Effects of Emulsifiers on Crystallization Behavior of Lipid Crystals in

Dec 6, 2005 - ABSTRACT: We observed the crystallization and polymorphic transformation processes of palm stearin (PS) dispersed in nanometer-...
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Effects of Emulsifiers on Crystallization Behavior of Lipid Crystals in Nanometer-Size Oil-in-Water Emulsion Droplets T.

Sonoda,†,‡

Y.

Takata,†

S.

Ueno,†

and K.

Sato*,†

Graduate School of Biological Science, Hiroshima UniVersity, Higashi-Hiroshima, Hiroshima 739-8528, Japan, and Pharmaceutics Department Laboratories, Tanabe Seiyaku Co., Ltd., Kashima-Yodogawa-ku, Osaka 532-8505, Japan

CRYSTAL GROWTH & DESIGN 2006 VOL. 6, NO. 1 306-312

ReceiVed February 3, 2005; ReVised Manuscript ReceiVed September 13, 2005

ABSTRACT: We observed the crystallization and polymorphic transformation processes of palm stearin (PS) dispersed in nanometersize oil-in-water emulsion droplets (nanometer-size droplets, average diameter 120 ( 30 nm) using DSC and in-situ synchrotron radiation X-ray diffraction (SR-XRD) techniques. The nanometer-size emulsion droplets were prepared by using four types of highly hydrophilic polyglycerine fatty-acid mono-esters, in which a polar moiety was made of 10 polymerized glycerins (decaglycerine, 10G) and nonpolar moieties were lauric, myristic, palmitic, and stearic fatty acids. When decaglycerin monolaurate (10G1L) was employed, crystallization temperatures (Tc’s) of high-melting and low-melting fractions of PS were 8 and 2 °C, which were decreased from 31 and 3 °C for the bulk PS. However, the Tc of the high-melting fraction of PS was increased as the fatty-acid moiety was changed to myristic (10G1M), palmitic (10G1P), and stearic (10G1S) acids; in particular, the Tc became 38 °C with 10G1S. Furthermore, the crystallization of the high-melting fraction with 10G1M, 10G1P, and 10G1S exhibited only wide-angle SR-XRD patterns without small-angle SR-XRD patterns, indicating that these crystals formed thin films in the nanometer-size emulsion droplets that did not diffract small-angle SR-XRD patterns. We did not observe these features in our previous work on PS crystallization in bulk and micrometer-size emulsion droplets (Sonoda, et al. J. Am. Oil Chem. Soc. 2004, 81, 365). It was assumed that the freezing of a high-melting emulsifier may act as a template for nucleation of a high-melting fraction of PS, whose effects are more remarkable in nanometer-size emulsion than in micrometer-size emulsion, due to the tight packing of the hydrophobic region of the interfacial emulsifier membrane of the nanometer-size emulsion. Introduction Polymorphism and crystallization of lipids in an emulsion state have attracted much interest in pharmaceuticals, foods, cosmetics, and agrochemicals. In pharmaceuticals, the oil-inwater (O/W) emulsion is used for the improvement of bioavailability and for controlled release and targeting of moderately water-soluble drugs, and lipid particles with a nanometer size have recently been investigated with a particular emphasis on the controlled release of lipophilic drugs.1-4 The O/W emulsions with lipid particle diameters of less than 200 nm (nanometersize emulsion) have been considered as drug delivery systems for parenteral,5 oral,6 topical,7 and transdermal8 drug administrations. This is because the increasing importance of biotechnologically prepared not only water-insoluble lipophilic drugs but also oil-insoluble drugs has been recognized, and these drug materials may be solubilized and delivered in nanometer-size O/W emulsion droplets.1 Physical properties of O/W emulsion droplets are tightly related to release control of drug materials solubilized in the emulsion droplets.9-11 When the oil phase of an emulsion is in a liquid state, release of the drug from the emulsion is not readily controlled because the drug release rate is too high. In contrast, when the oil phase of an emulsion contains high-melting lipid materials, the rate of drug release can be controlled because the rate of mass transfer of the drug materials incorporated in the crystalline or semicrystalline phases is reduced compared with the liquid phase.1 Furthermore, the drug release has been modified through polymorphic studies of lipid crystals2,9-11 and by the melting * To whom correspondence should be addressed. Phone number: +81824-24-7935. Fax number: +81-824-24-7910. E-mail: [email protected]. † Hiroshima University. ‡ Tanabe Seiyaku Co., Ltd.

