Synthesis and Characterization of PEGylated Gd2O3 Nanoparticles for

Mar 24, 2010 - †Division of Molecular Surface Physics and Nanoscience, and ‡ Division of ...... ticles tagged with Rhodamine taken both in dayligh...
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Synthesis and Characterization of PEGylated Gd2O3 Nanoparticles for MRI Contrast Enhancement )

Maria Ahren,† Linnea Selega˚rd,† Anna Klasson,§, Fredrik S€oderlind,‡ Natalia Abrikossova,† Caroline Skoglund,^ Torbj€orn Bengtsson,^,# Maria Engstr€om,§, Per-Olov K€all,‡ and Kajsa Uvdal*,†

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† Division of Molecular Surface Physics and Nanoscience, and ‡ Division of Chemistry, Department of Physics, Chemistry and Biology (IFM), Link€ oping University, SE-581 83 Link€ oping, Sweden, §Center for Medical Image Science and Visualization (CMIV), Link€ oping University, SE-581 85 Link€ oping, Sweden, Department of Medical and Health Sciences/Radiological Sciences, Link€ oping University, SE-581 85 oping Link€ oping, Sweden, ^Division of Drug Research, Department of Medical and Health Sciences, Link€ University, SE-581 85 Link€ oping, Sweden, and #Division of Clinical Medicine, Department of Biomedicine, € € Orebro University, SE-70182 Orebro, Sweden

Received June 18, 2009. Revised Manuscript Received January 28, 2010 Recently, much attention has been given to the development of biofunctionalized nanoparticles with magnetic properties for novel biomedical imaging. Guided, smart, targeting nanoparticulate magnetic resonance imaging (MRI) contrast agents inducing high MRI signal will be valuable tools for future tissue specific imaging and investigation of molecular and cellular events. In this study, we report a new design of functionalized ultrasmall rare earth based nanoparticles to be used as a positive contrast agent in MRI. The relaxivity is compared to commercially available Gd based chelates. The synthesis, PEGylation, and dialysis of small (3-5 nm) gadolinium oxide (DEG-Gd2O3) nanoparticles are presented. The chemical and physical properties of the nanomaterial were investigated with Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, transmission electron microscopy, and dynamic light scattering. Neutrophil activation after exposure to this nanomaterial was studied by means of fluorescence microscopy. The proton relaxation times as a function of dialysis time and functionalization were measured at 1.5 T. A capping procedure introducing stabilizing properties was designed and verified, and the dialysis effects were evaluated. A higher proton relaxivity was obtained for as-synthesized diethylene glycol (DEG)-Gd2O3 nanoparticles compared to commercial Gd-DTPA. A slight decrease of the relaxivity for as-synthesized DEG-Gd2O3 nanoparticles as a function of dialysis time was observed. The results for functionalized nanoparticles showed a considerable relaxivity increase for particles dialyzed extensively with r1 and r2 values approximately 4 times the corresponding values for Gd-DTPA. The microscopy study showed that PEGylated nanoparticles do not activate neutrophils in contrast to uncapped Gd2O3. Finally, the nanoparticles are equipped with Rhodamine to show that our PEGylated nanoparticles are available for further coupling chemistry, and thus prepared for targeting purposes. The long term goal is to design a powerful, directed contrast agent for MRI examinations with specific targeting possibilities and with properties inducing local contrast, that is, an extremely high MR signal at the cellular and molecular level.

Introduction The development of magnetic resonance imaging (MRI) contrast agents based on paramagnetic metal complexes started more than 20 years ago.1 The use of Gd-DTPA complexes as contrast agent in MRI exams was approved in 1988,2 and there are now several other Gd3þ based chelates on the market, for example, Dotarem, Omniscan, and Gadovist.3 The chelates have rather low relaxivity, compared to what would be theoretically possible for alternative materials, and must be administered in quite high doses to provide a good contrast.4 Considerable efforts have been devoted both to improve relaxivity and to minimize toxicity effects, for example, by linking multiple gadolinium complexes together and optimizing the molecular parameters that affect the

relaxivity.5,6 In recent years, intense research activities have been focused on nanosized particulate probes for MRI. Up to now, magnetic nanoparticles based on iron oxides (Fe2O3 or Fe3O4) are the only ones permitted for use in in vivo examinations of patients, but there is an interest in developing novel, more efficient systems, for example, nanoparticle contrast agents based on some of the highly magnetic rare earth metals. Among promising candidates to be used for MRI are Gd2O3, doped Gd2O3,7,8 and mixed rare earth/transition metal oxides.9,10 The main advantage of using a nanoparticle based system compared to chelate complexes is that the former is expected to yield a considerably higher relaxivity due to a higher density of magnetic ions.5,11 Other benefits would be less leakage of Gd ions and greater opportunities for functionalization and thereby possible targeting properties. Nanoparticles

*To whom correspondence should be addressed. E-mail: [email protected]. (1) Lauffer, R. B. Chem. Rev. 1987, 87, 901–927. (2) Caravan, P.; Ellison, J. J.; McMurry, T. J.; Lauffer, R. B. Chem. Rev. 1999, 99, 2293–2352. (3) Carlos, F. G. C.; Geraldes, S. L. Contrast Media Mol. Imaging 2009, 4, 1–23. (4) Raymond, K. N.; Pierre, V. C. Bioconjugate Chem. 2005, 16, 3–8. (5) Caravan, P. Chem. Soc. Rev. 2006, 35, 512–523. (6) Voisin, P.; Ribot, E. J.; Miraux, S.; Bouzier-Sore, A. K.; Lahitte, J. F.; Bouchaud, V.; Mornet, S.; Thiaudiere, E.; Franconi, J. M.; Raison, L.; Labrugere, C.; Delville, M. H. Bioconjugate Chem. 2007, 18, 1053–1063.

