Interactions Between Giant Unilamellar Vesicles and Charged Core

pubs.acs.org/Langmuir © 2010 American Chemical Society

Interactions Between Giant Unilamellar Vesicles and Charged Core-Shell Magnetic Nanoparticles Mathieu Laurencin, Thomas Georgelin, Bernard Malezieux, Jean-Michel Siaugue, and Christine M
enager* UPMC University of Paris 06-CNRS-ESPCI Laboratoire Physicochimie des Electrolytes, Colloı¨des et Sciences Analytiques PECSA UMR 7195, 4 place Jussieu, 75252 Paris, France Received June 10, 2010. Revised Manuscript Received September 1, 2010 This work combined two tools, giant unilamellar vesicles (GUVs) and core-shell magnetic nanoparticles (CSMNs), to develop a simplified model for studying interactions between the cell membrane and nanoparticles. We focused on charged functionalized CSMNs that can be either cationic or anionic. Using optical, electron, and confocal microscopy, we found that giant vesicle-nanoparticle interactions did not result from a simple electrostatic phenomenon because cationic CSMNs tended to bind to positively charged bilayers, whereas anionic CSMNs remained inert.

Introduction Many biological phenomena involve interactions between the cell plasma membranes and nanometric entities such as drugs, pathogens, protein complexes, or glycoconjugates. Although cell membranes comprise a wide variety of components, the major constituent is the lipid bilayer that acts as a physical barrier separating the cytoplasm from the outside environment but also regulating the movements of molecules into and out of the cells. The basic building blocks are amphipathic phospholipids, which assemble spontaneously in a continuous thin lipid bilayer. Giant unilamellar vesicles (GUVs) are often used to mimic the cell membrane’s behavior in a simplified environment.1 As their size ranges from 1 to 100 μm and they show a cell-like external curvature, GUVs are easily observable using light microscopy. Besides studies on their morphology or their mechanical properties, GUVs provide good models to investigate the interactions between lipid bilayers and nonlipid molecules such as DNA, surfactants, polymers, or peptides.2-5 Recently, GUVs have been revealed to be suitable tools to investigate interactions between nanoparticles and phospholipid bilayers. They are versatile models that allow the investigator to discriminate the forces that drive or even prevent the interactions. The ability of nanoparticles to target, label, and/or penetrate lipid bilayers has resulted in great interest in correlating these phenomena with nanoparticle penetration into cells or even with nanoparticle-related cytotoxicity. Currently, two mechanisms for the transport of nanoparticles into mammalian cells are being debated: endocytosis-mediated transport and passive transmembrane uptake. Most nanoparticle entry mechanisms have been claimed to be dependent on endocytotic pathways, as demonstrated by the use of specific inhibitors.6 Nevertheless, the situation is not clear because the uptake of *Corresponding author. E-mail: [email protected]. (1) Luisi, P. L.; Walde, P., Eds. Giant Vesicles, Perspectives in Supramolecular Chemistry; John Wiley and Sons, Ltd.: West Sussex, England, 2000; Vol. 6. (2) Angelova, M. I.; Tsoneva, I. Chem. Phys. Lipids 1999, 101(1), 123–137. (3) Babnik, B.; Miklavcic, D.; Kanduser, M.; H€agerstrand, H.; Kralj-Iglic, V.; Iglic, A. Chem. Phys. Lipids 2003, 125(2), 123–318. (4) Decher, G.; Kuchinka, E.; Ringsdorf, H.; Venzmer, J.; Bitter-Suermann, D.; Weisgerber, C. Angew. Makromol. Chem. 1989, 166(1), 71–80. (5) Yamashita, Y.; Masum, S. M.; Tanaka, T.; Yamazaki, M. Langmuir 2002, 18(25), 9638–9641. (6) Hillaireau, H.; Couvreur, P. Cell. Mol. Life Sci. 2009, 66(17), 2873–2896.

