- Email: [email protected]
In situ precipitation of magnetic fluid encapsulated in giant liposomes
Journal of Colloid and Interface Science 343 (2010) 396–399
Contents lists available at ScienceDirect
Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Short Communication
In situ precipitation of magnetic fluid encapsulated in giant liposomes Grégory Beaune, Christine Ménager * UPMC Univ Paris 06 UMR 7195 PECSA Physicochimie des Electrolytes Colloïdes et Sciences Analytique, CNRS ESPCI ParisTech, F-75005, Paris, France
a r t i c l e
i n f o
Article history: Received 14 September 2009 Accepted 5 November 2009 Available online 10 November 2009 Keywords: Liposomes Magnetic nanoparticles Magnetic fluid Precipitation Vesicles
a b s t r a c t It is well known that adding salt to a colloidal solution stabilized by electrostatic repulsions can induce its destabilization. When this colloidal solution is encapsulated inside liposomes an in situ precipitation can be induced by slightly modification of the environment: application of a magnetic field in the case of magnetic particles or increase of the temperature. It has been shown in a previous study that magnetic liposomes exhibit strong deformation under magnetic field. In this case the precipitation can be induced after their elongation under magnetic field and liposomes keep their shape even if the magnetic field is cut off. The following study is motivated by the desire to use the salt effect to precipitate magnetic nanoparticles inside giant liposomes. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction The self-assembly of nanoparticles into a variety of nano and microstructured morphologies is an important concept for the development of novel materials with unique properties. Different aggregated systems composed of nanoparticles have been explored such as gold [1,2], silver [2,3], magnetic [4–6] and silica nanoparticles [7]. The formation of nanoparticular aggregates with controlled size and shape may have useful applications. As an example, aggragates of silver nanoparticles have been studied for surface-enhanced Raman scattering on a hydrophobic surface [3]. The surface plasmon absorption band of metallic particles is dependent of their aggregation state [7]. This property is used for the development of colorimetric based assays with gold [8–10] and silver nanoparticles [11]. Concerning magnetic aggregates, different structures, in size and shape, have been synthesized through magnetic interactions and the application of a magnetic field [12–14]. For example, spherical superparamagnetic nanoparticles were assembled into needle-shaped structures at a macrometric scale under the application of a strong unidirectional magnetic flux [7]. In a previous article, it has been shown that the encapsulation of magnetic and fluorescent nanoparticles as a colloidal solution allowed the preparation of magnetic and fluorescent liposomes [15]. The nanoparticles are ferric oxides (maghemite c-Fe2O3) on which rhodamine molecules have been grafted. An increase of the ionic strength of the colloidal solution improved the encapsulation of nanoparticles in Giant Unilamellar Vesicles (GUVs). We proved by magnetophoresis experiments that GUVs encapsulating * Corresponding author. Fax: +33 1 44 27 36 75. E-mail address: [email protected] (C. Ménager). 0021-9797/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2009.11.016
magnetic nanoparticles with a high ionic strength exhibit better magnetic properties compared to GUVs encapsulated magnetic nanoparticles with a lower ionic strength. The strong magnetic properties and then the very important elongation of the magnetic liposome under a magnetic field can be explained by the formation of aggregates in the colloidal solution because the magnetic susceptibility is proportional to the number of magnetic objects and to the square of their volume. Indeed, it is known that the increase of the ionic strength by addition of salt induces the screening of electrostatic repulsions between particles and thus their aggregation [16]. The following study is motivated by the desire to understand the rule of the ionic strength on the properties of the giant liposomes encapsulated magnetic and fluorescent particles. The phase diagram of nanoparticles stabilized by electrostatic repulsions has been studied in the group [17,18] and it was found to be dependent on several parameters: volume fraction of nanoparticles in the colloidal solution, size of the nanoparticles, ionic strength and temperature. As an example, the destabilization of an ionic magnetic fluid by the addition of salt induces a phase separation between a concentrated phase rich in big particles in a diluted phase rich in small particles [19]. The concentrated phase may be a liquid one which exhibits deformation under the application of a magnetic field or a precipitated one with no deformation under magnetic field. In this paper we describe the destabilization under magnetic field of fluorescent and magnetic particles and we use this destabilization to precipitate magnetic particles inside giant liposomes. The liposome is used as a template to obtain an anisotropic shape especially interesting for magnetic applications. The precipitation of nanoparticles after encapsulation is also interesting for hyperthermia properties because the role of the interactions between
G. Beaune, C. Ménager / Journal of Colloid and Interface Science 343 (2010) 396–399
the particles may be important for the heating properties. Moreover this work is performed on giant liposomes (GUV) to follow the phenomenon by optical microscopy but it can be easily transposed to large vesicles (LUV). In fact, the encapsulation of magnetic particles has been described for these two kinds of vesicles [20,21].
