Sensitive Contrast-Enhanced MR Imaging of Orthotopic and Metastatic

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Sensitive Contrast-Enhanced MR Imaging of Orthotopic and Metastatic Hepatic Tumors by Ultralow Doses of Zinc Ferrite Octapods Lijiao Yang, Chengjie Sun, Hongyu Lin, Xuanqing Gong, Tiantian Zhou, Wen-Ting Deng, Zhong Chen, and Jinhao Gao Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.8b04760 • Publication Date (Web): 05 Feb 2019 Downloaded from http://pubs.acs.org on February 5, 2019

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Chemistry of Materials

Sensitive Contrast-Enhanced MR Imaging of Orthotopic and Metastatic Hepatic Tumors by Ultralow Doses of Zinc Ferrite Octapods

Lijiao Yang,1 Chengjie Sun,1 Hongyu Lin,1 Xuanqing Gong,1 Tiantian Zhou,2 Wen-Ting Deng,1 Zhong Chen,2 and Jinhao Gao1* 1State

Key Laboratory of Physical Chemistry of Solid Surfaces, The MOE Key Laboratory of

Spectrochemical Analysis & Instrumentation, The Key Laboratory for Chemical Biology of Fujian Province, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China. 2Department

of Electronic Science, Fujian Key Laboratory of Plasma and Magnetic Resonance,

College of Electronic Science and Technology, Xiamen University, Xiamen, 361005, China. *E-mail: [email protected]

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ABSTRCT: The contrast ability of magnetic nanoparticles (MNPs) is critical for sensitive and accurate diagnosis of cancer with magnetic resonance imaging (MRI). However, the low sensitivity of current contrast agents (CAs) remains a major limitation to meet clinical needs. Shape and composition engineering are two important means for improving the contrast ability of MNPs. Herein, we report a facile method incorporating both approaches to manufacture zinc ferrite octapods with large effective radius and various Zn doping ratios. The saturated magnetizations and T2 relaxivities of these engineered octapods both show an interesting trend with ascending zinc doping level. Particularly, the r2 value of ZnxFe3-xO4 (x = 0.44) octapods at 7.0 T is 989.1 mM-1s-1, the highest T2 relaxivity ever reported, which is 10.2 times higher than commercial sample Feraheme. With the aid of this octapod sample, contrast enhanced MRI (CE-MRI) for detecting orthotopic and metastatic hepatic tumors could be accomplished even at a dose as low as 0.2 mg/kg, 1/10 of the regular dose for commercial CAs. These ZnxFe3-xO4 octapods hold great potential for sensitive CE-MRI in detecting early hepatic tumors and tiny metastatic tumors with less dosages and side effects in clinic.


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INTRODUCTION The sensitive imaging of tiny tumors is essential for early diagnosis, proper disease management and personalized medicine. Among numerous clinical diagnostic techniques, magnetic resonance imaging (MRI) has been widely employed during the past two decades due to its safety and high spatial resolution for soft tissues.1-3 Magnetic resonance signal can be modulated with magnetic nanoparticles (MNPs) by accelerating the proton relaxation process of nearby water molecules under an external magnetic field.4-6 Therefore, MNPs can serve as contrast agents (CAs) to enhance the contrast between the detected region and the background, thus improve the sensitivity and accuracy of MRI. Iron oxide (IO) NPs have been well developed as T2 CAs,7-9 nevertheless, their low sensitivity still restricts their application in clinical accurate diagnosis. Researchers have explored various routes to improve their contrast ability, such as altering their morphology or composition.10, 11 Adjusting genre and quantity of metal doping could tune the magnetic property and contrast ability of ferrite NPs. Therefore, many stratagies on the regulation of IO’s composition12 (such as adjusting metal doping ratios of Mn,13-15 Zn,16 Co17,

