Colloids and Surfaces A: Physicochem. Eng.

Aspects 238 (2004) 8390

Effect of the hydrophilic surfactants on the preparation and encapsulation efciency in course and ne W/O/W type emulsions
Motohiro Shima , Yohei Kobayashi, Yukitaka Kimura, Shuji Adachi, Ryuichi Matsuno
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan Received 2 July 2003; accepted 26 February 2004 Available online 21 April 2004

Abstract Although the surfactant plays an important role in the preparation of a water-in-oil-in-water (W/O/W) emulsion, its concentration has been determined empirically. We investigated the location of a hydrophilic surfactant in a coarse W/O/W emulsion and a membrane-ltered ne emulsion. At rst, appropriate concentrations of some hydrophilic surfactants to prepare a W/O/W emulsion were investigated from the viewpoints of the median diameter of the oil droplets and the encapsulation efciency of a hydrophilic substance in the inner-water phase. The location of the surfactants and oil was examined for the W/O/W emulsions, prepared using polyglycerol monolaurate as a hydrophilic surfactant in the outer-phase solution at 110% (w/v) and octanoic acid triacylglycerol, which contained hexaglyceryl condensed ricinoleate as an oil-phase surfactant in the oil phase. The median diameter of the oil droplets in the W/O/W emulsion reached a minimum value at 3% (w/v) decaglycerol monolaurate (ML-750), and was constant over 4% (w/v). The excess decaglycerol monolaurate, that was not adsorbed on the oil-water interface was distributed to the outer-water and oil phases. The oil and hydrophobic surfactant were dissolved in the outer-phase solution at high concentrations of the hydrophilic surfactant. These results suggest that the hydrophilic-surfactant concentration appropriate for preparing the W/O/W emulsion would be 3% (w/v) in the outer-phase solution and that the location of the components in the W/O/W emulsion would also be useful in estimating the adequate concentration of the surfactant. 2004 Elsevier B.V. All rights reserved.
Keywords: W/O/W emulsion; Surfactant; Drug delivery system; Functional food; Encapsulation efciency

1. Introduction Practical application of bioactive substances or pharmaceuticals to foods or medication requires an efcient absorption system or a controlled-release system [1]. A water-in-oil-in-water (W/O/W) emulsion, which is an emulsion of oil droplets in which water droplets are included, is a candidate for a protective carrier or controlled-release container for these materials [2,3]. A W/O/W emulsion usually contains hydrophobic and hydrophilic surfactants in the oil and outer-water phases, respectively. A nonionic surfactant with a high HLB value is usually recommended to prevent leakage of the inner phase solution and to stabilize the oil droplets. A commonly used hydrophilic surfactant, such as Tween 20 (polyoxyethylene
Corresponding author. Tel.: +81-75-753-6288; fax: +81-75-753-6285. E-mail address: mshima@kais.kyoto-u.ac.jp (M. Shima). 0927-7757/$ see front matter 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2004.02.018

(20) sorbitan monolaurate) or a polyglycerol ester, consists of a polymer as the hydrophilic component and a fatty acid as the hydrophobic one. Because a commercially available polyglycerol is characterized by the hydroxyl value, it is a mixture of glycerol polymers with various degrees of polymerization. The hydrophilic surfactant, decaglycerol monolaurate (ML-750), used in this study is also a mixture of polyglycerols bound with lauric acid. The surfactant is called decaglycerol monolaurate because the molar ratio of the glycerol residue to lauric acid is about 10. The mixture itself is not always unsuitable for the preparation of emulsions, but it is reported that a mixture of some surfactants exhibits a stabilizing effect, for example, as a co-surfactant, in the preparation of the microemulsion [4]. Such an effect is not obtained by a puried surfactant. The hydrophilic and hydrophobic surfactants are thought to be adsorbed on the surface of the oil droplet and at the interface of the inner-water droplet, respectively. However,

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less attention has been paid to the real location of the surfactants in the W/O/W emulsion. Although many reports have been published on the preparation of the W/O/W emulsion, the relationship between the distribution of surfactants used in the preparation and the stability of the W/O/W emulsion has not been investigated. Therefore, we determined the concentrations of the surfactants in each phase using TLC-FID to discuss the location of the surfactants in the W/O/W emulsion. The stability of the W/O/W emulsion prepared at low surfactant concentrations was also examined.

