Magnetic Resonance Imaging of

May 29, 2019 - Water-soluble gadofullerene nanomaterials have been extensively investigated as magnetic resonance imaging (MRI) contrast agents, radic...
0 downloads 0 Views 5MB Size
Download PDF
Research Article Cite This: ACS Appl. Mater. Interfaces 2019, 11, 21343−21352

www.acsami.org

Positron Emission Tomography/Magnetic Resonance 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*,†,⊥ †

Department of Radiology, Center for Molecular Imaging, University of Michigan, Ann Arbor, Michigan 48109-2200, United States Key Laboratory of Molecular Nanostructure and Nanotechnology, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China § Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical University, Xuzhou, Jiangsu 221004, China ∥ College of Animal Sciences and Veterinary Medicine, Henan Agricultural University, Zhengzhou, Henan 450002, China ⊥ University of Michigan Comprehensive Cancer Center, Ann Arbor, Michigan 48109-0944, United States

Downloaded via MOUNT ALLISON UNIV on August 5, 2019 at 02:31:26 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

S Supporting Information *

ABSTRACT: Water-soluble gadofullerene nanomaterials have been extensively investigated as magnetic resonance imaging (MRI) contrast agents, radical scavengers, sensitizers for photodynamic therapy, and inherent antineoplastic agents. 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 being satisfactory, and its dynamic pharmacokinetics and long-term metabolic behaviors remain to be elucidated. Herein, Gd@C82-Ala was chemically modified with eight-arm polyethylene glycol amine 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 were properly characterized. Also, its glioblastoma cell targeting capacity was evaluated in vitro by flow cytometry, confocal fluorescence microscopy, and dynamic cellular interaction assays. Because of 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-Ala-PEGcRGD-NOTA-64Cu (NOTA stands for 1,4,7-triazacyclononane-triacetic acid) demonstrated much higher accumulation in U87MG tumor than its counterpart without cRGD attachment from in vivo PET observation, consistent with observation at the cellular level. In addition, Gd@C82-Ala-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 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

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 sponges,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 © 2019 American Chemical Society

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 Received: March 3, 2019 Accepted: May 29, 2019 Published: May 29, 2019 21343

DOI: 10.1021/acsami.9b03542 ACS Appl. Mater. Interfaces 2019, 11, 21343−21352

Research Article

ACS Applied Materials & Interfaces fluorescence imaging is a real-time and noninvasive 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. ICP-MS 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 pretreated with a 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, noninvasive, and quantifiable method for real-time monitoring of gadofullerenes in vivo. 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 Gd-diethylenetriaminepentaacetic acid, 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. The cyclic Arg-GlyAsp (cRGD) peptide is a small cyclic peptide that can selectively bound with integrin αvβ3, which is overexpressed on the 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 eight-arm PEGNH2 and then the targeting group, cRGD. PET isotopes such as 64Cu or 89Zr were subsequently incorporated to obtain Gd@ C82-Ala-PEG-cRGD-(NOTA-64Cu or Df-89Zr). The gadofullerene-cRGD conjugates demonstrated much higher accumulation in U87-MG tumor than gadofullerene without the 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-γ-maleimidobutyryl-oxysuccinimide ester (GMBS), 2mercaptoethanol, Chelex 100 resin (50−100 mesh) were all purchased from Sigma-Aldrich (St. Louis, MO). 64CuCl2 and 89Zr(C2O4)2 were acquired from the University of Wisconsin Cyclotron. Dialysis bags (M.W.cutoff = 3 kDa), ultrafiltration tubes (M.W.cutoff = 50 kDa and M.W.cutoff = 10 kDa) were all ordered from Thermo Fisher Scientific (Fair Lawn, NJ). Eight-arm PEG-NH2 (M.W. = 40 kDa) was purchased from Creative PEGWorks (Chapel Hill, NC). 2S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) and 1-(4-isothiocyanatophenyl)-3-[6,17dihydroxy-7,10,18,21-tetraoxo-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 International (Louisville, KY). The anti-integrin αvβ3 antibody was purchased from Abcam (Cat no. 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. PD-10 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 before its color turned to brown. Afterward, 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.cutoff = 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 of phosphate-buffered saline (PBS, pH = 6.0) to a concentration of 20 μM. After adding 0.4 mg of EDC·HCl and 0.6 mg of NHS, the mixture was stirred at R.T. for 15 min to activate the carboxyl groups on Gd@C82-Ala. 2-mercaptoethanol (1.4 μL) was then added to stop the activation. The obtained solution was passed through a centrifugation filter (M.W.cutoff = 10 kDa) for several times to remove the small-molecule reactants. The activated Gd@C82-Ala was resuspended in 1 mL of PBS (pH = 7.4) with 1.6 mg of eight-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.cutoff = 50 kDa). 2.4. Synthesis of Gd@C82-Ala-PEG-cRGD. Gd@C82-Ala-PEGNH2 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.cutoff = 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.cutoff = 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 21344

