Bioconjugate Chem. 2007, 18, 1053−1063
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Use of Lanthanide-Grafted Inorganic Nanoparticles as Effective Contrast Agents for Cellular Uptake Imaging Pierre Voisin,‡ Emeline Julie Ribot,‡ Sylvain Miraux,‡ Anne-Karine Bouzier-Sore,‡ Jean-Franc¸ ois Lahitte,†, Ve´ronique Bouchaud,‡ Ste´phane Mornet,§ Eric Thiaudie`re,‡ Jean-Michel Franconi,‡ Lydia Raison,† Christine Labruge`re,| and Marie-He´le`ne Delville*,†
‡
Institut de Chimie de la Matie`re Condense´e de Bordeaux, UPR CNRS 9048, Universite´ Bordeaux 1, 87 Avenue du Docteur A. Schweitzer, F-33608 Pessac cedex, France, Re´sonance Magne´tique des Syste`mes Biologiques, UMR CNRS 5536, 146 rue Le´o Saignat, F-33076 Bordeaux cedex France, Imagerie Mole´culaire et Nano-Bio-Technologie, IECB, UMR-CNRS 5471 Avenue des Faculte´s, Universite´ Bordeaux 1, F-33405 Talence, France, and Cecama, 87 Avenue du Docteur A. Schweitzer, F-33608 Pessac cedex, France. Received September 1, 2006; Revised Manuscript Received March 24, 2007
The improvement of commonly used Gd3+-based MRI agents requires the design of new systems with optimized in ViVo efficacy, pharmacokinetic properties, and specificity. To design these contrast agents, two parameters are usually considered: increasing the number of coordinated water molecules or increasing the rotational correlation time by increasing molecular weight and size. This has been achieved by noncovalent or covalent binding of low-molecular weight Gd3+ chelates to macromolecules or polymers. The grafting of these high-spin paramagnetic gadolinium chelates on metal oxide nanoparticles (SiO2, Al2O3) is proposed. This new synthetic strategy presents at least two main advantages: (1) a high T1-relaxivity for MRI with a 275% increase of the MRI signal and (2) the ability of nanoparticles to be internalized in cells. Results indicate that these new contrast agents lead to a huge reconcentration of Gd3+ paramagnetic species inside microglial cells. This reconcentration phenomenon gives rise to high signal-to-noise ratios on MR images of cells after particle internalization, from 1.4 to 3.75, using Al2O3 or SiO2 particles, respectively. The properties of these new particles will be further used to get new insight into gene therapy against glioma, using microglial cells as vehicles to simultaneously transport a suicide gene and contrast agents. Since microglia are chemoattracted to brain tumors, the presence of these new contrast agents inside the cells will lead to a better MRI determination of the in ViVo location, shape, and borders of the tumors. These Gd3+-loaded microglia can therefore provide effective localization of tumors by MRI before applying any therapeutic treatment. The rate of carcinoma remission following a suicide gene strategy is also possible.
INTRODUCTION Numerous strategies have been developed for cellular imaging (1, 2). Noninvasive imaging techniques such as magnetic resonance imaging (MRI) appear suitable since they can operate on soft tissues and cells in real time. When highly paramagnetic contrast agents are used, this technique allows for high contrast as well as spatial resolution (3, 4). Such an approach strongly improves the analysis of images and facilitates their interpretation (5-11). Further developments for specific cellular imaging require the synthesis of new contrast agents, which should be able on one hand to increase contrast and on the other hand, to target cells. Previous studies have demonstrated the utility of using circulating cells of the immunological system like lymphocytes, monocytes, and microglial cells after the uptake of contrast agents with a superparamagnetic core (12, 13). These contrast agents with various and controlled sizes are easily taken up. With their high susceptibility effect, iron oxide nanoparticles can be detected at very low concentration at high magnetic fields. With T2* weighted images, they allow the detection of single cells in Vitro (4). Nevertheless, in this situation, the signal due to the iron oxide nanoparticles can only be a hyposignal, and this appears as a limit in image analysis (12). Moreover, * Corresponding author. E-mail:
[email protected]. † Institut de Chimie de la Matie`re Condense´e de Bordeaux. ‡ Re´sonance Magne´tique des Syste`mes Biologiques. § Imagerie Mole´culaire et Nano-Bio-Technologie. | Cecama.
