Biomaterials 35 (2014) 7050e7057
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Indium-111 labeled gold nanoparticles for in-vivo molecular targeting Quinn K.T. Ng a, b,1, Cristina I. Olariu a, b,1, Marcus Yaffee a, Vincent F. Taelman a, b, Nicolas Marincek a, Thomas Krause a, b, Lorenz Meier a, b, Martin A. Walter a, b, c, * a
Institute of Nuclear Medicine, University Hospital Bern, Bern 3010-CH, Switzerland Department of Clinical Research, University Bern, Bern 3010-CH, Switzerland c Molecular and Medical Pharmacology, David Geffen School of Medicine, UCLA, Los Angeles, CA 90095-1722, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 April 2014 Accepted 24 April 2014 Available online 17 May 2014
The present report describes the synthesis and biological evaluation of a molecular imaging platform based on gold nanoparticles directly labeled with indium-111. The direct labeling approach facilitated radiolabeling with high activities while maintaining excellent stability within the biological environment. The resulting imaging platform exhibited low interference of the radiolabel with targeting molecules, which is highly desirable for in-vivo probe tracking and molecular targeted tumor imaging. The indium-111 labeled gold nanoparticles were synthesized using a simple procedure that allowed stable labeling of the nanoparticle core with various indium-111 activities. Subsequent surface modification of the particle cores with RGD-based ligands at various densities allowed for molecular targeting of the avß3 integrin in-vitro and for molecular targeted imaging in human melanoma and glioblastoma models invivo. The results demonstrate the vast potential of direct labeling with radioisotopes for tracking gold nanoparticles within biological systems. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: RGD avß3 integrin Molecular imaging Nanomedicine cell tracking
1. Introduction Radioisotopes are increasingly used in biomedical applications to help understanding of biological and pathological processes unattainable by other means [1,2]. For example, microdosing studies with radiolabeled drugs allow quantitative determination of the pharmacokinetics, drug metabolism and drugedrug interactions in humans, using doses below pharmacological concentration and prior to phase I clinical studies [3]. Additionally, tracking studies with radiolabeled cells facilitate monitoring of their migration and functionality, and provide paramount data for optimizing cell-based diagnosis and therapy [4,5]. Today, numerous radiotracers are clinically used [6], and the growing demand for imaging specific biological processes will further increase the development of new tracers. The efficiency of long-term imaging radiotracers depends on the optimization of several factors which include achieving high labeling yield, high labeling stability and low interference with the biological properties of the labeled molecule. However, few of the currently used clinical radiotracers fulfill these criteria.
* Corresponding author. University Hospital, 3010 Bern, Switzerland. Tel.: þ41 31 63 23542; fax: þ41 31 63 23137. E-mail address: [email protected] (M.A. Walter). 1 QKTN and CIO contributed equally to the manuscript. http://dx.doi.org/10.1016/j.biomaterials.2014.04.098 0142-9612/Ó 2014 Elsevier Ltd. All rights reserved.
Common radiolabeling techniques such as conjugation of tyrosine residues with iodine-123 [7], fluorination with fluorine-18 [8] and complexation with metal chelators [9] allow labeling with a single radioactive atom. Yet, actual conjugation and complexation ratios are commonly lower than one atom per molecule. Furthermore, complexation with radiometals can require harsh reaction conditions and has been shown to interfere with the biological properties of the labeled molecules [10]. Moreover, iodination [11] and complexation [12] are reversible, which limits quantitative imaging over long time periods. Consequently, clinical imaging with radioisotopes is mostly restricted to a few hours. To extend this window, imaging platforms that allow labeling of high activities at high labeling stability and low interference with its biological properties are highly desired. The present report describes the design, synthesis and biological evaluation of such an imaging tool. An indium-111 labeled gold nanoparticle platform, modified with the tumor targeting sequence arginine-glycine-aspartate (RGD, Fig. 1A) was developed and utilized for tumor cell targeting in-vitro and in-vivo. Particles were designed to be injected intravenously, travel to the tumor cell (Fig. 1B), internalize via avß3 integrin mediated endocytosis (Fig. 1C) and allow imaging via subsequent gamma emission (Fig. 1D). Gold nanoparticles were selected for use as platform cores due to their simple and efficient surface modification and reported use
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Fig. 1. Design of the indium-111 labeled gold nanoparticles. Gold nanoparticles were synthesized with the particle core stably labeled with the g-emitter indium-111 and the surface modified with linear and cyclic RGD ligands (A). The particles were designed to be injected intravenously, travel to the tumor cell (B), internalize via avß3 integrin mediated endocytosis (C) and allow imaging via g-emission (D). Variation of the core size was achieved by adjusting the tannic acid concentration during synthesis (E). Variation of the particles’ specific activity was achieved by varying the indium-111 activity during synthesis (F). Indium remained stably adsorbed before and after purification as well as after the ligand exchange reactions as derived from ICP-MS measurements (G).
in clinical applications [13,14]. Indium-111 was selected due to its FDA approval and its established clinical use for molecular imaging of somatostatin receptor [15] and CD20 expression [16]. Furthermore, the 2.8 day half-life of indium-111 allows for 3D whole-body imaging in humans over several days. Finally, RGD was selected, due to its multivalency effects for tumor targeting [17] and its successfully application for imaging of avß3 integrin expressing tumors in humans [18]. As a result, the design of the indium-111
labeled gold nanoparticles exclusively comprised components already in clinical use. 2. Materials & methods 2.1. Materials Hydrogen tetrachloroaurate trihydrate (99.9%), sodium citrate tribasic dehydrate (99%), indium chloride (99.9%) and diethylene triamine pentaacetic acid (DTPA, 99%) were purchased from SigmaeAldrich and used without further
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purification. Tannic acid with low molecular weight 1000-1500 was purchased from Lucerna-Chem. The PEGylated alkanethiol termed HS-C11-EG4-OH was obtained from ProChimia Surfaces. Linear and cyclic targeting sequences of the peptides CCVVVT-EG4-GRGDSP-NH2 (97%) (Cap-lRGD) and c[RGDfK(CCVVVT-EG4)] (96%) (Cap-cRGD) were purchased from CPC Scientific. Indium-111 chloride (111InCl3) solution in 0.05 M hydrochloric acid was purchased from Mallinckrodt. All buffers and solutions were prepared using MilliQ H2O. The human melanoma cell lines (M21 and M21-L) and the human glioblastoma cell line (U87-MG) were a gift from Prof. Urs F. Greber, University of Zurich and Prof. Erik Vassella, University of Bern, respectively. Dulbecco’s Modified Eagle Medium (DMEM) media, RPMI-1640 media, 1% Penicillin/Streptomycin solution were purchased from Invitrogen. Biodex TLC strips for radiochemical purity analysis were obtained from Raditec. The BCA total protein assay was purchased from Thermo Scientific. Six week old female athymic Nude-Foxn1nu mice were purchased from Harlan (Fullinsdorf/Itingen, Switzerland) and were delivered and housed in the animal facility at the University Hospital Bern. All animal manipulations were performed with sterile technique and were approved by the Bernese Animal Research Committee (Protocol BE95/11).
2.2. Synthesis of indium-111 labeled gold nanoparticles Indium-111 labeled gold nanoparticles were synthesized using a modified approach from Slot and Geuze [19]. First, all glassware and magnetic stirrer bars used for synthesis were carefully cleaned with aqua regia (HCl:HNO3 3:1, v/v), rinsed with triple distilled water (3d H2O) and dried prior to use. Then, under an argon atmosphere a solution of 100 mL of 1% HAuCl4 and 50 mL of varying 111InCl3 activities in 15.8 mL 3d H2O was heated to 60 C under constant stirring. A solution containing 800 mL of 1% sodium citrate, 150 mL of 1% tannic acid and 3.15 mL 3d H2O was heated to 60 C, quickly added to the 111InCl3/gold solution and stirred for 5 min. Then, temperature was increased to boiling and kept for 10 min. The indium-111 labeled gold nanoparticles were allowed to cool to room temperature and were purified three times by ultrafiltration (1500 g, MWCO 50 kDa) with 0.01% citrate solution. In addition, the influence of varying tannic acid concentrations on the particle size was investigated, and particles with non-radioactive indium-115 for characterization with ICP-MS, DLS, TEM and UVeVis were synthesized using the same protocol.