2. Materials and methods 2.1. Materials The cis-1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), dimercaptosuccinic acid (DMSA), rhodamine B, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), and cystamine were all purchased from Sigma–Aldrich.
2.2. Synthesis of rhodamine B magnetic fluid The magnetic and fluorescent nanoparticles were synthesized using a previously established process [22], and it was based on the modification of DMSA molecules by rhodamine maleimide before complexation on positively charged c-Fe2O3 particles. The DMSA was coupled to the particles by at least one carboxyl and one thiol group. Ungrafted thiol groups were available for subsequent coupling, and any unadsorbed carboxylate groups ensured that surface charges were present. Then, particles were submitted to successive precipitations with NaCl to eliminate any nonadsorbed rhodamine. Ultrafiltration was used to concentrate particles in the magnetic fluid with a MACROSEP filter (cutoff = 30 kD, Fisher Scientific Labosi) at 5000 rpm for 30 min. The ionic strength was measured from the conductivity. The properties of the fluorescent and magnetic fluid were as follows: r = 540 lS and [Fe] = 6.6 mol L 1.
2.3. Synthesis of magnetic and fluorescent liposomes The preparation of magnetoliposomes has been described elsewhere [23]. The liposomes have been loaded with magnetic particles by mixing preformed magnetic particles with phospholipid powder. The lipid film was thus obtained by shearing. The second step was to swell the obtained film with pure water. The ionic strength of the solution encapsulated inside liposomes was adjusted before encapsulation by adding salt. Sodium chloride (28 mg) was mixed with the magnetic fluid (100 lL) to obtain [NaCl] = 4.79 mol L 1 in the magnetic fluid. A small mass of perfectly dry DOPC powder (around 1 mg) was placed in a glass Petri dish. A total of 10 lL of the c-Fe2O3/NaCl mixture was added, and the mixture was spread and sheared with a glove finger to obtain a fat and oily orange film. This film was presumably a lamellar phase swelled with charged particles. Immediately following the shearing, 1.5 mL of distilled water was poured onto the fatty film to start the spontaneous swelling of the liposomes. The samples were placed in a water bath at 45 °C and observed after 30 min using an optical microscope.
2.4. Characterization The samples were imaged using bright field microscopy (inverted microscope Leica 40, numerical aperture (NA = 0.65) equipped with a 100 W mercury lamp. A fluorescence filter cube with a 546 nm excitation filter (BP546/10) and a 590 nm emission filter (LP 590) was used to visualize rhodamine fluorescence. Pictures from a charge-coupled-device camera were digitized with a frame grabber (LG-3, Scion Corp., Frederick, MD).