DOI: 10.1021/acsami.9b03542 ACS Appl. Mater. Interfaces 2019, 11, 21343−21352

Research Article

ACS Applied Materials & Interfaces

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) post-injection (p.i.). The three-dimensional ordered subset expectation maximization (3D-OSEM) algorithm was used for PET image reconstruction, and no attenuation or scatter correction was applied. Region-of-interest (ROI) analysis of PET images was done on the 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 wet-weighed). A WIZARD2 automatic γ-counter (PerkinElmer) was used to measure the radioactivity density (in the unit of % ID/g) in these organs and tissues to corroborate with PET findings. 2.10. PET/MRI Studies. Six-week-old female nude mice (n = 5) were anesthetized and injected with 200 μL of Gd@C82-Ala-PEGcRGD-NOTA-64Cu (1 mM, 16 MBq) in saline via a tail vein. At various time points, the test animals were placed in a prone position on the MRI mouse bed to keep the temperature at 37 °C, and a pressure transducer was placed under the mouse abdomen for respiratory monitoring. T1*-weighted FSET 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 average, 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 a 3D-OSEM 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 the sample size in each test was kept not less than 3 to maintain the statistical power. The unpaired two-tailed Student’s t test was applied to determine the statistical differences between test groups. P < 0.05 was considered to be statistically significant (indicating a confidence interval of >95%).

into the above solution. The mixture was under reaction for 2 h at R.T. Excessive p-SCN-Bn-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 the 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 0.25 M NH4OAc buffer (pH 5.5) as an ITLC eluent with TecControl 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-PEG-NOTA-64Cu or Gd@C82Ala-PEG-cRGD-NOTA-64Cu was suspended in PBS (pH = 7.4) or complete FBS at 37 °C for up to 24 h. A given amount of the sample (∼50 μL) was taken from the mixture at the set time points (15 min and 0.5, 1, 2, 4, 16, and 24 h) and passed through Amicon filters with a M.W.cutoff of 100 kDa (centrifuged at 12,000g for 10 min). The radioactivity from the filtrates was measured in a WIZARD2 γ-counter (PerkinElmer). The percentages of intact or stable 64Cu on Gd@C82Ala-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@C 82-Ala-PEG-Df was purified by repeated ultrafiltration (M.W.cutoff = 10 kDa). To incorporate 89Zr into Gd@C82-Ala-PEGDf, 89Zr-oxalate with a 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-PEGDf at a ratio of 0.81 μg of 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-PEGDf-89Zr was separated from 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 Tec-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 the incubation, the cells were harvested, washed repeatedly with cold PBS, and resuspended in PBS at a 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 fluorescence between treatment groups. To provide a further visual evidence, these cells were cultured on glass Petri dishes and undertaken the 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. A Inveon rodent microPET/CT scanner (Siemens Medical Solutions USA, Inc.) was used to acquire PET images from test animals. Gd@C82-Ala-PEGNOTA-64Cu or Gd@C82-Ala-PEG-cRGD-NOTA-64Cu with the

3. RESULTS 3.1. Material Preparation and Characterization. Gd@ C82-Ala was prepared through a solid−liquid reaction method, and the average molecular structure was determined as Gd@ C82(OH)13(NHCH2CH2COOH)6 by X-ray photoelectron spectrometry (XPS), thermogravimetric analysis (TGA), infrared spectroscopy (IR), and elemental analysis (Figures S1−S3 and Table S1). Gd@C82-Ala was further conjugated with eight-arm PEG amine, which not only rendered the nanoparticles with much longer blood circulation time but also introduced free amine residues that facilitated the subsequent surface modification of gadofullerenes. A glioblastomatargeting ligand, cRGD, was attached to the surface of Gd@ C82-Ala-PEG (Scheme 1). The UV−vis spectrum of Gd@C82Ala-PEG-cRGD is 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, 164, and 190 nm, respectively (Figure 1b). Because fullerene usually forms aggregates in the solution,4,35 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 ∼200 nm, which agreed well with the DLS results. 21345

