Gold Nanoparticles Spontaneously Generated in Onion-Type

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Gold Nanoparticles Spontaneously Generated in Onion-Type Multilamellar Vesicles. Bilayers-Particle Coupling Imaged by Cryo-TEM Oren Regev,‡,§ Re´nal Backov,† and Chrystel Faure*,† Centre de Recherche Paul Pascal (CNRS), U.P.R 8641, Av. du Docteur Schweitzer, 33600, Pessac, France, Laboratory of Polymer Chemistry, Technical University of Eindhoven, Post-bus 513, 5600MB Eindhoven, The Netherlands, and Department of Chemical Engineering, Ben-Gurion University of the Negev, 84105 Beer-Sheva, Israel Received May 12, 2004. Revised Manuscript Received September 17, 2004

We report the spontaneous, in-situ synthesis of gold nanoparticles within onion-type multilamellar vesicles (MLV) using a simple and mild strategy. Monoolein, one of the MLV components, was used as reductant, without any additional chemical. Two different preparative pathways were employed that resulted in gold particle formation as asserted by UV-vis spectroscopy and transmission electron microscopy (TEM). When onions were prepared from a lamellar phase containing gold ions, nanoparticles with a rice grain shape and narrow size distribution (6 × 10 nm*nm) were formed, suggesting synthesis within the vesicles. When preformed onions were dispersed in a KAuCl4 solution, TEM and cryogenic temperature-transmission electron microscopy (cryo-TEM) analysis reveal that both extraand intravesicular syntheses took place. Cryo-TEM imaging evidences the insertion of gold nanoparticles between MLV leaflets and the close coupling between particle morphology and the lamellar phase. A simple mechanism of particle growth within a lamellar phase is proposed that could explain the differences in nanoparticle size and shape observed between both preparative pathways.

Introduction It is well known that liposomes are good candidates for controlled reactions. As lipid-based colloids, they have been used to mimic biomineralization processes1,2 while as restricted volumes, they have been used as microreactors to synthesize nanoparticles such as metal oxides,3 metal hydroxides,4 metal sulfides,5 or zerovalent metal particles.6 Producing liposomes containing gold nanoparticles takes all its interest when, for example, one wants to trace liposomes inside living organisms. Indeed, gold particles, as electron-dense metal, can be used as probes to follow biological processes such as liposome internalization and transport inside cells or blood vessels through microscopic techniques.7,8 In this paper, gold nanoparticles were generated into oniontype multilamellar vesicles (MLV). Onions were chosen * Author to whom correspondence should be addressed. E-mail: [email protected]. Tel: (33)556845665. Fax: (33)556845600. † Centre de Recherche Paul Pascal. ‡ Technical University of Eindhoven. § Ben-Gurion University of the Negev. (1) Heywood, B. R.; Eanes, E. D. Calcified Tissue Int. 1992, 50, 149. (2) Mann, S.; Hannington, J. P.; Williams, R. J. P. Nature 1986, 324, 565. (3) Gauffre, F.; Roux, D. Langmuir 1999, 15, 3738. (4) Bhandarkar, S.; Bose, A. J. Colloid Interface Sci. 1990, 139, 541. (5) Khramov, M. I.; Parmon, V. N. J. Photochem. Photobiol., A 1993, 71, 279. (6) Faure, C.; Derre´, A.; Neri, W. J. Phys. Chem. B 2003, 107, 4738. (7) Hong, K.; Friend, D. S.; Glabe, C. G.; Papahadjopoulos, D. Biochim. Biophys. Acta 1983, 732, 320. (8) Thurston, G. J.; McLean, W.; Rizen, M.; Baluk, P.; Hakell, A.; Murphy, T. J.; Hanahan, D.; McDonald, D. M. J. Clin. Invest. 1998, 101, 1401.

