Langmuir 2006, 22, 2351-2357
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Maghemite Nanoparticles Protectively Coated with Poly(ethylene imine) and Poly(ethylene oxide)-block-poly(glutamic acid) Andreas F. Thu¨nemann,*,† Dagmar Schu¨tt,‡ Lutz Kaufner,§ Ulrich Pison,§ and Helmuth Mo¨hwald| Federal Institute for Materials Research and Testing, Richard-Willsta¨tter-Strasse 11, 12489 Berlin, Germany, Technical UniVersity Berlin, Institut fu¨r Werkstoffwissenschaften, Englische Strasse 20, 10587 Berlin, Germany, Charite´ , UniVersita¨tsmedizin Berlin, Spandauer Damm 130, 14050 Berlin, Germany, and Max Planck Institute of Colloids and Interfaces, Am Mu¨hlenberg 1, 14476 Potsdam, Germany ReceiVed NoVember 7, 2005. In Final Form: December 12, 2005 Superparamagnetic iron oxide particles (SPIO) of maghemite were prepared in aqueous solution and subsequently stabilized with polymers in two layer-by-layer deposition steps. The first layer around the maghemite core is formed by poly(ethylene imine) (PEI), and the second one is formed by poly(ethylene oxide)-block-poly(glutamic acid) (PEO-PGA). The hydrodynamic diameter of the particles increases stepwise from Dh ) 25 nm (parent) via 35 nm (PEI) to 46 nm (PEI plus PEO-PGA) due to stabilization. This is accompanied by a switching of their ζ-potentials from moderately positive (+28 mV) to highly positive (+50 mV) and finally slightly negative (-3 mV). By contrast, the polydispersity indexes of the particles remain constant (ca. 0.15). Mo¨ssbauer spectroscopy revealed that the iron oxide, which forms the core of the particles, is only present as Fe(III) in the form of superparamagnetic maghemite nanocrystals. The magnetic domains and the maghemite crystallites were found to be identical with a size of 12.0 ( 0.5 nm. The coated maghemite nanoparticles were tested to be stable in water and in physiological salt solution for longer than 6 months. In contrast to novel methods for magnetic nanoparticle production, where organic solvents are necessary, the procedure proposed here can dispense with organic solvents. Magnetic resonance imaging (MRI) experiments on living rats indicate that the nanoparticles are useful as an MRI contrast agent.
1. Introduction Superparamagnetic nanoparticles are used in biomedical applications for clinical diagnostics and for in vivo applications such as contrast agents for magnetic resonance imaging (MRI) in the diagnosis of tumors and cardiovascular diseases. Recent reviews on chemically prepared magnetic nanoparticles, their characteristics, and their applications in the biomedical sector have been published by Willard et al.1 and Rechenberg et al.2 A major problem is that the surfaces of the nanoparticles are not clearly defined and analytical tools for their precise characterization are rare. The term “superparamagnetism” is not very precise but is commonly used to stress the magnetic behavior of paramagnetic nanoparticles (arising from the coupling of several thousands of atoms) in comparison to the paramagnetism of a single atom. Superparamagnetic magnetization can reach nearly the saturation magnetization of ferromagnetic iron oxide, but in contrast to ferromagnetic iron the particles no longer show magnetic interactions after elimination of the magnetic field. Furthermore, the magnetization of superparamagnetic nanoparticles follows an external magnetic field without any hysteresis. The nanoparticles can be tracked in a magnetic field gradient (useful, e.g., for target specific drug delivery) and heated, the latter based on Ne´el relaxation (e.g., for magnetic fluid hyperthermia in brain cancer therapy). †
Federal Institute for Materials Research and Testing. Technical University Berlin. § Charite ´ , Universita¨tsmedizin Berlin. | Max Planck Institute of Colloids and Interfaces. ‡
(1) Willard, M. A.; Kurihara, L. K.; Carpenter, E. E.; Calvin, S.; Harris, V. G. Chemically prepared magnetic nanoparticles. Int. Mater. ReV. 2004, 49 (3-4), 125-170. (2) Neuberger, T.; Schopf, B.; Hofmann, H.; Hofmann, M.; von Rechenberg, B. Superparamagnetic nanoparticles for biomedical applications: Possibilities and limitations of a new drug delivery system. J. Magn. Magn. Mater. 2005, 293 (1), 483-496.