2.3. Surface modification of the indium-111 labeled gold nanoparticles The surface of the indium-111 labeled gold nanoparticles was modified adapting a method from Duchesne et al. [20]. A 200 nmol ligand mixture of HS-C11EG4-OH and varying mol percentages (0e40 mol%) of Cap-cRGD or Cap-lRGD dissolved in methanol were added to 1 mL of the indium-111 labeled gold nanoparticles in citrate solution and was reacted for 1 h at room temperature before the addition of 100 mL of PBS 10 and overnight incubation. Purification of the RGDmodified particles was carried out with PBS using 40 kDa MWCO spin columns according to manufactures protocols. Non-radioactive indium-115 labeled gold nanoparticles were modified using this procedure to evaluate the surface modifications by amino acid analysis.
2.4. Nanoparticle characterization Nanoparticle UVeVis spectra were recorded using a Genesys 10S system. Hydrodynamic diameters were measured via dynamic light scattering (DLS) with a Malvern Nano-S Zetasizer. Particle sizes were visualized using a JEOL-1010 transmission electron microscope (TEM) operating at 100 keV. Images were recorded with an AMT XR41-B CCD camera. TEM samples were drop casted onto a Formvar copper grid and dried in a vacuum desiccator. The size distribution was determined via ImageJ software from at least 100 nanoparticles. Negative staining with 2% uranyl acetate was used to enhance the particle shell after surface modification. Inorganic elemental analysis of nonradioactive indium-115 containing gold nanoparticles was performed at the Analytical Research Services, University Bern with inductively coupled plasma mass spectrometry (ICP-MS) to verify indium retention within the particles based on the gold-to-indium ratio. Thereby, indium and gold were quantified against palladium and platinum standard calibration curves. Amino acid analysis was performed at the Analytical Research Services core facility, University Bern, to quantify the particle-bound RGD peptides. Radioactivity of the particles was measured using a Veenstra VDC-405 dose calibrator. Radiochemical purity of the particles was measured via thin layer chromatography (Radio-TLC). TLC strips were spotted with 1 mL of each sample and placed in 0.1 M DTPA (pH 5.2) thereby, non-bound indium is transported at the front of the mobile phase whereas the nanoparticles remain immobilized. Developed TLC strips were placed in an autoradiography cassette with a multi-sensitive storage phosphor screen, which was then scanned using a Cyclone Plus phosphor imager (Perkin Elmer). Scans were quantified using the OptiQuant Acquisition and Analysis software. Radiochemical purity was measured via radio-TLC at 1, 4, 24, and 48 h. Serum stability was assessed by adding 50% human blood serum and 1% penicillin/streptomycin to the nanoparticle at room temperature. Colloidal serum stability was verified via changed in the UVeVis spectra at 1, 4, 24, and 48 h.
2.5. In-vitro studies with RGD-modified indium-111 labeled gold nanoparticles Nanoparticle uptake was studied in cell lines expressing various avb3 integrin levels. The high avb3 integrin expressing human melanoma cell line M21 was used along with low avb3 integrin expressing M21-L cells as a control cell line [21,22]. In addition, the avb3 integrin expressing human glioblastoma cell line U87-MG was used, and specific binding was tested with blocking experiments using excess of free RGD as controls [23]. Cells were plated at 200,000 cells per well on 12 well TC-plates in RPMI-1640 or DMEM, and kept overnight (37 C, 5% CO2). Then, cell media was removed and replaced with fresh serum-free culture media for 1 h. Afterwards, 40 mL PBS containing 3.8 1012 RGD-modified indium-111 labeled gold nanoparticles (calculated according to [24,25]) were added to each well and were allowed to incubate with the cells for 4 h (37 C, 5% CO2). Plates were then chilled on ice, media was removed and wells were washed 3 times with PBS. Media and PBS buffer were collected (noninternalized fraction). Then, glycine buffer (pH 2.8) was added to each well for 5 min on ice and wells were washed again 3 times with PBS. Glycine wash and PBS wash were collected (cell-bound fraction). Finally, RIPA buffer was added to each well for 5 min on ice. The resulting cell lysates (internalized fraction), collected media and washes were transferred to 5 mL conical tubes measured for radioactivity using a Perkin Elmer 2470 Wizard2 automatic gamma counter. Cell number per well was assessed from the cell lysate via BCA assay and was used for normalization. Nanoparticles without RGD-modification served as negative controls. Blocking experiments with addition of 0.1 mM of the cold RGD peptide GRGDSP 1 h prior to the nanoparticles were performed to verify specific internalization. 2.6. Biodistribution and in-vivo imaging with RGD-modified indium-111 labeled gold nanoparticles Six weeks old athymic Nude-Foxn1nu mice were subcutaneously injected in the femoral region with 1 106 M21, M21-L or U87-MG cells in 50 mL PBS. In-vivo studies were performed when the xenografts had reached approximately 5 mm3. At this point, 1 MBq of purified indium-111 labeled gold nanoparticles modified with 20% cyclic RGD in 50 mL PBS were injected via the tail vein. After 4 h, mice were sacrificed and organs and tumors were removed and weighed. Particle uptake in each organ was measured using a Perkin Elmer 2470 Wizard2 automatic gamma counter. Activity concentrations were computed as percent of decay-corrected injected activity per gram of tissue (%ID/g). Indium-111 labeled gold nanoparticles without RGD modification served as negative controls. Imaging studies were performed 4 h after injection of 1 MBq of the particles on a combined single photon emission tomography (SPECT)/computed tomography (CT) scanner (BrightView XCT, Philips) equipped with a medium energy collimator. CT scanning was performed with 5 mm slice thickness, 2.5 mm increment, 120 kV, 50 mAs/slice and 0.75 s rotation time. SPECT imaging was performed using the 0.171 and 0.245 MeV energy peaks of indium-111 with a 15% window. SPECT was acquired at 64 angles with 40 s per angle on a 128 x 128 matrix and was reconstructed with an Ordered Subset Expectation Maximization (OSEM) algorithm. The nuclear medicine application suite (Philips) software was used to display SPECT and CT images.
3. Results 3.1. Gold nanoparticles as a platform for stable labeling with the gemitter indium-111 A simple one-pot procedure allowed labeling of gold nanoparticles with the radioisotope indium-111 by caring out the synthesis in an aqueous system using tannic acid and sodium citrate, where both reagents reduce the gold to form nanoparticle cores. The particle core size was controlled by adjusting tannic acid concentration, while all other parameters including the indium-111 activity were kept constant. Overall, increasing tannic acid concentrations resulted in decreasing particle sizes (Fig. 1E). In addition, the particles’ specific activity could be varied by increasing the indium-111 activity during the synthetic steps (Fig. 1F). To confirm that the indium is stably bound to the gold nanoparticles during all procedures, non-radioactive gold nanoparticles labeled by indium115 were used. Gold-nanoparticle samples before and after purification as well as after the ligand exchange reaction demonstrated a stable elemental composition by elemental analysis (ICP-MS) (Fig. 1G). Indium-111 labeled gold nanoparticles with a size of 7 nm were selected for in-vitro and in-vivo evaluation as their size allows for background clearance by renal filtration [26]. Particles were
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Fig. 2. Characterization of the indium-111 labeled gold nanoparticles. TEM images and size distribution measurements of the indium-111 labeled gold nanoparticles demonstrated an average particle size of 6.9 0.9 nm (A). TEM images of the RGD-modified indium-111 labeled gold nanoparticles negatively stained with 2% uranyl acetate displayed the polymer layer visible as a light halo surrounding the particle gold core (B). Hydrodynamic size measurements showed narrowly distributed indium-111 labeled gold particles of w7e8 nm before and after purification (C). The absorbance spectra of the indium-111 labeled gold particles before and after purification showed a stable maximum at 524 nm (D). High serum stability of the indium-111 labeled gold particles was found in the presence of human blood serum at up to 48 h (E). The DLS showed that functionalization of the particle surface with RGD ligands resulted in a 3e4 nm increase of the hydrodynamic diameter (F). UVeVis spectroscopy of the RGD-modified indium-111 labeled gold particles in the presence of human blood serum revealed a stable SPR peak at up to 48 h, indicating the absence of aggregation (G).
