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Biodistribution Study of Nanometric Hybrid Gadolinium Oxide Particles as a Multimodal SPECT/MR/Optical Imaging and Theragnostic Agent David Kryza,*,^,†,‡ Jacqueline Taleb,^,‡,§ Marc Janier,†,‡ Laurence Marmuse,§ Imen Miladi,‡ Pauline Bonazza,‡ Cedric Louis,§ Pascal Perriat,|| Stephane Roux,‡ Olivier Tillement,‡ and Claire Billotey†,‡ †
Hospices Civils de Lyon, Lyon, France LPCML UMR 5620 CNRS, Universite Claude Bernard Lyon 1, Villeurbanne, France § NanoH, Saint-Quentin Fallavier, France UMR 5510 CNRS-INSA de Lyon, Villeurbanne, France
)
‡
ABSTRACT: Nanometric hybrid gadolinium oxide particles (Gado-6SiNP) for diagnostic and therapeutic applications (mean diameter 34 nm) were obtained by encapsulating Gd2O3 cores within a polysiloxane shell, which carries organic fluorophore (Cy 5) and is derivatized by a hydrophilic carboxylic layer. As residency time in the living body and methods of waste elimination are crucial to defining a good nanoparticle candidate and moving forward with steps for validation, this study was aimed at evaluating the biodistribution of these multimodal Gado-6Si-NP in rodents. Gado-6SiNP were imaged following intravenous injection in control Wistar rats and mice using MRI (7 T), optical fluorescent imaging, and SPECT. A clear correlation was observed among MRI, optical imaging, and SPECT regarding the renal elimination. Quantitative biodistribution using gamma-counting of each sampled organ confirmed that these nanoparticles circulated freely in the blood pool and were rapidly cleared by renal excretion without accumulation in liver and RES uptake. These results demonstrate that Gado-6Si-NP display optimal biodistribution properties, enabling them to be developed as multimodal agents for in vivo imaging and theragnostics, especially in oncological applications.
’ INTRODUCTION Explosive growth in the field of personalized medicine and nanotechnology has enabled advances in molecular imaging and therapies.1,2 Numerous strategies are explored in order to apply nanoparticles with multiple functions to clinical use.3 Some nanoparticles have multimodal properties that are interesting for medical imaging,46 while others are developed for therapeutic purposes only79 or for theragnostic use.1012 Whatever the nanoparticle’s composition, the hydrodynamic diameter and the surface of the nanoparticle are important parameters that potentially modify the biodistribution of the nanoparticle within the body. Most available nanoparticles have a hydrodynamic diameter greater than 10 nm. According to several studies,1316 different types of nanoparticles with a hydrodynamic diameter between 3 and 7 nm demonstrate optimal circulation lifetime with efficient clearance and the ability to be filtered through the kidneys. Ideally, these compounds should behave as closely as possible to molecules already used as contrast agents or for therapeutic purposes. Because of surface differences, nanoparticles with a small hydrodynamic diameter behave differently once injected in a living organism. Therefore, it is mandatory that we accurately and quantitatively evaluate the biodistribution of the nanoparticle studied. r 2011 American Chemical Society
Our group has developed a unique nanoparticle platform which is able to be detected regardless of the imaging modality used in humans, and which can be used for therapy as a radiosensitizer, or with a specific labeling as a tool for internal radiotherapy. These nanoparticles are composed of a core of rare-earth atoms (gadolinium), surrounded by a silica shell which can be used to covalently label specific targeting molecules. Because of their composition, no rapid degradation is expected, and it is therefore crucial to understand their behavior in a living organism. On a first move, the biodistribution of the nanoparticle platform should be evaluated and optimized as necessary, prior to developing targeted nanoparticles. A previous study17 has shown that these nanoparticles were filtered by the kidney, and that the nanoparticles were recovered without modification in the urine. Our study was aimed at assessing the exact repartition of the nanoparticles in control rats and mice over an 18-day period by labeling the nanoparticles with Indium 111.