18)

are developed. Meanwhile, the investigations of IO NPs with

different morphologies are never stagnated.19,

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However, up to now, reports on integrating

composition and morphology as a co-regulatory factor to achieve optimal contrast enhancement are still very destitute. Zinc doping can adjust the antiferromagnetic coupling of magnetic ions that occupy tetrahedral (Td) or octahedral (Oh) vacancies and regulate the saturation magnetization (Ms) of NPs.21 Based on extended X-ray absorption fine structure (EXAFS) analysis,16 zinc doping could induce structure transition from inverse spinel (Fe3O4) to spinel ((ZnxFe1-x)Fe2O4), resulting in highly improved T2 contrast effect. According to our previous study, octapod has the largest effective radius among classical morphologies.22 Its large magnetic field inhomogeneity and stray field gradient generated by the external magnetic field can extremely expedite the relaxation process of protons, leading to a considerably large saturated magnetization and strong T2 contrast ability. On the basis of these theories, we envisioned that zinc doped IO NPs of octapod shape could achieve optimal T2 imaging performance. 3 ACS Paragon Plus Environment


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In this work, we fabricated zinc-doped iron oxide (ZnxFe3-xO4) NPs of octapod shape with different zinc ratios by one-pot synthesis. Interestingly, the saturation magnetization (Ms) of ZnxFe3-xO4 octapods increase incipiently as the zinc doping proportion rises. When the proportion of zinc to iron reaches about 1/6 (x = 0.44), the ZnxFe3-xO4 octapods retain the typical magnetite structure with a surprising Ms of 89.0 emu/g and a remarkable r2 value of 989.1 mM-1s-1 at 7.0 T. However, when the doping ratio of zinc continues to increase, the Ms of ZnxFe3-xO4 decreases, resulting in diminished r2 values. In vivo experiments further confirm the robust T2 contrast-enhanced effect of ZnxFe3-xO4 (x = 0.44) octapods for orthotopic and metastatic tumors even at one tenth of the regular dose of Feraheme, indicating their potential as a new generation CA of low dosage for sensitive and accurate MRI.

Results and Discussion Characterization. We prepared ZnxFe3-xO4 octapods via thermal decomposition of ferric oleate and zinc oleate in 1-octadecene with oleic acid as a surfactant. ZnxFe3-xO4 octapods with different zinc contents (x = 0.21, 0.44, 0.76, and 0.99) and Fe3O4 (x = 0) octapods of similar size were produced by altering the proportions of the two precursors (see Table S1 and experimental sections for details). Transmission electron microscopy (TEM) images (Figure 1, Figure 2a and Figure S1 at low magnification) show that the NPs are all typical octapods with a narrow size distribution and high yield (>90%), the average edge length of two nearby arms is 30 nm and the corner angle is about 40°. ZnxFe3-xO4 (x = 0.44) octapods were chose as a representative sample for further structure characterization. The high-resolution transmission electron microscopy (HRTEM) image (Figure 2b) reveals their excellent crystallinity. The crossed lattice spacing distance of 0.30 nm is ascribed to the (220) facet of inverse spinel magnetite structure. This structure is further affirmed by selected area electron diffraction (SAED) patterns (Figure 2c). The energy-dispersive X-ray (EDX) line scanning analysis (Figure 2d), EDX mapping images (Figure 2e) both demonstrate that iron and zinc ions are evenly distributed in IO NPs. Structure and Magnetism. The diffraction peaks of X-ray diffraction (XRD) confirm the inverse spinel magnetite (JCPDS no. 01-088-0866) structure of these ZnxFe3-xO4 octapods (Figure 3a). The radius of Zn (II) ions (74 pm) is larger than those of Fe (II) ions (61 pm) and Fe (III) ions (55 pm), 4 ACS Paragon Plus Environment

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hence Zn doping results in slightly increased lattice distance and left-shifted peaks with the increment of Zn doping according to the previous investigation.23 The X-ray photoelectron spectroscopy (XPS) spectra of these octapods with different Zn doping ratios (Figure 3b and Figure S2) show clear peaks at 710.8 eV (Fe 2p3/2) and 724.0 eV (Fe 2p1/2),24 1021.5 eV (Zn 2p3/2) and 1044.7 eV (Zn 2p1/2),25,

