Magn Reson Mater Phy (2009) 22:77–87 DOI 10.1007/s10334-008-0147-x
RESEARCH ARTICLE
Synthesis and characterization of polyethylenimine-based iron oxide composites as novel contrast agents for MRI A. Masotti · A. Pitta · G. Ortaggi · M. Corti · C. Innocenti · A. Lascialfari · M. Marinone · P. Marzola · A. Daducci · A. Sbarbati · E. Micotti · F. Orsini · G. Poletti · C. Sangregorio
Received: 26 May 2008 / Revised: 4 September 2008 / Accepted: 22 September 2008 / Published online: 15 October 2008 © ESMRMB 2008
Abstract Object Use of polyethylenimines (PEIs) of different molecular weight and selected carboxylated-PEI derivatives (PEI-COOH) in the synthesis and stabilization of iron oxide nanoparticles, to obtain possible multifunctional contrast agents. Materials and methods Oxidation of Fe(II) at slightly elevated pH and temperature resulted in the formation of highly soluble and stable nanocomposites of iron oxides and polymer. Composites were characterized and studied by atomic force microscopy (AFM), transmission electron microscopy (TEM), X-ray diffractometry, AC and DC magnetometry, NMR relaxometry and magnetic resonance imaging (MRI). Results From AFM the dimensions of the aggregates were found to be in the ∼150–250 nm size region; the mean diameter of the magnetic core of the compounds named PEI-
25, PEI-500 and PEI-COOH60 resulted d ∼ 20 ± 5 nm for PEI-25, d ∼9.5 ± 1.0 nm for PEI-500 and d ∼6.8 ± 1.0 nm for PEI-COOH60. In PEI-COOH60 TEM and X-ray diffractometry revealed small assemblies of mineral magnetic cores with clear indications that the main constituents are maghemite and/or magnetite as confirmed by AC and DC SQUID magnetometry. For PEI-COOH60, the study of NMR-dispersion profiles revealed r1 and r2 relaxivities comparable to superparamagnetic iron-oxide commercial compounds in the whole investigated frequency range 7 ≤ ν ≤ 212 MHz. Conclusion PEI-25 was studied as possible MRI contrast agent (CA) to map the cerebral blood volume (CBV) and cerebral blood flow (CBF) in an animal model obtaining promising results. The reported compounds may be further functionalized to afford novel multifunctional systems for biomedical applications.
A. Masotti (B) · A. Pitta · G. Ortaggi Dipartimento di Chimica, SAPIENZA Università di Roma, P.le A.Moro 5, 00185 Rome, Italy e-mail:
[email protected]
Keywords Polyethylenimine · Iron oxide · Nanoparticles · Composites · Contrast agents · MRI
M. Corti · A. Lascialfari · E. Micotti Dipartimento di Fisica “A.Volta”, Università di Pavia, and CNR-INFM, Via Bassi 6, 27100 Pavia, Italy A. Lascialfari · M. Marinone · F. Orsini · G. Poletti Istituto di Fisiologia Generale e Chimica Biologica “G. Esposito”, Università di Milano, Via Trentacoste 2, 20134 Milan, Italy A. Lascialfari · M. Marinone S3-CNR-INFM, 41100 Modena, Italy P. Marzola · A. Daducci · A. Sbarbati Dipartimento di Scienze Morfologico-biomediche, Università di Verona, 37100 Verona, Italy C. Innocenti · C. Sangregorio Dipartimento di Chimica, Università degli Studi di Firenze, Via della Lastruccia 3, 50019 Sesto Fiorentino, Italy
Introduction In the last two decades magnetic resonance imaging (MRI) technique was developed with success for the diagnosis of different diseases, mainly because it is a non-invasive technique [1–3]. Images of materials and individuals are generated after measuring the nuclear (often the 1 H nucleus) spin density, the spin–spin (T2 ) and the spin–lattice (T1 ) nuclear relaxation times, that have different weights in different tissues. An increase in image contrast is achieved by the use of contrast agents (CAs) [3]. Usually the CAs reduce the relaxation times, T1 and T2 , of the tissues they reach and their efficacy is evaluated by measuring the longitudinal (r1 ) and transverse (r2 ) relaxivities that represent the increase of
123
78
the solvent (water or blood) relaxations in presence of 1 mM of magnetic center. Contrast agents can be distinguished in positive, or T1 -relaxing, and negative, or T2 -relaxing. Positive contrast agents locally enhance the signal (brilliant spots); negative agents partially or totally reduce the signal (black spots). To obtain a faster relaxation rate, the CA must be rich of uncoupled electrons, as happens, e.g., in paramagnets. The same results can be obtained by other kinds of CA, based on a superparamagnetic (SP) core, generally used as negative CA. Widely used commercial superparamagnetic CAs are Endorem (Feridex in USA) and Sinerem (Combidex in USA), that have a ferrite core (5–6 nm diameter) coated with a dextran shield, resulting in final diameters ∼150 and ∼15 nm, respectively. Particularly, researches on poly-amidoamine (PAMAM) dendrimers have suggested that these molecules can act effectively as organic matrices for the synthesis of inorganic nanoparticles [4–14]. Similar approaches using functionalized organic arrays have been previously used to form nanoparticles by encapsulation within protein cages [15–17] polymer matrices [18,19] and surfactant vesicles [20–22]. Recent works proposed different kind of coating and magnetic cores [22,23,32,33] to control and optimize the efficiency of MRI CA. On the other hand, polyethylenimine (PEI) and carboxylated derivatives can be used to synthesize inorganic nanoparticles as well [23]; in fact they present a structure with an architecture very similar to PAMAM dendrimers (roughly spherical in shape). It should be also noticed that they present a polyamine or a carboxylated surface on the outer spherical organic assembly and may be, in principle, used to form “magnetopolymers” in a way analogous to that reported for magnetodendrimers [24]. Finally, PEI and