Received: December 27, 2010 Revised: May 5, 2011 Published: May 06, 2011 1145
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’ MATERIALS AND METHODS Chemicals. Gadolinium chloride hexahydrate (GdCl3 3 6H2O, 99.99%) was purchased from Metall Rare Earth Limited. Sodium hydroxide (NaOH, 99.99%), tetraethyl orthosilicate (Si(OC2H5)4, TEOS, 98%), (3-aminopropyl)triethoxysilane (H2N(CH2)3 Si(OC2H5)3, APTES, 99%), Hepes solution (solution in water, pH 7.07.6), sodium chloride (NaCl), triethylamine (TEA, 99.5%), and nitric acid (HNO3, 65%), dimethyl sulfoxide anhydrous ((CH3)2SO, DMSO >99.9%) diethyl ether ((CH3CH2)2O > 99%) were purchased from Aldrich Chemical (France). Cy5 mono-NHS-ester was purchased from Amersham Bioscience. Diethylenetriamine pentaacetic dianhydride (C14H19N3O8, DTPA-ba >98.0%) was purchased from TCI Europe. Ethanol, diethylene glycol (DEG, 99%), and other organic solvents (reagent grade) were purchased from SDS (France) and used as received. For the preparation of an aqueous nanoparticle solution, Milli-Q water (F > 18 MΩ) was used. Ultrafiltration was carried out using Vivaspin 5000 Da purchased from GE Healthcare. Synthesis of NPs. The particles Gado-6Si-NP were provided from Nano-H SAS and prepared as previously described.17 Briefly, gadolinium oxide (Gd2O3) nanoparticles embedded in a polysiloxane shell whose inner part is functionalized by Cy5 and outer part by carboxylic acid were synthesized as follows. In the first step, gadolinium oxide particles were obtained via precipitation at temperatures below 200 C by applying a modified “polyol” synthesis.18 In the second step, controlled polysiloxane shell growth was induced on the gadolinium nano-oxide cores (six silicium/one gadolinium) via hydrolysis/condensation of a mixture of TEOS and APTES. At this stage, Cy5 could be grafted inside the polysiloxane shell at the desired percentage by introducing the reaction product between Cy5 NHS ester with APTES in dry DMSO.19 Finally, because of the presence of amine functions of APTES in the polysiloxane network, a postfunctionalization was carried out using DTPA-ba (via the formation of an amide linkage). Typically, a colloidal solution of hybrid nanoparticles in DEG (50 mL, [Gd] = 37 mM) was added into a suspension of DTPA-ba (4 equiv/Gd) in anhydrous DMSO (60 mL). The reaction mixture was stirred overnight at room temperature. Next, a mixture of acetone and diethyl ether (1:1, v:v) was poured into the reaction. A precipitate was formed, and a purification step was carried out via initial centrifugation at 6000 rpm for 5 min, and redispersion in ethanol and subsequent centrifugation (3-fold, 6000 rpm for 10 min). The nanoparticles were then redispersed in Milli-Q water at a high concentration. The colloidal solution was transferred into a Vivaspin 5000 Da, and ultrafiltration was performed. Finally, a freezedrying protocol of Gado-6Si-NP was carried out. NP Characterization. Direct measurements of particle size distribution were taken using the Zetasizer Nano S (Malvern Instruments, Malvern, U.K.). The hydrodynamic diameter of Gd2O3 core (in DEG) and Gado-6Si-NP (in water, pH = 7.4) was evaluated using photon correlation spectroscopy (PCS). Direct measurements of zeta potential of Gado-6Si-NP were performed using the Zetasizer Nano S (Malvern Instruments, Malvern, U.K.). Prior to the experiment, the solution was diluted in an aqueous solution containing 0.01 M NaCl and adjusted to the desired pH. Determination of the gadolinium content in a sample was performed using inductively coupled plasma-optical emission spectrometer (ICP-OES) analysis with a Varian 710ES. Before measuring Gd concentration, samples of lyophilizat
