A new method for the synthesis of magnetoliposomes

Claudio Sangregorio, Joan K. Wiemann, Charles J. OConnor, and Zeev Rosenzweig Citation: J. Appl. Phys. 85, 5699 (1999); doi: 10.1063/1.370256 View online: http://dx.doi.org/10.1063/1.370256 View Table of Contents: http://jap.aip.org/resource/1/JAPIAU/v85/i8 Published by the American Institute of Physics.

Related Articles
ZnS-nanocrystals/polypyrrole nanocomposite film based immunosensor Appl. Phys. Lett. 100, 053701 (2012) Polyaniline nano-composites with large negative dielectric permittivity AIP Advances 2, 012127 (2012) Multifunctional silicon inspired by a wing of male Papilio ulysse Appl. Phys. Lett. 100, 033109 (2012) Local domain sensing with nanostructured tunneling anisotropic magneto resistance probes Appl. Phys. Lett. 99, 202504 (2011) Adjustable stiffness of individual piezoelectric nanofibers by electron beam polarization Appl. Phys. Lett. 99, 193102 (2011)

Additional information on J. Appl. Phys.

Journal Homepage: http://jap.aip.org/ Journal Information: http://jap.aip.org/about/about_the_journal Top downloads: http://jap.aip.org/features/most_downloaded Information for Authors: http://jap.aip.org/authors

Downloaded 03 Feb 2012 to 150.140.255.30. Redistribution subject to AIP license or copyright; see http://jap.aip.org/about/rights_and_permissions

JOURNAL OF APPLIED PHYSICS

VOLUME 85, NUMBER 8

15 APRIL 1999

Bio/Chemical Magnetism

Zeev Rosenzweig, Chairman

A new method for the synthesis of magnetoliposomes

Claudio Sangregorio, Joan K. Wiemann, Charles J. OConnor, and Zeev Rosenzweiga)
Advanced Materials Research Institute, University of New Orleans, New Orleans, Louisiana 70148

A new method for the synthesis of magnetoliposomes, i.e., nanosized magnetic particles coated by a phospholipid membrane, is presented. Magnetoliposomes are prepared by directly using the phospholipid vesicles as nanoreactors for the precipitation of the magnetic particles. The magnetoliposomes have been characterized using transmission electron microscopy imaging and x-ray powder diffraction. The magnetic properties of the magnetoliposomes have been investigated with a superconducting quantum interference device magnetometer. Our results indicate that the magnetoliposomes contain approximately spherical maghemite nanoparticles averaging 25 nm in diameter. The occurrence of a phospholipid bilayer surrounding the magnetic particles is conrmed both by transmission electron micrographs of samples negatively stained with uranyl acetate and by digital uorescence imaging microscopy measurements of magnetoliposomes labeled with uorescein. The temperature dependence of the zero eld cooled and eld cooled susceptibilities of the magnetoliposomes is consistent with their expected superparamagnetic nature. 1999 American Institute of Physics. S0021-89799942808-7
I. INTRODUCTION

There is a growing interest in the synthesis and characterization of particles in the nanometer size region. Nanosized magnetic particles exhibit, in comparison to the conventional polycrystalline, coarse-grained materials, novel, and intriguing physical properties. By virtue of these properties, this relatively new class of materials has found use in a large variety of technological applications, ranging from magnetic data storage devices1 to anti-tumor drug carriers.2 Several different approaches to nanosized materials have been developed in the last years for a detailed review see Refs. 3 and 4. Examples of these techniques include evaporation and condensation processes, ionic implantation, pyrolysis, aerogel/xerogel processes, mechanical crushing of powders, and hydrothermal reactions. Among the others, precipitation inside the water core of vesicles5 or water in oil microemulsions6 has been proved to be one of the more efcient routes to the synthesis of nanophase materials. For example, microemulsions have been used to synthesize a variety of nanoparticles of silver halides, superconductors, and magnetic materials.7 Common problems of magnetic nanoparticles are their tendency to agglomerate once formed and their chemical instability with respect to oxidation in air. Magnetic nanoparticles have been coated with surfactants,8 polymeric materials,9 and thiol functional groups10 to overcome these problems. For biological applications, magnetic nanoparticles have been also encapsulated in phospholipid vesicles liposomes. Liposomes are vesicles in which an aqueous volume is entirely enclosed by a membrane composed of lipid molecules, usually phospholipids.11,12 Apart from their chemical constituents, which determine their membrane ua