2.2. Preparation of W/O/W emulsion Selection of a puried water as an outer-phase solution leads to the increased applicable hydrophilic surfactants, but the components of the outer-phase solution in the application elds are some kinds of solution, in many cases, a salt solution. The preparation prole of W/O/W emulsion using a physiological saline as an outer-phase solution would not follow a simple extrapolation from the result of the investigation using puried water as the outer-phase solution, because some of the hydrophilic surfactants act differently according to the ionic strength in the solution. Furthermore, we also planned for the investigation of the transport enhancement effect of the W/O/W emulsion using intestinal epithelial cell culture. The cells under investigation were usually maintained in the physiological saline to prevent the effect of an osmotic pressure. Therefore, we used Hanks solution as an outer-phase solution in consideration of its application. Hanks solution was a physiological saline and was used as the outer-water phase solution. The solution included KCl 0.4 g/l, KH2 PO4 0.06 g/l, NaCl 8 g/l, NaHCO3 0.35 g/ l, Na2 HPO4 0.0475 g/l, glucose 1 g/l, penicillin G 1 105 units/l, streptomycin 0.1 g titer/l, and HEPES 5.9578 g/l, and its pH was adjusted to 7.3 using a 4N NaOH solution. Decaglycerol monolaurate in Table 1 was mainly used as the hydrophilic surfactant. C8TG containing 10% (w/v) hexaglyceryl condensed ricinoleate was used as the oil-phase solution. Distilled water containing 104 M PTSA was used as the inner-water solution. A coarse W/O/W emulsion was prepared through a two-step homogenization using a rotor/stator homogenizer (Physcotron, Microtec, Tokyo, Japan) at 2.2 104 rpm for 2 min in the rst step and at 1.0 104 rpm for 1 min in the second step. A disposable membrane lter cartridge was connected to a disposable syringe, and the coarse W/O/W emulsion was loaded in the syringe [6]. The syringe was then sealed and pressurized by nitrogen gas to lter the coarse W/O/W emulsion to produce a ne W/O/W emulsion. 2.3. Median diameter of oil droplets The particle-size distribution of oil droplets in the W/O/W emulsion was measured with a laser particle-size analyzer

2. Materials and methods 2.1. Materials Decaglycerol monolaurate (SY-Glyster ML-750) was supplied by Sakamoto Yakuhin Kogyo (Osaka, Japan). Hexaglyceryl condensed ricinoleate (Sunsoft 818SX), pentaglycerol monolaurate (Sunsoft A-12E), pentaglycerol monomyristate (Sunsoft A-14E), decaglycerol monomyristate (Sunsoft Q-14S), and lysolecithin (Sunlecithin A) were gifts from Taiyo Kagaku (Yokkaichi, Japan). Octanoic acid triacylglycerol (abbreviated C8TG) was purchased from Sigma (St. Louis, MO, USA). A uorescent marker, 1,3,6,8-pyrenetetrasulfonic acid tetrasodium salt (PTSA, Mw 610.42, [5]), was purchased from Molecular Probes (Eugene, OR, USA). Decaglycerol monolaurate and decaglycerol monomyristate were prepared using mixtures of linear (mainly) and cyclic polyglycerols with different degrees of polymerization. Decaglycerol for the surfactants was designated based on the hydroxyl values of the polyglycerols used in their preparation. Pentaglycerol monolaurate and pentaglycerol monomyristate were manufactured using polyglycerols that contained ca. 60% pentaglycerol as a major component. Disposable membrane lter cartridges, DISMIC 25CS020AN, 25CS045AN and 25CS080AN, which held cellulose acetate membranes with pore diameters of 0.2, 0.45 and 0.8 m, respectively, were purchased from Advantec Toyo (Tokyo, Japan). A disposable syringe with a capacity of 30 ml (SS-30ES) was purchased from Terumo (Tokyo, Japan).
Table 1 HLB and surface excess of the hydrophilic surfactants Trade name Sunsoft A-12E Sunsoft A-14E SY-Glyster ML-750 Sunsoft Q-14S Sunlecithin A
a b

Compound Pentaglycerol monolaurate Pentaglycerol monomyristate Decaglycerol monolaurate Decaglycerol monomyristate Lysolecithin

HLBa 15.6 15.0 15 14.5 12

Surface excessb (108 g/cm2 ) 7.80 4.60 17.0 13.4 8.85

The values were provided by the manufacturers. Surface excess was determined for the interface between Hanks solution without Ca2+ and Mg2+ , and C8TG.