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were redispersed in 10 mL Milli-Q water, and 1 mL of this colloidal solution was diluted in 14 M HNO3 (1:1, v/v). After 30 min at 80 C, the solution was diluted in HNO3 (5%; 1:25, v/v). HRTEM was applied to obtain detailed structural and morphological information about the architecture of the core/ shell. It was carried out using a JEOL 2010 microscope operating at 200 kV. Samples were prepared by diluting nanoparticle solution and depositing a drop on a copper grid coated by a thin film of amorphous carbon, allowing the liquid to dry in air at room temperature. Determination of the number of DTPA molecules per particle was calculated from the N/Gd ratio obtained by chemical analyses (ICP-OES and energy dispersive X-ray) taking into account the particle size. Radiolabeling with Indium-111. For in vivo and quantitative biodistribution studies, hybrid gadolinium oxide nanoparticles were labeled using Indium-111 radionuclide (stability constant DTPA-In3þ close to 1029). Briefly, 370 MBq of high-purity 111Inchloride (specific activity >185GBq/μg indium) in diluted hydrochloric acid (Covidien, Petten, The Netherlands) was added to 1 mL of a saline solution of hybrid nanoparticles ([Gd] = 100 mM]). Indium-111 links to the DTPA hydrophilic layer of the nanoparticles via coordination bonds. The mixture was incubated for 30 min at room temperature. Gado-6Si-NP111In were separated from 111In-chloride by PD10 column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) as follows. The PD-10 column was first washed with 15 mL of 50 mM citrate buffer (pH = 5). The radiolabeling solution was then loaded on the PD-10 column, and fractions of 0.5 mL were eluted with 50 mM citrate buffer (pH = 5). Gado-6Si-NP111In were first eluted. The radioactivity of each fraction was counted using an ionization chamber (Capintec Radioisotope Calibrator CRC-15, Capintec Inc.). Finally, the four fractions with the highest radioactivity were pooled. The solution was then challenged with 200 μL of the complexing chelating agent diethylenetriaminepentaacetic acid (DTPA; 20 μmol) for 5 min to scavenge potential unbound 111In. Another elution was performed on a PD-10 column using 50 mM citrate buffer (pH = 5) as eluent. The DTPA complex was eluted following Gado6SiNP111In. Radiochemical purity was determined by instant thin layer chromatography (ITLC). ITLC of the purified Gado-6Si-NP111In was performed using silica gel plates (Gelman Science Inc., Ann Arbor, MI, USA) in 50 mM citrate buffer (pH = 5) as the solvent, and a TLC scanner (MiniGita, Raytest, Isotopenmessger€ate, GmbH, Straubenhardt, Germany) in order to determine the percentage of free 111In-chloride. Gado-6Si-NP111In remained at the origin, whereas residual 111In-chloride migrated with an Rf of 0.8. Stability of Gado-6Si-NP111In Nanoparticles. The purified Gado-6Si-NP111In were incubated in rat serum at 37 C and 20 mM DTPA solution (pH = 7.4) for 4 h, 24 h, and 7 days. Radiochemical purity was determined as described previously. Animals. All animal experiments were approved by the local animal ethics committee of University Claude Bernard Lyon 1, according to French legislation, and carried out in line with current guidelines. Adult male Wistar rats (200250 g) and nude Swiss mice (2025 g) were obtained from Charles River Laboratory (L’Arbresle, France). All animals were housed under standard environmental conditions (free access to food and water and a 12/12 h light/dark cycle), and acclimated for at least 48 h before experimentation. For all experiments except scintigraphic and optical dynamic acquisition, a gaseous anesthesia protocol was applied using 1146
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Biodistribution Study. Biodistribution of Gado-6Si-NP111In
Figure 1. (A) HRTEM images of gadolinium oxide core nanoparticles representative of the whole sample. (B) Zeta potential of Gado-6Si-NP (n = 3).
Figure 2. Size distribution determined by photon correlation spectroscopy (PCS) of gadolinium oxide core nanoparticles (a) and Gado-6SiNP (b).
