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Biological and Medical Applications of Materials and Interfaces

PET/MR Imaging of Glioblastoma using A Functionalized Gadofullerene Nanoparticle Daiqin Chen, Yue Zhou, Dongzhi Yang, Mirong Guan, Mingming Zhen, Weifei Lu, Marcian E. Van Dort, Brian D Ross, Chunru Wang, Chunying Shu, and Hao Hong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b03542 • Publication Date (Web): 29 May 2019 Downloaded from http://pubs.acs.org on May 30, 2019

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ACS Applied Materials & Interfaces

PET/MR Imaging of Glioblastoma Using A Functionalized Gadofullerene Nanoparticle Daiqin Chen,1# Yue Zhou2#, Dongzhi Yang,1,3 Mirong Guan2, Mingming Zhen2*, Weifei Lu1,4, Marcian E. Van Dort,1,5 Brian D. Ross,1,5 Chunru Wang2*, Chunying Shu2, Hao Hong1,5*

1

Department of Radiology, Center for Molecular Imaging, University of Michigan, Michigan

48109-2200, United States 2

Key Laboratory of Molecular Nanostructure and Nanotechnology, Institute of Chemistry,

Chinese Academy of Sciences, Beijing 100190, China. 3

Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical

University, Xuzhou, Jiangsu 221004, China 4 College of Animal Sciences and Veterinary Medicine, Henan Agriculture University, Zhengzhou,

Henan 450002, China 5 University # These

of Michigan Comprehensive Cancer Center, Michigan 48109-0944, United States

two authors contributed equally.

Corresponding Authors: * (M.Z.) Tel/Fax: +86-010-62561085; E-mail: [email protected] * (C.W.) Tel/Fax: 86-010-62652120; E-mail: [email protected] * (H.H.) Fax: 1-734-763-5447; Tel: 1-734-615-4634; E-mail: [email protected]

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Abstract Water-soluble gadofullerene nanomaterials have been extensively investigated as magnetic resonance imaging (MRI) contrast agents, radical scavengers, sensitizers for photo dynamic therapy (PDT) and inherent antineoplastic. Most recently, an alanine-modified gadofullerene nanoparticle (Gd@C82-Ala) with excellent anticancer activity has been reported; however, the absolute tumor uptake of Gd@C82-Ala is still far from satisfactory and its dynamic pharmacokinetic and long-term metabolic behaviors remain to be elucidated. Herein, Gd@C82-Ala was chemically modified with 8-arm polyethylene glycol amine (8-arm-PEG-NH2) to improve its biocompatibility and provide the active sites for the attachment of a tumor-homing ligand (cRGD) and positron emission tomography (PET) isotopes (i.e. 64Cu or 89Zr). The physical and chemical properties (e.g. size, surface functionalization condition, radiochemical stability etc.) of functionalized Gd@C82-Ala was properly characterized. Also, its glioblastoma cell targeting capacity was evaluated in vitro by flow cytometry, confocal fluorescence microscopy, and dynamic cellular interaction assays. Due to the presence of gadolinium ions, the gadofullerene conjugates can act simultaneously as T1 MRI contrast agents and PET probes. Thus, the pharmacokinetic behavior of functionalized Gd@C82-Ala was investigated by PET/MRI, which combines the merits of high resolution and excellent sensitivity. The functionalized Gd@C82-AlaPEG-cRGD-NOTA-64Cu (NOTA stands for 1,4,7-triazacyclononane-triacetic acid) demonstrated much higher accumulation in U87-MG tumor than its counterpart without cRGD attachment from in vivo PET observation, consistent with observation at the cellular level. In addition, Gd@C82Ala-PEG-Df-89Zr (Df stands for desferrioxamine) was employed to investigate the metabolic behavior of gadofullerene conjugates in vivo for up to 30 days. It was estimated that nearly 70% of Gd@C82-Ala-PEG-Df-89Zr was excreted from the test subjects primarily through renal


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pathways within 24 h. With proper surface engineering, functionalized Gd@C82-Ala nanoparticles can show an improved accumulation in glioblastoma. Pharmacokinetic studies also confirmed the safety of this nanoplatform, which can be used as an image-guidable therapeutic agent for glioblastoma.

Keywords: Gadofullerene, positron emission tomography, magnetic resonance imaging, tumor targeting, metabolic study.


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1. INTRODUCTION Gadofullerenes have attracted much attention for biomedical uses in the past decades. There have been extensive reports about the applications of gadofullerene derivatives as inherent antineoplastic agents,1-3 free radical sponge,4-5 photosensitizers,6 and magnetic resonance imaging (MRI) contrast agents.7-8 However, continuous safety concerns about these nanomaterials have been raised. Although fullerenes are generally considered as biocompatible, the toxicity from certain fullerene derivatives (including gadofullerenes) is still under continuous debate.9-10 The pharmacokinetics and metabolic behavior of nanoparticles in vivo are closely associated with their toxicity, thus it is imperative to investigate the distribution and metabolic performance of gadofullerene derivatives in vivo.11-12 Currently, the most widely adopted detection methods for gadofullerenes in vivo are fluorescence imaging and inductively coupled plasma mass spectrometry (ICP-MS).13-14 Although fluorescence imaging is a real-time and non-invasive detection tool, its inherent penetration limit hinders its detection of gadofullerenes in some deep visceral organs such as liver, spleen and kidneys.15-16 In addition, the quantification capacity of fluorescence in vivo is not optimal. ICPMS can quantify the Gd concentration in gadofullerenes, however it also suffers from some limitations.17-18 First, it is an invasive detection method and not very suitable for dynamic monitoring of gadofullerenes. Second, ICP-MS detects Gd ions rather than the gadofullerenes themselves. Biological samples (e.g. tissues) need to be pre-treated with strong acid for the destruction of carbon cages and the release of gadolinium ions. The efficiency of Gd escape from the fullerene “cage” and potential loss during sample transfer will compromise the accuracy of the final results. Hence, it is important to develop a reliable, non-invasive, and quantifiable method for real-time monitoring of gadofullerenes in vivo.