because they are hopeful systems for drug delivery. Indeed, this multilamellar system has demonstrated some abilities to deliver oligonucleotides to cells9 and is able to encapsulate a large amount of enzymes10 or DNA without the use of cationic molecules,11 which are cytotoxic molecules. It is also possible to put peptide groups on the surface of the colloids to target specific cells.12 The synthesis of gold nanoparticles from small unilamellar7,13 or multilamellar vesicles14 has already been reported. However, in all these approaches, (i) the addition of either a reductant13 or a basic species7,14 is required and (ii) gold particle formation in lipidic structures is asserted by the usual and conventional electron microscopy technique.7,13,14 As this technique requires vesicles drying and staining to enhance the contrast and reveal the lipids, electron-dense aggregates (artefacts) are formed that could be mistaken for metallic nanoparticles, making particle formation assertion uncertain and location difficult.7,14 Here, reduction occurs in a milder way, without adding any external, perturbing agent since onion components themselves were responsible for gold ion reduction, and the inter(9) Mignet, N.; Bup, A.; Degert, C.; Delord, B.; Roux, D.; He´le`ne, C.; Franc¸ ois, J. C.; Laversanne, R. Nucleic Acids Res. 2000, 28, 3134. (10) Olea, D.; Faure, C. J. Chem. Phys. 2003, 119, 12, 6111-6119 (11) Pott, T.; Roux, D. FEBS Lett. 2002, 511, 150 (12) Chenevier, P.; Delord, B.; Ame´de´e, J.; Bareille, R.; Ichas, F.; Roux, D. Biochim. Biophys. Acta 2002, 1593, 17. (13) Markowitz, M. A.; Dunn, D. N.; Chow, G. M.; Zhang, J. J. Colloid Interface Sci. 1999, 210, 73. (14) Meldrum, F.; Heywood, B. R.; Mann, S. J. Colloid Interface Sci. 1993, 161, 66.

10.1021/cm049251n CCC: $27.50 © 2004 American Chemical Society Published on Web 11/17/2004

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nalization of gold nanoparticles is evidenced by cryogenic temperature-transmission electron microscopy (cryo-TEM). The embedding of nanoparticles between lipid lamellae is clearly demonstrated, revealing a close coupling between the particle shape and the structure of the templating lamellar phase. Two preparative strategies were compared to induce nanoparticle formation within onions: (i) dispersion of preformed onions in a gold salt solution and (ii) formation of a gold salt-containing lamellar phase, subsequently sheared to produce onions, which were thereafter dispersed in water. The latter pathway avoids extravesicular synthesis, which is a real improvement when compared with conventional methods employed for particle synthesis in liposomes, where the internal concentration is not controlled, and a separation step is necessary to avoid extravesicular synthesis.2,4,7,13,15-17 The former pathway leads to both intra- and extravesicular synthesis. Significant differences in particle size and shape are evidenced by TEM and cryo-TEM techniques according to the chosen synthesis pathway. We intend to explain such differences by hypothesizing a simple mechanism of particle growth within multilamellar vesicles. Experimental Section Materials. S100 (mixture of phosphatidylcholine from fatfree soybean lecithin consisting mainly of linoleic phosphatidylcholine) was provided by (Lipoid GmbH). 1-Monooleyl-racglycerol (Monoolein) was purchased from (Sigma Chemical Co) and potassium tetrachloroaurate (III), KAuCl4, came from (Acros Organics). A 0.5-mL conic-shaped microcentrifuge tube and the fitted piston pellet used for the shearing step in onion preparation were from (Eppendorf AG) and (Polylabo), respectively. Onion Preparation and Dispersion. S100 (95 wt %) and Mo (5 wt %) were co-dissolved in cyclohexane, which was thereafter removed by lyophilisation. Roughly 5 mg of the dried mixture and the same amount of aqueous solution (dialyzed water for method A or 10-3 M KAuCl4 for method B) were weighed directly on a 0.5-mL conic-shaped microcentrifuge tube. The mixture was then sheared by manually rotating a piston pellet introduced in the microtube. A minimum of 40 rotations was performed. The obtained onions were dispersed either in 10-3 M KAuCl4 (Method A) or water (Method B) by mechanical rotation using a vortex stirrer (600 rpm). Onion density in the dispersion medium is fixed at 10 mg/mL. The diameter of the onions ranges from 0.2 to 2 µm with a population peak of around 0.3 µm. UV-Vis Spectroscopy. UV-vis absorbency measurements were performed at 25 °C with a UNICAM UV-4 spectrophotometer using cell with a 0.1-cm path length. Transmission Electron Microscopy. TEM measurements were performed on a JEOL 2000FX, working under an acceleration voltage of 200 kV. For the analysis, a drop of the multilamellar vesicle dispersion was deposited onto a carbon film supported by a copper grid and the solvent was allowed to evaporate at room temperature. For samples prepared by mixing directly lipids with 10-3 M KAuCl4 solution (method B), multilamellar vesicles were dispersed in water (3.5 mg of paste/1 mL of water) just before the preparation of the grids. Cryogenic Temperature-Transmission Electron Microscopy. The samples were prepared using a vitrification (15) Kurihara, K.; Fendler, J. H.; Ravet, I.; Nagy, J. B. J. Mol. Catal. 1986, 34, 325-335 (16) Li, T.; Deng, Y.; Song, X.; Jin, Z.; Zhang, Y. Bull. Korean Chem. Soc. 2003, 24, 957 (17) Tricot, Y. M.; Fendler, J. H. J. Phys. Chem. 1986, 90, 33693374

