Visualizing the Structure of Triglyceride Nanoparticles in Different

Feb 23, 2007 - Heike Bunjes,*,† Frank Steiniger,‡ and Walter Richter‡ ... Friedrich Schiller UniVersity Jena, Ziegelmühlenweg 1, 07740 Jena, Ge...
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Langmuir 2007, 23, 4005-4011

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Visualizing the Structure of Triglyceride Nanoparticles in Different Crystal Modifications Heike Bunjes,*,† Frank Steiniger,‡ and Walter Richter‡ Department of Pharmaceutical Technology, Institute of Pharmacy, Friedrich Schiller UniVersity Jena, Lessingstrasse 8, 07743 Jena, Germany and Center for Electron Microscopy of the Medical Faculty, Friedrich Schiller UniVersity Jena, Ziegelmu¨hlenweg 1, 07740 Jena, Germany ReceiVed October 3, 2006. In Final Form: December 26, 2006 Colloidal suspensions of triglycerides are under investigation as potential drug carrier systems. The properties of the matrix lipids are altered in the nanoparticles compared to those of the bulk material. For instance, the metastable R-modification of the triglycerides usually transforms into the stable β-polymorph quite rapidly in the colloidal particles. Recently, it was observed that the R-modification can be preserved for a considerable period of time in tristearin nanoparticles when the particles are stabilized with a blend of saturated long-chain phospholipids and the bile salt sodium glycocholate [Bunjes, H.; Koch, M. H. J. J. Controlled Release 2005, 107, 229-243]. As triglyceride nanoparticles in the R-modification may offer some advantages over those in the β-form with regard to drug delivery applications, the structure of the corresponding dispersions was investigated in more detail with differential scanning calorimetry, X-ray diffraction, and electron microscopy. The electron microscopic investigations confirmed a plateletlike, layered structure for particles in the β-modification and revealed a spheroidal shape with concentric layers for larger particles in the R-form. For the first time, not only was information on the internal structure of solid triglyceride nanoparticles obtained from freeze-fracture electron micrographs but also details were observed by cryoelectron microscopy.

Introduction Colloidal dispersions of solid lipids such as triglycerides, waxes, or fatty acids (solid lipid nanoparticles) are under intensive investigation as carrier systems for different routes of drug administration,1,2 for instance, for the intravenous or dermal delivery of lipophilic substances. Methods for the preparation of such dispersions include high-pressure homogenization of the melted matrix lipid in a hot aqueous phase and different precipitation techniques such as precipitation from warm microemulsions.1,2 As the lipidic substances used as matrix material usually occur in different polymorphic forms and since the preparation procedures typically include a crystallization step and thermal treatment, issues of polymorphism and polymorphic transitions have to be taken into consideration during the development of these nanodispersions.3-13 This is particularly important against * Corresponding author. Present address: Institute of Pharmaceutical Technology, TU Braunschweig, Mendelssohnstr. 1, 38106 Braunschweig, Germany; phone ++49 531 3915657; fax ++49 531 391 8108; e-mail [email protected]. † Department of Pharmaceutical Technology, Institute of Pharmacy. ‡ Center for Electron Microscopy of the Medical Faculty. (1) Mehnert, W.; Ma¨der, K. AdV. Drug DeliVery ReV. 2001, 47, 165. (2) Bunjes, H.; Siekmann, B. In Microencapsulation, 2nd ed.; Benita, S., Ed.; Marcel Dekker: New York, 2006; p 213. (3) Aquilano, D.; Cavalli, R.; Gasco, M. R. Thermochim. Acta 1993, 230, 29. (4) Cavalli, R.; Aquilano, D.; Carlotti, M. E.; Gasco, M. R. Eur. J. Pharm. Biopharm. 1995, 41, 329. (5) Cavalli, R.; Caputo, O.; Marengo, E.; Pattarino, F.; Gasco, M. R. Pharmazie 1998, 53, 392. (6) Siekmann, B.; Westesen, K. Colloids Surf., B 1994, 3, 159. (7) Bunjes, H.; Westesen, K.; Koch, M. H. J. Int. J. Pharm. 1996; 129, 159. (8) Westesen, K.; Bunjes, H.; Koch, M. H. J. J. Controlled Release 1997, 48, 223. (9) Bunjes, H.; Drechsler, M.; Koch, M. H. J.; Westesen, K. Pharm. Res. 2001, 18, 287. (10) Bunjes, H.; Koch, M. H. J.; Westesen, K. J. Pharm. Sci. 2003, 92, 1509. (11) Bunjes, H.; Koch, M. H. J. J. Controlled Release 2005, 107, 229. (12) Jenning, V.; Scha¨fer-Korting, M.; Gohla, S. H. J. Controlled Release 2000, 66, 115. (13) Schubert, M. A.; Schicke, B. C.; Mu¨ller-Goymann, C. C. Int. J. Pharm 2005, 298, 242.