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The existence of gadolinium ions in the carbon cage enables gadofullerenes to be used as magnetic resonance imaging (MRI) contrast agents. It has been reported that their contrast performance is even superior to the Gd-DTPA, which is a widely adopted MRI agent 19-20. A recent study has already used a peptide-conjugated fullerene derivative successfully for targeted MRI of tumor.21 The restriction of Gd ions in the carbon cage prohibits the leakage of free Gd ions, which prevents the potential toxicity from most Gd-based contrast agents.22-23 However, MRI suffers from its poor sensitivity in vivo. On the other hand, positron emission tomography (PET) imaging is a very powerful tool to study the pharmacokinetics and metabolism behaviors of various molecules in vivo 24-25. It is extremely sensitive and quantifiable while it is simultaneously limited by its low spatial resolution 26. Hence, it is widely accepted that the hybridization of PET and MRI can bring good spatial resolution and high sensitivity during the detection of in vivo molecular events 27-28. A desirable PET/MRI tracer should inherently bear PET isotopes and MRI contrast agents (T1* or T2*) simultaneously 29-30. By introducing isotopes, such as 64Cu (t1/2 = 12.7 h) or 89Zr (t1/2 = 78.4 h), radiolabeled gadofullerenes can serve as promising PET/MRI agents. As we have mentioned previously, the sensitivity of MRI is inherently limited and the tumor accumulation of most Gd@C82 derivatives is usually low, thus the concentration of gadofullerene derivatives should be increased in the tumor site to acquire sufficient MRI signals 31. One option is to simply increase the administration dose of gadofullerene derivatives, which can potentially bring safety concerns due to the elevated gadofullerene concentration in major organs (e.g. liver). Another more reasonable choice is to promote the accumulation of gadofullerene derivatives at the tumor site by the addition of active targeting ligands. Cyclic Arg-Gly-Asp peptide (cRGD) is a small cyclic peptide that can selectively bound with integrin αvβ3, which is overexpressed on the


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activated endothelial cells and the tumor cells in many tumors, especially in glioblastoma 32-33. It has been well investigated as an effective glioblastoma-targeted ligand 34. Herein, we functionalized Gd@C82-Ala with 8-arm PEG-NH2 and then the targeting group, cRGD. PET isotopes such as 64Cu or 89Zr, were subsequently incorporated to obtain Gd@C82-AlaPEG-cRGD-(NOTA-64Cu or Df-89Zr). The gadofullerene-cRGD conjugates demonstrated much higher accumulation in U87-MG tumor than gadofullerene without cRGD targeting group in vivo. The Gd@C82-Ala-PEG-cRGD-NOTA-64Cu nanoparticles were employed as a PET/MRI probe for the first time on U87-MG tumor bearing mice. Besides, the metabolic behavior of gadofullerene was investigated by PET imaging for up to 30 days with radiolabeling of

89Zr.

This study can

provide useful information for future optimization of fullerene-based biomedical agents in vivo.

2. MATERIALS AND METHODS 2.1. Materials. Gd@C82 (99% purity, as obtained from Xiamen Funano Co.). NaOH, β-alanine, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

hydrochloride

(EDC∙HCl),

N-

hydroxysuccinimide (NHS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), N-γmaleimidobutyryl-oxysuccinimide ester (GMBS), 2-mercaptoethanol, Chelex 100 resin (50 - 100 mesh) were all purchased from Sigma-Aldrich (St Louis, MO).

64CuCl 2

and

89Zr(C O ) 2 4 2

acquired from the University of Wisconsin Cyclotron. Dialysis bags (M.W.cut

off

were

= 3 kDa),

ultrafiltration tubes (M.W.cut off = 50 kDa and M.W.cut off = 10 kDa) were all ordered from Thermo Fisher Scientific (Fair Lawn, NJ). 8-Arm PEG-NH2 (MW: 40 kDa) was purchased from Creative PEGworks (Chapel Hill, NC). 2-S-(4-Isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7triacetic acid (p-SCN-Bn-NOTA) and 1-(4-isothiocyanatophenyl)-3-[6,17-dihydroxy-7,10,18,21tetraoxo-27-(N-acetylhydroxylamino)-6,11,17,22-tertraazaheptaeicosine]thiourea (p-SCN-Bn-Df) were purchased from Macrocyclics (TX, USA). c(RGDyC) was purchased from Peptide