PEI’s derivatives may be easily functionalized and are also cheaper than PAMAM. We recently reported the synthesis of a novel PEIbased vector functionalized with a near-infrared (NIR) dye for DNA delivery. Using this system DNA delivery in vivo was monitored with optical imaging devices [25]. Starting from this results, we are proceeding to the synthesis of a multifunctional system (superparamagnetic and fluorescent) for delivery of DNA and/or other biologically relevant molecules by simply combining the two synthetic procedures. The aim of the present work was to synthesize and characterize novel magnetic nanoparticles based on PEI and its PEI-COOH derivatives possibly useful as multifunctional contrast agents for MRI, and to investigate the efficacy of these functionalized architectures in controlling the synthesis of coated iron oxide minerals under mild biomimetic conditions as outlined in Fig. 1. The most important issue resides in the possibility to obtain multifunctional systems by an easy modification of the polymer surface. We have shown also that PEI and PEI-COOH polymers contribute to control the formation of well-defined iron oxide nanoparticles. In addition, the polymer slightly limits the interparticle
123
Magn Reson Mater Phy (2009) 22:77–87
Fig. 1 Schematic representation of the stabilization of maghemite/magnetite nanoparticles by means of PEI (or PEI-COOH)
aggregation, most likely due to a surface passivation phenomenon, resulting in a highly soluble composite material. Oxidative hydrolysis at the polymer-solution interface, under controlled and mild synthetic conditions, leads to the formation of composite assemblies of polymer and inorganic mineral (Fig. 1). These materials maintain their solubility over a wide range of conditions for extended periods of time. The magnetic properties of the new compounds were studied by means of AC susceptibility and DC magnetization measurements, while their relaxometric properties were investigated via the usual NMR-dispersion profile at room and physiological temperature. The magnetic behavior of these new materials whose magnetic core is mainly constituted by maghemite/magnetite, is consistent with the existence of single domain superparamagnetic particles whose moments freeze at the so-called blocking temperature Tb . With carboxylated (60% of COOH) PEI of molecular weight 25 kDa, the magneto-polymers were found to have high r1 and r2 NMR relaxivities and are currently being explored as the basis for a new generation of contrast agents for magnetic resonance imaging applications. As applicative example, we showed that the compound named PEI-25 (PEI with a molecular weight of 25 kDa) can be used in MRI bolus tracking experiments.
Magn Reson Mater Phy (2009) 22:77–87
79
Branched polyethylenimines of molecular weight 25 kDa (PEI-25) and 500 kDa (PEI-500), Fe(II) and Fe(III) salts were purchased from Sigma–Aldrich and used as received. Ultrafiltration was performed using an Amicon cell (Mod.8200) with regenerated cellulose membrane (Millipore, YM-10). 1 H and 13 C NMR spectra were recorded on a 300-MHz Varian instrument. PEI and PEI’s derivatives were analyzed by diluting a 45-mM stock solution (monomer aqueous solution) neutralized with HCl and filtered through a Millipore 0.2 mm membrane.
30% NH4 OH (15 ml) and maintained at 80◦ C for 1 h. Finally, the magnetopolymers were dialyzed exhaustively against double-distilled H2 O and concentrated by ultrafiltration (Amicon) using a 10 kDa Mw cut-off membrane. Polymer iron loading were calculated hydrolizing 100 ml of magnetopolymer solution with a mixture of concentrated HNO3 :HCl (1:3) (100◦ C, 1 h) and reducing all Fe(III) to Fe(II) by addition of an excess of solid sodium metabisulfite. The mixture was transferred in 25-ml volumetric flasks and made up to the mark with deionized water. An aliquot (100 ml) was mixed with 1,10-phenanthroline and the iron content was determined spectrophotometrically (reading the absorbance at 510 nm) interpolating the value from the standard curve.
Synthesis of carboxylated polyethylenimines
Transmission electron microscopy
PEI-COOH60. A solution of bromoacetic acid (9.90 g, 71.4 mmol) in water (400 cm3 ), was added over a period of 3 h to a solution of PEI-25(5 g, 0.2 mmol) in water (600 cm3 ). The resulting mixture was stirred at RT for 1 day, then ultrafiltered through a 10 kDa filter to remove the unreacted acid. The mixture was lyophilized affording PEI-COOH60 as a white solid. Yield 8.5 g (92%). Integration of the proton magnetic resonance (1 H NMR) spectrum of the product in D2 O indicated 60 mol% of bromoacetic acid groups (CH2 ; 3.5–3.7 ppm) per residue mol of ethylenimine unit (C2 H4 N; 2.2–3.2 ppm) in the polymer. The substituted polymer may be represented by the stoichiometric formula (C2 H4 N)m (C2 H3 O2 )0.60m , m = 595. Anal. Calcd: C, 49.60; H, 7.54; N, 18.08. Found: C, 49.53; H, 7.84; N, 17.97. 1 H NMR (D2 O): δ (ppm) 2.90 (b, −CH2 CH2 N–), 3.54 (b, –CH2 –COOH); 13 C NMR (D2 O): δ (ppm) 38.14, 45.18, 46.39, 47.88, 49.75, 49.99, 50.97, 51.69, 52.90, 54.19, 56.90, 58.78, 59.79, 171.60, 172.40. PEI-COOH75. Bromoacetic acid (12.5 g, 90 mmol) was added to PEI-25 (5 g, 0.2 mmol) following the previously reported procedure. The polymer can be represented by the stoichiometric formula: (C2 H4 N)m (C2 H3 O2 )0.75m , m = 595. Yield 9.15 g (89%). Anal. Calcd: C, 48.69; H, 7.30; N, 16.23. Found: C, 48.83; H, 7.54; N, 16.34. 1 H NMR (D2 O): δ (ppm) 2.90 (b,–CH2 CH2 N–), 3.54 (b, –CH2 –COOH); 13 C NMR (D2 O): δ (ppm) 38.23, 45.31, 46.24, 47.76, 49.57, 49.87, 51.13, 51.78, 52.76, 54.39, 56.98, 58.76, 59.55, 172.45, 172.87.