Chem. Mater., Vol. 16, No. 25, 2004 5281 robot (Vitrobot, FEI) in which the relative humidity is kept close to saturation. A 3-µL drop of the solution was placed on a carbon-coated lacey substrate supported by a TEM 300 mesh copper grid (Ted Pella). After automatic blotting, the grid was rapidly plunged into liquid ethane at its melting temperature. This resulted in a vitrified film. The vitrified specimen was then transferred under a liquid nitrogen environment to a cryoholder (model 626, Gatan Inc., Warrendale, PA) into the electron microscope, Tecnai 20, Sphera (FEI), operating at 200 kV with a nominal underfocus of 2-4 µm. The working temperature was kept below -175 °C, and the images were recorded on Gatan 794 MultiScan digital camera and processed with Digital Micrograph 3.6.

Results and Discussion Roux and co-workers have shown that shear causes a dynamic transition from the lamellar phase to multilamellar vesicles (MLV) called “onions”. These onions, in compact texture at first, can easily be dispersed in excess water to provide colloidal dispersions.18,19 In contrast to “conventional” MLV,20 they possess lamellar ordering up to the core of the vesicle21 (Figure 4b). The synthesis of inorganic particles has already been reported for onions composed of surfactant molecules3,6,22 (and not lipids), and an internal growth of the nanoparticles was assumed but without any direct evidence. In this study, we prepared onions from a mixture of lipids, namely, commercially available phosphatidylcholine (S100) and monoolein (Mo). Monoolein was chosen because of its nontoxicity for potential use in biological applications. To obtain hybrid organicinorganic systems, we used 10-3 M KAuCl4 solution as particle precursor. Two protocols already reported for silver nanoparticle synthesis in onions were performed.6 In method A, onion-type MLV are first prepared from shearing 50 wt % of lamellae-forming lipids (S100 and Mo) with 50 wt % water (see Materials and Method section) and are then dispersed in a 10-3 M KAuCl4 solution. In method B, S100 and Mo are mixed with a 10-3 M KAuCl4 solution and the mixture is immediately sheared. The sheared lamellar phase is let at rest for some time (from 1 h to 1 day) before dispersion in water. Synthesis of Gold Nanoparticles When Gold Ions Are Introduced in the Dispersion Medium (Method A). Evidence for Gold Synthesis. When onions are dispersed in KAuCl4 solution (Method A), a color change is observed within 20 min; onion suspension gradually changes from pale yellow to burgundy as shown and measured by UV-vis spectroscopy in Figure 1. The intense reddish-purple color observed after aging the onion suspension in gold salt for 180 min is indicative of isolated small gold particles synthesis.23 The color change results from the excitation of surface plasmon vibrations in the gold nanoparticle. Figure 1 shows the time evolution of the UV-vis absorption spectra of onions dispersed in 10-3 M KAuCl4 for 180 min. A progressive decrease of the absorption band occurs at 320 nm because of charge-transfer associated to the (18) Diat, O.; Roux, D.; Nallet, F. J. Phys. II 1993, 3, 1427. (19) Diat, O.; Roux, D. J. Phys. II 1993, 9. (20) Regev, O.; Khan, A. Prog. Colloid Polym. Sci. 1994, 97, 298. (21) Roux, D.; Gulik, T.; Dedieu, J.; Degert, C.; Laversanne, R. Langmuir 1996, 12, 4668. (22) Kim, D. W.; Oh, S. G.; Yi, S. C.; Bae, S. Y.; Moon, S. K. Chem. Mater. 2000, 12, 996. (23) Grabar, K. C.; Freeman, R. G.; Hommer, M. B.; Natan, M. J. Anal. Chem. 1995, 67, 735.

