Magnetic Multilamellar Liposomes Produced by In Situ Synthesis of

Jun 2, 2009 - Centre de Recherche Paul Pascal, CNRS−Université Bordeaux 1, Avenue du Dr Schweitzer, 33600 Pessac, France, Architecture de ...
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Magnetic Multilamellar Liposomes Produced by In Situ Synthesis of Iron Oxide Nanoparticles: “Magnetonions” Chrystel Faure,*,† Marie-Edith Meyre,† Sylvain Tre´pout,‡ Olivier Lambert,‡ and Eric Lebraud§ Centre de Recherche Paul Pascal, CNRS-UniVersite´ Bordeaux 1, AVenue du Dr Schweitzer, 33600 Pessac, France, Architecture de complexes membranaires et processus cellulaires, CBMN UMR CNRS 5248-UniVersite´ Bordeaux 1-IECB, Baˆtiment B8-AVenue des Faculte´s, F-33405 Talence, France, Institut de Chimie de la Matie`re Condense´e de Bordeaux, CNRS-UniVersite´ Bordeaux 1, 87, AVenue du Dr Schweitzer, 33608 Pessac Cedex, France ReceiVed: February 6, 2009; ReVised Manuscript ReceiVed: May 4, 2009

We report the formation of magnetic onion-type multilamellar vesicles. Iron oxide nanoparticles (np’s) were synthesized inside lipidic multilamellar vesicles by coprecipitation of vesicle-encapsulated Fe2+ and Fe3+ ions induced by HO- diffusion through vesicle lamellae. The iron ion encapsulation efficiency of onions was measured by potentiometry and UV-vis absorbance spectroscopy. Its high value (75 ( 5% for both Fe2+ and Fe3+) ensures an intravesicular synthesis, as confirmed by cryo-transmission electron microscopy (TEM) imaging. The as-grown nanoparticles are characterized by X-ray diffraction analysis and TEM, and magnetic onions are imaged by cryo-TEM. The np size, controlled by temperature and time, ranges from 3 to 6 nm and is shown to be a key parameter for onion stability. Introduction Iron oxides nanoparticles (np’s) have drawn considerable interest in biotechnology for the past 30 years because of their potential application in many medical and biological applications, such as cell separation,1-3 DNA purification,4 gene targeting,5 immunomagnetic assays,6 magnetic resonance imaging (MRI),7-12 and hyperthermia.13,14 They have also been considered as drug delivery systems.15-17 However, the latest application requires a careful design of the np’s. Nanoparticle surface modifications are necessary18 to, for example, avoid their aggregation (stabilization by dextran,19 chitosan,20 or synthetic polymer-21,22 have been exploited) or to bind the active drugs or the targeting ligands.18,23 Moreover, drawbacks such as desorption or limitation of grafted drug molecules by the number of ligands on the np surface constrain the development of magnetic np’s as drug delivery systems. In many points, “capsules” such as liposomes are more appealing for drug delivery applications. Liposomes are, indeed, chemically inert, and then biocompatible and biodegradable. They may encapsulate a large amount of drugs in their inner core, increasing the duration of the drug action, reducing drug toxicity, protecting them from factors that could inactivate them, and ensuring a prolonged release of the drugs.24 A challenge in drug delivery would then consist of combining the advantages of liposomes and magnetic np’s by making lipidbased vesicles containing magnetic np’s. Nanoparticles would add new functionalities to liposomes, allowing organ targeting by magnetic field induced-transport, vesicle imaging by MRI, and drug delivery triggering by hyperthermia, whereas the lipidbased vehicle would ensure biocompatibility, a large amount of transported drug, and drug protection. However, one of the main drawbacks of liposomes, as compared, for example, to * Corresponding author. Phone: 0033556845665. Fax: 0033556845600. E-mail: [email protected]. † Centre de Recherche Paul Pascal. ‡ Architecture de complexes membranaires et processus cellulaires. § Institut de Chimie de la Matie`re Condense´e de Bordeaux.