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(7) Louis, C.; Bazzi, R.; Marquette, C. A.; Bridot, J. L.; Roux, S.; Ledoux, G.; Mercier, B.; Blum, L.; Perriat, P.; Tillement, O. Chem. Mater. 2005, 17, 1673–1682. (8) Petoral, R. M.; S€oderlind, F.; Klasson, A.; Suska, A.; Fortin, M. A.; Abrikossova, N.; Selega˚rd, L. a.; K€all, P.-O.; Engstr€om, M.; Uvdal, K. J. Phys. Chem. C 2009, 113, 6913–6920. (9) Coroiu, I. J. Magn. Magn. Mater. 1999, 201, 449–452. (10) S€oderlind, F.; Fortin, M. A.; Petoral, R. M. J.; Klasson, A.; Veres, T.; Engstr€om, M.; Uvdal, K.; K€all, P.-O. Nanotechnology 2008, 085608. (11) LaConte, L.; Nitin, N.; Bao, G. Mater. Today 2005, 8, 32–38.

Published on Web 03/24/2010

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brought to specific sites would result in huge local contrast enhancement compared to chelate based contrast agents. The size of the nanoparticles is crucial. The nanomaterial must be small enough to be filtered out from the body and designed for specific targeting to enable interaction with biomolecules or cell surface receptors. It has been found that contrast agents based on superparamagnetic ion oxides (SPIOs) are quickly removed from the circulatory system, ending up in the liver and spleen.11 A natural consequence of this fast accumulation is hepatic imaging, one of the first applications of nanoparticle probes in clinical use. Access to biocompatible nanoparticles that can avoid the phagocyte system will extend the field of application and prolong the possible examination time. Moreover, as the rare earth cations cannot be naturally degraded in the living body as Fe, they are to some extent toxic and clearance must take place through the kidneys. Nanoparticle surface modification is of main importance to prevent nanoparticle aggregation prior to injection, decrease the toxicity, and increase the solubility and the biocompatibility in a living system.11 Carefully designed capping strategies can facilitate further coupling possibilities. For different nanoparticle applications in biology and medicine, hundreds of capping strategies have been studied. Many of these typically involve some kind of poly(ethylene glycol) (PEG) molecule12-16 or polysaccharide17-19 with the aim to achieve biocompatibility. In the case of PEG, an intervening siloxane layer is often used to attach the molecule to the nanoparticle, but several studies are also focused on functionalization with a pure silane layer20,21 for the purpose to study surface interaction or possible changes in the magnetic properties and size upon functionalization. Solid surfaces covered with PEG have shown to be nonimmunogenic, nonantigenic, and protein resistant,22 and pioneering in vivo imaging and biodistribution studies with mice show that PEGylated nanoparticles accumulate in the kidneys and bladder.14 Conjugation to targeting, biomolecules can be done provided that functional groups are available on the PEG. Often bifunctional PEG is used to enable coupling to the nanoparticle/siloxane and another molecule. In this Article, we report the MR properties of small Gd2O3 nanoparticles, synthesized via the polyol method20,23 and functionalized with a bifunctional PEG via a small silane to ensure a stable and biocompatible system (see Scheme 1). The choice of a bifunctional PEG was done to enable further tagging to diverse targets; that is, the nanomaterial is prepared for further coupling. Finally, PEG functionality was investigated, as a proof of concept, by coupling a flourophore to the NHS part of the molecule. (12) Zhang, Y.; Kohler, N.; Zhang, M. Biomaterials 2002, 23, 1553–1561. (13) Fortin, M.-A.; Petoral, R. M. P., Jr.; S€oderlind, F.; Klasson, A.; Engstr€om, M.; Veres, T.; K€all, P.-O.; Uvdal, K. Nanotechnology 2007, 18, 395501. (14) Bridot, J. L.; Faure, A. C.; Laurent, S.; Riviere, C.; Billotey, C.; Hiba, B.; Janier, M.; Josserand, V.; Coll, J. L.; VanderElst, L.; Muller, R.; Roux, S.; Perriat, P.; Tillement, O. J. Am. Chem. Soc. 2007, 129, 5076. (15) Kohler, N.; Fryxell, G. E.; Zhang, M. J. Am. Chem. Soc. 2004, 126, 7206– 7211. (16) Ja Young, P.; Eun Sook, C.; Myung, Ju, B.; Gang, Ho, L.; Seungtae, W.; Yongmin, C. Eur. J. Inorg. Chem. 2009, 2009, 2477–2481. (17) Lemarchand, C.; Gref, R.; Couvreur, P. Eur. J. Pharm. Biopharm. 2004, 58, 327–341. (18) Lacava, L. M.; Lacava, Z. G. M.; Da Silva, M. F.; Silva, O.; Chaves, S. B.; Azevedo, R. B.; Pelegrini, F.; Gansau, C.; Buske, N.; Sabolovic, D.; Morais, P. C. Biophys. J. 2001, 80, 2483–2486. (19) McDonald, M. A.; Watkin, K. L. Acad. Radiol. 2006, 13, 421–427. (20) Pedersen, H.; S€oderlind, F.; Petoral, J. R. M.; Uvdal, K.; K€all, P.-O.; Ojam€ae, L. Surf. Sci. 2005, 592, 124–140. (21) Yamaura, M.; Camilo, R. L.; Sampaio, L. C.; Macedo, M. A.; Nakamura, M.; Toma, H. E. J. Magn. Magn. Mater. 2004, 279, 210–217. (22) Harris, J. M. Polyethylene Glycol Chemistry; Plenum Press: New York, 1992. (23) Bazzi, R.; Flores-Gonzalez, M. A.; Louis, C.; Lebbou, K.; Dujardin, C.; Brenier, A.; Zhang, W.; Tillement, O.; Bernstein, E.; Perriat, P. J. Lumin. 2003, 102-103, 445–450.

5754 DOI: 10.1021/la903566y

Ahr en et al. Scheme 1. Reaction Scheme Showing the Functionalization Strategy When Capping Gd2O3 Nanoparticles with (3-Mercaptopropyl)trimethoxysilane (MPTS) and r-Maleinimido-ω-carboxysuccinimidyl Ester Poly(ethylene glycol) (Mal-PEG-NHS (1)); Nanoparticle Solution Is Added to the Intermediate Molecule (2), and the functionalized Nanoparticles (3) are Denoted GMPa

a

Note that the nanoparticle size is not to scale.

The properties of the nanoparticles and the capping were studied with Fourier transform infrared (FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM), dynamic light scattering (DLS), magnetic resonance imaging (MRI) and UV-vis spectrophotometry, and the morphology of neutrophils exposed to our capped and uncapped nanoparticles was studied using fluorescence microscopy. The aim of this study is to develop and design a contrast agent for MRI examination that is more powerful with respect to both MR signal enhancement and targeting functionality than what is commercially available today.

