Synthesis and Characterization of Tb3+-Doped Gd2O3 Nanocrystals

Apr 8, 2009 - Engström et. al8 reported that ultrasmall particles Gd2O3. (USPGO) of size 3-10 nm have doubled relaxivity compared to Gd-DTPA chelates...
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J. Phys. Chem. C 2009, 113, 6913–6920

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ARTICLES Synthesis and Characterization of Tb3+-Doped Gd2O3 Nanocrystals: A Bifunctional Material with Combined Fluorescent Labeling and MRI Contrast Agent Properties Rodrigo M. Petoral, Jr.,† Fredrik So¨derlind,‡ Anna Klasson,|,⊥ Anke Suska,§ Marc A. Fortin,† Natalia Abrikossova,† Linne´a Selegård,† Per-Olov Ka¨ll,‡ Maria Engstro¨m,|,⊥ and Kajsa Uvdal*,† DiVisions of Molecular Surface Physics and Nanoscience, Chemistry, and Applied Physics, Department of Physics, Chemistry and Biology (IFM), Linko¨ping UniVersity, SE-581 83 Linko¨ping, Sweden, Center for Medical Image Science and Visualization (CMIV), Linko¨ping UniVersity, SE-581 85 Linko¨ping, Sweden, and Department of Medical and Health Sciences (IMH)/Radiology, Linko¨ping UniVersity, SE-581 85 Linko¨ping, Sweden ReceiVed: NoVember 28, 2007; ReVised Manuscript ReceiVed: February 15, 2009

Ultrasmall gadolinium oxide nanoparticles doped with terbium ions were synthesized by the polyol route and characterized as a potentially bifunctional material with both fluorescent and magnetic contrast agent properties. The structural, optical, and magnetic properties of the organic-acid-capped and PEGylated Gd2O3:Tb3+ nanocrystals were studied by HR-TEM, XPS, EDX, IR, PL, and SQUID. The luminescent/fluorescent property of the particles is attributable to the Tb3+ ion located on the crystal lattice of the Gd2O3 host. The paramagnetic behavior of the particles is discussed. Pilot studies investigating the capability of the nanoparticles for fluorescent labeling of living cells and as a MRI contrast agent were also performed. Cells of two cell lines (THP-1 cells and fibroblasts) were incubated with the particles, and intracellular particle distribution was visualized by confocal microscopy. The MRI relaxivity of the PEGylated nanoparticles in water at low Gd concentration was assessed showing a higher T1 relaxation rate compared to conventional Gd-DTPA chelates and comparable to that of undoped Gd2O3 nanoparticles. 1. Introduction Molecular imaging with the aid of targeted contrast agents (e.g., fluorescent probes) has been increasingly improving since the past decade in terms of detection limits, imaging modalities, and engineered functionality. Imaging using, e.g., quantum dots (QDs), has overcome limitations of conventional organic dyes such as poor photostability and low quantum yield for specific applications in bioimaging.1 Recently, the development of engineered nanoparticles with multifunctional features has emerged. Remarkable successes of nanoparticle-based biolabels have shown vast potential not only for bioimaging2 but also for diagnostic3 and therapeutic4 purposes. Rare-earth-based nanoparticles5 are promising types of luminescent agents with similar properties as that of QDs. Iondoped lanthanide oxide nanoparticles are highly photostable and exhibit long luminescence lifetimes and narrow emission bands, comparable to QDs. Contrary to QDs, the emission color is not dependent on the size of the particle, but rather on the nature of the lanthanide ions. The use of rare-earth-based nanoparticles is, however, not restricted to fluorescent labeling. Emerging investigations of differently synthesized rare-earth oxide nanoparticles have been explored as potential magnetic resonance * To whom correspondence should be addressed. Phone: +46 (0)13 28 1208. Fax: +46 (0)13 28 8969. Email: [email protected]. † Division of Molecular Surface Physics and Nanoscience. ‡ Division of Chemistry. § Division of Applied Physics. | Center for Medical Image Science and Visualization (CMIV). ⊥ Department of Medical and Health Sciences (IMH)/Radiology.