397
3. Results and discussion 3.1. Destabilization of the colloidal solution The destabilization of the colloidal solution is induced by screening the electrostatic repulsions between particles. The controlled addition of salt (sodium chloride) may induce the destabilization. In the present study we added an important quantity of salt before the preparation of the liposomes. The ionic strength is mainly due to the added salt and it is 4.79 mol L 1 in colloidal solution. However this salt is diluted during the preparation because of the equilibrium of ions between the inside and the outside of the liposomes during the swelling. After swelling the calculated salt concentration taking into account the dilution factor is 32 mmol L 1. However a measure of the ionic strength made by conductivity measurements and based on a NaCl calibration corresponds to a salt concentration of 5 mmol L 1. This value, much lower than the mean ionic strength (32 mmol L 1) indicates that the salt concentration inside the GUVs is more important than the salt concentration outside. This fact is due to the viscosity of the sample because of the high volume fraction of magnetic particles (6.6 mol L 1 corresponds to 10.5% in volume fraction) and because of the addition of NaCl. High viscosity can really slow down the diffusion of ions occurring during the swelling. The limit of precipitation is studied in bulk samples and observed by optical microscopy (Fig. 1). In bulk samples, the precipitation is obtained for [NaCl] = 0.325 mol L 1 for an iron concentration of 1 mol L 1. For this concentration, aggregates of nanoparticles are formed with a spherical shape and a mean diameter of 1.04 ± 0.32 lm (Fig. 1a, for experimental data see supporting information). When a magnetic field is applied, spherical aggregates form magnetic chains in the direction of the field (Fig. 1b) which themselves come into contact to form magnetic bundles (Fig. 1c). It is important to quote that the obtained structures are irreversible. On Fig. 1c, the magnetic field is cut off. The turbidity of the sample for concentration lower than 0.325 mol L 1 indicates that the destabilization begins before this concentration and that small aggregates are already in solution. Even if the phenomenon observed in bulk samples are not the same in terms of concentration than in confined structures, the kind of observation is the same. However the aim of this study is not to build the phase diagram of the system but it is to use it to induce in situ precipitation of magnetic nanoparticles.
3.2. In situ precipitation Fig. 2a corresponds to optical microscopy pictures of liposomes loaded with magnetic and fluorescent nanoparticles. We can observe that the colloidal solution inside the GUV presents no microscopic destabilization, in bright field microscopy the liposome has a characteristic brown colour due to the magnetic fluid and in epifluorescence a red and homogeneous colour due to the emission of rhodamine molecules grafted on magnetic nanoparticles. Fig. 2 in the previous version of the paper has been placed at the end of the text for clarity. Under magnetic field, the thermal fluctuations due to the entropic behaviour of the bilayer are unfolded and the liposome elongates in the direction of the magnetic field (Fig. 2b). It is reminded that the prolate ellipsoid obtained is defined by the eccentricity, e, where e = [1 (b/a)2]1/2, a is the semiaxis of the liposome parallel to the magnetic field, and b is the value of the two other semiaxes orientated perpendicular to the magnetic field. It is the general behaviour of a magnetic liposome under magnetic field. The prolate deformation of the Rh-FF liposomes before (e = 0.69, Fig. 2b) and after the destabilization of Rh-FF (e = 0.79,
398
G. Beaune, C. Ménager / Journal of Colloid and Interface Science 343 (2010) 396–399
Fig. 1. Destabilization of magnetic and fluorescent nanoparticles under addition of salt [NaCl] = 0.325 mol L
1
(a) before and (b, c) after the application of a magnetic field.
Fig. 2. Optical microscopy images of a magnetic and fluorescent liposome (a) before the destabilization of Rh-FF and with H = 0 Oe, (b) before the destabilization of Rh-FF and with H = 202 Oe and (c) after the destabilization of Rh-FF and with H = 0 Oe.
Fig. 2c) is quite important compared to free salt liposomes encapsulating magnetic fluids (e = 0.55) under the same magnetic field [15]. However in the case of GUVs loaded with a high ionic strength magnetic fluid fifteen minutes after the application of the magnetic field we can noticed the apparition of ridges in the direction of the magnetic field corresponding to magnetic chains of nanoparticles as previously seen (Fig. 1). The magnetic field induces an in situ precipitation and leads to the rigidification of the liposome membrane in an anisotropic shape (Fig. 2c). The deformation of GUVs is kept when the magnetic field is cut off (Fig. 2c). Indeed, the precipitation of the nanoparticles induces the stiffness of the membrane. The magnetic liposomes with magnetic particles precipitated in their core behave as a permanent magnet and can be oriented in the desired direction in (Fig. 3a, b and c) or out of the plane (Fig. 3d). The destabilization can be observed also when observations in epifluorescence spend several minutes typically dozens of minutes. In this case the precipitation is due to the elevation of temperature caused by the beam light that is in accordance with the fact that the temperature is an important parameter for the stability of magnetic nanoparticles (Fig. 4). In this case the interesting fact is the shrinking induced by the precipitation. On Fig. 4b andd, we observe the formation of a small solid magnetic sphere representing 35% of the initial volume of the liposome. The liposome is thus divided in two parts, a colourless liquid one and a brown solid one, corresponding to the phase separation between a precipitate and a supernatant. The precipitation is concomitant with the swelling of the liposome, the volume of the liposome after the swelling is twice the initial one. This swelling is due to a diffusion of water inside the liposome indicating as we previously noted that the liposome is not in equilibrium. This fact is surprising according to the well known permeability properties of such lipid bilayer however the
Fig. 3. Orientation of the rigidified liposome obtained after precipitation of the nanoparticles in the three directions of space.