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Fig. 3. In-vitro targeting with RGD-modified indium-111 labeled gold nanoparticles. RGD-modified indium-111 labeled nanoparticles with various RGD densities were added to different cells for in-vitro uptake quantification. Experiments with (þ) and without () the conditions are indicated in the table below the graph. Nanoparticles with RGDmodification showed higher uptake than particles without RGD-modification, particles modified with cyclic RGD showed higher uptake than particles modified with linear RGD, and particles showed higher uptake at higher density of RGD-modification. RGD-modified nanoparticles showed higher uptake in melanoma cells with high avß3 integrin expression (M21) compared to melanoma cells with low avß3 integrin expression (M21-L, blue bars). Specific uptake was verified by blocking of uptake with excess of free RGD peptide in the glioblastoma model (red bars). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
synthesized with a narrow size distribution (6.9 0.9 nm), as evidenced by TEM and particle distribution analysis (Fig. 2A). In accordance with TEM, DLS revealed similar average hydrodynamic diameters before (7.7 0.2 nm) and after purification (7.1 0.3, Fig. 2C). UVeVis spectroscopy demonstrated a 524 nm surface plasmon resonance (SPR) peak before and after purification (Fig. 2D), consistent with spherical gold nanoparticles of sizes below 10 nm [27]. The absence of red-shifts or broadening of the SPR peak indicated the absence of aggregation during particle purification [28]. A stable red shift of the SPR peak from 524 to 527 nm was found for the indium-111 labeled gold nanoparticles in the presence of human blood serum at 1, 4, 24, and 48 h (Fig. 2E). In addition, radioTLC confirmed radiochemical purity yields over 95%, which maintained over 48 h in the presence of human blood serum (Supplementary Data, Fig. S1), confirming the stability of indium111 labeled gold nanoparticles also under physiological conditions. Furthermore, using strong chelating agents to capture any free radio-indium, radio-TLC confirmed that the indium-111 is indeed stably bound to the nanoparticles (Supplementary Data, Fig. S1).
3.2. Indium-111 labeled gold nanoparticles allow facile modification with tumor targeting ligands Capping peptides and PEGylated alkanethiols were used to link the targeting ligands onto the surface of gold nanoparticles. For this purpose, the surface of indium-111 labeled gold nanoparticles was modified with a mixture of HS-C11-EG4-OH and either Cap-cRGD or Cap-lRGD ligands (Fig. 1A). The 4 unit PEG spacer EG4 between the capping peptide sequence and the RGD targeting ligand was used to
extend the ligand from the nanoparticle surface. The ratio of either Cap-cRGD or Cap-lRGD ligands incorporated on the nanoparticle surface was varied from 0 to 40 mol% with respect to the HS-C11EG4-OH ligand. TEM images of the negatively stained RGD-modified indium-111 labeled gold nanoparticles revealed a light halo of low electron density surrounding the denser gold nanoparticle core (Fig. 2B), indicating the presence of a thin layer on the surface of the nanoparticle representing the surface modification [29,30]. Surface modification of the particle with the RGD ligands resulted in a 3e4 nm increase of the hydrodynamic diameter for all sets of synthesized nanoparticles in the DLS measurements (Fig. 2F). Ligand exchange reaction was confirmed by shifted SPR peaks from 524 for the citrate and tannic acid ligands (Fig. 2D) to 527 nm after replacement by thiol-containing ligands (Supplementary Data, Fig. S2). The absence of SPR peak broadening further confirmed the particle stability after the ligand exchange reactions. UVeVis spectroscopy of the RGD-modified indium-111 labeled gold particles in the presence of human blood serum revealed a stable SPR peak over a period of 48 h (Fig. 2G), indicating the effectiveness of the surface modification with thiol-containing ligands in preventing aggregation. Amino acid analysis revealed a linear correlation between the amount of RGD ligands used for the ligand exchange reaction and the amount finally present on the particle surface (slope: 0.53, Supplementary Data, Fig. S3).
3.3. RGD-modified indium-111 labeled gold nanoparticles allow targeting of avß3 integrin in-vitro Internalization experiments were performed with indium-111 labeled gold nanoparticles without (0%RGD) or with RGD-
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Fig. 4. In-vivo targeting with RGD-modified indium-111 labeled gold nanoparticles. Biodistribution studies were performed in nude mice bearing M21, M21-L and U87 tumor xenografts, and are displayed as percent injection dose per gram of organ weight (%ID/g). The organ (A) and tumor biodistribution (B) demonstrated higher tumor uptake with RGDmodified indium-111 labeled gold nanoparticles when compared with the non-targeted nanoparticles in the U87 tumor model. SPECT/CT imaging (C) demonstrated higher uptake of RGD-modified indium-111 labeled gold nanoparticles in the M21 tumor (left) compared to the M21-L tumor (right).
modifications of various amounts (10, 20, 30, 40% Cap-cRGD or 20% Cap-lRGD, Fig. 3). The highest uptake in M21 cells was found for particles with 30% surface modification of cyclic RGD (30%CapcRGD), while the highest uptake in U87-MG cells was found for particles with 40% surface modification of cyclic RGD (40%CapcRGD). At these conditions, RGD-modified particles showed higher uptake in the high avß3 integrin expressing M21 cells than in the low avß3 integrin expressing M21-L cells (p ¼ 0.04) and particle uptake in U87-MG could be blocked with an excess of cold linear RGD peptide (p ¼ 0.006), both indicating avß3 integrin-specific uptake. Particles modified with cyclic RGD (10, 20, 30, and 40% Cap-cRGD) generally showed higher uptake than particles modified with linear RGD (20%Cap-lRGD) or the negative controls, particles without RGD (0%RGD). The cell bound fraction was below 1% for all tested particles in each cell line. 3.4. RGD-modified indium-111 labeled gold nanoparticles for invivo imaging of avß3 integrin expressing tumors Tumor uptake and biodistribution of indium-111 labeled gold nanoparticles with and without RGD modification was assessed in 6 week old female athymic Nude-Foxn1nu mice in the human melanoma and glioblastoma model (Fig. 4A and B). Mice were bearing a M21 xenograft on the left leg and a M21-L xenograft on the right leg or were bearing a single U87-MG xenograft on the left leg. Corresponding to the in-vitro results, particles modified with cRGD showed higher uptake in the high avß3 integrin expressing tumors (M21, 0.52 0.21 %ID/g) than in the low avß3 integrin expressing tumors (M21-L, 0.39 0.14 %ID/g), indicating avß3 integrin-specific tumor binding. Coherently, U87-MG tumors showed higher uptake of nanoparticles with versus without cRGD-modification (0.93 0.14 %ID/g versus 0.37 0.04 %ID/g, p ¼ 0.003). Finally, in line with the biodistribution results, SPECT/CT imaging demonstrated higher uptake of cRGD-modified indium-111 labeled gold nanoparticles in the M21 tumor (left) compared to the M21-L tumor (right, Fig. 4C).