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nanoparticles has also been observed into cells lacking endocytotic capabilities, such as erythrocytes.7 Magnetic liposomes (giant or large vesicles containing maghemite nanoparticles) have been used unambiguously to illustrate the irreversible trapping of small nanoparticles inside vesicles.8 The leakage of such maghemite nanoparticles from the liposomes has been detected through the action of octyl-β-D-glucopyranoside, a surfactant responsible for inducing transient pore formation. Furthermore, no passive transmembrane transport across a lipid bilayer has been reported for gold nanoparticles entrapped into GUVs composed of phosphatidic acid (PA), phosphocholine (PC), and 30% cholesterol.9 Recent studies have demonstrated that preliminary electrostatic interactions are necessary to induce the adhesion of charged nanoparticles to GUVs, as described for small anionic peptidederived quantum dots that cover positively charged liposomes uniformly.10 In that case, the quantum dots remained tethered and adsorbed onto the outer surface of the GUVs. Another example has underlined that both anionic and cationic polystyrene nanoparticles can be adsorbed onto the zwitterionic PC headgroups of phospholipids, which provide particle-stabilized liposomes.11,12 Oppositely charged cationic and anionic polystyrene nanoparticles were described as having opposing effects on the fluidity of the membrane of pure 1,2-dioleoyl-sn-glycero3-phosphocholine (DOPC) vesicles.13 The nonspecific adsorption of the anionic polystyrene nanoparticles induced local gelation of the bilayers by increasing the tilt angle of the PC headgroups, which increases phospholipid density. By contrast, cationic nanoparticles tend to reduce lipid density by reducing the tilt angle of the PC headgroup. (7) Geiser, M.; Rothen-Rutishauser, B.; Kapp, N.; Sch€urch, S.; Kreyling, W.; Schulz, H.; Semmler, M.; Im Hof, V.; Heyder, J.; Gehr, P. Environ. Sci. Technol. 2006, 40(14), 4353–4359. (8) Lesieur, S.; Grabielle-Madelmont, C.; M
enager, C.; Cabuil, V.; Dadhi, D.; Pierrot, P.; Edwards, K. J. Am. Chem. Soc. 2003, 125(18), 5266–5267. (9) Banerji, S. K.; Hayes, M. A. Langmuir 2007, 23(6), 3305–3313. (10) Dif, A.; Henry, E.; Artzner, F.; Baudy-Floc’h, M.; Schmutz, M.; Dahan, M.; Marchi-Artzner, V. J. Am. Chem. Soc. 2008, 130(26), 8289–8296. (11) Zhang, L.; Granick, S. Nano Lett. 2006, 6(4), 694–698. (12) Yan, Y.; Anthony, S. M.; Zhang, L.; Bae, S. C.; Granick, S. J Phys. Chem. C 2007, 111(23), 8233–8236. (13) Wang, B.; Zhang, L.; Bae, S. C.; Granick, S. Proc. Natl. Acad. Sci. U.S.A. 2008, 105(47), 18171–18175.

Published on Web 09/24/2010

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It has also been proposed that strong nanoparticle interactions with the lipid membrane appear to serve as a prelude to translocation.14 The passive transport of core-shell γ-Fe2O3@SiO2 magnetic nanoparticles into neutral DOPC LUVs was described in this report and was assigned to the strong affinity between the silica-based nanoparticle surface and the lipid membrane. It is worth mentioning that internalization occurred only for those nanoparticles with a diameter exceeding 20 nm, whereas smaller ones remained outside the liposomes. Obviously, nanoparticle entry into cells is dependent on numerous interconnected factors, such as their charge, shape, size, or surface composition. Furthermore, comparisons between studies involving living cells remain equivocal because the experimental conditions (incubation conditions, cell treatment, or type of cell line) have varied.15 Here, we studied the interaction between core-shell magnetic nanoparticles (CSMNs) and GUVs. Besides their compatibility with cells, bifunctionalized CSMNs can be either positively or negatively charged, and GUVs offer the possibility to test various lipid mixtures, allowing us to tune the global net charge of the membrane.

Experimental Section Materials. 3-(N-Morpholino)-propanesulfonic acid (MOPS), tetraethylorthosilicate (TEOS), 3-aminopropyltriethoxysilane (APTS), rhodamine isothiocyanate (RITC), and fluorescein isothiocyanate (FITC) were purchased from Sigma-Aldrich (SaintQuentin Fallavier, France); 2-(methoxy(polyethyleneoxy)propyl)trimethoxysilane (PEOS) was purchased from Gelest, Inc., Morrisville, PA, USA). DOPC, 1,2-dioleoyl-sn-glycero-3-phospho(10 -rac-glycerol) (DOPG), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), and fluorescent 1,2-dioleoyl-sn-glycero3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rh-DOPE) were all purchased from Avanti Polar Lipids (Alabaster, AL, USA).