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Figure 1. Time evolution of UV-vis absorption spectrum of gold nanoparticles synthesized with method A and the corresponding color change. Onions were prepared from 95:5 (wt %/wt %) S100:Mo mixed with 50 wt % dialyzed water, subsequently dispersed in 10-3 M KAuCl4 for (a) 17, (b) 26, (c) 43, (d) 60, (e) 75, (f) 100, (g) 135, and 180 min. Photos of onion dispersions obtained after 17 and 180 min, respectively, are shown: (microtube 1) dispersion in 10-3 M KAuCl4, (microtube 2) dispersion in water.

anionic complex AuCl4-, concomitantly with an increase of the surface plasmon band around 550 nm.24 Both spectroscopic events depict the progressive disappearance of AuCl4- species, that is, the reduction of gold ions to gold nanoparticles. After 135 min of aging (curve g in Figure 1), the UV-vis spectra remain unchanged. It is noticeable that the global absorbance decreases as the gold particle forms. This could arise from the induced sedimentation of onions which density is changing when particles are growing or from the destruction of some onions. Gold Particle Imaging. Gold nanoparticles obtained from onions dispersed in 10-3 M KAuCl4 and aged for 1 day are imaged by TEM (Figure 2a and b). Isolated gold nanoparticles are obtained, polydisperse both in size and shape. Their size ranges from 8 to 105 nm. We can observe classical gold nanoparticle morphologies:25,26 multiply twinned particles such as decahedral types, with either (100) or (111) epitaxed (indicated (L) and (D) respectively in Figure 2), icosahedral crystals (I), flat (111) epitaxed triangular crystals with truncated corners (T). More ambiguous particle shapes whose resolution is not high enough to allow a clear morphology assignment are also observed: sphere-like (S) and elongated (E) particles. Gold Particle Location. Cryo-TEM is a direct imaging technique enabling one to visualize colloids in their native environment. Contrary to other electron microscopy techniques, neither staining nor drying is required when one wants to observe lipidic matrix. Images of onions dispersed in 10-3 M KAuCl4 for 2 and 12 h are displayed in Figure 3 and Figure 4, respectively. The in-situ generation and inclusion of particles between lamellae is unambiguously shown in Figure 4 where (24) Absorption and Scattering of Light by Small Particles; Bohren, C. F., Huffman, D. R., Eds.; Wiley: New York, 1983. (25) Marks, L. D.; Smith, D. J. J. Cryst. Growth 1981, 54, 425. (26) Smith, D. J.; Marks, L. D. J. Cryst. Growth 1981, 54, 433.

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confinement effect is depicted with (i) the presence of lamellar defects around nanoparticles (black arrows), (ii) the bending of bilayers which follow the nanoparticle shape (white arrows), and (iii) the faceting of nanoparticles tangent to the bilayer plane (diamond-shape black arrow). Whatever the dispersion duration, the number of nanoparticles per onion is rather low (1-7) and size is very different from one particle to another (36-107 nm for 12-h-aged onion dispersion). Another important feature is the appearance of classical gold morphologies such as icosahedral type (I in Figure 4 top) in large aqueous, unconstrained domains, that is, in onion domains of poor lamellarity, typically in the core of onions. Asymmetric and facetted particles grow in constrained regions, that is, between lamellae. Some elongated nanoparticles embedded in onions (diamondshape white arrows) are also visualized for a short time dispersion in Figure 3. Their shape is very close to that of nanoparticles synthesized by method B (Figure 2c and d) as will be depicted in the next section. Figure 5 shows that extravesicular gold synthesis also occurs using method A since a triangular, large, gold particle has formed at the MLV surface. Synthesis of Gold Nanoparticles When Gold Ions Are Introduced in the Lamellar Phase (Method B). Gold Particle Imaging. When KAuCl4 solution and lipids are directly sheared, kept 1 day in the onion compact phase, and then dispersed in water (method B), gold nanoparticles are also synthesized as evidenced by electron diffraction analysis (not shown), but both nanoparticle aspect ratio and size are drastically different. Here, nanoparticles are aggregated and have narrower size and shape distributions compared to method A. The majority of the particles are elongated, like rice grains, with an average size of 10 nm*6 nm (Figure 2c and d). Gold Particle Location. The “nonclassical” shape of the gold nanoparticles as well as their monodispersity and their gathering strongly suggest internal nanoparticle formation and growth as already reported for other metallic particles grown in onions.3,6 Moreover, a 6-nm d spacing was measured for the lamellar phase composed of 95:5 (wt %:wt %) S100:Mo and water,11 which suits the particle size supporting an internal synthesis. Eventually, the metallic salt content (50 wt %) is chosen to ensure that no excess aqueous phase coexists with the initial lamellar phase, that is, before the shearing step. Such a choice implies that the obtained onions are in close contact, not diluted, and thus the totality of the Au(III) ions are inside onions in contact with monoolein, the reductant (see next section). Monoolein is in large excess over Au(III) ions so that all the metallic ions are likely to be reduced inside onions before dispersion. The advantage of this method is thus twice: not only the internal content of onion-type MLV can be tuned but also extravesicular particle synthesis is prevented. This makes onions much more interesting than unilamellar vesicles or even classical multilamellar vesicles for particle synthesis. The formation of these vesicles indeed requires starting from a liquid state since sonication or mechanical stirring (usually with glass beads) is needed to produce unilamellar vesicles and MLV, respectively. Consequently, vesicle components are mixed with an excess solution of metallic salt so that

