane (0.3 mL) was added to 30 mL of pure water, and the mixture was sonicated for 3 min at 150 W without any surfactants. The obtained emulsion solution was stored at 4-10 °C overnight. The average size of an emulsion droplet was 780 ( 10 nm. The amount of (10) Hope, M. J.; Bally, M. B.; Webb, G.; Cullis, P. K. Biochim. Biophys. Acta 1985, 812, 55. (11) Bartlett, G. R. J. Biol. Chem. 1959, 234, 466.

10.1021/la991190y CCC: $19.00 © 2000 American Chemical Society Published on Web 02/18/2000

Aggregation Behavior of Hexadecane Emulsions emulsion was determined by a total organic carbon analyzer (TOC-500, Shimadzu Co., Japan). The emulsion solution was diluted a to concentration of 1/20 with a electrolyte aqueous solution for aggregation experiment and electrophoresis. Aggregation Measurements. The aggregation behavior of emulsion droplets was investigated by determining the enlarging size ratio (P) of emulsion. P is evaluated by P ) (Dt - D0)/D0. Here, Dt is the mean size of emulsion at time t after adding the PC vesicles to emulsion, D0 is the mean size of emulsion at the starting time (t ) 0). The mean size of emulsion droplets after adding PC vesicles was measured using a dynamic lightscattering apparatus (ELS-800; Otsuka Elect. Co., Japan). The apparatus was modified with a jet device for rapid and controlled mixing of emulsion and PC vesicle dispersion. Electrophoresis. The electrophoretic mobility of the PC vesicles and the oil emulsions was measured under various electrolyte conditions by a microelectrophoretic apparatus (Zeecom; Microtech Nichion. Co., Japan). The ζ-potentials were calculated from the mobility data by means of the O’Brien-White equation.12 The Leakage Test of Fluorescent Dyes. Egg yolk PC was dispersed in a mixed solution of ANTS (12.5 × 10-3 M) and DPX (45 × 10-3 M) by the same procedure as above. The dye-trapped vesicles were prepared by the extrusion method by using a 200 nm pore size nucleapore membrane filter and dialyzed to remove unentrapped dye and quencher with dialysis tubing (cut off MW ) 10 000 Da). The change of fluorescence intensity was measured by a fluorophotometer (F-3010; Hitachi Co., Japan). Details of this method are described in refs 13 and 14. Surface Tension Measurements. Surface tensions were measured by an electronic microbalance (LIBROR EB-50; Shimadzu Co., Japan) with a thin glass plate (24 × 24 × 0.3 mm) in a special glass cell. For each experiment, 50 mL of aqueous solution of 10-4 M LaCl3 was added into the cell at first and then the surface tension was measured to ensure the cleanness of the aqueous surface. The depth of the dipped plate in the solution was kept constant at about 1.0 mm from the aqueous surface, and after that 80 mL of hexadecane was added on it to form the O/W interface. One milliliter of vesicle dispersion, which contained 3.5 mM PC lipid, was injected into the water phase. The water phase was stirred well by a magnetic stirrer. The change in interfacial tension with time was obtained by a strip chart recording of the electrobalance output. The interfacial tension change (∆γ) reported here is defined by ∆γ ) γt - γ0, where γ0 is the initial value of interfacial tension and γt is that of interfacial tension at a time t. The measurements of interfacial tension were conducted three times for each experiment. The ambient temperature was 25 ( 1 °C.