The mineral particles isolated from these reactions were imaged by TEM using an EM10 electron microscope (Zeiss, Oberkocken, Germany). TEM images were analyzed using ImageJ 1.39 u (NIH, USA) and specifically the “Analyze Particles” Tool. Only particles having an area in the 25–2,000 nm2 range and a circularity factor of 0.8 were included in the quantitative analysis. At least 300 particles were measured in order to have a reliable estimate of the surface average.
Materials and methods Synthesis
Magnetopolymer preparation PEI-25, PEI-500 and carboxylated polymers (1 g each) were separately dissolved in deaerated water (600 cm3 ) and added to deaerated mixtures of FeCl3 · 6H2 O (9.20 g, 0.034 mmol) and FeSO4 · 7H2 O (4.73 g, 0.017 mmol) in water (400 cm3 ) under vigorous stirring. The mixtures were alkalinized with
X-ray diffraction X-ray diffraction patterns were collected on a Bruker D8 Advance diffractometer equipped with Cu Kα radiation and operating in θ –2θ Bragg Brentano geometry at 40 kV and 40 mA. Atomic force microscopy Atomic force microscopy imaging was performed by means of an AutoProbe CP Research atomic force microscope (ThermoMicroscopes, Sumyvale, CA, USA). The Glass coverslips supports were attached to stainless steel punches with double-sided adhesive tape (Ted Pella Inc., Redding, CA, USA) and magnetically fixed to the AFM sample holder. Standard rectangular silicon cantilevers (CSG01, NTMDT, Zelenograd, Moscow, Russia) with a 0.01 N/m spring constant and a conical silicon tip with a 10-nm curvature radius and a tip cone angle of 22◦ were used to scan the sample in air. The tip radius was previously evaluated by means of electron microscopy. Images of 512 × 512 pixels2 were collected at a scan rate of 1 Hz operating in contact mode (constant force conditions). The set point was manually adjusted and kept below as possible to obtain the best resolution. Topography and error signals were collected simultaneously. The use of the contact scanning mode was chosen in order
123
80
to obtain both higher resolution and error signal, which is useful for improved visualization of small features on the sample surface [26]. For the image processing, the Image Processing and Data Analysis 2.0 software (ThermoMicroscopes, Sunnyvale, CA, USA) was used. On collected images, a first order flattening has been performed, as usual and, where needed, a shading to sharpen boundaries. Different scan area have been used, 1.5×1.5 mm2 to evidence the shape and 4×4 mm2 to obtain a large number of nanoparticles and calculate size distribution. Samples have been diluted in distilled water and dried in air.
Magn Reson Mater Phy (2009) 22:77–87
The relaxivities r1 and r2 were calculated by the usual formula: (1/Ti )meas = (1/Ti )dia + ri · c i = 1, 2 where (1/T i )meas is the nuclear spin–lattice relaxation rate (NSLRR) measured on the samples in solution with chosen concentration c and (1/T i )dia is the NSLRR of the diamagnetic host (in this case mannitol; in most cases and physiological case is water). The (1/T i )dia values resulted for our samples much smaller than (1/T i )meas at any frequency and can be disregarded. The relaxivities at room and physiological temperatures resulted to be the same within 10%.
AC and DC magnetometry Magnetic resonance imaging Magnetization measurements were performed as a function of temperature and of the magnetic field by a Cryogenic Ltd SQUID commercial magnetometer both on the powder samples and on the solutions. All the data were corrected for the magnetism of the sample holder which was separately measured. The temperature dependence of the static susceptibility, M/H , was measured after zero field cooling (ZFC) and field cooling (FC) procedures with an applied magnetic field H = 5 mT. AC susceptibility measurements were performed with a home-made probe inserted in a Oxford cryostat. Data were collected at 10 frequencies log-spaced in the range 70–21,000 Hz, in the temperature range 5–250 K. NMR relaxometry The 1 H NMR measurements of nuclear transverse (T2 ) and longitudinal (T1 ) relaxation times were performed at room temperature in the frequency range 7 ≤ ν ≤ 212 MHz (i.e., magnetic fields 0.164 ≤ ν ≤ 5 T, utilizing a Bruker electromagnet and an Oxford superconducting magnet), by means of different pulse-FT spectrometers (Bruker-MSL200 and Apollo-Tecmag) on samples dissolved in isotonic d-mannitol solution with different concentrations c (mg/ml) of the magnetic center (Fe), contained in a quartz vial. The 1 H spectra were obtained by the Fourier transform of the second half of the echo signal, obtained from a Hahn-echo sequence π/2 − π . The full width at half maximum (FWHM) of spectra collected at different frequencies has values in the range 0.5 ≤ FWHM ≤ 22 kHz. Thus, choosing a π/2 pulse length between 1.5 and 18 ms, depending on the operating frequency of the spectrometer, the intensity of the radiofrequency field H1 was sufficiently strong to irradiate the whole NMR line. The spin–spin relaxation rates 1/T 2 were obtained by the CPMG or Hahn-echo sequences with varying delay times. The spin–lattice relaxation rates 1/T 1 were obtained from the recovery of the longitudinal component of the nuclear magnetization following a short sequence of saturating radio frequency pulses, preceding the echo π/2 − π sequence.