Electronic mail: zxrcm@uno.edu 5699

idity, charge density, and permeability, liposomes can be characterized by their size and shape. Typically round structured, the size of liposomes depends on the method of their preparation and varies between 15 nm and 300 m. Magnetoliposomes, i.e., magnetic nanoparticles coated with a phospholipid layer, are formed spontaneously when fatty acid coated magnetic nanoparticles are mixed with a pre-prepared liposome suspension.13,14 As mentioned previously, encapsulation of the magnetic nanoparticles in liposomes protects them from aggregation and oxidation. Furthermore, this approach offers some unique advantages when the magnetic nanoparticles are applied in biological systems. Encapsulation of the magnetic nanoparticles in liposomes increases their biocompatibility under physiological conditions, making them suitable for a large variety of biological applications. In fact, magnetoliposomes are currently employed for cell separation and sorting15 and in particle based immunoassays.16 In these applications, the bioactive molecules such as enzymes or antibodies are attached to the phospholipid membrane to provide them with biological site specicity and selectivity. This article describes a new and unique approach for the synthesis of magnetoliposomes. The liposomes are used in this technique as nanoreactors for the precipitation reaction and, as we will show, they provide a constrained domain, which limits the growth of the particles. The new method offers numerous advantages over previous methods for the synthesis of magnetoliposomes: rst, the protocol for the preparation of the magnetoliposomes is largely simplied; second, a higher homogeneity of size and shape is realized. The article describes in detail the new synthetic method for the preparation of magnetoliposomes and the characterization of their structural and magnetic properties.
1999 American Institute of Physics

0021-8979/99/85(8)/5699/3/$15.00

Downloaded 03 Feb 2012 to 150.140.255.30. Redistribution subject to AIP license or copyright; see http://jap.aip.org/about/rights_and_permissions

5700

J. Appl. Phys., Vol. 85, No. 8, 15 April 1999

Sangregorio et al.

II. EXPERIMENT Synthesis of Magnetoliposomes

Dimyristoylphos- phatidylcholine DMPC magnetoliposomes are prepared as follows: 1 ml of a 5:4:1 molar ratio, 50 mM solution of DMPC, cholesterol, and dihexadecyl phosphate in chloroform was dried under nitrogen until all the solvent is removed. The sample is immediately reconstituted in 1.25 ml of dry 2-propanol with rapid vortexing. This solution is then injected in 6 ml of a 0.5 M FeCl24H2O aqueous solution, under sonication in a Liposome Maker Laboratory Supplies Inc.. Under these conditions the liposomes form spontaneously encapsulating iron II ions. In order to remove the extravescicular ferrous ions, the lipid solution is dialyzed in nitrogen atmosphere for 24 h. After dialysis, 6 ml of a 10% ammonia solution is added under continuous stirring and the solution slowly turns to a yellowbrown color. The slow diffusion of hydroxide ions inside the ferrous containing vesicles causes the precipitation of nanosized particles in the aqueous core of the vesicles. The magnetoliposomes are separated from the still free phospholipid molecules upon application of a high gradient magnetic eld. The obtained brown powder is washed repeatedly with water and nally suspended in water. The structural characterization of the sample is carried out by transmission electron microscopy TEM and x-ray powder diffraction: the magnetic particles are observed in electron micrographs obtained with a Zeiss 10 C transmission electron microscope, while the x-ray diffraction XRD diffraction pattern is collected on a Philips XPERT Diffractometer System equipped with a Cu K wavelength, over the range 10 2 70. Digital uorescence image of uorescein doped magnetoliposomes are collected on an Olympus IX70 inverted uorescence microscope equipped with a charge coupled device camera Princeton Instruments, TK1 512 512 chip format. Static magnetic susceptibility is measured with a Quantum Design MPMS-5 superconducting quantum interference device SQUID magnetometer equipped with a superconducting magnet capable of producing elds up to 55 kOe. The temperature dependence of the susceptibility was investigated by cooling the sample in zero eld and then raising temperature and followed by cooling the sample with an external applied eld. Both the curves were collected with a measuring eld of 100 Oe.
III. RESULTS AND DISCUSSION TEM Characterization of the Magnetoliposomes

