- Email: [email protected]
Magnetic vesicles
MaterialsScience and Engineering: C 2 (1995) 197-203
Magnetic vesicles J.-C. Bacri a, V. Cabuil b, A. Cebers ‘, C. Mhager b, R. Perzynski a ’ Labo d’Acoustique et O,Dtiquede la MatiPre Condensee, Universite Paris 6, URA CNRS, Tour 13. Case 78, 75252 Paris Cedex OS, France b Lab0 de Physicochimie Inorganique, URA CNRS SRSI, Universite Paris 6, B&nent F, Case 63, 75252 Paris Cea’ex 05, France cInstitute of Physics, Latvian Academy of Sciences, Salaspils-1, LY-2169 Riga, Latvia
Abstract
Syntheses of two different kinds of magnetic vesicles are presented here: (a) 1 pm sized, non-fluctuating DDAB vesicles, chaining under magnetic field, and (b) larger 1110I.trn sized) fluctuating DOPCvesicles, both elongating and chaining under field. A magnetic fluid stabilized without any surfactant is incorporated inside the vesicles. Elongation of vesicles is interpreted in terms of bending elasticity of the membrane and chaining through dipole-dipole interaction between vesicles. Keywords: Magnetic vesicles;
Femofluids;Multiple emulsion; Phospholipids
1. Introduction Magnetic vesicles are a new and original system born from the association of two already complex media: uesicles, which are self-assemblies of tensioactive molecules [ 1,2], and ferrofuids, which are magnetic colloids, made of nanoscopic magnetic particles suspended in a liquid solvent [3]. The chemical synthesis and the study of the physical properties of magnetic vesicles are an up-to-date and very promising problem. The goal for the realization of such artificial materials is double-headed. There is first a biomedical goal, for these biocompatible structures could be used either as vectors of drugs in the organism or for the purpose of immunomagnetic separation [4]. The second goal is the realization of a microscopic model system for the physicist to study elastic constants of membranes through the effect of a very convenient external parameter; a magnetic field. From a historical point ‘of view, He& et al. [5] have successfully incorporated magnetic particles inside DODAC vesicles; the necessity of c:oating the nanoscopic particles with a double layer of small surfactant chains to stabilize the magnetic liquid heavily penalizes the colloidal stability of the final complex system: the global equilibrium of the surfactant is indeed modified Iwhen the magnetic particles are incorporated inside the vesicles. Later Mann and Hannington [ 61 proposed 1 pm vesicles as micro-reactors where a chemical synthesis of the magnetic particles could be directly performed, the purpose being a better control of the size dispersion of particles. These magnetic vesicles are always very dilute in magnetic material. A last example deals with 0928-4931/95/$09.50 0 1995Elsevier Science S.A. All rights reserved SSDIO928-4931(95)00076-3
the nanoscopic limit of such systems: De Cuyper and Joniau [7] have directly stabilized biocompatible magnetic particles, coating these with a phospholipid bilayer. We propose here two different chemical syntheses of magnetic vesicles. Their size ranges from 0.1 pm towards 40 pm; the magnetic liquid is an aqueous ferrofluid with particles electrostatically stabilized at pH 7, i.e. without any phospholipid nor surfactant chains. This clearly reduces all the problems of stability interference inside the system. Under magnetic field, these vesicles frequently associate in elongated chains aligned along the field. Isolated from each other, the largest giant vesicles largely fluctuate in zero field, and exhibit a shape deformation under a constant field, allowing a determination of the bending elastic constant of the membrane.
2. Synthesis and observation of magnetic vesicles Magnetic vesicles result from a combination of tensioactive bilayers and aqueous magnetic fluids. The aqueous magnetic fluids used here are colloidal solutions of nanometric particles of magnetic ferric oxide (magnetite Fe304, maghemite ‘y-Fe,03, or cobalt ferrite FeZCoO,). Such magnetic fluids are synthesized through a chemical process, now well known under the name of “Massart’s process” 181. This method consists in a controlled alkalinization of aqueous mixtures of ferric salts and salts of a divalent metal (Fe( II), Co( II), etc.). The magnetic particles thus obtained have specific magnetic properties (superparamagnetism) [ 31. As
198
J.-C. Bacri et al. /Materials Science and Engineering: C 2 (1995) 197-203
they carry surface charges, they are spontaneously dispersable in water without the use of surfactants, the stability of the solution being ensured by electrostatic repulsions between particles. According to the nature of the ions determining the charge, the pH range of stability of the solution may be monitored. It is now possible to have acidic magnetic fluids (cationic particles), alkaline magnetic fluids (anionic particles) and magnetic fluids of pH 7 (anionic particles, with citrate anions specifically adsorbed). These particles may also be coated by surfactants when it is necessary to disperse them in organic solvents [ 91. Provided aggregation between particles is correctly prevented, magnetic fluids are monophasic solutions, and may replace a usual aqueous or organic phase in organized systems of surfactant molecules. For example, if a cyclohexane-based magnetic fluid replaces cyclohexane in the quaternary system water/sodium dodecylsulfate/pentanol/cyclohexane, a ferrosmectic system is obtained in place of the ordinary lamellar phase, magnetic particles being confined in the cyclohexane phase [ lo]. In the same order of ideas, mixing aqueous magnetic fluids with surfactants in conditions suitable for the formation of vesicles seems the best way to get magnetic vesicles. Nevertheless, in practice the problem is not so easy, for many reasons: ( 1) The surfactant constituting the bilayer must not adsorb on the particles. (2) The composition of the magnetic fluid must not disturb the formation of the vesicles. The presence of the charged particles, like the presence of an extra ionic strength, will displace the boundaries of the phase diagram of the surfactant, and the domain of existence of vesicles may disappear. (3) The magnetic vesicles when synthesized may have size characteristics very different from those of the corresponding empty ones if the presence of the charged particles and of ionic species in solution avoid their swelling. (4) Finally, the problems that have to be faced: the localization of the particles has to be carefully checked and depends on the procedure used. Are the particles inside or outside the vesicle? Are they free to move, or stuck on the surface? We want to show here that the use of ionic magnetic fluids in which the sign of the particles’ surface charges may be monitored in a wide range of pH and which do not contain any undesired surfactant may be by themselves a response to all the problems mentioned above. In the process we propose, we encapsulate an aqueous magnetic fluid inside vesicles, the magnetic colloid staying liquid. 2.1. “Multiple emulsion' 'process “Multiple emulsion” processes are usually described to encapsulate solutions; for example, for pharmacological applications [ 111. The procedure is schematized in Fig. 1: the solution to encapsulate - in the present case, the aqueous magnetic fluid - is first dispersed in an organic solvent of
ether
I chloroform 43
aqueous
magnetic
fluid
magnetic
fluid
plus water in excess aqueous
ether
I chloroform
evaporation ’
1
aqueous
magnetic
fluid
“2O
Fig. 1. Preparation of magneticvesicles via the “multiple
emulsion” proc-
ess.