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International (Louisville, KY). Anti-integrin αvβ3 antibody was purchased from Abcam (Cat # ab78289, Cambridge, MA, USA). Secondary Cy3-conjugated rabbit anti-mouse antibodies were purchased from Jackson ImmunoResearch (St. Louis MO, USA). Millipore-grade water was used to prepare all the buffers used in this study, and buffers used in radiolabeling experiments were further treated with Chelex 100 resin to eliminate possible contamination from heavy metals. PD10 columns, which were used for size-exclusive separation of gadofullerene conjugates, were purchased from GE Healthcare (Piscataway, NJ). 2.2. Syntheses of Gd@C82-Ala. Gd@C82 (100 mg) was suspended in a mixed solution (60 mL) containing NaOH (12.5 mmol) and β-alanine (25 mmol). The mixture was heated to 80 °C and stirred (1500 rpm) for 2 h to before its color turned brown. Afterwards, the mixture was gently cooled to the room temperature (R.T.) before centrifugation (8000 rpm, 10 min) was taken to remove large aggregates. The supernatant was then loaded into a dialysis bag (M.W.cut off = 3 kDa) and dialyzed against Milli-Q water for 3 days to remove the small molecules. The obtained transparent brown solution was freeze dried to get the powder of Gd@C82-Ala. 2.3. Synthesis of Gd@C82-Ala-PEG-NH2. Gd@C82-Ala was dissolved in 1 mL phosphate buffered saline (PBS, pH = 6.0) to a concentration of 20 μM. After adding 0.4 mg EDC∙HCl and 0.6 mg NHS, the mixture was stirred at R.T. for 15 min to activate the carboxyl groups on Gd@C82Ala. 2-mercaptoethanol (1.4 μL) was then added to stop the activation. The obtained solution was passed through a centrifugation filter (M.W.cut off = 10 kDa) for several times to remove the smallmolecule reactants. The activated Gd@C82-Ala was resuspended in 1 mL PBS (pH = 7.4) with 1.6 mg 8-Arm PEG-NH2 added for a 2 h reaction at R.T. The reaction was stopped by the addition of glycine (5.7 mg) and the unreacted armed PEG was removed by repeated ultrafiltration (M.W.cut off

= 50 kDa).


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2.4. Synthesis of Gd@C82-Ala-PEG-cRGD. Gd@C82-Ala-PEG-NH2 was suspended in 0.2 mL of PBS (pH = 7.4) with 0.02 mL of GMBS solution (10 mM, dissolved in DMSO) added before a 0.5 h reaction was conducted. The product was purified by repeated ultrafiltration (M.W.cut off = 10 kDa). The intermediate product was suspended in 0.2 mL of PBS (pH=8.5) and cRGD-SH (0.07 mg) was added to react for 0.5 h. The product, Gd@C82-Ala-PEG-cRGD, was purified by repeated ultrafiltration (M.W.cut off = 10 kDa). 2.5. Synthesis of Gd@C82-Ala-PEG-(cRGD)-NOTA (FITC). Gd@C82-Ala-PEG-(cRGD)-NH2 was suspended in 0.2 mL of PBS (pH = 8.5). p-SCN-Bn-NOTA (1.3 mg) or FITC (1.2 mg) was added into the above solution. The mixture was under reaction for 2 h at R.T. Excessive p-SCNBn-NOTA (or FITC) was removed by repeated ultrafiltration (M.W.cut off = 10 kDa). 2.6. 64Cu-labeling and stability evaluation. 64CuCl2 (∼74 MBq, in 0.1 M hydrochloric acid) was diluted with 0.1 M sodium acetate (pH 6.5) buffer and added to Gd@C82-Ala-PEG-NOTA or Gd@C82-Ala-PEG-cRGD-NOTA (100 mM, 40 μl), and the final pH of reacting mixture was adjusted to be between 4 and 5.5. The reaction took place at 37 °C for 0.5-1 h with constant gentle shaking. Gd@C82-Ala-PEG-NOTA-64Cu or Gd@C82-Ala-PEG-cRGD-NOTA-64Cu was purified by a PD-10 column (PBS (pH 7.4) was used as the eluent phase). Instant thin layer chromatography (ITLC) was used to evaluate the radiolabeling efficiency and product purity with 0.25M NH4OAc buffer (pH 5.5) as ITLC eluent with Tech-Control chromatography strips (Biodex). The solution containing Gd@C82-Ala-PEG-NOTA or Gd@C82-Ala-PEG-cRGD-NOTA was passed through a 0.2 μm syringe filter prior to in vivo administration. For stability evaluation, Gd@C82-Ala-PEGNOTA-64Cu or Gd@C82-Ala-PEG-cRGD-NOTA-64Cu was suspended in PBS (pH = 7.4) or complete FBS at 37 °C for up to 24 h. Given amount of the sample (~50 μL) was taken from the mixture at the set time points (15 min, 0.5, 1, 2, 4, 16, and 24 h) and passed through Amicon filters


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with M.W. cutoff of 100 kDa (centrifuged at 12,000 × g for 10 min). The radioactivity from the filtrates was measured in a WIZARD2 gamma counter (Perkins-Elmer). The percentages of intact or stable 64Cu on Gd@C82-Ala-PEG-NOTA-64Cu or Gd@C82-Ala-PEG-cRGD-NOTA-64Cu were calculated by the equation [(total radioactivity - radioactivity in filtrate)/total radioactivity × 100%]. 2.7. Preparation of Gd@C82-Ala-PEG-Df and

89Zr-labeling.

Gd@C82-Ala-PEG-NH2 was

resuspended in 200 μl of HEPES (pH = 8.5) buffer. p-SCN-Bn-Df (1 mg) was added to the above solution and to allow 0.5 – 1 h reaction at 37 °C with constant gentle shaking. Gd@C82-Ala-PEGDf was purified by repeated ultrafiltration (M.W.cut off = 10 kDa). To incorporate

89Zr

into

Gd@C82-Ala-PEG-Df, 89Zr-oxalate with the radioactivity amount of 111–185 MBq (volume ~ 200 μL) was neutralized with sodium carbonate buffer (2 mol/L, volume ~90 μL) before its reaction with Gd@C82-Ala-PEG-Df at the ratio of 0.81 μg Gd@C82-Ala-PEG-Df per 1 MBq of

89Zr.