123
Longitudinal (r1 ) and transversal (r2 ) relaxivities were measured using imaging sequences in order to compare data obtained from images with those obtained by spectroscopic sequences. All MRI experiments were carried out using a Biospec Tomograph System (Bruker, Karlsruhe, Germany) equipped with a 4.7 T, 33 cm bore horizontal magnet (Oxford Ltd, UK) and with a gradient insert of 20 G/cm strength. A 72 mm i.d. birdcage coil was used. Samples containing different concentrations (from 0.07 to 0.75 mM) of contrast agents (either PEI-25 or Endorem ) were prepared in physiological saline. The transversal relaxation times were measured using a standard Spin-Echo Multi-Echo sequence with the following parameters: TR/TE = 1,000/20 ms, FOV = 70 × 70 mm2 , matrix size = 256 × 192, slice thickness = 5 mm, number of echoes = 30. For the measurement of the longitudinal relaxation time, a fast T1 mapping technique based on a IR-SNAPSHOT sequence was used [27]. The acquisition parameters were: FOV = 35 × 35 mm2 , matrix size = 128 × 128, slice thickness = 1 mm, TR/TE = 10/3.6 ms, excitation pulse angle = 5◦ , 5 ms Sech inversion pulse. The acquisition was segmented in 8 steps in order to obtain enough time resolution to monitor the recovery of the longitudinal magnetization. The acquisition time for T1 maps was about 2 min. Longitudinal and transversal relaxation rates (1/T 1 and 1/T 2 ) were plotted as a function of iron concentration and r1 and r2 relaxivities obtained by the slope of the fitting straight line. Longitudinal and transversal relaxivities determined as above described amounted to: r1 = 0.17 mM−1 s−1 , r2 = 53.07 mM−1 s−1 and r1 = 2.90 mM−1 s−1 , r2 = 94.83 mM−1 s−1 for PEI-25 and Endorem respectively, in good agreement with the values obtained from spectroscopic sequences (see also Fig. 8). In vivo experiments were performed in order to characterize the performances of PEI-25 as a contrast agent for mapping of cerebral blood volume (CBV) and cerebral blood flow (CBF). CBF and CBV measurements are considered robust methods for the characterization of the perfusion reduction in cerebral ischemia both in humans [28] and in animal models
Magn Reson Mater Phy (2009) 22:77–87
First-passage images were acquired using an Echo Planar Imaging (EPI) sequence with the following parameters: matrix size 64×64, FOV 38×38 mm2 , slice thickness 2 mm, 4 EPI shots × 50 ms (time resolution of 5 frames/s). Images were continuously acquired for 30 s. Steady-state images were acquired using a Gradient Echo sequence with the following parameters: TR/TE = 350/15 ms, flip angle = 30◦ , matrix size = 256×192, FOV = 35×35 mm2 (corresponding to 0.137×0.182 mm2 in-plane resolution), slice thickness = 1 mm, number of excitation = 2. The acquisition time for a single image was 134 s; images were acquired before and 268 s after the injection of contrast agents. A phantom containing 1 mM Gd-DTPA in saline was inserted in the field of view and used as external reference standard, in order to normalize possible spectrometer drifts during the acquisition.
Results The mineral particles isolated from the reported reactions revealed fairly homogeneous cluster sizes. As an example, we obtained an average area of 69 ± 45 nm2 for PEI-500 (TEM), corresponding to a diameter of approximately 9.4 ± 3.8 nm (Fig. 2). The XRD pattern of PEI-COOH60 is shown in Fig. 3. The low-theta part of the spectrum is dominated by a very broad peak (not shown) attributable to the amorphous organic matrix embedding the iron oxide nanoparticles. The pattern observed above 2θ = 20◦ can be instead unambiguously attributed to a cubic spinel structure corresponding to both maghemite (γ -Fe2 O3 ) or magnetite (Fe3 O4 ), which could
Fig. 2 TEM image of PEI-500. The average area is 69 ± 45 nm2 that corresponds to particles’ diameter of 9.4 ± 3.8 nm. Scale bar represents 200 nm
Intensity
[29]. All experiments were performed in comparison with Endorem . In in vivo experiments the signal was received through a rat helmet-shaped surface coil actively decoupled from a 72 mm i.d. birdcage coil that was used for transmission. Experiments were performed using a total of six Sprague Dawley rats weighing 250–300 g. Three animals were used for first passage experiments and three animals were used for steady state experiments. Each animal received PEI-25 and Endorem (24 h later). PEI-25 was injected at 6 and 12 mg Fe/kg that corresponds to about 5 and 10 mg/kg of polymer. Such dosages are respectively 400 and 200 times lower than the reported LD50 in rats [30]. Quantitative evaluations of CBF and CBV can be performed by bolus tracking method: images are acquired with high time resolution during the first passage of the contrast agent in the brain [28,29]. Regional CBV can be also measured using blood pool contrast agents, at the steady state concentration of contrast agent in blood, by acquiring T2∗ images before and after contrast agent injection using the following relationship [31,32]: S Ipre rCBV = k · ln S Ipost
81
(220)
(311) (400) (511) (440)
20
30
40
50
60
70
2θ (°)
Fig. 3 XRD pattern of PEI-COOH-60. The reference pattern of magnetite is shown for comparison
not be discriminated owing to the significant line broadening. The average crystallite size of the magnetic core was estimated from the line broadening of (311) and (400) peaks using the Scherrer formula, and was found 6.8 ± 1.0 nm. A similar pattern but with sharper peaks was observed for PEI-500. In this case the analysis of the line broadening provided an average crystallite size of 9.5 ± 1.0 nm while for PEI-25 the size was 20 ± 5 nm. A more complex pattern was instead observed for PEI-25, owing to the presence of some inorganic salts. The analysis of PEI-25 spectrum suggests the presence of different iron oxide phases as beside broad peaks consistent with magnetite/maghemite (2θ = 30.01, 35.31, 56.91, 62.50) also sharper peaks attributable to goethite, FeOOH (2θ = 21.26, 33.2, 36.71) and hematite, α-Fe2 O3 (2θ = 33.2, 35.69, 49.4) were observed. In the pattern of PEI-500 the main iron oxide crystalline phases found is goethite with a small amount of a spinel phase, while no traces of hematite were observed.