low boiling point, containing the tensioactive that is intended to constitute the double layer, at room temperature under ultrasonic stirring to get a water-in-oil emulsion (W/O). This emulsion is introduced slowly in an excess of ultra-pure water to get a multiple emulsion W/O/W. Vesicles are then obtained by evaporation of the organic phase from the microscopic oil spherules, the mixture being kept in a warm water bath under magnetic stirring to keep the spherules suspended. The nature of the ferrofluid to encapsulate has to be chosen according to the nature of the polar head of the surfactant constituting the double layer. For example, in the case of cationic surfactants such as didodecyl dimethyl ammonium bromide (DDAB) , the particles have to wear positive surface charges, in order that the surfactant does not adsorb on the particles through electrostatic interactions. If this happens, the particles become surfactant-coated and no longer stay in the water phase. This is immediately detectable, although to form a W/O emulsion a magnetic precipitate is observed. For tensioactives such as phospholipids, anionic particles stabilized at pH 7, i.e. coated by citrate species, have to be used. Illustrations of these two cases are given below. It should be noted that the use of surfactant-coated particles dispersed in the oily phase could be a way to incorporate the particles specifically in the membrane. 2.2. Vesicle observation and characterization Each step of the synthesis is controlled by optical microscopic observation to check if the magnetic fluid is really
J.-C. Bacri et al. /Materials Science and Engineering: C 2 (1995) 197-203
199
confined, first in the drople.ts of the emulsion, second in the vesicles. The magnetic fluid has a characteristic orange colour and thus may be easily localized on the preparation. To test their magnetic properties, a magnetic field of up to IO3 Oe can be applied to the samples. Negative-stain electron microscopy is performed according to the procedure described in Ref. [ 11: a drop of the solution of vesicles is deposited on the microscope grid, initially covered by a carbon film. 2 min are allowed for the vesicles to attach to the film, and the excess of solution is drawn! off and replaced with a drop of colourant (a solution of uranyl acetate for DDAB vesicles, a solution of ammonium molybdate for phospholipid vesicles). After a few minutes, the excess of colourant is again drawn off in order to get a thin film of heavy metal in which the vesicles are embedded. 2.3. DDAB magnetic vesicles DDAB (didodecyl dimethyl ammonium bromide) is a double-chain cationic surfactant which swells spontaneously in the presence of water at room temperature. The binary system water/DDAB has been described in Ref. [ 121, and presents in the dilute regime a domain of vesicles for a weight concentration in DDAB of the order of 0.2%. As the tensioactive is cationic, cationic particles have to be used, and the aqueous magnetic fluid encapsulated is an acidic one. The ionic magnetic fluid is constituted by cationic maghemite particles ( 3/-Fe$J3) dispersed in water. Magnetite particles are synthesized according to Massart’s procedure [ 81, then oxidized to maghemite by ferric nitrate in nitric acid medium. These particles have a mean diameter of about 9 nm. They carry positive surface charges, the surface charge density being of the order of 0.2 C rnd2 [ 91, and the associated counter-ions being nitrate anions. By dispersion in deionized water, they form ,a stable magnetic colloidal solution (ferrofluid) which is acid (pH < 4). The volume fraction of particles in such a solution may range from 0 to 10%. Stability is ensured by screened electrostatic repulsions. Ionic strength is thus reduced as much as possible, but free nitric acid always remains in solution. The maximum concentration of free acid is 0.2 mol l- ‘. The synthesis of magnetllc DDAB vesicles is performed according to the following lprocedure: the oily phase in the multiple emulsion is a mixture of ether ( 1.25 ml) and cyclohexane (0.25 ml). DDAB (2 wt.%) is solubilized in this phase. The aqueous acidic magnetic fluid, in which the volume fraction of the particles, may range up to lo%, is added (0.02 ml) at room temperature to the oily phase under ultrasonic stirring. The emulsion thus obtained (0.75 ml) is introduced slowly (in 20 s) into water ( 15 ml). The ethercyclohexane mixture is evaporated at 45-50 “C under a nitrogen stream, the flask being kept in a warm water bath under magnetic stirring to keep the spherules suspended. The complete evaporation of the ether/cyclohexane mixture is indicated by a noticeable decrease of turbidity and takes about 30 min.
Fig. 2. Optical micrograph images: (a) emulsion of the aqueous magnetic fluid (dark droplets of typical size 2 pm) in a cyclohexane/ether mixture; (b) multiple emulsion.
The different steps of the synthesis of these magnetic vesicles have been followed by optical microscopy (Fig. 2). Fig. 2(a) corresponds to the first step of the synthesis, i.e. the synthesis of the emulsion of the aqueous magnetic fluid in a mixture of ether/cyclohexane. The magnetic fluid appears as orange droplets in a colourless background. Fig. 2(b) corresponds to the multiple emulsion: the droplets of magnetic fluids are inside larger droplets of oily phase, themselves dispersed in water. After evaporation of the oily phase, the double layers are formed and the vesicles synthesized (see Fig. 3 (a) ) . They appear as almost spherical orange droplets. Their diameters range from about 0.5 pm to 5 pm. They seem rather rigid: very few of them present noticeable membrane fluctuations. When a magnetic field is applied, they align along the direction of the field (Fig. 3(b) ) , but few of them present shape modifications. 2.4. Giant magnetic liposomes To get giant vesicles, phospholipidic tensioactives have to be used. We choose as phospholipids dioleoylphosphatidylcholine (DOPC) , for which the melting temperature is 20 “C. For this tensioactive, acidic magnetic fluid is not well suited for encapsulation via a multiple emulsion process: it is impossible to form the emulsion, because of the interactions between the cationic particles and DOPC. Therefore we use a magnetic fluid of pH 7, constituted by the same maghemite particles used in the case of the DDAB system (mean diameter around 9 nm), but coated by citrate anions, and thus carrying negative surface charges. The multiple-emulsion process is performed according to the following procedure: the aqueous magnetic fluid, with a
200
J.-C. Bacri et al. /Materials Science and Engineering: C 2 (1995) 197-203
(b) Fig. 4. Optical micrograph images of magnetic liposomes: (a) without magnetic field (Ro= 9 pm); (h) subjected to a 40 Oe magnetic field (a/ b=1.27).