non equilibrium is due to the high density and viscosity of the initial colloidal solution which can slow down the diffusion rate of water inside. After the phase separation, the difference in osmotic pressure between the inside of the liposome constituted by a highly salty solution in equilibrium with a precipitate and the outside induces the water diffusion. The swelling in not observed in the experiences under magnetic field because in this case the membrane is tense and there is no excess of membrane to permit the swelling of the liposome.
G. Beaune, C. Ménager / Journal of Colloid and Interface Science 343 (2010) 396–399
399
4. Summary By using liposomes as templates it is possible to make controlled aggregation in size, shape and volume fraction of nanoparticles. The aggregates may be retrieve and use for other applications. We also prove that the encapsulation of a salty magnetic colloidal solution inside liposomes can lead to the formation of aggregates responsible of their unique magnetic properties, as we previously described, and also to their in situ precipitation. The precipitation is induced by a magnetic field or by increasing temperature. This phenomenon may be extended to a lot of aqueous dispersion of nanoparticles. Acknowledgments The authors thank Aude Michel for chemical synthesis, Delphine Talbot for chemical analysis, and Andrejs Cebers for helpful discussions. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jcis.2009.11.016. References Fig. 4. Optical microscopy images of two liposomes encapsulating magnetic nanoparticles before (a and c) and after in situ precipitation (b and d).
Fig. 5. Optical microscopy image of magnetic and fluorescent aggregates obtained after GUV solubilization with octylglucoside.
The magnetic globules can be extracted from the liposomes by addition of solubilizing molecules like octylglucoside (OG) (Fig. 5).
[1] A.K. Boal, F. Ilhan, J.E. Derouchey, T. Thurn-Albrecht, T.P. Russell, V.M. Rotello, Nature 404 (2000) 746. [2] C.L. Schofield, A.H. Haines, R.A. Field, D.A. Russell, Langmuir 22 (2006) 6707. [3] M. Culha, M. Kahraman, N. Tokman, G. Turkoglu, J. Phys. Chem. C 112 (2008) 10338. [4] R. Abu-Much, A. Gendanken, J. Phys. Chem. C 112 (2008) 35. [5] K.F.C. Yiu, C.H. Yu, H. Tang, H. He, S.C. Tsang, K.Y. Tam, J. Phys. Chem. C 112 (2008) 7599. [6] H.-B. Xia, J. Yi, P.-S. Foo, B. Liu, Chem. Mater. 19 (2007) 4087. [7] F. Gaboriaud, A. Nonat, D. Chaumont, A. Craievich, J. Colloid Interface Sci. 253 (2002) 140. [8] N.T.K. Thanh, Z. Rosenzweig, Anal. Chem. 74 (2002) 1624. [9] Y. Kim, R.C. Johnson, J.T. Hupp, Nano Lett. 1 (2001) 165. [10] J. Liu, Y. Lu, Angew. Chem. Int. Ed. 45 (2006) 90. [11] M. Sastry, N. Lala, V. Patil, S.P. Chavan, A.G. Chittiboyina, Langmuir 14 (1998) 4138. [12] J.C. Love, A.R. Urbach, M.G. Prentiss, G.M. Whitesides, J. Am. Chem. Soc. 125 (2003) 12696. [13] Z.Y. Tang, N. Kotov, Adv. Mater. 17 (2005) 951. [14] Jao B Zhang, X. Zhang, B. Xu, Angew. Chem. Int. Ed. 45 (2006) 1220. [15] G. Beaune, V. Cabuil, C. Menager, J. Phys. Chem. B 112 (2008) 7224. [16] E. Dubois, V. Cabuil, F. Boué, J.C. Bacri, R. Perzynski, Eur. Phys. J. E 104 (1997) 173. [17] F. Cousin, E. Dubois, V. Cabuil, Phys. Rev. E 68 (2003) 1. [18] G. Mériguet, F. Cousin, E. Dubois, F. Boué, A. Cebers, B. Farago, R. Perzynski, J. Phys. Chem. B 110 (2006) 4378. [19] R. Massart, E. Dubois, V. Cabuil, E. Hasmoney, J. Mag. Mag. Mater. 149 (1995) 1. [20] C. Ménager, D. Guemghar, R. Perzynski, S. Lesieur, V. Cabuil, Langmuir 24 (2008) 4968. [21] M.S. Martina, J.P. Fortin, C. Ménager, O. Clément, G. Barratt, C. GrabielleMadelmont, F. Gazeau, V. Cabuil, S. Lesieur, J. Am. Chem. Soc. 127 (2005) 10676. [22] F. Bertorelle, C. Wilhelm, J. Roger, F. Gazeau, C. Ménager, V. Cabuil, Langmuir 22 (2006) 5385. [23] O. Sandre, C. Ménager, J. Prost, V. Cabuil, J.C. Bacri, A. Cebers, Phys. Rev. E 62 (2000) 3865.
Contents lists available at ScienceDirect
Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Short Communication