Synthesis and Characterizations of Cationic AminoPoly(ethylene glycol) (PEG) Core-Shell Nanoparticles and Anionic Carboxy-PEG Core-Shell Nanoparticles. Magnetic maghemite nanoparticles (γ-Fe2O3, 7 nm mean diameter) were prepared following the procedure described by Massart and were coated subsequently with citrate anions.16,17 A first silica shell was prepared in ethanolic medium in the presence of ammonia by condensing TEOS.18 Two fluorescent dyes bearing an isothiocyanate functional group (FITC or RITC) were coupled previously to the amino group of APTS through an addition reaction. During the first step, stable fluorescent dyes were incorporated within the first silica shell by cohydrolyzing (co-condensation) with TEOS. The silica shell was made using a second step by simultaneous condensation of APTS and a silica PEG-derived compound, PEOS, (molar ratio APTS/PEOS = 1), with an appropriate amount of TEOS to generate a cross-linked silica shell.19,20 This synthesis produced a colloidally stable CSMN type with a mean diameter of 34 ( 6 nm as measured by transmission electron microscopy (TEM; JEOL 100CX) and a þ13 mV ζ potential in 150 mM MOPS buffer at pH 7.4 (Zetasizer, Malvern Instruments, Ltd., Malvern, Worcestershire, UK). The relative amine coverage (14) Le Bihan, O.; Bonnafous, P.; Marak, L.; Bickel, T.; Tr
epout, S.; Mornet, S.; De Haas, F.; Talbot, H.; Taveau, J. C.; Lambert, O. J. Struct. Biol. 2009, 168(3), 419–425. (15) Verma, A.; Stellacci, F. Small 2010, 6(1), 12–21. (16) Massart, R. IEEE Trans. Magn. 1981, 17(2), 1247–1248. (17) Fauconnier, N.; Bee, A.; Roger, J.; Pons, J. N. Prog. Colloid Polym. Sci. 1996, 100, 212–216. (18) Lu, Y.; Yin, Y.; Mayers, B. T.; Xia, Y. Nano Lett. 2002, 2(3), 183–186. (19) Maurice, V.; Georgelin, T.; Siaugue, J. M.; Cabuil, V. J. Magn. Magn. Mater. 2009, 321, 1408–1413. (20) Georgelin, T.; Maurice, V.; Malizieux, B.; Siaugue, J. M.; Cabuil, V. J. Nanopart. Res. 2009, 12, 675–680.