By definition, superparamagnetic iron oxide particles are generally classified with regard to their size into superparamagnetic iron oxide particles (SPIO), displaying hydrodynamic diameters larger than 30 nm, and ultrasmall superparamagnetic iron oxide particles (USPIO), with hydrodynamic diameters smaller than 30 nm.3 Tailored surfaces play a key role in the future development of advanced superparamagnetic nanoparticle systems for biomedical applications. Such surfaces should (i) stabilize the nanoparticles in biological surroundings with a pH of about 7.4, particularly at physiological salt concentration, (ii) provide functional groups at the surface for further derivatization (e.g., with receptor ligands for cell specific uptake), and (iii) suppress the uptake by the reticuloendothelial system. Various methods of stabilization for SPIOs are nowadays available,4 and one of the most attractive ones is their stabilization in the presence of biocompatible polymers.2 Suitable here are synthetic polymers including poly(alkylcyanoacrylate)s, poly(-caprolactone)s, and poly(lactic acid)s as well as natural polymers such as polysaccharides and proteins. However, little is known of whether synthetic poly(amino acid)s and their block copolymers, especially those with poly(ethylene oxide), are suitable for the development of SPIO systems with defined surface properties. This is surprising because the coating of particles with poly(ethylene oxide)s to avoid their uptake by the reticuloendothelial system is under intensive investigation (often referred to as PEGylation).5 The aim of this work is to apply the (3) Lawaczeck, R.; Menzel, M.; Pietsch, H. Superparamagnetic iron oxide particles: contrast media for magnetic resonance imaging. Appl. Organomet. Chem. 2004, 18 (10), 506-513. (4) Kim, D. K.; Mikhaylova, M.; Zhang, Y.; Muhammed, M. Protective coating of superparamagnetic iron oxide nanoparticles. Chem. Mater. 2003, 15 (8), 16171627. (5) Bhadra, D.; Bhadra, S.; Jain, P.; Jain, N. K., Pegnology: a review of PEG-ylated systems. Pharmazie 2002, 57 (1), 5-29.
10.1021/la052990d CCC: $33.50 © 2006 American Chemical Society Published on Web 01/19/2006
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Figure 1. Sketch of polymer coated maghemite nanoparticles and chemical structures of the polymers. The branched poly(ethylene imine) (PEI) was used for the first layer (red), and poly(ethylene oxide)-b-poly(glutamic acid) (PEO-PGA) was used for the second layer (blue and green, respectively).
layer-by-layer technique6-11 for the coating of SPIOs with polymers to approximate nanoparticles that meet the requirements mentioned above. Here, we report the synthesis of superparamagnetic nanoparticles surrounded by a first layer of poly(ethylene imine) (PEI) and a second layer of poly(ethylene oxide)-blockpoly(glutamic acid) (PEO-PGA). The chemical structures of the polymers and a sketch of the target particle morphology are shown in Figure 1. 2. Materials and Methods 2.1. Materials. Ammonium hydroxide, nitric acid, methanol, hexane, diethyl ether, tetrahydrofuran (THF), ferric chloride hexahydrate (FeCl3‚6H2O), ferrous chloride tetrahydrate (FeCl2‚4H2O), sodium bicarbonate, magnesium sulfate, chloroform, γ-benzyl-Lglutamic acid, and triphosgene were obtained from Fluka. Resovist was obtained from Schering. Mono amino terminated methoxy poly(ethylene oxide) (Mw ) 5000 g mol-1, Mw/Mn ) 1.05) was purchased from Rapp. N,N-Dimethylformamide (DMF; water free), ethyl acetate (anhydrous), and palladium catalyst (palladium, 10% (w/w) on activated carbon) were obtained from Aldrich. All chemicals were used without further purification. Dialyzing tubes (molecular weight cutoff was 4000-6000 g mol-1) were purchased from Roth. Poly(ethylene imine) (PEI) was purchased from BASF (Mw ) 2.5 × 104 g mol-1) and was purified by ultrafiltration prior to use (molecular weight cutoff was 104 g mol-1) and freeze-dried. The molecular weight characteristics of the purified PEI were Mw ) 4 × 104 g (6) Decher, G.; Hong, J. D.; Schmitt, J. Buildup of Ultrathin Multilayer Films by a Self-Assembly Process. 3. Consecutively Alternating Adsorption of Anionic and Cationic Polyelectrolytes on Charged Surfaces. Thin Solid Films 1992, 210 (1-2), 831-835. (7) Decher, G.; Eckle, M.; Schmitt, J.; Struth, B. Layer-by-layer assembled multicomposite films. Curr. Opin. Colloid Interface Sci. 1998, 3 (1), 32-39. (8) Sukhorukov, G. B.; Donath, E.; Lichtenfeld, H.; Knippel, E.; Knippel, M.; Budde, A.; Mohwald, H. Layer-by-layer self-assembly of polyelectrolytes on colloidal particles. Colloids Surf., A: Physicochem. Eng. Aspects 1998, 137 (13), 253-266. (9) Schonhoff, M. Self-assembled polyelectrolyte multilayers. Curr. Opin. Colloid Interface Sci. 2003, 8 (1), 86-95. (10) Vinogradova, O. I. Mechanical properties of polyelectrolyte multilayer microcapsules. J. Phys.: Condens. Matter 2004, 16 (32), R1105-R1134. (11) Caruso, F. Nanoengineering of particle surfaces. AdV. Mater. 2001, 13, (1), 11.