other candidates for drug delivery, such as polymer capsules, is their poor stability due to the thin hydrophobic layer forming the liposome membrane,25,26 thus limiting their biological use. Such a drawback can be circumvented using multilamellar vesicles, such as onions, which present a sharply increased in vivo stability27 conferred by the succession of membranes up to their core,28-30 while keeping all the liposome advantages. Encapsulation of different types of biomolecules (DNA,31 enzymes,32 oligonucleotides,33 etc.) has, indeed, been performed using onion-type multilamellar vesicles, and their biological appeal has largely been shown.34-39 If many works have been reported on the design of magnetic np’s wrapped by a lipid membrane, the so-called “magnetoliposomes”,40-48 more seldom are the results of the production of lipidic vesicles containing magnetic np’s. Recently, Martina et al. succeeded in producing such unilamellar lipid-based vesicles containing magnetic np’s.49 To get them, they started from preformed np’s (ferrofluid) they encapsulated into unilamellar vesicles. In the 1990s, Mann et al. developed another method to get np-loaded liposomes, inspired by the classical chemical pathway to synthesize iron oxide np’s. They used preformed unilamellar liposomes in which iron ions were encapsulated and then used a pH gradient to achieve transmembrane transport of OH- to induce precipitation and formation of the iron oxide np’s into the vesicles.50 Here, we report for the first time how to produce onion-type multilamellar vesicles containing magnetic np’s, the so-called “magnet-onions”. Nanoparticles are grown inside the onions by the method initially developed by Mann et al.: that is, by coprecipitation of Fe3+ and Fe2+ ions encapsulated into the onions due to the diffusion of alkaline species from the external phase. The presence of np’s inside onions, as well as the multilamellar structure of the inorganic-organic hybrids, is clearly evidenced by cryo-TEM imaging.

10.1021/jp901105c CCC: $40.75  2009 American Chemical Society Published on Web 06/02/2009

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Figure 1. (a) Image of Fe3+-containing onions after centrifugation. Fe3+ ions solutions absorb at 330 nm (enlarged graph in b), and calibration curves have been made from the supernatant (b) and from the sediment (d) after TX100 addition to measure Fe3+ concentration outside and inside the onions, respectively. For Fe2+ ions, a potentiometric titration was performed using the oxidation reaction of Fe2+ by MnO42- ions (c).

Materials and Methods Materials. S100 (PC) is a mixture of phosphatidylcholine (ca. 94%), lysophosphatidylcholine (ca. 3%), and phosphore (ca. 3%) from fat-free soybean lecithin, consisting mainly of linoleic phosphatidylcholine. It was provided by Lipoid GmbH, Ludwigshafen. 1-Monooleyl-rac-glycerol (monoolein) was purchased from Sigma Chemical Co, St Quentin Fallavier. Iron(II) chloride nonahydrate (FeCl2 · 9H2O) and iron(III) chloride

hexahydrate (FeCl3 · 6H2O) came from Sigma-Aldrich, Steinheim. Sodium hydroxide (NaOH) was from SDS, Peypin. Permanganate potassium (KMnO4) was from Merck, Darmstadt. Nanoparticle-Containing Onion Preparation. Preparation of onion-type multilamellar vesicles has been described in detail elsewhere.30,51 It mainly consists of shearing a lamellar phase using a homemade Couette cell. A tube (1 mL, 3 mm diameter, Polylabo, Fontenay-sous-bois) is filled with lamellar phase

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Figure 2. (a) Typical TEM image of magnetic nanoparticles grown inside onions. Size distribution of np’s grown in onions at (b) 4 °C (sample L9), and (c) 25 °C (sample H9).

(roughly 50 mg) and placed inside the stator chamber. The stator is then vertically translated to insert the rotor inside the tube, defining a gap between the stator and rotor of 15 µm. The rotor speed may be changed from 100 to 1500 rpm, the maximum shearing rate being 3.2 × 104 s-1. Shearing of the lamellar phase is applied for 5 min at 2.5 × 104 s-1, and the resulting onions are then diluted/dispersed in an excess of the aqueous phase. Here, the lamellar phase consists of a mixture of lipids (S100monoolein, 90-10 mol %) hydrated, in the proportion 55/45 wt % (lipids/aqueous phase), by a buffered solution (pH ) 4.85) containing Fe2+ and Fe3+ ions. The concentration of iron ions in the buffer is 0.1 M, and the Fe2+/Fe3+ molar ratio is fixed either to 2/3 or 1. Buffer is composed of acetic acid and acetate ion in equal amounts (0.5 M). The dispersion solution consists of a basic NaOH (10-3 M) solution (pH ) 10). The osmotic pressure of the internal and external/dispersing phases was measured: 1750 and 1950 mOsmol for the iron molar ratio of 2/3 and 1, respectively. A NaCl solution (ca. 1 M) was added in the external phase to ensure osmolar equilibrium and avoid onion destruction.