imaging (MRI) contrast agents. One of the most popular nanoparticulate contrast agents is superparamagnetic iron oxide (SPIO) particles.6 Contrast in MRI is governed by longitudinal (T1) and transverse (T2) relaxation times of water protons. Relaxivity, ri, is the proportionality constant between 1/Ti (i ) 1, 2) and the concentration of the contrast agent. Enhancement of the contrast in MR images is dependent on the relaxivity of the agents. Negative contrast agents like SPIOs (e.g., Resovist) have high transverse relaxivity (r2), while Gd-based contrast agents (e.g., Magnevist, a Gd-DTPA chelate) are considered positive contrast agents possessing high longitudinal relaxivity (r1). To our knowledge, there is no nanoparticulate positive contrast agent that is commercially available so far. Nanoparticles based on rare-earth oxides, e.g., Gd2O3, have been recently studied as potential MRI contrast agents. Small nanoparticles Gd2O3 (SPGO) of size 20-40 nm were investigated by McDonald and Watkin,7 who showed that the relaxivity of the particles is comparable to that of Gd-DTPA chelates. Engstro¨m et. al8 reported that ultrasmall particles Gd2O3 (USPGO) of size 3-10 nm have doubled relaxivity compared to Gd-DTPA chelates. We also currently communicated a poly(ethylene glycol) (PEG)-stabilized USPGO having similar relaxivity values.9 Furthermore, by combining the magnetic property of the nanoparticle matrix (Gd2O3) and the fluorescent property of the doping ions (Tb3+), multifunctional nanoparticles can be tailored. In this study, we report the synthesis and characterization of the core of the rare-earth oxide Gd2O3 nanoparticles doped with Tb3+ ions, capped with a set of organic acids. The particles are

10.1021/jp808708m CCC: $40.75  2009 American Chemical Society Published on Web 04/08/2009

6914 J. Phys. Chem. C, Vol. 113, No. 17, 2009 in a second step PEG functionalized. The structural, optical, and magnetic properties of the core as well as capping of Tb3+ Gd2O3 nanoparticles are investigated using TEM, XPS, IR, photoluminescence, magnetic measurements, and confocal laser scanning microscopy (CLSM) analysis. Detailed information is obtained regarding the core and capping properties as chemical state, composition of the core, binding strength of the capping, and surface states of the particles. This is of main importance for development of nanoparticles to be used for MR contrast enhancement. In the second step PEG-containing molecules were coupled to the organic-acid-capped particles, as a polymer shield, in order to obtain a material more robust to the surrounding physioloical environment, which is a prerequisite for an optimal MRI contrast agent. Since a polymer coating of the particle is expected to strongly affect the relaxation properties, the MR data collected are all obtained for PEG-coated particles. 2. Experimental Section Materials. The following materials were used and purchased from Sigma-Aldrich: GdCl3 · 6H2O, TbCl3 · 6H2O, diethylene glycol (DEG), NaOH, citric acid monohydrate (CA), dinicotinic acid (DA), dimercaptosuccinic acid (DMSA), 1-ethyl-3-[3dimethylaminopropyl]carbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS). PEGylated molecules such as poly(ethylene glycol)-R-maleimide-ω j - NHS ester (NHS-PEGMal; 5000 Da) and methoxy poly(ethylene glycol) silane (PEGsilane, 5000 Da) were purchased from NEKTAR, Alabama, while R-mercapto-ω j -amino poly(ethylene glycol) hydrochloride (HS-PEG-NH2*HCl, 5000 Da) was purchased from Iris Biotech GmbH, Germany. All chemicals used were as-received of synthesis grade or higher (g98%). Nanocrystal Synthesis and Functionalization. Terbiumdoped gadolinium oxide nanoparticles were synthesized by a slightly modified version of the “polyol” method developed by Bazzi et al.5b For the 5% Tb-doped Gd2O3 nanocrystals, 5.7 mmol GdCl3 · 6H2O and 0.3 mmol TbCl3 · 6H2O were dissolved in 30 mL of DEG, constantly stirred, and heated in a silicon oil bath at 140-160 °C for 1 h. Then, 7.5 mmol NaOH dissolved in 30 mL of DEG was added. After complete dissolution of the reactants, the solution was refluxed at 180 °C for 4 h under vigorous stirring. For comparison the synthesis of 20% Tb-doped Gd2O3 was also synthesized, using the same method as above (but adding 1.1 mmol of TbCl3 · 6H2O). To obtain a powdered form of the product, exchange of the capping molecule was done using organic acids.10 The as-synthesized suspension was first centrifuged-filtered with a 0.22 µm membrane (poly(ether sulfone), Vivaspin) for 30 min at 25 °C until complete collection of the fluid. This step was done to remove any large sized agglomeration of the particles. Exchange of the capping DEG layer, to obtain the product in powdered form, was done using organic acids.10 The filtered suspension was heated to 140-160 °C under stirring, and 0.5 mmol NaOH together with 0.5 mmol of the above-mentioned organic acid dissolved in a small amount of DEG (∼2 mL) were added. The solution was then refluxed at 180 °C for 30 min under strong stirring, yielding a “dirty” whitish (for CA- and DA-capped) or dark brownish (for DMSAcapped) dispersion/precipitate. After washing and centrifuging in methanol for several times and subsequent drying in air, a powdered form of the nanoparticles was collected. Further functionalization of the nanocrystals with PEGcontaining molecules was achieved by grafting the CA- and DMSA-capped particles with HS-PEG-NH2*HCl and NHSPEG-Mal molecules, respectively. PEGylation was performed