FIG. 1. TEM of the product sample taken at 185.000 magnication. The average size of the particles is 25 nm.

surrounding the magnetic particles is obtained by a uorescence microscopy study of vesicles labeled with a uorescent phospholipids. For this purpose a DMPC liposomes solution is prepared with the same procedure described in the experimental section, with the addition of 5% in weight amount of uorescein-DHPE Molecular Probes. In this way it is possible to detect the stability of the liposomes throughout all the steps of the synthetic procedure. A digital uorescent image of the powder sample dissolved in water, after separation in the magnetic eld, is shown in Fig. 2. The images show the occurrence of small isolated spots that are due to the uorescein doped liposomes. Since the particle has been washed several times we can exclude the occurrence of free liposomes. Thus, the uorescent spots are originated from molecules adsorbed on the particles surface, conrming that the particles are actually coated. Due to the inherent limitation of the resolution of uorescence microscopy, no information on the thickness of the coating can be drawn. XRD AnalysisThe XRD pattern of the powdered sample displays the characteristic peaks of a spinel phase, which can be attributed either to maghemite ( -Fe2O3) or to magnetite (Fe3O4). Due to peak broadening, we are unable

A typical TEM image of a magnetoliposome sample is shown in Fig. 1. The micrograph clearly shows the occurrence of relatively uniform, spherical shaped nanosized particles. The particles are well separated and their average size is of 25 nm, with a very small size variation. TEM evidence of the occurrence of the phospholipid coating is obtained by transmission electron micrographs of samples stained with a 1% uranyl acetate solution. The images show the occurrence of a thin layer surrounding each particle, which is attributed to the liposomes bilayer membrane. A further indication of the occurrence of a phospholipid bilayer

FIG. 2. Digital uorescence image of uorescein labeled magnetoliposomes.

Downloaded 03 Feb 2012 to 150.140.255.30. Redistribution subject to AIP license or copyright; see http://jap.aip.org/about/rights_and_permissions

J. Appl. Phys., Vol. 85, No. 8, 15 April 1999

Sangregorio et al.

5701

to distinguish between the two iron oxide phases. However, since the precipitation reaction is carried out in air, it is reasonable to expect that our sample contains maghemite particles, as also suggested by the light brown color of the obtained powder. The width of the peaks of the x-ray pattern is much larger than expected for an assembly of 25 nm maghemite particles. To get further information on the effective crystallite size, the experimental powder pattern is compared with simulated patterns of nanometric maghemite particles of different size. We point out that, since the patterns are almost identical, the results of the simulation are the same either for the case of maghemite or magnetite. The maghemite patterns are simulated using a pseudo-Voigt prole with a Cauchy factor of 10. No lattice contraction or expansion is included and the patterns are corrected for K 2 wavelength. The comparison indicates that the size of the particle is slightly lower than 10 nm, which is smaller than the diameter of the particles measured from TEM micrographs. A reason for this discrepancy may lay in a particle strain. This effect, often observed in nanoparticles, is known to give line broadening and has not been included into the simulation. However, this discrepancy may also suggest a partial crystallinity of the particles which might be described by a crystalline core surrounded by an amorphous iron oxide layer.
Characterization of the Magnetic Properties of the Magnetoliposomes

FIG. 4. Field dependence of the magnetization measured at 5 K.

hysteretic behavior, as is shown in Fig. 4, where the eld dependence of the magnetization measured at T 5 K is reported. The measured coercive eld is 350 Oe. Note that the sample is still far from being saturated, even at the highest measuring eld of 50 kOe.
IV. CONCLUSIONS