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(SALD-2100, Shimadzu) using 0.1% (w/v) sodium dodecyl sulfate solution as a dispersion medium. The median diameter was evaluated from the volume-based distribution. 2.4. Encapsulation efciency of a marker substance The encapsulation efciency of PTSA in the inner-water phase of a W/O/W emulsion was estimated according to our previous method [6]. The efciency in percentage was dened by (m0 mout )/m0 100, where m0 is the amount of the marker loaded into the inner-water phase and mout is the amount of the marker leaked into the outer-water phase. The PTSA concentration was determined with a Shimadzu RF-1500 spectrouorometer (Kyoto, Japan) at excitation and emission wavelengths of 374 and 404 nm, respectively. 2.5. Measurement of interface tension Interface tension was measured by an automatic surface tension meter (CBVP-A3, Kyowa Interface Science, Tokyo), which employed Wilhelmy method [7]. The instrument uses a clean platinum plate hanged from a precision balance. The plate is touched on the interface or surface of a target solution and the edge of the meniscus of the target solution at the plate pull it down. The power of the gravity plus interface or surface tension is measured by the balance, and the effect of gravity is excluded by initial zero setting. To measure an interface tension on the oil droplet in the W/O/W emulsion, the outer-water phase solution was carefully loaded in a clean sample bottle to avoid babble on the surface, and the oil-phase solution was also carefully loaded on the solution. The platinum plate was immersed in the oil-phase and the buoyant force to the plate in the oil phase was avoided by the setting indicated by the manufacturer. The plate was contacted at the interface of the two solutions. The sample solutions were maintained at 25 C using water jacket for 1 h and then the interface tension was measured. 2.6. Measurement of osmotic pressure Osmotic pressures in the inner- and outer-water phase solutions were measured by an osmometer (OM-802, Vogel, Germany). 2.7. Estimation of outer-phase solution volume

(800 l) were mixed with 400 l of 2 106 M PTSA dissolved in Hanks solution. The mixture is called a sample solution. Aliquots of the emulsion (800 l) were also mixed with 400 l of the Hanks solution not containing PTSA, and the mixture is designated a reference solution A. Hanks solution (800 l) was mixed with 400 l of 2 106 M PTSA in the Hanks solution. The mixture is called reference solution B, which was used for assessment of the handling and apparatus derived errors. The sample solution, reference solutions A and B were gently shaken and stored at 4 C overnight. The oil droplets in the sample solution and reference solution A oated to form cream layers. The outer-water phases of the sample solution and reference solution A were separately withdrawn by a syringe with a needle and were ltered through a disposable lter cartridge equipped with a lter of a pore size of 0.1 m (Millex-VV (SLVVR25LS), Millipore, Bedford, USA). The ltered solutions (400 l) and 400 l of the reference solution B were separately diluted by an addition of 3600 l of distilled water. The PTSA concentrations in the sample and reference A and B solutions, CS , CR1 and CR2 , were determined. The PTSA amounts in the sample and reference A and B solutions are given by (V + VA ) CS , (V + VA ) CR1 and (VH + VA ) CR2 , respectively. V, VH and VA are the volume of the outer-phase solution in the W/O/W emulsion, the volume of the Hanks solution in the reference solution B (800 l) and the volume of the PTSA solution added to the sample solution or the reference solution B, respectively. VA also means the volume of the solution added to the reference solution A, which was the same volume as the solution added to the sample solution and reference solution B, but without PTSA. The sample solution contained PTSA leaked from the inner-water phase and added after emulsication. The reference solution A contained PTSA leaked only from the inner-water phase. The reference solution B contained PTSA only added later as a PTSA solution. Based on the above procedures, the following equation should be considered: (V + VA )CS = (V + VA )CR1 + (VH + VA )CR2 (1)