Petoral et al. by first dissolving the 5Tb:Gd-CA (or 5Tb:Gd-DMSA) powdered samples with an ample amount of NaOH (or HCl) followed by ultrasonication of the dispersion. The pH of the CA-capped (or DMSA-capped) dispersion was adjusted by first adding a 2-(N-morpholino)ethanesulfonic acid (MES) buffer and then followed by HCl (or NaOH) until pH 6-7 was achieved. EDC/NHS in MES solution was added to the CA-capped particle suspension. Half an hour sonication and in-between vortexing was done to completely react the EDC/NHS with the CA-capped particles. Addition of SH-PEG-NH2 was followed by vortexing. For the DMSA-capped particle suspension, Mal-PEG-NHS was directly reacted followed by vortexing. Both reactions were performed in a 2 mL Eppendorf tube overnight by placing the tubes on a mixer. The suspensions were then dialyzed using a 10 000 molecular weight cutoff (MWCO) membrane (SpectraPor 6, Spectrum Laboratories) against water for >70 h. After collection of the dialyzed suspensions, ultracentrifugation (12 000 rpm, 15 min) was done to separate the precipitate, which contained aggregated particles and polymerized molecules. The supernatant was then collected and immediately subjected to MRI studies. Another PEG molecule called PEG-silane was also directly grafted onto the DEG-capped particles. Direct functionalization with PEG-silane of the as-synthesized nanoparticle was carried out according to ref 9. Cell Preparation. THP-1 cell, which is a monocytic cell line originally from a one-year-old boy with acute monocytic leukemia, was cultured in a cell culture flask in 20-30 mL of RPMI 1640 medium (GIBCO, Invitrogen) with 10% fetal calf serum (FCS), 2 mM L-glutamine, 50 µg/mL streptomycin, and 50 units/mL penicillin. The cells were kept at 37 °C and with 5% CO2. This cell type can be differentiated to macrophagelike cells. This was done by incubating the cells with 200 nM phorbol 12-myristate 13-acetate (PMA) for 72 h. The differentiated cells had become adherent and had to be loosened from the culture flasks using a cell scraper prior to the experiment. Loosened cells were incubated for 2 h with 1.0 mM 5%Tb: Gd2O3 nanoparticle (5Tb:Gd-DEG) capped with citric acid, to yield a ratio of 1 million cells/sample. Two hours incubation of cells with 0.25 mL of 5Tb:Gd-DEG (95 mM Gd) nanoparticles in 2.75 mL of cell culture medium, with one million cells per sample, was performed,. After incubation the cells were washed twice in cell culture medium (RPMI 1640) and twice in phosphate-buffered saline (PBS, 1X solution). In each washing step, the cells were centrifuged for 8 min at 1050 rpm. After washing, the cell pellets were resuspended in PBS, at 1 million cells/mL. The cells were then mounted on an adhesion slide (Marienfel Laboratory Glassware), with 20 µL of the suspension in each well, and fixated with 4% PFA. The cells were also treated with antifade reagent prior to microscopy examination. Xenopus laeVis dermal fibroblast cells were also used in the experiment. Cells of an immortal cell line were cultured and maintained as previously described.11 The fibroblasts were detached from culture flasks with 0.05% trypsin and 0.53 mM EDTA in 70% PBS, centrifuged at 500 RFC for 5 min, and resuspended in culture media at a concentration of 100 cells/ µL. Incubation of 40 µL of nanoparticle suspension of 5%Tb: Gd2O3 capped with CA (5Tb:Gd-CA, at pH 6-7 with concentration of about 22 mM Gd) with 40 µL of cell media containing fibroblast cells was done for 12 h. The cell-particle mixture was spread on poly-L-lysin-coated glass slides and incubated for 12 h at 27 °C. After incubation, the cells were washed with

