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PET imaging with multimodal upconversion nanoparticles† Juan Gallo,a,b Israt S. Alam,a Jiefu Jin,c Yan-Juan Gu,d Eric O. Aboagye,a Wing-Tak Wong*d and Nicholas J. Long*a,b A series of new upconversion nanoparticles have been functionalised with tumour-targeting molecules and metal chelates, prepared following standard peptidic and thiol chemistry. The targeting strategy has been delivered via the αvβ3 integrin, which is a heterodimeric cell surface receptor that is up-regulated in a variety of cancers, such as melanoma and breast cancer. The well-known DOTA (1,4,7,10-tetraazacyclo-
Received 1st November 2013, Accepted 7th February 2014
dodecane-1,4,7,10-tetraacetic acid) motif allows coordination to the radionuclide
68
Ga. Radiolabelling
DOI: 10.1039/c3dt53095g
experiments were optimised under relatively mild conditions, and are rare amongst nanoparticulate materials. In vivo application of these probes in mouse tumour models revealed their potential as specific
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cancer contrast agents for PET imaging.
Introduction Within molecular imaging, an aspect that is gaining increasing attention is the integration of two or more imaging modalities into a single probe.1–4 Motivation for this research interest comes from the fact that the performance of individual imaging modalities is often not completely satisfactory. For example, magnetic resonance imaging (MRI) gives good spatial resolution but its sensitivity is low; positron emission tomography (PET) and single-photon emission computed tomography (SPECT) have excellent sensitivity, but resolution can be an issue; optical imaging has great sensitivity and ex vivo resolution, but in vivo studies can present problems with tissue penetration and resolution.5,6 Accordingly, a contrast agent able to produce a signal within two or more of these modalities could integrate the strengths of each whilst helping to overcome their individual limitations.1 Although there have been reports on the preparation of multimodal probes with small molecules,1,7–12 much of the work in this area has focused on the development of nanoparticle based multimodal contrast agents.6,13–25 The inherent imaging properties of
a Comprehensive Cancer Imaging Centre, Department of Surgery and Cancer, Hammersmith Campus, Imperial College London, Du Cane Road, London, W12 0NN, UK. E-mail: [email protected] b Department of Chemistry, Imperial College London, South Kensington, London, SW7 2AZ, UK c Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong, SAR China d Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, SAR China † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c3dt53095g
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certain types of nanocrystals (e.g. magnetic nanoparticles, upconverting nanoparticles, quantum dots) together with the large number of surface ligands or reactive groups per nanoparticle, allows for a relatively easy integration of a second reporter moiety onto a single platform. However, the integration of a further modality usually requires complex design and chemistry.26 Upconversion nanoparticles (UCNPs) show anti-Stokes luminescence, where NIR (near-infrared) photons are absorbed in a successive manner for conversion into visible light. Biological tissues present minimal absorption, scattering and emission of NIR wavelengths (in the range 700 to 1000 nm). Also, these wavelengths present deeper tissue penetration and little tissue photo-damage.27 Thus, UCNPs can be functionalised with targeting molecules and used not only for diagnostic purposes but also as a fluorescent guide in surgical interventions.28–36 The availability of 68Ga from in-house modern 68Ge/68Ga generators, independent of an on-site cyclotron, has made this isotope an attractive alternative to 18F radiolabelling.37–42 Its half-life, of around 68 min, makes it ideal from a chemistry point of view, as 2 or 3 elutions can be performed per day (with 3–5 hours separation between each elution). In this paper, we present a radiolabelling extension to our previously-reported αvβ3 integrin targeted dual-MRI/optical probe. The αvβ3 integrin is a heterodimeric cell surface receptor that is up-regulated in a variety of cancers, such as melanoma and breast cancer.43 These receptors bind to extracellular matrix components such as fibronectin, via recognition of the arginine-glycine-aspartic acid (RGD) sequence.44 The preparation of this multimodal probe, rather than involving complex chemistry, keeps the design as simple and
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versatile as possible, allowing facile interchange between magnetic and radiochemical centres. The UCNPs functionalised with tumour-targeting molecules and metal chelates were prepared following standard peptidic and thiol chemistry. Radiolabelling with 68Ga as the radioactive positron emitter tracer was optimised under relatively mild conditions, and is rare amongst nanoparticulate materials. In vivo application of these probes in mouse tumour models revealed their potential as specific cancer contrast agents for PET imaging.
Materials and methods Yttrium nitrate hexahydrate (Y(NO3)3·6H2O, 99.9%), sodium nitrate (NaNO3, ≥99.0%), PEI ( polyethyleneimine, branched, Mw ∼ 25 kDa), and 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were all purchased from SigmaAldrich. Ytterbium oxide (Yb2O3, 99.9%) and erbium oxide (Er2O3, 99.9%) were purchased from STREM. Nitric acid (HNO3, 69.5%, for trace analysis) and hydrochloride acid (HCl, 37%, for trace analysis) were purchased from Fluka. Ethylene glycol (EG, ≥99.0%) and ammonium fluoride (NH4F, laboratory reagents) were purchased from Acros Organics and BDH Chemicals, respectively. 1,4,7,10-Tetraazacyclododecane1,4,7,10-tetraacetic acid mono (N-hydroxysuccinimide ester) (DOTA-NHS) was purchased from Macrocyclics. COOH-PEG-Fmoc (5 kDa) was purchased from Iris Biotech. 3-Maleimidobenzoic acid N-hydroxysuccinimide ester and Pierce BCA Protein Assay Kit were all purchased from Pierce. Cyclo (RGDyK) peptide was purchased from AnaSpec. All of the chemicals were used as received without further purification. Transmission electron microscopy (TEM) measurements were carried out on a transmission electron microscope (Philips, Tecnai 20) equipped with an energy-dispersive X-ray spectrometer (EDX; Hitachi HF-2000). The operating voltage of the microscope was 200 kV. Upconversion fluorescent spectra were obtained on LS-55B fluorescence spectrophotometer (PerkinElmer Corp., Forster City, CA), where an external 0–2 W adjustable laser integrated with an optical fibre (980 nm, Beijing Hi-Tech Optoelectronic Co., China) was used as the excitation source to replace the original Xenon source in the spectrophotometer. Dynamic laser scanning (DLS) measurements were performed on Malvern Zetasizer Nano ZS90. X-ray power diffraction (XRD) spectra were recorded on a Bruker D8 ADVANCE X-ray diffractometer. ICP-MS measurement was conducted on Agilent 7500 series ICP-MS (Agilent Technologies). Preparation of UCNP-NH2, UCNP-DO3A, and UCNP-DO3A-RGD UCNP-NH2 was prepared via a previously reported hydrothermal method with slight modification.45 Briefly, Yb2O3 (85.1 mg, 0.216 mmol), Er2O3 (6.94 mg, 0.018 mmol), and Tm2O3 (2.32 mg, 0.006 mmol) were dissolved in 10% hot HNO3 and the acidic solution was allowed to evaporate to dryness. Upon cooling, EG (80 mL) was poured in, followed by addition of Y(NO3)3·6H2O (0.7354 g, 1.92 mmol), branched PEI (1.0 g, 1.0 wt% solution in EG) and NaNO3 (0.408 g,