Materials and Methods Chemicals and Synthesis of Nanoparticles. All chemicals were used as received: gadolinium(III) chloride hexahydrate (Sigma, 99%), sodium hydroxide (Merck, 99.98%), diethylene glycol (DEG) (Fluka, g 98%), (3-mercaptopropyl)trimethoxysilane (Aldrich, 95%; herein denoted MPTS), R-maleinimido-ωcarboxysuccinimidyl ester poly(ethylene glycol), 5000 D (IRIS biotech; Mal-PEG-NHS), and Rhodamine 123 hydrate (Sigma Aldrich). When ethanol was used as solvent, 99.5% purity grade was chosen. For dialysis and preparation of water based solutions, Milli-Q water (F > 18.2 MΩ) was used. The following chemicals were used for the cell study: bodipy-phallacidin (Invitrogen), paraformaldehyde (Sigma), lysophosphatidylcholine (Sigma), 12-O-tetradecanoylphorbol 13-acetate (PMA) (Sigma), phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 6.7 mM Na2HPO4 3 2H2O, and 1.5 mM KH2PO4, pH 7.3), and Krebs-Ringer glucose buffer (KRG; 120 mM NaCl, 4.9 mM KCl, 1.2 mM MgSO4, 1.7 mM KH2PO4, 8.3 mM Na2HPO4, 1.1 mM CaCl2, and 10 mM glucose, pH 7.3). KRG was also prepared without the addition of CaCl2. The Gd2O3 samples in this study were synthesized via the polyol route.20,23 In short, 6 mmol GdCl3 and 7.5 mmol NaOH were dissolved in 30 mL of DEG. The two solutions were mixed, magnetically stirred, and heated to about 140 C. The temperature was held constant for 1 h and then increased to and retained at 180 C for the next 4 h. Prepared samples were stored in refrigerator. Langmuir 2010, 26(8), 5753–5762

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Filtering and Dialysis. All samples were centrifuge filtered after synthesis to exclude large aggregates prior to further treatment. Vivaspin filters (polyethersulphone (PES), Vivascience Sartorius) with pore size 0.2 um were used, and the process was performed at 1750 rpm for about 30 min or until nearly all fluid had passed through the filter. Dialysis (1000 MWCO, Spectra/ Por, Spectrum Laboratories Inc.) was performed for 24 h to exclude free ions and potentially small nonmature clusters still left after filtering and to replace DEG in the samples with water. Further dialysis (10 000 MWCO, Spectra/Por, Spectrum Laboratories Inc.) was performed after functionalization of the nanoparticles to remove excess of noncoupled ligands. The dialysis procedure was done against Milli-Q water with a relative sample to water volume ratio of at least 1:1000. Water was refreshed three times a day during the first two days and twice a day at later times. Magnetic stirring was used to increase the circulation close to the dialysis membrane to ensure efficiency of the dialysis process. Nanoparticle Surface Modification. A solution of 15 mg of Mal-PEG-NHS dissolved in 300 μL of ethanol, to which 0.5 μL of MPTS was added, was placed in an ultrasonic bath for 1 h. A total of 1 mL of filtered and dialyzed (24 h) nanoparticle suspension was added to the solution, which was again ultrasonicated for 2 h. Finally, the tube was placed on a mixer-table for incubation overnight. Further dialysis was followed to remove unreacted precursors. MPTS and PEG capped Gd2O3 nanoparticles are hereafter denoted GMP or PEGylated Gd2O3. When fluorophore coupling was studied, 1 mg of Rhodamine was added to the MPTS Mal-PEG-NHS mixture in the first step. The same dialyisis procedure was performed as for solely PEGylated Gd2O3 nanoparticles. As-synthesized nanoparticle samples in this paper are denoted DEG-Gd2O3, whereas dialyzed samples are denoted dialyzed DEG-Gd2O3 or Gd2O3. Capped or functionalized samples in general refer to Gd2O3 with MPTS and PEG. Isolation of Neutrophil Granulocytes and Staining of the Actin Cytoskeleton. Peripheral blood was drawn from apparently healthy, nonmedicated donors at the Link€ oping University Hospital, and neutrophils were isolated according to B€ oyum and others.24,25 Briefly, whole blood was layered onto an equal volume of a density gradient (Polymorphprep), and the tubes were centrifuged at 480g for 40 min at room temperature in accordance to the manufacturer’s instructions. The neutrophil-containing fraction was collected and washed in PBS at 480g for 10 min. Any remaining erythrocytes were removed by brief hypotonic treatment performed twice in ice-cold distilled water for 35 s and by washing in PBS containing 3.4% NaCl and calcium-free Krebs-Ringer glucose buffer (400g at 4 C). After the isolation procedure, neutrophils were resuspended in KRG supplemented with 1.1 mM CaCl2 (pH 7.3). Isolated neutrophils were counted in a b€ urkner chamber, the cell concentration was adjusted to 2  106 cells/mL, and cells were kept on ice until exposed to Gd2O3 nanoparticles ([Gd] in sample = 0.11 mM) or PEGylated Gd2O3 nanoparticles ([Gd] in sample = 0.19 mM) for 30 min under stirring condition at 37 C. Samples were then fixed in 4% paraformaldehyde for 30 min, washed three times in PBS, permeabilized and stained for F-actin by incubation with 100 μg/mL lysophosphatidylcholine and 0.6 μg/mL bodipy-phallacidin in PBS for 30 min at room temperature in darkness.26 After triplicate washing, the cells were visualized in the fluorescence microscope. Instrumentation. XPS measurements were carried out in a Microlab 310F instrument with a hemispheric analyzer using unmonochromatized Al KR photons (1486.6 eV). The pressure in the analysis chamber was approximately 4  10-8 mbar during measurement, and the resolution was determined from the full width at half-maximum of the Au 4f7/2 line which was 1.6 eV. Substrates for XPS measurements were prepared by evaporating (24) Boyum, A. Scand. J. Clin. Lab. Invest. Suppl. 1968, 97, 77–89. (25) Ferrente, A.; Thong, Y. H. J. Immunol. Methods 1980, 36, 109–117. (26) Bengtsson, T.; Zalavary, S.; Stendahl, O.; Grenegard, M. Blood 1996, 87, 4411–4423.