4. Discussion The present work demonstrates the feasibility of molecular targeting with directly radiolabeled gold nanoparticles. It describes the design and synthesis of a modular gold nanoparticle platform stably labeled with indium-111, the surface modification with targeting moieties and that such modified particles can be used for molecular targeted imaging in a melanoma and a glioblastoma model. The direct incorporation of indium-111 allowed stable radiolabeling of the gold nanoparticle with varying activities. The modification with targeting motifs, which were spatially separated from the radioisotope, enabled the use of the nanoparticle platform for molecular targeting in-vitro and in-vivo. Direct radiolabeling of the gold nanoparticle cores by radioisotopes represents a concept that allows stable radiolabeling without the necessity of additional surface modifications to accommodate chelators for subsequent labeling. Previous approaches with surface modifications for radiolabeling as DTPA, DOTA or desferroxamin required harsh radiolabeling conditions [31-34], which are incompatible with the use of commonly used peptide or antibody based targeting motifs and can cause colloidal instability. Furthermore, surface modifications for radiolabeling have shown to interfere with the particles’ pharmacological profile [34]. Direct integration of radioisotopes into gold nanoparticles represents an efficient way to avoid the use of metal chelators and harsh radiolabeling procedures and to spare the entire particle surface for modifications with targeting motifs. The feasibility of directly integrating radioisotopes into gold particles was recently demonstrated using gold-198 and copper-64 [35,36]. The copper-64 labeled gold nanoparticles accumulated in breast cancer xenografts due to enhanced permeability and retention, while the gold-198 labeled gold nanoparticles were directly injected into prostate cancer xenografts. The present results confirm the feasibility of the direct labeling approach with indium-111, a long half-life g-emitter that is clinically established for molecular imaging. The long half-life of the radioisotope used in
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this study allowed the modification of the spared nanoparticle surface with targeting motifs. The results of the in-vivo studies demonstrate the potential of gold nanoparticles with integrated radioisotopes for molecular targeting after intravenous injection in two independent tumor models. The indium-111 labeled gold nanoparticles that were suitable for intravenous injection were synthesized using a simple procedure and the stability of the indium-111 integrated with the gold nanoparticles was confirmed by inorganic elemental analysis before and after surface modification with RGD-containing ligands. Colloidal stability of the surface modified gold nanoparticles was demonstrated by DLS. Overall, the synthetic procedure allowed controlling the specific radioactivity of the indium-111 labeled gold nanoparticles by varying the initial activity of the 111InCl3 during synthesis, which allows tailoring the particles’ activity to the biological application. The in-vitro studies demonstrated the molecular targeting capabilities of the RGD-modified indium-111 labeled gold nanoparticles in two different human cell lines expressing the avß3 integrin. Specificity of avß3 integrin mediated uptake was demonstrated by reduced uptake with particles not modified with RGD, in cells with low avß3 integrin expression and by blocking experiments using an excess of cold RGD, respectively. The absence of chelating groups for radiolabeling allowed testing a range of different RGD densities on the nanoparticle surface, thereby higher uptake was found with increasing amounts of RGD present on the nanoparticle surface. These results indicate that indium-111 labeled gold nanoparticles represent a valuable universal platform for refining the use of various targeting motifs, potentially facilitating the use of targeting concepts that could not be successfully used with nanoparticles yet. The biodistribution studies confirmed the targeting capabilities of the RGD-modified indium-111 labeled gold nanoparticles in-vivo. RGD-modified indium-111 labeled gold nanoparticles showed higher uptake than the non-targeted nanoparticles in the glioblastoma model. Additionally adrenal glands, which physiologically express high amounts of avß3 integrin [37], as well as the liver and spleen, known for opsonization and mononuclear phagocytosis, showed uptake of the nanoparticle [38,39]. Finally, the RGDmodified indium-111 labeled gold nanoparticles were successfully used for in-vivo imaging of tumors overexpressing avß3 integrins after intravenous administration. Non-radioactive gold nanoparticles are gaining increasing attention for their clinical use as drug carriers, photo-thermal agents, contrast agents and radio-sensitizers [13,14]. However, their pharmacokinetics and biodistribution are key issues that need to be clarified before they will enter routine clinical use. The use of indium-111 is already clinically established to assess pharmacokinetics and biodistribution e.g. of CD20 antibodies, as work-up for CD20-based immune-radiotherapy in patients with non-Hodgkin’s lymphoma. Similarly, indium-111 labeled gold nanoparticles might represent a powerful tool to image and quantify the pharmacokinetics and biodistribution of gold nanoparticles and facilitate a faster transition into the clinic. 5. Conclusion The present work describes the design, synthesis and biological evaluation of gold nanoparticles with stably integrated indium-111 as a universal modular platform for tracking and molecular targeted imaging. The platform fulfills key criteria for successful molecular imaging, such as high labeling yield, high labeling stability and low interference with the biological properties of the targeting molecule. The half-life of indium-111 allowed for modification of the nanoparticle with targeting motifs, and the animal studies
demonstrated the feasibility of molecular targeting with directly radiolabeled gold nanoparticles. Acknowledgments The authors gratefully acknowledge funding from the Swiss National Science Foundation (PZ00P3/126616) and the Bernese Cancer League. Furthermore, the authors are grateful to Andrew Q. Tran for his support in designing the figures and for the input of Andrea Grotzky, Renzo Cescato and Piotr Radojewski into the manuscript. All authors declare no potential conflict of interest. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2014.04.098. References [1] Jaffer FA, Weissleder R. Molecular imaging in the clinical arena. J Am Med Assoc 2005;293:855e62. [2] Velikyan I. Molecular imaging and radiotherapy: theranostics for personalized patient management. Theranostics 2012;2:424e6. [3] Bergstrom M, Grahnen A, Langstrom B. Positron emission tomography microdosing: a new concept with application in tracer and early clinical drug development. Eur J Clin Pharmacol 2003;59:357e66. [4] Frangioni JV, Hajjar RJ. In vivo tracking of stem cells for clinical trials in cardiovascular disease. Circulation 2004;110:3378e83. [5] Janowski M, Bulte JWM, Walczak P. Personalized nanomedicine advancements for stem cell tracking. Adv Drug Deliv Rev 2012;64:1488e507. [6] Van de Wiele C, Lahorte C, Oyen W, Boerman O, Goethals I, Slegers G, et al. Nuclear medicine imaging to predict response to radiotherapy: a review. Int J Radiat Oncol Biol Phys 2003;55:5e15. [7] Marchalonis J. An enzymic method for the trace iodination of immunoglobulins and other proteins. Biochem J 1969;113:299e305. [8] Varagnolo L, Stokkel MPM, Mazzi U, Pauwels EKJ. 18F-labeled radiopharmaceuticals for PET in oncology, excluding FDG. Nucl Med Biol 2000;27:103e12. [9] Reichert DE, Lewis JS, Anderson CJ. Metal complexes as diagnostic tools. Coord Chem Rev 1999;184:3e66. [10] Reubi JC, Schar JC, Waser B, Wenger S, Heppeler A, Schmitt JS, et al. Affinity profiles for human somatostatin receptor subtypes sst1-sst5 of somatostatin radiotracers selected for scintigraphic and radiotherapeutic use. Eur J Nucl Med 2000;27:273e82. [11] Adam MJ, Wilbur DS. Radiohalogens for imaging and therapy. Chem Soc Rev 2005;34:153e63. [12] Walrand S, Barone R, Pauwels S, Jamar F. Experimental facts supporting a red marrow uptake due to radiometal transchelation in 90Y-DOTATOC therapy and relationship to the decrease of platelet counts. Eur J Nucl Med Mol Imaging 2011;38:1270e80. [13] Libutti SK, Paciotti GF, Byrnes AA, Alexander Jr HR, Gannon WE, Walker M, et al. Phase I and pharmacokinetic studies of CYT-6091, a novel PEGylated colloidal gold-rhTNF nanomedicine. Clin Cancer Res 2010;16:6139e49. [14] Weintraub K. Biomedicine: the new gold standard. Nature 2013;495:S14e6. [15] Hicks RJ. Use of molecular targeted agents for the diagnosis, staging and therapy of neuroendocrine malignancy. Cancer Imaging 2010;10(Spec no A): S83e91. [16] Lin FI, Iagaru A. Current concepts and future directions in radioimmunotherapy. Curr Drug Discov Technol 2010;7:253e62. [17] Montet X, Funovics M, Montet-Abou K, Weissleder R, Josephson L. Multivalent effects of RGD peptides obtained by nanoparticle display. J Med Chem 2006;49:6087e93. [18] Gaertner FC, Kessler H, Wester HJ, Schwaiger M, Beer AJ. Radiolabelled RGD peptides for imaging and therapy. Eur J Nucl Med Mol Imaging 2012;39(Suppl. 1):S126e38. [19] Slot JW, Geuze HJ. A new method of preparing gold probes for multiplelabeling cytochemistry. Eur J Cell Biol 1985;38:87e93. [20] Duchesne L, Gentili D, Comes-Franchini M, Fernig DG. Robust ligand shells for biological applications of gold nanoparticles. Langmuir 2008;24:13572e80. [21] Felding-Habermann B, Fransvea E, O’Toole T, Manzuk L, Faha B, Hensler M. Involvement of tumor cell integrin avb3 in hematogenous metastasis of human melanoma cells. Clin Exp Metastasis 2002;19:427e36. [22] Cheresh DA, Spiro RC. Biosynthetic and functional properties of an Arg-GlyAsp-directed receptor involved in human melanoma cell attachment to vitronectin, fibrinogen, and von Willebrand factor. J Biol Chem 1987;262: 17703e11. [23] Li Z-B, Cai W, Cao Q, Chen K, Wu Z, He L, et al. 64Cu-labeled tetrameric and octameric RGD peptides for small-animal PET of tumor avb3 integrin expression. J Nucl Med 2007;48:1162e71.