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(0.85 amines per nm2) was estimated with the ninhydrin colorimetric assay as described.21 These CSMNs were further characterized using capillary electrophoresis, and a direct correlation between electrophoretic mobility and relative amine coverage was found. The amine-modified particles were converted to carboxylic acid-modified nanoparticles via an overnight reaction with 700 equivalents of succinic anhydride (relative to the amine function quantity) in 150 mM MOPS buffer. The mixture was then purified with a PD-10 desalting column (Sigma-Aldrich) equilibrated with 150 mM MOPS buffer to remove the excess of succinic anhydride. This chemical modification produced colloidally stable carboxylic acid-modified CSMNs with a 35 nm mean diameter as measured by TEM (JEOL 100 CX) and a -36 mV ζ potential in 150 mM MOPS buffer at pH 7.4 (Zetasizer, Malvern). GUV Preparation. GUVs were made using electroformation, which produces GUVs with high efficiency and very few multilamellar aggregates.22 The lipids (Avanti polar lipids) were neutral DOPC, negative DOPG, positive DOTAP, and fluorescent RhDOPE, all of which are in the fluid phase at room temperature and form vesicles in water. A solution of desired lipids (1 mg/mL in a 2:1 chloroform/methanol mixture) was deposited on both windows of a homemade preparation chamber composed of two transparent electrodes (formed by glass plates coated with indium tin oxide) separated by a 1 mm thick Teflon spacer. The lipid film was dried overnight in a vacuum oven and then filled with ∼1.5 mL of 1 mM MOPS buffer (pH 7.4). The alternating current voltage was set immediately to initiate gentle swelling of the lipid film and avoid spontaneous vesiculation. GUV formation involved three stages: the first step was an increase of the field amplitude from 0.2 to 1.2 V at 10 Hz over 1 h, the second was a swelling period of 3 h during which electric field parameters were constant (1.2 V at 10 Hz), and the final step involved “rebounding” (20 min at 1.4 V at 4 Hz). The vesicles were allowed to “relax” overnight in the preparation chamber and were then drawn out carefully. This stock solution contained GUVs of 5-100 μm diameter with ∼0.1 mg/mL lipid. Three different lipid mixtures were used in this study: zwitterionic GUVs composed of pure DOPC, anionic GUVs made of a 4/1 (w/w) ratio of DOPC/DOPG, and positive GUVs using a 9/1 (w/w) ratio of DOPC/DOTAP. After having checked that it did not alter the interactions, we added 1% (w/w) of Rh-DOPE to each lipid mixture to enable subsequent fluorescence microscopy. Interactions between CSMNs and GUVs. A solution of CSMNs at [Fe] = 2  10-3 mol/L, which corresponds to a concentration of 4.4  1016 CSMN/L (in 1 mM MOPS/NaOH buffer pH 7.4), was mixed with iso-osmolar solutions of neutral DOPC GUVs (GUV), negative DOPC/DOPG GUVs (GUV-), or positive DOPC/DOTAP GUVs (GUVþ). The nanoparticles diffused rapidly according to the concentration gradient, and the fluorescence of liposomes was checked immediately by fluorescence microscopy using an inverted microscope (Zeiss, Axiovert 200) equipped with a 40 objective. Fluorescent dyes (FITC and RITC) were excited using filter set 15 (for RITC, excitation 552 nm and emission 577 nm) and filter set 09 (for FITC, excitation 495 nm and emission 517 nm) with a fluorescent lamp (Zeiss, HBO 103). The localization of the nanoparticles was checked using confocal microscopy with an inverted microscope (Leica, DMI 6000) equipped with a 63 objective and a Leica SP-5 acquisition system. For these experiments, 100 μL of CSMN solution was mixed with 100 μL of GUV solution. The resulting mixture (40 μL) was then placed on a slide and the coverslip was sealed with paraffin wax. TEM. An aliquot of 1 mL of CSMN solution at [Fe] = 2.10-3 mol/L was added slowly to 1 mL of GUV solution. The mixture (21) d’Orly
e, F.; Varenne, A.; Georgelin, T.; Siaugue, J. M.; Teste, B.; Descroix, S.; Gareil, P. Electrophoresis 2009, 30(14), 2572–2582. (22) Angelova, M. I. Liposome electroformation. In Giant Vesicles, Perspectives in Supramolecular Chemistry; Luisi, P. L., Walde, P., Eds.; John Wiley and Sons, Ltd.: West Sussex, England, 2000, 6, 27-36.

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Figure 1. General reaction scheme for the synthesis and the modification of cationic (a) and anionic (b) CSMNs. The insert is an electron micrograph of cationic CSMNs stained with 2% uranyl acetate. The dark spots inside each sphere correspond to magnetic nanoparticles.

Figure 2. Confocal micrographs of negatively charged giant vesicles (DOPC/DOPG 4/1 w/w with 1% Rh-DOPE) covered with cationic CSMNs (labeled with FITC). (a) Red fluorescence of the membrane from Rh-DOPE, (b) green fluorescence caused by cationic CSMNs adsorbed on the membrane, (c) merged images of a and b. (d) Plot profile corresponding to the orange dotted line on (c). was then vortexed, and GUVs were collected by magnetic separation (20 min). After three washes with 1 mM MOPS buffer, the majority of unbound nanoparticles had been removed. The sample was observed using TEM (JEOL 100CX).

Results We investigated the behavior of cationic or anionic charged CSMNs (Figure 1) versus neutral, positive, and negative GUVs. CSMNS were selected because these multifunctional magnetic nanoparticles can be grafted covalently with various therapeutic agents for potential clinical applications. Interactions between Cationic CSMNs and GUVs. When an aqueous solution of fluorescent cationic FITC-PEG-amine CSMN was added to an iso-osmolar solution of negatively charged GUVs (GUV: DOPC/DOPG), the membrane of the GUV vesicles became homogeneously fluorescent at the interface between nanoparticles and vesicles. This phenomenon was instantaneous, and the more the fluorescent front of nanoparticles diffused, the more the vesicles appeared labeled with green fluorescence. The same applied when the two solutions were mixed Langmuir 2010, 26(20), 16025–16030