In situ precipitation of magnetic fluid encapsulated in giant liposomes Grégory Beaune, Christine Ménager * UPMC Univ Paris 06 UMR 7195 PECSA Physicochimie des Electrolytes Colloïdes et Sciences Analytique, CNRS ESPCI ParisTech, F-75005, Paris, France
a r t i c l e
i n f o
Article history: Received 14 September 2009 Accepted 5 November 2009 Available online 10 November 2009 Keywords: Liposomes Magnetic nanoparticles Magnetic fluid Precipitation Vesicles
a b s t r a c t It is well known that adding salt to a colloidal solution stabilized by electrostatic repulsions can induce its destabilization. When this colloidal solution is encapsulated inside liposomes an in situ precipitation can be induced by slightly modification of the environment: application of a magnetic field in the case of magnetic particles or increase of the temperature. It has been shown in a previous study that magnetic liposomes exhibit strong deformation under magnetic field. In this case the precipitation can be induced after their elongation under magnetic field and liposomes keep their shape even if the magnetic field is cut off. The following study is motivated by the desire to use the salt effect to precipitate magnetic nanoparticles inside giant liposomes. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction The self-assembly of nanoparticles into a variety of nano and microstructured morphologies is an important concept for the development of novel materials with unique properties. Different aggregated systems composed of nanoparticles have been explored such as gold [1,2], silver [2,3], magnetic [4–6] and silica nanoparticles [7]. The formation of nanoparticular aggregates with controlled size and shape may have useful applications. As an example, aggragates of silver nanoparticles have been studied for surface-enhanced Raman scattering on a hydrophobic surface [3]. The surface plasmon absorption band of metallic particles is dependent of their aggregation state [7]. This property is used for the development of colorimetric based assays with gold [8–10] and silver nanoparticles [11]. Concerning magnetic aggregates, different structures, in size and shape, have been synthesized through magnetic interactions and the application of a magnetic field [12–14]. For example, spherical superparamagnetic nanoparticles were assembled into needle-shaped structures at a macrometric scale under the application of a strong unidirectional magnetic flux [7]. In a previous article, it has been shown that the encapsulation of magnetic and fluorescent nanoparticles as a colloidal solution allowed the preparation of magnetic and fluorescent liposomes [15]. The nanoparticles are ferric oxides (maghemite c-Fe2O3) on which rhodamine molecules have been grafted. An increase of the ionic strength of the colloidal solution improved the encapsulation of nanoparticles in Giant Unilamellar Vesicles (GUVs). We proved by magnetophoresis experiments that GUVs encapsulating * Corresponding author. Fax: +33 1 44 27 36 75. E-mail address: [email protected] (C. Ménager). 0021-9797/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2009.11.016
magnetic nanoparticles with a high ionic strength exhibit better magnetic properties compared to GUVs encapsulated magnetic nanoparticles with a lower ionic strength. The strong magnetic properties and then the very important elongation of the magnetic liposome under a magnetic field can be explained by the formation of aggregates in the colloidal solution because the magnetic susceptibility is proportional to the number of magnetic objects and to the square of their volume. Indeed, it is known that the increase of the ionic strength by addition of salt induces the screening of electrostatic repulsions between particles and thus their aggregation [16]. The following study is motivated by the desire to understand the rule of the ionic strength on the properties of the giant