the precise anatomic localization of this iron contrast agent is limited by the breakdown of spatial resolution due to a high local susceptibility effect. Consequently, the detection of a defined cellular population in a localized area in ViVo becomes a major difficulty. Therefore, there is a real need to design MRI contrast agents, which can produce detectable changes in the MR signal intensity of the target tissue or organ by changing their MR relaxation properties (14, 15). Among them, the Gd3+ DTPA chelate was the first FDA approved contrast agent in clinical use (16). These contrast agents require (i) a large number of paramagnetic centers (seven unpaired electrons for Gd3+) selectively bound to the target tissue and (ii) an adapted molecular weight of the MRI agents in order to extend their vascular retention (17, 18) and to slow their tissue clearance (19). Over the past decade, various approaches have been used to address both of these issues such as the conjugation of paramagnetic chelates to macromolecules (20) and linear polymers (21). Recent reports are on polymeric paramagnetic liposomes (22, 23), micelles (24), and dendrimer-based metal chelates (25). These studies have shown that Gd-DTPA (DTPA: diethylenetriaminepentaacetic acid)-based contrast agents of high molecular weight have extended circulation times and can accumulate at specific sites. The liposomes are made by incorporating DTPA-conjugated lipids into liposomes. The conjugated lipids form chelating sites for gadolinium ions. Because of its excellent metal-chelating properties, DTPA is one of the most widely used organic ligands in MRI and positron emission tomography (PET) (26-28). This approach can be
10.1021/bc060269t CCC: $37.00 © 2007 American Chemical Society Published on Web 05/19/2007
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extended to the MR imaging of any receptors once the targeting ligands are conjugated to the lipids. A new class of polymeric metal-loaded nanoparticles with core-shell morphology has been developed and shows a great potential as a contrast agent for medical imaging (29). The Gd3+loaded nanoparticles could reduce relaxation times in Vitro, and provide excellent contrast when used to image the heart and the gastrointestinal tract in rat animal models. However, the utility of these contrast agents might be limited. First, the relaxivity of the entrapped paramagnetic species is lowered because of the limited exchange of bulk water with the contrast agents (30, 31). Indeed, this exchange is dependent on the permeability of the liposome or polymeric membrane to water (29, 32). For this reason, the development of biocompatible nanoparticles with an external shell of high-spin paramagnetic lanthanide contrast agents like gadolinium chelate appears to be advantageous for targeted imaging (33). The gadolinium nucleus generates a hypersignal with a T1-weighted sequence, even at high magnetic field and, consequently, a much easier interpretation of the MR images. However, these low molecular weight gadolinium chelates accumulate in the extra cellular space only where the “blood-brain barrier” breakdown took place. They undergo rapid diffusion through the interstitial as well as renal elimination and therefore have the limitation of providing a timedependent image of tumor margins (34). The chemical grafting of such contrast agents on metal oxide nanoparticles will be an alternative route to change their biodistribution and increase their half-lives, since they are internalized by macrophages. Moreover, they will also exhibit a more pronounced hypersignal with a T1-weighted sequence. Indeed, silica, for example, would be an excellent carrier for the lanthanide chelates, as it is porous enough for water to freely move in and out of the frame. The size of the particles will slow the rotational movement of the chelates and improve the relaxation of water. Additionally, the biocompatible silica particles (35, 36) can be easily derivatized, and targeted contrast agents can be synthesized (37). These nanoparticles could be radio-opaque and thus show X-ray contrast due to the presence of these electron-dense metal ions. Nanosized particles of about 20 nm, are small enough to pass through the body, and above all are able to enter cells since they can be easily internalized (pynocytosis, phagocytosis, or both). In the following experiments, we have modified SiO2 nanoparticles and alumina nanoparticles with an aminosilane to bind Gd3+-DTPA on their surface. Then, in Vitro microglial cells were used to validate the increased MRI contrast, owing to internalized gadolinium particles and not free particles located in the intercellular space. These experiments were performed with the particles linked to tetramethyl rhodamine isothiocyanate (TRITC) as a fluorochrome and analyzed by microscopy and flow cytometry before the MRI characterizations and observations.