behavior of semisolid lipids composed of low-melting and highmelting fractions.12,13 Therefore, polymorphic crystallization of lipids in nanometer-size O/W emulsion droplets is of great significance. Many researchers have studied the factors affecting lipid crystallization properties in the emulsion droplets, and their results have been summarized in recent reviews.14-16 The following may be pointed out as the main factors: influences of emulsion droplet size, effect of emulsifiers, droplet-droplet interactions, effects of additives present in the oil phase, fat polymorphism, and subsequent temperature history. Basic research on the nanometer-size O/W emulsion used simply shaped triacylglycerols (TAGs).17-19 In this work, we used palm stearin (PS) as the model lipid dispersed in the micrometer-size emulsion. We prepared the PS by fractionating the high-melting part of palm oil. Due to the multicomponent property of PS, melting temperatures ranged from -5 to 53 °C and crystallization temperatures ranged from 3 to 31 °C in the bulk state. Both ranges are wider than those of palm oil and palm mid-fraction (PMF). We expect that this property will provide opportunities for using PS in solid-lipid drug-delivery particles. In our previous work, we examined polymorphic crystallization and transformation of PS in bulk and micrometer-size emulsion droplets.20 In this study, we examined the crystallization kinetics of PS in the nanometersize emulsion using different types of emulsifiers. Bunjes demonstrated that emulsifiers affect the physical properties of lipids in nanometer-size emulsion remarkably, using different types of emulsifiers with polar and nonpolar moieties.21 Sakamoto et al. showed that adding hydrophobic polyglycerine fattyacid esters accelerated the crystallization of PMF in micrometersize emulsion, in which acceleration was more enhanced with increasing hydrophobicity of the emulsifier and increasing melting point of the fatty-acid chains of the emulsifiers.22

10.1021/cg050045h CCC: $33.50 © 2006 American Chemical Society Published on Web 12/06/2005

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Figure 1. Size distribution of nanometer-size emulsion of PS using 10G1L.

Therefore, the present study employed four types of polyglycerine fatty-acid esters with the same polar group but different saturated fatty-acid chains for the emulsification. Materials and Methods PS was provided by Fuji Oil Co., Ltd (Osaka Japan), and its fatty acid compositions and main triacylglycerol components are provided in a previous report.20 In brief, the major fatty acids are palmitic (∼55%), stearic (∼5%), oleic (∼32%), and linoleic (∼7%), which comprise different types of TAGs forming high-melting and low-melting fractions. The two fractions revealed different melting, crystallization, and polymorphic transformation behaviors in both bulk and micrometersize emulsion droplets, as precisely examined in our previous work.20 We employed four types of polyglycerol fatty-acid esters in the formation of the O/W emulsion droplets: decaglycerin-mono-stearic acid ester (10G1S), decaglycerin-mono-palmitric acid ester (10G1P), decaglycerin-mono-myristric acid ester (10G1M), and decaglycerinmono-lauric acid ester (10G1L), provided by Sakamoto Pharmaceuticals Co., Ltd (Osaka, Japan). A polar group of the four emulsifiers was made of 10 polymerized glycerin molecules, and the average degree of esterification of the fatty acids is 1. Therefore, the four emulsifiers are highly hydrophilic. Nanometer-sized O/W emulsion droplets (20 wt % oil phase, 10 wt % emulsifier, and 70 wt % distilled water phase) were prepared by the following two-stage method. In the first stage, the three components were mixed using a homogenizing mixer (pre-emulsification). The second stage was performed by using a Debee 2000 (Bee International, Inc., America) at 30 000 psi for 5 min. The average droplet size was measured by using a laser particle-size analyzer (ELS 8000, Otsuka Electric, Osaka, Japan). Figure 1 depicts the distribution of the droplet diameters with an average value of 120 ( 30 nm for emulsions prepared with 10G1L. The average diameter of emulsion droplets using 10G1S, 10G1P, and 10G1M were also 120 ( 30 nm (data not shown). We conducted the DSC experiments using DSC-8230 (Rigaku, Tokyo, Japan). The sample was sealed in an aluminum pan, and Al2O3 was used as a reference material. A fixed rate (2 °C/min) of cooling and heating was applied. Samples were cooled from 60 to -5 °C, held for 10 min at -5 °C, and heated to 60 °C. Time-resolved in situ smallangle and wide-angle X-ray diffraction using synchrotron radiation (SRSAXS/WAXS) were carried out at the Photon Factory (beam lines 15A and 9C) in the KEK Institute, Tsukuba, Japan. The methods of the SR-SAXS/WAXS experiments are fully described elsewhere.20 The temperature program of the SR-SAXS/WAXS consisted of heating from room temperature to 60 °C, cooling from 60 to -5 °C, and then heating again from -5 to 60 °C, all at a constant rate of 2 °C/min. The samples (∼24 mg) were placed in a 4 × 4 × 1.5 mm3 cell. To confirm the stability of the nanometer-size PS emulsion droplets under the heating-cooling-reheating conditions, we placed the sample in a glass ampule, stored it at -5 °C for 4 h, and then heated it to 60 °C before visual observation at room temperature.