UV-Visible Spectroscopy. UV-visible absorbance measurements were performed at 25 °C with a Unicam UV-4 spectrophotometer operating at λ ) 330 nm, using a 1 cm path length cell. This technique was used to determine the Fe3+ ions concentration inside and outside onions. To perform these measurements, onions were separated from the dispersing medium by centrifugation (7000 rpm, 20 min, 4 °C). Both the supernatant and sediment were then treated with a Triton X-100 solution (TX100) to solubilize onions to get a clear Fe3+containing solution. Fe3+ concentrations in the supernatant (i.e., in the external phase) and in the sediment (i.e., in the internal phase) were deduced from calibration curves. Those curves were obtained from Fe3+ solutions containing TX-100 in the concentration needed for onion solubilization. Potentiometric Measurements. Fe2+ ions were assayed by potentiometric titration. The working electrode was a platinum electrode, and the reference electrode was a saturated calomel electrode. Potassium permanganate solution (65 µM) was added to the Fe2+-containing solution, and the electric potential (E) of the solution was monitored as a function of the added

Magnetic Multilamellar Liposomes: “Magnetonions” K2MnO4 volume (V). From the equivalence volume determined on the E-V titration curve, the molar number of Fe2+ ions in the assayed solution is deduced. Here, onions were separated from the dispersing medium by centrifugation (7000 rpm, 20 min, 4 °C), and only the supernatant (containing nonencapsulated ions) was assayed. Indeed, onions (and even the TX-100 solubilized onions) impede potential measurements, certainly because of the adsorption of lipids (or surfactants, or both) onto the working electrode surface. From the amount of nonencapsulated Fe2+ ions and knowing the total amount of Fe2+ ions used to prepare the onion dispersion, the amount of encapsulated ions was deduced. Transmission Electron Microscopy (TEM). TEM measurements were performed on a JEOL 2000FX, working under an acceleration voltage of 200 kV. For nanoparticle imaging, onion dispersions (3.5 mg/1 mL) were submitted to ultrasound (duty cycle 80%, power 10 W, time 20 min) to destroy the onions and liberate the entrapped np’s. One drop of the ultrasonicated solution was then deposited onto a carbon film supported by a copper grid; water was partly absorbed with a soft tissue and then allowed to evaporate at room temperature. Observations were performed just after grid preparation. Size distributions of iron oxide nanoparticles were determined considering at least 100 particles to ensure a correct statistical analysis. Preparation of Frozen Hydrated Specimens and CryoTEM. Multilamellar vesicles were diluted in water (2 mg/mL). A 5 µL sample was deposited onto a perforated, carbon-coated, copper grid; the excess was blotted with a filter paper; and the grid was plunged into a liquid ethane bath cooled with liquid nitrogen (Leica EM CPC). Specimens were observed at a temperature of -170 °C using a cryo holder (Gatan, Evry), with a FEI Tecnai F20 electron microscope operating at 200 kV. Low-dose electron micrographs were recorded at a nominal magnification of ×50 000 on a 2k × 2k slow scan CCD camera (Gatan, Evry). X-ray Diffraction. The in situ synthesized nanoparticles were characterized by X-ray diffraction. Onion dispersions were freeze-dried so that powders were analyzed. The X-ray diffraction experiments were performed on a theta-theta Bragg-Brentano geometry diffractometer equipped with a secondary graphite monochromator. The samples were prepared by depositing the powder on a sample holder and using a copper anode (wavelength: 1.5418 Å). Each measurement lasted 50 min and was done over the angular range 25-95° (2θ) with 0.02° (2θ) steps. The phase identification was performed with Bruker AXS Diffracplus EVA software (Version 11.0.0.2, Karlsruhe, Germany, 2007) Results and Discussion 1. Fe 2+ and Fe3+ Ions Encapsulation Efficiency. The encapsulation efficiency of Fe2+ and Fe3+ ions (ratio of the number of ions contained in the internal volume of onions to the total number of ions used to prepare onions) was assessed to ensure that most of the nanoparticles would grow within the onions. Fe2+ encapsulation efficiency was measured by electrochemical assay; the Fe3+ one, by absorbance spectroscopy. In both cases, onions were separated from the extravesicular solution by centrifugation (see Materials and Methods). Figure 1a shows an image of a dispersion of Fe3+-containing onions after centrifugation: a brown precipitate composed of onions (checked by phase contrast microscopy) is visible in the bottom of the flask; the supernatant does not present such a color. The brown tint is due to FeCl3 solution and indicates