123
82
Fig. 4 AFM images: a PEI-COOH60—topography on 1.5 × 1.5 mm2 ; b PEI-COOH60—error signal on 4 × 4 mm2
Results obtained with AFM give an average diameter of 242 ± 60 nm for PEI-25, 228 ± 44 nm for PEI-500 and 146 ± 40 nm for PEI-COOH60 (see Fig. 4 for PEI-COOH60). Magnetization measurements for PEI-25, PEI-500 and PEI-COOH60 solutions are shown in Fig. 5. PEI-COOH60 displays the typical thermal irreversibility of a set of single domain nanoparticles. The average blocking temperature, evaluated as the temperature corresponding to the maximum of the ZFC curve is 45 K, while the splitting temperature between the ZFC and FC curves is well above, at ca. 130 K, indicating that the particle size is not perfectly monodisperse, as indeed observed by TEM. A similar behaviour can be attributed to the other two samples, by taking into account a much broader size distribution; the blocking temperature was estimated to be Tb = 105 ± 10 K for PEI-500 and Tb = 190 ± 10 K for PEI-25. No significant differ-
123
Magn Reson Mater Phy (2009) 22:77–87
ences were observed on the corresponding powder samples apart from a broadening of the ZFC curves whose maxima slightly move to higher temperatures. This result suggests that interparticle interactions do not play a determinant role in the reorientation process of the magnetization. A sample of Endorem was also measured in the same conditions and compared to our samples. The shape of ZFC/FC curves of Endorem and PEI-COOH60 present very similar features, as shown in Fig. 5. Hysteresis curves M(H) (Fig. 6) were collected at T = 2.5 K in the range −6.5 < H < 6.5 T. As the metal oxide concentration of both powder and solutions was unknown the data were normalized to the saturation value obtained by extrapolating the magnetization data to 1/H → 0. The coercive field HC is found to be similar for all the samples (HC = 47 mT, for PEI-25, HC = 40 mT for PEI-500, HC = 48 mT for PEI-COOH60) and larger than that directly measured for Endorem (HC = 32 mT). The HC values are of the order of bulk value but larger than those reported in the literature for magnetite nanoparticles of size in the 6–150 nm range. The remnant magnetization is 0.31 ± 0.03 for all the samples. This value is slightly lower than the typical one, 0.5, expected for a set of isolated uniaxial nanoparticles whose easy axis are isotropically orientated. In Fig. 7 the in phase component, χ , of a PEI-COOH60 solution (Fig. 7b) is compared to that of Endorem (Fig. 7a). In both samples the positions of the maxima, Tmax , which are related to the blocking temperature observed at the characteristic time τ = (2π υ)−1 , are frequency dependent, moving to higher temperatures with increasing frequency. The variation of Tmax occurs in the same range of temperature for the two samples, despite Endorem displays a larger variation with frequency (Tmax increases from 61 to 79 K for Endorem while for PEI-COOH60 it varies from 61 to 71 K in the same frequency range). A tentative fit to the Arrhenius law τ = τ0 exp( E/kB T ) where E is the energy barrier for the reorientation of the magnetization which is equal to the product of the magnetic anisotropy, K, and the particle volume, and τ0 is the attempt time, requires the analysis of the outof-phase component of the AC susceptibility. Unfortunately for PEI-COOH60 these curves were too noisy hampering a reasonable estimation of the temperature of the maxima. On the contrary the data measured for Endorem could be nicely fit to this law, with best fit parameters E = 915 K and τ0 = 10−13 s. In Fig. 8 we report the relaxivities of PEI-based compounds at the same concentration of Fe (11.2 mg/ml corresponding to the one used in the commercial compound Endorem ), but with a variable molecular weight (25 and 500 kDa) and/or a different carboxylation degree. For comparison we report the r1 and r2 curve of Endorem . As can be noted, ri of PEI-COOH60 is comparable to the values of Endorem .
83
M (a.u.)
M(a.u.)
Magn Reson Mater Phy (2009) 22:77–87
0
100
200
300
0
100
Temperature (K) Fig. 5 Left side Magnetization data as a function of temperature collected in an applied DC field H = 50 Oe on solution of PEICOOH-60 (empty circles), PEI-500 (full triangles) and PEI-25 (empty
(a)
1.0
300
diamonds). Right side comparison between PEI-COOH-60 (empty circles) and Endorem (full circles). Data have been rescaled for clarity
(a)
Endorem
χ' (a.u.)
0.5
M/MS
200
Temperature (K)
ν
0.0
T =2.5 K
-0.5
0
50
100
150
250
(b)
-1.0 -8
-6
-4
-2
0
2
4
6
8
Magnetic Field (T)
Pei -COOH 60
χ ' (a.u.)
(b)
200
0.50
M/MS
0.25
0 0.00
100
150
200
250
T (K) Fig. 7 AC susceptibility data at different frequencies as a function of temperature. The data were collected at 10 log-spaced frequencies from 70 to 21,000 kHz (the arrow indicates increasing frequencies). a Endorem; b PEI-COOH 60
-0.25
-0.50 -0.10
50
-0.05
0.00
0.05
0.10
Magnetic Field (T) Fig. 6 Full hysteresis loop of a PEI-COOH-60 solution at T = 2.5 K; the curve is representative of all the investigated samples. b Enlargement of the 2.5 K hysteresis loops of PEI-COOH-60 (empty circles), PEI-500 (full triangles), PEI-25 (empty diamonds) and Endorem (full circles). All data were normalized to the saturation value
Data on PEI-COOH75 and PEI-25 at different Fe concentration showed a linear behaviour of 1/T1 and 1/T2 as a function of c, thus indirectly proving the homogeneity of our samples (data not shown).