Tb3+-Doped Gd2O3 Nanocrystals PBS, fixed 15 min at room temperature in 4% paraformaldehyde in PBS, and mounted in Vectashield (Vector laboratories). Instrumentation. High-resolution transmission electron microscopy (HRTEM) studies were carried out with a Philips CM20 electron microscope, operated at 200 kV. X-ray photoelectron spectroscopy (XPS) analysis was performed in a VG instrument equipped with CLAM2 analyzer using unmonochromatized Al KR photons (1486.6 eV). Energy dispersive X-ray (EDX) measurements were executed using an Oxford LINK ISIS system with a Ge detector, which is linked to a scanning electron microscope (SEM) system (LEO Gemini 1550 FEG). Infrared (IR) spectra were measured on a Bruker IFS48 FTIR spectrometer in transmission mode, under vacuum condition, using pressed KBr pellets. The optical properties were characterized by photoluminescence (PL) measurements with the 266 nm line of an Ar-ion laser as excitation source. The PL signals were analyzed with a double-grating monochromator, together with a GaAs photomoultiplier, using standard lock-in techniques. Magnetic characterization of the nanocrystals (in powder form) was performed with a Quantum Design model MPMS magnetic property system (SQUID magnetometer) using the extraction method with a sensitivity of 1 × 10-4 emu for DC measurements. The temperature dependence of magnetization was measured in an applied magnetic field of 100 Oe between 5 and 300 K, using zero-field-cooling (ZFC) and field-cooling (FC) procedures. Magnetization measurements were performed in a magnetic field range of (-50 000)-(50 000) Oe. Fluorescence analysis in water-based solutions was carried out using a Jobin-Yvon spexFluoroMax-2 apparatus. The excitation wavelength was set to 350 nm, and the emission spectra were recorded from 450 to 650 nm. Optical images of the fibroblasts and THP-1 cells were obtained using confocal laser scanning microscopy (CLSM) with a Zeiss LSM5 confocal microscope (Zeiss, Oberkochen, Germany) and a Plan-Apochromat 63×/ 1.4 Oil DIC objective (Zeiss, Oberkochen, Germany). The 5Tb: Gd-CA-particles were excited with the 488 nm wavelengh argon laser and detected using a LP 505 filter; optical slices taken were 0.6 µm thick. Cells treated with particles and control cells without particle exposure were mounted on the same slide and imaged using the same settings. Two central acquisitions were merged (Divisor 2) using Paint Shop Pro Photo X2 (Corel, Ottawa, Canada). Proton relaxation times were measured with a 1.5 T Philips Achieva whole body scanner using the head coil. A 2D mixed spin-echo (SE) sequence interleaved with an inversion recovery (IR) sequence was used for the relaxivity measurements.12 Imaging time parameters and other MR settings are reported in earlier published studies of similar nanoparticles..8,9 The measurements were performed in water at room temperature (21-23 °C). An inductively coupled plasma sector field mass spectroscopy (ICP-SFMS) instrument was used to measure the Gd concentration of the samples subjected for MRI, performed at Analytica AB (Sweden). 3. Results and Discussions Structural, Optical, and Magnetic Properties of Nanoparticles. HRTEM was used to examine the average size, shape, and crystallinity of the synthesized nanoparticles. The Tb-doped Gd2O3 nanoparticles are approximately of a spherical, faceted shape (Figure 1a). A bar graph of the particle size distribution, based on HRTEM measurements performed on >100 particles, is shown in Figure 1b. The average particle size was estimated to be 4.3 nm and with similar size distribution for both the 5% and 20% Tb-doped particles. As shown in Figure 1c, the particles appear as a regular crystalline lattice, exhibiting the