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4.8 mmol). The reaction mixture was heated to 80 °C for 10 min under vigorous stirring, and then an EG (10 mL) solution of NH4F (0.71 g, 19.2 mmol) was added drop-wise. After ageing at 80 °C for 10 min, the reaction mixture was transferred to a 100 mL Teflon-lined stainless steel autoclave and treated at 180 °C for 3 hours. After cooling to room temperature, the products were collected by centrifugation (10 min at 10 000 RPM), purified by the addition of ethanol to precipitate the particles followed by centrifugation and final redispersion in water. This process was repeated three times, and then the sample was dried overnight in a vacuum oven at 80 °C. For the preparation of UCNP-DOTA, 1 mg of UCNP-NH2 was sequentially reacted with a 435 μg (8.7 × 10−5 mmol) of Fmoc-PEG5000NHS, 35 μg (8.7 × 10−5 mmol) of DOTA-NHS and 20 μg (6.5 × 10−5 mmol) of MBS in 0.1 M HEPES buffer solution ( pH = 7.2) at room temperature (r.t.). The molar ratio of NH2/PEG/DOTA/ MBS was chosen as 1 : 1 : 1 : 0.75. The reaction mixture was subjected to purification by ultracentrifugation (Centriprep YM-10, 10 kDa, Millipore) to remove any unreacted reagent residues. Then UCNP-DOTA could be obtained as a white solid after purification and lyophilization. For the preparation of UCNP-DOTA-RGD, half of the UCNP-DOTA powder was redispersed in 0.1 M HEPES ( pH = 7.2) buffer solution and then further reacted with excess RGD-SH. UCNP-DOTA-RGD was obtained as a white solid after ultracentrifugation purification and lyophilization. Preparation of RGD-SH 168 μg (3.64 × 10−4 mmol) of CO2H-PEG4-SPDP were activated with EDC (77 μg, 4.00 × 10−4 mmol) and NHS (96 μg, 8.37 × 10−4 mmol) in water (500 μL). After 90 min the solution was added onto a water solution (500 μL) of the cRGDfK peptide (200 μg, 3.31 × 10−4 mmol) and the mixture was allowed to react overnight. Finally, to ‘deprotect’ the thiol group in the SPDP group, NaBH4 in water (14 μg, 3.64 × 10−4 mmol) was added into the solution right before the reaction with the nanoparticles. The product was used without further purification. 68
Ga radiolabelling of UCNPs-DO3A and UCNPs-DO3A-RGD
Both targeted and control nanoparticles were labelled in the same way. 300 μg of UCNPs in 15 μL distilled H2O were mixed with 100 μL of 0.2 M NaOH solution. 300 μL of 68GaCl3 solution in 0.1 M HCl (250 μCi) was added and the pH was corrected to the desired value with 0.2 M NaOH. The sample was incubated at 90 °C for 10 min and then purified by centrifugal filtering (MWCO 100 000 Da). After centrifugation, the activity of both filter and filtrate were measured. Labelled UCNPs were recovered from the filter and reconstituted in saline. Finally the activity of product, and filter were measured again to calculate radiochemical yields. Cell culture Human melanoma M21 cells (kind donation from Dr Amin Hajitou, Imperial College London, UK) were grown in DMEM
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media, supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U mL−1 penicillin and 100 μg mL−1 streptomycin (Invitrogen, Paisley, Refrewshire, UK). M21 cells are a αvβ3 integrin expressing line.44 Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2. Human glioblastoma U87MG cells and human prostate carcinoma PC-3 cells were maintained in MEM (Eagle’s Minimum Essential Medium, Invitrogen), supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin at 37 °C and in a humidified atmosphere of 5% CO2. Upconversion confocal cell imaging U87MG (or PC-3) cells were seeded on sterile glass coverslips in glass-bottomed 35 mm tissue culture dish. Upconversion confocal cell imaging was conducted on Leica TCS SP5. The excitation at 980 nm was provided from a femtosecond Ti:sapphire pulsed laser. The green emissions of 520–580 nm and red emissions of 630–680 nm were acquired by PMT. The incubation concentrations of Y in all the samples were set at 1 mM. After washing with PBS, cells-adhered cover-slips were mounted onto slides and immersed with CO2 independent medium (Invitrogen) for imaging experiments. In vivo PET imaging Dynamic 68Ga imaging scans were carried out on a dedicated small animal PET scanner (Siemens Inveon PET module, Siemens Medical Solutions USA, Inc., Malvern, PA, USA) following a bolus i.v. injection in tumor-bearing mice of ∼2 MBq. Dynamic scans were acquired in list-mode format over 60 min. The acquired data were then sorted into 0.5 × 0.5 × 0.5 mm bins and 19 time frames for image reconstruction (4 × 15 s, 4 × 60 s, and 11 × 300 s), which was done by 2D-ordered subset expectation maximization (OSEM) reconstruction. The Siemens Inveon Research Workplace software was used for visualization of radiotracer uptake in the tumor; 30 to 60 min cumulative images of the dynamic data were employed to define 3-dimensional (3D) regions of interest (ROIs). For each animal ROIs encompassing the tumor and other tissues were manually drawn on the summed PET image and used to compute time-activity-curves (TACs). Both tumor and tissue TACs were normalized to injected dose, measured by a VDC-304 dose calibrator (Veenstra Instruments, Joure, The Netherlands), and expressed as percentage injected dose per mL of tissue (%ID mL−1). Biodistribution studies For the biodistribution study, mice were maintained under anesthesia following their PET scan and sacrificed by exsanguination via cardiac puncture at 60 min post radiotracer injection to obtain blood, plasma, urine, heart, lung, liver, kidney, muscle and tumour. Tissue radioactivity was determined on a gamma counter (Cobra II Auto-Gamma counter, Packard Biosciences Co, Pangbourne, UK) and decay corrected. Data were expressed as percent injected dose per gram of tissue.
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Results and discussion UCNPs preparation High quality upconversion nanoparticles were prepared as previously described.46 A hydrothermal method was followed using Yb2O3, Er2O3, Tm2O3, Y(NO3)3·6H2O, NaNO3 and NH4F as starting materials. Polyethyleneimine (PEI) was used as capping ligand. The resulting UCNPs were characterised by ICP-MS and EDXS to be formulated as NaYF4:Yb/Er/Tm (18 : 1.5 : 0.5 mol%). Electron microscopy analysis showed that the particles were highly crystalline, quasi-spherical, and presented an average diameter of 36 nm (Fig. 1). Bulk hexagonal NaYF4 has been reported to be the most efficient host material for green (Yb3+/Er3+ doped) upconversion fluorescence,47 but green emission is not ideal for deep tissue imaging.27 Therefore, PEI was selected as the coating agent as it suppresses NaYF4 hexagonal phase by competing for fluorides against lanthanides,45 and so NIR-to-deep-red energy transfer48 can be harvested with higher yields. Deep red fluorescence (660 nm for Er3+) lies in the optimal spectrum region for biomedical applications as it is both visible to the naked eye and approachable to deep tissues. XRD (X-ray diffraction) and SAED (selective area electron diffraction) demonstrate that the NaYF4 structure corresponds to a cubic phase and not a hexagonal one (JCPDS card no. 77-2042). Surface functionalization chemistry Before further functionalization, the number of available amines per nanoparticle was characterised by reaction with SPDP (3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester). After purification and reduction, release of pyridine2-thione was followed by UV-Vis spectroscopy (λ = 343 nm, ε = 8080 M cm−1). In this way the number of available amines per nanoparticle was calculated to be around 6000. The nanoparticles were then functionalised with PEG molecules, DOTA chelates and RGD peptides. PEG molecules have been reported to enhance the blood half-life of particles in vivo49 and DOTA can be used to chelate reporter metals for MRI (Gd3+) and PET (68Ga3+). In addition, the RGD-integrin αvβ3 system has been widely explored for the labelling of tumours. Firstly, pre-activated DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, with only one of the four carboxylic groups activated) and carboxy-PEG molecules with a molecular weight of 5000 Da, were coupled to the amine groups on the surface of the nanoparticles. After purification, the remaining free amine groups from the PEI coating of the particles were reacted with the small molecule MBS (3-maleimidobenzoic acid N-hydroxysuccinimide ester). This molecule has an activated carboxylic group on one side ready to react with any amine, while on the other side of the molecule it displays a maleimide group that can react with any free thiol. Meanwhile in a different reaction vessel, cRGDfK peptides were modified with a short PEG molecule (PEG4) ending in a SPDP (3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester) group. The SPDP presents a disulfur bond that upon reduction provides a free thiol group and releases a pyridine-2-thione
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Fig. 1 (A) Overview TEM micrograph of PEI protected UCNPs. (B) TEM micrograph of UCNPs-DO3A-RGD. Inset, SAED pattern obtained from the same sample. (C) XRD spectra of as prepared PEI protected UCNPs compared to the line pattern of the calculated cubic phase of NaYF4 (JCPDS no. 77-2042). (D) Overview TEM micrograph of DOTA functionalised PEI protected UCNPs (UCNPs-DO3A-RGD). (E) TEM micrograph of UCNPs-DO3A. (F) Hydrodynamic sizes measurement before (dotted line) and after (solid line) the functionalisation of PEI protected UCNPs with DOTA and RGD peptides.