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indicating the existence of Fe (III) and Zn (II). Then we measured the

magnetic properties of these ZnxFe3-xO4 (x = 0, 0.21, 0.44, 0.76 and 0.99) NPs under an external magnetic field of 5 T at 300 K. The samples were all prepared as powder after ethanol washing and plasma cleaning. The magnetic hysteresis loops (M-H) (Figure 3c) suggest that they all exhibit typical superparamagnetic properties with negligible coercive forces (less than 20 Oe, Table S2). The Ms values (Figure 3d) of these samples are 70.5 (x = 0), 74.7 (x = 0.21), 88.9 (x = 0.44), 69.3 (x = 0.76) and 58.9 (x = 0.99) emu/g (emu/total mass of all metal and oxygen atom). With the further improvement of Zn doping level, Ms gradually descends to 58.9 emu/g (x = 0.99). This phenomenon is attributed to that Zn (II) ions have no single electron. With low proportion of zinc doping, Zn (II) ions occupy tetrahedral (Td) sites in the inverse spinel structure of magnetite, which diminishes the antiferromagnetic coupling interactions of Fe (III) ions at Td and octahedral (Oh) sites. Therefore, magnetic moment and Ms value are increamental as the increase of zinc doping. However, after Zn doping ratio reaches a critical point, the antiferromagnetic coupling of Fe (III) ions at each Oh site becomes primary, which leads to the dwindling of net magnetic moment even the zinc doping ratio continues to increase. Stability, Cytotoxicity and Biocompatibility. Diverse surface modifications affect NPs’ distribution in tissues and in vivo fate.27-30 On the other hand, previous studies suggest that NPs less than 50 nm could escape the phagocytosis of cells in a way.31-33 Hence, we chose sodium citrate as the agent for phase transfer due to that this small molecule with good biocompatibility cause little augments in the hydrodynamic diameter of NPs. Dynamic light scattering (DLS) analysis (Figure 4a and Figure S3) demonstrates that both IO and ZnxFe3-xO4 NPs after sodium citrate coating have narrow size distribution with a small hydrated diameter. The polydispersity coefficient (PDI) and surface zeta potential (ζ) both corroborate that the samples are stable after storage in PBS for more than six months (Table S3). Besides, the hydrated diameter altered little after incubation with fetal bovine serum (FBS) (Figure S4), indicating sodium citrate could effectively stabilize NPs against the 5 ACS Paragon Plus Environment


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agglomeration of protein fouling. Moreover, the diameters of the particles in PBS and FBS barely changed for a long time (Figure S5), which indicates the good stabilities of these sodium citrate coated octapods. The cytotoxicity of ZnxFe3-xO4 octapods with different Zn doping ratios were measured by 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) assays. After incubation with human hepatoma SMMC-7721 cells for 24 h, no palpable cytotoxicity was observed even at the highest concentration (120 μg [Zn + Fe]/mL) (Figure 4b and Figure S6). The MTT assay with hepatic primary cell culture also indicates the good biocompatibility of these samples (Figure S7). H&E staining indicates no apparent tissue injury or inflammation of major organs two weeks after venous injection of ZnxFe3-xO4 octapods (at a dose of 2.0 mg/kg [Zn + Fe]) for mice (Figure 4c and Figure S8). We also did the hemolysis assay of these ZnxFe3-xO4 octapods with sodium citrate coating (Figure S9). As expected, there was no spontaneous red blood cells (RBCs) lysis after incubation with these samples even at a high concentration of 120 μg/mL. These results all attest to the excellent biocompatibility of sodium citrate modified ZnxFe3-xO4 octapods. T1 and T2 Relaxivities. We then evaluated the contrast abilities of ZnxFe3-xO4 octapods with different Zn doping levels. We first assessed the T2 contrast effects of these samples at low magnetic fields (Figure 5a and Figure S10). T2 relaxation rate reveals an interesting and obvious tendency as the Zn ratio rises (Table S4). The T2 relaxivity of Fe3O4 (x = 0) was 424.2 ± 15.3 mM-1s-1 (relative to the iron concentration, [Fe]). For ZnxFe3-xO4 octapods (x = 0.21, 0.44, 0.76, and 0.99), their r2 values were 553.0 ± 16.8, 647.4 ± 18.7, 400.8 ± 12.4 and 357.9 ± 10.1 mM-1s-1 (referring to total metal ions, [Zn + Fe]) at 0.5 T, respectively. At 1.5 T, the T2 relaxivities of Fe3O4 (x = 0) and ZnxFe3-xO4 octapods (x = 0.21, 0.44, 0.76, and 0.99) were 551.0 ± 38.1, 756.4 ± 11.2, 803.5 ± 12.1, 534.8 ± 53.6 and 476.7 ± 45.0 mM-1s-1, respectively. According to the quantum mechanical outer-sphere theory,34-36 the saturation magnetization has a profound effect on the transverse relaxation37 and NPs with large Ms possess high r2 values. As a result, the T2 relaxivity has the same trend as the saturation magnetization (Figure 5b). The sample with a short T2 relaxation time (i.e., high r2 value) presents a dark signal in the T2-weighted MR images. The T2-weighted phantom imaging of these samples further confirms the trend of their T2 contrast abilities (Figure 5c).