DOI: 10.1021/acsami.9b03542 ACS Appl. Mater. Interfaces 2019, 11, 21343−21352

Research Article

ACS Applied Materials & Interfaces

(Figure 1e). On the basis of the specific activity of 64Cu, the Cu/Gd molar ratio in Gd@C82-Ala-PEG-(cRGD)-NOTA-64Cu was calculated to be 32/1,000,000 (the 89Zr/Gd molar ratio in Gd@C82-Ala-PEG-(cRGD)-Df-89Zr was calculated to be 133/1,000,000). A PET/MRI phantom study was conducted to examine the detectability of Gd@C82-Ala-PEGcRGD-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) are shown in Figure 1d. The intensities 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@C82-Ala-PEG-cRGDNOTA-64Cu. 3.2. Stability Evaluation. The stability of Gd@C82-AlaPEG-NOTA-64Cu and Gd@C82-Ala-PEG-cRGD-NOTA-64Cu was tested in phosphate-buffered saline (PBS, pH = 7.4) and 100% fetal bovine 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 γ-irradiation via the PET isotope (i.e., 64Cu); thus, the high stability of Gd@C82 conjugates

Scheme 1. Scheme of employing Gd@C82-Ala-PEG-cRGDNOTA-64Cu as a PET/MRI probe for the targeted imaging of xenografted glioblastoma tumor.

64

Considering the distinct difference on sensitivity between PET imaging and MRI, the molar ratio of Gd@C82 and 64Cu needs to be optimized before their integration. After radiolabeling Gd@C82-Ala-PEG-(cRGD)-NOTA with 64Cu, the specific activity of Gd@C 82-Ala-PEG-(cRGD)-NOTA-64Cu was calculated to be 14.8 MBq/μmol, and the decay-corrected radiochemical yield was 81 ± 3% (n = 3)

Figure 1. Characterization of Gd@C82-Ala-PEG-cRGD-NOTA-64Cu. (a) UV−vis spectra of Gd@C82-Ala, Gd@C82-Ala-PEG, and Gd@C82-AlaPEG-cRGD. (b) DLS measurements of Gd@C82-Ala, Gd@C82-Ala-PEG, and Gd@C82-Ala-PEG-cRGD. (c) TEM image of Gd@C82-Ala-PEGcRGD. Scale bar: 200 nm. (d) The correlation between the signals from PET and MRI and the concentration of Gd@C82-Ala-PEG-cRGDNOTA-64Cu. (e) The elution curve of Gd@C82-Ala-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). 21346

DOI: 10.1021/acsami.9b03542 ACS Appl. Mater. Interfaces 2019, 11, 21343−21352

Research Article

ACS Applied Materials & Interfaces

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. U87MG-bearing mice were injected with Gd@C82-Ala-PEGNOTA-64Cu and Gd@C82-Ala-PEG-cRGD-NOTA-64Cu. 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 are shown in Figure 3. Organ distribution

excluded the possibility that PET signals might be from free 64 Cu 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−). In 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@C82-Ala-PEG-FITC in U87-MG cells, and the fluorescence from these two Gd@C82 conjugates was substantially weaker in MCF-7 cells (Figure 2a and Figure

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-cRGDNOTA-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-NOTA-64Cu, 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.

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-FITC-cRGD was strongly dependent on the specific interaction between integrin αvβ3 and the 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-Ala-PEGcRGD-NOTA-64Cu on the surface was then internalized into U87-MG cells within 15 min; then, the cell uptake of Gd@C82Ala-PEG-cRGD-NOTA-64Cu reached the plateau at the following time points. The efflux rate of Gd@C82-Ala-PEGcRGD-NOTA-64Cu from U87-MG cells was continuously growing within the first 0.5 h and then reached a plateau. The efflux rate of Gd@C82-Ala-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-PEG-cRGD-NOTA-64Cu

information of Gd@C82-Ala-PEG-NOTA-64Cu and Gd@C82Ala-PEG-cRGD-NOTA-64Cu was deduced from ROI analysis of PET images and is shown in Figure 4. Gd@C82-Ala-PEGcRGD-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). Meanwhile, 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-cRGDNOTA-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-Ala-PEG-cRGD-NOTA-64Cu could cause a sustainable tumor uptake reduction for Gd@C82-Ala-PEG-cRGDNOTA-64Cu to 0.7 ± 0.2% ID/g (n = 5) at 48 h p.i (Figure 3). It confirmed that the integrin αvβ3 specificity of Gd@C82-AlaPEG-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 the cRGD peptide, as shown in Figure 4c. Calculated from the accumulation levels of Gd@ C82 conjugates in U87-MG tumors in different test groups over 21347