Magnetic measurements performed on the powdered sample reveal the single domain nature of the particles, as expected for nanosized magnetic particles. The temperature dependence of the zero eld cooled ZFC and eld cooled FC susceptibilities of the powder are shown in Fig. 3. At high temperature the two curves coincide and the susceptibility follows, in rst approximation, a CurieWeiss law, while at lower temperature they start to separate and the ZFC magnetization exhibits a maximum. Such behavior is characteristic of superparamagnetism17 and is due to the progressive blocking of the magnetic moment of smaller and smaller particles when the temperature is decreased. The temperature of the maximum of the ZFC curve blocking temperature, T B , corresponds to the blocking of particles with the average volume and is 20 K. The hysteresis loop measured in the 50 kOe range, at room temperature displays no coercivity. On the contrary, below the blocking temperature, the particles give rise to an

A new method for the synthesis of magnetoliposomes is presented. The synthetic method is largely simplied over the other synthetic procedures previously employed to prepare magnetoliposomes. TEM micrographs and the XRD pattern indicate that this method offers the possibility to synthesize magnetic particles with size in the nanometric range and with high uniformity. The control over the size is achieved by constraining the growth of the particle to the limited size of the phospholipid vesicles, used here as nanoreactors. The magnetic properties are consistent with their expected superparamagnetic nature. Future studies will focus on further optimization of the structural properties of the magnetoliposomes and on their application in magnetic based immunosensors. The authors gratefully acknowledge the support of this work by the Advanced Materials Research Institute through DOD/DARPA Grant No. MDA972-97-1-0003.
Science and Technology of Nanostructured Magnetic Materials, NATO ASI Series B: Physics Vol. 259, edited by G. C. Hadjipanays and G. Prinz 1991. 2 A. R. Schweneder, Chimia 46, 69 1992. 3 D. L. Leslie-Pelecky and R. D. Rieke, Chem. Mater. 8, 1770 1996. 4 K. J. Klabude et al., in NATO ASI Series E, Vol. 260, edited by G. C. Hadjipanayis and R. W. Siegel Kluwer, Dordrecht, 1994, pp. 119, and references therein. 5 I. I. Yaacob, A. C. Nunes, and A. Bose, J. Colloid Interface Sci. 171, 73 1995. 6 C. Petit, P. Lixon, and M. P. Pileni, J. Phys. Chem. 94, 1528 1990. 7 V. Pillai, P. Kumar, M. J. Hou, P. Ayyub, and D. O. Shah, Adv. Colloid Interface Sci. 55, 241 1995. 8 N. S. Kommareddi et al., Chem. Mater. 8, 801 1996. 9 N. Fauconnier, J. N. Pons, J. Roger, and A. Bee, J. Colloid Interface Sci. 194, 423 1997. 10 K. Adzamli, R. B. Dorshow, M. R. Hynes, D. L. Nosco, and M. D. Adams, Acta Radiol., Suppl. 412, 73 1997. 11 R. R. C. New, LiposomesA Practical Approach Oxford University Press, Oxford, United Kingdom, 1989, pp. 33104. 12 M. Babincova and P. Babinec, Cell. Mol. Biol. Lett. 2, 3 1997. 13 M. De Cuyper and M. Joniau, Eur. Biophys. J. 15, 311 1988. 14 M. De Cuyper and M. Joniau, Langmuir 7, 647 1991. 15 A. D. Gee, in Cell Separation Methods and Applications, edited by D. Recktenwald and A. Radbrush Marcel Dekker, Inc., New York, 1997, p. 175. 16 S. E. Wright and H. Leaf, J. Lipid Res. 2, 257 1992. 17 A. H. Morrish, The Physical Principles of Magnetism Wiley, New York, 1965.
1

FIG. 3. Temperature dependence of the ZFC and FC magnetic susceptibilities of a powdered sample of magnetoliposomes. Data points are were collected with a measuring eld of 50 Oe.

Downloaded 03 Feb 2012 to 150.140.255.30. Redistribution subject to AIP license or copyright; see http://jap.aip.org/about/rights_and_permissions