the background that different polymorphic forms of the matrix lipid have different physicochemical properties and that there are indications that they may interact with incorporated drugs in a different way.8,12 Triglyceride nanoparticles prepared by crystallization from high-pressure melt-homogenized emulsion droplets, for instance, typically crystallize in the metastable R-form, which subsequently transforms into the stable β-modification upon thermal treatment or upon storage (sometimes via the stage of other metastable modifications).7,8,11 As the rate of polymorphic transitions is much higher in triglyceride nanoparticles than in the bulk,6,13,14 the metastable R-modification is difficult to obtain in pure form in small colloidal particles. In spite of some promise with regard to application, it has, therefore, not yet received much attention. The only more detailed investigations on triglyceride nanoparticles in the metastable R-form have so far been obtained on dispersions stabilized with the bile salt sodium glycocholate.10 Electron microscopic investigations on the particles revealed deviations of the particle shape from the plateletlike appearance typically observed for β-form triglyceride nanoparticles and a pronounced dependence of the particle shape and internal structure of the R-form nanoparticles on their thermal history. Dispersions that are stabilized solely with bile salts do, however, not appear particularly promising for pharmaceutical applications due to the comparatively high irritative potential of the bile salts. Recently, it was observed that triglyceride nanoparticles stabilized with a combination of the bile salt sodium glycocholate and fully saturated long-chain phospholipids show an unusually high (albeit not absolute) stability of the R-modification, in particular for long-chain triglycerides like tristearin.11 These particles may thus indicate new possibilities for pharmaceutical applications and, in particular, they offer the opportunity for detailed studies of the particles in the metastable R-modification for a composition that is more (14) Westesen, K.; Drechsler, M.; Bunjes, H. In Food Colloids: Fundamentals of Formulation; Dickinson, E., Miller, R., Eds.; Royal Society of Chemistry: Cambridge, U.K., 2001; p 103.

10.1021/la062904p CCC: $37.00 © 2007 American Chemical Society Published on Web 02/23/2007