Q.K.T. Ng et al. / Biomaterials 35 (2014) 7050e7057 [24] Haiss W, Thanh NTK, Aveyard J, Fernig DG. Determination of size and concentration of gold nanoparticles from UV-Vis spectra. Anal Chem 2007;79: 4215e21. [25] Lewis DJ, Day TM, MacPherson JV, Pikramenou Z. Luminescent nanobeads: attachment of surface reactive Eu(III) complexes to gold nanoparticles. Chem Commun; 2006:1433e5. [26] Choi HS, Liu W, Misra P, Tanaka E, Zimmer JP, Ipe BI, et al. Renal clearance of quantum dots. Nat Biotechnol 2007;25:1165e70. [27] Rouhana LL, Jaber JA, Schlenoff JB. Aggregation-resistant water-soluble gold nanoparticles. Langmuir 2007;23:12799e801. [28] Albanese A, Chan WCW. Effect of gold nanoparticle aggregation on cell uptake and toxicity. ACS Nano 2011;5:5478e89. [29] Qian X, Peng XH, Ansari DO, Yin-Goen Q, Chen GZ, Shin DM, et al. In vivo tumor targeting and spectroscopic detection with surface-enhanced Raman nanoparticle tags. Nat Biotechnol 2007;26:83e90. [30] Alexandridis P, Tsianou M. Block copolymer-directed metal nanoparticle morphogenesis and organization. Eur Polym J 2011;47:569e83. [31] Alric C, Miladi I, Kryza D, Taleb J, Lux F, Bazzi R, et al. The biodistribution of gold nanoparticles designed for renal clearance. Nanoscale 2013;5:5930e9. [32] Zhang G, Yang Z, Lu W, Zhang R, Huang Q, Tian M, et al. Influence of anchoring ligands and particle size on the colloidal stability and in vivo biodistribution of
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Indium-111 labeled gold nanoparticles for in-vivo molecular targeting Quinn K.T. Ng a, b,1, Cristina I. Olariu a, b,1, Marcus Yaffee a, Vincent F. Taelman a, b, Nicolas Marincek a, Thomas Krause a, b, Lorenz Meier a, b, Martin A. Walter a, b, c, * a
Institute of Nuclear Medicine, University Hospital Bern, Bern 3010-CH, Switzerland Department of Clinical Research, University Bern, Bern 3010-CH, Switzerland c Molecular and Medical Pharmacology, David Geffen School of Medicine, UCLA, Los Angeles, CA 90095-1722, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 April 2014 Accepted 24 April 2014 Available online 17 May 2014
The present report describes the synthesis and biological evaluation of a molecular imaging platform based on gold nanoparticles directly labeled with indium-111. The direct labeling approach facilitated radiolabeling with high activities while maintaining excellent stability within the biological environment. The resulting imaging platform exhibited low interference of the radiolabel with targeting molecules, which is highly desirable for in-vivo probe tracking and molecular targeted tumor imaging. The indium-111 labeled gold nanoparticles were synthesized using a simple procedure that allowed stable labeling of the nanoparticle core with various indium-111 activities. Subsequent surface modification of the particle cores with RGD-based ligands at various densities allowed for molecular targeting of the avß3 integrin in-vitro and for molecular targeted imaging in human melanoma and glioblastoma models invivo. The results demonstrate the vast potential of direct labeling with radioisotopes for tracking gold nanoparticles within biological systems. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: RGD avß3 integrin Molecular imaging Nanomedicine cell tracking
1. Introduction Radioisotopes are increasingly used in biomedical applications to help understanding of biological and pathological processes unattainable by other means [1,2]. For example, microdosing studies with radiolabeled drugs allow quantitative determination of the pharmacokinetics, drug metabolism and drugedrug interactions in humans, using doses below pharmacological concentration and prior to phase I clinical studies [3]. Additionally, tracking studies with radiolabeled cells facilitate monitoring of their migration and functionality, and provide paramount data for optimizing cell-based diagnosis and therapy [4,5]. Today, numerous radiotracers are clinically used [6], and the growing demand for imaging specific biological processes will further increase the development of new tracers. The efficiency of long-term imaging radiotracers depends on the optimization of several factors which include achieving high labeling yield, high labeling stability and low interference with the biological properties of the labeled molecule. However, few of the currently used clinical radiotracers fulfill these criteria.
* Corresponding author. University Hospital, 3010 Bern, Switzerland. Tel.: þ41 31 63 23542; fax: þ41 31 63 23137. E-mail address: [email protected] (M.A. Walter). 1 QKTN and CIO contributed equally to the manuscript. http://dx.doi.org/10.1016/j.biomaterials.2014.04.098 0142-9612/Ó 2014 Elsevier Ltd. All rights reserved.
Common radiolabeling techniques such as conjugation of tyrosine residues with iodine-123 [7], fluorination with fluorine-18 [8] and complexation with metal chelators [9] allow labeling with a single radioactive atom. Yet, actual conjugation and complexation ratios are commonly lower than one atom per molecule. Furthermore, complexation with radiometals can require harsh reaction conditions and has been shown to interfere with the biological properties of the labeled molecules [10]. Moreover, iodination [11] and complexation [12] are reversible, which limits quantitative imaging over long time periods. Consequently, clinical imaging with radioisotopes is mostly restricted to a few hours. To extend this window, imaging platforms that allow labeling of high activities at high labeling stability and low interference with its biological properties are highly desired. The present report describes the design, synthesis and biological evaluation of such an imaging tool. An indium-111 labeled gold nanoparticle platform, modified with the tumor targeting sequence arginine-glycine-aspartate (RGD, Fig. 1A) was developed and utilized for tumor cell targeting in-vitro and in-vivo. Particles were designed to be injected intravenously, travel to the tumor cell (Fig. 1B), internalize via avß3 integrin mediated endocytosis (Fig. 1C) and allow imaging via subsequent gamma emission (Fig. 1D). Gold nanoparticles were selected for use as platform cores due to their simple and efficient surface modification and reported use
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Fig. 1. Design of the indium-111 labeled gold nanoparticles. Gold nanoparticles were synthesized with the particle core stably labeled with the g-emitter indium-111 and the surface modified with linear and cyclic RGD ligands (A). The particles were designed to be injected intravenously, travel to the tumor cell (B), internalize via avß3 integrin mediated endocytosis (C) and allow imaging via g-emission (D). Variation of the core size was achieved by adjusting the tannic acid concentration during synthesis (E). Variation of the particles’ specific activity was achieved by varying the indium-111 activity during synthesis (F). Indium remained stably adsorbed before and after purification as well as after the ligand exchange reactions as derived from ICP-MS measurements (G).
in clinical applications [13,14]. Indium-111 was selected due to its FDA approval and its established clinical use for molecular imaging of somatostatin receptor [15] and CD20 expression [16]. Furthermore, the 2.8 day half-life of indium-111 allows for 3D whole-body imaging in humans over several days. Finally, RGD was selected, due to its multivalency effects for tumor targeting [17] and its successfully application for imaging of avß3 integrin expressing tumors in humans [18]. As a result, the design of the indium-111
labeled gold nanoparticles exclusively comprised components already in clinical use. 2. Materials & methods 2.1. Materials Hydrogen tetrachloroaurate trihydrate (99.9%), sodium citrate tribasic dehydrate (99%), indium chloride (99.9%) and diethylene triamine pentaacetic acid (DTPA, 99%) were purchased from SigmaeAldrich and used without further
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purification. Tannic acid with low molecular weight 1000-1500 was purchased from Lucerna-Chem. The PEGylated alkanethiol termed HS-C11-EG4-OH was obtained from ProChimia Surfaces. Linear and cyclic targeting sequences of the peptides CCVVVT-EG4-GRGDSP-NH2 (97%) (Cap-lRGD) and c[RGDfK(CCVVVT-EG4)] (96%) (Cap-cRGD) were purchased from CPC Scientific. Indium-111 chloride (111InCl3) solution in 0.05 M hydrochloric acid was purchased from Mallinckrodt. All buffers and solutions were prepared using MilliQ H2O. The human melanoma cell lines (M21 and M21-L) and the human glioblastoma cell line (U87-MG) were a gift from Prof. Urs F. Greber, University of Zurich and Prof. Erik Vassella, University of Bern, respectively. Dulbecco’s Modified Eagle Medium (DMEM) media, RPMI-1640 media, 1% Penicillin/Streptomycin solution were purchased from Invitrogen. Biodex TLC strips for radiochemical purity analysis were obtained from Raditec. The BCA total protein assay was purchased from Thermo Scientific. Six week old female athymic Nude-Foxn1nu mice were purchased from Harlan (Fullinsdorf/Itingen, Switzerland) and were delivered and housed in the animal facility at the University Hospital Bern. All animal manipulations were performed with sterile technique and were approved by the Bernese Animal Research Committee (Protocol BE95/11).