Thu¨nemann et al. mol-1 and Mw/Mn ) 1.5 as determined by analytical ultracentrifugation. The PEI is highly branched, with molar ratios of 34:40:26 (primary to secondary to tertiary) amino groups. 2.2. Syntheses. Poly(ethylene oxide)-block-poly(glutamic acid). Monomer Synthesis. γ-Benzyl-L-glutamic acid N-carboxy anhydride (BLG-NCA) was synthesized by phosgenation of γ-benzyl-Lglutamic acid using triphosgene.12 γ-Benzyl-L-glutamic acid (25 g, 0.1 mol) was suspended in 350 mL of anhydrous ethyl acetate in an argon atmosphere and heated to 96 °C. A solution of triphosgene (12.3 g, 0.04 mol) in 50 mL of ethyl acetate was added dropwise over a period of 4 h while argon was flushed continuously through the suspension. The reaction was finished when the suspension became a clear solution. The solution was chilled and washed 5 times with 50 mL of sodium bicarbonate solution (10 wt %) at 0 °C, dried with magnesium sulfate, and filtered. Water-free hexane was added until crystallization started, and the mixture was stored at -20 °C overnight. The crystalline product was filtered off and dried in a vacuum. Polymerization. The poly(ethylene oxide)-block-poly(γ-benzylL-glutamic acid) (PEO-PBLG) was polymerized by ring-opening polymerization of γ-benzyl-L-glutamic acid N-carboxy anhydride using R-methoxy-ω-amino-poly(ethylene oxide) (PEO) as a macroinitiator. PEO (3.00 g, 7.2 × 10-4 mol) was dissolved under stirring in 140 mL of water-free DMF in an argon atmosphere at 35 °C. BLG-NCA (2.36 g, 9 × 10-3 mol) was added, and the solution was stirred for 5 days at 35 °C. The solvent was removed in a vacuum, the residue was dissolved in 50 mL of chloroform, and PEO-PBLG was isolated by precipitation in diethyl ether and dried under vacuum. The composition of the block copolymer was determined by 1H NMR from the peak intensity ratios of the methylene protons of PEO and the phenyl protons of the γ-benzyl groups (400 MHz in DMSO-d6 at room temperature): δ ) 3.5 (s, OCH2CH2); δ ) 5.0 (m, 2 H, benzyl); δ ) 7.3 (m, 5 H, aryl). The benzyl groups of PEO-PBLG were removed by hydrogenation with a palladium catalyst (Pd/C, 10%) and H2 in THF/methanol at room temperature for 24 h. The palladium catalyst was filtered off and the solvent removed in a vacuum. The residue was dissolved in deionized water and dialyzed in deionized water for 4 days. Poly(ethylene oxide)-block-poly(L-glutamic acid) was isolated by freezedrying. Complete deprotection was confirmed by 1H NMR (D2O, pH 8, room temperature); the polymerization degree was determined to be 13. 1H NMR of poly(ethylene oxide)-block-poly(L-glutamic acid) in D2O, 400 MHz δ ) 3.5 (s, PEO). No signals of benzyl groups could be detected, indicating a complete removal of the protecting groups. Parent Particles. The magnetic particles were synthesized according to Bee et al.13 Briefly, ammonium hydroxide solution was added to an acidic solution of iron(II) chloride and iron(III) chloride ([Fetotal] ) 0.13 mol L-1 with Vtotal ) 1000 mL and [Fe(II)]/[Fe(III)] ) 0.5). The precipitate was isolated by centrifugation, washed twice by stirring in distilled water (50 mL), and isolated again by centrifugation (15 min, 2500 rpm). Afterward, the precipitate was stirred in nitric acid (40 mL, 2 mol L-1) for 30 min at room temperature, centrifuged again, and suspended by stirring in 50 mL of distilled water. The pH of the resulting suspension was about 2. The solid content of the suspension was determined to 1.95% (w/w) by gravimetric analysis. One-Layer-Coated Particles. A 1.24 g sample of the magnetic particle suspension was diluted with 5.22 g of water under stirring, and 0.42 g of a PEI solution (5% (w/w) in water) was added. The suspension was stirred for 10 min at room temperature. Two-Layer-Coated Particles. After preparation of the one-layer particles, 5.14 g of a PEO-PGA solution (3% (w/w) in water) was added under stirring. The pH was adjusted to 7.4 with 1 M HCl. 2.3. Methods. Dynamic Light Scattering (DLS). Dynamic light scattering measurements were performed with a Malvern Instruments (12) Penczek, S. Models of Biopolymers by Ring-Opening Polymerization; CRC Press: Boca Raton, FL, 1990; p 388. (13) Bee, A.; Massart, R.; Neveu, S., Synthesis of very fine maghemite particles. J. Magn. Magn. Mater. 1995, 149, 6-9.