HEPES buffer (0.5 mol/L, pH = 7.4) was used to adjust the total reaction volume to ~1.5 mL and the final reaction pH was maintained at 6.8–7.2. The radiolabeling reaction usually lasted for 1 h at R.T. with constant shaking at 350 rpm. Gd@C82-Ala-PEG-Df -89Zr was separated from the unreacted 89Zr by PD-10 using a mixed mobile phase of 0.25 M sodium acetate + 5 mg/mL gentisic acid (pH = 5.5). ITLC was used to evaluate the radiolabeling efficiency and product purity with 20 mM citric acid (pH = 4.9–5.1) as the eluent with Tech-Control chromatography strips (Biodex). The solution containing Gd@C82-Ala-PEG-Df -89Zr was passed through a 0.2 μm syringe filter prior to in vivo administration. 2.8. Flow cytometry and confocal fluorescence microscopy. Two cell lines were adopted to test the interactions between Gd@C82 conjugates and cellular integrin αvβ3: U87-MG (integrin αvβ3+) and MCF-7 (integrin αvβ3-). These cells were first incubated with Gd@C82-Ala-PEG-FITC or Gd@C82-Ala-PEG-cRGD-FITC (concentration: 1 μg/ml based on FITC) for 30 min at R.T. After


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the incubation, the cells were harvested, washed repeatedly with cold PBS, and re-suspended in PBS at the density of 2 × 106 cells/ml. Cellular fluorescence was analyzed using a BD LSR Fortessa cytometer equipped with a 488-nm laser (Becton-Dickinson, San Jose, CA). FlowJo (X.0.7, Tree Star, Ashland, OR) was used to calculate and compare cellular between each treatment group. To provide a further visual evidence, these cells were cultured on class petri dishes and undertaken same Gd@C82 incubation/wash procedures, before they were examined under a Nikon A1 confocal microscope with a magnitude of 200×. No cell fixation was used in these studies. 2.9. PET Imaging and Organ Distributions. Inveon rodent microPET/CT scanner (Siemens Medical Solutions USA, Inc.) was used to acquire PET images from test animals. Gd@C82-AlaPEG-NOTA-64Cu or Gd@C82-Ala-PEG-cRGD-NOTA-64Cu with the radioactivity of between 5 and 10 MBq was injected into the U87MG tumor-bearing mice via the tail vein. Static PET scans (4 × 107 events per scan) were recorded at preset time points (0.5, 3, 18, 24, and 48 h) postinjection (p.i.). Three-dimensional ordered subset expectation maximization (3D-OSEM) algorithm was used for PET images reconstruction and no attenuation or scatter correction was applied. Region-of-interest (ROI) analysis of PET images was done on a vendor-developed software - Inveon Research Workshop (IRW, v4.2.0.8) with decay corrections to count the tissue radioactivity density. The ratio between tissue radioactivity density (in the unit of MBq/g) and total administered radioactivity was the percentage of injected dose per gram of tissue (%ID/g) for each organ/tissue. After the last PET scans at 48 h p.i., the tumor-bearing mice were euthanized to collect their major organs/tissues along with blood and tumors (all these organs/tissues were wetweighed). A WIZARD2 automatic gamma-counter (Perkin-Elmer) was used to measure radioactivity density (in the unit of %ID/g) in these organs and tissues to corroborate with PET findings.


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2.10. PET/MRI studies. Six-week-old female nude mice (n = 5) were anesthetized and injected with 200 μL of Gd@C82-Ala-PEG-cRGD-NOTA-64Cu (1 mM, 16 MBq) in saline via tail vein. At various time points, the test animals were placed in prone position on the MRI mouse bed to keep the temperature at 37 OC and a pressure-transducer was placed under the mouse abdomen for respiratory monitoring. FSET T1*-weighted was performed without the use of triggering. Imaging parameters were as follows: repetition time (TR) = 720 ms, echo time (TE) = 11 ms; field of view (FOV) = 60 × 60 mm; matrix size = 256 × 252; slice thickness = 1 mm; 1 averages, 12 slices. Short axis and coronal views of the tumor of the mouse were acquired. The mouse was then subjected to static PET scans (4 × 107 events per scan). The images were also reconstructed using 3DOSEM algorithm (no attenuation or scatter correction applied). The PET and MR images were merged using the VivoQuant software (Invicro, Boston, MA). 2.11. Statistical analysis. All the measurements and tests were done at least three times and sample size in each test was kept as no less than 3 to maintain the statistical power. The unpaired twotailed Student ’ s t-test was applied to determine the statistical differences between test groups. P 95%).

3. RESULTS 3.1. Material preparation and characterization. Gd@C82-Ala was prepared through a solidliquid

reaction

method

and

the

Gd@C82(OH)13(NHCH2CH2COOH)6

average by

molecular

X-ray

structure

photoelectron

was

determined

spectrometry

as

(XPS),

thermogravimetric analysis (TGA), infrared spectroscopy (IR) and elemental analysis (Figure S1S3, Table S1). Gd@C82-Ala was further conjugated with 8-armed amine PEG, which not only rendered the nanoparticles with much longer blood circulation time, but also introduced free amine residues which facilitated the subsequent surface modification of gadofullerenes. A glioblastoma-