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Figure 1. TEM micrographs and particle size distribution of Gd2O3 doped with 5% Tb3+ (5Tb:Gd) or 20% Tb3+ (20Tb:Gd). (a) TEM image of as-synthesized DEG-capped 5Tb:Gd (5Tb:Gd-DEG). (b) Size distribution based on TEM images of 5Tb:Gd-DEG and citric-acidcapped 20Tb:Gd (20Tb:Gd-CA). (Note: The frequency unit represents the total fraction of the particle sample in a given diameter range, and the sum of the columns of each type is equal to 1.) (c) As-synthesized nanocrystals revealing the (222) lattice planes of Tb-doped cubic Gd2O3.

(222) planes with the interplanar distance d ≈ 3.1 Å, in good agreement with that for cubic phase Gd2O3.5b The chemical composition of the nanoparticle samples was analyzed with XPS and EDX. Results from the two techniques indicated a 0.055 ( 0.004 and 0.226 ( 0.031 Tb to Gd atom ratio for the 5%Tb- and 20%Tb-doped particles, respectively, with the estimated errors e15% based on several measurements. ICP-MS analysis of the 5% Tb-doped Gd2O3-PEGylated nanoparticle suspension yielded a Tb:Gd ratio of 0.054 ( 0.001. XPS analysis corroborated the presence of gadolinium oxide based on the line shape and the binding energy peak positions of the Gd (3d) peaks, consistent with earlier published data on Gd2O3 powder pressed into an In sheet.13 The Tb (3d5/2) binding energy peak position is found at 1242 eV (Figure 2a), which differs slightly from that earlier published for pure Tb2O3 where the Tb 3d5/2 peak is reported at about 1241 eV.14 This supports the notion of Tb substituting for Gd in the Gd2O3 lattice and that terbium has entered into the gadolinium oxide lattice as an extrinsic dopant. Whether some of the Tb also might be present as terbium oxide(s), in particular for the 20% doped samples, cannot be determined from the data present. The presence of an organic (capping) surface layer of citric or dinicotinic acid on nanoparticles after dialysis and separation was evidenced by both XPS and IR analysis. The as-synthesized particles capped with DEG capping exhibit a core level C (1s) main peak around 286.5 eV, assignable to the ethylene groups

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Figure 2. Core-level XPS and IR spectra of various organic-capped Tb:Gd2O3 nanoparticles. (a) Representative Tb (3d) and Gd (3d) XP spectrum of as-synthesized Tb-doped Gd2O3 nanoparticles. (b) C (1s) XP spectra of differently capped particles showing the replacement of DEG with organic acids such as citric acid (CA) and dinicotinic acid (DA). The bottom spectrum shows the dimercaptosuccinic acid (DMSA)-capped particles grafted with Mal-PEG-NHS molecule with main peak assigned to the grafted PEG functionality. (c) Transmission IR spectra of reference organic capping molecules compared to the capped nanoparticles.

