A new method for the synthesis of magnetoliposomesClaudio Sangregorio, Joan K. Wiemann, Charles J. O’Connor, 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 ArticlesZnS-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

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JOURNAL OF APPLIED PHYSICS VOLUME 85, NUMBER 8 15 APRIL 1999

Bio/Chemical Magnetism Zeev Rosenzweig, Chairman

A new method for the synthesis of magnetoliposomesClaudio Sangregorio, Joan K. Wiemann, Charles J. O’Connor, 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 bya phospholipid membrane, is presented. Magnetoliposomes are prepared by directly using thephospholipid vesicles as nanoreactors for the precipitation of the magnetic particles. Themagnetoliposomes have been characterized using transmission electron microscopy imaging andx-ray powder diffraction. The magnetic properties of the magnetoliposomes have been investigatedwith a superconducting quantum interference device magnetometer. Our results indicate that themagnetoliposomes contain approximately spherical maghemite nanoparticles averaging 25 nm indiameter. The occurrence of a phospholipid bilayer surrounding the magnetic particles is confirmedboth by transmission electron micrographs of samples negatively stained with uranyl acetate and bydigital fluorescence imaging microscopy measurements of magnetoliposomes labeled withfluorescein. The temperature dependence of the zero field cooled and field cooled susceptibilities ofthe magnetoliposomes is consistent with their expected superparamagnetic nature. ©1999American Institute of Physics.@S0021-8979~99!42808-7#

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I. INTRODUCTION

There is a growing interest in the synthesis and chaterization of particles in the nanometer size region. Nasized magnetic particles exhibit, in comparison to the cventional polycrystalline, coarse-grained materials, novand intriguing physical properties. By virtue of these propties, this relatively new class of materials has found uselarge variety of technological applications, ranging fromagnetic data storage devices1 to anti-tumor drug carriers.2

Several different approaches to nanosized materials hbeen developed in the last years~for a detailed review seeRefs. 3 and 4!. Examples of these techniques include evaration and condensation processes, ionic implantation,rolysis, aerogel/xerogel processes, mechanical crushinpowders, and hydrothermal reactions. Among the othprecipitation inside the water core of vesicles5 or water in oilmicroemulsions6 has been proved to be one of the moreficient routes to the synthesis of nanophase materials.example, microemulsions have been used to synthesizeriety of nanoparticles of silver halides, superconductors,magnetic materials.7

Common problems of magnetic nanoparticles are thtendency to agglomerate once formed and their chemicastability with respect to oxidation in air. Magnetic nanopaticles have been coated with surfactants,8 polymericmaterials,9 and thiol functional groups10 to overcome theseproblems. For biological applications, magnetic nanopticles have been also encapsulated in phospholipid vesiclliposomes. Liposomes are vesicles in which an aqueousume is entirely enclosed by a membrane composed of lmolecules, usually phospholipids.11,12 Apart from theirchemical constituents, which determine their membrane

a!Electronic mail: zxrcm@uno.edu

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idity, charge density, and permeability, liposomes cancharacterized by their size and shape. Typically round strtured, the size of liposomes depends on the method of tpreparation and varies between 15 nm and 300mm.

Magnetoliposomes, i.e., magnetic nanoparticles coawith a phospholipid layer, are formed spontaneously whfatty acid coated magnetic nanoparticles are mixed witpre-prepared liposome suspension.13,14 As mentioned previ-ously, encapsulation of the magnetic nanoparticles in lisomes protects them from aggregation and oxidation. Fthermore, this approach offers some unique advantages wthe magnetic nanoparticles are applied in biological systeEncapsulation of the magnetic nanoparticles in liposomescreases their biocompatibility under physiological contions, making them suitable for a large variety of biologicapplications. In fact, magnetoliposomes are currently eployed for cell separation and sorting15 and in particle basedimmunoassays.16 In these applications, the bioactive moecules such as enzymes or antibodies are attached tophospholipid membrane to provide them with biological sspecificity and selectivity.