Fig. 3. Optical micrograph images: (a) final DDAB magnetic vesicles in zero magnetic field; (b) the same vesicles subjected to a lo3 Oe magnetic field (they align along the direction of the field).
volume fraction of magnetic particles which may reach 6% (0.02 ml) is first dispersed in a chloroform/ether mixture ( 1.5 ml) containing DOPC (purchased from Avanti Polar Lipids, and used as received, 2 wt.%) at room temperature under ultrasonic stirring to get the water-in-oil emulsion. This emulsion is introduced slowly in an excess of ultrapure water (15 ml) to get the multiple emulsion W/O/W. Vesicles are obtained by evaporation of the chloroform/ether mixture from the microscopic oil spherules at 50 “C under a nitrogen stream, the flask being kept in a warm water bath under magnetic stirring to keep the spherules suspended. By this procedure, large vesicles are obtained, besides agglomerates of phospholipids. In the absence of a magnetic field, the vesicles appear generally as spheres with a diameter ranging from 10 pm to 40 pm (Fig. 4(a) ) . Some of them clearly present membrane fluctuations and go out of shape when a magnetic field is applied (Fig. 4(b) ) . Chaining of these vesicles is also observed (Fig. 5). Negative-stain electron microscopy was performed according to the procedure described above, the colourant being a solution of ammonium
molybdate. Fig. 6 is a typical electron micrograph image of a magnetic DOPC liposome. It is stuck to an empty vesicle. For the filled liposome, the magnetic nanoparticles, identified by electron diffraction, appear as clearly encapsulated. Note that the vesicles observed by this method often appear smaller than when observed by optical microscopy.
3. Response to a magnetic field A stable magnetic colloid being incorporated inside the vesicles, the goal is then to measure their physical characteristics through their specific response to an external magnetic field. An analysis of the shape deformations of magnetic vesicles can lead to a determination of the viscoelastic constants of the membrane. On the other hand, the chaining of vesicles can lead through the magnetic dipole-dipole interaction to the parameters of membrane-membrane interaction. Typically, two elastic constants determine the viscoelastic properties of the external bilayer: the bending elastic constant Kc accounting for the local curvature of the membrane, and the extensional elastic constant K, accounting for the stretching of the membrane in the inflated regimes. In zero magnetic field, the shape of deflated vesicles is driven by a competition between the energy of thermal fluctuations k,T and elastic energy. In an optical microscope observation, it is only a
J.-C. Bacri et al. /Materials
201
Science and Engineering: C 2 (1995) 197-203
Fig. 5. Chaining of DOPC vesicles under a 500 Oe magnetic field.
external electric field [ 15,161. Here, in a similar way, the applied magnetic field H modifies the stress experienced by the membrane and induces a shape deformation of the vesicle, which lengthens along its direction. The mean shape is assumed to be a prolate ellipsoid, defined by its axis ratio a/ b (a is the long semi-axis and b the short semi-axis of the ellipsoid). Fig. 7 presents the variation of a/b as a function of magnetic field for two different and isolated DOPC vesicles of initial radius R. = 9 pm. The axis ratio a/b is an increasing function of the field. Experimentally, inter-vesicle interaction limits such measurements: if a second vesicle comes close to the first one, they chain together and mutually prevent their elongation. It is shown in Ref. [ 171 that, in our experimental conditions of low field and low magnetization (the magnetic permeability cc,of the colloid inside the vesicle is of the order of 1.1) , T is proportional to Hz. Eq. ( 1) then becomes equal to: 1.6~
1.6
, :
Fig. 6. Electron micrograph image (negative-stain microscopy) of a magnetic DOPC vesicle.
L
a/b
0
projected area So, smaller than the real membrane surface, which is measured. Se is the mean projected area of the fluctuating vesicle, the membrane being under its initial stress ro. If a supplementary external stress T is applied to the membrane, the projected area S, increases. Different authors [ 13-151 have derived similar expressions for the variations of 5, as a function of r. If the fluctuations are small enough, we have:
1.4-
1.3;
-
fi
A
Ab
0
:
0
O
- 1.5
A
A
0
- 1.4
- 1.3
1.2- - &2
- 1.2
l.l-
- 1.1
(1)
WW 1
With non-magnetic vesicles, several experiments under variable external stress have already been performed, either changing the pressure inside the vesicle through a micropipette [ 141 and thus modifying 7, or monitoring T with an
0
0
1.5-
I-
~“‘,““,““,“‘,,,,,.,,,.,,,..,,,~..~ 0
50
100
150
200
250
300
350
1
400
Fig. 7. Axis ratio a/b of elongated DOPC vesicles as a function of applied magnetic field H. The. two symbols correspond to hvo different vesicles of identical size (R. = 9 pm).