2.2. Synthesis of indium-111 labeled gold nanoparticles Indium-111 labeled gold nanoparticles were synthesized using a modified approach from Slot and Geuze [19]. First, all glassware and magnetic stirrer bars used for synthesis were carefully cleaned with aqua regia (HCl:HNO3 3:1, v/v), rinsed with triple distilled water (3d H2O) and dried prior to use. Then, under an argon atmosphere a solution of 100 mL of 1% HAuCl4 and 50 mL of varying 111InCl3 activities in 15.8 mL 3d H2O was heated to 60 C under constant stirring. A solution containing 800 mL of 1% sodium citrate, 150 mL of 1% tannic acid and 3.15 mL 3d H2O was heated to 60 C, quickly added to the 111InCl3/gold solution and stirred for 5 min. Then, temperature was increased to boiling and kept for 10 min. The indium-111 labeled gold nanoparticles were allowed to cool to room temperature and were purified three times by ultrafiltration (1500 g, MWCO 50 kDa) with 0.01% citrate solution. In addition, the influence of varying tannic acid concentrations on the particle size was investigated, and particles with non-radioactive indium-115 for characterization with ICP-MS, DLS, TEM and UVeVis were synthesized using the same protocol.
2.3. Surface modification of the indium-111 labeled gold nanoparticles The surface of the indium-111 labeled gold nanoparticles was modified adapting a method from Duchesne et al. [20]. A 200 nmol ligand mixture of HS-C11EG4-OH and varying mol percentages (0e40 mol%) of Cap-cRGD or Cap-lRGD dissolved in methanol were added to 1 mL of the indium-111 labeled gold nanoparticles in citrate solution and was reacted for 1 h at room temperature before the addition of 100 mL of PBS 10 and overnight incubation. Purification of the RGDmodified particles was carried out with PBS using 40 kDa MWCO spin columns according to manufactures protocols. Non-radioactive indium-115 labeled gold nanoparticles were modified using this procedure to evaluate the surface modifications by amino acid analysis.
2.4. Nanoparticle characterization Nanoparticle UVeVis spectra were recorded using a Genesys 10S system. Hydrodynamic diameters were measured via dynamic light scattering (DLS) with a Malvern Nano-S Zetasizer. Particle sizes were visualized using a JEOL-1010 transmission electron microscope (TEM) operating at 100 keV. Images were recorded with an AMT XR41-B CCD camera. TEM samples were drop casted onto a Formvar copper grid and dried in a vacuum desiccator. The size distribution was determined via ImageJ software from at least 100 nanoparticles. Negative staining with 2% uranyl acetate was used to enhance the particle shell after surface modification. Inorganic elemental analysis of nonradioactive indium-115 containing gold nanoparticles was performed at the Analytical Research Services, University Bern with inductively coupled plasma mass spectrometry (ICP-MS) to verify indium retention within the particles based on the gold-to-indium ratio. Thereby, indium and gold were quantified against palladium and platinum standard calibration curves. Amino acid analysis was performed at the Analytical Research Services core facility, University Bern, to quantify the particle-bound RGD peptides. Radioactivity of the particles was measured using a Veenstra VDC-405 dose calibrator. Radiochemical purity of the particles was measured via thin layer chromatography (Radio-TLC). TLC strips were spotted with 1 mL of each sample and placed in 0.1 M DTPA (pH 5.2) thereby, non-bound indium is transported at the front of the mobile phase whereas the nanoparticles remain immobilized. Developed TLC strips were placed in an autoradiography cassette with a multi-sensitive storage phosphor screen, which was then scanned using a Cyclone Plus phosphor imager (Perkin Elmer). Scans were quantified using the OptiQuant Acquisition and Analysis software. Radiochemical purity was measured via radio-TLC at 1, 4, 24, and 48 h. Serum stability was assessed by adding 50% human blood serum and 1% penicillin/streptomycin to the nanoparticle at room temperature. Colloidal serum stability was verified via changed in the UVeVis spectra at 1, 4, 24, and 48 h.
2.5. In-vitro studies with RGD-modified indium-111 labeled gold nanoparticles Nanoparticle uptake was studied in cell lines expressing various avb3 integrin levels. The high avb3 integrin expressing human melanoma cell line M21 was used along with low avb3 integrin expressing M21-L cells as a control cell line [21,22]. In addition, the avb3 integrin expressing human glioblastoma cell line U87-MG was used, and specific binding was tested with blocking experiments using excess of free RGD as controls [23]. Cells were plated at 200,000 cells per well on 12 well TC-plates in RPMI-1640 or DMEM, and kept overnight (37 C, 5% CO2). Then, cell media was removed and replaced with fresh serum-free culture media for 1 h. Afterwards, 40 mL PBS containing 3.8 1012 RGD-modified indium-111 labeled gold nanoparticles (calculated according to [24,25]) were added to each well and were allowed to incubate with the cells for 4 h (37 C, 5% CO2). Plates were then chilled on ice, media was removed and wells were washed 3 times with PBS. Media and PBS buffer were collected (noninternalized fraction). Then, glycine buffer (pH 2.8) was added to each well for 5 min on ice and wells were washed again 3 times with PBS. Glycine wash and PBS wash were collected (cell-bound fraction). Finally, RIPA buffer was added to each well for 5 min on ice. The resulting cell lysates (internalized fraction), collected media and washes were transferred to 5 mL conical tubes measured for radioactivity using a Perkin Elmer 2470 Wizard2 automatic gamma counter. Cell number per well was assessed from the cell lysate via BCA assay and was used for normalization. Nanoparticles without RGD-modification served as negative controls. Blocking experiments with addition of 0.1 mM of the cold RGD peptide GRGDSP 1 h prior to the nanoparticles were performed to verify specific internalization. 2.6. Biodistribution and in-vivo imaging with RGD-modified indium-111 labeled gold nanoparticles Six weeks old athymic Nude-Foxn1nu mice were subcutaneously injected in the femoral region with 1 106 M21, M21-L or U87-MG cells in 50 mL PBS. In-vivo studies were performed when the xenografts had reached approximately 5 mm3. At this point, 1 MBq of purified indium-111 labeled gold nanoparticles modified with 20% cyclic RGD in 50 mL PBS were injected via the tail vein. After 4 h, mice were sacrificed and organs and tumors were removed and weighed. Particle uptake in each organ was measured using a Perkin Elmer 2470 Wizard2 automatic gamma counter. Activity concentrations were computed as percent of decay-corrected injected activity per gram of tissue (%ID/g). Indium-111 labeled gold nanoparticles without RGD modification served as negative controls. Imaging studies were performed 4 h after injection of 1 MBq of the particles on a combined single photon emission tomography (SPECT)/computed tomography (CT) scanner (BrightView XCT, Philips) equipped with a medium energy collimator. CT scanning was performed with 5 mm slice thickness, 2.5 mm increment, 120 kV, 50 mAs/slice and 0.75 s rotation time. SPECT imaging was performed using the 0.171 and 0.245 MeV energy peaks of indium-111 with a 15% window. SPECT was acquired at 64 angles with 40 s per angle on a 128 x 128 matrix and was reconstructed with an Ordered Subset Expectation Maximization (OSEM) algorithm. The nuclear medicine application suite (Philips) software was used to display SPECT and CT images.
3. Results 3.1. Gold nanoparticles as a platform for stable labeling with the gemitter indium-111 A simple one-pot procedure allowed labeling of gold nanoparticles with the radioisotope indium-111 by caring out the synthesis in an aqueous system using tannic acid and sodium citrate, where both reagents reduce the gold to form nanoparticle cores. The particle core size was controlled by adjusting tannic acid concentration, while all other parameters including the indium-111 activity were kept constant. Overall, increasing tannic acid concentrations resulted in decreasing particle sizes (Fig. 1E). In addition, the particles’ specific activity could be varied by increasing the indium-111 activity during the synthetic steps (Fig. 1F). To confirm that the indium is stably bound to the gold nanoparticles during all procedures, non-radioactive gold nanoparticles labeled by indium115 were used. Gold-nanoparticle samples before and after purification as well as after the ligand exchange reaction demonstrated a stable elemental composition by elemental analysis (ICP-MS) (Fig. 1G). Indium-111 labeled gold nanoparticles with a size of 7 nm were selected for in-vitro and in-vivo evaluation as their size allows for background clearance by renal filtration [26]. Particles were
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Fig. 2. Characterization of the indium-111 labeled gold nanoparticles. TEM images and size distribution measurements of the indium-111 labeled gold nanoparticles demonstrated an average particle size of 6.9 0.9 nm (A). TEM images of the RGD-modified indium-111 labeled gold nanoparticles negatively stained with 2% uranyl acetate displayed the polymer layer visible as a light halo surrounding the particle gold core (B). Hydrodynamic size measurements showed narrowly distributed indium-111 labeled gold particles of w7e8 nm before and after purification (C). The absorbance spectra of the indium-111 labeled gold particles before and after purification showed a stable maximum at 524 nm (D). High serum stability of the indium-111 labeled gold particles was found in the presence of human blood serum at up to 48 h (E). The DLS showed that functionalization of the particle surface with RGD ligands resulted in a 3e4 nm increase of the hydrodynamic diameter (F). UVeVis spectroscopy of the RGD-modified indium-111 labeled gold particles in the presence of human blood serum revealed a stable SPR peak at up to 48 h, indicating the absence of aggregation (G).