This article describes a new and unique approach forsynthesis of magnetoliposomes. The liposomes are usethis technique as nanoreactors for the precipitation reacand, as we will show, they provide a constrained domawhich limits the growth of the particles. The new methooffers numerous advantages over previous methods forsynthesis of magnetoliposomes: first, the protocol forpreparation of the magnetoliposomes is largely simplifisecond, a higher homogeneity of size and shape is realiThe article describes in detail the new synthetic methodthe preparation of magnetoliposomes and the charactetion of their structural and magnetic properties.

9 © 1999 American Institute of Physics

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II. EXPERIMENT

Synthesis of Magnetoliposomes

Dimyristoylphos- phatidylcholine~DMPC! magnetolipo-somes are prepared as follows: 1 ml of a 5:4:1 molar ra50 mM solution of DMPC, cholesterol, and dihexadecphosphate in chloroform was dried under nitrogen untilthe solvent is removed. The sample is immediately recontuted in 1.25 ml of dry 2-propanol with rapid vortexing. Thsolution is then injected in 6 ml of a 0.5 M FeCl2•4H2Oaqueous solution, under sonication in a Liposome Ma~Laboratory Supplies Inc.!. Under these conditions the liposomes form spontaneously encapsulating iron~II ! ions. Inorder to remove the extravescicular ferrous ions, the lisolution is dialyzed in nitrogen atmosphere for 24 h. Afdialysis, 6 ml of a 10% ammonia solution is added undcontinuous stirring and the solution slowly turns to a yellobrown color. The slow diffusion of hydroxide ions inside thferrous containing vesicles causes the precipitation of nasized particles in the aqueous core of the vesicles. The mnetoliposomes are separated from the still free phosphomolecules upon application of a high gradient magnetic fieThe obtained brown powder is washed repeatedly with wand finally suspended in water.

The structural characterization of the sample is carrout by transmission electron microscopy~TEM! and x-raypowder diffraction: the magnetic particles are observedelectron micrographs obtained with a Zeiss 10 C transmsion electron microscope, while the x-ray diffraction~XRD!diffraction pattern is collected on a Philips X’PERT Diffractometer System equipped with a CuKa wavelength, over therange 10,2u,70. Digital fluorescence image of fluorescedoped magnetoliposomes are collected on an Olympus Iinverted fluorescence microscope equipped with a chacoupled device camera~Princeton Instruments, TK1 513512 chip format!.

Static magnetic susceptibility is measured with a Qutum Design MPMS-5 superconducting quantum interferedevice ~SQUID! magnetometer equipped with a supercoducting magnet capable of producing fields up to 55 kOThe temperature dependence of the susceptibility was intigated by cooling the sample in zero field and then raistemperature and followed by cooling the sample with anternal applied field. Both the curves were collected withmeasuring field of 100 Oe.

III. RESULTS AND DISCUSSION

TEM Characterization of the Magnetoliposomes

A typical TEM image of a magnetoliposome sampleshown in Fig. 1. The micrograph clearly shows the occrence of relatively uniform, spherical shaped nanosized pticles. The particles are well separated and their averageis of ;25 nm, with a very small size variation. TEM evdence of the occurrence of the phospholipid coating istained by transmission electron micrographs of sampstained with a 1% uranyl acetate solution. The images shthe occurrence of a thin layer surrounding each partiwhich is attributed to the liposomes bilayer membrane.further indication of the occurrence of a phospholipid bilay

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surrounding the magnetic particles is obtained by a fluorcence microscopy study of vesicles labeled with a fluorcent phospholipids. For this purpose a DMPC liposomeslution is prepared with the same procedure described inexperimental section, with the addition of 5% in weigamount of fluorescein-DHPE~Molecular Probes!. In thisway it is possible to detect the stability of the liposomthroughout all the steps of the synthetic procedure. A digfluorescent image of the powder sample dissolved in waafter separation in the magnetic field, is shown in Fig. 2. Timages show the occurrence of small isolated spots thatdue to the fluorescein doped liposomes. Since the parhas been washed several times we can exclude the ocrence of free liposomes. Thus, the fluorescent spots are onated from molecules adsorbed on the particles surface,firming that the particles are actually coated. Due toinherent limitation of the resolution of fluorescence microcopy, no information on the thickness of the coating candrawn.