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promising with regard to pharmaceutical applications. The present study was aimed at investigating the shape and structure of these novel R-form tristearin particles in comparison to that previously observed in glycocholate-stabilized systems and to that of the β-form. Moreover, we wanted to elucidate the processes occurring during the crystallization of triglyceride nanoparticles stabilized with the aid of saturated phospholipids in more detail, as these particles display a more complex crystallization behavior than typically observed in such systems.11 Materials and Methods Materials. Tristearin (Dynasan 118, D118) was kindly provided by Condea Chemie GmbH, D-Witten. According to the manufacturer’s specification, the purity of the fatty acid fraction is about 99% for this triglyceride quality and the hydroxyl value as a measure for the content of partial glycerides is below 5. The purified soybean lecithin Lipoid S100 (S100) and the hydrogenated soybean lecithin Lipoid S100-3 (S100-3) were a kind gift of Lipoid KG, D-Ludwigshafen. According to manufacturer’s specifications both phospholipid qualities contain g94% phosphatidylcholine and the fatty acid fraction consists of 12-17% C16:0, 2-5% C18:0, 11-15% C18:1, 59-70% C18:2, and 3-7% C18:3 (S100) or 12-16% C16:0, 85-88% C18:0, e2% C18:1, and e1 % C18:2 (S100-3), respectively. The lipids as well as sodium glycocholate (SGC, Sigma-Aldrich Chemie, D-Taufkirchen), glycerol (Solvay Chemicals, D-Hannover), and thiomersal (Synopharm, D-Barsbu¨ttel) were used as received. The water for the dispersions was purified by reverse osmosis (Alpha Q, Direct Q 5, Millipore). Methods: Preparation of Dispersions. The dispersions were prepared from 10% tristearin, 2.4% phospholipid (S100 or S100-3), and 0.6% sodium glycocholate (concentrations w/w) in an aqueous phase containing 2.25% glycerol and 0.01% thiomersal (concentrations w/v). The SGC serves as a costabilizer in the dispersions, which prevents gelation of the systems during recrystallization of the nanoparticles.15 Phospholipid and glycocholate were dispersed/ dissolved in the aqueous phase by magnetic stirring overnight; dispersions containing S100-3, which has a phase transition above room temperature (around 52-53 °C in the glycocholate-containing aqueous phase according to DSC), were transiently heated above this phase transition temperature to facilitate dispersion. After dispersion was completed, the stabilizer dispersion was heated to ∼90 °C. The hot liquid was added to the molten triglyceride, which had been heated to the same temperature, and the mixture was predispersed by probe sonication (Bandelin Desintegrator HD200, D-Berlin). The hot predispersion was passed through a heated (∼90 °C) high-pressure homogenizer (Micron Lab 40, APV Gaulin, D-Lu¨beck) for 5 cycles at 800 bar. Cooling was performed at 0.5 °C/min from 65 to 23 °C in a thermostat, and the S100-3/SGCcontaining samples were refrigerated (∼2-8 °C) during long-term storage. To obtain S100-3/SGC-stabilized nanoparticles in the β- or R-modification for electron microscopic investigations, a fraction of cold-stored S100-3-containing dispersion was aged at elevated temperature (∼1 h at 45 °C followed by ∼0.5 h at 50 and 55 °C each) or freshly crystallized by controlled cooling (see above) after heating to 90 °C, respectively. For comparison, particles in the R-modification were also prepared by flash cooling (a small amount of heated (90 °C) sample was poured into a large glass vial precooled to 5 °C in a thermostat). The electron microscopic investigations on the S100/ SGC-stabilized dispersion were carried out after storage at 23 °C for less than 4 weeks. Particle Size Analysis. An estimate of the mean particle size was obtained by photon correlation spectroscopy at 25 °C and a scattering angle of 173° (Zetasizer Nano ZS, Malvern Instruments, D-Herrenberg). Prior to measurement, the samples were diluted with demineralized, filtered (0.22 µm) water to an almost clear optical appearance. An estimate of the mean particle size was obtained by analyzing the data by the cumulants method with the assumption of spherical particles. Accordingly, the results are given as the z-average (15) Westesen, K.; Siekmann, B. Int. J. Pharm. 1997, 151, 35.

Figure 1. DSC heating (10 °C/min) and cooling (5 °C/min) curves of tristearin nanoparticles stabilized with phospholipid/bile salt blends containing unmodified (top) or hydrogenated soybean lecithin (bottom) shortly after preparation. The dashed arrow in the bottom panel indicates the thermal range of the exothermic event prior to crystallization. diameter (intensity weighted mean diameter) and the polydispersity index (PI) as a measure for the relative width of the particle size distribution. Values given are means of three successive measurements of 5 min run duration each (obtained after an equilibration period of 5 min). Differential Scanning Calorimetry. Thermograms were recorded with a Pyris 1 DSC (Perkin-Elmer) at scan rates of 5 °C/min (cooling) or 10 °C/min (heating). About 10-15 mg of dispersion was accurately weighed into standard aluminum pans, which were tightly sealed. An empty pan was used as reference. X-ray Diffraction. Small- and wide-angle X-ray diffraction patterns were recorded with a SWAX camera based on a Kratky collimator system (Hecus M. Braun-Graz X-ray Systems) on a conventional X-ray source (Seifert generator ID3003, Cu KR1,2, Ni filter). Electron Microscopy. (A) Freeze-Fracture. A perforated gold grid (BAL-TEC) was wetted with the dispersion and placed between two sandwich copper holders (BAL-TEC). The sample was rapidly frozen in liquid propane (JFD 030, BAL-TEC) and fractured with a freeze-fracture unit (BAF 600, BAL-TEC) at -150 °C and 5 × 10-8 mbar. The fractured samples were shadowed under 45° with 2 nm platinum/carbon and stabilized by perpendicular deposition of 20 nm pure carbon for replica production. Replicas were cleaned with chloroform/methanol (1:1 v/v) and viewed with a transmission electron microscope (Zeiss, EM 900, CEM 902A) operated at 80 kV. (B) Cryopreparation. Three microliters of dispersion was placed on a copper grid with perforated carbon film (Quantifoil R 1.2/1.3), and excess liquid was blotted automatically for 2 s between two strips of filter paper. Subsequently, the samples were rapidly plunged into liquid ethane (cooled to ∼ -175 °C) in a cryobox (Zeiss, Oberkochen). Excess ethane was removed with a piece of filter paper. The sample was transferred with a liquid nitrogen-cooled holder (Gatan 626) into the cryo-TEM (Philips CM 120) and investigated at 120 kV. The micrographs were generated by a TietzFast Scan CCD camera.