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Article 25 A˚ Ti followed by 2000 A˚ Au on Si(100) surfaces cleaned in a 5:1:1 mixture of Milli-Q water, 25% hydrogen peroxide, and 30% ammonia at 80 C and rinsed in Milli-Q water. Drops of sample were spin-coated on the surfaces. High resolution TEM studies were performed with a FEI Tecnai G2 electron microscope operated at 200 kV. Sample preparation prior to TEM measurements was done by carefully drying 3-5 drops of sample solution on amorphous carboncovered copper grids. IR spectroscopy studies were carried out on a Bruker Vertex 70 FT-IR instrument using CaF2 tablets. A few drops of sample were placed on a CaF2 disk and dried with N2 to remove the solvent. Measurements were performed in the range 400-4800 cm-1. Dynamic light scattering measurements were performed on an ALV/DLS/SLS-5022F system from ALV-GmbH, Langen, Germany, using a HeNe laser at 632.8 nm with 22 mW output power. Analyzed samples were diluted 10 times with deionized water before measurements, and the temperature was stabilized in a thermostat bath at 21.5 C. The scattering angle was 150, and each sample was studied at least in six repetitive measurements. All test tubes were rinsed in ethanol and dried with nitrogen before being utilized. The behavior of nanoparticles in salt solution was studied by DLS by adding 100 μL of 1 M NaCl to 1 mL of the diluted samples. Absorbance studies were carried out on an UV-2450 UV-vis spectrophotometer from Shimadzu. The measurements were performed in semimicro UV cuvettes (Brand GmbH) in the range 200-700 nm. Relaxation times were measured with a Philips Achieva 1.5 T whole body MR scanner using the head coil. The resolution was 0.78  0.78  5.0 mm3. Prior to MRI measurements, samples were diluted in deionized water to different Gd concentrations in the approximate range 0-2 mM. The samples were placed in a tube holder, immersed in a saline filled bowl, and placed in the head coil of the MRI instrument. The sample pH was approximately 7, and the measurements were performed at a temperature of 22 ( 1 C. T1 was measured with inversion recovery pulse sequences. In order to cover the entire concentration range of the samples, two different TR parameter settings were used. For samples with very low Gd concentrations (generating 5  T1 > 3000 ms), TR was set to 10 000 ms, and for samples with higher concentrations (generating 5  T1 < 3000 ms) TR = 3000 ms. The remaining parameter settings were TE = 7.5 ms and IR = 100-2600, and seven measure points were used. T2 was measured with a multiecho sequence with TE = 20 ms and TR = 1000 ms. The number of echoes was 16, and a 70 flip angle was used. Linear regression analysis of the MRI data was done using Kaleidagraph 4.0 software. The Gd concentrations of the samples were determined by inductively coupled plasma sector field mass spectroscopy (ICP-SFMS, Analytica AB (Sweden)). Fluorescence microscopy images were acquired using an Axiovert 200 fluorescence microscope with an oil immersion 63/ 1.4NA objective (Carl Zeiss GmbH, Germany), and digital images were obtained using an Axiocam MRm camera and the AxioVision software (version 4.6.3.0).

Results and Discussion In this Article, small Gd2O3 nanoparticles are synthesized, functionalized, dialyzed, and characterized. The aim is to design and develop a more powerful MRI contrast agent, with respect to the induced MR signal, than what is commercially available today. This type of nanoparticles is expected to deliver an excellent contrast enhancement many times higher than that of the commercially available ionic based contrast agents. The main benefits are potential site specificity and a high number of Gd atoms per nanoparticle. The particles are synthesized via the polyol route and capped with a bifunctional PEG to ensure a biocompatible and stable system and facilitate further tagging. A DOI: 10.1021/la903566y

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Figure 1. Relative amount of Gd (%) left after different times of dialyis of filtered DEG-Gd2O3 nanoparticle samples (A) and PEGylated Gd2O3 samples (GMP) (B) against Milli-Q water, and corresponding hypothetical fitted curves.

silane linker molecule is used to couple the PEG to the nanoparticle. The nanoparticles are investigated with respect to elemental composition and stability of the nanoparticle core and the coupling and function of the capping layer. This work is also focused on studying the dialysis in which the excess of ions and other reaction precursors are removed. The properties of the nanomaterial and the capping layer are mainly studied with IR spectroscopy, XPS, TEM, and DLS. Relaxivity measurements are performed on pure nanoparticle samples as well as capped particles dissolved in water to be able to evaluate the power of the material as a future contrast agent. Filtering of the nanoparticle solutions is done to remove large aggregates or clusters. The Gd concentration of the samples was measured before and after filtering, but no significant Gd concentration change was found. The result indicates that no detectable amounts of particles larger than 200 nm can be found in newly synthesized nanoparticle suspensions. Particle clusters or aggregates of smaller sizes cannot be excluded. The dialysis of the nanoparticle samples in this study is of major importance as a purification step to produce a useful MR contrast agent. Besides being dissolved in DEG, the as-synthesized nanoparticles are coated with a layer of diethylene glycol that is supposed to minimize the risk of particle aggregation. The presence of DEG complicates functionalization of the nanoparticle surface, and the presence of DEG is not suitable in MRI measurements. These newly synthesized samples furthermore contain an excess of ion precursors that must be removed before further treatment. Dialysis is a tool to exchange DEG to either water or buffer and to remove ions. Free ligands and reaction precursors (MPTS and PEG) are also present in the capped nanoparticle samples. To clear these, further dialysis was performed after functionalization. The dialysis procedure must be optimized regarding the choice of membrane, volume, and time, since this may have a direct effect on the nanoparticle sample and on the relaxivity. To evaluate the dialysis effects, the concentration of Gd in nanoparticle samples as a function of dialysis time was studied. By comparing dialysis graphs for pure DEG-Gd2O3 nanoparticles (Figure 1A) and surface modified Gd2O3 nanoparticles (GMP) (Figure 1B), interesting information can be achieved. A linear relationship can be fitted to the data for uncapped samples, and the particles appear to be totally dissolved during long periods of dialysis. This major loss of Gd is expected when looking at the solubility product or stability constant for 5756 DOI: 10.1021/la903566y