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Meanwhile, we also evaluated the T1 contrast ability of ZnxFe3-xO4 octapods with different zinc ratios at 1.5 T (Figure S11). The T1 relaxivities of Fe3O4 (x = 0) and ZnxFe3-xO4 (x = 0.21, 0.44, 0.76 and 0.99) were 49.4 ± 1.1, 45.3 ± 1.7, 37.2 ± 0.8, 29.9 ± 0.6 and 23.9 ± 0.3 mM-1s-1, respectively (Table S5). The results manifest that the r1 values decrease gradually with the increase of zinc fractions. Iron ions on the surface of ZnxFe3-xO4 NPs are gradually replaced by Zn2+ ions as the zinc doping level rises. Because Zn2+ ions have no unpaired electrons, the chemical exchange between metal ions and water molecules would be greatly surpressed.38 Therefore, ZnxFe3-xO4 NPs with high Zn doping ratios would have low r1 values according to SBM theory,39-41 which is consistent with the experimental results. The r2/r1 proportion is a critical parameter to assess that whether a given CA has T1 or T2 dominated MRI ability.42-44 We calculated the r2/r1 ratios of ZnxFe3-xO4 NPs with different Zn levels at the clinical field of 1.5 T. The r2/r1 ratios were 11.2, 16.7, 21.6, 17.9 and 19.9 for Fe3O4 (x = 0) and ZnxFe3-xO4 (x = 0.21, 0.44, 0.76 and 0.99), respectively (Table S5). The high r2/r1 ratios (> 10) affirm that ZnxFe3-xO4 octapods could serve as powerful T2 CAs in clinic.45 Before in vivo experiments at 7 T, we performed T2 imaging on a 7 T MRI scanner (Figure 5d). High magnetic field enhances the sensitivity of MR imaging,46 thus T2 relaxivities increase at 7 T. Consistent with the trend at 1.5 T, the r2 values of Fe3O4 (x = 0) and ZnxFe3-xO4 (x = 0.21, 0.44, 0.76 and 0.99) were 636.2 ± 18.4, 834.5 ± 16.6, 989.1 ± 22.2, 621.9 ± 31.9, and 542.2 ± 13.3 mM-1s-1, peaking at x = 0.44 (Figure 5e and Table S4). The T2-weighted phantom imaging clearly shows the transition of the contrast effects of ZnxFe3-xO4 (Figure 5f), indicating that x = 0.44 is the optimal zinc doping level. Notably, the T2 relaxivity of ZnxFe3-xO4 (x = 0.44) reaches an unprecedent value of 989.1 ± 22.2 mM-1s-1, which is about tenfold higher than that of Feraheme (ferumoxytol, a commercial IO sample, the r2 value is 96.9 mM-1s-1 at 7 T, Figure S12). This high value attributes to the octapod shape with a large effective radius and the optimal zinc doping ratio. Owing to the ultrahigh transverse relaxation rate, the image exhibits a distinctly attenuated signal even at 0.025 mM and shows a totally dark signal at only 0.1 mM (referring to total metal ions). These results indicate that ZnxFe3-xO4 (x = 0.44) octapods could be employed as a powerful T2 CA for sensitive imaging with MRI. In Vivo T2-Weighted MRI of Liver. We conducted T2-weighted MRI of liver by using healthy 7 ACS Paragon Plus Environment