12% isoflurane (Laboratoire Bellamont, Boulogne Billancourt, France) in a mixture of O2/N2 (25/75%) at 0.8 L/min after an induction process. To avoid the absence of renal excretion by isoflurane gaseous anesthesia, scintigraphic and optical dynamic acquisition were performed immediately after chemical anesthesia (Ketamine1000, Virbac, Carros, France) at a dose of 0.15 mg per gram of body weight by IM injection, followed with medetomidine (Domitor, Orion Pharma, Espoo, Finland) at a dose of 0.5 (in rats) to 1 (in mice) μg per gram of body weight by IP. Anesthesia was monitored using a small animal respiratory monitoring system (Sa Instrument, Inc., NY, USA).
was studied in order to evaluate the potential of the multimodal nanohybrid agent. Radiolabeled nanoparticles in HEPES buffer containing 150 mM NaCl were injected intravenously through a caudal vein in rats after anesthesia had been administered. Rats were sacrificed at 2 h, 24 h, 72 h, 10 days, and 18 days postinjection, and organs of interest, such as the heart, lungs, spleen, liver, brain, kidneys, bones, muscles, skin, digestive tract, and cadaver were collected. Urine and feces were also collected each day and weighed and counted for 5 min in an automatic gamma scintillation counter (Wizard gamma counter, Perkin-Elmer, USA). Tissue distribution was expressed as the percentage injected dose per gram tissue (% ID/g) and as the percentage injected dose per organ (%ID/ organ). The injected dose was calculated by adding activity of all organs, cadaver, and excretions (feces and urine). Renal and hepatobiliary eliminations were evaluated by measuring respectively urine and feces activities and expressed as cumulated radioactivity under total injected radioactivity. Blood Circulation Half-Life of Radiolabeled Gado-6Si-NP. After IV injection of 10 MBq of Gado-6Si-NP111In in rats (n = 3), blood samples were removed at suitable time intervals (5 min, 10 min, 20 min, 30 min, 60 min, 120 min, and 240 min). Samples were weighed and counted for 5 min in an automatic gamma scintillation counter. Data points were fit to an exponential decay function to determine blood circulation half-life. Imaging Studies. Dynamic Scintigraphy and SPECT. Acquisitions were obtained using a small animal Nano-SPECT/CT system (Bioscan, Washington, DC, USA). This system consists of four detectors (215 230 mm2 NaI, 33 PMTs) equipped with interchangeable multipinhole apertures. Dynamic and SPECT/ CT acquisitions were performed after IV injection of Gado-6SiNP111In in 17 rats. Double phase (12 10 s images and 116 30 s images) dynamic acquisition was initiated immediately following the IV injection of Gado-6Si-NP111In in rats in supine position placed directly on the planar low-energy ultrahigh-resolution collimator set in order to verify the quality of the injection and analyze early organ nanoparticle uptake. X-ray CT (tube voltage of 55 kVp, exposure time of 500 ms, and 180 projections) and SPECT acquisitions were performed in rats in a supine position, placed in a temperature-controlled bed (Minerve, Esternay, France), in order to maintain body temperature (set to 37 C). Afterward, the SPECT (20 000 cps per projection/20 projections) were acquired using a rat whole-body high-resolution aperture. All image data was reconstructed and analyzed using InVivoScope (Bioscan, Washington, DC, USA). In Vivo and Ex Vivo Optical Imaging. Fluorescence images as