Results The tristearin dispersions were macroscopically homogeneous milky systems with a PCS z-average particle size/polydispersity

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Figure 2. Characteristics of tristearin dispersions stabilized with the S100-3/SGC mixture in dependence on thermal history. The panel to the left displays wide-angle X-ray diffractograms and DSC heating curves (top and bottom curve, respectively) corresponding to the respective systems. Cryo- and freeze-fracture transmission electron micrographs are shown in the middle and right panel, respectively (bars represent 100 nm). Top row: Original dispersion cold-stored for about 9 months containing spheroidal (s) and anisometric (a), platelet-like particles (the fraction of platelet-like particles was higher in some other images; the anisometric particle displayed in the inset is in the R-modification according to its layer distance). Middle row: Particles in the R-modification obtained by slow cooling from the melt; large particles appear spheroidal but anisometric (a) structures are also observed, particularly for smaller particle sizes. In freeze-fractured samples, some of the spheroidal particles are fractured out of plane and stand out of the image (S-o), others are cross-fractured (S-c) and display a “fingerprint-like” internal lining (see also inset) which can also be identified in the cryoelectron micrographs. Bottom row: Particles in the β-modification (obtained by heating to slightly elevated temperatures) are anisometric, sharply edged nanocrystals with a planar layered internal structure (l), which tend to pack in stacks at this concentration.

index of 198 nm/0.22 (S100-containing dispersion) and 167 nm/0.13 or 148 nm/0.18 (S100-3-containing dispersions). The dispersed tristearin formed the desired solid nanoparticles under the chosen preparation conditions, as reflected in DSC melting transitions recorded shortly after preparation (Figure 1). While the dispersion stabilized with the aid of the unmodified soybean lecithin S100 had already almost completely transformed into the β-modification, the use of the hydrogenated, fully saturated soybean lecithin S100-3 led to the occurrence of a predominant fraction of the metastable R-form. The dispersed

triglyceride crystallized at a temperature much below its melting point (∼68-69 °C), reflecting the high supercooling tendency of triglycerides in the colloidal state7,16 (Figure 1). Stabilization with unmodified soybean lecithin (S100) led to a much lower crystallization temperature than stabilization with the fully saturated lecithin S100-3. Moreover, crystallization of the triglyceride was preceded by a complex exothermal event in the (16) Bunjes, H.; Siekmann, B.; Westesen, K. In Submicron Emulsions in Drug Targeting and DeliVery; Benita, S., Ed.; Harwood Academic Publishers: Chur, Switzerland, 1998; p 175.

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Figure 3. Small- and wide-angle X-ray diffractograms of S1003/SGC-stabilized dispersions obtained by slow or rapid cooling.