liposomes encapsulated magnetic and fluorescent particles. The phase diagram of nanoparticles stabilized by electrostatic repulsions has been studied in the group [17,18] and it was found to be dependent on several parameters: volume fraction of nanoparticles in the colloidal solution, size of the nanoparticles, ionic strength and temperature. As an example, the destabilization of an ionic magnetic fluid by the addition of salt induces a phase separation between a concentrated phase rich in big particles in a diluted phase rich in small particles [19]. The concentrated phase may be a liquid one which exhibits deformation under the application of a magnetic field or a precipitated one with no deformation under magnetic field. In this paper we describe the destabilization under magnetic field of fluorescent and magnetic particles and we use this destabilization to precipitate magnetic particles inside giant liposomes. The liposome is used as a template to obtain an anisotropic shape especially interesting for magnetic applications. The precipitation of nanoparticles after encapsulation is also interesting for hyperthermia properties because the role of the interactions between
G. Beaune, C. Ménager / Journal of Colloid and Interface Science 343 (2010) 396–399
the particles may be important for the heating properties. Moreover this work is performed on giant liposomes (GUV) to follow the phenomenon by optical microscopy but it can be easily transposed to large vesicles (LUV). In fact, the encapsulation of magnetic particles has been described for these two kinds of vesicles [20,21].
2. Materials and methods 2.1. Materials The cis-1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), dimercaptosuccinic acid (DMSA), rhodamine B, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), and cystamine were all purchased from Sigma–Aldrich.
2.2. Synthesis of rhodamine B magnetic fluid The magnetic and fluorescent nanoparticles were synthesized using a previously established process [22], and it was based on the modification of DMSA molecules by rhodamine maleimide before complexation on positively charged c-Fe2O3 particles. The DMSA was coupled to the particles by at least one carboxyl and one thiol group. Ungrafted thiol groups were available for subsequent coupling, and any unadsorbed carboxylate groups ensured that surface charges were present. Then, particles were submitted to successive precipitations with NaCl to eliminate any nonadsorbed rhodamine. Ultrafiltration was used to concentrate particles in the magnetic fluid with a MACROSEP filter (cutoff = 30 kD, Fisher Scientific Labosi) at 5000 rpm for 30 min. The ionic strength was measured from the conductivity. The properties of the fluorescent and magnetic fluid were as follows: r = 540 lS and [Fe] = 6.6 mol L 1.
2.3. Synthesis of magnetic and fluorescent liposomes The preparation of magnetoliposomes has been described elsewhere [23]. The liposomes have been loaded with magnetic particles by mixing preformed magnetic particles with phospholipid powder. The lipid film was thus obtained by shearing. The second step was to swell the obtained film with pure water. The ionic strength of the solution encapsulated inside liposomes was adjusted before encapsulation by adding salt. Sodium chloride (28 mg) was mixed with the magnetic fluid (100 lL) to obtain [NaCl] = 4.79 mol L 1 in the magnetic fluid. A small mass of perfectly dry DOPC powder (around 1 mg) was placed in a glass Petri dish. A total of 10 lL of the c-Fe2O3/NaCl mixture was added, and the mixture was spread and sheared with a glove finger to obtain a fat and oily orange film. This film was presumably a lamellar phase swelled with charged particles. Immediately following the shearing, 1.5 mL of distilled water was poured onto the fatty film to start the spontaneous swelling of the liposomes. The samples were placed in a water bath at 45 °C and observed after 30 min using an optical microscope.