of DEG, while the peak is absent, or very weak, in the powdered CA- and DA-capped samples. Instead, a peak around 289 eV is observed corresponding to the carboxylic acid groups of the organic acid capping (see Figure 2b). A similar C (1s) spectrum was also observed for the DMSA-capped particles (not shown). The peak at about 290 eV, more evident on the CA-capped particles, is related to carboxylate/ester groups likely to be formed as a byproduct during synthesis. XPS also reveals the PEGylation of the powdered particles. The PEG functionality is verified by the presence of the dominant peak about 286.5 eV in the C 1s spectra of the PEGylated DMSA-capped particle. IR spectroscopy was used to further verify the presence and properties of the capping molecules. IR spectra for pure CA, DA, and DMSA molecules show distinct sharp peaks between 1800 and 1650 cm-1 that are assigned to the free and hydrogenbonded CdO stretches of the carboxylic acids, while in the IR spectra for the capped particles, these stretching bands are absent. A small peak at about 1728 cm-1 for the CA-capped particles is assigned to carboxylate/ester in consistency with the XPS results. The peak at 1640 cm-1 on the DA-capped particles can be assigned to uncoordinated carboxylates forming dimers in the dicarboxylic acid. Significant IR peaks of the capped

nanoparticles at 1569, 1581, and 1585 cm-1 are assigned to the antisymmetric carboxylate stretches νas (COO-), while the bands between 1420 and 1300 cm-1 are assigned to the symmetric carboxylate stretches νs (COO-). The bands observed between 1620 and 1570 cm-1 for the NA-capped particles may overlap with the ring stretches present for the pyridine ring. The organic acids are suggested to be coordinated or bound to the surface metal cations via the carboxylate group in a monodentate, bidentate (chelating), or bridging fashion, as reported in our previous work.,10a b. The PEGylated nanoparticles were also confirmed by IR (spectra not shown). The emission spectra for the nanocrystalline powders are depicted in Figure 3. The PL spectra, under excitation at 266 nm, display four emission peaks between 460 and 640 nm assigned to the 4f f 4f transitions within the Tb3+ ions, in accordance with Louis et. al’s5a work on Tb-doped Gd2O3 nanoparticles. The spectra exhibit the transition lines 5D4 f 7FJ (J ) 3, 4, 5, 6) of the Tb3+ with the strongest emission for J ) 5 at 544 nm.15 The fine structures present in the highly resolved PL spectrum (Figure 3a) suggest that the Tb3+ ions are located in the crystalline environment of the Gd2O3 nanocrystals. It is also suggested from Figure 3a (inset) that the type of capping molecules used, i.e., citric acid or dinicotinic acid, does not

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Figure 3. (a) Photoluminescence spectra of citric acid (CA)- and dinicotinic acid (DA)-capped Tb-doped Gd2O3 nanoparticles in powder form exhibiting transition lines 5D4 f 7FJ (J ) 3, 4, 5, 6) of the Tb3+ with the strongest emission for J ) 5 at 544 nm. (b) Fluorescence spectra of citric acid (CA)- and dinicotinic acid (DA)-capped Tb:Gd2O3 dissolved in water, showing the same Tb3+ emission peaks observed in powder form.

significantly affect the emission intensities in this region. Fluorescence of nanoparticles suspended/dissolved in water was also measured. Figure 3b shows the fluorescence spectra of assynthesized nanoparticles (5Tb:Gd-DEG) suspended in water and CA-coated particles dissolved in basic aqueous solution. The emission spectra of the colloids excited at 350 nm reflect the four emission peaks typical for Tb3+ luminescence. The differences in background and intensities can probably be attributed to the different coatings of the nanoparticles. The fluorescence intensity of the particle coated with CA is lower compared with the as-synthesized DEG-coated ones. In the former, hydroxides may have formed on the particle surface resulting in partly quenching of the luminescence.16 It is known from earlier work on Ln-doped glasses that OH is a quenching group. Figure 4a illustrates the close similarity in magnetization behavior of the Tb-doped with similarly synthesized pure Gd2O3 nanoparticles measured at 5 K, showing a saturation magnetization in the range 65-70 emu/g. As expected, the magnetic behavior is dominated by the Gd2O3 host and not very much by the Tb3+ ions, the magnetic moment of which is 22% larger than that of Gd3+. The nonhysteric but sigmoidal M-H curves