Maghemite Nanoparticles Coated by PEI and PEO-PGA particle sizer (HPPS-ET 5002) (Malvern Instruments, UK) equipped with a He-Ne laser (λ ) 632.8 nm). The scattering data were recorded at 25 ( 0.1 °C in backscattering mode at a scattering angle of 2θ ) 173°. A 40 µL volume of the samples was diluted with 1000 µL of filtrated water (the cutoff of the filter was 0.45 µm) and placed in a 10 × 10 mm quartz cuvette. ζ-Potential. The ζ-potential of the nanoparticles was determined with a Zetamaster (Malvern Instruments) from an average of five measurements. (For those measurements, 400 µL of a sample was diluted with 4.6 mL of a millimolar sodium chloride solution of pH 7.4.) X-ray Diffraction. Samples studied with X-ray diffraction (XRD) were prepared by drying the nanoparticle dispersion on a glass substrate in a vacuum at 50 °C. XRD data were collected using Co KR radiation (λ ) 0.178 892 nm) with a Siemens D-5000 diffractometer. The modulus of the scattering vector is given as q ) 4 π/λ sin(θ). Mo¨ssbauer Spectroscopy. For Mo¨ssbauer spectroscopy we used the same samples as for XRD. Mo¨ssbauer spectra were recorded at 293 and 78 K with a constant acceleration Mo¨ssbauer spectrometer using a Mo¨ssbauer source of 57Co in chromium matrix with an activity of about 0.3 GBq. A proportional counter was used with a voltage of 1800 V to detect the spectrum. The samples (100 mg) were placed in a sealable cylindrical polyethylene sample compartment with an inner diameter of 19 mm. The Mo¨ssbauer spectra were recorded with a maximum velocity of 3.1 mm/s and referenced to a standard R-Fe foil (Goodfellow). Raman Spectroscopy. Raman spectra were collected in backscattering geometry at room temperature in ambient atmosphere using a Dilor XY spectrometer equipped with a liquid nitrogen cooled multichannel detector. The samples were excited with an argon ion laser (λ ) 514.5 nm), which was focused on the sample to a spot of about 2 µm. Laser powers of 2-10 mW were used for excitation, yielding a power density at the sample surface of 0.2-1.0 mW cm-2. Spectra were recorded using an integration time of 100 s (2to 10-fold accumulation) with a spectral resolution of 1 cm-1. Magnetic Resonance Imaging. Maghemite nanoparticles were evaluated as a contrast enhancer for magnetic resonance imaging after intravenous application in 250-300 g Wistar rats (Harlan Winkelmann, Borchen, Germany). Before the application of particles, the rats were anesthetized by an intraperitoneal administration of 90 mg/kg body weight ketamine hydrochloride (Serumwerk, Bernburg, Germany) and 10 mg/kg body weight of Xylazinhydrochlorid 2% (Bayer, Leverkusen, Germany). The magnetic resonance imaging was performed in a 3 T magnetic resonance scanner (Sigma 3T94, General Electric Healthcare, Milwaukee, WI). Axial scans of the rat liver were obtained in a knee coil (General Electric) before and after 10 min intravenous application of the particles in a dose of 0.6 mg of Fe/kg of body weight. For high-resolution imaging of the liver we used T2-weighted gradient echo sequences with a repetition time (TR) of 3500 ms, an echo train length (ET) of 12, and an effective echo time (TE) of 80 ms. In a field of view (FOV) of 10 × 6 cm and a slice thickness of 3 mm, we received six axial liver slices at a scan time of 4 min 37 s and in-plane resolution of 0.25 × 0.16 mm.
3. Results and Discussion 3.1. Particle Sizes and Stability. The hydrodynamic diameter (Dh) is a useful quantity to control the steps of nanoparticle preparation and to develop structural models for the particles. Therefore, we determined Dh after each step of preparation by dynamic light scattering. First, the parent particles were synthesized by coprecipitation of iron(II) and iron(III) chloride in water by adding ammonium hydroxide as described by Bee et al.13 The reaction was carried out under conventional conditions in the absence of organic stabilizers such as dextran and carboxydextran.3 The resulting particle size distribution is shown in Figure 2. It can be seen that the maximum for the parent particles is found at Dh ) 25 nm (curve 1) and that 95% of the
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Figure 2. Volume-weighted distributions of hydrodynamic diameters of nanoparticles as determined by dynamic light scattering. The numbers denote the particles after different steps of preparation: (1) bare maghemite; (2) coated with a layer of poly(ethylene imine); (3) coated with a layer of poly(ethylene imine) and PEO-PGA; (4) adjusted to a salt concentration of 0.15 M sodium chloride. The maxima are at 25 (1), 35 (2), 46 (3), and 43 nm (4), respectively. The inset displays the data as cumulative volume-weighted distributions. 95% of the particles display sizes in the range of 15-50 (1), 20-75 (2), and 25-110 nm (3,4). The corresponding DLS polydispersity indexes are 0.14 (1), 0.22 (2), 0.16 (3), and 0.15 (4).