J.-C. Bacri et al. /Materials Science and Engineering: C 2 (1995) 197-203
202
4. Future prospects and conclusion
&-So =GW
so
ln[ (H/H*)‘]
(2)
c
where H* is a characteristic field de ndent on both initial stress r,, and A. The eccentricity e = * 1 - (b/a) of the ellipsoidal shape is always small, and (S,- S,) /S, = 2e4/45. The membrane of DDAB vesicles, here synthesized, does not fluctuate in zero magnetic field. One reason to put forward is the rather small size of these vesicles: the fluctuations are proportional to the surface of the membrane. These nonfluctuating vesicles do not elongate significantly in our experimental range of magnetic fields. In contrast, larger DOPC vesicles which largely fluctuate in zero field also elongate along the field (see Fig. 4). A fit of IQ. (2) to experimental results [ 171 leads to Kc = lOk,T, in good agreement with the other kinds of measurements. At high magnetic field, we expect another regime coupled to the extensional elastic constant. The variations of projected surface area are then [ 141: (3) In this regime, the magnetic stress r can be written [ 171 as r= b( tiRo/4e2), M being the magnetization of the magnetic fluid inside the vesicle. Typically the order of magnitude of K, is some 100 mJ m-*. To perform a K, measurement, a stress r larger than K, is necessary. Magnetic vesicles filled with a more magnetic colloid would be necessary: it is impossible to reach the required magnetization (M= 100 kA m- ’ for a/b = 1.6) with a magnetic colloid of H = 1.1. Another interesting phenomenon of magnetic vesicles submitted to a constant field is the formation of chains. Figs. 3 (b) and 5 show this chaining for DDAB vesicles and DOPC vesicles respectively. Whatever the nature of the external bilayer, each magnetic vesicle of volume V behaves in a magnetic field as a macrodipole of magnetic moment 31*1-l li;=---_vG
CL,+2
(4)
Two aligned magnetic dipoles experience an attractive interaction. If the two dipoles are identical, the energy of interaction is Edd= - p& m2/d3), where d is the inter-vesicle distance, centre to centre. At the shortest distance of approach (d- 2Ro if elongation is neglected), the dipolar energy becomes Edd= -h[3(~,.1)/(~,+2)]*VH*. It is of the order of lo- I5 J for our vesicles. We must point out that this energy is much larger than k,T. It means that the interaction between two vesicles kills completely the membrane fluctuations: it is indeed observed experimentally when two vesicles come too close to each other. On the other hand, this dipolar energy is too small to have access to the stretching elastic constant: the elastic energy of the order of Kso2 is equal to some lo- I1 J . Exactly as in the previous problem, to measure K, with this method we need a magnetic liquid of larger k inside the vesicles.
A genuine synthesis of magnetic vesicles, filled with a new generation of magnetic colloid without surfactant, is reported here. Two different kinds of external membrane (DDAB and DOPC) lead to two different typical sizes of vesicles (respectively 0.5 to 5 pm and 10 to 40 pm), and thus to two wellseparated behaviours. Strong membrane fluctuations are only observed with the largest vesicles. Their shape deformation under a magnetic field allows the determination of the bending modulus of the membrane. Determination of the stretching modulus will require a more concentrated magnetic fluid inside the vesicles. Future prospects for the study of such systems are numerous. It would be interesting to analyse membrane fluctuations under a static field, but also under an alternating one that would force a given wave vector. Would magnetic vesicles exhibit shape instabilities as spectacular as droplets of simple magnetic fluids? Anyway, this work is a first step towards the biomechanics of the future: a muscle at the scale of 10 Frn! For example, this system could be used to measure DNA elasticity: clinging on to a DNA macromolecule one extremity of a magnetic vesicle, both of these already being caught at a solid wall from the other extremity. From a biological point of view, determination of the elastic constants of membranes is of paramount importance: it is a measure of the sensitivity of biological cells to external agents such as antibodies, proteins or some drugs which could attack the cell membrane. The present magnetic vesicles are an important step in this direction, but unfortunately DDAB and DOPC membranes are artificial and do not reflect the exact properties of true biological membranes. A natural trend would then be to build up a dual system with a real biological cell embedded in a magnetic fluid. Problems of biocompatibility of the magnetic fluid are in principle now soluble with colloidal magnetic particles coated with citrate ligands. Another attractive idea is to trap the magnetic particles either on or inside the lipidic bilayer. Magnetic particles trapped inside a membrane would be a convenient system, more or less equivalent to biological membrane proteins, that could be suitably probed with an external magnetic field. Magnetic particles externally stuck to the bilayer, either inside or outside the cell, would make the membrane assymmetric, leading to spontaneous curvatures. Magnetic vesicles are thus a rich and very promising system, with large prospects of attractive applications.
Acknowledgments The authors are greatly indebted to M. Lavergne, who performed the electron micrographs in the Groupement Regional de Mesures Physiques (Universite Pierre et Marie Curie). They also thank M. Dubois and T. Zemb for discussions concerning DDAB vesicles, and X. Michalet from Laboratoire de Physique des Solides, Ecole Normale Sup& rieure, Paris, for introducing them to phospholipids.
J.-C. Bacri et al. /Materials Science and Engineering: C 2 (1995) 197-203
References [I I R.R.C. New, in R.R.C. New (ed), Liposomes: a PracticalApproach, IRL Press, New York, 1990, p. 140. [2] R. Lipowsky, Nature. 349 (1991) 475. [3] R.E. Rosensweig, Ferrohya’rodynamics. Cambridge University Press, New York, 1985. [4] D.T. Grow, S.V. Sonti, A. B,ose and K. Raj, J. Magn. Magn. Mat.. 122 (1993) 343. [5] P. Hervb, F. Nome and J.H Fendler, J. Am. Chem. Sot., 106 ( 1984) 8291. [6] S. Mann and J.P. Hannington, J. Colloid Interface Sci., 122 (1988) 326. [7] M. De Cuyper and M. Joniau, Langmuir, 7 ( 1991) 647.
203
[8] R. Massart. IEEE Trans. Magn., 17 (1981) 1247.
[9] J.-C. Bacri, R. Perzynski, D. Salin, V. Cabuil and R. Massart, J. Magn. Magn. Mat., 85 (1990) 27. [ 101 P. Fabre, C. Casagrande, M. Veyssie, V. Cabuil and R. Massart, Phys. Rev. L.ett., 64 (1990) 539.
[ 111 S. Kim, S. Michell, M.S. Turker, E.Y. Chi, S. Sela and G.M. Martin, Biochim. Biophys. Acta, 728 (1983) 339.
[ 121 M. Dubois and Th. Zemb, Langmuir, 7 (1991) 1352. [ 131 S. Milner and S.A. Safran, Phys. Rev. A, 36 (1987) 4371. [ 141 E. Evans and W. Rawicz, Phys. Rev. Left, 64 ( 1990) 2094. [ 151 M. Kummrow and W. Helfrich, Phys. Rev. A, 44 (1991) 8356. [16] M.D. Mitov, P. M&&d, M. Winterhalter, MI. Angelova and P. Bothorel, Phys. Rev. E., 48 ( 1993) 628. [17] J.-C. Bacri, V. Cabuil, A. Cebers, C. Menager and R. Perzynski, Deformation of ferro-vesicles by a magnetic field, in preparation.