Results Figure 2 illustrates the DSC cooling and heating thermo peaks of crystallization, polymorphic transformation, and melting of

Figure 2. DSC cooling and heating thermopeaks of nanometer-size PS emulsion.

PS in the nanoemulsions prepared with each of the four emulsifiers. When 10G1L was employed, the first crystallization occurred at 8 °C as expressed in a large exothermic peak, and the second crystallization was observed around 2 °C. The former and the latter were due to the crystallization of the high-melting and low-melting fractions of PS, which also exhibited a similar splitting of DSC exothermic peaks in the micrometer-size emulsion.20 On heating, a broad exothermic peak appeared around 20 °C, and broad endothermic peaks appeared between 35 and 45 °C. A broad endothermic peak around -5 °C was also detectable on cooling, but this peak was due to the melting of the very low-melting fraction of PS, which was disregarded in the present study. When 10G1M, 10G1P, and 10G1S were employed for emulsification, the following remarkable changes in the cooling DSC patterns were detectable: (a) The crystallization temperature (Tc) of the high-melting fraction of PS increased from 8 °C (10G1L) to 19 °C (10G1M), 28 °C (10G1P), and 36 °C (10G1S), as expressed by the exothermic peak highest temperatures. (b) Tc of the low-melting fraction decreased from 2 °C (10G1L) to -2 °C (10G1M, 10G1P, and 10G1S). (c) The DSC patterns of the crystallization of the high-melting fraction became broader as the emulsifier varied from 10G1L to 10G1S. In the heating process, it was observed that no major change was detectable in the melting behavior of PS among the nanometer-sized emulsion particles prepared with the four emulsifiers. However, the broad exothermic peak at ∼20 °C (10G1L) changed to ∼22 °C (10G1M), ∼24 °C (10G1P), and ∼26 °C (10G1S). This result indicated that the temperature of the exothermic peak appearing during the heating process increased as the fatty-acid chain length of the emulsifier increased. Visual observation confirmed the stability of the nanoemulsion of PS during the thermal thawing process between 60, -5, and 60 °C, in which PS was crystallized and melted in the droplets. It was confirmed that all emulsions using the four emulsifiers assumed a liquid state after cooling to -5 °C for 4 h and after reheating to 60 °C. From these results, it was obvious that gelation of the crystallized PS particles due to droplet coalescence and oil-water separation did not occur during the thermal thawing process. Figure 3 shows the SR-XRD patterns of the nanometer-size emulsion of PS prepared with 10G1L taken during the cooling (60 to -10 °C) and heating (-10 to 60 °C) processes. During

308 Crystal Growth & Design, Vol. 6, No. 1, 2006

Figure 3. SR-X-ray diffraction long and short spacing patterns of the nanometer-size PS emulsion using 10G1L during (A) cooling from 60 to -5 °C and (B) heating from -5 to 60 °C.

the cooling process (Figure 3A), the WAXS pattern of a short spacing value of 0.42 nm and the SAXS pattern of 4.9 nm both appeared at 8 °C, and their intensity increased as the temperature decreased. The polymorphic form of this crystal was R. On heating (Figure 3B), the SAXS pattern changed from 4.9 to 4.2 nm around 20 °C, and the WAXS pattern also changed from 0.42 to 0.46 nm around 24 °C. These conversions corresponded to polymorphic transformation from R to β, in accordance with the WAXS patterns of 0.46 nm (β). It is obvious that the DSC exothermic peak around 20 °C shown in Figure 2B for 10G1L also corresponds to this conversion. Continuous conversion in the SAXS and WAXS patterns and the corresponding exothermic DSC peak of the R-β transformation mean that this transformation occurred in a solid state and not via melt mediation, which must be accompanied by endothermicexothermic DSC peaks. The β form melted around 45 °C as indicated by the disappearance of the SAXS and WAXS patterns in Figure 3, corresponding with the DSC result (Figure 2B). Figure 4A,B shows the SR-XRD patterns of the PS emulsion prepared with 10G1S, taken during the cooling and heating processes. On cooling, the WAXS pattern of 0.42 nm appeared at 38 °C, corresponding to the R form. However, a corresponding SAXS pattern was not detectable at this temperature. When the cooling proceeded, the intensity of the WAXS pattern of 0.42 nm increased and its diffraction 2θ angle increased slightly, but no corresponding SAXS pattern was detectable until -2 °C. At -2 °C, the SAXS pattern of 4.9 nm appeared, and the corresponding WAXS pattern decreased its 2θ angle with a short spacing value of 0.425 nm. The single WAXS pattern corre-

Sonoda et al.