The use of the reference solution B enabled us to directly estimate the volume of the outer-phase solution, V, by Eq. (2). V = (VH + VA )CR2 VA (CS CR1 ) (2)

2.8. Analysis of components in the outer-water phase Leakage of the inner-water phase to the outer-water phase or incorporation of the outer-water phase into the oil phase results in a change in the volume of the outer-water phase. Because an exact volume of the outer-water phase was required to estimate the location of the hydrophilic surfactant, it was determined as given further. A coarse W/O/W emulsion including PTSA in the inner-water phase was prepared. Aliquots of the emulsion Two W/O/W emulsions, a coarse W/O/W emulsion and a 0.2 m-ltered one, were used in this study. Aliquots of a W/O/W emulsion were centrifuged at 17,600g for 10 min. The lower phase was collected using a syringe with a needle and was ltered with a disposable lter of 0.1 m pore diameter to eliminate the oil droplets. C8TG and the hydrophilic and hydrophobic surfactants in the ltrate were

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60 2 40 1 20

2.9. Statistical analysis

0

1.32
0 5 10 0

Error bars in the gures show the standard deviations of the measurements. Their numbers are shown in the gure captions.

(B) Hydrophilic surfactant concentration [% (w/v)]

Fig. 1. Median diameter of oil droplets in the (A) coarse and (B) membrane-ltered W/O/W emulsions prepared using different hydrophilic surfactants. Symbols, ( ), ( ), ( ), ( ), and (), indicate that pentaglycerol monolaurate, decaglycerol monolaurate, pentaglycerol monomyristate, decaglycerol monomyristate, and lysolecithin were used as the hydrophilic surfactant, respectively. The numbers of measurement are two for pentaglycerol monolaurate, two or one for pentaglycerol monomyristate and lysolecithin, and one for decaglycerol monolaurate and decaglycerol monomyristate.

3. Results and discussion 3.1. Effect of the hydrophilic-surfactant concentration on the median diameter of oil droplets Fig. 1A and B show the median diameters of the coarse and membrane-ltered W/O/W emulsions, respectively. The median diameter decreased along with the increase in the surfactant concentration. The difference in the median diameters of the oil droplets in W/O/W emulsions prepared using different polyglycerol esters shown in Fig. 1A would be insignicant, because of the broad distribution of the diameter of the oil droplets in the coarse W/O/W emulsion as shown in the previous report [6]. The diameters of the oil droplets in the W/O/W emulsions prepared using lysolecithin were much smaller than those of the droplets in the emulsion prepared using polyglycerol esters (Fig. 1B). The median diameter of the oil droplets in the emulsion prepared with lysolecithin also decreased with the increase in the surfactant concentration, but it was difcult to handle at a concentration higher than 2% (w/v) because the solution became viscous. Fig. 2A and B show the distribution of the oil droplet diameters in the W/O/W emulsions prepared at different concentrations of decaglycerol monolaurate and lysolecithin, respectively. The distributions of the oil-droplet diameters in the W/O/W emulsions prepared at 210% (w/v) decaglycerol monolaurate were narrow and the median diameter was smaller at the higher concentration. The distribution

for the emulsion prepared at 1% (w/v) decaglycerol monolaurate was wide and had two peaks. This suggested that the concentration was too low to prepare a W/O/W emulsion. Fig. 2B shows that the oil-droplet distributions for the emulsions prepared using lysolecithin. The distributions for the emulsions prepared at 0.1 to 1% (w/v) were also wide and had two peaks. A membrane ltration at the low hydrophilic-surfactant concentration thus produced a W/O/W emulsion with both relatively small and large oil droplets. The large droplets would be formed by the coalescence of the small droplets. This would be applicable to the emulsion prepared using 1% (w/v) decaglycerol monolaurate shown in Fig. 2A. 3.2. Change in the encapsulation efciency The encapsulation efciency decreased as the surfactant concentration increased (Fig. 3). Decaglycerol and pentaglycerol monolaurates showed relatively low encapsulation efciency compared to pentaglycerol and decaglycerol monomyristates at a high concentration. The difference between the two groups is the hydrophobic acyl-chain length, i.e., the former two surfactants have a 12-carbon-chain