J. Phys. Chem. B, Vol. 113, No. 25, 2009 8555 TABLE 1: Iron Oxide Nanoparticle Size Determined by TEM for Samples Differing in Fe(II)/Fe(III) Molar Ratio, Incubation Time (Age), and Temperature sample

nFe(II)/nFe(III)

T (°C)

age (days)

np size (nm)

H1 H9 L9 B6 B9

2/3 2/3 2/3 1 1

25 25 4 4 4

1 9 9 6 9

3(1 6(1 4(1 3(1 4(1

that Fe3+ ions are mainly present within the onions. The absorption band of Fe3+ ions is located at 330 nm, as shown in the UV-vis spectrum shown in Figure 1b. Calibration curves were made from different FeCl3 solutions, all of them containing the surfactant TX-100 in concentrations used to solubilize onions either in the supernatant (Figure 1b) or in the sediment (Figure 1d). TX-100 addition indeed induces a slight change of the calibration curve slope. From these curves, one can deduce the number of Fe3+ ions that are present in the supernatant and the precipitate after onion destruction by TX-100. From these measurements, the encapsulation efficiency was found to be 77 ( 5%. The encapsulation efficiency of Fe2+ was deduced from potentiometric measurements (see Material and Methods). The encapsulated Fe2+ ions could not have been assayed directly on the sediment, since the large quantity of surfactants (onion components and TX-100) adsorb onto the electrode surface, impeding any direct electrochemical measurements. On the contrary, a classical titration curve of the supernatant after TX100 treatment was obtained (Figure 1c). From the equivalent volume, one can determine Fe2+ concentration in the supernatant, and knowing the global concentration in the sample, one can deduce the encapsulated Fe2+ concentration and thus the encapsulation efficiency. A value of 75% was found. Let us note that this value is underestimated, since the smallest onions may have remained in the supernatant. For both types of iron ions, the percentage of encapsulated ions is high, roughly 75%. This high value can be due to the strong affinity between lipid headgroups and metal ions.44,50,52,53 This affinity has already been evidenced in the case of dimyristoylphosphatidylcholine molecules interacting with Fe2+ and Fe3+ ions.50,54 Here, onions are composed mainly of S100, a mixture of phosphatidylcholine that bears a phosphate (PO42-) group. Phosphate groups may then display attractive Coulombic interactions with Fe2+ and Fe3+ ions, forming complex lipid-iron ions, as already suggested.55,56 Because phosphate groups are in large excess over iron ionssthe molar ratio between S100 and iron ions is 15smost of the ions are trapped inside onions, ensuring high encapsulation efficiency, and then an essentially intravesicular growth of the iron oxide np’s. 2. Effect of Aging, Temperature and Fe2+/Fe3+ Molar Ratio on Nanoparticle Size. Table 1 shows the average size of the iron oxide nanoparticles synthesized inside the onions under different experimental conditions. Nanoparticle size distributions were determined from TEM imaging after onion destruction by ultrasound. In all cases, quasispherical nanoparticles have been synthesized, as shown in Figure 2a. It is noteworthy that np’s are not aggregated but isolated, suggesting that onion components are present around np’s, probably forming a bilayer. Generally, such structures, called “magnetoliposomes”, are obtained by applying ultrasound on a solution containing both preformed np’s and lipids41,42,46 or by molecular exchange between surfactants covering the np surface and lipids introduced via small unilamellar vesicles.43-45 Applying ultra-

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Figure 3. Time evolution of size distributions (a, c) and normalized size distributions (b, d) for nanoparticles grown at 4 °C (a, b) and 25 °C (c, d).

sound directly on np-containing onions could then constitute another pathway to get such magnetic np’s wrapped by lipid membranes. Nanoparticle size depends on temperature and sample aging and is slightly affected by the Fe2+/Fe3+ molar ratio of the solution used for np synthesis, as seen in Table 1. However, in all cases, nanoparticles are smaller than the smectic distance of the onion lamellar phase (i.e., 6 nm30,51) suggesting an intralamellar synthesis. This effect has already been observed in the case of gold np’s grown into onions by spontaneous reduction of the encapsulated gold precursor.30 This has also been shown in other constrained media, such as microemulsions in which the size of the iron oxide np’s is controlled by the size and, thus, the volume of the droplets when the coprecipitation method is employed to synthesize np’s.57,58 Comparing L9 with H9 shows that the average np size, as well as their size dispersity, increases with synthesis temperature. This is clearly shown in Figure 2, where the np size distribution is given for np’s grown at 4 °C (Figure 2b) and 25 °C (Figure 2c). This result is not surprising and has already been reported