S100-3-containing dispersion. Reinvestigation of one of the S1003-containing dispersions after 9 months of storage by DSC and X-ray diffraction still revealed the presence of a considerable amount of residual R-modification (∼70% according to DSC, Figure 2, top left). Aging the sample at elevated temperatures close to the R-melting point transformed the particles into the β-modification, as verified with DSC and X-ray diffraction (Figure 2, bottom left). On the other hand, remelting the particles and subjecting them to the same cooling program as after melt-homogenization brought them back into the metastable R-modification (Figure 2, middle left). Rapid cooling of a small sample fraction to 5 °C after remelting of the particles also led to the formation of R-modification, according to DSC and X-ray diffraction. Compared to the slowly cooled sample, the rapidly cooled sample displayed a weaker small-angle X-ray reflection, whereas the broad wide-angle reflection was comparable to that of the slowly cooled sample (Figure 3). Moreover, the enthalpy involved in the recrystallization transition after melting of the R-modification was typically smaller by a factor of about 2-3 in rapidly cooled samples compared to slowly cooled ones (e.g., ∼ -1.2 compared to ∼ -3.1 J/g). The tristearin long spacings (corresponding to the d001 reflection) derived from the small-angle X-ray diffractograms were 5.1 nm for the samples in the R-modification (irrespective of the cooling conditions) and 4.5 nm for the sample containing nanoparticles in the β-form, which is in good agreement with the values reported by Chapman (5.06 and 4.50 nm for the R- and β-forms, respectively17). The ultrastructure of the long-term-stored S100-3-containing dispersion as well as that of the fractions that had been transformed into the R- or β-modification, respectively, by slow cooling or by heating was investigated with different electron microscopic techniques (Figure 2). For particles in the β-modification, freezefracture TEM revealed a platelet-like shape with layered internal structure (Figure 2, bottom right). The layers were orientated in parallel to the large surfaces of the platelets. Anisometric particles were also observed in cryo-TEM (Figure 2, bottom middle). Usually, the particles were orientated with their large surfaces in parallel to the plane of observation (“top view”) and appeared as large, circular to elongated shadows. At some points of the sample, however, the particles appeared edge-on and packed in small stacks. These particles in side view had a striated appearance with fine, regular dark stripes in the interior of the particles and, in some cases, very dark, broader stripes marking the large surfaces (see also inset). Measurements on selected particles revealed a repeat distance of about 4.6 nm (4.57 nm ( 0.16 nm SD, n ) 6; Figure 4). The boundary at the small, lateral surfaces of these particles was of low contrast and rather diffuse. Similar structures as in the S100-3/SGC-stabilized dispersion containing particles in the β-form were observed in the sample prepared with the (17) Chapman, D. Chem. ReV. 1962, 62, 433.

Figure 4. Repeat distances determined from cryoelectron micrographs for selected S100-3/SGC-stabilized particles in different polymorphic forms. The graph displays single measurement values ([), mean values (b) with standard deviations, and literature values for X-ray d-spacings (O) according to ref 17.

unmodified soybean lecithin S100, which also contains particles in the β-modification according to DSC (Figure 5). The appearance of the S100-3/SGC-stabilized dispersion with particles in the R-modification in freeze-fracture TEM (Figure 2, middle right) was dominated by circular or nearly circular structures that often seemed to be fractured along their surface layers. Others were, however, completely cross-fractured, revealing a concentrically layered internal structure with “fingerprintlike” appearance (such structures were also observed in a parallel investigation on a rapidly cooled sample). The concentric layers often seemed to be disturbed toward the center of the particles. The smaller particles in the sample appeared more anisometric, and sometimes a planar layered structure was observed. In cryo-TEM, larger particles appeared mainly as circular to polyhedral, and a concentrically ordered internal structure could be observed in many of the particles (Figure 2, center). Also here, the concentric layering often seemed to be disturbed in the center of the particles accompanied by a very high electron density which is due to the large thickness of the (spheroidal) particles in these areas. The smaller particles appeared more anisometric, and a planar multilayered internal structure could be observed for some of them. A comparison of the electron micrographs of the slowly and rapidly crystallized particles did not reveal much obvious difference (Figure 6). The mean distance of the stripes was in the range between 5.2 and 5.3 nm (5.28 ( 0.19 nm SD, n ) 20 for the slowly cooled sample and 5.21 ( 0.19 nm, n ) 6 for the rapidly cooled sample; Figure 4). As a trend, the particles in the rapidly cooled sample appeared more round and the fraction of spheroidal particles might be larger. Moreover, spheroidal particles with only a few peripheral concentric layers were sometimes observed that were not prominent in the slowly cooled samples (other particles appeared, however, highly structured, including their center). A quantitative impression of these phenomena can, unfortunately, not easily be obtained as the structures were very sensitive toward electron irradiation and quickly started to disintegrate (“bubble”) upon observation, in particular at higher magnifications. The original, long-term-stored dispersion (still predominantly containing particles in the R-modification) displayed the concentrically layered circular as well as anisometric structures,

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Figure 5. Electron micrographs of the dispersion stabilized with the S100/SGC mixture. (Left) Cryoelectron micrograph (bars represent 100 nm); (right) micrograph of the freeze-fractured specimen [bars represent 200 nm (large image) and 100 nm (inset)].