Median diameter for lysolecithin [m]

Median diameter [m]

extracted by mixing the ltrate (500 l) in a mixture of chloroform and methanol (1:1 by vol., 500 l) with a lab-mixer for 30 s. The mixture was centrifuged at 17,600 g for 10 min, and 1 l of the lower layer solution was applied to a chromarod (Dia-iatron, Tokyo, Japan), which is a glass rod coated with silica-gel thin-layer for TLC-FID analysis. The chromarod was rst developed with a mixture of benzene, chloroform and acetic acid (50:20:0.7 by vol.). After the development, the upper half of the rod was analyzed using Iatroscan MK-5 (Dia-iatron) to determine the C8TG concentration. The rod was again developed with a mixture of benzene, chloroform, diethyl ether and acetic acid (50:10:10:0.2 by vol.), and the upper half of the rod was analyzed to determine the concentration of the hydrophobic surfactant, hexaglyceryl condensed ricinoleate. The rod was further developed using a mixture of chloroform and methanol (65:5 by vol.) and the entire rod was analyzed to determine the concentration of the hydrophilic surfactant, decaglycerol monolaurate.

50 Median diameter [m] 40 30 20 10 0

0 5 10 (A) Hydrophilic Surfactant concentration [% (w/v)]

80

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40 Encapsulation efficiency [%]

100 98 96 94 92 90 0.1 1 10 Diameter [m] 100

(A)

Frequency [%]

30

20

10

0
(A)

10

12

Hydrophilic surfactant concentration [% (w/v)]

35 30 Frequency [%] 25 20 15 10 5 0
(B)

100 Encapsulation efficiency [%] 98 96 94 92 90 0.1 1 10 Diameter [m]

Fig. 3. Encapsulation efciency for the (A) coarse and (B) membraneltered W/O/W emulsions prepared using the different hydrophilic surfactants. Symbols, ( ), ( ), ( ), ( ), and (), indicate that pentaglycerol monolaurate, decaglycerol monolaurate, pentaglycerol monomyristate, decaglycerol monomyristate, and lysolecithin were used as the hydrophilic surfactant, respectively. The numbers of measurement are two for pentaglycerol monolaurate, two or one for pentaglycerol monomyristate and lysolecithin, and one for decaglycerol monolaurate and decaglycerol monomyristate.

100

1000

10

12

(B)

Hydrophilic surfactant concentration [% (w/v)]

Fig. 2. The distribution of oil-droplet diameters of the W/O/W emulsions ltered by a 0.2 m pore-size membrane. (A) Symbols, ( ), ( ), (), ( ), and ( ), represent 1, 2, 3, 4, and 10% (w/v) decaglycerol monolaurate, respectively. (B) Symbols, ( ), ( ), ( ), and ( ), represent lysolecithin concentrations of 0.1, 0.5, 1, and 2% (w/v), respectively.

length and the latter ones have a 14-carbon-chain length. The use of surfactants with longer acyl-chains enabled the production of an emulsion with higher encapsulation efciency. The encapsulation efciency of the emulsions was slightly decreased after membrane emulsication (Fig. 3). The encapsulation efciencies of the membrane-ltered emulsions prepared with decaglycerol monolaurate and decaglycerol monomyristate at lower concentrations (14% (w/v)) were lower than those of the coarse emulsions. The encapsulation efciencies of the membrane-ltered emulsions prepared with decaglycerol monolaurate and decaglycerol monomyristate were also lower than those of the other two. These results suggested that the property of the hydrophilic moiety of the surfactant would also affect the encapsulation efciency of the W/O/W emulsions. The encapsulation efciency became constant at the high surfactant concentrations except for pentaglycerol monolaurate. The W/O/W emulsion prepared using lysolecithin showed a sharp decline in the encapsulation efciency with an increase in its concentration. These results suggested that the emulsication method affected the relationship between the types of surfactants and the encapsulation efciency of the W/O/W emulsion. Suzuki et al. [8] reported that the hydrophilicity of the