Figure 4. SR-X-ray diffraction long and short spacing patterns of the nanometer-size PS emulsion using 10G1S during (A) cooling from 60 to -5 °C and (B) heating from -5 to 60 °C.

sponds to the R form, and therefore it can be concluded that the occurrence of the new SAXS and WAXS patterns at -2 °C was due to the crystallization of the low-melting fraction of PS. It may be worth noting that the DSC exothermic peak observed around 36 °C (for 10G1S in Figure 2) was accompanied by the corresponding WAXS pattern but not the SAXS in Figure 4A. Figure 4B illustrates the conversions in the SAXS (4.9 to 4.2 nm) and WAXS (0.42 to 0.46 nm) patterns due to the R-β transformation of PS and melting of the β form, which are basically the same as the nanoemulsion prepared with 10G1L. The transformation from R to β observed in Figure 4B occurred around 26 °C, corresponding to the exothermic peak in Figure 2B. The same transformation occurred around 20 °C in the nanoemulsion droplets with 10G1L as shown in Figure 3B and Figure 2. This result indicated that the crystallization and transformation behaviors of PS in the nanoemulsion droplets were largely influenced by the emulsifiers with different fatty acid chains that were employed for emulsification. To further clarify this, we examined SR-SAXS/WAXS patterns of the PS nanometer-size emulsion droplets prepared with 10G1M and 10G1P. Figure 5 depicts the SR-XRD WAXS patterns of PS emulsion with 10G1M (Figure 5A) and 10G1P (Figure 5B) taken during the cooling process. In Figure 5A, the WAXS pattern of 0.42 nm, corresponding to the crystallization of the R form, occurred around 20 °C, whereas no SAXS patterns were detectable at this temperature (data not shown). Further cooling increased

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Figure 5. SR-X-ray diffraction short spacing patterns of the nanometersize PS emulsion using (A) 10G1M and (B) 10G1P during cooling from 60 to -5 °C.

the intensity of the WAXS pattern of 0.425 nm at a slightly decreased diffraction angle corresponding to the crystallization of the low-temperature R form at -2 °C. At this temperature, the SAXS pattern of 4.9 nm appeared (data not shown). These properties were identical to those observed in the emulsion with 10G1S (Figure 4A). Figure 5B shows the SR-XRD WAXS patterns of the PS emulsion with 10G1P taken during the cooling process. The WAXS pattern of 0.42 nm appeared at 30 °C without a corresponding SAXS pattern (data not shown), and further cooling showed the same changes in the WAXS and SAXS patterns around -2 °C as those of the emulsions with 10G1S and 10G1M. To summarize the crystallization behavior of the R form at high temperatures, Tc was 20 °C for 10G1M, 30 °C for 10G1P, and 38 °C for 10G1S. Figure 6 shows temperature variations of the intensity of the SR-XRD WAXS patterns of the R form (0.42 nm) and β form (0.46 nm) of PS crystals in the nanometer-size emulsion droplets prepared by the four emulsifiers taken during the heating process from -10 to 60 °C. In all cases, the intensity of the R form gradually decreased and that of the β form increased at the expense of R, due to the polymorphic transformation from R to β. Crossover temperatures, at which the SAXS pattern intensity of β became greater than that of R, were 16 °C (10G1L), 19 °C (10G1M), 22 °C (10G1P), and 26 °C (10G1S). This variation was in good agreement with the exothermic DSC peaks shown in Figure 2B, indicating that the solid-state transformation from R to β was inhibited by the increasing chain length of the

Figure 6. Temperature dependence of the intensity of WAXS peak diffraction (peak top) of nanometer-size emulsion of PS using (A) 10G1L, (B) 10G1M, (C) 10G1P, and (D) 10G1S taken during the heating process.

emulsifier employed for the emulsification. Although a high noise level was observed at high temperatures, the melting of the β form is shown as the decrease in its WAXS pattern intensity around 45 °C, irrespective of the emulsifiers. Discussion Table 1 summarizes the results of the DSC and SR-XRD experiments for the nanometer-size emulsion of PS prepared with the four polyglycerine fatty-acid esters, together with the same experiments in bulk, and the micrometer-size emulsion