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targeting ligand, cRGD, was attached to the surface of Gd@C82-Ala-PEG. The UV-vis spectrum of Gd@C82-Ala-PEG-cRGD was shown in Figure 1a. Dynamic light scattering (DLS) showed that the diameters of Gd@C82-Ala, Gd@C82-Ala-PEG and Gd@C82-Ala-PEG-cRGD were 122 nm, 164 nm and 190 nm, respectively (Figure 1b). Since fullerene usually forms aggregates in the solution,35-36 the particle size change does not reflect directly the successful conjugation of PEG or cRGD. The incorporation of PEG and cRGD was further validated in the cellular studies. In the transmission electron microscopy (TEM) image (Figure S4), the size of Gd@C82-Ala-PEG-cRGD was measured to be ca. 200 nm, which agreed well with the DLS results. Considering the distinct difference on sensitivity between PET imaging and MRI, the molar ratio of Gd@C82 and 64Cu need to be optimized before their integration. After radiolabeling Gd@C82Ala-PEG-(cRGD)-NOTA with 64Cu, the specific activity of Gd@C82-Ala-PEG-(cRGD)-NOTA64Cu

was calculated to be 14.8 MBq/μmol, and the decay-corrected radiochemical yield was 81 ±

3% (n = 3) (Figure 1e). Based on the specific activity of 64Cu, the 64Cu/Gd molar ratio in Gd@C82Ala-PEG-(cRGD)-NOTA-64Cu was calculated to be 32/1000,000 (the

89Zr/Gd

molar ratio in

Gd@C82-Ala-PEG-(cRGD)-Df-89Zr was calculated to be 133/1000,000. A PET/MRI phantom study was conducted to examine the detectability of Gd@C82-Ala-PEG-cRGD-NOTA-64Cu in both PET and MRI. PET and MRI images of Gd@C82-Ala-PEG-cRGD-NOTA-64Cu (0 -30 μM, in a 96-well plate) were shown in Figure 1d. The intensity of signals from PET and MRI were both strongly dependent on the concentration of Gd@C82-Ala-PEG-cRGD-NOTA-64Cu, which suggested that both signals from PET and MRI should truly reflect the concentration of Gd@C82Ala-PEG-cRGD-NOTA-64Cu. 3.2. Stability evaluation. The stability of Gd@C82-Ala-PEG-NOTA-64Cu and Gd@C82-Ala-PEGcRGD-NOTA-64Cu was tested in phosphate buffer saline (PBS, pH = 7.4) and 100% fetal bovine


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serum (FBS). After incubation in these media for 24 h, more than 90% of the 64Cu stayed intact on these two Gd@C82 conjugates (Figure 1f). In PET imaging, the signal detected came from γirradiation via the PET isotope (i.e. 64Cu), thus the high stability of Gd@C82 conjugates excluded the possibility that PET signals might from free

64Cu

dissociated from these two Gd@C82

conjugates, guaranteeing that the PET signal could truly reflect the distribution of these two Gd@C82 conjugates. 3.3. In vitro tumor cell targeting. We selected two cell lines to evaluate the Gd@C82 conjugates, U87-MG cells (integrin αvβ3+) and MCF-7 breast cancer cells (integrin αvβ3-). From the flow cytometry and confocal laser scanning microscopy (CLSM) examinations, we could conclude that the fluorescence from Gd@C82-Ala-PEG-FITC-cRGD was much stronger than that from Gd@C82Ala-PEG-FITC in U87-MG cells, while the fluorescence from these two Gd@C82 conjugates was substantially weaker in MCF-7 cells (Figure 2a & S6). All these observations indicated that the uptake of Gd@C82-Ala-PEG-FITC-cRGD was promoted as the result of cRGD incorporation in integrin αvβ3 overexpressed U87-MG cells, suggesting the uptake of Gd@C82-Ala-PEG-FITCcRGD was strongly dependent on the specific interaction between integrin αvβ3 and cRGD group. To better understand the dynamic interaction between Gd@C82-Ala-PEG-cRGD and U87-MG cells, we incubated Gd@C82-Ala-PEG-cRGD-NOTA-64Cu with U87-MG cells and the cell suspension was withdrawn at different time points to analyze their cell internalization and efflux rate. It showed that Gd@C82-Ala-PEG-cRGD-NOTA-64Cu could quickly attach onto the surface of U87-MG cells, reaching the peak value within 15 min (Figure 2b). Nearly half of Gd@C82-AlaPEG-cRGD-NOTA-64Cu on the surface was then internalized into U87-MG cells within 15 min, then the cell uptake of Gd@C82-Ala-PEG-cRGD-NOTA-64Cu reached the plateau at the following time points. The efflux rate of Gd@C82-Ala-PEG-cRGD-NOTA-64Cu from U87-MG cells was


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continuously growing within the first 0.5 h and then reached a plateau. The efflux rate of Gd@C82Ala-PEG-cRGD-NOTA-64Cu was calculated to be 25% on the plateau, which was relatively low for nanoparticles (Figure 2c). The high cell uptake and low cell efflux rate of Gd@C82-Ala-PEGcRGD-NOTA-64Cu proved that it possessed good affinity to U87-MG cells, which prepared it for the subsequent PET/MRI imaging of U87-MG tumors in vivo. 3.4. In vivo tumor targeting and PET imaging. U87-MG bearing mice were injected with Gd@C82-Ala-PEG-NOTA-64Cu and Gd@C82-Ala-PEG-cRGD-NOTA-64Cu, respectively. PET images were recorded at different time points of 0.5, 3, 18, 24, 48 h post injection (p.i.). Representative two-dimensional coronal images of U87-MG tumor bearing mice with different treatments were shown in Figure 3. Organ distribution information of Gd@C82-Ala-PEG-NOTA64Cu