in the case of bulk synthesis of magnetite particles59 and for magnetite/maghemite nanoparticles synthesized inside reverse micelles.60 This temperature effect is usually attributed to an acceleration of the collisions between nuclei, precursors, or both, leading to an accelerated growth of nanoparticles. Comparing H1 with H9, and B6 with B9, shows that the average np size also increases with sample aging: the longer onions have been in contact with the HO--containing solution, the larger the np’s are. More precisely, the smallest particles disappear for the benefit of the largest ones with time (Figure 3 a, c). To assess whether Ostwald ripening is responsible for np growth, the normalized size distributions were plotted. Figure 3 displays size distributions (a, c) and the corresponding normalized size distributions (b, d) for both series of dispersions (B6/B9, H1/H9). In both cases, normalized size distributions are superimposed (Figure 3b, d), revealing that nanoparticle growth is due mainly to Ostwald mechanism.61-63 This indicates that HO- diffusion does not influence np size at these time scales. Eventually, comparing L9 with B9 indicates that the molar ratio between both types of ions does not seem to significantly

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Figure 5. Image showing the attraction of nanoparticle-containing onions induced by a 0.2 T magnet (sample B6). Figure 4. Typical XRD spectrum obtained on iron oxides-containing onions after freeze-drying. Peaks are indicated for NaCl (solid arrows), goethite (asterisks), and maghemite or magnetite (open arrows).

affect np size. These high molar ratios (2/3 and 1) were chosen because some Fe2+ ions oxidize spontaneously during synthesis.44 3. X-ray Analysis of the In Situ Grown Nanoparticles. To identify nanoparticles, X-ray diffraction analysis was carried out on all types of dispersions after onion destruction (see Material and Methods). Whatever the synthesis conditions (i.e. whatever sample age, iron ions molar ratio, and temperature synthesis), the as-grown nanoparticles display the same XRD pattern (Figure 4). Six peaks correspond to NaCl crystals (indicated by full arrows): the corresponding angles are 31.7°, 45.5°, 56.6°, 66.4°, 75.3°, and 84.0° which correspond to {200}, {220}, {222}, {400}, {420}, and {422} planes, respectively. NaCl is, indeed, added into the external basic solution to ensure osmolar equilibrium (see Material and Methods). Five other reflection peaks (indicated by stars) are detected at 26.4°, 40.0°, 47.7°, 48.9°, and 50.1°. These diffraction angles can be attributed to goethite, R-FeOOH.64 The XRD pattern of the powdered sample also displays the characteristic peaks of a spinel phase at 31°, 35,7°, 43.3°, and 53.8° (indicated by white arrows), which correspond to the {220}, {311}, {400}, and {422} planes, respectively, of the spinel phase.65,66 These peaks can be attributed either to maghemite (γ-Fe2O3) or to magnetite (Fe3O4). Due to peak broadening, we are unable to distinguish between the two iron phases. However, since the precipitation reaction is carried out in air, it is reasonable to expect that our samples contain maghemite particles. Peak broadening can be attributed to the presence of the lipid phase around np, the poor crystallinity of the particles, or both. Such broadening is typically observed for np synthesized in an organic matrix.46,48,52,57,65 Different crystalline phases are then detected in our samples: goethite, magnetite or maghemite, or both. Maghemite usually results from the O2-induced oxidation of magnetite, which itself results from the precipitation of ferrous and ferric ions in the stoichiometric molar ratio of 0.5 (Fe2+/Fe3+). Here, we worked in oxidizing conditions (no particular attention has been paid to remove O2 from water and air). It is not then surprising that maghemite was detected in our samples. Maghemite can also be synthesized if the molar ratio between both iron ions is smaller than 0.5. In our case, the initial molar ratio was 2/3 or