Gd2O3. Ksp = [Gd3þ]2[OH-]6) is of the order ∼10-31, as calculated from thermodynamical data,27 and the oxide is thus not completely stable in water at neutral pH, although the solubility kinetics is quite slow. For capped samples, that is, GMP, the concentration decrease is high the first day of dialysis but lower at later stages of dialysis. This indicates that there are free ions in the sample when starting dialysis of functionalized nanoparticles but also that capping has stabilizing effects. It should be noted that the dialysis of samples dissolved in DEG involves a 3-fold increase in volume as a consequence of water uptake. The initial value of about 100 mM Gd in freshly synthesized filtered samples decreases to about 10 mM after 1 day of dialysis. With the volume increase of the dialysis membrane taken into account, the Gd content decreases to about one-third of the initial value in uncapped samples. Ions are removed as well as fragments of nonmature nanoparticles or small Gd2O3 clusters that are dissolved during dialysis. A comparative study of a 24 h dialysis of GdCl3 dissolved in DEG was done to verify the efficiency of the dialysis procedure. The same initial Gd3þ concentration (100 mM) was used, and after dialysis a Gd content of about 4 μM was found. Hence, the free ion content in dialyzed samples is a negligible fraction of the total Gd content immediately after completed dialysis. Hydrodynamic Radius and Stability. DLS was primarily used to study the stability of the nanoparticles but also to estimate the hydrodynamic radius of the nanoparticles. Samples that contain mainly DEG are very viscous, and no reproducible results could be obtained with DLS. Dialyzed uncapped nanoparticle samples (Gd2O3) and functionalized and dialyzed samples (GMP) were more straightforward to investigate, since these are dissolved in deionized water. The hydrodynamic radius of filtered and dialyzed (24 h) nanoparticles was estimated to be 3.1 ( 0.9 nm. A considerably larger radius (6.3 ( 0.5) was obtained when studying the corresponding samples before dialysis. These nanoparticles contain a larger amount of DEG with a water attracting effect that is likely to generate the larger hydrophilic radius. In general, the particle size distribution was broader after functionalization, and the fits were not as reproducible as those for uncapped dialyzed samples. Freshly synthesized nanoparticle samples are stable for weeks at room temperature. Dialyzed samples are more vulnerable, probably since some or most of the DEG capping layer is removed (27) Handbook of Chemistry and Physics (internet version), 89th ed.; Lide, D. R., Ed.; CRC Press/Taylor and Francis: Boca Raton, FL, 2009.

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stored for 3 weeks in room temperator, refrigerator, and freezer, and freshly dialyzed DEG-Gd2O3 nanoparticle sample.

Figure 4. Transmission electron microscope images of pure DEGGd2O3 nanoparticles dialyzed for 1 day (lower left corner and upper left corresponding overview), PEGylated Gd2O3 nanoparticles (GMP) (lower right corner and upper right corresponding overview), and the related fast Fourier transform (FFT) for the PEGylated nanoparticles (GMP). The overviews show several clearly separated particles.

Figure 3. Intensity correlation functions of dialyzed DEG-Gd2O3

crystalline planes (d ≈ 3.1 A˚). Furthermore the (400), (440), and (622) planes can be seen in the figure as well. Molecular Composition Analysis. Infrared spectroscopy was used to study the functionalization of the nanoparticles. IR spectra of filtered and dialyzed nanoparticles were compared with spectra for GMP nanoparticles primarily to verify successful PEG functionalization. A reference spectrum of pure PEG dissolved in Milli-Q water was also studied as a reference. The results are summarized in Figure 5 where all peaks are normalized to the largest intensity peak in each spectrum. Filtered and dialyzed samples show no distinct vibrations in the fingerprint region, which is consistent with pure samples with no or only very small amounts of DEG present. Peaks characteristic for NHS are observed at 1740, 1782, and 1815 cm-1 in the Mal-PEG-NHS reference spectrum.29 These are strongly suppressed in the spectra for functionalized samples, indicating hydrolysis of the NHS group during reaction and dialysis, as expected.30 The peak at 1709 cm-1 is assigned to the antisymmetric vibration of the OdC-N-CdO group of the five membered cyclic maleimide.31 The PEG backbone chain gives rise to numerous peaks in the fingerprint area. Most of these are assigned to different vibrational modes of CH2. Peaks assigned to the antisymmetric and symmetric stretches of CH2 are found at around 2900 cm-1.30 Peaks arising from scissoring, wagging, and twisting CH2 vibrational modes are found at around 1470, 1340, and 1240 cm-1, respectively.32,33 Furthermore, the most prominent peak in the spectrum, at about 1100 cm-1, is assigned to PEG chain C-O-C vibrations.32 The MPTS contribution to the GMP spectrum is, as expected, not observed. The SH vibration peak would be located at around 2550 cm-1,34,35 but the intensity is far too low, compared to the

Figure 2. Intensity correlation functions of dialyzed DEG-Gd2O3

nanoparticles, GMP, GMP exposed to NaCl, and Gd2O3 nanoparticles exposed to NaCl.

during dialysis, and unfunctionalized dialyzed samples can neither be stored in the freezer (Figure 2) nor be treated with NaCl (Figure 3) without aggregating to clusters. Functionalized samples (GMP), on the other hand, are stable against both freezing and NaCl treatment (Figure 3). This strongly supports that functionalization of the nanoparticles introduces stabilizing properties. Size and Crystallinity. TEM was used to study the size and crystallinity of the samples. As shown previous13,28 the size of DEG-Gd2O3 nanoparticles synthesized with the polyol method is about 2-5 nm in diameter. TEM studies of dialyzed samples show crystalline particles in the same size range (Figure 4; lower left corner). After functionalization, a lot of organic material is part of the samples. This results in poor contrast and more complicated evaluation. Crystalline particles about 5 nm in size were still found (Figure 4; lower right corner) in these images but also a few aggregates. In the lower middle position of Figure 4, a fast Fourier transform of a TEM image of functionalized nanoparticles can be seen. We suggest the clearest light dot to represent the (222) (28) S€oderlind, F.; Pedersen, H.; Petoral, J. R. M.; K€all, P.-O.; Uvdal, K. J. Colloid Interface Sci. 2005, 288, 140–148.