well as black and white pictures were acquired via a backthinned CCD-cooled camera (ORCAIIBT-512G, Hamamatsu phonics, Massy, France) using a colored glass long-pass RG 665 filter (Melles Griot, Voisins les Bretonneaux, France), which cuts off all excitation light. Ventral, dorsal, and lateral views were acquired prior to Gado-6Si-NP injection in order to estimate the self-fluorescence level. Immediately after direct IV injection of 50 μL of Gado-6Si-NP in HEPES buffer containing 150 mM NaCl ([Gd] = 100 mM) in two nude Swiss mice under chemical anesthesia, a set of 2 s ventral (in one mouse) or dorsal (in the another mouse) images was acquired over 60 min. Ventral, dorsal, and lateral static views were then obtained using the parameters applied prior to injection. Euthanasia was performed 2 h after injection, and the liver, kidneys, brain, spleen, lungs, heart, skin, muscle, bone, and intestine were 1147
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Table 1. Biodistribution Data of Gado-6Si-NP111In in Wistar Ratsa 2 h (n = 3)
3 days (n = 4)
10 days (n = 3)
18 days (n = 4) 1.18 ( 0.17
21.67 ( 7.45
4.40 ( 1.08
3.68 ( 3.10
1.50 ( 0.54
brain
0.01 ( 0.00
0.00 ( 0.00
0.05 ( 0.03
0.00 ( 0.00
0.00 ( 0.00
digestive tract
3.32 ( 0.79
1.58 ( 0.68
ND ( --
0.10 ( 0.04
0.09 ( 0.03
liver
2.97 ( 0.45
0.95 ( 0.33
1.14 0.41
0.33 ( 0.12
0.27 ( 0.03
heart
0.08 ( 0.01
0.01 ( 0.01
ND ( --
0.00 ( 0.00
0.00 ( 0.00
kidney
35.15 ( 7.77
17.73 ( 1.82
18.60 ( 1.12
6.77 ( 1.21
3.95 ( 0.41
lung
0.31 ( 0 0.04
0.03 ( 0.01
0.10 ( 0.09
0.02 ( 0.01
0.02 ( 0.01
muscles bones
0.12 ( 0.07 0.05 ( 0.03
0.00 ( 0.00 0.01 ( 0.01
ND ( -0.07 ( 0.04
0.00 ( 0.00 0.00 ( 0.00
0.00 ( 0.00 0.00 ( 0.00
skin
0.01 ( 0.01
0.00 ( 0.00
ND ( --
0.00 ( 0.00
0.00 ( 0.00
spleen
0.47 ( 0.23
0.28 ( 0.14
0.05 ( 0.02
0.09 ( 0.01
0.03 ( 0.01
cadaver
a
1 day (n = 3)
Data are expressed as percentage injected dose per gram of tissue (%ID/organ, mean ( standard deviation).
’ RESULTS
Figure 3. Elimination of Gado-6Si-NP111In after IV injection in Wistar rats. Values are expressed as percentage of cumulative activity measured in urine (circle) and feces (triangle) per injected dose (%ID). Data was expressed as mean ( standard deviation.
then collected in order to be imaged and compared with the same ex vivo organs of noninjected mice. In Vivo MR Imaging. Two positioned supine Wistar rats were imaged using a 7-T system (Biospec; Bruker, Ettlingen, Germany) equipped with a 400 mT/m gradient under gaseous anesthesia. Three direction localizers were first used to localize kidneys, liver, and bladder. The imaging protocol consisted of a dynamic series of two coronal slices centered on the kidneys, bladder, and liver, and obtained using a T1-weighed MSME sequence (180 flip angle, 118/10.6 [repetition time ms/echo time msec], 3 mm section thickness, 100 mm field of view, 256256 matrix, and 16.6kHz bandwidth) and respiratory triggered (Rapid Biomedical GmbH, Rimpor, Germany). Acquisition was initiated, and during the fourth repetition, bolus IV injection of 50 μL of Gado-6Si-NP in HEPES buffer containing 150 mM NaCl ([Gd] = 100 mM) was performed. Duration of each repetition was approximately 3 min. Rat body temperature was regulated at 37 C. Positive enhancement of the signal (EHC) in each tissue was calculated as (St S0)/S0, where St was the signal intensity value measured at each time after injection, and S0 the signal value prior to injection.