particles possess a diameter between 15 and 52 nm. Their ζ-potential of +28 mV is as positive as expected. After addition of the first layer (PEI), the hydrodynamic particle diameter increased to Dh ) 35 nm (95% are in the range of 18-82 nm). These PEI coated particles have a ζ-potential of +50 mV. We interpret the increase of the size and the high positive ζ-potential to be a result of adsorption of the cationic PEI to the positively charged particle surface. It seems contradictory at first sight that a cationic polymer adsorbs to positively charged particles. However, PEI is the cationic polyelectrolyte with the highest known charge density (formally every third atom in the polymer backbone can carry a positive charge), and thus PEI is able to adsorb at very different surfaces within a broad pH range.14 In particular, PEI can even adsorb at already highly positively charged surfaces and thereby increase the surface charge further. In our case this adsorption leads to an increase of +22 mV of the ζ-potential (from +28 to +50 mV) and is accompanied by an increase of +10 nm of the mean diameter. After addition of the second polymer layer (PEO-PGA), Dh increased to 46 nm (95% of the particls are in the range of 25100 nm). The final nanoparticles coated with PEI-(PEO-PGA) then display a slightly negative ζ-potential of -3 mV. This can be explained by adsorption of the anionic PEO-PGA to the cationic PEI on the surface, entailing almost complete charge compensation with only a slight overcharging, a phenomenon which is known from layer-by-layer coating of nanoparticles.15 The final particles are no longer stabilized by charges but are stabilized sterically by PEO chains which are covalently bound to the PGA, acting like “glue”. A crucial point in our studies was whether the steric stabilization is strong enough to prevent precipitation of the particles in solutions of high salt concentration such as physiological sodium chloride solution (0.15 M). This was tested, and indeed we found that Dh did not change significantly in 0.15 M sodium chloride solution (cf. Figure 2), but rather a slight reduction in Dh of ca. 3 nm toward smaller (14) Schneider, M.; Brinkmann, M.; Mohwald, H. Adsorption of polyethylenimine on graphite: An atomic force microscopy study. Macromolecules 2003, 36 (25), 9510-9518. (15) Shi, X. Y.; Shen, M. W.; Mohwald, H. Polyelectrolyte multilayer nanoreactors toward the synthesis of diverse nanostructured materials. Prog. Polym. Sci. 2004, 29 (10), 987-1019.
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Figure 3. Hydrodynamic diameters (maximum of the volume distributions) as a function of storage time at 25 °C for bare magnetite (squares), PEO-PGA coated maghemite in water (triangles), and PEO-PGA coated maghemite in 0.15 M sodium chloride solution (circles). The straight lines are guides to the eye.
Figure 4. Mo¨ssbauer spectra taken at low temperature (78 K, left) and room temperature (293 K, right). The symbols are measured data; the lines are calculated curves. The low-temperature spectrum is fitted with a single sextet. The room-temperature curve is fitted with two doublets (curves 1 and 2) and a sextet (curve 3) resulting in the sum spectrum (curve 4).
diameters was observed. The latter is assumed to originate from a slightly more compact conformation of the polymeric chains in the presence of the salt. Regarding the ζ-potential under physiological conditions, it is well-known that a meaningful measurement of the ζ-potential is not possible because of the high shielding of surface charges due to the ions. The polydispersity indexes obtained by DLS (corresponding to the particle size distributions) have low values of 0.14 (parent), 0.22 (PEI coated), 0.16 (PEI plus PEO-PGA coated), and 0.15 (in salt solution), suggesting that the narrowness of the particle size distribution is negligibly changed by the different steps of preparation (cf. also the inset of Figure 2 for comparison). This is a clear indication for a stepwise two-layer adsorption of the polymers to individual particles. An alternative explanation that polymer chains act as bridges between different particles followed by particle aggregation is very unlikely. Much larger polydispersity indexes and also larger particle sizes would be expected in such a scenario. For comparison, we measured a larger polydispersity index of about 0.25 and Dh ) 37 nm for Resovist, a carboxydextran stabilized SPIO, which is commercially available and used as a contrast agent in magnetic resonance tomography.3 Stability of the nanoparticles during long-term storage is an important factor for potential medical applications. It should be stressed that especially in solution many nanoparticle systems, stabilized with polyelectrolytes and typically prepared by the layer-by-layer technique, are stable without salt but aggregate fast when salt is added. The structure and the properties of the polyelectrolyte multilayers strongly depend on the salt concentration.9,10,16 This is a consequence of the reduction in electrical stabilization by the low molecular weight ions. Only a few examples are known where long-term stability is reported at salt concentrations as high as in solutions with a physiological sodium chloride level. Therefore, to evaluate the colloidal stability as a function of time, we stored the nanoparticles in 0.15 M sodium chloride solution at 25 °C and measured the hydrodynamic diameters after different storage times. A comparison of the time dependency of the Dh of the parent particles in water, the coated particles in water, and the coated particles in salt solution, respectively, is shown in Figure 3. Obviously, no change of the particle sizes is detectable during the 30 days of storage time evaluated here. In addition, the particles were stored for a further 6 months in 0.15 M sodium chloride solution. Again, we found
Table 1. d Spacing Values Calculated from XRD Patterns (d ) 2π/qmax) of Maghemite and Polymer Stabilized Maghemite Nanoparticles in Figure 5a
(16) Kovacevic, D.; van der Burgh, S.; de Keizer, A.; Stuart, M. A. C. Kinetics of formation and dissolution of weak polyelectrolyte multilayers: Role of salt and free polyions. Langmuir 2002, 18 (14), 5607-5612.