Our previous results [33] examined the cytotoxicity of PEI and other PEI’s derivatives for in vitro DNA delivery experiments. The careful dilution of the polymer and solution buffering prior to its use, is a necessary step for the minimization of toxic effects and the maximization of DNA delivery. Both PEI-COOH75 and PEI-25 iron oxide composites were used at a dosage corresponding to a polymer amount of 5–10 mg/kg, a range well below the reported LD50 in rats [30]. Moreover, iron oxides are generally well tolerated by the animal. Therefore, we may assume that the assayed compounds are relatively low toxic.
123
84
Magn Reson Mater Phy (2009) 22:77–87
(a)
Endorem PEI-25 PEI-COOH60 PEI-COOH75 PEI-500
r1 (mM-1 s -1)
10
1
0.1 0
100
200
ν (MHz)
Figure 10 shows representative pre-contrast and post-contrast images acquired 268 s after injection of PEI-25 and Endorem respectively (dosage 12 mg Fe/kg). We selected PEI-25 for MRI instead of PEI-COOH60 since the amount of the latter compound was too small for these in vivo experiments. In this steady-state conditions Endorem produces signal drop substantially higher than PEI-25 [average (±SD) signal drop amounted to −36 ± 7 and −14 ± 4%, respectively, when measured in cerebral cortex]. Moreover postcontrast images and rCBV maps obtained with Endorem depicted very well the space arrangement of cerebral vessels, that was not clearly detectable in images obtained using PEI-25. Images and maps reported in Fig. 10 clearly indicate that acquiring steady state rCBV maps using PEI-25 would be disadvantageous compared to Endorem .
Endorem
PEI-25
r2 (mM-1 s -1)
(b)
PEI-25 (after one year) PEI-COOH60 PEI-500
150
100
50
0 0
100
200
ν (MHz) Fig. 8 r1 a and r2 b relaxivities (per millimole of Fe) of PEI-based compounds at Fe concentration 11.2 mg/ml, T = 297 K. For comparison, r1 and r2 data of Endorem are reported. The r2 values of PEI-25 after 1 year are shown for comparison: slight variations are all within the experimental error
Figure 9 shows representative first-passage curves obtained in two region-of-interest selected in differently vascularized brain regions for PEI-25 and Endorem ; contrast agents were injected at 6 mgFe/Kg dosages. Data have been normalized using the following relationship: normalized SI% =
SI(t) − SI(0) · 100 SI(0)
where SI(0) was the average signal intensities before injection and SI(t) the signal intensity at time t. r oi 1 was placed over the brain cortex, while r oi 2 was placed below the cortex in a more vascularized region. Figure 9 shows that the signal drop produced by Endorem is slightly higher than that produced by PEI-25 in corresponding brain areas; however data clearly show that first-passage curve can be properly acquired by using PEI-25.
123
Discussion Using mild conditions of slightly elevated temperature (80◦ C) and pH (pH = 10), resulted in the formation of stable deep black-brown solutions. Equal relaxivity and magnetization data were obtained for samples stored at room temperature for up to 1 year (Fig. 8b). In the absence of polymers, the mixture of Fe(II) and Fe(III) rapidly formed a black precipitate of iron oxides. Both the polyamine polymers and some of their carboxylated derivatives are therefore very effective in stabilizing iron oxide nanoparticles under the reaction conditions we have explored. The polymer could act to stabilize the particles in a number of ways. By virtue of the high charge density of its functionalized surface, the polymer is expected to assist nucleation through stabilization of embryonic aggregates. This would result in the formation of small particles. In addition, polymer-particle interactions might act to passivate the iron oxide surface and thus limit particle growth as well as block the interparticle interaction and aggregation as suggested in Fig. 1. These novel compounds have a strong effect on T1 and T2 relaxation of solvent water protons, therefore candidating for further exploration as magnetic resonance imaging contrast agents. To establish the efficiency of the newly synthesized materials as possible MRI contrast agents and to understand their magnetic behavior, we analyzed the susceptibility, magnetization, relaxometry and MRI data. The magnetic properties of our materials measured by SQUID were compared with the ones of Endorem . This material has been shown to be very effective contrast agents for magnetic resonance imaging [23,34]. In agreement with the average diameters of the magnetic core of the particles the blocking temperature above which the particle exhibits superparamagnetism of Endorem [34] and PEI-COOH60, is sensibly lower than that of PEI-25, and PEI-500, which, in addition, present a broad distribution. As a consequence of these two facts,
Magn Reson Mater Phy (2009) 22:77–87
85
Fig. 9 First-passage signal intensity (SI) versus time in rat brain
Fig. 10 Rat brain images acquired before (a, d) and 268 s after administration of PEI-25 (b) and Endorem (e) with corresponding rCBV maps (c, f)
Endorem and PEI-COOH60 exhibits a larger decrease of the magnetic moment at high temperatures, corresponding to a more effective superparamagnetic behaviour. Looking at the NMRD profiles of our samples, it can be noted that the r1 relaxivity of PEI-COOH60 is comparable to that of Endorem over the whole investigated frequency range, thus resulting in a comparable efficiency. On the other hand the r2 relaxivity of PEI-COOH60 is again comparable to that of Endorem, showing the typical almost flat behaviour.
Both 1/T1 and 1/T2 data are thus consistent with the ones typical of large oligomeric structures [35–37], where the dimensions of the magnetic nanoparticle as a whole is dominating the mechanism of relaxation. As a further confirmation, it can be noted that the behaviour of r1 (ν) in the range 7 < ν < 200 MHz is qualitatively the same of Endorem sample, suggesting that the driving mechanism of nuclear relaxation at ν > 10 MHz is the Curie relaxation [38]. Further investigation at lower frequencies (ν < 5 MHz) are in
123
86
plan to understand if also in this case the magnetic anisotropy dominates the relaxation in such frequency range. Finally it should be noted that the increase of the percentage of COOH groups (see Fig. 6, PEI-COOH60 compared to PEICOOH75) does not result in more efficiency, despite the relatively high values of r1 and r2 in PEI-COOH75. As general remark on magnetic and relaxometry data, we can conclude that with the magnetic core of our samples, we got nuclear relaxation efficiencies very near to the commercial samples that contain superparamagnetic particles. As regards MRI data, we can observe that PEI-25 can be used in bolus tracking experiments while, probably due to a rapid washout from blood, it seems to be of difficult use in steady state acquisitions.