NP Characterization. The nanoparticles used are coreshell particles with a core of gadolinium oxide. Gadolinium content was estimated to approximately 16% per particle. According to HRTEM (Figure 1), this core was well-crystallized with a bcc structure belonging to the Ia3 space group and with a size between 1.4 and 2 nm. This size is in line with the hydrodynamic diameter measured for the noncoated particles (Figure 2), of 1.2 ( 0.3 nm. After the hydrolysiscondensation, functionalization steps leading to the growth of a polysiloxane shell were evidenced by TEM and chemical analysis (EDX), and correlated with hydrodynamic diameter measured by DLS as 3.8 ( 0.1 nm (Figure 2). As previously stated, organic fluorophores conjugated to APTES were included during this coating step. This confers fluorescence properties to the hybrid gadolinium oxide nanoparticles.20 Zeta potential measurements of Gado-6Si-NP in the pH range from 4 to 10 are shown in Figure 1. The iso-electric point was evaluated at pH 78. Radiolabeling of Nanoparticles with 111In. Gado-6Si-NP nanoparticles were labeled with 111InCl3 in citrate buffer at pH 5. Nonspecifically bound 111In was removed from Gado-6SiNP111In by adding excess DTPA, followed by a purification step using a PD10 column. Nevertheless, a very small amount of radioactivity was separated from the nanoparticles by DTPA, indicating efficient and stable labeling. Following purification, radiochemical purity exceeded 99%. Radiochemical yield was 35 ( 3% (n = 10). Solution Stability. Negligible radioactivity was lost from Gado-6Si-NP111In nanoparticles after 4 h incubation in both solutions. After 24 h and 7 days incubation, radiochemical purity was greater than 93% and 91%, respectively. This indicated that Gado-6Si-NP111In remained stable for at least 7 days in rat serum or aqueous 20 mM DTPA solutions. Biodistribution Studies. When injecting a nontargeted nanoparticle using IV administration, optimal biodistribution should show dominant renal elimination, associated with the absence of reticulo-endothelial system uptake and the absence of significant uptake in other tissues. Quantitative tissue distribution was performed over 18 days at 5 intervals (2 h, 24 h, 72 h, 10 days, and 18 days) following IV injection in order to evaluate the biodistribution of Gado-6Si-NP labeled with indium 111. Tissue distribution of Gado-6Si-NP expressed as the percentage injected dose per gram tissue as well as the percentage injected dose per organ are presented in 1148
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Figure 4. Biodistribution of Gado-6Si-NP111In in Wistar rats. Group of rats (n = 3 except at days 3 and 18, n = 4) were sacrificed at 2 h, 1 day, 3 days, 10 days, and 18 days after injection, and selected organs were sampled and measured for 111In activity. Left axis: data is expressed as percent injected dose per gram of tissue (%ID/g). Right axis: data is expressed as percent injected dose per gram of kidney (shaded area).
Figure 5. SPECT/CT in vivo imaging in Wistar rats. Planar sequential scintigraphic images acquired (a) during IV injection of 25 MBq of Gado-6SiNP111In, (b) 15 min, (c) 30 min, and (d) 60 min after injection. Tomographic scintigraphy and X-ray CT of the whole body of the rat acquired at day 2, posterior projection (e1) and transversal slice (e2). LK: left kidney; RK: right kidney; B: bladder. UC: urine collector.
Figure 4 and Table 1. High levels of radioactivity were quickly observed in the kidneys after IV administration (35.2%ID/organ at 2 h), and were reduced from 18.6% at 3 days to 3.9% at 18 days. The blood circulation half-life of labeled Gado-6Si-NP was evaluated at 42 ( 3 min. Renal and liver excretion of Gado6Si-NP were evaluated by measuring urine and feces activity, and expressed as cumulated radioactivity under total injected radioactivity (Figure 3). Gado-6Si-NP were dominantly and rapidly eliminated through renal excretion with 67 ( 4%ID and 84 ( 3% ID, compared to liver excretion with 4 ( 2%ID and 10 ( 3%ID after 1 day and 18 days, respectively, as demonstrated by the quantitative biodistribution study. Similar results were observed in scintigraphic imaging (Figure 5), MR imaging (Figure 6), and
optical imaging (Figure 7), where injection signal increased rapidly in the kidneys, with initial increase in the cortex, followed by an increase in the pelvis and then the bladder. MRI dynamic acquisition (Figure 6) evidenced a moderately increased EHC signal (38% at 39 min) in the area of the liver, but this was not due to RES uptake, as no significant Gado-6Si-NP accumulation has been observed in the quantitative biodistribution analysis. This increase seems to be related to liver elimination of the Gado-6SiNP, as radioactivity was found in the feces at later time points. The absence of Gado-6Si-NP accumulation in the liver, as well as in the spleen, lung, and bones (medulla) supported the absence of RES uptake (Table 1). This finding was confirmed using scintigraphic and optical imaging, where no significant signal was 1149
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Figure 6. T1w.seq. coronal images acquired before and at 3, 15, 30, and 60 min after IV injection of 50 μL of Gado-6Si-NP, centered on kidneys (a) and bladder (b).