hkl
d spacing from JCPD data [nm]
calcd d spacing [nm]
111 220 311 400 422 511 440
0.4852 0.2967 0.2532 0.2099 0.1714 0.1615 0.1484
0.4835 0.2959 0.2524 0.2089 0.1744 0.1610 0.1475
a Standard atomic spacing for maghemite and respective hkl indices are from JCPDS (Card No. 19-0629).
that the particle sizes do not change within this time interval. This proves that our two-step layer-by-layer procedure results in nanoparticles that are permanently stable in physiological sodium chloride. 3.2. Magnetic Phase Assignment. Identification of the phase structure of the produced iron oxide nanoparticles was performed by X-ray diffraction (XRD). Here, it is important to note that a discrimination between hematite (R-Fe2O3) on one hand and magnetite (Fe3O4) and maghemite (γ-Fe2O3) on the other hand is straightforward. However, maghemite and magnetite cannot easily be distinguished because they have a very similar inverse spinel structure. Maghemite can be seen as an iron-deficient form of magnetite, with the structural formula Fe21.333+02.67O322where 02.67 means 2.67 vacancies in octahedral spinel sites.17 Therefore, the reflex positions and intensities are also similar. In the case of nanoparticles it is even impossible to distinguish between the two phases because of the strong peak broadening that results from the small crystallite sizes. Therefore, we used Mo¨ssbauer spectroscopy, which is very sensitive to the oxidation state of iron, to determine the iron oxide phase. Spectra for the PEI-(PEO-PGA) coated nanoparticles, measured at low temperature (78 K) and at room temperature (293 K), are shown in Figure 4. It can be seen that the spectra contain the typical Mo¨ssbauer doublets and sextets, which were characterized according to Prene et al.18 The low-temperature spectrum exhibits a magnetically split pattern, which was fitted with a single sextet. The fit results in an isomer shift of 0.44 mm s-1, a quadrupole splitting of 0.009 mm-1, and a hyperfine field of 50 T. In this (17) Ferguson, G. A.; Hass, M. Magnetic Structure and Vacancy Distribution in Gamma-Fe2O3 by Neutron Diffraction. Phys. ReV. 1958, 112 (4), 1130-1131. (18) Prene, P.; Tronc, E.; Jolivet, J. P.; Dormann, J. L. In Mo¨ssbauer spectra of gamma-Fe2O3; ICAME-95 Conference Proceedings, 1996; pp 485-488.
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Table 2. Mo1 ssbauer Parameters of Coated Nanoparticles at Low Temperature and Room Temperaturea Mo¨ssbauer hyperfine parameters
a
low temperature (78 K) -1
isomer shift
0.44 mm s
quadrupole splitting
0.009 mm s-1
hyperfine field amount of Fe(III)
50 T 100% magnetically ordered
room temperature (293 K) 0.38 mm s-1 (doublet, curve 1) 0.35 mm s-1 (doublet, curve 2) 0.33 mm s-1 (sextet, curve 3) 0.54 mm s-1 (doublet, curve 1) 0.94 mm s-1 (doublet, curve 2) 0.03 mm s-1 (sextet, curve 3) 44 T (sextet, curve 3) 10% paramagnetic Fe(III) (doublet, curve 1) 7% paramagnetic Fe(III) (doublet, curve 2) 83% magnetically ordered (sextet, curve 3)
The corresponding fitted curves are displayed in Figure 4.
Figure 5. Wide-angle X-ray scattering diagrams of parent maghemite nanoparticles (a), stabilizing block copolymer PEO-PGA (b), and maghemite nanoparticles stabilized with PEO-PGA. The size of the maghemite crystallites is 12.0 ( 0.5 nm in (a) and (c) (before and after stabilization, respectively). Positions of the two indices (511 and 440) used for determination of the size of the crystals are indicated by arrows. The scattering vector is given as 4 π/λ sin(θ).