before observation. In the case of subsequent mixing of the two solutions, all of the negatively charged vesicles remained uniformly labeled with green fluorescent nanoparticles. With the addition of 1% Rh-DOPE to the lipid mixtures, it was possible to confirm the colocalization of the phospholipid membrane and the nanoparticles by red fluorescence. Confocal microscopy documented the perfect correlation between red and green fluorescence. As shown in Figure 2, the red fluorescence of Rh-DOPE inserted within the membrane (Figure 2a) and the green fluorescence displayed by positive nanoparticles (Figure 2b) appear superimposed, and the merged fluorescent images appear yellow (Figure 2c, green þ red). These merged confocal images confirmed the accumulation of positive nanoparticles at the surface of the negative vesicles as evidenced by the two fluorescence intensity maxima observed on the plot profile (Figure 2d). The strong interaction between cationic CSMNs and anionic GUVs resulted in the formation of hybrid giant vesicles covered with a densely packed layer of CSMNs without any observable vesicle rupture. The vesicles also appeared rigid when put in contact with cationic CSMNs. Using differential interference contrast (DIC) DOI: 10.1021/la1023746

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Figure 3. Optical micrographs (DIC mode) of GUV (DOPC/ DOPG 4/1 w/w), (a) without and (b) covered with cationic CSMNs.

microscopy, anionic liposomes showed increased contrast when they were labeled with CSMNs (Figure 3b) compared with free vesicles (Figure 3a). This was caused by the intrinsic high optical density of the nanoparticles on the vesicle surfaces. By comparing these two cases, we ascertained that the vesicles maintained their integrity during the nanoparticle adsorption step even though the entropic fluctuations of the membrane had vanished. Electrostatic interaction is the most efficient nonspecific interaction to induce nanoparticle adhesion onto a fluid membrane. To check whether these bifunctionalized CSMNs behaved in the same way, cationic FITC-PEG-amine CSMNs were mixed with positively charged vesicles (GUVþ: DOPC/DOTAP). Although identically charged, the two entities interacted strongly, showing homogeneous green fluorescence of giant vesicles (image not shown). Thus, the nanoparticles were adsorbed identically on the membrane of vesicles bearing either a positive or a negative net charge. As mentioned for anionic liposomes, vesicles covered with nanoparticles appeared to have higher optical contrast when imaged by DIC microscopy and were rigid, with no observable fluctuations in size. Identical observations were made with neutral pure DOPC GUVs, cationic FITC-PEG-amine CSMNs, and red fluorescent RITC-PEG-amine CSMNs. CSMNs covered the lipid vesicles homogeneously without affecting liposome integrity, leading to the formation of hybrid vesicles. Other relevant information was provided by fluorescence microscopy observations of multivesicular vesicles (MVVs). During electroformation, some GUVs are trapped inside other larger vesicles, as shown in Figure 4a. When the fluorescent nanoparticle front first interacted with these MVVs, only the membrane of the outer vesicle exhibited green fluorescence (Figure 4b). When the two images were merged, the membrane of the outer biggest GUV appeared yellow (Figure 4c), whereas the internal GUVs remained red from Rh-DOPE fluorescence. Moreover, when the MVVs were observed using DIC microscopy, only the outer membrane appeared optically high contrast and rigid (Figure 4d). These observations confirmed that the nanoparticles did not cross the lipid membrane passively. The presence of cationic CSMNs on vesicles was confirmed by TEM. Electron micrographs of anionic GUVs covered with positive PEG-amine-CSMNs are presented in Figure 5. In fact, there are always smaller vesicles in preparations of GUVs than can be imaged using TEM. GUVs appeared as big circular objects exhibiting high electronic density with sizes ranging from 500 nm to 3 μm. High magnification showed the presence of densely packed CSMNs on their surface. These micrographs illustrate that liposomes act as templates for CSMNs. They also demonstrate that it is possible to separate free nanoparticles from adsorbed ones by magnetic decantation, because the background is clear of nanoparticles. TEM images did not permit us to corroborate the exclusive localization of the nanoparticles at the surface of the vesicles because the samples were first dried; 16028 DOI: 10.1021/la1023746

Figure 4. Optical fluorescence pictures of multilamellar vesicles (DOPC/DOPG 4/1 w/w with 1% Rh-DOPE). (a) Red fluorescence of the membrane caused by Rh-DOPE staining. (b) Green fluorescence caused by the adsorption of cationic CSMNs on the outer membrane. (c) Merged images of a and b. (d) DIC micrograph showing the enhancement of optical contrast on the outer membrane.