membrane inuenced the properties of the O/W emulsions produced using a porous glass membrane. The surface excesses of decaglycerol monolaurate and decaglycerol monomyristate were larger than those of the other two polyglycerol esters, as shown in Table 1. The HLB of these surfactants are almost the same, i.e., 15 (decaglycerol monolaurate), 14.5 (decaglycerol monomyristate), 15.6 (pentaglycerol monolaurate), and 15.0 (pentaglycerol monomyristate). The decrement in the encapsulation efciency due to the membrane-emulsication at the low concentration of decaglycerol monolaurate and decaglycerol monomyristate (lower than 4% (w/v)) would be ascribed mainly to the difference in the polyglycerol residue of these surfactants, because their hydrophobic residues are the same as those of pentaglycerol monolaurate and pentaglycerol monomyristate, respectively. The relatively large hydrophilic residues of the decaglycerol monolaurate and decaglycerol monomyristate would interact with the hydrophilic membrane used in the membrane ltration to prepare a ne W/O/W emulsion. The encapsulation efciency of the W/O/W emulsion prepared using lysolecithin, which was more hydrophobic than the other four surfactants (Table 1), was

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Volume of the outer-phase solution [L/mL W/O/W emulsion]

not altered by the membrane emulsication. These results suggested that the encapsulation efciency of the W/O/W emulsion prepared using the high HLB surfactants at a low concentration was more affected by the membrane-ltration than that of the emulsion prepared using the relatively low HLB surfactants. 3.3. Location of surfactant in the W/O/W emulsion An adequate concentration of a surfactant was determined empirically, but the actual location of the surfactant during the preparation process was scarcely investigated. In order to determine the behavior of the surfactant during the process, the location of the surfactant during the preparation of the W/O/W emulsion was investigated. We reported previously [6] that an outer-phase inclusion occurred in the homogenization process. The outer-phase inclusion means the incorporation of the outer-phase solution in the oil droplet during the preparation of a W/O/W emulsion. Wan and Zhang [9] have recently discussed that this phenomenon is ascribed to swelling of the W/O/W emulsion prepared by the liquid membrane method, which is called entrainment swelling [9]. In our preparation procedures, the observation of the W/O/W emulsions using uorescent microscopy showed that the included water droplets diminished during the ltration of the coarse W/O/W emulsion [6]. The outer-phase volume, which was estimated based on the PTSA concentration in the outer-phase solution in this study, increased greatly during the preparation of the coarse W/O/W emulsion. The difference in the osmotic pressure between the outer-water phase (0.533 0.004 osm/kg, n = 3) and inner-water phase (0.005 0.000 osmol/kg, n = 3) would be the main reason for this phenomenon, and the volume of the solution released from the inner-phase to the outer-phase by the osmotic pressure was larger than the volume of the outer-phase solution incorporated in the oil phase (Fig. 4). Based on the outer-phase volume observed above and the following three assumptions, the locations of the surfactants and oil in a W/O/W emulsion were estimated. (1) The outer surface of an oil droplet was fully covered by the hydrophilic surfactant. The residual area per molecule at the interface of Hanks solution and the oil phase was measured by the Wilhelmy method. (2) Distribution of the hydrophilic surfactant to the oil phase was completed during the homogenization period. (3) The volume of the outer-phase incorporated in the oil phase was negligibly small compared with the entire volume of the outer-phase solution after membrane emulsication. Based on these assumptions, the amounts of the hydrophilic surfactant in the outer-water, oil and inner-water phases were estimated from the following equations: m0 = mA + mB + mC + mD m0 = mA + mB + mC (3) (4)

800 700 600 500 400 300

0 2 4 6 8 10 12 Hydrophilic surfactant concentration [% (w/v)]

Fig. 4. Volume of the outer-phase solution of the coarse W/O/W emulsions. A dotted line at 500 l/ml W/O/W emulsion indicates the original outer-phase solution volume and a broken line at 650 l/ml W/O/W emulsion represents the sum of the volume of the outer- and inner-phase solutions. Measurement was triplicated at each concentration, except at 10% (w/v) that was replicated ve times.

m A = Vp C p mA = Va Ca

(5) (6)

where m0 is the amount of decaglycerol monolaurate used in the emulsication, mA is the amount of decaglycerol monolaurate in the outer-phase solution before membrane emulsication, mB is the amount in the oil-phase solution, mC is

Fig. 5. Schematic diagram of the distribution of the hydrophilic ( ) and hydrophobic surfactants ( ) in the W/O/W emulsion. The upper gure represents the location of the surfactants in the coarse W/O/W emulsion, and the lower one represents that of the surfactant in the ne W/O/W emulsion.