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(29) Xiao, S.-J.; Wieland, M.; Brunner, S. J. Colloid Interface Sci. 2005, 290, 172–183. (30) Dordi, B.; Schonherr, H.; Vancso, G. J. Langmuir 2003, 19, 5780–5786. (31) Xiao, S. J.; Textor, M.; Spencer, N. D.; Sigrist, H. Langmuir 1998, 14, 5507– 5516. (32) Larsson, A.; Ekblad, T.; Andersson, O.; Liedberg, B. Biomacromolecules 2007, 8, 287–295. (33) Zhou, Y.; Valiokas, R.; Liedberg, B. Langmuir 2004, 20, 6206–6215. (34) Andrade, G.; Barbosa-Stancioli, E. F.; Mansur, A. A. P.; Vasconcelos, W. L.; Mansur, H. S. Biomed. Mater. 2006, 1, 221–234. (35) Li, Y.-S.; Wang, Y.; Tran, T.; Perkins, A. Spectrochim. Acta, Part A 2005, 61, 3032–3037.

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Figure 5. FTIR spectra of the wavenumber range 2600-3100 cm-1 (A), 1400-1900 cm-1 (B), and 1000-1550 cm-1 (C) of (a) Mal-PEGNHS, (b) Gd2O3 nanoparticles capped with MPTS and Mal-PEG-NHS (GMP), and (c) filtered and dialyzed DEG-Gd2O3 nanoparticles.

Figure 6. Gd (3d) XPS spectra for dialyzed DEG-Gd2O3 nanoparticles and PEGylated Gd2O3 nanoparticles (GMP).

characteristic PEG peaks, to be observed. The vibrations associated with Si (Si-O-Si, Si-O-C) are overlapping with C-O-C vibrations of the PEG backbone around 1100 cm-1 or are weak and located at lower wavenumbers (around 450 and 600 cm-1)34,35 out of range for the instrument in use. Overall, the reference spectra and the GMP spectra correspond well to each other. XPS measurements were used to verify the functionalization of MPTS and PEG and to be able to calculate the relative abundance of sulfur, silicon, and nitrogen. The measurements were performed on TL1 cleaned gold surfaces (for details, see Materials and Methods). Figure 6 shows the Gd(3d) spectra of dialyzed DEG-Gd2O3 and PEGylated Gd2O3 nanoparticles. A XPS spectrum of pure gadolinium involves a spin-orbit coupled 3d5/2 and 3d3/2 doublet with a binding energy position of 1186 and 1218 eV, respectively. However, due to the oxygen in Gd2O3, a shift to a higher binding energy occurs. The peak positions of Gd(3d) in the upper curve in Figure 6 are at 1188 and 1220 eV, in good agreement with earlier published data.36 The weak peaks in the Gd(3d) spectrum of GMP are a result of a relatively thick capping (36) Raiser, D.; Deville, J. P. J. Electron Spectrosc. Relat. Phenom. 1991, 57, 91– 97.

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Figure 7. S(2p) XPS spectrum for PEGylated Gd2O3 nanoparticles (GMP).

layer shielding the Gd in the very surface sensitive XPS measurement. The Gd signal can be regained by heating the gold surface, removing organic materials. At higher binding energies in the GMP spectra, some broad background peaks from the Au surface can be seen (Auger lines NNV). The S(2p) XPS spectra of GMP are shown in Figure 7. A curve fit was done, and the best-fit curve of S(2p) shows that the sulfur peak consists of two spin-orbit split doublets with splitting 1.2 eV and a 2:1 peak area ratio. The highest energy doublet (black curves) consists of 2p3/2 and 2p1/2 peaks with binding energy 163.8 and 165 eV, respectively. The gray curve fit has the corresponding peaks at 162.3 and 163.5 eV. We cannot fully establish whether the higher energy doublet (black) around 164 eV corresponds to unbound sulfur (SH) or the thioether bond (C-S-C) between MPTS and the maleimide unit in PEG, but most likely both of these are present. No oxidized sulfur species were found. The presence of the lower energy doublet states that an amount of MPTS molecules have the sulfur atom bound either to the gold substrate or to the particles.37,38 (37) Senkevich, J. J.; Mitchell, C. J.; Yang, G. R.; Lu, T. M. Colloids Surf., A 2003, 221, 29–37. (38) Castner, D. G.; Hinds, K.; Grainger, D. W. Langmuir 1996, 12, 5083–5086.

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Figure 8. Si(2p) XPS spectrum for PEGylated Gd2O3 nanoparticles (GMP).

Figure 9. N(1s) XPS spectra for PEGylated Gd2O3 nanoparticles (GMP) and the capping molecule Mal-PEG-NHS.

Figure 8 shows a reference subtracted spectrum of Si(2p), meaning that a gold reference spectrum is subtracted from the GMP spectrum. The peak fit of the reference subtracted data shows that there are two types of silicon: one peak fit is colored in gray and one in black. Each silicon species shows a spin-orbit coupled 2p1/2, 2p3/2 doublet. The doublet around 103 eV corresponds to SiO2 from the silane molecules forming a network with each other and with the particles. The doublet around 101 eV on the other hand is probably the Si-O-C bond indicating that free silane molecules are present.37 The ratio between silicon and sulfur was calculated. The area under the peaks was calculated from S(2p) and Si(2p) peak-fitted curves. The Si/S ratio is approximately 1.7, which agrees quite well with the expected value. As MPTS involves one sulfur atom and one silicon atom, there should be a 1:1 ratio between these two elements. Figure 9 shows two spectra, one corresponding to Mal-PEG-NHS and one to GMP. They are both peak-fitted and (39) Xiao, S. J.; Textor, M.; Spencer, N. D.; Wieland, M.; Keller, B.; Sigrist, H. J. Mater. Sci.: Mater. Med. 1997, 8, 867–872.