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Fig. 3. In-vitro targeting with RGD-modified indium-111 labeled gold nanoparticles. RGD-modified indium-111 labeled nanoparticles with various RGD densities were added to different cells for in-vitro uptake quantification. Experiments with (þ) and without () the conditions are indicated in the table below the graph. Nanoparticles with RGDmodification showed higher uptake than particles without RGD-modification, particles modified with cyclic RGD showed higher uptake than particles modified with linear RGD, and particles showed higher uptake at higher density of RGD-modification. RGD-modified nanoparticles showed higher uptake in melanoma cells with high avß3 integrin expression (M21) compared to melanoma cells with low avß3 integrin expression (M21-L, blue bars). Specific uptake was verified by blocking of uptake with excess of free RGD peptide in the glioblastoma model (red bars). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
synthesized with a narrow size distribution (6.9 0.9 nm), as evidenced by TEM and particle distribution analysis (Fig. 2A). In accordance with TEM, DLS revealed similar average hydrodynamic diameters before (7.7 0.2 nm) and after purification (7.1 0.3, Fig. 2C). UVeVis spectroscopy demonstrated a 524 nm surface plasmon resonance (SPR) peak before and after purification (Fig. 2D), consistent with spherical gold nanoparticles of sizes below 10 nm [27]. The absence of red-shifts or broadening of the SPR peak indicated the absence of aggregation during particle purification [28]. A stable red shift of the SPR peak from 524 to 527 nm was found for the indium-111 labeled gold nanoparticles in the presence of human blood serum at 1, 4, 24, and 48 h (Fig. 2E). In addition, radioTLC confirmed radiochemical purity yields over 95%, which maintained over 48 h in the presence of human blood serum (Supplementary Data, Fig. S1), confirming the stability of indium111 labeled gold nanoparticles also under physiological conditions. Furthermore, using strong chelating agents to capture any free radio-indium, radio-TLC confirmed that the indium-111 is indeed stably bound to the nanoparticles (Supplementary Data, Fig. S1).
3.2. Indium-111 labeled gold nanoparticles allow facile modification with tumor targeting ligands Capping peptides and PEGylated alkanethiols were used to link the targeting ligands onto the surface of gold nanoparticles. For this purpose, the surface of indium-111 labeled gold nanoparticles was modified with a mixture of HS-C11-EG4-OH and either Cap-cRGD or Cap-lRGD ligands (Fig. 1A). The 4 unit PEG spacer EG4 between the capping peptide sequence and the RGD targeting ligand was used to
extend the ligand from the nanoparticle surface. The ratio of either Cap-cRGD or Cap-lRGD ligands incorporated on the nanoparticle surface was varied from 0 to 40 mol% with respect to the HS-C11EG4-OH ligand. TEM images of the negatively stained RGD-modified indium-111 labeled gold nanoparticles revealed a light halo of low electron density surrounding the denser gold nanoparticle core (Fig. 2B), indicating the presence of a thin layer on the surface of the nanoparticle representing the surface modification [29,30]. Surface modification of the particle with the RGD ligands resulted in a 3e4 nm increase of the hydrodynamic diameter for all sets of synthesized nanoparticles in the DLS measurements (Fig. 2F). Ligand exchange reaction was confirmed by shifted SPR peaks from 524 for the citrate and tannic acid ligands (Fig. 2D) to 527 nm after replacement by thiol-containing ligands (Supplementary Data, Fig. S2). The absence of SPR peak broadening further confirmed the particle stability after the ligand exchange reactions. UVeVis spectroscopy of the RGD-modified indium-111 labeled gold particles in the presence of human blood serum revealed a stable SPR peak over a period of 48 h (Fig. 2G), indicating the effectiveness of the surface modification with thiol-containing ligands in preventing aggregation. Amino acid analysis revealed a linear correlation between the amount of RGD ligands used for the ligand exchange reaction and the amount finally present on the particle surface (slope: 0.53, Supplementary Data, Fig. S3).
3.3. RGD-modified indium-111 labeled gold nanoparticles allow targeting of avß3 integrin in-vitro Internalization experiments were performed with indium-111 labeled gold nanoparticles without (0%RGD) or with RGD-
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Fig. 4. In-vivo targeting with RGD-modified indium-111 labeled gold nanoparticles. Biodistribution studies were performed in nude mice bearing M21, M21-L and U87 tumor xenografts, and are displayed as percent injection dose per gram of organ weight (%ID/g). The organ (A) and tumor biodistribution (B) demonstrated higher tumor uptake with RGDmodified indium-111 labeled gold nanoparticles when compared with the non-targeted nanoparticles in the U87 tumor model. SPECT/CT imaging (C) demonstrated higher uptake of RGD-modified indium-111 labeled gold nanoparticles in the M21 tumor (left) compared to the M21-L tumor (right).
modifications of various amounts (10, 20, 30, 40% Cap-cRGD or 20% Cap-lRGD, Fig. 3). The highest uptake in M21 cells was found for particles with 30% surface modification of cyclic RGD (30%CapcRGD), while the highest uptake in U87-MG cells was found for particles with 40% surface modification of cyclic RGD (40%CapcRGD). At these conditions, RGD-modified particles showed higher uptake in the high avß3 integrin expressing M21 cells than in the low avß3 integrin expressing M21-L cells (p ¼ 0.04) and particle uptake in U87-MG could be blocked with an excess of cold linear RGD peptide (p ¼ 0.006), both indicating avß3 integrin-specific uptake. Particles modified with cyclic RGD (10, 20, 30, and 40% Cap-cRGD) generally showed higher uptake than particles modified with linear RGD (20%Cap-lRGD) or the negative controls, particles without RGD (0%RGD). The cell bound fraction was below 1% for all tested particles in each cell line. 3.4. RGD-modified indium-111 labeled gold nanoparticles for invivo imaging of avß3 integrin expressing tumors Tumor uptake and biodistribution of indium-111 labeled gold nanoparticles with and without RGD modification was assessed in 6 week old female athymic Nude-Foxn1nu mice in the human melanoma and glioblastoma model (Fig. 4A and B). Mice were bearing a M21 xenograft on the left leg and a M21-L xenograft on the right leg or were bearing a single U87-MG xenograft on the left leg. Corresponding to the in-vitro results, particles modified with cRGD showed higher uptake in the high avß3 integrin expressing tumors (M21, 0.52 0.21 %ID/g) than in the low avß3 integrin expressing tumors (M21-L, 0.39 0.14 %ID/g), indicating avß3 integrin-specific tumor binding. Coherently, U87-MG tumors showed higher uptake of nanoparticles with versus without cRGD-modification (0.93 0.14 %ID/g versus 0.37 0.04 %ID/g, p ¼ 0.003). Finally, in line with the biodistribution results, SPECT/CT imaging demonstrated higher uptake of cRGD-modified indium-111 labeled gold nanoparticles in the M21 tumor (left) compared to the M21-L tumor (right, Fig. 4C).