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the amount on the surface of the oil droplet, and mD is the amount in the included outer-phase solution (Fig. 5). mA , mB and mC are the amounts of decaglycerol monolaurate in the outer-water and oil phases and on the surface of the oil droplet, respectively, after membrane ltration. According to the assumption (1), mC and mC can be calculated by dividing the surface areas of the oil droplets in the emulsion before and after membrane ltration, respectively, by the residual area per molecule calculated from the surface excess at the interface between outer-water and oil phase solution from Fig. 6 (8.8 105 g/m2 ). From the assumption (2), mB is equal to mB . Therefore, mB can be estimated from mC using Eqs. (4) and (6). mD was estimated from mB and mC based on Eqs. (3) and (5). Fig. 7 shows the concentrations of (A) hexaglyceryl condensed ricinoleate in the outer-phase solution before and after the membrane emulsication, (B) C8TG in the outer-phase solution before and after the membrane emulsication, (C) decaglycerol monolaurate before membrane emulsication, and (D) decaglycerol monolaurate after membrane emulsication, at decaglycerol monolaurate concentrations of 110% (w/v). The concentration of hexaglyceryl condensed ricinoleate in the outer-phase solution slightly increased after the membrane emulsication and almost linearly increased with
0.004

2 Surface tension [mN/m]

0 -12

-10

-8

-6

-4

-2

Decaglycerol monolaurate [log(M)]

Fig. 6. The Symbol ( ) represented the interface tension between a Hanks solution containing indicated decaglycerol monolaurate and C8TG containing 10% (w/v) hexaglyceryl condensed ricinoleate. The dotted line represents the interface tension (1.5 [mN/m]) when the concentration of decaglycerol monolaurate equals zero. The horizontal line represents the interface tension (0.475 [mN/m]) over CMC. The diagonal line represents the tting line, slope is 0.238 [mN/m], intercept is 1.010 [mN/m], with a correlation factor (R2 ) is 0.999. Replication run numbers were one.

the decaglycerol monolaurate concentration (Fig. 7A). The hexaglyceryl condensed ricinoleate, which could be passed through the pores of the 0.1 m membrane, would be solubilized by the decaglycerol monolaurate in the outer-water
0.05
ML-750 [g/mL emulsion]
1.E-03 ML-750 [g/mL emulsion] ML-750 [g/mL emulsion] 8.E-04 6.E-04 4.E-04 2.E-04 0.E+00

Sunsoft 818SX [g/mL emulsion]

0.003

0.04 0.03 0.02 0.01 0

0.002

0.001

10 12

8 10 12

(A)

Decaglycerol monolaurate [% (w/v)]

(C) Decaglycerol monolaurate [% (w/v)]

0.01 0.008 0.006 0.004 0.002 0

0.05
ML-750 [g/mL emulsion]

1.E-03 8.E-04 6.E-04 4.E-04 2.E-04 0.E+00

C8TG [g/mL emulsion]

0.04 0.03 0.02 0.01 0 0 2 4 6

10 12
(D)

8 10 12

(B)

Decaglycerol monolaurate [% (w/v)]

Decaglycerol monolaurate [% (w/v)]

Fig. 7. Effect of the concentration of a hydrophilic surfactant, decaglycerol monolaurate (ML-750), on the concentration of each component in the W/O/W emulsions. Symbols, ( ) and ( ), in A and B represent the concentrations of the hexaglyceryl condensed ricinoleate (Sunsoft 818SX) or C8TG in the outer-phase solution before and after membrane emulsication using a 0.2 m pore-size membrane, respectively. Symbols, ( ), ( ), ( ), and ( ), in C and D show the concentrations of decaglycerol monolaurate in the outer-water phase, oil-phase, outer-phase incorporated into the oil-phase, and on the oil-water interface in the W/O/W emulsion, respectively. Figures (C and D) are for the coarse and membrane-ltered emulsions, respectively. Replication run numbers were from one to three.