molecule that is enabled to bind to thiols or maleimides. In a final step, the modified cRGDfK peptide was mixed with the nanoparticles under reducing conditions (NaBH4) to obtain the final multimodal RGD targeted upconversion nanoparticles. The yield of this final step was measured, using standard protein characterisation protocols (Bradford method50), to be above 95%, meaning that there were an average of 102 RGD peptides per particle (Scheme 1). ICP-MS measurements were used to estimate the number of DO3A and PEG molecules per particles.46 On average there were around 4500 PEG molecules and 1200 DO3A molecules per particle. TEM micrographs acquired after the functionalization showed no changes in the UCNPs in shape or size (Fig. 1). Finally the hydrodynamic diameter of both the initial UCNPs-NH2 and the final targeted UCNPs-DO3A-RGD were measured by dynamic light scattering in water (Fig. 1F). According to the results, after the functionalization of the particles their hydrodynamic size increased from an average of 83.8 nm to 93.6 nm.
at 300 mW, and both solutions, UCNP-NH2 and UCNPDO3A-RGD, were prepared to contain the same weight concentration of UCNP core (1 mg mL−1). Both solutions emitted orange light upon irradiation coming from the combined green and red luminescence from Er3+ ions plus the blue luminescence from Tm3+ ions. The upconversion luminescence spectra of UCNP-NH2 and UCNP-DO3A-RGD solutions are shown in Fig. 2. The typical upconversion luminescence peaks of Er3+ and Tm3+ can be observed. 4S3/2–4I15/2 and 4F9/2–4I15/2 transitions of doped Er3+ ions account for the green peak at 543.5 nm and red peak at 658.1 nm respectively, while 1G4–3H6 and 3H4–3H6 transitions of doped Tm3+ are responsible for the blue peak at 475.7 nm and NIR peak at 795.8 nm, respectively.45 Compared with UCNP-NH2, the emission intensity of Er3+ in UCNP-DO3A-RGD was slightly lower, while the emission intensity of Tm3+ remains unchanged. It is thought that this is due to Er3+ emissions of UCNPs being more vulnerable to non-radiative energy transfer and vibrational quenching by the various ligands on the surface of the nanocrystals.
Upconversion measurements
Radiochemistry
The upconversion properties of UCNP-NH2 and UCNPDO3A-RGD were characterised in solution. For these studies, an optic-fibre coupled diode laser emitting at 980 nm was set
Once the nanoparticles were fully characterised, their ability to complex 68Ga was tested. To date, only a few reports have been published on multimodal imaging (including PET) with
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Scheme 1
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Preparation of the targeted multimodal upconversion nanoparticles.
Fig. 2 (A) Upconversion luminescence spectra of PEI-protected upconversion nanoparticles (UCNP-NH2), and DOTA and PEG-functionalised upconversion nanoparticles (UCNP-DO3A-RGD) in water solutions.
UCNPs. Some of these reports have used 18F as the radioisotope.51,52 Only a few examples have shown the application of nanoprobes for 68Ga chelation. Stelter and co-workers53 directly adsorbed 68Ga onto the surface of silica nanoparticles. Both Hwang et al.26 and Jeon et al.54 used NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) as 68Ga chelator on silica nanoparticles, and Lee et al. also used NOTA as chelator on QDs. While the direct unspecific adsorption of 68Ga is not the best option for future potential translation into human health (due to stability concerns), NOTA has been reported to form very stable complexes with Ga3+ (log K = 30.9855). In this work, we have chosen to use DOTA instead of NOTA because although it forms less stable chelates with Ga3+ (log K =
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21.3355), they are still stable enough to be used in vivo. DOTA presents the extra advantage in that it is already approved for its use in human health application, in particular as Gd3+ chelator for MRI applications.7 The first attempts to label the nanoparticles with 68Ga were not successful. Standard labelling conditions for the labelling of DOTA on small molecules56 were used: sample dissolved in 0.2 M pH 4 acetate buffer, 68Ga in 0.1 M HCl solution, 90 °C for 10 min. With the low volumes used (15 μL sample in water, 150 μL acetate buffer, and 300 μL 68 Ga elute in HCl), the pH of the solution during the incubation was around 1. At this pH, the amine groups on the nanoparticles become protonated57 and an electrostatic repulsion occurs between the –NH3+ groups on the nanoparticles and the 68 Ga3+. This effect presumably accounts for the very low labelling yields. The labelling reaction was therefore, studied at different pH to determine the optimal conditions for the radiolabelling (Table 1). The yield of the labelling reaction consistently increased with a rise in pH from around 5% at pH 1 to 87% at pH 6. Although pH 6 gave the highest yield, pH 5 was selected for the labelling studies as at pH values above 6.3,58 Ga3+ forms Ga(OH)3 which is insoluble in water. These experiments were carried out with control nanoparticles i.e. functionalised in the way as described above, but without RGD
Table 1 Yields of the radiochemical labelling of DOTA functionalised UCNPs, and control UCNPs (without DOTA) at different pH
pH
% Labelling DO3A UCNPs
% Labelling control UCNPs
1 3 4 5 6
5.56 ± 0.52 10.14 ± 1.53 20.46 ± 0.20 38.42 ± 0.87 87.49 ± 2.36
4.62 ± 0.63 5.25 ± 0.26 4.99 ± 0.62 5.31 ± 0.82 8.72 ± 1.25
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peptides. The final corrected radiochemical yield for these control nanoparticles was 38.4% with a specific activity of 8 × 1012 μCi mol−1, while the corrected radiochemical yield of the targeted nanoparticles was lower, at 15.2% with a specific activity of 5.1 × 1012 μCi mol−1. In vitro confocal imaging The ability of functionalised nanocrystal to target and label cells was assessed in vitro. Two different carcinoma cell lines with different expression levels of αvβ3 integrin, U87MG and PC-3 cells, were used. Live U87MG cells were incubated with the nanoprobes at 1 mM Y for 4 h. Green and red upconversion luminescence and bright-field micrographs are shown in Fig. 3. First, PC-3 cells were used as control. As compared with U87MG cells, PC-3 cells express a low level of αvβ3 integrin. This low expression of integrin correlates well with the confocal images acquired after the incubation with the UCNPs showing no difference in signal between RGD and non-RGD functionalised particles. On the other hand, images obtained from U87MG cells incubated under the same conditions with
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UCNP-DO3A-RGD (Fig. 3, third column) present numerous bright green and red luminescent spots, while the signal intensity coming from the same cells when incubated with UCNP-DO3A (Fig. 3, fourth column) is significantly lower. These results suggest that the cellular labelling of UCNPDO3A-RGD is greatly enhanced due to the selective targeting of integrin αvβ3 receptors on U87MG cell surface. To further support this finding, U87MG cells were incubated at 4 °C in the presence of either UCNP-DO3A or UCNP-DO3A-RGD, and subsequently imaged after washing. The images (Fig. S1†) show higher signal intensity in the case of RGD functionalised UCNPs, as they are able to target the integrin on the surface of the cells, in a process independent of cell internalisation. In vivo PET imaging Following the promising in vitro results, the nanoparticles were tested in vivo as targeted PET probes. In this case a wellestablished mouse model for integrin expression was used.59 Fig. 4A and 4B depicts typical transverse PET image slices demonstrating localisation of the targeted and control tracer
Fig. 3 Upconversion confocal live cell imaging. Green (upper row) and red (middle row) upconversion luminescence and bright-field (lower row) images of PC-3 (first and second columns) and U87MG cells (third and fourth columns) treated with UCNP-DO3A-RGD (first and third columns) or UCNP-DO3A (second and fourth columns) at 37 °C for 4 h. The incubation concentration of Y was set at 1 mM for all the samples (40× oil lens, zoom = 1, scale bar = 50 μm).