Figure 6. Cryoelectron micrographs of tristearin particles in the R-phase obtained from a dispersion stabilized with S100-3/SGC by slow (left) or rapid (right) cooling. All scale bars represent 100 nm.

including planar layered particles (Figure 2). A fast Fourier transform analysis of an electron micrograph of a long-termstored dispersion (Figure 7) indicated the presence of two characteristic structural distances in the sample corresponding to about 5.21 nm (spherical particles) and 4.45 nm (anisometric particles), which is in agreement with the layer distances obtained on single particles in the images of samples containing only one type of triglyceride modification.

Discussion As described earlier,11 stabilization of tristearin nanoparticles with an emulsifier mixture containing hydrogenated soybean phospholipid (with fully saturated acyl chains) increases the crystallization temperature and delays the R- to β- polymorphic transition compared to the use of unmodified soybean lecithin (containing a high fraction of unsaturated fatty acid chains). The differences in crystallization and polymorphic transition have been attributed to the formation of a shell of solidified phospholipid (possibly with the participation of some triglyceride) in the emulsifier monolayer surrounding the triglyceride core of the nanoparticles. In addition to the phospholipid, this monolayer contains SGC molecules and potentially also a certain fraction of phospholipid hydrolysis products that might form, for example, due to the stress conditions upon preparation. The formation of the solidified phospholipid shell (which cannot occur in the system containing unsaturated soybean lecithin due to the low phase transition temperature of this phospholipid) is reflected in a complex exothermal event preceding the main crystallization transition of the triglyceride in the DSC curve. The solidified

phospholipid chains would then serve as nucleation template, enhancing triglyceride crystallization, and would counteract polymorphic transitions. The R-form stability of tristearin nanoparticles stabilized with the S100-3/SGC combination is sufficiently high to allow electron microscopic investigations on the morphology of the R-form nanoparticles and compare it to that of nanoparticles in the β-form. For the β-form, the well-known platelet-like shape10,15,18-20 and stack formation in concentrated dispersions21,22 was also observed for the particles under investigation here. For the first time, the molecular layering in these particles not only was observed in freeze-fractured specimens but also could clearly be derived from cryoelectron micrographs, which displayed “stripes” in particles that were projected edge-on. The repeating unit determined for the stripes is in reasonably good agreement with the spacing for the d001 X-ray reflection, which reflects the stacking of individual layers of triglyceride molecules. The dark stripes in the interior of the particles seem to be correlated with the position of the glycerol backbone of the triglycerides within the crystal lattice. This assumption is supported by the observation that the “bubbling” artifacts by electron beam damage appeared to originate from the bright areas in the structure (which would correspond to the region of the acyl chains according to this (18) Siekmann, B.; Westesen, K. Pharm. Pharmacol. Lett. 1992, 1, 123. (19) Bunjes, H.; Koch, M. H. J.; Westesen, K. Langmuir 2000, 16, 5234. (20) Schubert, M. A.; Mu¨ller-Goymann, C. C. Eur. J. Pharm. Biopharm. 2005, 61, 77. (21) Unruh, T.; Westesen, K.; Bo¨secke, P.; Lindner, P.; Koch, M. H. J. Langmuir 2002, 18, 1796. (22) Illing, A.; Unruh, T.; Koch, M. H. J. Pharm. Res. 2004, 21, 592.

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Figure 7. Filtered cryoelectron micrograph of an S100-3/SGCstabilized, long-term-stored (>1 year) dispersion containing spherical and anisometric particles. A fast Fourier transformed image (inset) shows a circular feature corresponding to a distance of about 5.21 nm as well as an anisometric feature (arrows) corresponding to a distance of about 4.45 nm (bar represents 200 nm).