Results and Discussion In view of the nature of the surfaces of PC vesicle and emulsion droplet, the electrostatic force is expected to greatly govern the aggregation behavior of emulsion droplets caused by PC vesicles. Therefore, we have investigated the ζ-potentials of vesicles and emulsions in the aqueous solution of three kinds of electrolytes with various concentrations. In Figure 1a, the PC vesicles showed a negative ζ-potential at very low electrolyte concentration (in pure water, the ζ-potential of PC vesicle is -62.5 mV). This is due to the fact that egg yolk PC sample includes a small amount of acidic lipid impurities.15 The negative ζ-potentials of PC vesicles decreased with increasing electrolyte concentration and reversed to positive ones over certain concentrations for LaCl3 and MgCl2. This is due to the binding of cations to the phospholipid headgroups. (12) O’Brien, R. W.; White, L. R. J. Chem. Soc., Faraday Trans. 1978, 74, 1607. (13) Elens, H.; Bentz, T.; Szoka, F. C. Biochemistry 1985, 24, 3099. (14) Dimitrova, M. N.; Matsumura, H. J. Colloids Surf. B 1997, 8, 287. (15) Matsumura, H.; Mori, F.; Kawahara, K.; Obata, C.; Furusawa, K. J. Colloids Surf., A 1994, 92, 87.

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Figure 1. ζ-potentials of PC vesicles (a) and emulsions (b) vs concentration of electrolyte: (2) NaCl, (9) MgCl2, (b) LaCl3.

According to previous studies,16-19 the cations are adsorbed on phospholipid vesicles surface by an electrostatic attraction, and then, the cations penetrate between the lipid headgroups, and coordination complexes with one or more lipid phosphate groups are formed. Generally, the binding strength of trivalent cations (La3+) to phospholipid is larger than those of the other divalent or monovalent cations (Mg2+, Na+). In the case of emulsion droplets (Figure 1b), the oil/ water interface kept a negative electrical potential (in pure water, the ζ-potential of emulsion is -100 mV), which has been also reported in previous studies. The origin of the negative ζ-potential at the oil/water interface can be explained by the adsorption of OH- to the surface. It can be due to the strong dipole momentum or hydrogen bonding of OH- ions with the hydrogen atoms of the interfacial water molecules.20-21 The strong decrease of the ζ-potential and the reverse change of its sign from negative to positive with increasing concentration of La3+ can be due to the strong adsorption of the trivalent cation at the interface. At first, the effects of concentration and electrolyte type on the aggregation behavior were investigated. As shown in Figure 2, the size of flocs was maximum around 10-4 M of LaCl3; i.e., at 10-4 M of LaCl3 there was a maximum aggregation of emulsion droplets as a function of the PC (16) Lehrmann, R.; Seelig, J. Biochim. Biophys. Acta 1994, 1189, 89. (17) Sundler, R.; Papahadjopoulos, D. Biochim. Biophys. Acta 1981, 649, 743. (18) Hammoudah, M. M.; Nir, S.; Bentz, J.; Stewart, T. P.; Hui, S. W.; Kurland, R. J. Biochim. Biophys. Acta 1981, 645, 102. (19) Hammoudah, M. M.; Nir, S.; Isac, T.; Kornhauser, R.; Stewart, T. P.; Hui, S. W.; Vaz, W. L. C. Biochim. Biophys. Acta 1979, 558, 338. (20) Taylor, A. J.; Wood, F. W. Trans. Faraday Soc. 1957, 53, 523. (21) Marinova, K. G.; Alargova, R. G.; Denkov, N. D.; Velev, O. D.; Petsev, D. N.; Ivanov, I. B.; Borwankar, R. P. Langmuir 1996, 12, 2045.

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Figure 2. Plot of enlarging rate of emulsion size (P) vs LaCl3 concentration at 4 × 10-3 mM PC concentration at 15 min after mixing. Here, a PC vesicle with a diameter of 200 nm was used.