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Fig. 4 Representative axial 3D-OSEM PET-CT images of targeted UCNP (A) and control UCNP (B) in M21 tumour bearing mice, showing 30–60 minutes summed activity. The axial image of the targeted tracer (A) shows higher activity in the tumour compared to the axial view of the control tracer (B). The tumour margins, as identified by CT, are outlined in red. Corresponding coronal and sagittal 3D-OSEM PET-CT images of the targeted UCNP in the same M21 tumour bearing mice (C). The location of the heart (H), liver (L) and bladder (B) are indicated.
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Fig. 5 (A) Time versus radioactivity curves (TAC) obtained for the tumour from 60 min dynamic PET imaging for the targeted (diamonds) and control UCNP group (circles). Data represents mean %ID mL−1 values ± SEM, n = 3 per group. (B) Representative time versus radioactivity curves (TAC) obtained for targeted 68Ga-UCNP in a tumour-bearing mouse from 60 min dynamic PET imaging.
in M21 tumours (outlined in red). The highest tumour localisation was observed for the targeted agent characterised by a heterogeneous pattern of tracer uptake. In contrast to the targeted agent, uptake for the control agent is low and relatively homogeneous. Time versus radioactivity curves (TACs) obtained from ROI analysis of the tumours show that higher uptake is indeed observed with the RGD targeted nanoparticle (Fig. 5A, diamonds) than the control (Fig. 5A, circles). Mean NUV60 values were 5.53 ± 0.70 and 4.24 ± 0.17 for targeted and control nanoparticle respectively ( p = 0.0724). Both tracers show accumulation over the 60 minutes of imaging with no washout, and greater retention is observed with the targeted species. There is greater variation observed in the TACs and overall retention (%ID mL−1 values) of the individual subjects in the targeted group than observed in the control group. This accounts for the larger standard error bars seen in the mean TAC for the targeted UCNP (Fig. 5A, diamonds). Fig. 5B shows representative TACs for the targeted nanoparticle in tissues/organs. Time versus radioactivity curves (TACs) obtained for the targeted nanoparticle shows accumulation in the bladder, tumour and liver over time. Despite high activity in the bladder and accumulation of the tracer in the bladder over time, the kidney is not visualised in the PET images pointing to the likelihood of inefficient filtration of the radioactive nanoparticle complex, together with a cleavage event leading to elimination of a radiolabelled cleaved product into the bladder (Fig. 4C). Biodistribution The PET data were corroborated by ex vivo gamma counting of excised tissues, which demonstrated significantly higher uptake of the targeted UCNP in the tumour compared to control non-targeted UNCP (*P < 0.05) (Fig. 6C). The results show that the percentage injected dose per gram of tissue (%ID g−1) values at 60 minutes were 10.37 ± 1.58 and 5.61 ± 0.26 for the targeted and control nanoparticles respectively, a 1.84-fold difference. High activity in the urine is suggestive of
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renal excretion. High activity in plasma is indicative of long circulation times as expected from the PEG functionalization of the nanoparticles. Additionally %ID g−1 values were obtained for the liver, spleen, lung and kidney. High activity in the liver is visualised clearly in the PET-CT images (Fig. 4C). The presence of radioactive signal in the bladder at 60 min raised some concerns about the stability of nanoparticles. Due to the large size of these nanoprobes they are not expected to undergo renal excretion (the size limit for kidney excretion has been reported to be around 10 nm60,61). To assess that both components of the nanoparticles (UCNP core and DOTA-68Ga) were still together after the PET imaging, some of the organs were harvested, digested and analysed by ICP-MS to determine their Yb content coming from the UCNP core, having already demonstrated that the tissues contained 68Ga. The results, Fig. 6D, show increased levels of this lanthanide in the tissues, indication that the UCNPs also accumulated in those tissues. The RGD-targeted agent shows higher accumulation in integrin-positive melanoma tumours, thereby confirming functionality. There is larger variation in the %ID mL−1 and tracer accumulation for the targeted species but not the control agent. This may well be due to variation in integrin expression in the tumour as observed in previous studies using the same tumour model. The control agent also shows relatively high % ID mL−1 values in the tumour, likely to be due to EPR effects. The combination of PET imaging results, radioactivity biodistribution and ICP-MS organ analysis confirms the presence of both the UCNP cores and the PET reporter moiety in the tissues. Further experiments will be required to explain the levels of activity in the bladder and urine. The activity seems to leak slowly through the kidneys as they are not highlighted in the PET images.
Conclusions In this work we have demonstrated that nanoprobes are a valid platform for the chelation of radionuclides, such as 68Ga, to
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Fig. 6 Biodistribution data of (A) 68Ga-RGD targeted and (B) control UCNP in M21 tumour bearing mice 60 minutes post injection of tracer. SInt, small intestine; Lint, large intestine. C, 68Ga-RGD targeted and control UCNP retention in M21 tumours. (Data points represent mean ± SEM, N = 3, *P < 0.05.) D, results of the ICP-MS analyses for the content of Yb in tumour, spleen and liver of animals injected with targeted UCNPs.
multimodal MRI/optical nanoparticles. The incubation conditions were optimised to obtain effective radiochemical yields, while keeping preparation times as short as possible, and using low levels of radiation. The presence of DOTA-like chelates on the surface of the nanoparticles allows the versatile application of these nanocrystals either/both as MRI contrast agents (DO3A-Gd3+) or/and as PET tracers (DO3A-68Ga3+). The upconversion abilities of the nanoparticle core are confirmed, via histological analysis, and via MRI/PET to be diagnostic, and subsequently, have the potential to guide surgical interventions. Moreover, we have shown that the functionalization of these probes with targeting peptides enables their use as specific cancer diagnostic agents. In this case, cycloRGD peptides were chosen as cancer targeting agents, but the simple chemistry involved in their preparation allows the use of other biomolecules as targeting agents to broaden the field of application of these nanoprobes.
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Acknowledgements Funding for this project was provided from CRUK, EPSRC, MRC and the Department of Health, grant (C2536/A10337).