Figure 8. Schematic representation of the preferential adsorption of phospholipid and glycocholate at the different nanoparticle faces as suggested in ref 15, as well as the organization of the triglyceride lattice within the nanoparticle (following ref 23).

interpretation). The high contrast often observed at the large surfaces of the β-form nanoparticles projected in side view might be caused by the presence of adsorbed phospholipids, in particular by their phosphate groups. It has been proposed earlier that phospholipids adsorb preferably at the large (001) faces of triglyceride nanocrystals, whereas the small, lateral surfaces are mainly covered by cosurfactants like SGC15 (Figure 8). This would be consistent with our observation of low contrast at the small, lateral faces of the nanoparticles, which might, however, also be related to the curved outline of the nanoparticles that does not allow projection along a huge array of adsorbed emulsifier molecules. The matter of electron microscopic appearance of the nanoparticle outline does, however, remain to be further investigated to be fully resolved. For instance, lines of higher electron density along particle surfaces would also be expected for the R-form tristearin nanoparticles according to the above considerations but could not be observed in the present study. Tristearin nanoparticles in the R-modification did also display layered structures in freeze-fracture as well as in cryo-TEM. As

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expected, the repeat distance of the layers was larger than in the β-form nanoparticles. Although the deviation from the literature value was slightly higher than for the β-form particles, the values still seem to be in reasonable agreement considering the uncertainties of the electron microscopic determination. Large R-form particles assume a spheroidal, sometimes also somewhat more polyhedral shape with concentric triglyceride layers, the packing of which often seems to be disturbed toward the center of the sphere. This spheroidal shape of the larger R-form particles, which is stable upon cold storage, is assumed to originate from the shape of the original emulsion droplets that is transferred to the solidified shell of phospholipids, which later serves as a template for triglyceride crystallization. Spherical R-form particles have been previously observed in a freeze-fracture TEM study of tripalmitin nanoparticles stabilized solely with sodium glycocholate, but only when they were prepared by rapid cooling.10 Within these particles, no layering was observed and the triglyceride molecules were assumed to exist in a state of high mosaicity as reflected in the almost complete absence of the small-angle X-ray reflection.24 In contrast, slowly cooled particles crystallized in a nonspherical shape and did display layering. Also, for the nanoparticles stabilized solely with glycocholate and crystallized by either slow or rapid cooling, there were differences in the enthalpy of recrystallization after melting of the R-form during the DSC scans. This phenomenon was tentatively attributed to the fact that a layered organization of the triglycerides in the R-form might promote recrystallization into the stable β-modification after melting of the R-form during heating. For the differently crystallized S100-3/SGC-stabilized dispersions in the present investigation, smaller differences in the X-ray diffraction patterns were detected and concentrically layered particles were observed in both rapidly and slowly cooled samples. The dependence of the structure formation of these particles on the thermal history thus seems to be lower than previously observed for triglyceride dispersions containing SGC as the only stabilizer. The differences in morphology between S100-3/SGC-stabilized tristearin and solely SGC-stabilized tripalmitin nanoparticles formed upon slow cooling may result from the fact that SGC alone as a surfactant is unable to form a shell of solidified material around the liquid triglyceride core, so that the spherical shape of the emulsion droplet can only be preserved at a very high cooling rate. On the other hand, as the rigid shell of phopholipid is assumed to act as nucleation template in the S100-3-containing dispersions, it may impose the layering even in rapidly cooled samples. The disturbance of concentric layering in the center of the spheroidal S100-3/SGC-stabilized particles is not unexpected because crystalline triglycerides preferentially assume a planar layered structure. A completely concentric layering is thus hardly possible as it would force the innermost triglyceride layers into an arrangement of extremely high curvature. The orientation of the lamellae is, therefore, different in the center of the particles where it is assumed to be more flat. This may be the reason why the internal part of many particles appears unstructured: The lamellae will appear as stripes in the electron micrographic projection only when the plane of observation is almost perpendicular to the lamellar planes. For the rapidly cooled particles, the results from X-ray diffraction support, however, the assumption of a somewhat less ordered state. The high packing (23) Skoda, W.; Hoekstra, L. L.; van Soest, T. C.; Bennema, P.; van den Tempel, M. Kolloid Z. Z. Polym. 1967, 219, 149. (24) These earlier investigations were performed on nanoparticles containing tripalmitin as matrix material. The phenomenon does, however, also occur in tristearin nanoparticles as confirmed by X-ray investigations (see Supporting Information).

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the crystallization of which seems to proceed from the surface to the center of the nanoparticles. In the nanoparticle center, due to spatial constraints, the concentric layering changes into a lamellar packing of a preferred orientation, allowing a more straight arrangement of the lamellae. Upon cold storage, the transformation into β-form particles, which have a completely different morphology, occurs only very slowly. This may partly result again from the rigidity of the phopholipid shell: major conformational changes of the triglyceride molecules are assumed to occur during polymorphic transitions,28 and the mixed phospholipid/bile salt monolayer may not provide the working space necessary for the required molecular rearrangements. The transformation can, however, easily be induced by raising the temperature of the sample.