Conclusions In this paper we have shown an alternative method to obtain iron-oxide based possible novel contrast agents. The most remarkable results are: (a) the synthesis of stable suspensions of nanoparticles with or without the presence of carboxylated polymers. (b) the fundamental parameter in determining the signal disappearance and so the image contrast (i.e., the r2 relaxivity) for sample PEI-COOH60 is comparable to that of the commercially available compounds (e.g., Endorem ), in the whole frequency range 7 ≤ ν ≤ 212 MHz ; (c) the r1 relaxivity for PEI-COOH60 is comparable to that of commercial compounds; (d) the compound PEI-25 resulted a good CA for MRI bolus tracking experiments (rCBF measurements). Even if the novel synthesized PEI-coated iron oxide compounds are less efficient than the commercially available contrast agent Endorem , only small differences have been reported. Nevertheless, the versatility of these polymers (and their derivatives) resides in their ability to complex and deliver DNA in vitro and in vivo, as reported in our previous works [30,33], and their ability to be monitored by non-invasive optical imaging techniques [25] if conjugated with a proper dye. Therefore, PEI is a versatile polymer to use if a multifunctional system with improved properties (superparamagnetic and fluorescent) is to be obtained. Moreover, efforts have been made to render this molecules “tissue-specific” for DNA delivery (targeted delivery systems) by coupling with suitable monoclonal antibodies. We think that the minor efficiency of the reported compounds may be well compensated by the additional multifunctional properties to be added in a near future [39]. Work is in progress to determine: (a) the role of the aggregation of the magnetic cores; (b) the possibility to decrease further the molecular weight MWCOOH to understand if the tendency of r1 and r2 to increase with diminishing the per-
123
Magn Reson Mater Phy (2009) 22:77–87
centage of COOH groups leads to more efficient (higher r1 and r2 ) contrast agents; (c) the toxicity and bio-compatibility of the proposed compounds; (d) study the distribution of PEI-COOH60 in vivo on animal model. Acknowledgments Authors dedicate this work to the memory of Prof. Alessandro Bencini, emeritus professor, recently and suddenly disappeared. His clever reasoning and patient teaching will always remain in our memory as a limpid example of perseverance in this scientific discipline. Authors thank Dr. Paola Vicennati and Dr. Emanuela Picchi for their support and discussions. The work was carried out also within the framework of the national projects FIRB no. RBNE01YLKN2001, PRIN 2005-prot. No. 2005039758 and the european project NoE MAGMANET (NMP3-CT-2005 515767). This study was also partially supported by ECSIN (European Center for Sustainable Impact of Nanotechnology).
References 1. Rinck PA (1993) Magnetic resonance in medicine, 3rd edn. Blackwell, Oxford 2. Laurent S, Elst LV, Roch A, Muller RN (2007) In: Carretta P, Lascialfari A (eds) NMR–MRI, mSR and Mossbauer spectroscopies in molecular magnets. Springer, Italy, pp 71–88 3. Lascialfari A, Corti M (2007) In: Carretta P, Lascialfari A NMRMRI, mSR and Mossbauer spectroscopies in molecular magnets. Springer, Italy, pp 89–110 4. Zhao M, Sun L, Crooks RM (1998) Preparation of Cu nanoclusters within dendrimer templates. J Am Chem Soc 120:4877–4878 5. Balogh L, Tomalia DA (1998) Poly(amidoamine) dendrimertemplated nanocomposites I. Synthesis of zero-valent copper nanoclusters. J Am Chem Soc 120:7355–7356 6. Zhao M, Sun L, Crooks RM (1999) Dendrimer-encapsulated transition metal nanocluster: synthesis, characterization, and applications to catalysts. Polym Prepr (Am Chem Soc Div Polym Chem) 40:400–401 7. Garcia ME, Baker LA, Crooks RM (1999) Preparation and characterization of dendrimer-gold colloid nanocomposites. Anal Chem 71:256–258 8. Zhao M, Crooks RM (1999) Dendrimer-encapsulated Pt nanoparticles: synthesis, characterization, and applications to catalysis. Adv Mater 11:217–220 9. Sooklal K, Hanus LH, Ploehn HJ, Murphy CJ (1998) A blue emitting CdS-dendrimer nanocomposite. Adv Mater 10:1083–1087 10. Sooklal K, Huang J, Murphy CJ, Hanus L, Ploehn HJ (1999) Inorganic quantum dot-organic dendrimer nanocomposite material. Mater Res Soc Symp Proc 576:439–444 11. Huang JM, Murphy CJ (1999) Luminescence of CdS nanoparticles doped and activated with foreign ions. Mater Res Soc Symp Proc 560:33–38 12. Groehn F, Bauer BJ, Akpalu YA, Jackson CL, Amis EJ (2000) Dendrimers as nanotemplates for the formation of inorganic colloids. Macromolecules 33:6042–6050 13. Keki S, Torok J, Deak G, Daroczi L, Zsuga M (2000) Silver nanoparticles by PAMAM-assisted photochemical reduction of Ag+. J Colloid Interface Sci 229:550–553 14. Esumi K, Hosoya T, Suzuki A, Torigoe K (2000) Formation of gold and silver nanoparticles in aqueous solution of sugar-persubstituted poly(amidoamine) dendrimers. J Colloid Interface Sci 226:346– 352 15. Meldrum FC, Heywood BR, Mann S (1999) Magnetoferritin: in vitro synthesis of a novel magnetic protein. Science 257:522–523