Figure 7. Ventral (a1,2,3,4) and dorsal (b1,2,3,4) planar fluorescence reflectance images acquired at the end of IV injection of Gado-6Si-NP (a1, b1), at 15 min (a2, b2), 30 min (a3, b3), and 60 min (a4, b4) postinjection. c 1 to 12: Fluorescence reflectance imaging of major organs after dissection of a nude mouse. Dissection was performed 2 h after IV injection of Gado-6Si-NP. d 1 to 12: Fluorescence reflectance imaging of some organs after dissection of a control nude mouse. Each image was acquired with an exposure time of 2 s.
seen in spleen, lung, and bones as shown in Figures 5 and 7. Finally, our study demonstrated the absence of brain uptake.
’ DISCUSSION Quantitative in vivo biodistribution has shown that multimodal Gado-6Si-NP display excellent properties as contrast agents, with excellent renal filtration, and can be detected using different imaging modalities such as optical (fluorescence), MR, or isotopic (SPECT) imaging. This was an important step, as it is essential in determining the biodistribution and clearance of nanoparticles that may be used for imaging and theragnostic applications prior to their clinical use. Such a long-term quantitative distribution of silica nanoparticles has not yet been extensively reported.21 Overall, radiolabeling nanoparticles allows for whole-body imaging with high sensitivity and quantitative ex vivo biodistribution, which is considered to be a robust and reliable method for
evaluating the in vivo behavior of a nanoprobe.22 Nevertheless, the stability of the radiolabeling should be carefully controlled in vitro and in vivo, in order to ensure the accurate evaluation of in vivo biodistribution. No significant release of Indium 111 has been observed in vivo, as no significant bone uptake was measured, taking into account that trivalent radiometals such as indium are known to be taken up by bones23 indicating an in vivo stability of the radiolabeling. Stability of the radiolabeling was also confirmed in vitro; due to their multimodal properties, it was possible to compare the biodistribution obtained using three different modalities in order to confirm the efficiency and stability of radiolabeling. In our study, indium 111 with a half-life of 2.8 days was used for this long-term biodistribution quantitative analysis over 18 days. Other radioisotopes could have been used to complex DTPA, such as technetium 99m (half-life: 6 h) or PET emitter gallium 68 (half-life: 1.13 h). However, due to their short half-lives, they were not suitable for long-term biodistribution assessment. 1150
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Bioconjugate Chemistry Biodistribution and clearance of nanoparticles depend on their chemical and biochemical properties such as size, surface functionality, and charge. In general, it is well-established that nanoparticles are taken up by the RES system following IV administration, leading to the clearance of nanoparticles from systemic circulation.24 In this study, a high rate of renal elimination of Gado-6Si-NP following IV injection was evidenced with more than 95% of Gado-6Si-NP eliminated 18 days after injection. Our γ-counting data revealed that Gado-6Si-NP showed minimal particle retention in major organs (especially RES tissue), and the major route of elimination was renal excretion (Table 1), which is consistent with results obtained from optical imaging, where the fluorescence signal could be observed in the urinary bladder and kidneys. This is attributed to the presence of hydrophilic molecules (DTPA), which functionalize these hybrid gadolinium oxide nanoparticles, as generally observed for other PEG-capped nanoparticles.2527 The derivatization of Gado-6Si-NP noticeably decreased liver retention and increased renal filtration into the bladder, whereas underivatized nanoparticles frequently accumulated in the liver and spleen.17 This demonstrates that biodistribution and pharmacokinetics of Gado-6Si-NP were wellcontrolled. No significant Gado-6Si-NP accumulation was observed in lungs (Table 1). This result demonstrated