Figure 5. TEM micrographs of anionic GUVs (DOPC/DOPG 4/ 1 w/w) covered with cationic CSMNs. Overview (left) and high magnification of the nanoparticle clusters (right).

nevertheless, it is important to note that the nanoparticle clusters were particularly dense. This finding was compatible with a uniform adsorption. Langmuir 2010, 26(20), 16025–16030

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Article Scheme 1. Schematic Representation of the Interaction between Cationic CSMNs and Neutral GUVs (Top) and Anionic CSMNs and Neutral GUVs (Bottom)a

Figure 6. Anionic CSMNs mixed with giant cationic vesicles GUVsþ (9/1 w/w ratio of DOPC/DOTAP with 1% Rh-DOPE) (a) Red fluorescence of the membrane from Rh-DOPE staining. (b) Green fluorescence caused by anionic CSMNs.

In conclusion, cationic CSMNs were similarly adsorbed on the three types of GUVs in this study: neutral, positive, and negative. Clearly, this interaction did not depend on the overall charge of the vesicle. It is worth noting that the major constituent of the three lipid mixtures was the zwitterionic phospholipid DOPC. The selectivity toward the adsorption of cationic CSMNs on phospholipid bilayers seems to be ruled by the affinity of the phospholipid headgroups for the silica surface. Interactions between Anionic CSMNs and GUVs. Anionic carboxylic acid-modified nanoparticles were generated by a simple chemical modification of cationic FITC-PEG-amine CSMNs. All amino groups were converted in carboxylic acid by reacting with a large excess of succinic anhydride, and all the unreacted anhydride was removed by desalting chromatography. Whatever the charge of the GUVs (neutral, negative, or positive), no fluorescence was observed on giant liposomes (Figure 6a,b), showing the absence of interactions between anionic CSMNs and phospholipid bilayers. This absence of interaction between anionic nanoparticles and liposomes was supported by the lack of enhancement of vesicle contrast in DIC mode, and membrane fluctuations were not affected. These observations emphasize that CSMN/GUV interactions cannot be reduced to a simple electrostatic attraction.

Discussion Engineered nanoparticles could represent hazards, so a better understanding of nanoparticle-membrane interactions depending on numerous parameters such as the size, the charge, and the surface composition of the nanoparticle and the biomembrane constituents may generate crucial information. The aim of this study was to evaluate the role played by the electric charges in the interaction between bifunctionalized γ-Fe2O3@SiO2 CSMNs and lipid bilayers. After different series of experiments in which the charge of liposomes was modulated versus the charge of nanoparticles, our main surprising conclusion is that the interaction is not ruled by a global attraction between two oppositely charged species (GUV and CSMNs). Instead, the nanoparticle surface properties appeared to rule the interactions. Therefore, in the following discussions, the selected model to represent GUVs is exclusively comprised of lipids with a zwitterionic phosphatidylcholine headgroup. Here, we demonstrated the marked affinity of cationic aminoderived CSMNs for GUVs, leading to a fast uniform covering of giant liposomes. Before attempting to explain the behavior of these cationic nanoparticles, it is necessary to focus on the pathway selected to produce them. The starting material comprised maghemite/silica core-shell nanoparticles that were simultaneously functionalized with PEG chains and with amino groups cross-linked with an amount of TEOS. The resulting grafted nanoparticles (35 nm mean diameter) presented with a positive ζ potential value (þ13 mV). Despite the presence of PEG chains and amino groups (0.85 per nm2),21 the major available surface of Langmuir 2010, 26(20), 16025–16030

a Red dotted circles underline the interactions between silanol groups and phosphatidylcholine headgroups.