case, assignment to Fe(III) located to 100% in ordered maghemite phase with ferromagnetic coupling is straightforward.19 At room temperature the Mo¨ssbauer spectrum is very different. Intense doublets are present in addition to a sextet, which are typical for superparamagnetic particles. The room-temperature Mo¨ssbauer spectrum is very similar to that of commercial SPIO particles (e.g., the spectrum of Resovist reported by Lawaczeck et al.)3. Calculating the room-temperature spectrum gives a best fit with the sum of two doublets and a sextet (curves 1, 2, and 3, respectively, in curve 4 in Figure 4; Mo¨ssbauer parameters are listed in Table 2). Again, solely Fe(III) was detected in maghemite, but the small size of the particles results in a loss of the ferrimagnetic coupling when compared to the low-temperature phase and this is accompanied by the appearance of the paramagnetic doublets. The amount of the paramagnetic Fe(III) at room temperature is 17% while 83% are magnetically ordered. The phase analysis was also performed by X-ray powder diffraction (XRD). It can be seen in Figure 5 that the reflex positions are in agreement with the known reflex positions of maghemite (cf. Table 1). No difference of the reflex positions is found for parent and coated particles (curves a and c, respectively, in Figure 5). The d spacing data are listed in Table 1. It can further be seen that for the 511 and 440 reflections of maghemite there is no overlapping of the XRD curves between the reflections of the bare polymer and the polymer coated nanoparticles (curves b and c, respectively, in Figure 5), and therefore these reflections are most suitable to get information on the maghemite core of the particles. For determination of the (19) Shenoy, G. K.; Wagner, F. E. Mo¨ssbauer Isomer Shifts; North-Holland: Amsterdam, 1978.
Figure 6. Raman spectra of parent maghemite nanoparticles (a), PEO-PGA (b), and coated maghemite nanoparticles (c) in the region of 270-1800 cm-1. Arrows in curve a indicate the characteristic bands of maghemite at 350, 500, and 700 cm-1. The band at 1670 cm-1 in curves b and c is the CdO stretching mode of the adsorbed carboxylate functions of the PGA blocks.
crystal size, we performed a Scherrer analysis and a WilliamsonHall analysis. The Scherrer analysis takes into account the reflection broadening resulting from the small crystal sizes, while the Williamson-Hall analysis also considers the reflection broadening induced by strain effects. We found that the crystal sizes are the same 12.0 ( 0.5 nm for the parent maghemite particles, the one-layer-coated nanoparticles, and the two-layercoated nanoparticles. No difference was found after storage of the particles in distilled water and in physiological salt solution at a pH of 7.4. No indications were found that strain effects influence the structures of the crystallites. Maghemite (γ-Fe2O3) in its macroscopic form is only a metastable, low-temperature Fe2O3 modification, and its phase transition to hematite (R-Fe2O3) is reported at temperatures above 300 °C.20 However, in nanocrystalline material it is possible that a metastable form becomes thermodynamically stable.21 A detailed study of the phase transition of nanocrystalline maghemite to hematite indicates that maghemite is more stable for crystal sizes below 16 nm than hematite.22 The sizes of the produced hematite crystals were above 35 nm, and growth of the maghemite crystals was not observed. A further indication for the stabilization of maghemite in nanocrystalline form is the fact that only maghemite is produced with the reverse micelle method when the crystal size is below 30 nm and only hematite when the size is larger.23 (20) Cornell, R. M.; Schwertmann, U. The Iron Oxides; VCH: Weinheim, 1996. (21) Garvie, R. C. Stabilization of the Tetragonal Structure in Zirconia Microcrystals. J. Phys. Chem. 1978, 82, 218-224. (22) Schimanke, G.; Martin, M. In situ XRD study of the phase transition of nanocrystalline maghemite (gamma-Fe2O3) to hematite (alpha-Fe2O3). Solid State Ionics 2000, 136, 1235-1240.
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Figure 7. Magnetic resonance signal intensity of liver parenchyma of rats in T2-weighted sequences. Images were acquired before (A and C), 10 min after intravenous injection of maghemite nanoparticles in a concentration of 0.6 mg of Fe/kg of body weight (B), and 10 min after intravenous injection of Resovist in a concentration of 0.6 mg of Fe/kg of body weight (D).
Even if the final proof about the thermodynamic stability of maghemite in general has not yet been reported, it can be assumed that at least nanocrystalline maghemite is thermally more stable than its coarse-grained counterpart.24 Therefore, our finding that we detected exclusively maghemite in the nanoparticles and not hematite even at long storage time is in agreement with the literature. We performed Raman spectroscopy in backscattering symmetry on the dried nanoparticles to confirm our results from Mo¨ssbauer spectroscopy and XRD. The Raman spectrum of the parent particles displays broad bands around 350, 500, and 700 cm-1 (indicated by arrows, curve a in Figure 6), which are not present in any other spectrum of iron oxide, neither in magnetite nor in hematite.25 Also, the very broad features around 1400 and 1580 cm-1 are characteristic for maghemite.26 This confirms our conclusion that exclusively maghemite is present. The spectrum of the coated particles is more complex (curve c). It can be seen that in addition to the broad bands of maghemite numerous sharp bands are present. By comparing the spectrum with that of PEOPGA (curve b), the bands at 278, 831, 859, 930, 1057, 1123, 1228, 1279, 1394, and 1481 cm-1 can be assigned to crystalline (23) Ziolo, R. F.; Giannelis, E. P.; Weinstein, B. A.; Ohoro, M. P.; Ganguly, B. N.; Mehrotra, V.; Russell, M. W.; Huffman, D. R., Matrix-Mediated Synthesis of Nanocrystalline Gamma-Fe2O3sa New Optically Transparent Magnetic Material. Science 1992, 257 (5067), 219-223. (24) Ye, X. S.; Lin, D. S.; Jiao, Z. K.; Zhang, L. D. The thermal stability of nanocrystalline maghemite Fe2O3. J. Phys. D: Appl. Phys. 1998, 31 (20), 27392744. (25) deFaria, D. L. A.; Silva, S. V.; deOliveira, M. T. Raman microspectroscopy of some iron oxides and oxyhydroxides. J. Raman Spectrosc. 1997, 28 (11), 873-878. (26) Sousa, M. H.; Tourinho, F. A.; Rubim, J. C. Use of Raman microspectroscopy in the characterization of M(II)Fe2O4 (M ) Fe, Zn) electric double layer ferrofluids. J. Raman Spectrosc. 2000, 31 (3), 185-191.