vesicle concentration. However, in the cases of NaCl and MgCl2, a clear aggregation was not observed as anticipated from the ζ-potential measurements. Thus, we selected an optimum condition of aggregation at 10-4 M of LaCl3 for further studies. At this electrolyte concentration, the ζ-potentials of the hexadecane emulsion and PC vesicles were -21.4 and 43.0 mV, respectively. Figure 2 shows the effect of LaCl3 concentration on the emulsion aggregation at 4.0 × 10-3 mM PC, which can be explained by the effects of ζ-potential of PC vesicle and emulsion under different ionic strengths (see Figure 1a,b). At the low LaCl3 concentration (10-6 M), both the PC vesicle and the emulsion have negative ζ-potentials, which indicates that the repulsion force will operate between emulsion droplets and also between emulsion-vesicle particles. With increasing LaCl3 concentration up to the range of 10-4 M, the PC vesicles’ ζ-potential reversed to high positive values and emulsions kept negative ζ-potentials. Then, PC vesicles adsorbed preferentially on the emulsion surface by the electrostatic attraction, and the adsorption induced emulsion aggregation. Over the 10-3 M LaCl3 concentration, however, the PC vesicles and the emulsions possessed positive ζ-potentials, and a repulsive force is expected to operate between the emulsion and PC vesicles. Therefore, the aggregation caused by adsorbed PC vesicle was hindered under this bulk condition, but the homoaggregation of emulsion droplets can occur, because the repulsion force between the emulsion droplets is very weak (see Figure 1b). A set of aggregation experiments were carried out by using three different PC vesicles of different size (d ) 100, 200, and 300 nm) under various PC concentrations at 10-4 M LaCl3. In Figure 3, the enlarging size ratios (P) of emulsion droplets after adding PC vesicles (200 nm) at different PC concentrations are plotted as a function of elapsed time. The similar curves were obtained for 100 and 300 nm vesicle systems. As seen from the figure, the P values depend on the amounts of PC vesicle and there is an optimum amount of PC vesicles for aggregation of the emulsion. To clarify this reason, we plotted the P value at 15 min after adding the vesicles against the PC concentration (see Figure 4). The P value shows a maximum at a certain PC concentration for respective PC vesicles with different sizes. Here, we call this maximum value the “maximum aggregation concentration (MAC)” for the respective PC vesicles. Interestingly, for the three sizes of PC vesicles, the behavior of the P value for the PC concentration showed a similar tendency; only

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Figure 3. Change of enlarging rate of emulsion size (P) vs elapsed time after mixing PC vesicles (d ) 200 nm) with different concentrations under 10-4 M LaCl3 solution: 1, 0 mM; 2, 4 × 10-4 mM; 3, 8 × 10-4 mM; 4, 1.3 × 10-3 mM; 5, 2 × 10-3 mM; 6, 4 × 10-3 mM; 7, 5 × 10-3 mM; 8, 8 × 10-3 mM; 9, 2 × 10-2 mM; 10, 4 × 10-2 mM; 11, 8 × 10-2 mM; 12, 4 × 10-1 mM.

Figure 4. Change of enlarging rate of emulsion size (P) vs concentration of PC vesicles with three different particle sizes at 15 min after mixing under 10-4 M LaCl3 solution: (b) d ) 100 nm, (0) d ) 200 nm, (9) d ) 300 nm.

Figure 5. ζ-potential of emulsion in 10-4 M LaCl3 aqueous solution as a function of PC vesicle concentration with three different vesicle sizes at 15 min after mixing: (b) 100 nm, (O) 200 nm, (2) 300 nm.

the MAC value shifted toward higher PC concentration with increasing vesicle size. Figure 5 shows the ζ-potentials of emulsion droplet after adding three different sized PC vesicles (d ) 100, 200, and 300 nm) under different vesicle concentrations. The ζ-potential of emulsion suspension decreased quickly with increasing PC concentration and reversed to a positive value at a very low PC concentration, indicating that the positively charged PC vesicles by La3+ adsorption adhered strongly on the negatively charged emulsion surface. The data of ζ-potential corresponding to the MAC and the MAC

Aggregation Behavior of Hexadecane Emulsions

Figure 6. Schematic picture of the aggregation mechanism of emulsion droplets after adding PC vesicles: (a) at a low PC vesicle concentration, (b) at a high PC vesicle concentration.