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Cite this: Dalton Trans., 2014, 43, 5535
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PET imaging with multimodal upconversion nanoparticles† Juan Gallo,a,b Israt S. Alam,a Jiefu Jin,c Yan-Juan Gu,d Eric O. Aboagye,a Wing-Tak Wong*d and Nicholas J. Long*a,b A series of new upconversion nanoparticles have been functionalised with tumour-targeting molecules and metal chelates, prepared following standard peptidic and thiol chemistry. The targeting strategy has been delivered via the αvβ3 integrin, which is a heterodimeric cell surface receptor that is up-regulated in a variety of cancers, such as melanoma and breast cancer. The well-known DOTA (1,4,7,10-tetraazacyclo-
Received 1st November 2013, Accepted 7th February 2014
dodecane-1,4,7,10-tetraacetic acid) motif allows coordination to the radionuclide
68
Ga. Radiolabelling
DOI: 10.1039/c3dt53095g
experiments were optimised under relatively mild conditions, and are rare amongst nanoparticulate materials. In vivo application of these probes in mouse tumour models revealed their potential as specific
www.rsc.org/dalton
cancer contrast agents for PET imaging.
Introduction Within molecular imaging, an aspect that is gaining increasing attention is the integration of two or more imaging modalities into a single probe.1–4 Motivation for this research interest comes from the fact that the performance of individual imaging modalities is often not completely satisfactory. For example, magnetic resonance imaging (MRI) gives good spatial resolution but its sensitivity is low; positron emission tomography (PET) and single-photon emission computed tomography (SPECT) have excellent sensitivity, but resolution can be an issue; optical imaging has great sensitivity and ex vivo resolution, but in vivo studies can present problems with tissue penetration and resolution.5,6 Accordingly, a contrast agent able to produce a signal within two or more of these modalities could integrate the strengths of each whilst helping to overcome their individual limitations.1 Although there have been reports on the preparation of multimodal probes with small molecules,1,7–12 much of the work in this area has focused on the development of nanoparticle based multimodal contrast agents.6,13–25 The inherent imaging properties of
a Comprehensive Cancer Imaging Centre, Department of Surgery and Cancer, Hammersmith Campus, Imperial College London, Du Cane Road, London, W12 0NN, UK. E-mail: [email protected] b Department of Chemistry, Imperial College London, South Kensington, London, SW7 2AZ, UK c Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong, SAR China d Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, SAR China † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c3dt53095g
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certain types of nanocrystals (e.g. magnetic nanoparticles, upconverting nanoparticles, quantum dots) together with the large number of surface ligands or reactive groups per nanoparticle, allows for a relatively easy integration of a second reporter moiety onto a single platform. However, the integration of a further modality usually requires complex design and chemistry.26 Upconversion nanoparticles (UCNPs) show anti-Stokes luminescence, where NIR (near-infrared) photons are absorbed in a successive manner for conversion into visible light. Biological tissues present minimal absorption, scattering and emission of NIR wavelengths (in the range 700 to 1000 nm). Also, these wavelengths present deeper tissue penetration and little tissue photo-damage.27 Thus, UCNPs can be functionalised with targeting molecules and used not only for diagnostic purposes but also as a fluorescent guide in surgical interventions.28–36 The availability of 68Ga from in-house modern 68Ge/68Ga generators, independent of an on-site cyclotron, has made this isotope an attractive alternative to 18F radiolabelling.37–42 Its half-life, of around 68 min, makes it ideal from a chemistry point of view, as 2 or 3 elutions can be performed per day (with 3–5 hours separation between each elution). In this paper, we present a radiolabelling extension to our previously-reported αvβ3 integrin targeted dual-MRI/optical probe. The αvβ3 integrin is a heterodimeric cell surface receptor that is up-regulated in a variety of cancers, such as melanoma and breast cancer.43 These receptors bind to extracellular matrix components such as fibronectin, via recognition of the arginine-glycine-aspartic acid (RGD) sequence.44 The preparation of this multimodal probe, rather than involving complex chemistry, keeps the design as simple and
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versatile as possible, allowing facile interchange between magnetic and radiochemical centres. The UCNPs functionalised with tumour-targeting molecules and metal chelates were prepared following standard peptidic and thiol chemistry. Radiolabelling with 68Ga as the radioactive positron emitter tracer was optimised under relatively mild conditions, and is rare amongst nanoparticulate materials. In vivo application of these probes in mouse tumour models revealed their potential as specific cancer contrast agents for PET imaging.
Materials and methods Yttrium nitrate hexahydrate (Y(NO3)3·6H2O, 99.9%), sodium nitrate (NaNO3, ≥99.0%), PEI ( polyethyleneimine, branched, Mw ∼ 25 kDa), and 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were all purchased from SigmaAldrich. Ytterbium oxide (Yb2O3, 99.9%) and erbium oxide (Er2O3, 99.9%) were purchased from STREM. Nitric acid (HNO3, 69.5%, for trace analysis) and hydrochloride acid (HCl, 37%, for trace analysis) were purchased from Fluka. Ethylene glycol (EG, ≥99.0%) and ammonium fluoride (NH4F, laboratory reagents) were purchased from Acros Organics and BDH Chemicals, respectively. 1,4,7,10-Tetraazacyclododecane1,4,7,10-tetraacetic acid mono (N-hydroxysuccinimide ester) (DOTA-NHS) was purchased from Macrocyclics. COOH-PEG-Fmoc (5 kDa) was purchased from Iris Biotech. 3-Maleimidobenzoic acid N-hydroxysuccinimide ester and Pierce BCA Protein Assay Kit were all purchased from Pierce. Cyclo (RGDyK) peptide was purchased from AnaSpec. All of the chemicals were used as received without further purification. Transmission electron microscopy (TEM) measurements were carried out on a transmission electron microscope (Philips, Tecnai 20) equipped with an energy-dispersive X-ray spectrometer (EDX; Hitachi HF-2000). The operating voltage of the microscope was 200 kV. Upconversion fluorescent spectra were obtained on LS-55B fluorescence spectrophotometer (PerkinElmer Corp., Forster City, CA), where an external 0–2 W adjustable laser integrated with an optical fibre (980 nm, Beijing Hi-Tech Optoelectronic Co., China) was used as the excitation source to replace the original Xenon source in the spectrophotometer. Dynamic laser scanning (DLS) measurements were performed on Malvern Zetasizer Nano ZS90. X-ray power diffraction (XRD) spectra were recorded on a Bruker D8 ADVANCE X-ray diffractometer. ICP-MS measurement was conducted on Agilent 7500 series ICP-MS (Agilent Technologies). Preparation of UCNP-NH2, UCNP-DO3A, and UCNP-DO3A-RGD UCNP-NH2 was prepared via a previously reported hydrothermal method with slight modification.45 Briefly, Yb2O3 (85.1 mg, 0.216 mmol), Er2O3 (6.94 mg, 0.018 mmol), and Tm2O3 (2.32 mg, 0.006 mmol) were dissolved in 10% hot HNO3 and the acidic solution was allowed to evaporate to dryness. Upon cooling, EG (80 mL) was poured in, followed by addition of Y(NO3)3·6H2O (0.7354 g, 1.92 mmol), branched PEI (1.0 g, 1.0 wt% solution in EG) and NaNO3 (0.408 g,