Magnetic vesicles J.-C. Bacri a, V. Cabuil b, A. Cebers ‘, C. Mhager b, R. Perzynski a ’ Labo d’Acoustique et O,Dtiquede la MatiPre Condensee, Universite Paris 6, URA CNRS, Tour 13. Case 78, 75252 Paris Cedex OS, France b Lab0 de Physicochimie Inorganique, URA CNRS SRSI, Universite Paris 6, B&nent F, Case 63, 75252 Paris Cea’ex 05, France cInstitute of Physics, Latvian Academy of Sciences, Salaspils-1, LY-2169 Riga, Latvia
Abstract
Syntheses of two different kinds of magnetic vesicles are presented here: (a) 1 pm sized, non-fluctuating DDAB vesicles, chaining under magnetic field, and (b) larger 1110I.trn sized) fluctuating DOPCvesicles, both elongating and chaining under field. A magnetic fluid stabilized without any surfactant is incorporated inside the vesicles. Elongation of vesicles is interpreted in terms of bending elasticity of the membrane and chaining through dipole-dipole interaction between vesicles. Keywords: Magnetic vesicles;
Femofluids;Multiple emulsion; Phospholipids
1. Introduction Magnetic vesicles are a new and original system born from the association of two already complex media: uesicles, which are self-assemblies of tensioactive molecules [ 1,2], and ferrofuids, which are magnetic colloids, made of nanoscopic magnetic particles suspended in a liquid solvent [3]. The chemical synthesis and the study of the physical properties of magnetic vesicles are an up-to-date and very promising problem. The goal for the realization of such artificial materials is double-headed. There is first a biomedical goal, for these biocompatible structures could be used either as vectors of drugs in the organism or for the purpose of immunomagnetic separation [4]. The second goal is the realization of a microscopic model system for the physicist to study elastic constants of membranes through the effect of a very convenient external parameter; a magnetic field. From a historical point ‘of view, He& et al. [5] have successfully incorporated magnetic particles inside DODAC vesicles; the necessity of c:oating the nanoscopic particles with a double layer of small surfactant chains to stabilize the magnetic liquid heavily penalizes the colloidal stability of the final complex system: the global equilibrium of the surfactant is indeed modified Iwhen the magnetic particles are incorporated inside the vesicles. Later Mann and Hannington [ 61 proposed 1 pm vesicles as micro-reactors where a chemical synthesis of the magnetic particles could be directly performed, the purpose being a better control of the size dispersion of particles. These magnetic vesicles are always very dilute in magnetic material. A last example deals with 0928-4931/95/$09.50 0 1995Elsevier Science S.A. All rights reserved SSDIO928-4931(95)00076-3
the nanoscopic limit of such systems: De Cuyper and Joniau [7] have directly stabilized biocompatible magnetic particles, coating these with a phospholipid bilayer. We propose here two different chemical syntheses of magnetic vesicles. Their size ranges from 0.1 pm towards 40 pm; the magnetic liquid is an aqueous ferrofluid with particles electrostatically stabilized at pH 7, i.e. without any phospholipid nor surfactant chains. This clearly reduces all the problems of stability interference inside the system. Under magnetic field, these vesicles frequently associate in elongated chains aligned along the field. Isolated from each other, the largest giant vesicles largely fluctuate in zero field, and exhibit a shape deformation under a constant field, allowing a determination of the bending elastic constant of the membrane.
2. Synthesis and observation of magnetic vesicles Magnetic vesicles result from a combination of tensioactive bilayers and aqueous magnetic fluids. The aqueous magnetic fluids used here are colloidal solutions of nanometric particles of magnetic ferric oxide (magnetite Fe304, maghemite ‘y-Fe,03, or cobalt ferrite FeZCoO,). Such magnetic fluids are synthesized through a chemical process, now well known under the name of “Massart’s process” 181. This method consists in a controlled alkalinization of aqueous mixtures of ferric salts and salts of a divalent metal (Fe( II), Co( II), etc.). The magnetic particles thus obtained have specific magnetic properties (superparamagnetism) [ 31. As
198
J.-C. Bacri et al. /Materials Science and Engineering: C 2 (1995) 197-203
they carry surface charges, they are spontaneously dispersable in water without the use of surfactants, the stability of the solution being ensured by electrostatic repulsions between particles. According to the nature of the ions determining the charge, the pH range of stability of the solution may be monitored. It is now possible to have acidic magnetic fluids (cationic particles), alkaline magnetic fluids (anionic particles) and magnetic fluids of pH 7 (anionic particles, with citrate anions specifically adsorbed). These particles may also be coated by surfactants when it is necessary to disperse them in organic solvents [ 91. Provided aggregation between particles is correctly prevented, magnetic fluids are monophasic solutions, and may replace a usual aqueous or organic phase in organized systems of surfactant molecules. For example, if a cyclohexane-based magnetic fluid replaces cyclohexane in the quaternary system water/sodium dodecylsulfate/pentanol/cyclohexane, a ferrosmectic system is obtained in place of the ordinary lamellar phase, magnetic particles being confined in the cyclohexane phase [ lo]. In the same order of ideas, mixing aqueous magnetic fluids with surfactants in conditions suitable for the formation of vesicles seems the best way to get magnetic vesicles. Nevertheless, in practice the problem is not so easy, for many reasons: ( 1) The surfactant constituting the bilayer must not adsorb on the particles. (2) The composition of the magnetic fluid must not disturb the formation of the vesicles. The presence of the charged particles, like the presence of an extra ionic strength, will displace the boundaries of the phase diagram of the surfactant, and the domain of existence of vesicles may disappear. (3) The magnetic vesicles when synthesized may have size characteristics very different from those of the corresponding empty ones if the presence of the charged particles and of ionic species in solution avoid their swelling. (4) Finally, the problems that have to be faced: the localization of the particles has to be carefully checked and depends on the procedure used. Are the particles inside or outside the vesicle? Are they free to move, or stuck on the surface? We want to show here that the use of ionic magnetic fluids in which the sign of the particles’ surface charges may be monitored in a wide range of pH and which do not contain any undesired surfactant may be by themselves a response to all the problems mentioned above. In the process we propose, we encapsulate an aqueous magnetic fluid inside vesicles, the magnetic colloid staying liquid. 2.1. “Multiple emulsion' 'process “Multiple emulsion” processes are usually described to encapsulate solutions; for example, for pharmacological applications [ 111. The procedure is schematized in Fig. 1: the solution to encapsulate - in the present case, the aqueous magnetic fluid - is first dispersed in an organic solvent of
ether
I chloroform 43
aqueous
magnetic
fluid
magnetic
fluid
plus water in excess aqueous
ether
I chloroform
evaporation ’
1
aqueous
magnetic
fluid
“2O
Fig. 1. Preparation of magneticvesicles via the “multiple
emulsion” proc-
ess.