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phase. A similar phenomenon was reported by Lezer et al. [10] as a solubilization of the hydrophobic surfactant, which stabilized the oil droplet to prevent swelling breakdown. The solubilization of the hydrophobic surfactant by the hydrophilic one could reduce the concentration of the hydrophobic surfactant in the oil droplet, although the effect seemed to be small in this study because the ratio of the amount of the surfactant solubilized in the outer-phase solution to that presented in the entire system was below ca. 0.9% of the entire hydrophobic surfactant and the osmotic pressure was greater in the outer-phase solution than in the inner-phase solution. The C8TG concentration also increased after the membrane emulsication. The concentration became a minimum value at a decaglycerol monolaurate concentration of 23% (w/v), and the increase in the C8TG concentration after the membrane emulsication was large at the low and high decaglycerol monolaurate concentrations (Fig. 7B). As shown in Fig. 7C, the amount of decaglycerol monolaurate on the oil-droplet surface was very small, and the amount of decaglycerol monolaurate in the outer-phase inclusion was almost constant at concentrations over 2% (w/v). The concentration of decaglycerol monolaurate in the oil phase increased at over 3% (w/v) decaglycerol monolaurate. Fig. 7D shows the location of decaglycerol monolaurate after membrane emulsication. The amount of decaglycerol monolaurate located on the surface of the oil droplet ( ) increased after membrane emulsication, and the concentration in the oil phase was constant because of assumption (2). The amount of the surfactant included into the outer-phase was not considered due to assumption (3). Over the concentration of 23% (w/v) in Fig. 7C and D, the decaglycerol monolaurate partitioned from the outer-water phase to the oil phase and the amount of decaglycerol monolaurate located on the interface between outer-phase solution and oil droplet remained constant. When the surfactant located on the interface was supposed to be effective for stabilization of the emulsion, a lower concentration of decaglycerol monolaurate was sufcient for the preparation of the emulsion. The distribution of the oil droplet diameters was, however, broad in the W/O/W emulsion prepared at 1% (w/v) decaglycerol monolaurate, and the distribution became narrow at decaglycerol monolaurate concentrations higher than 2% (w/v) (Fig. 2). These results suggested that the most effective concentration of decaglycerol monolaurate was 23% (w/v). At concentrations higher than 4% (w/v), the encapsulation efciency and median diameter were almost constant as shown in Figs. 3 and 2, although the amount of decaglycerol monolaurate distributed in the oil phase increased. The distributed decaglycerol monolaurate would form reverse micelles, and the micelles would transport the hydrophilic compound in the inner-water phase to the outer-water phase. Although other factors such as the cost and operationality should be considered in selection of a hydrophilic surfactant, the location of the surfactant would be a criterion in the selection.

4. Conclusions In order to estimate a minimum concentration of hydrophilic surfactant to successfully prepare a W/O/W emulsion, the emulsion was prepared at different concentrations of several polyglycerol fatty acid esters and lysolecithin. The location of the decaglycerol monolaurate was investigated to obtain its optimum concentration. The concentration estimated from the location of the surfactant was in accord with the minimum concentration at which the median diameter and encapsulation efciency of the emulsions remained constant. Concentrations lower than 2% (w/v) were too low to prepare a W/O/W emulsion with a narrow oil-droplet distribution. Distribution of the hydrophilic and hydrophobic surfactants to the oil-phase and outer-water phase became signicant at concentrations higher than 4% (w/v). Acknowledgements This work was supported by a grant-in-aid for scientic research from the Japan Society for the Promotion of Science. The surfactants, SY-Glyster ML-750 and Sunsoft 818SX, were gifts from Sakamoto Yakuhin Kogyo (Osaka, Japan) and Taiyo Kagaku (Yokkaichi, Japan), respectively. References
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