indicate the paramagnetic behavior of the terbium-doped Gd2O3 nanoparticles.17 From Figure 4b, which shows representative magnetization curves of a Tb-doped Gd2O3 nanoparticle at different temperatures (5-300 K), it can be observed that the curvature and hysteresis of the magnetization progressively disappear above 50 K. The temperature dependence of the magnetization may suggest magnetically isolated paramagnetic particles.18 The superposition of the magnetization versus H/T curves (Figure 4c) taken at different temperatures may also be interpreted as to indicate the presence of noninteracting paramagnetic particles.17 Fluorescence Labeling of Macrophages and Fibroblasts. Pilot studies were performed to investigate the capacity of the Tb-doped nanoparticles for fluorescent labeling of living cells. The nanoparticles were incubated with two different cell lines in culture, namely, THP-1 macrophage-like cells and dermal fibroblast cells from X. laeVis. In order to examine the interaction of the nanoparticles capped with CA, 5Tb:Gd CA, with the cells, CLSM analysis was performed. THP-1 cells were incubated for 2 h with a particlesolution containing 1 mM Gd (atomic concentration) (Figure 5a) and fibroblasts for 12 h with a 5Tb:Gd-CA particles solution

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Figure 5. CLSM analysis of (a) THP-1 cells incubated for 2 h with 1 mM citric acid-capped 5%Tb:Gd2O3 (5Tb:Gd-CA) particles, (b) THP01 negative control cells, (c) X. laeVis fibroblasts incubated for 12 h with 10 mM 5Tb:Gd-CA particles, (d) and fibroblast negative control cells. Two central 0.6 µm thick optical slices have been merged.

Figure 4. Magnetization curves for the citric acid (CA)- and dinicotinic acid (DA)-capped Tb:Gd2O3 nanoparticles. (a) Comparable nonhysteretic and sigmoidal M-H curves of the Tb-doped (Tb:Gd-CA and Tb:Gd-DA) and pure (Gd-CA) Gd2O3 nanoaparticles indicating similar paramagnetic characteristics. (b) Representative M-H curve of Tb: Gd2O3 showing the temperature dependence of magnetization. (c) Superimposition of magnetization curves of Tb:Gd2O3 particles at indicated temperatures.

containing 10 mM Gd ions (Figure 5b). Due to the preliminary nature of this study, a direct comparison of the two cell line cannot be made. Nevertheless, in both cell lines which were treated with the nanoparticles, green fluorescence signals could be found contrasting to the dim autofluorescence of the negative controls (cells without the addition of particles). Recent literature

indicated that the uptake of nanoparticles into living cells is size dependent19,20 with a size of about 50 nm showing optimal uptake. The presented results can show that gadolinium oxide nanoparticles doped with terbium ions with a size of 4.3 ( 1 nm can be taken up by living cells and that the intracellular concentration of these particles allows fluorescent labeling. The fluorescent signals were uniformly distributed in the cytoplasm; however, especially in the fibroblasts, the particles seem to accumulate in the perinuclear region, a feature also found with other nanoparticle-loaded cells.21 Heterogeneously distributed intensely fluorescent spots, a feature common to cells loaded with fluorescent nanoparticles,22,23 could not be observed in the present study. These fluorescent dots are attributed to collections of particles in intracellular vesicles. Since we do not observe these characteristic dots, we assume that in our study the 5Tb: Gd-CA particles containing vesicles do not contain large enough numbers of fluorescent particles to be seen individually. For the determination of the exact particle distribution in the cells, transmission electron microscopy analysis should be performed. The 5Tb:Gd CA loaded fibroblasts seem smaller than the control cells (Figure 5c versus d), which might be due to a cytotoxic effect of the particles, the presence of toxic Gd3+ ions, or changes of the ionic strength in the culture media. Indeed, the higher the dilution factor of the medium with the nanoparticle solution (to increase the concentration of nanoparticles), the greater cytotoxic effects could be observed (unpublished data). In THP-1 no such size difference is observed. In this macrophage-like cell type, cytotoxic effects might not be essential due to the nature of this cell line. It should also be noted that a smaller concentration of 5Tb:Gd CA nanoparticles has been used, contributing to the improved fitness of the THP-1 cells. Systematic cytotoxicity studies are scheduled. MRI Relaxation Studies. To investigate the potential of the Tb-containing particles as MRI contrast agents, proton relaxivity measurements were performed. PEGylated Tb-doped Gd2O3 nanoparticles were subjected to MRI relaxivity studies in water