that hydrophilic carboxylic DTPA coating prevents Gado-6Si-NP aggregation under in vitro and in vivo conditions, as usually observed with PEG coating.28 Moreover, hydrodynamic particle size is known to be a key factor in the biodistribution of long-circulating nanoparticles,16,29,30 and large nanoparticles tend to accumulate in the liver.31 Burns et al.15 have shown that the optimal probe size to obtain a reasonable circulation lifetime and efficient clearance is between 3 and 7 nm in hydrodynamic diameter, as the capillary pore diameter is estimated to be around 5 μm. The small size of these Gado-6Si-NP nanoparticles (mean diameter 34 nm) reduced plasma protein adsorption, opsonization, and hepatic filtration. Gado-6Si-NP can be effectively cleared out by renal excretion with more than 75%ID eliminated after 48 h, which is consistent with similar-sized probes.13,14,32 Finally, another very interesting finding was the absence of significant uptake in any other tissues. Since these nanoparticles are multimodal, they can be used with different imaging modalities, each modality having its own advantages and drawbacks.33 MRI produces excellent spatial resolution, though sensitivity and specificity are critical issues. Optical imaging provides good sensitivity, but penetration of near-infrared light in tissues is limited to a few centimeters34 with a high autofluorescence background, and with limited abilities to provide quantitative biodistribution in vivo. Isotopic imaging displays excellent sensitivity but low spatial resolution. Interests in multimodality imaging are numerous and offer many opportunities as multimodality imaging provides complementary information that cannot be discerned easily from one type of image modality alone, combining the benefits of each modality. The presence of gadolinium ions inside the organic shell, which is one the most efficient contrast agents for MRI, provides a strong positive contrast and allows for high-resolution soft tissue images in vivo (Figure 6), as predicted in a previous publication.17 In addition to imaging, the presence of gadolinium (atomic number Z = 64), which has a huge neutron capture cross section, provides opportunities for radiosensitization mainly based on the action of Auger electrons and γ-rays generated by 155,157Gd after neutron capture.35 Due to the high number of gadolinium ions
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per particle (about 40), irradiation of these hybrid gadolinium oxide nanoparticles has been shown to generate an in vitro cytotoxic effect.36 Finally, these nanoparticles exhibit multiple DTPA molecules (about 160 per particle) and can be functionalized with a large number of ligands, increasing the affinity to their biological target. The theragnostic approach of these nanoparticles, guided by multimodal imaging, allows us to envisage their application in oncology. Targeting these nanoparticles is currently being researched.
’ CONCLUSION This study reports the in vivo biodistribution of hybrid gadolinium oxide nanoparticles using different imaging modalities and γ ex vivo counting. The biodistribution of these nanohybrid agents by MRI, optical and radionuclide, demonstrates that Gado-6Si-NP circulate freely in the blood pool, without RES uptake, and are eliminated by renal excretion. The understanding of the biodistribution and pharmacokinetics of nanoparticles is a key factor in the process of successfully developing new multimodal agents. Multimodal and quantitative biodistribution of this new category of nanoparticles shows promising properties that may enable us to develop them as targeted or nontargeted contrast media or therapeutic agents. ’ AUTHOR INFORMATION Corresponding Author
*David KRYZA, Federation Medecine Nucleaire - Radiopharmacie, H^opital Edouard Herriot, 5 place d’Arsonval, 69437 Lyon cedex 03 - France. E-mail:
[email protected], Tel: þ33 472 68 46 17, Fax: þ33 472 11 69 57. Author Contributions ^
These authors contributed equally to this work.
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