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Figure 7. Fluorescence intensity vs time curves of PC vesicle (d ) 200 nm) dispersion with emulsion (b) and without emulsion (0) at 10-4 M LaCl3.

Table 1. ζ-Potential at MAC and MAC Value for Three Different PC Vesicles at 10-4 M Aqueous Solution of LaCl3 PC vesicle size (nm)

MAC value (mM)

ζ-potential at MAC (mV)

100 200 300

2.2 × 10-3 3.5 × 10-3 8.9 × 10-3

11.0 14.0 21.0

values are summarized in Table 1. As seen from Table 1, the ζ-potentials at MAC are positive and greatly shifted from the zero ζ-potential. This behavior in the last system cannot be explained by a simple charge neutralization mechanism, and the “particle bridging” mechanism of PC vesicles between emulsion droplets must be taken into account. The PC vesicles have positive surface charges on the headgroup of the PC molecule, and the emulsion surface has negative charges at LaCl3 10-4 M, so the attractive force arises between the adsorbed PC vesicle on one emulsion droplet and the bare surface of other emulsion droplet. Under these experimental conditions, the repulsive force between emulsion droplets can be ignored because the value of vesicle diameter (100, 200, and 300 nm) is much larger than the thickness of the electric double layer (k-1) on the emulsion surface under 10-4 M LaCl3 concentrated solution. Here, k is the DebyeHu¨ckel reciprocal length parameter, and the value of k-1 is 12.4 nm.22 The schematic image of “interparticle bridging” is shown in Figure 6. When the PC concentration increases, the number of bridging site increases, which causes a substantial increase in the aggregation behavior. However, when the negative surface charges on the emulsion surface are neutralized gradually and finally reversed to positive sign with the adsorbed PC vesicles, the attractive electrostatic force between the adsorbed PC vesicles and emulsion becomes weak. When adsorption of PC vesicles proceeds over a certain value, the emulsion surface is covered with vesicles and subvesicles (in some cases, vesicle forms are partially destroyed) and shows a positive potential. In this concentration range, the repulsion between the PC vesicles on each emulsion surface becomes predominant over the bridging effect. So, the aggregation is prevented by the electrostatic repulsion. Consequently, there is an optimum PC concentration on the aggregation behavior.

As to the effect of particle size of PC vesicles, the MACs for three sizes of PC vesicles were found to decrease in the order 100 nm < 200 nm < 300 nm (Figure 4 and Table 1). This can be explained by the adsorption rate for different sizes of PC vesicles onto the emulsion surface and the increased number of vesicle particles contributing to “interparticle bridging”. Under the same PC molecular concentration, the vesicle number for three sizes can be evaluated by the Harry method.23 From this method, the number concentration of vesicle particles increases with decreasing particle size. So, the collision probability between small vesicles and emulsion droplets is higher than that for large vesicles. As a result, the small PC vesicle strongly induces emulsion aggregation. The adhesion of PC vesicles to the emulsion surface can lead to two final states: (1) a vesicle monoparticle layer and (2) a lipid molecular monolayer formation at the oil/ water interface. The latter hypothesis was proved by fluorescent, electrophoresis, and surface tension experiments. As shown in Figure 7, the fluorescence intensity (I) increased gradually with the elapsed time in the system with emulsion droplets. On the other hand, no change in the intensity (I) was observed from the system without

(22) Israelachvili, J. N. Intermolecular and Surface Forces; Academic Press: London, 1992; Chapter 12.

(23) Harry. G. E.; Philipps. S. Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 145.

Figure 8. ζ-potential vs elapsed time curves of emulsion at 10-4 M LaCl3 after adding PC vesicles (d ) 200 nm) with different concentrations: (b) 4.05 × 10-3 mM, (O) 4.05 × 10-1 mM.