low boiling point, containing the tensioactive that is intended to constitute the double layer, at room temperature under ultrasonic stirring to get a water-in-oil emulsion (W/O). This emulsion is introduced slowly in an excess of ultra-pure water to get a multiple emulsion W/O/W. Vesicles are then obtained by evaporation of the organic phase from the microscopic oil spherules, the mixture being kept in a warm water bath under magnetic stirring to keep the spherules suspended. The nature of the ferrofluid to encapsulate has to be chosen according to the nature of the polar head of the surfactant constituting the double layer. For example, in the case of cationic surfactants such as didodecyl dimethyl ammonium bromide (DDAB) , the particles have to wear positive surface charges, in order that the surfactant does not adsorb on the particles through electrostatic interactions. If this happens, the particles become surfactant-coated and no longer stay in the water phase. This is immediately detectable, although to form a W/O emulsion a magnetic precipitate is observed. For tensioactives such as phospholipids, anionic particles stabilized at pH 7, i.e. coated by citrate species, have to be used. Illustrations of these two cases are given below. It should be noted that the use of surfactant-coated particles dispersed in the oily phase could be a way to incorporate the particles specifically in the membrane. 2.2. Vesicle observation and characterization Each step of the synthesis is controlled by optical microscopic observation to check if the magnetic fluid is really
J.-C. Bacri et al. /Materials Science and Engineering: C 2 (1995) 197-203
199
confined, first in the drople.ts of the emulsion, second in the vesicles. The magnetic fluid has a characteristic orange colour and thus may be easily localized on the preparation. To test their magnetic properties, a magnetic field of up to IO3 Oe can be applied to the samples. Negative-stain electron microscopy is performed according to the procedure described in Ref. [ 11: a drop of the solution of vesicles is deposited on the microscope grid, initially covered by a carbon film. 2 min are allowed for the vesicles to attach to the film, and the excess of solution is drawn! off and replaced with a drop of colourant (a solution of uranyl acetate for DDAB vesicles, a solution of ammonium molybdate for phospholipid vesicles). After a few minutes, the excess of colourant is again drawn off in order to get a thin film of heavy metal in which the vesicles are embedded. 2.3. DDAB magnetic vesicles DDAB (didodecyl dimethyl ammonium bromide) is a double-chain cationic surfactant which swells spontaneously in the presence of water at room temperature. The binary system water/DDAB has been described in Ref. [ 121, and presents in the dilute regime a domain of vesicles for a weight concentration in DDAB of the order of 0.2%. As the tensioactive is cationic, cationic particles have to be used, and the aqueous magnetic fluid encapsulated is an acidic one. The ionic magnetic fluid is constituted by cationic maghemite particles ( 3/-Fe$J3) dispersed in water. Magnetite particles are synthesized according to Massart’s procedure [ 81, then oxidized to maghemite by ferric nitrate in nitric acid medium. These particles have a mean diameter of about 9 nm. They carry positive surface charges, the surface charge density being of the order of 0.2 C rnd2 [ 91, and the associated counter-ions being nitrate anions. By dispersion in deionized water, they form ,a stable magnetic colloidal solution (ferrofluid) which is acid (pH < 4). The volume fraction of particles in such a solution may range from 0 to 10%. Stability is ensured by screened electrostatic repulsions. Ionic strength is thus reduced as much as possible, but free nitric acid always remains in solution. The maximum concentration of free acid is 0.2 mol l- ‘. The synthesis of magnetllc DDAB vesicles is performed according to the following lprocedure: the oily phase in the multiple emulsion is a mixture of ether ( 1.25 ml) and cyclohexane (0.25 ml). DDAB (2 wt.%) is solubilized in this phase. The aqueous acidic magnetic fluid, in which the volume fraction of the particles, may range up to lo%, is added (0.02 ml) at room temperature to the oily phase under ultrasonic stirring. The emulsion thus obtained (0.75 ml) is introduced slowly (in 20 s) into water ( 15 ml). The ethercyclohexane mixture is evaporated at 45-50 “C under a nitrogen stream, the flask being kept in a warm water bath under magnetic stirring to keep the spherules suspended. The complete evaporation of the ether/cyclohexane mixture is indicated by a noticeable decrease of turbidity and takes about 30 min.
Fig. 2. Optical micrograph images: (a) emulsion of the aqueous magnetic fluid (dark droplets of typical size 2 pm) in a cyclohexane/ether mixture; (b) multiple emulsion.
The different steps of the synthesis of these magnetic vesicles have been followed by optical microscopy (Fig. 2). Fig. 2(a) corresponds to the first step of the synthesis, i.e. the synthesis of the emulsion of the aqueous magnetic fluid in a mixture of ether/cyclohexane. The magnetic fluid appears as orange droplets in a colourless background. Fig. 2(b) corresponds to the multiple emulsion: the droplets of magnetic fluids are inside larger droplets of oily phase, themselves dispersed in water. After evaporation of the oily phase, the double layers are formed and the vesicles synthesized (see Fig. 3 (a) ) . They appear as almost spherical orange droplets. Their diameters range from about 0.5 pm to 5 pm. They seem rather rigid: very few of them present noticeable membrane fluctuations. When a magnetic field is applied, they align along the direction of the field (Fig. 3(b) ) , but few of them present shape modifications. 2.4. Giant magnetic liposomes To get giant vesicles, phospholipidic tensioactives have to be used. We choose as phospholipids dioleoylphosphatidylcholine (DOPC) , for which the melting temperature is 20 “C. For this tensioactive, acidic magnetic fluid is not well suited for encapsulation via a multiple emulsion process: it is impossible to form the emulsion, because of the interactions between the cationic particles and DOPC. Therefore we use a magnetic fluid of pH 7, constituted by the same maghemite particles used in the case of the DDAB system (mean diameter around 9 nm), but coated by citrate anions, and thus carrying negative surface charges. The multiple-emulsion process is performed according to the following procedure: the aqueous magnetic fluid, with a
200
J.-C. Bacri et al. /Materials Science and Engineering: C 2 (1995) 197-203
(b) Fig. 4. Optical micrograph images of magnetic liposomes: (a) without magnetic field (Ro= 9 pm); (h) subjected to a 40 Oe magnetic field (a/ b=1.27).