2.4. Characterization The samples were imaged using bright field microscopy (inverted microscope Leica 40, numerical aperture (NA = 0.65) equipped with a 100 W mercury lamp. A fluorescence filter cube with a 546 nm excitation filter (BP546/10) and a 590 nm emission filter (LP 590) was used to visualize rhodamine fluorescence. Pictures from a charge-coupled-device camera were digitized with a frame grabber (LG-3, Scion Corp., Frederick, MD).
397
3. Results and discussion 3.1. Destabilization of the colloidal solution The destabilization of the colloidal solution is induced by screening the electrostatic repulsions between particles. The controlled addition of salt (sodium chloride) may induce the destabilization. In the present study we added an important quantity of salt before the preparation of the liposomes. The ionic strength is mainly due to the added salt and it is 4.79 mol L 1 in colloidal solution. However this salt is diluted during the preparation because of the equilibrium of ions between the inside and the outside of the liposomes during the swelling. After swelling the calculated salt concentration taking into account the dilution factor is 32 mmol L 1. However a measure of the ionic strength made by conductivity measurements and based on a NaCl calibration corresponds to a salt concentration of 5 mmol L 1. This value, much lower than the mean ionic strength (32 mmol L 1) indicates that the salt concentration inside the GUVs is more important than the salt concentration outside. This fact is due to the viscosity of the sample because of the high volume fraction of magnetic particles (6.6 mol L 1 corresponds to 10.5% in volume fraction) and because of the addition of NaCl. High viscosity can really slow down the diffusion of ions occurring during the swelling. The limit of precipitation is studied in bulk samples and observed by optical microscopy (Fig. 1). In bulk samples, the precipitation is obtained for [NaCl] = 0.325 mol L 1 for an iron concentration of 1 mol L 1. For this concentration, aggregates of nanoparticles are formed with a spherical shape and a mean diameter of 1.04 ± 0.32 lm (Fig. 1a, for experimental data see supporting information). When a magnetic field is applied, spherical aggregates form magnetic chains in the direction of the field (Fig. 1b) which themselves come into contact to form magnetic bundles (Fig. 1c). It is important to quote that the obtained structures are irreversible. On Fig. 1c, the magnetic field is cut off. The turbidity of the sample for concentration lower than 0.325 mol L 1 indicates that the destabilization begins before this concentration and that small aggregates are already in solution. Even if the phenomenon observed in bulk samples are not the same in terms of concentration than in confined structures, the kind of observation is the same. However the aim of this study is not to build the phase diagram of the system but it is to use it to induce in situ precipitation of magnetic nanoparticles.
3.2. In situ precipitation Fig. 2a corresponds to optical microscopy pictures of liposomes loaded with magnetic and fluorescent nanoparticles. We can observe that the colloidal solution inside the GUV presents no microscopic destabilization, in bright field microscopy the liposome has a characteristic brown colour due to the magnetic fluid and in epifluorescence a red and homogeneous colour due to the emission of rhodamine molecules grafted on magnetic nanoparticles. Fig. 2 in the previous version of the paper has been placed at the end of the text for clarity. Under magnetic field, the thermal fluctuations due to the entropic behaviour of the bilayer are unfolded and the liposome elongates in the direction of the magnetic field (Fig. 2b). It is reminded that the prolate ellipsoid obtained is defined by the eccentricity, e, where e = [1 (b/a)2]1/2, a is the semiaxis of the liposome parallel to the magnetic field, and b is the value of the two other semiaxes orientated perpendicular to the magnetic field. It is the general behaviour of a magnetic liposome under magnetic field. The prolate deformation of the Rh-FF liposomes before (e = 0.69, Fig. 2b) and after the destabilization of Rh-FF (e = 0.79,
398
G. Beaune, C. Ménager / Journal of Colloid and Interface Science 343 (2010) 396–399
Fig. 1. Destabilization of magnetic and fluorescent nanoparticles under addition of salt [NaCl] = 0.325 mol L
1
(a) before and (b, c) after the application of a magnetic field.