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have three peaks each. The high intensity peak around 400.5 eV corresponds to the nitrogen in the maleimide unit,39 whereas the peak around 402.1 eV is from the NHS unit.40-42 The ratio between the NHS and the Mal peak is 3:4 in PEG and approximately 2:5 in GMP. This ratio difference clearly shows that the NHS group is partly deactivated during functionalization, which is also shown with IR spectroscopy. The third peak in the spectrum has an intensity of 20% compared to the maleimide intensity. The XPS measurements give clear evidence that both PEG and MPTS are bound to the particles. MRI Relaxation Studies. The goal of this study was to develop and design an alternative Gd based contrast agent for MRI examination more powerful than those commercially available at present. MRI measurements were performed to study the MR signal response and the dialysis effects of the relaxivity prior to and after surface modification. The results are presented in Figures 10 and 11 and in Table 1. A corresponding MRI intensity image for PEGylated Gd2O3 nanoparticle samples (GMP) dialyzed for 6 days is also included in Figure 12. The image shows the contrast of six samples with decreasing concentrations of Gd. Note that the Gd concentration in all graphs is given as the number of Gd atoms (instead of number of nanoparticles) to facilitate comparison with commercially available Gd-DTPA chelates (one ion per complex). The nondialyzed filtered samples have, prior to surface modification, the highest proton relaxivity with r1 and r2 values of 9.5 and 10.6 s-1 mM-1, respectively (Figure 10 and Table 1). The corresponding relaxivity for Gd-DTPA in water has previously been reported to be 4.7 s-1 mM-1 for r1 and 5.3 s-1 mM-1 for r2 (1.5 T magnetic field).43 This shows that the relaxivity is roughly doubled for nondialyzed filtered nanoparticles compared to the Gd-DTPA chelates, in good agreement with earlier results.13 In general, the capped nanoparticles have higher relaxivity than uncapped nanoparticles do. For the dialyzed uncapped samples, the r1 relaxivity is decreasing as a function of dialysis time, and after 3 days of dialysis the relaxivity has decreased to less than 1/3 of the initial value. Relaxivity is very dependent on molecular motion, and the rotational dynamics of the material is crucial for all MRI contrast agents. Earlier theoretical and experimental studies show that longer rotational correlation time involves a faster relaxation rate.5 Strategies to improve the relaxivity of MRI contrast agents involve therefore attachment of larger molecules to paramagnetic species. The lower relaxivity for dialyzed uncapped particles is thus probably due to the loss of DEG molecules on the particle surface leading to a faster rotational time, which is unfavorable for contrast enhancement. The conclusion is that the amounts of DEG and Gd ions in the samples have a major influence on the material, and the idea is also supported by reference measurements on pure GdCl3. When dissolved in DEG and diluted in water, GdCl3 ends up in slightly higher relaxivities compared with GdCl3 solely diluted in water (Table 1). Furthermore, the absolute r1 and r2 relaxivity values of these ion samples are generally higher than the relaxivities for the uncapped nanoparticles. A large number of transient binding sites for water increases the possibilities for water exchange and consequently also the relaxivity. The number of coordinated (40) Chow, E.; Wong, E. L. S.; B€ocking, T.; Nguyen, Q. T.; Hibbert, D. B.; Gooding, J. J. Sens. Actuators, B 2005, 111-112, 540–548. (41) B€ocking, T.; James, M.; Coster, H. G. L.; Chilcott, T. C.; Barrow, K. D. Langmuir 2004, 20, 9227–9235. (42) Delamarche, E.; Sundarababu, G.; Biebuyck, H.; Michel, B.; Gerber, C.; Sigrist, H.; Wolf, H.; Ringsdorf, H.; Xanthopoulos, N.; Mathieu, H. J. Langmuir 1996, 12, 1997–2006. (43) Engstr€om, M.; Klasson, A.; Pedersen, H.; Vahlberg, C.; K€all, P.-O.; Uvdal, K. Magn. Reson. Mater. Phys., Biol. Med. 2006, 19, 180–186.

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Figure 10. Experimentally measured longitudinal (A) and transverse (B) relaxation rates and the corresponding calculated regression slopes for filtered DEG-Gd2O3 nanoparticle samples dialyzed for different time lengths: (a) nondialyzed, (b) 3 days dialysis, and (c) 6 days dialysis.

Figure 11. Experimentally measured longitudinal (A) and transverse (B) relaxation rates and the corresponding calculated regression slopes for GMP samples dialyzed for different time lengths: (a) 1 day dialysis, (b) 2 days dialysis, (c) 3 days dialysis, and (d) 6 days dialysis. Table 1. Relaxivities r1 and r2 for Different Dialysis Time Lengths for Gd2O3 Nanoparticle Samples, PEGylated Gd2O3 Samples (GMP), and Gd3þ Ion Samples Dissolved in Milli-Q or DEG and Diluted in Milli-Q sample

dialysis time

r1

r2

r2/r1

Gd2O3 Gd2O3 Gd2O3

0 days 3 days 6 days

9.5 2.7 0.9

10.6 3.7 2.5

1.1 1.4 2.8

GMP GMP GMP GMP

1 day 2 days 3 days 6 days

5.7 7.8 10 22.8

6.4 9 12.9 31.2

1.1 1.2 1.3 1.4

13.8 11.7

16.1 14.5

1.2 1.2

GdCl3 (DEG/MQ) GdCl3 (MQ/MQ)

water molecules, q, for Gd3þ is 8 or 9,1 which is a high value causing relatively high relaxivites for ion samples. Chelating Gd3þ with DTPA enhances the rotational conditions, but it also reduces the number of binding sites for water to q = 1. On the contrary to the uncapped samples, MRI measurements on surface modified Gd2O3 nanoparticles show that the relaxivity 5760 DOI: 10.1021/la903566y

Figure 12. T1-weighed MRI intensity images for PEGylated nanoparticles (GMP) dialyzed for 6 days ordered with decreasing concentration (0.39, 0.19, 0.10, 0.05, 0.02, 0.01 mM) from left to right. The images were acquired using a spin echo sequence with TE = 20 ms and TR = 1000 ms.