the obtained nanoparticles remained as silica (see Figure 1). It is known that such a surface is of great importance in adsorption and ion exchange because of the presence of negatively charged silanol groups at a physiological pH.23 Taking this into account, it is reasonable to assume that the simple presence of silanol groups constitutes the driving force responsible for the strong attraction with GUV membranes. A fast reaction occurs when cationic CSMNs are in the presence of GUVs and the nanoparticles are irreversibly attracted at the surface of the membrane until saturation. The particularly strong affinity between silica-based moieties and phosphatidylcholine has been reported widely because it is involved in the mechanism of the lung disease silicosis.24 Moreover, amorphous silica particles are known to cause hemolysis of mammalian erythrocytes.25 Although the precise mechanism of membranolysis is still being debated, the demonstrated high affinity of surface silanol groups for binding phospholipid headgroups seems to play a preponderant role. This high affinity was historically first attributed to hydrogen-bonding interactions of silanol groups with phosphate groups and then was attributed to strong interactions between SiO- moieties and the positively charged R-N(CH3)3þ of PC headgroups.24,26 These two contrasting models are still studied regularly using (23) Iler, R. K. The Chemistry of Silica; Wiley: New York, 1979; Chapter 6. (24) Nash, T.; Allison, A. C.; Harington, J. S. Nature 1966, 210(5033), 259–261. (25) Gerashchenko, B. I.; Gun’ko, V. M.; Gerashchenko, I. I.; Mironyuk, I. F.; Leboda, R.; Hosoya, H. Cytometry 2002, 49(2), 56–61. (26) Depasse, J.; Warlus, J. J. Colloid Interface Sci. 1976, 56(3), 618–621.

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techniques such as nuclear magnetic resonance (NMR) or theoretical chemistry. For amorphous silica nanoparticles, it was recently shown that their membranolytic effect can be prevented by covering the silica surface with organosilanes such as APTS, carboxylic acid, sulfonic acid-derived silanes, or PEOS.27,28 All of these results support the idea that the hemolytic activity of silica nanoparticles depends on the concentration of accessible negatively charged silanol groups at their surfaces.29 Starting from the hypothesis that GUVs comprise PC headgroups exclusively and that cationic CSMNs present accessible silanol moieties, we propose a simplified anchoring mechanism as depicted in Scheme 1 (top). It is also possible that electrostatic attractions between the negative phosphate groups of the bilayer and the amine groups of the nanoparticles might also reinforce any previous binding because of the similar length of their respective chains. The adsorption of cationic CSMNs induces an extinction of membrane fluctuations, as observed in the case of anionic peptidederived quantum dots.6 Once the nanoparticles are adsorbed to the surface of vesicles, GUVs appear perfectly spherical, and the membrane appears to stiffen. Silanol/PC affinity, as well as possible supplementary interactions, could increase the tilt angle of the PC headgroups, leading to an enhancement of phospholipid density. This was described previously, but only for anionic nanoparticles, and can help explain the observed gelation of liposomal membranes after nanoparticle adsorption.9 Previous research dealing with internalization has shown that very small γ-Fe2O3@SiO2 nanoparticles (15-20 nm) remain

anchored at the outer surface of liposomes, while those larger than 30 nm can be translocated.14 It is worth mentioning that nanoparticles with a mean diameter of 35 nm remained strongly linked at the GUV surface, illustrating that the bifunctionalization with PEG and amino groups prevents any translocation process. One proof of the absence of translocation of the coreshell used in this study is our observation on MVVs showing that nanoparticles remained at the outer bilayer. Anionic nanoparticles were produced by conversion of the positive CSMNs by the reaction of succinic anhydride on the amine groups. Compared with the starting material, the resulting nanoparticles exhibited extended organosilanes bearing negatively charged carboxylic acidic groups. This chemical modification seems to be sufficient to prevent further interactions; in this case PC headgroups could not approach the inner silanol units (Scheme 1, bottom). Some recent reports showed that core-shell Fe2O3@SiO2 nanoparticles exhibit a similar behavior toward human red blood cells (hRBCs). Anionic nanoparticles are inert toward hRBCs, whereas cationic ones are strongly adsorbed on the surface (they cover the surface of these cells uniformly). Even if hRBCs are much more complex systems than the simple phospholipid bilayer of giant liposomes; with a rich lipid mixture and the presence of proteins and sugars; they do not have endocytotic capacity. The surface properties of nanomaterials play a preponderant role in nanoparticle-cell interactions, and the nanoparticle surface must be considered more precisely. Here, we demonstrated that surface chemical composition could also greatly affect the type of interaction, in addition to the global surface charge.

(27) Slowing, I. I.; Wu, C. W.; Vivero-Escoto, J. L.; Lin, V. S. Small 2009, 5(1), 57–62. (28) Lin, Y. S.; Haynes, C. L. Chem. Mater. 2009, 21(17), 3979–3986. (29) Murashov, V.; Harper, M.; Demchuk, E. J. Occup. Environ. Hyg. 2006, 3(12), 718–723.

Acknowledgment. This work was supported by grants from the French National Research Agency, ANR-PCV Prob-DOM. The authors thank Aude Michel for assistance with electron microscopy.

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