310 Crystal Growth & Design, Vol. 6, No. 1, 2006 Table 1. Correspondence of DSC and SR-XRD Data of PS in Nanometer-Size Emulsion, Bulk, and Micrometer-Size Emulsion emulsifier 10G1L

10G1M 10G1P 10G1S

-

cycle

Nanometer-Size Emulsion 8 (L), 2 (S) -5-20 (M) 20 (M) 35-46 (M) cooling 19 (M), -2 (M) heating 22 (M) 37-44 (M) cooling 28 (M), -2 (M) heating 24 (M) 39-44 (M) cooling 38 (M), -2 (M) heating 26 (M) 39-46 (M) cooling heating

SR-XRD analysisb R (c) R (m) R f β (t) β (m) R (c) R f β (t) β (m) R (c) R f β (t) β (m) R (c) R f β (t) β (m)

Bulk 27-31 (S) 21 (M) 3 (M) 3 (M) 8-37 (M) 53 (L)

R (c) β′ (c) R (c) R (m) R f β′ (t), β′ f β (t) β (m)

Micrometer-Size Emulsion cooling 15-27 (M) 3 (M) heating -3-10 (M) 15-43 (M) 51 (L)

R (c) R (c) R (m) R f β′ (t), β′ f β (t) β (c)

cooling heating

10G1S

DSC thermopeaks (°C)a

a L indicates large peak, M indicates medium peak, and S indicates small peak. b c indicates crystallization, m indicates melting, and t indicates transformation.

droplets that were formed with 10G1S.20 In Table 1, the melting at -5 °C is disregarded. The following major remarks should be noted regarding Table 1: (a) In the cooling process, the highmelting and low-melting fractions of PS were crystallized in the metastable R polymorph at different temperatures for the bulk, micrometer-size emulsion, and nanometer-size emulsion systems. Tc in the high-melting fraction was 8 °C in the nanometer-size emulsion using 10G1L, whereas Tc was 31 °C in bulk and 27 °C in micrometer-size emulsion. However, Tc of PS in the nanometer-size emulsion increased with increasing chain length of the fatty-acid moiety of the emulsifiers. In particular, the use of 10G1S caused the crystallization of R at 38 °C, which is higher than the micrometer-size emulsion (32 °C) and even higher than the bulk PS (27-31 °C). In contrast, the R form of the low-melting fraction occurred at 2-3 °C in bulk, in the micrometer-size emulsion, and in the nanometersize emulsion using 10G1L. However, Tc of this fraction was -2 °C in the nanometer-size emulsion using 10G1M, 10G1P, and 10G1S. (b) Occurrence of the SR-XRD patterns of the highmelting fraction of PS exhibited extraordinary behavior. In the nanometer-size emulsion using 10G1L, the SAXS and WAXS patterns appeared at the same time when the R form was crystallized at 2 °C. The same behavior was observed in the bulk and micrometer-size emulsions.20 However, the crystallization of R of the high-melting fraction in the nanometer-size emulsion using the emulsifiers of 10G1M, 10G1P, and 10G1S did not produce the SAXS pattern during the cooling process, while only exhibiting the WAXS pattern of 0.42 nm. The SAXS pattern appeared when the R form of the low-melting fraction was crystallized at -2 °C. This SAXS pattern continued to appear during further cooling to -10 °C and the following heating process to 60 °C, until β melted, exhibiting the conversion from R (4.9 nm) to β (4.2 nm) upon heating. (c) During the heating process, the R form of the high-melting