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4.8 mmol). The reaction mixture was heated to 80 °C for 10 min under vigorous stirring, and then an EG (10 mL) solution of NH4F (0.71 g, 19.2 mmol) was added drop-wise. After ageing at 80 °C for 10 min, the reaction mixture was transferred to a 100 mL Teflon-lined stainless steel autoclave and treated at 180 °C for 3 hours. After cooling to room temperature, the products were collected by centrifugation (10 min at 10 000 RPM), purified by the addition of ethanol to precipitate the particles followed by centrifugation and final redispersion in water. This process was repeated three times, and then the sample was dried overnight in a vacuum oven at 80 °C. For the preparation of UCNP-DOTA, 1 mg of UCNP-NH2 was sequentially reacted with a 435 μg (8.7 × 10−5 mmol) of Fmoc-PEG5000NHS, 35 μg (8.7 × 10−5 mmol) of DOTA-NHS and 20 μg (6.5 × 10−5 mmol) of MBS in 0.1 M HEPES buffer solution ( pH = 7.2) at room temperature (r.t.). The molar ratio of NH2/PEG/DOTA/ MBS was chosen as 1 : 1 : 1 : 0.75. The reaction mixture was subjected to purification by ultracentrifugation (Centriprep YM-10, 10 kDa, Millipore) to remove any unreacted reagent residues. Then UCNP-DOTA could be obtained as a white solid after purification and lyophilization. For the preparation of UCNP-DOTA-RGD, half of the UCNP-DOTA powder was redispersed in 0.1 M HEPES ( pH = 7.2) buffer solution and then further reacted with excess RGD-SH. UCNP-DOTA-RGD was obtained as a white solid after ultracentrifugation purification and lyophilization. Preparation of RGD-SH 168 μg (3.64 × 10−4 mmol) of CO2H-PEG4-SPDP were activated with EDC (77 μg, 4.00 × 10−4 mmol) and NHS (96 μg, 8.37 × 10−4 mmol) in water (500 μL). After 90 min the solution was added onto a water solution (500 μL) of the cRGDfK peptide (200 μg, 3.31 × 10−4 mmol) and the mixture was allowed to react overnight. Finally, to ‘deprotect’ the thiol group in the SPDP group, NaBH4 in water (14 μg, 3.64 × 10−4 mmol) was added into the solution right before the reaction with the nanoparticles. The product was used without further purification. 68
Ga radiolabelling of UCNPs-DO3A and UCNPs-DO3A-RGD
Both targeted and control nanoparticles were labelled in the same way. 300 μg of UCNPs in 15 μL distilled H2O were mixed with 100 μL of 0.2 M NaOH solution. 300 μL of 68GaCl3 solution in 0.1 M HCl (250 μCi) was added and the pH was corrected to the desired value with 0.2 M NaOH. The sample was incubated at 90 °C for 10 min and then purified by centrifugal filtering (MWCO 100 000 Da). After centrifugation, the activity of both filter and filtrate were measured. Labelled UCNPs were recovered from the filter and reconstituted in saline. Finally the activity of product, and filter were measured again to calculate radiochemical yields. Cell culture Human melanoma M21 cells (kind donation from Dr Amin Hajitou, Imperial College London, UK) were grown in DMEM
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media, supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U mL−1 penicillin and 100 μg mL−1 streptomycin (Invitrogen, Paisley, Refrewshire, UK). M21 cells are a αvβ3 integrin expressing line.44 Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2. Human glioblastoma U87MG cells and human prostate carcinoma PC-3 cells were maintained in MEM (Eagle’s Minimum Essential Medium, Invitrogen), supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin at 37 °C and in a humidified atmosphere of 5% CO2. Upconversion confocal cell imaging U87MG (or PC-3) cells were seeded on sterile glass coverslips in glass-bottomed 35 mm tissue culture dish. Upconversion confocal cell imaging was conducted on Leica TCS SP5. The excitation at 980 nm was provided from a femtosecond Ti:sapphire pulsed laser. The green emissions of 520–580 nm and red emissions of 630–680 nm were acquired by PMT. The incubation concentrations of Y in all the samples were set at 1 mM. After washing with PBS, cells-adhered cover-slips were mounted onto slides and immersed with CO2 independent medium (Invitrogen) for imaging experiments. In vivo PET imaging Dynamic 68Ga imaging scans were carried out on a dedicated small animal PET scanner (Siemens Inveon PET module, Siemens Medical Solutions USA, Inc., Malvern, PA, USA) following a bolus i.v. injection in tumor-bearing mice of ∼2 MBq. Dynamic scans were acquired in list-mode format over 60 min. The acquired data were then sorted into 0.5 × 0.5 × 0.5 mm bins and 19 time frames for image reconstruction (4 × 15 s, 4 × 60 s, and 11 × 300 s), which was done by 2D-ordered subset expectation maximization (OSEM) reconstruction. The Siemens Inveon Research Workplace software was used for visualization of radiotracer uptake in the tumor; 30 to 60 min cumulative images of the dynamic data were employed to define 3-dimensional (3D) regions of interest (ROIs). For each animal ROIs encompassing the tumor and other tissues were manually drawn on the summed PET image and used to compute time-activity-curves (TACs). Both tumor and tissue TACs were normalized to injected dose, measured by a VDC-304 dose calibrator (Veenstra Instruments, Joure, The Netherlands), and expressed as percentage injected dose per mL of tissue (%ID mL−1). Biodistribution studies For the biodistribution study, mice were maintained under anesthesia following their PET scan and sacrificed by exsanguination via cardiac puncture at 60 min post radiotracer injection to obtain blood, plasma, urine, heart, lung, liver, kidney, muscle and tumour. Tissue radioactivity was determined on a gamma counter (Cobra II Auto-Gamma counter, Packard Biosciences Co, Pangbourne, UK) and decay corrected. Data were expressed as percent injected dose per gram of tissue.
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Results and discussion UCNPs preparation High quality upconversion nanoparticles were prepared as previously described.46 A hydrothermal method was followed using Yb2O3, Er2O3, Tm2O3, Y(NO3)3·6H2O, NaNO3 and NH4F as starting materials. Polyethyleneimine (PEI) was used as capping ligand. The resulting UCNPs were characterised by ICP-MS and EDXS to be formulated as NaYF4:Yb/Er/Tm (18 : 1.5 : 0.5 mol%). Electron microscopy analysis showed that the particles were highly crystalline, quasi-spherical, and presented an average diameter of 36 nm (Fig. 1). Bulk hexagonal NaYF4 has been reported to be the most efficient host material for green (Yb3+/Er3+ doped) upconversion fluorescence,47 but green emission is not ideal for deep tissue imaging.27 Therefore, PEI was selected as the coating agent as it suppresses NaYF4 hexagonal phase by competing for fluorides against lanthanides,45 and so NIR-to-deep-red energy transfer48 can be harvested with higher yields. Deep red fluorescence (660 nm for Er3+) lies in the optimal spectrum region for biomedical applications as it is both visible to the naked eye and approachable to deep tissues. XRD (X-ray diffraction) and SAED (selective area electron diffraction) demonstrate that the NaYF4 structure corresponds to a cubic phase and not a hexagonal one (JCPDS card no. 77-2042). Surface functionalization chemistry Before further functionalization, the number of available amines per nanoparticle was characterised by reaction with SPDP (3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester). After purification and reduction, release of pyridine2-thione was followed by UV-Vis spectroscopy (λ = 343 nm, ε = 8080 M cm−1). In this way the number of available amines per nanoparticle was calculated to be around 6000. The nanoparticles were then functionalised with PEG molecules, DOTA chelates and RGD peptides. PEG molecules have been reported to enhance the blood half-life of particles in vivo49 and DOTA can be used to chelate reporter metals for MRI (Gd3+) and PET (68Ga3+). In addition, the RGD-integrin αvβ3 system has been widely explored for the labelling of tumours. Firstly, pre-activated DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, with only one of the four carboxylic groups activated) and carboxy-PEG molecules with a molecular weight of 5000 Da, were coupled to the amine groups on the surface of the nanoparticles. After purification, the remaining free amine groups from the PEI coating of the particles were reacted with the small molecule MBS (3-maleimidobenzoic acid N-hydroxysuccinimide ester). This molecule has an activated carboxylic group on one side ready to react with any amine, while on the other side of the molecule it displays a maleimide group that can react with any free thiol. Meanwhile in a different reaction vessel, cRGDfK peptides were modified with a short PEG molecule (PEG4) ending in a SPDP (3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester) group. The SPDP presents a disulfur bond that upon reduction provides a free thiol group and releases a pyridine-2-thione
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Fig. 1 (A) Overview TEM micrograph of PEI protected UCNPs. (B) TEM micrograph of UCNPs-DO3A-RGD. Inset, SAED pattern obtained from the same sample. (C) XRD spectra of as prepared PEI protected UCNPs compared to the line pattern of the calculated cubic phase of NaYF4 (JCPDS no. 77-2042). (D) Overview TEM micrograph of DOTA functionalised PEI protected UCNPs (UCNPs-DO3A-RGD). (E) TEM micrograph of UCNPs-DO3A. (F) Hydrodynamic sizes measurement before (dotted line) and after (solid line) the functionalisation of PEI protected UCNPs with DOTA and RGD peptides.