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Figure 2. TEM images of gold nanoparticles synthesized by the two preparative methods. Gold nanoparticles were synthesized in onions prepared from 95:5 (wt %/wt %) S100:Mo mixed with 50 wt % of either dialyzed water or KAuCl4 aqueous solution. (a, b) (method A) Surfactants are mixed with dialyzed water, dispersed in 10-3 M KAuCl4 aqueous solution, and then aged for 1 day. (c, d) (method B) Surfactants are mixed with 10-3 M KAuCl4 aqueous solution and aged for 1 day before dispersing in dialyzed water. Bars: (a, b) ) 100 nm, (c) ) 50 nm, (d) ) 10 nm.

Figure 3. Cryo-TEM image of embedded, elongated gold nanoparticles (diamond-shape white arrows) grown in onions dispersed in gold salt for 2 h. Onions were prepared from 95:5 (wt %/wt %) S100:Mo mixed with 50 wt % of dialyzed water, subsequently dispersed in 10-3 M KAuCl4 aqueous solution (method A). Bar ) 200 nm.

not only the exact internal concentration is unknown, but also extravesicular synthesis cannot be avoided,16,17

unless a separation step (either by column chromatography2,4,15 or dialysis14) is performed to remove metallic ions which are not encapsulated. Particle Nucleation and Growth. No external reductant was needed for Au(0) production in both reported methods, suggesting that onions’ surfactants themselves are responsible for the reduction of Au(III) ions. The spontaneous reduction of Au(III) to Au(0), that is, without addition of an external reducing agent, has already been reported in other confined media: fumed silica treated to produce HO groups,24 OH-containing poly(amidoamine) dendrimers,27 multilamellar vesicles.14 Esumi et al. proposed that the reduction of gold ions occurs through oxidation of alcohol groups into carbonyl ones.27 This hypothesis is valid in our case since we checked that monoolein, the onion supplier of OH groups, acts as a reductant for the gold salt: R-CH(OH)-CH2(OH) being oxidized in R-CH(OH)-CO2H when Au(III) ions are reduced to Au(0).28 According to the colloidal particle nucleation and growth model proposed by La Mer and Dinegar, nucleation occurs when ion concentration reaches supersaturation concentration.29 This condition is likely to be met when gold salt is directly mixed with the lipids (method B, Figure 2c and d) since as already discussed, the advantage of this pathway is that all metallic ions are indeed in onions and thus available for nucleation to occur. Nucleation may thus occur simultaneously at (27) Esumi, K.; Hosoya, T.; Torigoe, K. Langmuir 2000, 16, 2978. (28) Meyre, M.-E, Desbat, B.; Faure, C. Centre de Recherche Paul Pascal (CNRS), (unpublished results). (29) La Mer, V. K.; Dinegar, R. H. J. Am. Chem. Soc. 1950, 72, 4847.

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Regev et al. Scheme 1. Simple Representation of Particle Growth between Bilayersa-d

a (a) Nucleation at bilayer interfaces and “bulklike” growth when particle size is smaller than the lamellar d spacing. (b) Once “transversal” particle size is larger than the lamellar d spacing, growth is slowed by the bilayers, transversal Au(III) flux being limited, leading to elongated particle shapes. (c) When the constraint exerted by particles onto the bilayers is too high, restructuring of lamellae in, e.g., liposomes or oblate-shaped vesicles occurs. (d) Classical multiply twinned particles essentially grown on the onion surface or within onion defect areas such as onion core.

Figure 4. Cryo-TEM image of gold nanoparticles grown in onions dispersed in gold salt for 12 h (method A). Diamondshape black arrows: faceting of particles, black arrows: lamellar defects around nanoparticles, white arrows: bending of bilayers, diamond-shape black arrows: nanoparticles faceting tangent to the bilayer plane. Icosahedral particle (I) is observed in onion core. Bar ) 200 nm.