and Gd@C82-Ala-PEG-cRGD-NOTA-64Cu was deduced from ROI analysis of PET images

and shown in Figure 4. Gd@C82-Ala-PEG-cRGD-NOTA-64Cu demonstrated a sustained accumulation in U87-MG tumors and reached its peak at 48 h p.i., with tumor uptake of 2.4 ± 0.2 %ID/g (n = 5). While the tumor uptake of Gd@C82-Ala-PEG-NOTA-64Cu was much lower than that of Gd@C82-Ala-PEG-cRGD-NOTA-64Cu, with peak values of 1.2 ± 0.2 %ID/g (n = 5). To further confirm the in vivo integrin αvβ3 specificity of Gd@C82-Ala-PEG-cRGD-NOTA-64Cu, a competitive study with high-dose cRGD peptide co-injection was conducted. The coadministration of ~5 mg/kg cRGD peptide (served as the “blocking” dose) with Gd@C82-AlaPEG-cRGD-NOTA-64Cu could cause a sustainable tumor uptake reduction for Gd@C82-Ala-PEGcRGD-NOTA-64Cu, to 0.7 ± 0.2 %ID/g (n = 5) at 48 h p.i (Figure 3). It confirmed the integrin αvβ3 specificity of Gd@C82-Ala-PEG-cRGD-NOTA-64Cu in vivo. Radioactivity captured in other major organs (e.g. liver, blood etc.) was not significantly affected by the blocking dose of cRGD peptide, as shown in Figure 4c. Calculated from the accumulation levels of Gd@C82 conjugates in


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U87-MG tumors in different test groups over time, Gd@C82-Ala-PEG-cRGD-NOTA-64Cu showed the strongest tumor accumulation (P < 0.05 in all cases, n = 5). 3.5. Ex vivo organ distribution studies. The U87-MG tumor bearing mice in different groups were sacrificed at 48 h p.i. and the organs were harvested to measure the radioactivity by γ counter. The ex vivo organ distribution results matched well with the quantitative organ distribution data by PET imaging (Figure 4d). A tumor uptake of 2.2 ± 0.3 %ID/g was achieved for Gd@C82-AlaPEG-cRGD-NOTA-64Cu with a tumor/muscle ratio of 18.6 ± 4.0 at this time (n = 5). Organ distribution profiles from both PET measurement and γ counting confirmed integrin αvβ3-targeting specificity of Gd@C82-Ala-PEG-cRGD-NOTA-64Cu in vivo. 3.6. Tumor targeting is mediated from integrin αvβ3. U87-MG tumors from mice injected with Gd@C82-Ala-PEG-FITC-cRGD and Gd@C82-Ala-PEG-FITC were frozen cut into 6 μm slices for further histological analysis. The green fluorescence (from FITC) in the U87-MG tumor was more intense in Gd@C82-Ala-PEG-FITC-cRGD treated group when compared with that in Gd@C82Ala-PEG-FITC (Figure 4e). Simultaneously, green fluorescence in Gd@C82-Ala-PEG-FITCcRGD group exhibited good overlay with integrin αvβ3 (red fluorescence), indicating that interactions with integrin αvβ3 in vivo contribute to the elevated uptake of Gd@C82-Ala-PEGFITC-cRGD in U87-MG tumors. 3.7. PET/MRI studies. In vivo PET/MRI studies with Gd@C82-Ala-PEG-cRGD-NOTA-64Cu were carried out on U87-MG bearing mice in a PET/MRI hybridization machine (MRS-PET, MR solutions, Guildford, United Kingdom). First, T1*-weighted MR images of the upper abdominal area and legs of the U87-MG bearing mouse injected with Gd@C82-Ala-PEG-cRGD-NOTA-64Cu at 24 h p.i. were obtained, and the tumor was precisely located and the boundary was clearly delineated. Both the coronal and axis MR images showed that the tumor site was significantly


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brighter than the peripheral tissue, suggesting a specific accumulation of Gd@C82-Ala-PEGcRGD-NOTA-64Cu at the tumor site. Then the mouse was subjected to PET scanning and the coronal and axis images were obtained, respectively. Finally, the MRI and PET images were merged using the VivoQuant software (3.5rc23, Christian Lackas, inviCRO LLC.) accordingly. Figure 5 showed the representative two-dimensional coronal and axis PET/MRI image of U87MG tumor bearing mice at 24 h p.i. From the image, the outline of the tumor was clearly delineated with the MR image and the PET signals showed an obvious accumulation of Gd@C82-Ala-PEGNOTA-64Cu-cRGD at the tumor site. Liver that possessed high uptake of Gd@C82-Ala-PEGNOTA-64Cu-cRGD could be clearly seen in the image as well. 3.8. Metabolic behavior of Gd@C82-Ala-PEG-Df-89Zr in vivo. The metabolism behavior of Gd@C82-Ala-PEG was monitored in vivo for up to 30 days, since the decay half-life of