DOI: 10.1021/acsami.9b03542 ACS Appl. Mater. Interfaces 2019, 11, 21343−21352

Research Article

ACS Applied Materials & Interfaces

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, muscle, bladder, and spleen were shown post-injection of (a) Gd@C82-Ala-PEG-cRGD-NOTA-64Cu, (b) Gd@C82-Ala-PEG-NOTA-64Cu, and (c) Gd@C82-Ala-PEG-cRGD-NOTA-64Cu with excessive amount of cRGD peptide blocking. (d) Organ distribution data obtained by tissue γcounting were also given at 48 h p.i. of Gd@C82 conjugates. (e) Immunohistological staining of U87-MG tumors from mice injected with Gd@C82Ala-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.

into 6 μm slices for further histological analysis. The green fluorescence (from FITC) in U87-MG tumors was more intense in the Gd@C82-Ala-PEG-FITC-cRGD-treated group when compared with that in Gd@C82-Ala-PEG-FITC (Figure 4e). Simultaneously, green fluorescence in the Gd@C82-AlaPEG-FITC-cRGD group exhibited a good overlay with integrin αvβ3 (red fluorescence), indicating that interactions with integrin αvβ3 in vivo contribute to the elevated uptake of Gd@C82-Ala-PEG-FITC-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, U.K.). First, T1*weighted MR images of the upper abdominal area and legs of the U87-MG-bearing mouse injected with Gd@C82-AlaPEG-cRGD-NOTA-64Cu at 24 h p.i. were obtained; the tumor

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. 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 the 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-Ala-PEG-cRGD-NOTA-64Cu with a tumor/muscle ratio of 18.6 ± 4.0 at this time (n = 5). Organ distribution profiles from both the PET measurement and γcounting confirmed the 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-PEGFITC-cRGD and Gd@C82-Ala-PEG-FITC were frozen cut 21348

DOI: 10.1021/acsami.9b03542 ACS Appl. Mater. Interfaces 2019, 11, 21343−21352

Research Article

ACS Applied Materials & Interfaces

possessed high uptake of Gd@C82-Ala-PEG-NOTA-64CucRGD could be clearly seen in the image also. 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 because 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 U87MG-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, and 720 h) are listed in Figure 6a. At times 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-PEGDf-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-PEG-Df-89Zr nanoparticle (∼200 nm), the renal clearance is not originally anticipated. However, the Gd@C82Ala-PEG-Df-89Zr nanoparticle was a self-assembly from thousands of Gd@C82-Ala-PEG-Df-89Zr molecules. It was highly likely that this nanostructure could disassemble into smaller pieces, which may pass through renal filtration in vivo. After 24 h, the liver was the only tissue that has obvious uptake of Gd@C82-Ala-PEG-Df-89Zr, and the accumulation remained almost the same (∼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 the γ-counter. In good

was precisely located, and the boundary was clearly delineated. Both the coronal and axis MR images showed that the tumor site was significantly 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. Finally, the MRI and PET images were merged using the VivoQuant software (3.5rc23, Christian Lackas, Invicro, LLC) accordingly. Figure 5 shows the representative

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.

two-dimensional coronal and axis PET/MRI image of U87MG tumor-bearing mice at 24 h p.i. In the image, the outline of the tumor was clearly delineated with the MR image, and the PET signals showed an obvious accumulation of Gd@C82Ala-PEG-NOTA-64Cu-cRGD at the tumor site. The liver that

Figure 6. (a) Representative coronal PET images of U87-MG tumor-bearing mice at different time points post-injection of Gd@C82-Ala-PEGDf-89Zr. (b) Organ distribution data obtained by tissue γ-counting were also given at 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. 21349

DOI: 10.1021/acsami.9b03542 ACS Appl. Mater. Interfaces 2019, 11, 21343−21352

Research Article

ACS Applied Materials & Interfaces

admit that the absolute tumor accumulation (