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fraction transformed to the β form and exhibited two peculiar properties in the nanometer-size emulsion compared with those in the bulk and micrometer-size emulsion states. In the first, it is the solid-state transformation, whereas melt-mediated transformation from R to β through β′ was observed in the bulk and micrometer-size emulsions. The second property was the R f β transformation temperature in the nanometer-size emulsion, which increased with increasing chain length of the emulsifier from 20 °C (10G1L) to 22 °C (10G1M), 24 °C (10G1P), and 28 °C (10G1S) as observed by DSC (Figure 2B) and from 16 °C (10G1L), to 19 °C (10G1M), 22 °C (10G1P), and 26 °C (10G1S) as observed in the SR-XRD WAXS patterns (Figure 6). (d) Polymorphs of R and β were observed in the nanometersize emulsion, whereas the R, β′, and β forms were separately crystallized and transformed in the bulk state. In the micrometersize emulsion, R and β were crystallized, and the transformation of R f β′ f β occurred during the heating process. (e) Finally, the melting temperatures of β of the high-melting fraction of PS were 53 °C (bulk), 51 °C (micrometer-size emulsion), and 46 °C (nanometer-size emulsion), revealing the reduction with decreasing size of the emulsion droplets. Generally, it has been recognized that a smaller emulsion size yields a lower the crystallization temperature, as widely observed for various crystalline materials in micrometer-size14 and nanometer-size emulsion systems.17-21 This reduction in the crystallization temperature is due to the decreased nucleation rate of crystals in the emulsion, which is most probably caused by dilution of nucleation-catalytic impurities by emulsification.14 In the present case of the nanometer-size emulsion, the reduced crystallization of the high-melting fraction of PS using 10G1L may be caused by this mechanism, since the 10G1L might not have any template effect for the nucleation of PS crystals (see below). As for the polymorphic occurrence and transformation, it was observed for trilauryl-glycerol in the nanometer-size emulsion20 that the transformation from R to β through β′ occurred much faster than that in the bulk phase. This phenomenon was interpreted by taking into account the increased crystal surface area compared to the volume fraction, since polymorphic transformation is initiated from the crystal surface. Furthermore, lattice defects of crystals occurring in the nanometer-size emulsion may be increased, causing the initiation of the polymorphic transformation from the lattice defects where crystal free energy is increased. There are basically two types of lattice defects formed in crystals; grain boundaries and dislocations.23 The former defect is caused when many crystals growing from different nucleating points caused by a polynuclei mechanism (many nuclei are formed in a droplet) may collide with each other. By contrast, such defects may not be formed substantially when one nucleus is formed and grows in a droplet (a mononucleus mechanism). Kashchiev discussed the two types of nucleation mechanisms in emulsion crystallization.24 In the present case, we consider that the polynuclei model is applied to the crystallization in the emulsion using the emulsifiers catalyzing the interfacial heterogeneous nucleation such as 10G1S and 10G1P. In such a case, minimized curvature of the radius of a droplet may cause enhanced anisotropy of orientation of crystals that are heterogeneously nucleated from the interface, and thereby density of lattice defects due to grain boundaries may increase. As to the dislocation-type lattice defects, it is rather difficult to relate the nucleation mechanisms, since such dislocations may be present in any type of crystals, yet few observations of dislocation have been done on fat crystals. The above influences of crystal surface and lattice defects

Effects of Emulsifiers on Transformation Behavior

Figure 7. (A) Two types of nucleation processes in emulsion and (B) detailed illustration of fat crystallization at the template films.

may explain results d and e above regarding the absence of the β′ form and the reduction in the melting point of β. The solidstate transformation from R to β in the nanometer-size emulsion, instead of the melt-mediated transformation that occurred in the bulk and micrometer-size emulsion states, may also be due to this instability of the crystal structure. The most peculiar crystallization properties of PS in the nanometer-size emulsion examined in the present work are the increased Tc of the high-melting fraction of PS with the use of 10G1M, 10G1P, and 10G1S. These were accompanied only by WAXS patterns, not SAXS patterns. This property was not observed in the micrometer-size emulsion, in which the SAXS and WAXS patterns were clearly observed during the crystallization and transformation processes of R, β′, and β.20 In the present case, however, the fact that Tc increased almost linearly with the increasing number of carbon atoms of the fatty-acid moiety of the emulsifiers strongly indicates the interactions of the emulsifier membrane of the nanometer-size emulsion with the TAG molecules of the high-melting fraction of PS. The same interactions have been observed in the crystallization properties of n-alkanes and natural fats in the micrometer-size emulsion with high-melting emulsifier additives.25-28 In these studies, it was assumed that the additive may play the role of a template that induces the crystallization of lipids in the emulsion droplets. It was clearly shown that the effects of additives of high-melting polyglycerine fatty-acid esters on the acceleration of crystallization of palm mid-fraction were more manifest with increasing chain length of the fatty-acid moiety. Dickinson et al. also demonstrated the influence of emulsifiers on the crystallization of n-alkane in the micrometer-size emulsion.29 Figure 7A shows two types of heterogeneous nucleation of crystals in the emulsion. Volume heterogeneous nucleation triggered by nucleation-catalytic impurity must occur not only in bulk but also in emulsion states. Interfacial heterogeneous nucleation is caused by the triggering of interactions between the emulsifier membrane and the crystallizing materials at the oil-water interface of the emulsion. In the emulsion droplets, it is reasonable to assume that dilution of nucleation-catalytic particles occurs, and thereby the possibility of volume heterogeneous nucleation is lowered and nucleation rate is decreased in comparison with the bulk state. Krog and Larsson discussed interfacial heterogeneous nucleation occurring at water-oil interfacial membranes.30 Such heterogeneity may be brought up by two cases, (a) adding surface-reacting reagents in the oil phase and (b) using emulsifier having surface-reacting properties for emulsification. As to case a, our group has been working