89Zr

is

sufficiently long (The injection dose per animal was increased to 15-20 MBq for long-term decay purposes). Representative two dimensional coronal PET images of U87-MG bearing mice injected with Gd@C82-Ala-PEG-Df-89Zr at different time points (0.5, 3, 20, 24, 48, 72, 96, 120, 168, 216, 288, 336, 432, 528, 624, 720 h) were listed in Figure 6a. At the time of 0.5 h p.i. and 3 h p.i., the strong PET signals in the bladder indicated that Gd@C82-Ala-PEG-Df-89Zr had a significant excretion via the renal pathway. It was calculated that nearly 70% of Gd@C82-Ala-PEG-Df-89Zr was eliminated from the body within 24 h, suggesting that Gd@C82-Ala-PEG-Df-89Zr was clearable in vivo. It has reported that only small nanoparticles (smaller than 5 nm) could be eliminated from the body through the renal pathway. Considering the size of Gd@C82-Ala-PEGDf-89Zr nanoparticle (ca. 200 nm), the renal clearance is not originally anticipated. However, Gd@C82-Ala-PEG-Df-89Zr nanoparticle was a self-assembly from thousands of Gd@C82-AlaPEG-Df-89Zr molecules. It was highly likely that this nanostructure could disassemble into smaller


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pieces which may pass through renal filtration in vivo. After 24 h, liver was the only tissue that have obvious uptake of Gd@C82-Ala-PEG-Df-89Zr and the accumulation remained almost the same (ca. 20 %ID/g) during the rest time frame (Figure 6c). The Gd@C82-Ala-PEG-Df-89Zr injected mice were sacrificed at 720 h p.i. and the organs were harvested to measure the radioactivity by γ counter. In good agreement with the PET imaging results, most of the Gd@C82Ala-PEG-Df-89Zr was accumulated in the liver and the distribution in other organs was negligible (Figure 6b). It has been reported that the free 89Zr ions have significant accumulation in the bone; however, the uptake of Gd@C82-Ala-PEG-Df-89Zr in bone was less than 3% at 720 p.i., indicating that the PET signal should be from Gd@C82-Ala-PEG-Df-89Zr instead of free 89Zr during the whole monitoring time frame. 4. DISCUSSION The data obtained in this study confirmed that the tumor targeting efficacy of gadofullerene derivative, Gd@C82-Ala, could be significantly improved post proper surface modification strategy. More importantly, the real-time pharmacokinetic behavior of Gd@C82-Ala can be monitored by PET/MRI (64Cu) or PET (89Zr and 64Cu) for up to 30 days post the incorporation of PET isotopes with good sensitivity, which provided very reliable information for dynamic toxicological evaluation of gadofullerene derivatives. As we previously stated, this information can give some insights on the in vivo safety of gadofullerene derivatives, which is a significant concern for these materials despite their broad biomedical applications. The physical/chemical properties of gadofullerene materials can be adjusted in a more efficient manner with the information acquired. There are two reasons for us to adopt two isotopes (89Zr and 64Cu) in this study: firstly, 89Zr is much more costive than Cu-64 and its chelation and purification process are much more time-consuming than those of Cu-64. Secondly, the radiation dose imposed by Cu-64


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is also significantly lower than Zr-89. Thus, we chose Cu-64 for short-term in vivo study and cellular evaluation. Since the good radiochemical stability of radiolabeled Gd@C82-Ala has been validated in this study (Figure 1f), the early renal clearance of Gd@C82-Ala-PEG-NOTA-64Cu observed from PET studies (Figure 3 and 4) may indicate the partial disruption of gadofullerene nanoparticles in vivo. The truth is, fullerene derivatives usually assemble (or aggregate) into “micellar” structures in the aqueous environment 35-36, thus these micellar structure can be dissembled (at least partially) in vivo once the material concentration is below the critical micelle concentration (CMC). This disruption of gadofullerene nanoparticles does not cause the leakage of Gd from fullerene structure, thus should not lead to safety or toxicity concerns. Combined with the fact that more than 70% of the total gadofullerene particles can be excreted from the test subjects within 24 h (Figure 6), we believe that the gadofullerene material used in this study will not cause significant safety concern, since fast clearance and minimal interaction with the mononuclear phagocytic system (MPS, e.g. liver, spleen etc.) are preferred characters for nanomaterials used in an imaging/therapeutic application

37.

However, we have to be aware that the diagnostic or therapeutic efficacy of

gadofullerene nanoparticles results from a balance between material clearance and target tissue (glioblastoma in this case) retention. Thus, the degradation speed of Gd@C82-Ala derivatives should be further investigated to meet the requirement of different scenarios. For example, a biodegradable silica shell can be formed as an protective “shell” for gadofullerene nanoparticles to slow down their degradation speed and increase their circulation time to further increase the accumulation into the target tissue 38. Although the uptake of Gd@C82-Ala in U87MG tumors was significantly enhanced after attachment of cRGD, we have to admit that the absolute tumor accumulation (< 3 %ID/g at all


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time points examined, Figure 3 and 4) still needs further optimization. One of the reasons may be due to the medium expression level of integrin αvβ3 on glioblastoma cells (~105 per cell). Other receptors with higher abundance will be pursued as targets of interest in the future (e.g. nucleolin 39,

follicle-stimulating hormone receptor 40 etc.), with the hope of further boosting tumor uptake.