Figure 5. Cryo-TEM image of a triangular gold nanoparticle grown on the surface of a MLV. Method A was used and the onion dispersion was 12-h aged.

many sites of onion walls and many particles can grow within bilayers. In contrast, in method A, AuCl4- ions diffuse slowly down their chemical potential gradient into onions through successive bilayers acting as a barrier, resulting in a low number of nucleation events and low nanoparticle density. A good indication that nucleation is heterogeneous, that is, it occurs at the bilayer interface, is the fact that nanoparticles display

flat facets tangentially to the bilayers and that external nanoparticles seem to have been synthesized on onion surface (Figure 5). The size of the particle will depend on the nucleation time, which is probably later at the inner part of the onion. This could account for the wide size distribution of particles and the fact that smaller particles seem to have been formed in the inner part of the onion (Figure 4). The comparison of particles synthesized by both preparative strategies leads us to propose a qualitative and hypothesized mechanism for nanoparticle growth. At the initial, unconstrained growth stage, that is, when the particle size is smaller than the d spacing of the onions, particle growth is likely to occur like in bulk solution, resulting in classical morphologies (Scheme 1a). The same pattern is also found when growth occurs in domains of poor lamellarity, for example, in onion core (Scheme 1d, see the icosahedral particle in Figure 4 top). Bulklike growth may continue until particle size becomes closer to the lamellar d spacing. Then, the interfering bilayers may inhibit growth along the direction perpendicular to their planes, gold ion flux being limited perpendicularly to the bilayer plane because of low transmembrane diffusion rate (Scheme 1b). This might explain the gold nanoparticle elongated shape observed for method A for short dispersion time (Figure 3) and method B (Figure 2c and d), where particle growth is certainly stopped at the initial stage because of complete consumption of gold ions. When gold ions are still available, the particle growth goes on, even in the direction perpendicular to the bilayer plane, resulting in bending of the bilayers to accommodate the growing particles as observed in Figure 4, in agreement with the fact that membrane fluidity might help in accommodating particle sizes.30,31 Solid particles whose size is larger than the bilayer spacing cannot be inserted in a lamellar phase by direct mixing.32,33 Above a certain (30) Cooper, S. J.; Sessions, R. B.; Lubetkin, S. D. Langmuir 1997, 13, 7165. (31) Backov, R.; Lee, C. M.; Khan, S. R.; Fanucci, G. E.; Talham, D. R. Langmuir 2000, 16, 6013 (32) Fabre, P.; Casagrande, C.; Veyssie, M.; Cabuil, V.; Massart, R. Phys. Rev. Lett. 1990, 64, 539.

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particle size, the cost in elastic energy for the surrounding bilayers is likely to be too high resulting in bilayer collapse to liposomes or oblate-shaped vesicles (Scheme 1c, Figure 4). It is probable that too many lamellar defects induced by too many large particles would result in onion destruction. That is maybe why only onions with a low number of particles are observed by cryoTEM. When gold salt is introduced in the dispersion medium, gold synthesis may occur on the surface of the outer membrane of onions (Figure 5) giving rise to particles with classical morphologies (Scheme 1d). Conclusion Gold nanoparticles have been successfully generated inside onion-type multilamellar vesicles without any external agent (neither reductant nor basic species); only gold salt and onions were necessary. Two different preparative strategies were compared. When nanoparticles are formed by gold salt diffusion within preformed onions, both internal and external syntheses occur. The number of nanoparticles per onion (33) Wang, W.; Efrima, S.; Regev, O. J. Phys. Chem. B 1999, 103, 5613.

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is quite low (1-7) while they are quite large and polydisperse in both size (35-105 nm) and shape. They have been unambiguously located between MLV leaflets by cryo-TEM experiments. This technique reveals a close coupling between in-situ synthesized particles and lamellae. TEM observations on onions made by direct mixing of gold salt with S100-monoolein mixture show aggregates of smallest nanoparticles (10*6 nm*nm), monodisperse both in size and shape suggesting an internal gold nanoparticle formation. Comparison of TEM and cryo-TEM observations of gold particles grown by both preparative strategies led us to propose a simple mechanism of particle growth within MLV. Acknowledgment. Israel-France Arc en Ciel program (Project 38) is acknowledged for providing traveling allowance (O.R.). This research was partially supported by the Center of Excellence, “Origin of Ordering and Functionality in Mesostructured Hybrid Materials”. Supported by The Israel Science Foundation (under Grant No. 800301-1). CM049251N