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on interfacial heterogeneous nucleation induced by adding highmelting hydrophobic emulsifiers in the oil phase.22, 25-28 The crystallization in the nanometer-size emulsion using 10G1L was probably due to volume heterogeneous nucleation, since lauric acid chains of the 10G1L were in a liquid state at the membrane for the range of temperatures examined and therefore 10G1L probably did not cause interfacial heterogeneous nucleation. In this case, the crystallization temperature was reduced much more remarkably due to emulsification than that in the micrometer-size emulsion. However, in the nanometer-size emulsion droplets using the emulsifiers with longer fatty-acid chains, interfacial heterogeneous nucleation based on the above mechanism b may be caused by partial freezing of the fatty-acid chains prior to the crystallization of the highmelting fraction of PS. The extent of chain freezing of the 10G1S membrane is greater than that for the other two emulsifier membranes, and therefore Tc of the high-melting fraction of PS became higher than those of the other nanometersize emulsion droplets. PS is a semisolid lipid at ambient temperature. The templateinduced interfacial heterogeneous nucleation of PS may result in partial fractionation of the high-melting and low-melting fractions. The former is composed of tripalmitoyl-glycerol (PPP, ∼20%) and dipalmitoyl-oleoyl-glycerols (POP and PPO, ∼35%), and the latter involves the TAGs containing oleic and linoleic acids. The melting points of the most stable polymorphic forms of pure samples are 64 °C (PPP), 36 °C (POP), and 35 °C (PPO). It is reasonable to assume that the melting of POP and PPO in PS must occur below the melting points of the pure samples, because of the coexistence of the liquid fraction in PS. Therefore, the melting at highest temperatures in bulk, micrometer-size emulsion, and nanometer-size emulsion is due to the PPP present in PS, which crystallizes at higher temperatures than the other fractions during the cooling process. This assumption is in good agreement with the long spacing values of the R form (4.1 nm) and β form (4.6 nm), both of which are double chain length. The long spacing values of the β form of POP and the β′ form of PPO are 6.1 and 6.5 nm, both of which are triple chain length. We assumed that the PPP crystals in the high-melting fraction were crystallized from the outer area of the emulsion droplets in close contact with the emulsifier membrane by the interfacial heterogeneous nucleation. Povey postulated the occurrence of cocrystallization of the TAGs at the membrane interface when the van der Waals interactions among the hydrophobic tails were significant.14 We can calculate the volume fraction (∆V/V) of the crystallized part adherent to the oil-water interface (∆V) in the emulsion droplet of volume V with radius R as ∆V/V ) 3∆R/R. Taking R ) 60 nm for the average diameter and ∆V/V ) 0.3 (high-melting fraction of PS), we obtain a calculated thickness of the crystallized PS fraction, ∆R ) 6 nm. This means that the PPPs in the high-melting fraction of PS which are all crystallized at the interface may form 6 nm thick layers, which equals 1.5 times the long spacing of the TAGs comprising PS, as illustrated in Figure 7B. These crystalline layers can diffract the WAXS patterns, but not the SAXS patterns, since 1.5 layers are too short to form the coherence length for the diffraction of SAXS. This may be the reason for the disappearance of the SAXS patterns of the high-melting fraction of PS caused by the template of the emulsifier membrane. PS might be crystallized by the template film in the micrometer-emulsion, but those crystals must be thick enough to produce the SAXS patterns, as actually observed. The partial fractionation of PS thus caused by the template film may reduce Tc of the low-melting fraction