In addition, the impact of surface ligand (e.g. cRGD) density, surface charges, the length of linker (PEG in our case), or interactions with plasma proteins, will be investigated in more details for future studies. Therapeutic study will also be initiated once optimal tumor retention is achieved. PET isotopes (89Zr and

64Cu)

were incorporated onto the structure of gadofullerene

nanoparticles by the interaction with their chelators (Df or NOTA attached on surface PEG) in this study. One potential limitation is that the amide bond between PEG and Gd@C82-Ala can be broken down by internal proteases, which can compromise the accurate quantification of Gd@C82Ala nanoparticles in vivo since PET only detects the 511-keV γ-photon pairs emitted from the isotopes. Future “chelator-free” labeling of gadofullerene can be pursued to increase the overall integrity of gadofullerene in vivo

41,

while other radiometals (e.g. manganese-52) can be

incorporated into the structure of fullerene to develop new metal-fullerene for other biomedical utilization. 5. CONCLUSIONS A water-soluble Gd@C82 fullerene derivative, Gd@C82-Ala-PEG-cRGD-NOTA-64Cu as a PET/MRI tracer was synthesized and characterized. It can serve as a simultaneous MRI and PET contrast agent. It can target the glioblastoma as the presence of cRGD, which can specifically bind with αvβ3 integrin. The PET/MRI images of U87-MG tumor bearing mice injected with Gd@C82Ala-PEG-cRGD-NOTA-64Cu were recorded for the first time and used to study the pharmacokinetics and biodistribution in vivo. By employing a much longer half-life isotope, 89Zr,


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we can monitor the metabolism behavior of Gd@C82-Ala-PEG up to 30 days. The PET/MRI hybrid imaging modalities provide a reliable tool to study the fate of Gd@C82-Ala-PEG in vivo, which may extend to other Gd@C82 derivatives. ASSOCIATED CONTENT Supporting Information. Some material characterization data (e.g. XPS, TGA, and IR spectroscopy) were included in the Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. ACKNOWLEDGMENT This work is supported, in part, by the University of Michigan Department of Radiology, NIH/NCI P01 CA085878, the National Natural Science Foundation of China (81201696), and Jiangsu Government Scholarship for Overseas Studies. COMPETING FINANCIQAL INTEREST STATEMENT The authors declare no competing financial interests.

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Figure Legends Scheme 1 The scheme of employing Gd@C82-Ala-PEG-cRGD-NOTA-64Cu as a PET/MRI probe for the targeted imaging of xenografted glioblastoma tumor. Figure 1 The characterization of Gd@C82-Ala-PEG-cRGD-NOTA-64Cu. (a) UV-vis spectra of Gd@C82-Ala, Gd@C82-Ala-PEG and Gd@C82-Ala-PEG-cRGD. (b) DLS measurements of Gd@C82-Ala, Gd@C82-Ala-PEG and Gd@C82-Ala-PEG-cRGD. (c) TEM image of Gd@C82-AlaPEG-cRGD. Scale bar: 200 nm. (d) The correlation between the signals from PET and MRI and the concentration of Gd@C82-Ala-PEG-cRGD-NOTA-64Cu. (e) The elution curve of Gd@C82Ala-PEG-cRGD-NOTA-64Cu. (f) Stability evaluation of Gd@C82-Ala-PEG-cRGD-NOTA-64Cu and Gd@C82-Ala-PEG-NOTA-64Cu in various media (FBS and PBS). Figure 2 In vitro evaluations of Gd@C82-Ala-PEG conjugates. (a) Representative confocal fluorescence microscopy images of U87-MG (integrin αvβ3+) and MCF-7 cells (integrin αvβ3-) incubated with Gd@C82-Ala-PEG-FITC-cRGD and Gd@C82-Ala-PEG-FITC (both containing 1 μg/mL of FITC). Scale bar: 20 μm. (b) The evaluation of cell internalization and surface bound Gd@C82-Ala-PEG-cRGD-NOTA-64Cu in U87-MG cells. (c) The efflux behavior of Gd@C82Ala-PEG-cRGD-NOTA-64Cu from U87-MG cells. Figure 3 Representative coronal PET images of MDA-MB-231 tumor bearing mice at different time points post-injection of Gd@C82-Ala-PEG-cRGD-NOTA-64Cu, Gd@C82-Ala-PEG-NOTA64Cu,

and Gd@C82-Ala-PEG-cRGD-NOTA-64Cu with excessive amount of cRGD peptide

blocking. The location of tumors was identified by white dashed circles and arrows. Figure 4 Quantitative region-of-interest (ROI) analysis of PET data and organ distribution data. Time-activity curves of the liver, U87-MG tumor, blood, and muscle were shown post-injection of (a) Gd@C82-Ala-PEG-cRGD-NOTA-64Cu, (b) Gd@C82-Ala-PEG-NOTA-64Cu, and (c)


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Gd@C82-Ala-PEG-cRGD-NOTA-64Cu with excessive amount of cRGD peptide blocking. (d) Organ distribution data obtained by tissue gamma counting were also given at and 48 h p.i. of Gd@C82 conjugates. (e) Immunohistological staining of U87-MG tumors from mice injected with Gd@C82-Ala-PEG-FITC-cRGD and Gd@C82-Ala-PEG-FITC. Integrin αvβ3 (red) expression profile was compared with FITC (green) distribution pattern from both of the Gd@C82 conjugates. Scale bar: 100 μm. Figure 5 PET/MRI image of U87-MG tumor bearing mice at 24 h p.i. The tumor was identified by white dashed circle. (a) Coronal PET, MRI, and PET/MRI merged images. (b) Cross-sectional PET, MRI, and PET/MRI merged images. Figure 6 (a) Representative coronal PET images of U87-MG tumor bearing mice at different time points post-injection of Gd@C82-Ala-PEG-Df-89Zr. (b) Organ distribution data obtained by tissue gamma-counting were also given at and 720 h p.i. of Gd@C82-Ala-PEG-Df-89Zr. (c) Time-activity curves of the liver of the mice post-injection of Gd@C82-Ala-PEG-Df-89Zr.


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Scheme 1


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Figure 1


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Figure 2


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Figure 3


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Figure 4


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Figure 5


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Figure 6


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TOC figure 74x41mm (300 x 300 DPI)


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Magnetic Resonance Imaging of

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