is increasing as a function of dialysis time (Figure 11). The excess of free capping agents from the surface modification reaction, which possibly block water molecules from coming close to the nanoparticles and/or deteriorate the rotational dynamics in the sample, is removed during dialysis.5 Prolonged dialysis results in less free molecules and better water accessibility, and after an extended dialysis the samples mainly contain well functionalized particles and a very high relaxivity can be achieved. For extensively dialyzed (6 days) GMP samples, the overall highest longitudinal relaxivity was obtained (r1=22.8 s-1 mM-1). This value is remarkably high, more than four times higher than r1 for the Gd-DTPA chelate. The r2/r1 ratio for functionalized Langmuir 2010, 26(8), 5753–5762

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Figure 13. Fluorescence microscopy images of neutrophils stained for F-actin exposed to (A) buffer (negative control cells), (B) Gd2O3 nanoparticles (0.11 mM Gd), (C) PEGylated Gd2O3 nanoparticles (GMP, 0.19 mM Gd), and (D) 0.1 μM PMA, positive control cells. Bars indicate 20 μm.

samples increases slightly for samples dialyzed for a long time but is still close to one; that is, positive contrast is achieved. Thus, PEG capping seems to introduce properties well suited for positive MRI contrast. The results of the relaxivity measurements indicate that the dialysis markedly has a great impact on the purity of the final sample and that still after days of dialysis free capping precursors can be removed. The results also show that surface modification of Gd2O3 nanoparticles enhances not only the stability and biocompatibility but also the relaxivity of the material. Microscopy Study of Neutrophil Granulocyte Activation. In order to evaluate the Gd2O3 nanoparticle induced inflammatory response, we carried out morphological examination of neutrophil granulocytes upon their exposure to Gd2O3 and functionalized Gd2O3 nanoparticles (GMP). The results are presented in Figure 13. Neutrophil granulocytes, or neutrophils, are white blood cells forming an integral part of the immune system. Neutrophils react rapidly to tissue injury and are the hallmark of acute inflammation. The actin cytoskeleton of neutrophils is very dynamic and may quickly be rearranged to facilitate movement toward and phagocytosis of invading bacteria. Unstimulated neutrophils show a round morphology, whereas they upon activation form pseudopodia (extensions), spread, and become polarized.44,45 By staining the filamentous actin (F-actin), we clearly visualize the morphology of the neutrophils, and the activation state of the cells can be studied. As shown in Figure 13, a clear change in cell morphology, indicating activation, is observed for neutrophils exposed to Gd2O3 nanoparticles (0.11 mM Gd) (B) compared to negative control cells (A). No such extensive activation is observed when neutrophils are exposed to PEGylated Gd2O3 nanoparticles (0.19 mM Gd), as shown in (C), in spite of a higher Gd concentration. This shows that PEGylation of the nanoparticles enhances the cell compatibility for neutrophils, which are active players in our immune system. Complete studies on the effect of these nanoparticles on other physiological relevant properties of neutrophils are currently under investigation (manuscript in preparation). Fluorophore Tagged Nanoparticles. PEG functionality was studied by coupling Rhodamine to the NHS part of the PEG (44) Sanchez-Madrid, F.; Angel del Pozo, M. EMBO J. 1999, 18, 501–511. (45) Howard, T. H.; Oresajo, C. O. J. Cell Biol. 1985, 101, 1078–1085.

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Figure 14. Absorbance spectra of PEGylated nanoparticles (GMP), PEGylated nanoparticles successfully coupled to Rhodamine, and a reference spectrum for Rhodamine in water dialyzed for 2 days against water. Photos in the upper right corner show the corresponding samples taken in daylight and when irradiated with UV light.

molecule. Rhodamine was added together with MPTS and PEG in the Gd2O3 nanoparticle functionalization process. The absorbance results are shown in Figure 14 and include spectra for PEGylated nanoparticles (GMP), PEGylated nanoparticles equipped with Rhodamine, and pure Rodamine sample after dialysis, as dialysis reference. The latter spectrum proves that nonbonded Rhodamine can easily be removed by dialysis. In the upper right corner of Figure 14, two photos are inset showing cuvettes with PEGylated nanoparticles and PEGylated nanoparticles tagged with Rhodamine taken both in daylight and when irradiated with a UV lamp. The cuvette with PEGylated nanoparticles tagged with Rhodamine is clearly colored in daylight and fluorescent in UV while the solely PEgylated nanoparticles are colorless. These results clearly show that the Rhodamine fluorophore has coupled to the NHS part of the PEG and that the bifunctional PEG construction works. These results also indicate that the particles can be further tagged with diverse targeting molecules depending on the purpose and are thus presented as a proof of concept, showing that our PEGylated nanoparticles are available for further coupling chemistry. Preliminary relaxation measurements on PEGylated Gd2O3 nanoparticles further functionalized with Rhodamine indicate that the longitudinal and transverse relaxivities are increased with approximately 10% upon Rhodamine functionalization. This change is considered to be tag specific, and other tags could probably have a more enhanced beneficial effect on the relaxivity.

Conclusions This study clearly shows that functionalized Gd2O3 nanoparticles, synthesized by the polyol method, have great potential as a future contrast agent in magnetic resonance imaging. An enhanced stability to salt exposure and a prolonged shelf life compared to freshly dialyzed nanoparticle samples were obtained by using silanes as the innermost stabilizer layer and an outer layer of bifunctional PEG. For nonfunctionalized samples, the r1 relaxivity decreased as a function of dialysis time. The surface modified nanoparticle samples showed considerably increased relaxation rates compared to uncapped samples, especially after extensive dialysis to DOI: 10.1021/la903566y

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remove free capping agents. In general, dialysis appeared to have a great effect on the relaxivity; the longer the dialysis of functionalized nanoparticles, the higher r1 and r2. A more extensive activation of neutrophil granulocytes was observed when exposed to Gd2O3 nanoparticles compared to PEGylated Gd2O3 nanoparticles. In this work, the nanoparticles are equipped with Rhodamine, turning the nanomaterial into a flouroscent probe. This is done to prove that a tag, in this case a fluorescent dye, can be coupled to our PEGylated nanoparticles, and the results verify

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that the functionalized nanomaterial is prepared for targeting purposes. Acknowledgment. The present work is financed by the Swedish Foundation for Strategic Research (SSF) within the Nano-X programme/NanoSense Grant No. SSF[A3 05:204], the Centre in Nano science and technology at LiTH (CeNano), and Center for Medical Image Science and Visualization (CMIV) at Link€ oping University.

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