XRD Analysis—The XRD pattern of the powderesample displays the characteristic peaks of a spinel phwhich can be attributed either to maghemite (g-Fe2O3) or tomagnetite (Fe3O4). Due to peak broadening, we are unab

FIG. 1. TEM of the product sample taken at 185.0003 magnification. Theaverage size of the particles is;25 nm.

FIG. 2. Digital fluorescence image of fluorescein labeled magnetolisomes.

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5701J. Appl. Phys., Vol. 85, No. 8, 15 April 1999 Sangregorio et al.

to distinguish between the two iron oxide phases. Howesince the precipitation reaction is carried out in air, it is resonable to expect that our sample contains maghemiteticles, as also suggested by the light brown color of thetained powder. The width of the peaks of the x-ray patternmuch larger than expected for an assembly of 25maghemite particles. To get further information on the efftive crystallite size, the experimental powder pattern is copared with simulated patterns of nanometric maghemiteticles of different size. We point out that, since the patteare almost identical, the results of the simulation aresame either for the case of maghemite or magnetite.maghemite patterns are simulated using a pseudo-Voigtfile with a Cauchy factor of 10. No lattice contractionexpansion is included and the patterns are corrected forKa2

wavelength. The comparison indicates that the size ofparticle is slightly lower than 10 nm, which is smaller thathe diameter of the particles measured from TEM micgraphs. A reason for this discrepancy may lay in a partstrain. This effect, often observed in nanoparticles, is knoto give line broadening and has not been included intosimulation. However, this discrepancy may also suggepartial crystallinity of the particles which might be describby a crystalline core surrounded by an amorphous iron oxlayer.

Characterization of the Magnetic Properties of theMagnetoliposomes

Magnetic measurements performed on the powdesample reveal the single domain nature of the particlesexpected for nanosized magnetic particles. The temperadependence of the zero field cooled~ZFC! and field cooled~FC! susceptibilities of the powder are shown in Fig. 3.high temperature the two curves coincide and the suscebility follows, in first approximation, a Curie–Weiss lawwhile at lower temperature they start to separate and themagnetization exhibits a maximum. Such behavior is chateristic of superparamagnetism17 and is due to the progressive blocking of the magnetic moment of smaller and smaparticles when the temperature is decreased. The temperof the maximum of the ZFC curve~blocking temperatureTB!, corresponds to the blocking of particles with the avage volume and is;20 K.

The hysteresis loop measured in the650 kOe range, atroom temperature displays no coercivity. On the contrabelow the blocking temperature, the particles give rise to

FIG. 3. Temperature dependence of the ZFC and FC magnetic suscepties of a powdered sample of magnetoliposomes. Data points are werelected with a measuring field of 50 Oe.

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hysteretic behavior, as is shown in Fig. 4, where the fidependence of the magnetization measured atT55 K is re-ported. The measured coercive field is 350 Oe. Note thatsample is still far from being saturated, even at the highmeasuring field of 50 kOe.

IV. CONCLUSIONS

A new method for the synthesis of magnetoliposomespresented. The synthetic method is largely simplified othe other synthetic procedures previously employed to ppare magnetoliposomes. TEM micrographs and the Xpattern indicate that this method offers the possibility to sthesize magnetic particles with size in the nanometric raand with high uniformity. The control over the sizeachieved by constraining the growth of the particle to tlimited size of the phospholipid vesicles, used here as noreactors. The magnetic properties are consistent with texpected superparamagnetic nature. Future studies will foon further optimization of the structural properties of tmagnetoliposomes and on their application in magnebased immunosensors.

The authors gratefully acknowledge the support of twork by the Advanced Materials Research Institute throuDOD/DARPA Grant No. MDA972-97-1-0003.

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

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