Conclusion

Figure 9. Schematic model of the proposed processes occurring during particle solidification.

frustration in concentric triglyceride layers expected for small spheres is probably the reason why smaller particles seem to preferably obtain a nonspherical shape with planar layering. Observations pointing to similar conclusions were made on phospholipid/SGC-stabilized cholesterol ester nanoparticles in the thermotropic smectic state, which are assumed to exist in either concentrically layered spherical or planar layered cylindrical form. Also those particles seem to favor the geometrically more “relaxed” planar layered structure, in particular when they have a small size.25 Geometrical considerations may also be the cause for the deviations from an ideally spherical shape of the large R-form particles, which were particularly observed in the cryoelectron micrographs of slowly cooled dispersions: A polyhedral shape would be more favorable for the arrangement of phospholipids with solidified chains, which can hardly assume a perfectly smooth curvature. Also for phospholipid vesicles, a polyhedral shape is observed below the phospholipid phase transition temperature.26,27 In addition, the usually straight crystal structure of the triglyceride R-form may impose a more polyhedral shape to the tristearin particles. In summary (and with the results of previous investigations on these dispersions11 taken into consideration), the process of crystallization in S100-3/SGC-stabilized tristearin particles may proceed as schematically illustrated in Figure 9: In a first step upon cooling, the phospholipid chains in the mixed S100-3/SGC monolayer on the particle surface solidify (possibly with participation of some triglyceride material). In the next step, the rigid phospholipid layer serves as a nucleation template for triglyceride crystallization in the R-modification and at the same time imposes a concentric arrangement of the triglyceride layers, (25) Kuntsche, J.; Koch, M. H. J.; Drechsler, M.; Bunjes, H. Colloids Surf., B 2005, 44, 25. (26) Johnsson, M.; Edwards, K. Biophys. J. 2003, 85, 3839. (27) Ickenstein, L.; Sandstro¨m, M. C.; Mayer, L. D.; Edwards, K. Biochim. Biophys. Acta 2006, 1758, 171.

The comparatively high stability of the R-modification in tristearin nanoparticles stabilized with the aid of saturated soybean lecithin in combination with sodium glycocholate allows detailed investigations on their ultrastructure. Electron microscopic investigations reveal that larger particles in the R-modification obtain a spheroidal shape with concentric layers of triglyceride molecules, whereas after transformation into the β-polymorph the typical platelet-like shape is observed. The spheroidal, layered structures of the R-form nanoparticles can be observed even after a long period of storage. For the first time, cryoelectron microscopic investigations on solid triglyceride nanoparticles not only allowed conclusions on the particle shape but also revealed details of the internal structure of the nanoparticles. The observation of striations that reflect the repeating unit of the single triglyceride layers within the particles opens up the possibility to assign the corresponding polymorphic form to single particles in the electron micrographs if the repeating units can be measured accurately. Although a considerable scatter of single values was observed in our study, reliable measurements seem to be possible against a sufficient set of reference values, as no overlap occurred between the data for the different polymorphic forms (Figure 4). With consideration also of the information on the thermal behavior upon crystallization of the nanoparticles, a schematic model of the process is proposed that describes crystallization of the triglyceride as proceeding from the shell of solidified phospholipid chains forming a mixed monolayer with bile salt molecules around the nanoparticles toward the center of the particles. With regard to pharmaceutical applications, it remains to be investigated whether this special type of crystallization process may influence the intraparticular distribution of incorporated drugs. Moreover, the potential influence of differences in molecular packing and particle shape of the different polymorphic forms of triglyceride nanoparticles on the drug incorporation capacity, as well as the effects of drug incorporation on the crystalline structure of the nanoparticles, require further investigation. Acknowledgment. We thank S. Richter for the preparation of the freeze-fractured specimen and J. Kuntsche for providing the drawing in Figure 8. Supporting Information Available: X-ray diffractograms of slowly and rapidly cooled tristearin particles stabilized solely with SGC. This material is available free of charge via the Internet at http://pubs.acs.org. LA062904P (28) Hagemann, J. W.; Rothfus, J. A. J. Am. Oil Chem. Soc. 1993, 70, 211.