1, but part of the Fe2+ ions are likely to be oxidized by O2. What’s more, as already mentioned, iron ions are complexed by the phosphatidylcholine headgroup so that their mobility is drastically reduced as compared to the bulk conditions. The local concentration of both ions is not controlled at all, and different stoichiometries in iron ions could then be found within onions. Concerning the formation of goethite inside onions, one can refer to the work of Mann,50 who produced such oxide from Fe3+-containing liposomes. One can guess that this crystalline structure results from precipitation occurring in Fe3+-rich areas of the onions. 4. Internal Character of the np Synthesis and Stability of Nanoparticles/Onion Hybrids. An easy way to ensure that magnetic nanoparticles have been synthesized inside vesicles is to observe their behavior under a magnet-induced field. A dispersion of np-loaded onions was then centrifuged to sediment and concentrate them, and Figure 5 confirms their attraction by a magnet (0.2 T), revealing the magnetic properties of npcontaining onions. To strengthen this indirect evidence of intravesicular synthesis, a direct imaging of onions was performed using cryoTEM. This noninvasive technique is also ideal to study the influence of nanoparticles on the stability of onions. Figure 6 shows cryo-TEM images of onions in which nanoparticles were grown for different incubation times. As seen in Figure 6, the onion size is about 200 nm. It is noteworthy that no np’s are observed outside the onions. On the contrary, np’s are always present inside the onions. These np’s are either isolated and then hardly visible because of their tiny size (black arrows), or they are aggregated inside the onions (white arrows). The enlarged image of Figure 6a shows that np’s grow mainly into the aqueous compartments of the lamellar phase, distorting the lamellae for them to accommodate aggregates. Lamellar distortion has already been observed in the case of large, gold np’s grown inside onions.67 The influence of time on onion stability is enhanced in Table 2. Table 2 reports the percentage of vesicles (either unilamellar, multilamellar, or onion-type vesicles) that contain np’s, as well as the percentage of onions amid all these np-containing vesicles as a function of sample age. The first remark concerns the efficiency of this method to produce intravesicular synthesis, since all the observed vesicles contain nanoparticles. Second, the longer the contact time between the preformed onions and

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Faure et al. and the more disturbed the internal lamellarity, then driving to a lower percentage of onion-type vesicles. Conclusions We succeeded in producing onion-type multilamellar vesicles containing magnetic nanoparticles. These hybrids have been produced by coprecipitation of iron ions encapsulated inside the vesicles, induced by OH- diffusion through onion lamellae. The high encapsulation efficiency of both ions (75%) ensures an intravesicular synthesis. Different iron oxides are producedsmagnetite, maghemite, goethitescertainly because of an inhomogeneous repartition of Fe2+ and Fe3+ ions inside onions. Cryo-TEM imaging reveals np growth occurs between lamellae, mainly into the aqueous compartments of the lamellar phase. Nanoparticle size increases with time, and Ostwald ripening is believed to be responsible for np growth. Stability of such hybrids is affected by time, suggesting an effect of np size on the internal lamellar structure. These np-loaded onions are currently under investigation to characterize their magnetic properties and to evaluate their potential interest for hyperthermia and MRI applications. Acknowledgment. This work has been supported by the “FAME” European Network of Excellence of the EU 6th FP. The authors thank E. Sellier from the CREMEM (Bordeaux) for performing TEM imaging. References and Notes

Figure 6. Cryo-TEM images of np-containing onions after dispersion in a NaOH solution for (a) 2 and (b) 9 days. The scale bar is for both images. Black arrows indicate “isolated” np’s; white arrows, “aggregated” nanoparticles.

TABLE 2: Effect of Aging on Stability of np-Containing Onions (Molar Ratio ) 1, T ) 4 °C) age (days)

np-containing vesicles (%)

np-containing onions

2 6 9

100 100 100

61 46 34

the basic solution (the so-called sample age), the less numerous the onions are. In other words, the internal lamellarity of onions is disturbed by nanoparticle growth giving unilamellar or, more generally, multilamellar vesicles. This is observed in Figure 6b, where the core of the onions is destroyed, forming a multilamellar vesicle, as compared to Figure 6a, where onions are visible, since bilayers are present up to the vesicle center. The influence of np size on onion stability has already been enhanced in the case of gold np’s spontaneously generated inside onions.30,67 We showed by X-ray analysis that the internal structure of onions is affected when the internally grown np reaches a size similar, or larger than the smectic distance of the onion lamellae. For S100-monoolein-based lamellae, a 6.3 nm periodic distance was measured.30,51 This distance does not seem to be drastically affected by salt addition, since a value of ∼6 nm is found from cryo-TEM images and corresponds to the np size after 9 days of incubation in the basic solution (Figure 6b). In conclusion, the more aged the samples, the larger the np size

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