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of PS from 2 to -2 °C, since the crystallization of the lowmelting fraction is not caused by the template and volume heterogeneous nucleation would be operating. The effects of emulsifiers on increased polymorphic transformation temperatures from R to β during the heating process in the nanometer-size emulsion may be caused by their retardation effects, which were observed in many fats in bulk and emulsion states.31 In summary, our study demonstrated that the crystallization and polymorphic transformation of PS in nanometer-size emulsions can be modified by using polyglycerine fatty-acid esters. This result may be useful in applying the nanometersize emulsion to loading ability and the controlled release of lipophilic drug materials. Acknowledgment. The authors would like to thank Prof. Yoshiyuki Amemiya of the University of Tokyo and Prof. Masaharu Nomura of KEK for cooperating in the preparation of the beam lines BL-15A and 9C, respectively, at the Photon Factory, KEK Institute, Tsukuba, Japan. References (1) Muller, R. H.; Keck, C. M. J. Biotechnol. 2004, 113, 151-170. (2) Heurtault, B.; Sauliner, P.; Pech, B.; Proust, J. E.; Benoit, J.-P. Biomaterials 2003, 24, 4283-4300. (3) Kim, S. J.; Choi, H.; Suh, S.; Lee, Y. Eur. J. Pharm. Sci. 2002, 15, 497-502. (4) Bunjes H.; Siekmann, B.; Westesen, K. In Submicron Emulsions in Drug Targeting and DeliVery; Benita, S., Ed.; Harwood Academic Publishers: Singapore, 1998; pp 175-204. (5) Lundberg, B. B. J. Pharm. Pharmacol. 1997, 49, 16-21. (6) Baluom, M.; Friedman, D. I.; Rubinstein, A. Int. J. Pharm. 1997, 154, 235-243. (7) Youenang, M. P.; Korner, D.; Benita, S.; Marty, J. P. J. Controlled Release 1999, 5, 177-187. (8) Schwarz, J. S.; Weisspapir, M. R.; Friedmam, D. I. Pharm. Res. 1995, 12, 687-692. (9) Jenning, V.; Schafer-Korting, M.; Gohla, S. H. J. Controlled Release 2000, 66, 115-126. (10) Bunjes, H.; Westesen, K.; Koch, M. H. Int. J. Pharm. 1996, 129, 159-175

Sonoda et al. (11) Jenning, V.; Gysler, A.; Schafer-Korting, M.; Gohla, S. H. Eur. J. Pharm. Biopharm. 2000, 49, 211-218. (12) Jenning, V.; Thunemann, A. F.; Gohla, S. H. Int. J. Pharm. 2000, 199, 167-177. (13) Jenning, V.; Marder, K.; Gohla, S. H. Int. J. Pharm. 2000, 205, 1521. (14) Povey, M. J. W. In Crystallization Processes in Fats and Lipid Systems; Garti, N., Sato, K., Eds.; Marcel Dekker: New York, 2001; pp 251-228. (15) Coupland, J. N. Curr. Opin. Colloid Interface Sci. 2002, 7, 445450. (16) Sato, K. In Nanoscale Structure and Assembly at Solid-Fluid Interfaces, Vol. II: Assembly in Hybrid and Biological Systems; Liu, X. Y., De Yoreo, J. J., Eds.; Kluwer Academic/Plenum Publishing: New York, 2004; pp 83-104. (17) Bunjes H.; Westesen, K.; Koch, M. H. Int. J. Pharm. 1996, 129, 159-173. (18) Bunjes, H.; Koch, M. H.; Westesen, K. Langmuir 2000, 16, 51345241. (19) Higami, M.; Ueno, S.; Segawa, T.; Iwanami, K.; Sato, K. J. Am. Oil Chem. Soc. 2003, 80, 731-739. (20) Sonoda, T.; Takata, Y.; Ueno, S.; Sato, K. J. Am. Oil Chem. Soc. 2004, 81, 365-373. (21) Bunjes, H.; Koch, M. H.; Westesen, K. J. Pharm. Sci. 2003, 92, 1509-1520. (22) Sakamoto, M.; Ohba, A.; Kuriyama, J.; Maruo, K.; Ueno, S.; Sato, K. Colloids Surf., B 2004, 37, 27-33. (23) Mullin, J. W. Crystallization, 4th ed.; Butterworth-Heinemann: England, 2001. (24) Kashchiev, D.; Kaneko, N.; Sato, K. J. Colloid Interface Sci. 1998, 208, 167-177. (25) Ueno, S.; Hamada, Y.; Sato, K. Cryst. Growth Des. 2003, 3, 935939. (26) Hamada, Y.; Kobayashi, I.; Nakajima, M.; Sato, K. Cryst. Growth Des. 2002, 2, 579-584. (27) Awad, T.; Sato, K. Colloids Surf., B. 2002, 25, 45-53. (28) Awad, T.; Sato, K. J. Am. Oil Chem. Soc. 2001, 78, 837-842. (29) Dickinson, E.; McClements, D. J.; Povey, M. J. W. J. Colloid Interface Sci. 1991, 142, 103-110. (30) Krog, N.; Larsson, K. Fat Sci. Technol. 1992, 94, 55-57. (31) Garti, N.; Yano, J. In Crystallization Processes in Fats and Lipid Systems; Garti, N., Sato, K., Eds.; Marcel Dekker Inc.: New York, 2001; pp 211-250.

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