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Table 2. Adsorption Ratio for Different PC Vesicle Concentrations in 10-4 M LaCl3 Aqueous Solution theoretical number of number of concentration PC vesicles PC vesicles of PC adsorbed adsorbed vesicles (mM) per emulsion per emulsion 4.05 × 10-3 (6.68 × 109)a 4.05 × 10-1 (6.68 × 1011)a a

fraction coverage of adsorbed vesicles (θ)

75

296

0.25

7500

296

1 (over saturation)

The number concentration of PC vesicles (number/mL).

hexadecane emulsion. This implies that the fluorescence dye leaked from the inside of vesicles to the bulk solution, which can be due to the gradual destruction of vesicle structure when the vesicles encountered emulsion droplets. Figure 8 shows the time dependence of ζ-potential of hexadecane emulsion after adding PC vesicles. At low PC concentration (4.05 × 10-3 mM), the ζ-potentials increased rapidly with the elapsed time and reached saturation after 100 min. The saturation value of ζ-potential was very close to the value (26.0 mV) at large PC concentration (4.05 × 10-1 mM). To explain this result, the maximum number of vesicles adsorbed on one emulsion surface was calculated,24 and the vesicle number (N) per emulsion and adsorption ratio (θ) were calculated and are listed in Table 2. From Table 2, the adsorption ratios (θ) of PC vesicles were 0.25 and 1 (over saturation), at 4.05 × 10-3 and 4.05 × 10-1 mM of PC concentration, respectively. Interestingly, the saturation value of ζ-potential possesses the same value for the two different concentrations. This means that the PC vesicles adsorbed on the emulsion surface are collapsed. The collapsing process appears to be relatively slow (about 100 min). Furthermore, from the surface tension experiments (see Figure 9), the destruction of vesicles was also proved. It was confirmed that the decrease in the interfacial tension at the hexadecane/water interface, induced by addition of PC vesicles, was close to the value of a monolayer state of PC molecules at the hexadecane/water interface (∆γ ) -52.0 mJ/m2). These results indicate that the PC vesicles can be destroyed gradually from vesicle form to a monolayerlike structure at the oil/water interface after an encounter with the emulsion droplets (Figure 10). All those data show that the PC vesicles adsorb rapidly on the emulsion surface and are destroyed slowly from the vesicle form to a monolayerlike structure on the emulsion surface. However, the aggregation process is rather rapid, compared with the destroying process of vesicles, so the aggregation is due to vesicle-induced particle bridging. (24) Hansen. K. F.; Matijevic. E. J. Chem. Soc., Faraday Trans 1 1980, 76, 1240.

Figure 9. Change of interfacial tension (∆γ) at the oil/water (10-4 M LaCl3) interface vs elapsed time after adding PC vesicles with different diameters ((b) 100 nm, (9) 200 nm, (O) 300 nm) into the bulk. At saturation, the PC concentration was 7 × 10-2 mM.

Figure 10. Schematic picture of a monolayerlike structure on the emulsion surface formed by PC vesicles.

Conclusions The aggregation of emulsion droplets by adding PC vesicles into the medium was observed within a certain range of LaCl3 concentration, where the PC vesicles and emulsions had opposite charges on the surface. The mechanism of the aggregation of emulsion induced by PC vesicles is as follows. The PC vesicles adsorb on emulsion surfaces by electrostatic attraction. At low PC concentration, the emulsion droplets aggregate by the vesicle-induced “particle-bridging” mechanism owing to the electrostatic attraction between the vesicles and the emulsion surface. At large PC concentration, the emulsion surface is covered with many PC vesicles, so the surface charge of emulsion shifts to a positive one, and, hence, the aggregation is prevented by the electrostatic repulsion. This behavior only occurs in the initial stage, after mixing the vesicle dispersion and emulsion suspension. The PC vesicles, however, undergo gradual destraction on the emulsion surface and form a monolayerlike structure with elapsed time. LA991190Y