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BALB/c mice as a model at 7.0 T. We chose liver as the region of interest, because most of NPs could be expeditiously engulfed by mononuclear phagocyte system (MPS) and easily accumulated in hepatic Kupffer cells.47-49 Therefore, the accumulation of ZnxFe3-xO4 NPs with this unique octapod shape in the liver showed a distinct contrast effect for hepatic MRI. Meanwhile, the negative charge of citrate coated NPs (Table S3) could reduce the undesirable clearance by the reticuloendothelial system (RES) and improve the blood compatibility.50 The images of transverse (Figure 6a) and coronal planes (Figure S13) were obtained before and after intravenous injection of Fe3O4 (x = 0) and ZnxFe3-xO4 (x = 0.21, 0.44, 0.76 and 0.99) at a dose of only 1 mg [Fe]/kg or 1 mg [Zn + Fe]/kg (relative to the body weight of mice, n = 3/group), respectively. We observed significant signal depression in the liver region for all groups at 0.5, 1, 2, and 4 h after injection. The signal began to dim at 0.5 h, reached the darkest at 1 h, recovered marginally at 2 h, and became slightly brighter at 4 h, implying that the NPs are degraded and excreted from the body, which is in agreement with former reports.51-54 Previous studies reporting the process and degradation mode of metal oxide NPs in living cells suggest that the size plays an important role in biological distribution and blood circulation, and NPs smaller than 50 nm possess many superiorities in biomedical applications.30, 33 The results show that ZnxFe3-xO4 octapods not only have a rapid T2 contrast-enhanced effect, but also offer a relative long diagnostic time window for several hours, which will provide more useful information for accurate diagnosis and imaging-mediated therapy. To quantitatively analyze the contrast effects of these ZnxFe3-xO4 octapods with different zinc doping levels, we calculated the signal-to-noise ratio (SNR) changes for the liver region in the transverse and coronal planes for each group (Figure 6b). The signal changes (∆SNR%) of Fe3O4 (x = 0) and ZnxFe3-xO4 (x = 0.21, 0.44, 0.76 and 0.99) in the transverse plane at 1 h are 61.5 ± 4.2, 72.2 ± 1.9, 82.1 ± 2.4, 57.5 ± 2.5 and 54.8 ± 2.3, respectively (Table S6). Their ∆SNR (%) in the coronal plane are 60.9 ± 3.5, 73.5 ± 2.1, 81.8 ± 2.9, 58.3 ± 2.6 and 56.1 ± 1.9 (Table S7), respectively. In particular, the maximum ∆SNR in the transverse and coronal planes at 1 h are up to 82.1 ± 2.4% and 81.8 ± 2.9%, suggesting that ZnxFe3-xO4 (x = 0.44) has the optimal T2 contrast-enhanced effect. Besides, the liver uptake of total metal ions in all groups are comparable by the inductively coupled plasma mass spectroscopy (ICP-MS) analysis (Figure 6c and Table S8), further indicating that the excellent contrast ability of ZnxFe3-xO4 (x = 0.44) is due to its high T2 relaxivity. 8 ACS Paragon Plus Environment