molecule that is enabled to bind to thiols or maleimides. In a final step, the modified cRGDfK peptide was mixed with the nanoparticles under reducing conditions (NaBH4) to obtain the final multimodal RGD targeted upconversion nanoparticles. The yield of this final step was measured, using standard protein characterisation protocols (Bradford method50), to be above 95%, meaning that there were an average of 102 RGD peptides per particle (Scheme 1). ICP-MS measurements were used to estimate the number of DO3A and PEG molecules per particles.46 On average there were around 4500 PEG molecules and 1200 DO3A molecules per particle. TEM micrographs acquired after the functionalization showed no changes in the UCNPs in shape or size (Fig. 1). Finally the hydrodynamic diameter of both the initial UCNPs-NH2 and the final targeted UCNPs-DO3A-RGD were measured by dynamic light scattering in water (Fig. 1F). According to the results, after the functionalization of the particles their hydrodynamic size increased from an average of 83.8 nm to 93.6 nm.
at 300 mW, and both solutions, UCNP-NH2 and UCNPDO3A-RGD, were prepared to contain the same weight concentration of UCNP core (1 mg mL−1). Both solutions emitted orange light upon irradiation coming from the combined green and red luminescence from Er3+ ions plus the blue luminescence from Tm3+ ions. The upconversion luminescence spectra of UCNP-NH2 and UCNP-DO3A-RGD solutions are shown in Fig. 2. The typical upconversion luminescence peaks of Er3+ and Tm3+ can be observed. 4S3/2–4I15/2 and 4F9/2–4I15/2 transitions of doped Er3+ ions account for the green peak at 543.5 nm and red peak at 658.1 nm respectively, while 1G4–3H6 and 3H4–3H6 transitions of doped Tm3+ are responsible for the blue peak at 475.7 nm and NIR peak at 795.8 nm, respectively.45 Compared with UCNP-NH2, the emission intensity of Er3+ in UCNP-DO3A-RGD was slightly lower, while the emission intensity of Tm3+ remains unchanged. It is thought that this is due to Er3+ emissions of UCNPs being more vulnerable to non-radiative energy transfer and vibrational quenching by the various ligands on the surface of the nanocrystals.
Upconversion measurements
Radiochemistry
The upconversion properties of UCNP-NH2 and UCNPDO3A-RGD were characterised in solution. For these studies, an optic-fibre coupled diode laser emitting at 980 nm was set
Once the nanoparticles were fully characterised, their ability to complex 68Ga was tested. To date, only a few reports have been published on multimodal imaging (including PET) with
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Scheme 1
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Preparation of the targeted multimodal upconversion nanoparticles.
Fig. 2 (A) Upconversion luminescence spectra of PEI-protected upconversion nanoparticles (UCNP-NH2), and DOTA and PEG-functionalised upconversion nanoparticles (UCNP-DO3A-RGD) in water solutions.
UCNPs. Some of these reports have used 18F as the radioisotope.51,52 Only a few examples have shown the application of nanoprobes for 68Ga chelation. Stelter and co-workers53 directly adsorbed 68Ga onto the surface of silica nanoparticles. Both Hwang et al.26 and Jeon et al.54 used NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) as 68Ga chelator on silica nanoparticles, and Lee et al. also used NOTA as chelator on QDs. While the direct unspecific adsorption of 68Ga is not the best option for future potential translation into human health (due to stability concerns), NOTA has been reported to form very stable complexes with Ga3+ (log K = 30.9855). In this work, we have chosen to use DOTA instead of NOTA because although it forms less stable chelates with Ga3+ (log K =
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21.3355), they are still stable enough to be used in vivo. DOTA presents the extra advantage in that it is already approved for its use in human health application, in particular as Gd3+ chelator for MRI applications.7 The first attempts to label the nanoparticles with 68Ga were not successful. Standard labelling conditions for the labelling of DOTA on small molecules56 were used: sample dissolved in 0.2 M pH 4 acetate buffer, 68Ga in 0.1 M HCl solution, 90 °C for 10 min. With the low volumes used (15 μL sample in water, 150 μL acetate buffer, and 300 μL 68 Ga elute in HCl), the pH of the solution during the incubation was around 1. At this pH, the amine groups on the nanoparticles become protonated57 and an electrostatic repulsion occurs between the –NH3+ groups on the nanoparticles and the 68 Ga3+. This effect presumably accounts for the very low labelling yields. The labelling reaction was therefore, studied at different pH to determine the optimal conditions for the radiolabelling (Table 1). The yield of the labelling reaction consistently increased with a rise in pH from around 5% at pH 1 to 87% at pH 6. Although pH 6 gave the highest yield, pH 5 was selected for the labelling studies as at pH values above 6.3,58 Ga3+ forms Ga(OH)3 which is insoluble in water. These experiments were carried out with control nanoparticles i.e. functionalised in the way as described above, but without RGD
Table 1 Yields of the radiochemical labelling of DOTA functionalised UCNPs, and control UCNPs (without DOTA) at different pH
pH
% Labelling DO3A UCNPs
% Labelling control UCNPs
1 3 4 5 6
5.56 ± 0.52 10.14 ± 1.53 20.46 ± 0.20 38.42 ± 0.87 87.49 ± 2.36
4.62 ± 0.63 5.25 ± 0.26 4.99 ± 0.62 5.31 ± 0.82 8.72 ± 1.25
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peptides. The final corrected radiochemical yield for these control nanoparticles was 38.4% with a specific activity of 8 × 1012 μCi mol−1, while the corrected radiochemical yield of the targeted nanoparticles was lower, at 15.2% with a specific activity of 5.1 × 1012 μCi mol−1. In vitro confocal imaging The ability of functionalised nanocrystal to target and label cells was assessed in vitro. Two different carcinoma cell lines with different expression levels of αvβ3 integrin, U87MG and PC-3 cells, were used. Live U87MG cells were incubated with the nanoprobes at 1 mM Y for 4 h. Green and red upconversion luminescence and bright-field micrographs are shown in Fig. 3. First, PC-3 cells were used as control. As compared with U87MG cells, PC-3 cells express a low level of αvβ3 integrin. This low expression of integrin correlates well with the confocal images acquired after the incubation with the UCNPs showing no difference in signal between RGD and non-RGD functionalised particles. On the other hand, images obtained from U87MG cells incubated under the same conditions with
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UCNP-DO3A-RGD (Fig. 3, third column) present numerous bright green and red luminescent spots, while the signal intensity coming from the same cells when incubated with UCNP-DO3A (Fig. 3, fourth column) is significantly lower. These results suggest that the cellular labelling of UCNPDO3A-RGD is greatly enhanced due to the selective targeting of integrin αvβ3 receptors on U87MG cell surface. To further support this finding, U87MG cells were incubated at 4 °C in the presence of either UCNP-DO3A or UCNP-DO3A-RGD, and subsequently imaged after washing. The images (Fig. S1†) show higher signal intensity in the case of RGD functionalised UCNPs, as they are able to target the integrin on the surface of the cells, in a process independent of cell internalisation. In vivo PET imaging Following the promising in vitro results, the nanoparticles were tested in vivo as targeted PET probes. In this case a wellestablished mouse model for integrin expression was used.59 Fig. 4A and 4B depicts typical transverse PET image slices demonstrating localisation of the targeted and control tracer
Fig. 3 Upconversion confocal live cell imaging. Green (upper row) and red (middle row) upconversion luminescence and bright-field (lower row) images of PC-3 (first and second columns) and U87MG cells (third and fourth columns) treated with UCNP-DO3A-RGD (first and third columns) or UCNP-DO3A (second and fourth columns) at 37 °C for 4 h. The incubation concentration of Y was set at 1 mM for all the samples (40× oil lens, zoom = 1, scale bar = 50 μm).