Fig. 3. Optical micrograph images: (a) final DDAB magnetic vesicles in zero magnetic field; (b) the same vesicles subjected to a lo3 Oe magnetic field (they align along the direction of the field).
volume fraction of magnetic particles which may reach 6% (0.02 ml) is first dispersed in a chloroform/ether mixture ( 1.5 ml) containing DOPC (purchased from Avanti Polar Lipids, and used as received, 2 wt.%) at room temperature under ultrasonic stirring to get the water-in-oil emulsion. This emulsion is introduced slowly in an excess of ultrapure water (15 ml) to get the multiple emulsion W/O/W. Vesicles are obtained by evaporation of the chloroform/ether mixture from the microscopic oil spherules at 50 “C under a nitrogen stream, the flask being kept in a warm water bath under magnetic stirring to keep the spherules suspended. By this procedure, large vesicles are obtained, besides agglomerates of phospholipids. In the absence of a magnetic field, the vesicles appear generally as spheres with a diameter ranging from 10 pm to 40 pm (Fig. 4(a) ) . Some of them clearly present membrane fluctuations and go out of shape when a magnetic field is applied (Fig. 4(b) ) . Chaining of these vesicles is also observed (Fig. 5). Negative-stain electron microscopy was performed according to the procedure described above, the colourant being a solution of ammonium
molybdate. Fig. 6 is a typical electron micrograph image of a magnetic DOPC liposome. It is stuck to an empty vesicle. For the filled liposome, the magnetic nanoparticles, identified by electron diffraction, appear as clearly encapsulated. Note that the vesicles observed by this method often appear smaller than when observed by optical microscopy.
3. Response to a magnetic field A stable magnetic colloid being incorporated inside the vesicles, the goal is then to measure their physical characteristics through their specific response to an external magnetic field. An analysis of the shape deformations of magnetic vesicles can lead to a determination of the viscoelastic constants of the membrane. On the other hand, the chaining of vesicles can lead through the magnetic dipole-dipole interaction to the parameters of membrane-membrane interaction. Typically, two elastic constants determine the viscoelastic properties of the external bilayer: the bending elastic constant Kc accounting for the local curvature of the membrane, and the extensional elastic constant K, accounting for the stretching of the membrane in the inflated regimes. In zero magnetic field, the shape of deflated vesicles is driven by a competition between the energy of thermal fluctuations k,T and elastic energy. In an optical microscope observation, it is only a
J.-C. Bacri et al. /Materials
201
Science and Engineering: C 2 (1995) 197-203
Fig. 5. Chaining of DOPC vesicles under a 500 Oe magnetic field.
external electric field [ 15,161. Here, in a similar way, the applied magnetic field H modifies the stress experienced by the membrane and induces a shape deformation of the vesicle, which lengthens along its direction. The mean shape is assumed to be a prolate ellipsoid, defined by its axis ratio a/ b (a is the long semi-axis and b the short semi-axis of the ellipsoid). Fig. 7 presents the variation of a/b as a function of magnetic field for two different and isolated DOPC vesicles of initial radius R. = 9 pm. The axis ratio a/b is an increasing function of the field. Experimentally, inter-vesicle interaction limits such measurements: if a second vesicle comes close to the first one, they chain together and mutually prevent their elongation. It is shown in Ref. [ 171 that, in our experimental conditions of low field and low magnetization (the magnetic permeability cc,of the colloid inside the vesicle is of the order of 1.1) , T is proportional to Hz. Eq. ( 1) then becomes equal to: 1.6~
1.6
, :
Fig. 6. Electron micrograph image (negative-stain microscopy) of a magnetic DOPC vesicle.
L
a/b
0
projected area So, smaller than the real membrane surface, which is measured. Se is the mean projected area of the fluctuating vesicle, the membrane being under its initial stress ro. If a supplementary external stress T is applied to the membrane, the projected area S, increases. Different authors [ 13-151 have derived similar expressions for the variations of 5, as a function of r. If the fluctuations are small enough, we have:
1.4-
1.3;
-
fi
A
Ab
0
:
0
O
- 1.5
A
A
0
- 1.4
- 1.3
1.2- - &2
- 1.2
l.l-
- 1.1
(1)
WW 1
With non-magnetic vesicles, several experiments under variable external stress have already been performed, either changing the pressure inside the vesicle through a micropipette [ 141 and thus modifying 7, or monitoring T with an
0
0
1.5-
I-
~“‘,““,““,“‘,,,,,.,,,.,,,..,,,~..~ 0
50
100
150
200
250
300
350
1
400
Fig. 7. Axis ratio a/b of elongated DOPC vesicles as a function of applied magnetic field H. The. two symbols correspond to hvo different vesicles of identical size (R. = 9 pm).