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Sensitive CE-MRI of Orthotopic and Metastatic Hepatic Tumors with Low Doses. In order to investigate the imaging performance of ZnxFe3-xO4 (x = 0.44) octapods for hepatocellular carcinoma (HCC), we performed T2-weighted MRI of BALB/c mice bearing orthotopic H22 tumors at 7 T. Sagittal images (Figure 7a) were acquired before and at 0.5, 1, 2, and 4 h after intravenous injection of ZnxFe3-xO4 (x = 0.44) (at a dose of 1.0 mg [Zn + Fe]/kg, 0.5 mg [Zn + Fe]/kg or 0.2 mg [Zn + Fe]/kg) or Feraheme (at a dose of 2.0 mg [Fe]/kg, as a control). Because the amount of effective Kupffer cells and macrophages in tumor is less than that in normal tissues of liver,55 hepatic tumors could not effectively accumulate NPs.56 Therefore, they show pseudopositive signals in images compared with normal liver tissues. The signals of hepatic tumors for mice injected with ZnxFe3-xO4 (x = 0.44) octapods became positive at 0.5 h, reached the brightest at 1 h, dimed slightly at 2 h, and turned dark at 4 h. The signals of hepatic tumors for mice injected with Feraheme became positive at 0.5 h, reached the brightest at 2 h, and declined gradually at 4 h. The brightest signal of Feraheme is at 2 h after injection while that of octapods is at 1 h, demonstrating that the fast contrast-enhanced ability of ZnxFe3-xO4 (x = 0.44) octapods. In addition, ZnxFe3-xO4 (x = 0.44) octapods show a brighter signal than Feraheme at a half (1.0 mg [Zn + Fe]/kg), a quarter (1.0 mg [Zn + Fe]/kg), or even a one-tenth (0.2 mg [Zn + Fe]/kg) of the injected dose (2.0 mg [Fe]/kg) of Feraheme. The MR contrast-to-noise ratio (CNR) changes of tumor (Figure 7b and Table S9) further validate their high sensitivity for tumor imaging. The ∆CNR% of ZnxFe3-xO4 (x = 0.44, at a dose of 1.0 mg [Zn + Fe]/kg) are 30.7 ± 5.4, 49.5 ± 2.5, 41.9 ± 3.1, and 26.2 ± 2.8, respectively. The ∆CNR% of ZnxFe3-xO4 (x = 0.44, at a dose of 0.5 mg [Zn + Fe]/kg) are 16.8 ± 3.6, 31.5 ± 3.1, 25.0 ± 4.4, and 12.4 ± 2.7, respectively. The ∆CNR% of ZnxFe3-xO4 (x = 0.44, at a dose of 0.2 mg [Zn + Fe]/kg) are 6.4 ± 4.1, 14.9 ± 3.9, 9.9 ± 5.4, and 7.0 ± 3.5, respectively. The ∆CNR% of Feraheme (at a dose of 2.0 mg [Fe]/kg) are 3.9 ± 4.5, 6.1 ± 3.2, 9.5 ± 3.6, and 5.3 ± 5.1, respectively. We also conducted a quantitative analysis for the uptake of total metal ions in liver and tumor by ICP-MS (Figure S14 and Table S10). The uptake of NPs in liver or tumor has the same trend with the injected dose instead of being correlative with the injected material, which indicates that the significantly enhanced effect is owing to the ultrahigh T2 relaxivity of the ZnxFe3-xO4 octapods. These results confirm the excellent T2 contrast-enhanced ability of ZnxFe3-xO4 (x = 0.44) octapods for liver tumors even at one tenth of the regular dose due to their ultra-high T2 relaxivity, suggesting that ZnxFe3-xO4 (x = 0.44) octapods 9 ACS Paragon Plus Environment


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is an efficient CA for sensitive diagnosis of tumor. ZnxFe3-xO4 (x = 0.44) octapods could also be exploited to detect liver metastatic tumors, which is the most frequent cause of death for patients with liver cancer. The T2-weighted images of liver in the sagittal plane were collected before and at 1 h after intravenous injection of ZnxFe3-xO4 (x = 0.44) at a dose of 0.5 mg [Zn + Fe]/kg or 0.2 mg [Zn + Fe]/kg (Figure 7c). Apart from the orthotopic tumor, visible metastatic liver tumors with high intensity were detected after intravenous injection. Three metastatic tumors could be observed clearly in the image for the mouse injected at a dose of 0.5 mg [Zn + Fe]/kg, the sizes of which range from 0.3 to 0.8 mm. In the T2-weighted image for the mouse injected at a dose of 0.2 mg [Zn + Fe]/kg, the sizes of the two metastatic tumors are 0.7 and 1.1 mm (see Figure S15 for precise measurements). We conducted H&E histology to verify the suspected metastatic tumors consideration of other liver lesions, such as cysts and hemangiomas, may also exhibit hyperintensity in T2-weighted images.57 The H&E staining images of these liver lesions exhibited the characteristics of large pleomorphic nuclei, delicate vesicular chromatin and prominent nucleoli.58 Besides, the diameters of lesions are in agreement with the sizes of suspected metastases in MR images (Figure S15 e,f). In brief, these results confirm that these lesions are multiple liver metastases. These detection limits are considerably lower than those of IO-based CAs in previous reports.59, 60 These results demonstrate the outstanding capability of ZnxFe3-xO4 (x = 0.44) octapods in hypersensitive imaging for detecting metastatic tumors, which is of great importance for early diagnosis and accurate prognosis in cancer management. Furthermore, they could meet the diagnostic needs even at low doses, which is critical for reducing the costs and side effects in clinic.