Fig. 2. Optical microscopy images of a magnetic and fluorescent liposome (a) before the destabilization of Rh-FF and with H = 0 Oe, (b) before the destabilization of Rh-FF and with H = 202 Oe and (c) after the destabilization of Rh-FF and with H = 0 Oe.
Fig. 2c) is quite important compared to free salt liposomes encapsulating magnetic fluids (e = 0.55) under the same magnetic field [15]. However in the case of GUVs loaded with a high ionic strength magnetic fluid fifteen minutes after the application of the magnetic field we can noticed the apparition of ridges in the direction of the magnetic field corresponding to magnetic chains of nanoparticles as previously seen (Fig. 1). The magnetic field induces an in situ precipitation and leads to the rigidification of the liposome membrane in an anisotropic shape (Fig. 2c). The deformation of GUVs is kept when the magnetic field is cut off (Fig. 2c). Indeed, the precipitation of the nanoparticles induces the stiffness of the membrane. The magnetic liposomes with magnetic particles precipitated in their core behave as a permanent magnet and can be oriented in the desired direction in (Fig. 3a, b and c) or out of the plane (Fig. 3d). The destabilization can be observed also when observations in epifluorescence spend several minutes typically dozens of minutes. In this case the precipitation is due to the elevation of temperature caused by the beam light that is in accordance with the fact that the temperature is an important parameter for the stability of magnetic nanoparticles (Fig. 4). In this case the interesting fact is the shrinking induced by the precipitation. On Fig. 4b andd, we observe the formation of a small solid magnetic sphere representing 35% of the initial volume of the liposome. The liposome is thus divided in two parts, a colourless liquid one and a brown solid one, corresponding to the phase separation between a precipitate and a supernatant. The precipitation is concomitant with the swelling of the liposome, the volume of the liposome after the swelling is twice the initial one. This swelling is due to a diffusion of water inside the liposome indicating as we previously noted that the liposome is not in equilibrium. This fact is surprising according to the well known permeability properties of such lipid bilayer however the
Fig. 3. Orientation of the rigidified liposome obtained after precipitation of the nanoparticles in the three directions of space.
non equilibrium is due to the high density and viscosity of the initial colloidal solution which can slow down the diffusion rate of water inside. After the phase separation, the difference in osmotic pressure between the inside of the liposome constituted by a highly salty solution in equilibrium with a precipitate and the outside induces the water diffusion. The swelling in not observed in the experiences under magnetic field because in this case the membrane is tense and there is no excess of membrane to permit the swelling of the liposome.
G. Beaune, C. Ménager / Journal of Colloid and Interface Science 343 (2010) 396–399
399
4. Summary By using liposomes as templates it is possible to make controlled aggregation in size, shape and volume fraction of nanoparticles. The aggregates may be retrieve and use for other applications. We also prove that the encapsulation of a salty magnetic colloidal solution inside liposomes can lead to the formation of aggregates responsible of their unique magnetic properties, as we previously described, and also to their in situ precipitation. The precipitation is induced by a magnetic field or by increasing temperature. This phenomenon may be extended to a lot of aqueous dispersion of nanoparticles. Acknowledgments The authors thank Aude Michel for chemical synthesis, Delphine Talbot for chemical analysis, and Andrejs Cebers for helpful discussions. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jcis.2009.11.016. References Fig. 4. Optical microscopy images of two liposomes encapsulating magnetic nanoparticles before (a and c) and after in situ precipitation (b and d).
Fig. 5. Optical microscopy image of magnetic and fluorescent aggregates obtained after GUV solubilization with octylglucoside.
The magnetic globules can be extracted from the liposomes by addition of solubilizing molecules like octylglucoside (OG) (Fig. 5).
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