J.-C. Bacri et al. /Materials Science and Engineering: C 2 (1995) 197-203
202
4. Future prospects and conclusion
&-So =GW
so
ln[ (H/H*)‘]
(2)
c
where H* is a characteristic field de ndent on both initial stress r,, and A. The eccentricity e = * 1 - (b/a) of the ellipsoidal shape is always small, and (S,- S,) /S, = 2e4/45. The membrane of DDAB vesicles, here synthesized, does not fluctuate in zero magnetic field. One reason to put forward is the rather small size of these vesicles: the fluctuations are proportional to the surface of the membrane. These nonfluctuating vesicles do not elongate significantly in our experimental range of magnetic fields. In contrast, larger DOPC vesicles which largely fluctuate in zero field also elongate along the field (see Fig. 4). A fit of IQ. (2) to experimental results [ 171 leads to Kc = lOk,T, in good agreement with the other kinds of measurements. At high magnetic field, we expect another regime coupled to the extensional elastic constant. The variations of projected surface area are then [ 141: (3) In this regime, the magnetic stress r can be written [ 171 as r= b( tiRo/4e2), M being the magnetization of the magnetic fluid inside the vesicle. Typically the order of magnitude of K, is some 100 mJ m-*. To perform a K, measurement, a stress r larger than K, is necessary. Magnetic vesicles filled with a more magnetic colloid would be necessary: it is impossible to reach the required magnetization (M= 100 kA m- ’ for a/b = 1.6) with a magnetic colloid of H = 1.1. Another interesting phenomenon of magnetic vesicles submitted to a constant field is the formation of chains. Figs. 3 (b) and 5 show this chaining for DDAB vesicles and DOPC vesicles respectively. Whatever the nature of the external bilayer, each magnetic vesicle of volume V behaves in a magnetic field as a macrodipole of magnetic moment 31*1-l li;=---_vG
CL,+2
(4)
Two aligned magnetic dipoles experience an attractive interaction. If the two dipoles are identical, the energy of interaction is Edd= - p& m2/d3), where d is the inter-vesicle distance, centre to centre. At the shortest distance of approach (d- 2Ro if elongation is neglected), the dipolar energy becomes Edd= -h[3(~,.1)/(~,+2)]*VH*. It is of the order of lo- I5 J for our vesicles. We must point out that this energy is much larger than k,T. It means that the interaction between two vesicles kills completely the membrane fluctuations: it is indeed observed experimentally when two vesicles come too close to each other. On the other hand, this dipolar energy is too small to have access to the stretching elastic constant: the elastic energy of the order of Kso2 is equal to some lo- I1 J . Exactly as in the previous problem, to measure K, with this method we need a magnetic liquid of larger k inside the vesicles.
A genuine synthesis of magnetic vesicles, filled with a new generation of magnetic colloid without surfactant, is reported here. Two different kinds of external membrane (DDAB and DOPC) lead to two different typical sizes of vesicles (respectively 0.5 to 5 pm and 10 to 40 pm), and thus to two wellseparated behaviours. Strong membrane fluctuations are only observed with the largest vesicles. Their shape deformation under a magnetic field allows the determination of the bending modulus of the membrane. Determination of the stretching modulus will require a more concentrated magnetic fluid inside the vesicles. Future prospects for the study of such systems are numerous. It would be interesting to analyse membrane fluctuations under a static field, but also under an alternating one that would force a given wave vector. Would magnetic vesicles exhibit shape instabilities as spectacular as droplets of simple magnetic fluids? Anyway, this work is a first step towards the biomechanics of the future: a muscle at the scale of 10 Frn! For example, this system could be used to measure DNA elasticity: clinging on to a DNA macromolecule one extremity of a magnetic vesicle, both of these already being caught at a solid wall from the other extremity. From a biological point of view, determination of the elastic constants of membranes is of paramount importance: it is a measure of the sensitivity of biological cells to external agents such as antibodies, proteins or some drugs which could attack the cell membrane. The present magnetic vesicles are an important step in this direction, but unfortunately DDAB and DOPC membranes are artificial and do not reflect the exact properties of true biological membranes. A natural trend would then be to build up a dual system with a real biological cell embedded in a magnetic fluid. Problems of biocompatibility of the magnetic fluid are in principle now soluble with colloidal magnetic particles coated with citrate ligands. Another attractive idea is to trap the magnetic particles either on or inside the lipidic bilayer. Magnetic particles trapped inside a membrane would be a convenient system, more or less equivalent to biological membrane proteins, that could be suitably probed with an external magnetic field. Magnetic particles externally stuck to the bilayer, either inside or outside the cell, would make the membrane assymmetric, leading to spontaneous curvatures. Magnetic vesicles are thus a rich and very promising system, with large prospects of attractive applications.
Acknowledgments The authors are greatly indebted to M. Lavergne, who performed the electron micrographs in the Groupement Regional de Mesures Physiques (Universite Pierre et Marie Curie). They also thank M. Dubois and T. Zemb for discussions concerning DDAB vesicles, and X. Michalet from Laboratoire de Physique des Solides, Ecole Normale Sup& rieure, Paris, for introducing them to phospholipids.
J.-C. Bacri et al. /Materials Science and Engineering: C 2 (1995) 197-203
References [I I R.R.C. New, in R.R.C. New (ed), Liposomes: a PracticalApproach, IRL Press, New York, 1990, p. 140. [2] R. Lipowsky, Nature. 349 (1991) 475. [3] R.E. Rosensweig, Ferrohya’rodynamics. Cambridge University Press, New York, 1985. [4] D.T. Grow, S.V. Sonti, A. B,ose and K. Raj, J. Magn. Magn. Mat.. 122 (1993) 343. [5] P. Hervb, F. Nome and J.H Fendler, J. Am. Chem. Sot., 106 ( 1984) 8291. [6] S. Mann and J.P. Hannington, J. Colloid Interface Sci., 122 (1988) 326. [7] M. De Cuyper and M. Joniau, Langmuir, 7 ( 1991) 647.
203
[8] R. Massart. IEEE Trans. Magn., 17 (1981) 1247.
[9] J.-C. Bacri, R. Perzynski, D. Salin, V. Cabuil and R. Massart, J. Magn. Magn. Mat., 85 (1990) 27. [ 101 P. Fabre, C. Casagrande, M. Veyssie, V. Cabuil and R. Massart, Phys. Rev. L.ett., 64 (1990) 539.
[ 111 S. Kim, S. Michell, M.S. Turker, E.Y. Chi, S. Sela and G.M. Martin, Biochim. Biophys. Acta, 728 (1983) 339.
[ 121 M. Dubois and Th. Zemb, Langmuir, 7 (1991) 1352. [ 131 S. Milner and S.A. Safran, Phys. Rev. A, 36 (1987) 4371. [ 141 E. Evans and W. Rawicz, Phys. Rev. Left, 64 ( 1990) 2094. [ 151 M. Kummrow and W. Helfrich, Phys. Rev. A, 44 (1991) 8356. [16] M.D. Mitov, P. M&&d, M. Winterhalter, MI. Angelova and P. Bothorel, Phys. Rev. E., 48 ( 1993) 628. [17] J.-C. Bacri, V. Cabuil, A. Cebers, C. Menager and R. Perzynski, Deformation of ferro-vesicles by a magnetic field, in preparation.