PEO. The band positions are in agreement with the data reported in the literature.27,28 The amide I band (CO stretch vibration) of the glutamic acid units is located at ∼1670 cm-1, which is at the same position and as broad as reported for a PGA homopolymer with charged carboxylate groups.29 Other expected PGA bands are too low in intensity for detection because of the high amount of PEO. It is important is that PEO-PGA has no bands in the region around 700 cm-1, where the most intense band of maghemite is located. In the spectrum of the coated nanoparticles it can be seen that this band of maghemite is split into two bands, located at 665 and 710 cm-1. An appearance of these two bands has been observed also for bare maghemite,26 and we conclude that this is not an effect of the coating. The broader band at 1670 cm-1, as indicated by an arrow in Figure 6c, of the coated particles verifies the presence of the PGA on the particle surface. For clarity, the intensity of PEI on the particles was too low for detection. 3.3. Magnetic Imaging Contrast of the Particles in Vivo. To test their potential for imaging applications, the maghemite nanoparticles were subjected to magnetic resonance imaging (MRI) experiments in living rats. Figure 7 shows spin-echo abdomen images of a living rat acquired before and after intravenous injection of the nanoparticles in physiological salt (27) Kozielski, M.; Muhle, M.; Blaszczak, Z.; Szybowicz, M. Raman and Rayleigh scattering study of crystalline polyoxyethyleneglycols. Cryst. Res. Technol. 2005, 40 (4-5), 466-470. (28) Begum, R.; Matsuura, H. Conformational properties of short poly(oxyethylene) chains in water studied by IR spectroscopy. J. Chem. Soc., Faraday Trans. 1997, 93 (21), 3839-3848. (29) Mikhonin, A. V.; Myshakina, N. S.; Bykov, S. V.; Asher, S. A. UV resonance Raman determination of polyproline II, extended 2.5(1)-helix, and beta-sheet psi angle energy landscape in poly-L-lysine and poly-L-glutamic acid. J. Am. Chem. Soc. 2005, 127 (21), 7712-7720.
Maghemite Nanoparticles Coated by PEI and PEO-PGA
solution. The injected dose was 0.6 mg of Fe/kg of body weight. This amount is similar to the amount recommended for commercial contrast agents such as Resovist. We found strong reduction of the magnetic resonance signal intensity of the liver parenchyma in the T2-weighted sequences 10 min after intravenous application. The rat recovered from anesthesia spontaneously after the experiment and lived normally, indicating that the current sample was not toxic. This suggests further that the nanoparticles prepared by the current organic solvent free approach possess potential as new MRI contrast agents. It has to be mentioned that PEI is known to be toxic in its noncomplexed state because of its high cationic charge. However, in contrast to this, PEI is extensively discussed in the development of efficient nonviral gene carriers, where it has the function to encapsulate foreign genetic material by complexation.30 The rats (n ) 5) are still alive. No difference was found between the treated rats and the control group, but a further study (rats and cell cultures) with a detailed investigation on the toxicity of the particles when different amounts of PEI are used is underway. (30) El-Aneed, A. An overview of current delivery systems in cancer gene therapy. J. Controlled Release 2004, 94 (1), 1-14.
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4. Conclusions In summary, superparamagnetic maghemite nanoparticles were prepared via a two-step layer-by-layer technique using poly(ethylene imine) as a first layer and poly(ethylene oxide)-blockpoly(glutamic acid) as a second layer. The resultant nanoparticles have a slightly negative ζ-potential and display excellent stability in physiological salt solution. The combination of Mo¨ssbauer spectroscopy and X-ray diffraction reveals that the size of the maghemite domains is 12 nm and that only maghemite is present. Our preliminary MRI experimental results show that the particles cause a strong MRI contrast and indicate that they possess biocompatibility. Acknowledgment. The authors thank M. Menzel for help with the Mo¨ssbauer spectroscopy, W. Kraus and B. Peplinski for XRD, K.-W. Brzezinka for Raman spectroscopy, H. Bruhn for MRI, and K. Rurack for correcting the manuscript. Financial support was from the EU Project Nano-Carrier (No. 2000/2006 2u¨/1) and the “Kompetenznetzwerk Biokompatible Oberfla¨chen Berlin”. LA052990D