Magn Reson Mater Phy (2009) 22:77–87 16. Meldrum FC, Wade VJ, Nimmo DL, Heywood BR, Mann S (1991) Synthesis of inorganic nanophase materials in supramolecular protein cages. Nature 349:684–687 17. Douglas T, Young M (1998) Host–guest encapsulation of materials by assembled virus protein cages. Nature 393:152–155 18. Ziolo RF, Giannelis EP, Weinstein BA, O’Horo MP, Ganguly BN, Mehrotra V, Russell MW, Huffman DR (1992) Matrix-mediated synthesis of nanocrystalline Fe2 O3 : a new optically transparent magnetic material. Science 257:219–223 19. Tang BZ, Geng Y, Lam JWY, Li B, Jing X, Wang X, Wang F, Pakhomov AB, Zhang XX (1999) Processible nanostructured materials with electrical conductivity and magnetic susceptibility: preparation and properties of maghemite/polyaniline nanocomposite films. Chem Mater 11:1581–1589 20. Mann S, Hannington JP, Williams RJP (1986) Phospholipid vesicles as a model system for biomineralization. Nature 324:565–567 21. De Cuyper M, Joniau M (1988) Magnetoliposomes. Formation and structural characterization. Eur Biophys J 15:311–319 22. Bulte JWM, de Cuyper M, Despres D, Frank JA (1999) Preparation, relaxometry, and biokinetics of PEGylated magnetoliposomes as MR contrast agent. J Magn Magn Mater 194:204–209 23. Corti M, Lascialfari A, Marinone M, Masotti A, Micotti E, Orsini F, Ortaggi G, Poletti G, Innocenti C, Sangregorio C (2008) Magnetic and relaxometric properties of polyethylenimine-coated superparamagnetic MRI contrast agents. J Magn Magn Mater 320:e316–e319 24. Bulte J, Douglas T, Witwer B, Zhang S-C, Strable E, Lewis BK, Zywicke H, Miller B, Van Gelderen P, Moskowitz BM, Duncan ID, Frank JA (2001) Magnetodendrimers allow endosomal magnetic labeling and in vivo tracking of stem cells. Nat Biotechnol 19:1141–1147 25. Masotti A, Vicennati P, Boschi F, Calderan L, Sbarbati A, Ortaggi G (2008) A novel near-infrared indocyanine dye-polyethylenimine conjugate allows DNA delivery imaging in vivo. Bioconjug Chem 19(5):983–987 26. Putman CAJ, van der Werft K, de Grooth BG, van Hulst NF, Greve J, Hansma PK (1992) New imaging mode in the atomic force microscopy based on the error signal. Proc SPIE 1639:198–204 27. Deichmann R, Haase A (1992) Quantification of T1 values by SNAPSHOT-FLASH NMR imaging. J Magn Reson 96:608–612 28. Rowley HA, Roberts TPL (2004) Clinical perspectives in perfusion: neuroradiologic applications. Top Magn Reson Imaging 15:28–40 29. Haraldseth O, Jones RA, Muller TB, Fahlvik AK, Oksendal AN (1996) Comparison of dysprosium DTPA BMA and superparamagnetic iron oxide particles as susceptibility contrast agents for perfusion imaging of regional cerebral ischemia in the rat. J Magn Reson Imaging 6:714–717
87 30. Masotti A, Ortaggi G (2008) Peptide nucleic acid (PNA)-polyethylenimine (PEI) conjugates. Promising multifunctional therapeutic tools for the future. Oligonucleotides 18(3): 301–303 31. Hamberg LM, Boccalini P, Stranjalis G, Hunter GJ, Huang Z, Halpern E, Weisskoff RM, Moskowitz MA, Rosen BR (1996) Continuous assessment of relative cerebral blood volume in transient ischemia using steady state susceptibility-contrast MRI. Magn Reson Med 35:168–173 32. Sbarbati A, Reggiani A, Lunati E, Arban R, Nicolato E, Marzola P, Asperio RM, Bernardi P, Osculati F (2000) Regional cerebral blood volume mapping after ischemic lesions. Neuroimage 12:418–424 33. Masotti A, Moretti F, Mancini F, Russo G, Di Lauro N, Checchia P, Marianecci C, Carafa M, Santucci E, Ortaggi G (2007) Physicochemical and biological study of selected hydrophobic polyethylenimine-based polycationic liposomes and their complexes with DNA. Bioorg Med Chem 15(3):1504–1515 34. Gamarra LF, Brito GES, Pontuschka WM, Amaro E, Parma AHC, Goya GF (2005) Biocompatible superparamagnetic iron oxide nanoparticles used for contrast agents: a structural and magnetic study. J Magn Magn Mater 289:439–441 35. Bulte JWM, Vymazal J, Brooks RA, Pierpaoli C, Frank JA (1993) Frequency dependence of MR relaxation times. II. Iron oxides. J Magn Reson Imaging 3:641–648 36. Bulte JWM, Brooks RA (1997) Scientific and clinical applications of magnetic carriers. In: Häfeli U (ed) Plenum Press, New York, pp 527–542 37. Boni A , Marinone M, Innocenti C, Sangregorio C, Corti M, Lascialfari A, Mariani M, Orsini F, Poletti G, Casula MF (2008) Magnetic and relaxometric properties of Mn-ferrites. J Phys D Appl Phys 14:134021/1–134021/6 38. Roch A, Muller RN, Gillis P (1999) Theory of proton relaxation induced by superparamagnetic particles. J Chem Phys 110:5403– 5411 39. Masotti A, Vicennati P, Ortaggi G (2008) Multifunctional delivery vehicles for biomedical applications: polyethylenimine as a multipurpose polymer. In: Colombo GP, Ricci S (eds) Medicinal chemistry research progress, chap 6. Nova Science Publishers, New York
123