EXPERIMENTAL PROCEDURES Characterizations. TEM was performed at room temperature on a JEOL JEM-2000 FX transmission electron microscope using an accelerating voltage of 200 kV. DRIFT characterizations were performed using a Bruker IFS Equinox 55FTIR spectrometer (signal averaging 64 scans at a resolution of 4 cm-1 in KBr pellets containing ca. 2 mass % of material). The zeta potential of the nanoparticles was assessed by using a Zetasizer 3000HSA setup (Malvern Instruments) equipped with a HeNe laser (50 mW, 532 nm). The zeta potential measurement based on laser Doppler interferometry was used to assess the electrophoretic mobility of nanoparticles. Measurements were performed for 20 s using a standard capillary electrophoresis cell. The dielectric constant was set to 80.4 and the Smolu-
Voisin et al.
chowsky constant f(ka) was 1.5. Thermogravimetric analyses (TGA) were performed using a SETARAM apparatus (5 °C min-1 from 30 to 600 °C, air with a 2-h plateau at 170 °C). X-ray photoelectron spectra were recorded with a VG 220i-XL ESCALAB spectrometer. Irradiation was a non-monochromatized Al source operating at 110W (hν ) 1486.6 eV). Acquisition was done at constant pass energy (i.e., 40 eV for high resolution) with a flood gun, and analyzed area was about 150 µm diameter. Spectra were fitted with the AVANTAGE software by THERMO Election. Fluorescence analysis by flow cytometry was performed on a FACScan cytometer (BD BioSciences, Le pont de Claix, France) using Cell Quest Pro software. X-ray photoelectron spectra were obtained using a VG 220iXL Escalab spectrometer, employing non-monochromatized Mg KR source (hν ) 1253.6 eV). The X-ray gun was operated at 10 kV and 20 mA (pressure: 10-8 bar). Powdered samples were pressed onto small indium foils and survey spectra obtained in the constant pass energy mode of 150 eV. The C1s contamination line (BE ) 284.6 eV) was used to calibrate all binding energy values. MR images were acquired on a 4.7 T Biospec system (Bruker, Ettlingen, Germany). The system was equipped with a 6-cm BG6 gradient system capable of 950 mT/m maximum strength. Measurements were performed with a birdcage resonator (35mm diameter and 80-mm length) tuned at 200.3 MHz. Longitudinal relaxation time constants were measured with the inversion recovery technique (48 points, inversion delay from 20 to 5000 ms) and transverse relaxation time using the CPMG pulse sequence (32 echoes) on different nanoparticle concentrations (20.4, 17, 14.57, 12.75, 11.33, 10.2, 6.8, 5.1 µM). 2D images were acquired with a T1-weighted sequence (FLASH sequence, TE/TR ) 2/75 ms, flip angle: 75°, 8 slices, FOV ) 27 × 27 mm, matrix ) 128 × 128, average of 16, total acquisition time ) 2 min 35 s) on microglial cell phantoms before and after internalization of the MRI contrast agent particles. For Al2O3 particles, TE/TR was 1.7/100 ms and the flip angle was 90°. T1-relaxation time was also evaluated on these two samples. The lanthanide content has been measured by inductively coupled plasma mass spectrometer ICP-MS (HP 4500, Agilent Technologies) equipped with a crossflow nebulizer. The instrumental conditions have been optimized with a 10 µg/L solution of lithium, yttrium, and bismuth. Plasma gas flow rate: 15 L/min; auxiliary gas flow rate: 0.8 L/min; nebulizer gas flow rate: 1.1 L/min; power: 1350 W; oxide (CeO/Ce):