4. Discussion The present work demonstrates the feasibility of molecular targeting with directly radiolabeled gold nanoparticles. It describes the design and synthesis of a modular gold nanoparticle platform stably labeled with indium-111, the surface modification with targeting moieties and that such modified particles can be used for molecular targeted imaging in a melanoma and a glioblastoma model. The direct incorporation of indium-111 allowed stable radiolabeling of the gold nanoparticle with varying activities. The modification with targeting motifs, which were spatially separated from the radioisotope, enabled the use of the nanoparticle platform for molecular targeting in-vitro and in-vivo. Direct radiolabeling of the gold nanoparticle cores by radioisotopes represents a concept that allows stable radiolabeling without the necessity of additional surface modifications to accommodate chelators for subsequent labeling. Previous approaches with surface modifications for radiolabeling as DTPA, DOTA or desferroxamin required harsh radiolabeling conditions [31-34], which are incompatible with the use of commonly used peptide or antibody based targeting motifs and can cause colloidal instability. Furthermore, surface modifications for radiolabeling have shown to interfere with the particles’ pharmacological profile [34]. Direct integration of radioisotopes into gold nanoparticles represents an efficient way to avoid the use of metal chelators and harsh radiolabeling procedures and to spare the entire particle surface for modifications with targeting motifs. The feasibility of directly integrating radioisotopes into gold particles was recently demonstrated using gold-198 and copper-64 [35,36]. The copper-64 labeled gold nanoparticles accumulated in breast cancer xenografts due to enhanced permeability and retention, while the gold-198 labeled gold nanoparticles were directly injected into prostate cancer xenografts. The present results confirm the feasibility of the direct labeling approach with indium-111, a long half-life g-emitter that is clinically established for molecular imaging. The long half-life of the radioisotope used in
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this study allowed the modification of the spared nanoparticle surface with targeting motifs. The results of the in-vivo studies demonstrate the potential of gold nanoparticles with integrated radioisotopes for molecular targeting after intravenous injection in two independent tumor models. The indium-111 labeled gold nanoparticles that were suitable for intravenous injection were synthesized using a simple procedure and the stability of the indium-111 integrated with the gold nanoparticles was confirmed by inorganic elemental analysis before and after surface modification with RGD-containing ligands. Colloidal stability of the surface modified gold nanoparticles was demonstrated by DLS. Overall, the synthetic procedure allowed controlling the specific radioactivity of the indium-111 labeled gold nanoparticles by varying the initial activity of the 111InCl3 during synthesis, which allows tailoring the particles’ activity to the biological application. The in-vitro studies demonstrated the molecular targeting capabilities of the RGD-modified indium-111 labeled gold nanoparticles in two different human cell lines expressing the avß3 integrin. Specificity of avß3 integrin mediated uptake was demonstrated by reduced uptake with particles not modified with RGD, in cells with low avß3 integrin expression and by blocking experiments using an excess of cold RGD, respectively. The absence of chelating groups for radiolabeling allowed testing a range of different RGD densities on the nanoparticle surface, thereby higher uptake was found with increasing amounts of RGD present on the nanoparticle surface. These results indicate that indium-111 labeled gold nanoparticles represent a valuable universal platform for refining the use of various targeting motifs, potentially facilitating the use of targeting concepts that could not be successfully used with nanoparticles yet. The biodistribution studies confirmed the targeting capabilities of the RGD-modified indium-111 labeled gold nanoparticles in-vivo. RGD-modified indium-111 labeled gold nanoparticles showed higher uptake than the non-targeted nanoparticles in the glioblastoma model. Additionally adrenal glands, which physiologically express high amounts of avß3 integrin [37], as well as the liver and spleen, known for opsonization and mononuclear phagocytosis, showed uptake of the nanoparticle [38,39]. Finally, the RGDmodified indium-111 labeled gold nanoparticles were successfully used for in-vivo imaging of tumors overexpressing avß3 integrins after intravenous administration. Non-radioactive gold nanoparticles are gaining increasing attention for their clinical use as drug carriers, photo-thermal agents, contrast agents and radio-sensitizers [13,14]. However, their pharmacokinetics and biodistribution are key issues that need to be clarified before they will enter routine clinical use. The use of indium-111 is already clinically established to assess pharmacokinetics and biodistribution e.g. of CD20 antibodies, as work-up for CD20-based immune-radiotherapy in patients with non-Hodgkin’s lymphoma. Similarly, indium-111 labeled gold nanoparticles might represent a powerful tool to image and quantify the pharmacokinetics and biodistribution of gold nanoparticles and facilitate a faster transition into the clinic. 5. Conclusion The present work describes the design, synthesis and biological evaluation of gold nanoparticles with stably integrated indium-111 as a universal modular platform for tracking and molecular targeted imaging. The platform fulfills key criteria for successful molecular imaging, such as high labeling yield, high labeling stability and low interference with the biological properties of the targeting molecule. The half-life of indium-111 allowed for modification of the nanoparticle with targeting motifs, and the animal studies
demonstrated the feasibility of molecular targeting with directly radiolabeled gold nanoparticles. Acknowledgments The authors gratefully acknowledge funding from the Swiss National Science Foundation (PZ00P3/126616) and the Bernese Cancer League. Furthermore, the authors are grateful to Andrew Q. Tran for his support in designing the figures and for the input of Andrea Grotzky, Renzo Cescato and Piotr Radojewski into the manuscript. All authors declare no potential conflict of interest. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2014.04.098. References [1] Jaffer FA, Weissleder R. Molecular imaging in the clinical arena. J Am Med Assoc 2005;293:855e62. [2] Velikyan I. Molecular imaging and radiotherapy: theranostics for personalized patient management. Theranostics 2012;2:424e6. [3] Bergstrom M, Grahnen A, Langstrom B. Positron emission tomography microdosing: a new concept with application in tracer and early clinical drug development. Eur J Clin Pharmacol 2003;59:357e66. [4] Frangioni JV, Hajjar RJ. In vivo tracking of stem cells for clinical trials in cardiovascular disease. Circulation 2004;110:3378e83. [5] Janowski M, Bulte JWM, Walczak P. Personalized nanomedicine advancements for stem cell tracking. Adv Drug Deliv Rev 2012;64:1488e507. [6] Van de Wiele C, Lahorte C, Oyen W, Boerman O, Goethals I, Slegers G, et al. Nuclear medicine imaging to predict response to radiotherapy: a review. Int J Radiat Oncol Biol Phys 2003;55:5e15. [7] Marchalonis J. An enzymic method for the trace iodination of immunoglobulins and other proteins. Biochem J 1969;113:299e305. [8] Varagnolo L, Stokkel MPM, Mazzi U, Pauwels EKJ. 18F-labeled radiopharmaceuticals for PET in oncology, excluding FDG. Nucl Med Biol 2000;27:103e12. [9] Reichert DE, Lewis JS, Anderson CJ. Metal complexes as diagnostic tools. Coord Chem Rev 1999;184:3e66. [10] Reubi JC, Schar JC, Waser B, Wenger S, Heppeler A, Schmitt JS, et al. Affinity profiles for human somatostatin receptor subtypes sst1-sst5 of somatostatin radiotracers selected for scintigraphic and radiotherapeutic use. Eur J Nucl Med 2000;27:273e82. [11] Adam MJ, Wilbur DS. Radiohalogens for imaging and therapy. Chem Soc Rev 2005;34:153e63. [12] Walrand S, Barone R, Pauwels S, Jamar F. Experimental facts supporting a red marrow uptake due to radiometal transchelation in 90Y-DOTATOC therapy and relationship to the decrease of platelet counts. Eur J Nucl Med Mol Imaging 2011;38:1270e80. [13] Libutti SK, Paciotti GF, Byrnes AA, Alexander Jr HR, Gannon WE, Walker M, et al. Phase I and pharmacokinetic studies of CYT-6091, a novel PEGylated colloidal gold-rhTNF nanomedicine. Clin Cancer Res 2010;16:6139e49. [14] Weintraub K. Biomedicine: the new gold standard. Nature 2013;495:S14e6. [15] Hicks RJ. Use of molecular targeted agents for the diagnosis, staging and therapy of neuroendocrine malignancy. Cancer Imaging 2010;10(Spec no A): S83e91. [16] Lin FI, Iagaru A. Current concepts and future directions in radioimmunotherapy. Curr Drug Discov Technol 2010;7:253e62. [17] Montet X, Funovics M, Montet-Abou K, Weissleder R, Josephson L. Multivalent effects of RGD peptides obtained by nanoparticle display. J Med Chem 2006;49:6087e93. [18] Gaertner FC, Kessler H, Wester HJ, Schwaiger M, Beer AJ. Radiolabelled RGD peptides for imaging and therapy. Eur J Nucl Med Mol Imaging 2012;39(Suppl. 1):S126e38. [19] Slot JW, Geuze HJ. A new method of preparing gold probes for multiplelabeling cytochemistry. Eur J Cell Biol 1985;38:87e93. [20] Duchesne L, Gentili D, Comes-Franchini M, Fernig DG. Robust ligand shells for biological applications of gold nanoparticles. Langmuir 2008;24:13572e80. [21] Felding-Habermann B, Fransvea E, O’Toole T, Manzuk L, Faha B, Hensler M. Involvement of tumor cell integrin avb3 in hematogenous metastasis of human melanoma cells. Clin Exp Metastasis 2002;19:427e36. [22] Cheresh DA, Spiro RC. Biosynthetic and functional properties of an Arg-GlyAsp-directed receptor involved in human melanoma cell attachment to vitronectin, fibrinogen, and von Willebrand factor. J Biol Chem 1987;262: 17703e11. [23] Li Z-B, Cai W, Cao Q, Chen K, Wu Z, He L, et al. 64Cu-labeled tetrameric and octameric RGD peptides for small-animal PET of tumor avb3 integrin expression. J Nucl Med 2007;48:1162e71.
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