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J. Phys. Chem. C, Vol. 113, No. 17, 2009 6919 cent properties of the particles originate principally from the Tb3+ dopant, which exhibits a strong emission peak at ∼544 nm. Similar emission spectra were observed for colloidal dispersion of the particles using fluorescent spectroctroscopy. The bifunctional characters of the nanoparticles, i.e., its luminescent and paramagnetic properties, were explored. We have demonstrated the potential of the Tb3+:Gd2O3 nanoparticles to be used both as fluorescent contrast agents in imaging fibroblast cells and macrophages and as MRI contrast agents. CLSM images indicate that citric-acid-capped 5%Tb:Gd2O3 particles are taken up by two different cell types, THP-1 cells and fibroblasts, showing fluorescent signals distributed throughout the cytoplasm. MRI relaxivity measurement studies of Tbdoped particles showed doubled relaxivity compared with GdDTPA. This high T1 relaxation rate at low Gd concentration is comparable to that of very small pure Gd2O3 nanoparticles we have studied earlier.8,9 The specific (bio)functionalization of the synthesized nanoparticles for specific targeting in different cell lines is in progress. Studies extended for in vivo live animal systems will also be addressed.

Figure 6. MRI relaxation rate (1/T1) and images for PEGylated 5%Tb: Gd2O3 nanoparticles in water. (a) T1 relaxation rate of PEGylated Tbdoped particles compared to PEGylated undoped Gd2O3 (Gd-PEG) nanoparticles and Gd-DTPA. (Note: Broken lines represent extrapolated fit.) (b) T1-weighted spin-echo MR image of various concentrations of PEGylated DMSA- and CA-capped Tb-doped Gd2O3 nanoparticles in water. The concentrations correspond to the values in (a).

Acknowledgment. This work was supported by the Swedish Research Council, Carl Tryggers Foundation, Materials in Medicine, and Accelerator I Linko¨ping AB. We thank Dr. Mats Larsson and Prof. Per-Olof Holtz (IFM-Material Science, Linko¨ping University) for their assistance with PL measurements. Help from Dr. Peter Åsberg (IFM, Linko¨ping University) in some aspects of acquiring fluorescent images is appreciated. Roselyn Emergo and Prof. Judy Wu (Department of Physics, Kansas University) are gratefully acknowledged for the SQUID magnetic measurements. References and Notes

media and compared to undoped Gd2O3 nanoparticles and GdDTPA chelate. Relaxivity (r1) was calculated from the slope of the plot of the relaxation rate (1/T1) as a function of Gd concentration, shown in Figure 6a. The relaxivity values of the 5%Tb-doped Gd2O3 nanoparticles stabilized with PEG-silane, Mal-PEG-NHS (DMSA-capped), and SH-PEG-NH2 (CAcapped) were 12.0 ( 0.5, 14.2 ( 0.5, and 7.8 ( 0.9 mM-1 s-1, respectively. The above-mentioned relaxivity values are comparable to that of recently reported PEG-stabilized USPGO (9.4 mM-1 s-1)9 and 2-3 times higher than commercially available contrast agent such as Magnevist (4.7 ( 0.1 mM-1 s-1).8 In our experiment, the particles initially capped with DMSA or CA were run at lower concentration, as compared to PEG-silane capped particles, due to the multiple step process involved in the functionalization resulting in the lowering of Gd concentration. The MR images (T1-weighted) of solutions of various concentrations of PEG-capped nanoparticles are shown in Figure 6b. The images become brighter with higher concentration, which is analogous to when Gd-DTPA chelates are used. 4. Conclusions In summary, we have synthesized and functionalized by wet chemistry ultrasmall Gd2O3 nanoparticles doped with terbium. TEM micrographs showed dispersed, ∼4 nm sized, and crystalline particles. The oxidation state and dopant level of the Tb3+ ion were verified by XPS and EDX for the powdered sample. The polyol-route-synthesized nanocrystals, which were subsequently capped with organic acids and stabilized with PEGylated molecules, were confirmed by XPS and IR. The photolumines-

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