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Fig. 4 Representative axial 3D-OSEM PET-CT images of targeted UCNP (A) and control UCNP (B) in M21 tumour bearing mice, showing 30–60 minutes summed activity. The axial image of the targeted tracer (A) shows higher activity in the tumour compared to the axial view of the control tracer (B). The tumour margins, as identified by CT, are outlined in red. Corresponding coronal and sagittal 3D-OSEM PET-CT images of the targeted UCNP in the same M21 tumour bearing mice (C). The location of the heart (H), liver (L) and bladder (B) are indicated.
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Fig. 5 (A) Time versus radioactivity curves (TAC) obtained for the tumour from 60 min dynamic PET imaging for the targeted (diamonds) and control UCNP group (circles). Data represents mean %ID mL−1 values ± SEM, n = 3 per group. (B) Representative time versus radioactivity curves (TAC) obtained for targeted 68Ga-UCNP in a tumour-bearing mouse from 60 min dynamic PET imaging.
in M21 tumours (outlined in red). The highest tumour localisation was observed for the targeted agent characterised by a heterogeneous pattern of tracer uptake. In contrast to the targeted agent, uptake for the control agent is low and relatively homogeneous. Time versus radioactivity curves (TACs) obtained from ROI analysis of the tumours show that higher uptake is indeed observed with the RGD targeted nanoparticle (Fig. 5A, diamonds) than the control (Fig. 5A, circles). Mean NUV60 values were 5.53 ± 0.70 and 4.24 ± 0.17 for targeted and control nanoparticle respectively ( p = 0.0724). Both tracers show accumulation over the 60 minutes of imaging with no washout, and greater retention is observed with the targeted species. There is greater variation observed in the TACs and overall retention (%ID mL−1 values) of the individual subjects in the targeted group than observed in the control group. This accounts for the larger standard error bars seen in the mean TAC for the targeted UCNP (Fig. 5A, diamonds). Fig. 5B shows representative TACs for the targeted nanoparticle in tissues/organs. Time versus radioactivity curves (TACs) obtained for the targeted nanoparticle shows accumulation in the bladder, tumour and liver over time. Despite high activity in the bladder and accumulation of the tracer in the bladder over time, the kidney is not visualised in the PET images pointing to the likelihood of inefficient filtration of the radioactive nanoparticle complex, together with a cleavage event leading to elimination of a radiolabelled cleaved product into the bladder (Fig. 4C). Biodistribution The PET data were corroborated by ex vivo gamma counting of excised tissues, which demonstrated significantly higher uptake of the targeted UCNP in the tumour compared to control non-targeted UNCP (*P < 0.05) (Fig. 6C). The results show that the percentage injected dose per gram of tissue (%ID g−1) values at 60 minutes were 10.37 ± 1.58 and 5.61 ± 0.26 for the targeted and control nanoparticles respectively, a 1.84-fold difference. High activity in the urine is suggestive of
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renal excretion. High activity in plasma is indicative of long circulation times as expected from the PEG functionalization of the nanoparticles. Additionally %ID g−1 values were obtained for the liver, spleen, lung and kidney. High activity in the liver is visualised clearly in the PET-CT images (Fig. 4C). The presence of radioactive signal in the bladder at 60 min raised some concerns about the stability of nanoparticles. Due to the large size of these nanoprobes they are not expected to undergo renal excretion (the size limit for kidney excretion has been reported to be around 10 nm60,61). To assess that both components of the nanoparticles (UCNP core and DOTA-68Ga) were still together after the PET imaging, some of the organs were harvested, digested and analysed by ICP-MS to determine their Yb content coming from the UCNP core, having already demonstrated that the tissues contained 68Ga. The results, Fig. 6D, show increased levels of this lanthanide in the tissues, indication that the UCNPs also accumulated in those tissues. The RGD-targeted agent shows higher accumulation in integrin-positive melanoma tumours, thereby confirming functionality. There is larger variation in the %ID mL−1 and tracer accumulation for the targeted species but not the control agent. This may well be due to variation in integrin expression in the tumour as observed in previous studies using the same tumour model. The control agent also shows relatively high % ID mL−1 values in the tumour, likely to be due to EPR effects. The combination of PET imaging results, radioactivity biodistribution and ICP-MS organ analysis confirms the presence of both the UCNP cores and the PET reporter moiety in the tissues. Further experiments will be required to explain the levels of activity in the bladder and urine. The activity seems to leak slowly through the kidneys as they are not highlighted in the PET images.
Conclusions In this work we have demonstrated that nanoprobes are a valid platform for the chelation of radionuclides, such as 68Ga, to
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Fig. 6 Biodistribution data of (A) 68Ga-RGD targeted and (B) control UCNP in M21 tumour bearing mice 60 minutes post injection of tracer. SInt, small intestine; Lint, large intestine. C, 68Ga-RGD targeted and control UCNP retention in M21 tumours. (Data points represent mean ± SEM, N = 3, *P < 0.05.) D, results of the ICP-MS analyses for the content of Yb in tumour, spleen and liver of animals injected with targeted UCNPs.
multimodal MRI/optical nanoparticles. The incubation conditions were optimised to obtain effective radiochemical yields, while keeping preparation times as short as possible, and using low levels of radiation. The presence of DOTA-like chelates on the surface of the nanoparticles allows the versatile application of these nanocrystals either/both as MRI contrast agents (DO3A-Gd3+) or/and as PET tracers (DO3A-68Ga3+). The upconversion abilities of the nanoparticle core are confirmed, via histological analysis, and via MRI/PET to be diagnostic, and subsequently, have the potential to guide surgical interventions. Moreover, we have shown that the functionalization of these probes with targeting peptides enables their use as specific cancer diagnostic agents. In this case, cycloRGD peptides were chosen as cancer targeting agents, but the simple chemistry involved in their preparation allows the use of other biomolecules as targeting agents to broaden the field of application of these nanoprobes.
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Acknowledgements Funding for this project was provided from CRUK, EPSRC, MRC and the Department of Health, grant (C2536/A10337).
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