CONCLUSION In this study, we synthesized ZnxFe3-xO4 octapods with different zinc doping levels via a facile method. We investigated the magnetic properties of these ZnxFe3-xO4 octapods and found that both the saturation magnetizations (Ms) and transverse relaxivities (r2) of ZnxFe3-xO4 octapods increase firstly as the doping ratio rises, but reduce after the doping ratio reaches a critical point. Among these octapods, ZnxFe3-xO4 (x = 0.44) octapods possess a considerable Ms of 89.0 emu/g and a remarkable r2 of 989.1 mM-1s-1, which is a promising negative CA for sensitive MRI. In vivo T2-weighted MRI with BALB/c mice reveals the eminent T2 contrast-enhanced capability of ZnxFe3-xO4 (x = 0.44) for 10 ACS Paragon Plus Environment

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sensitive detection of orthotopic and metastatic hepatic tumors as small as 0.7 mm even at a low dose (one tenth of the regular dose), demonstrating their auspicious potential as a next-generation CA with less cost and side effects. This work provides an enlightening approach for developing high-performance T2 CAs by integrating doped IO with high Ms and unique shape with large effective radius.

EXPERIMENTAL SECTION Materials. Iron (III) chloride anhydrous (>97%), zinc chloride, sodium oleate, trisodium citrate dehydrate, hexane, ethanol, and acetone were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 1-Octadecene (tech, 90%) was purchased from Acros Organics. Oleic acid (tech, 90%) was purchased from Alfa Aesar. Feraheme (ferumoxytol injection, polyglucose sorbitol carboboxymethylether iron oxide formulated with mannitol) was purchased from AMAG Pharmaceuticals (Waltham). All the reagents were utilized as received without further purification. Characterization. Transmission electron microscopy (TEM) and high-resolution TEM (HRTEM) images were both acquired on a JEM-2100 microscope (JEOL, accelerating voltage is 200 kV). X-ray powder diffraction (XRD) patterns of octapods were collected on a Rigaku Ultima IV system (Cu-Kα, 15 mA, 30 kV, scan angle from 20 to 70, Sampling. W = 0.02, DivSlit =1/2 Deg, Div. HL. Slit = 10 mm). Energy-dispersive X-ray (EDX) element mapping experiments were performed on a Tecnai F30 microscope with an accelerating voltage of 300 kV. Hysteresis loops (M-H curves) at 300 K were obtained with superconducting quantum interference device (SQUID). The samples for magnetic measurements were prepared as powder after ethanol washing and plasma cleaning. Hydrated diameters of octapods were analyzed by the dynamic light scattering (DLS) (Malvern Zetasizer nano ZS instrument). Metal concentrations of the samples were quantified with inductively coupled plasma atomic emission spectroscopy (ICP-AES) and inductively coupled plasma mass spectroscopy (ICP-MS). The relaxivity measurements and phantom imaging at 0.5 T were all performed on an NMI20-Analyst system (Suzhou Niumag Analytical Instrument Corporation). Synthesis of Ferric Oleate and Zinc Oleate. Metal oleate complexes were prepared by the reaction of metal chlorides and sodium oleate according to a classical method with minor 11 ACS Paragon Plus Environment


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modification. For ferric oleate: 0.811 g of ferric chloride (5 mmol) and 4.567 g of sodium oleate (15 mmol) were added into a 100 mL flask containing 25 mL of ethanol and 25 mL of distilled water, then the mixture was heated to 70 °C and stirred for 4 h. After cooled to room temperature, the upper layer was discarded, and the red oil wax at the bottom of the flask was dissolved with hexane and transferred to an open dish. The red-brown waxy iron oleate complex was obtained after natural volatilization of hexane. For zinc oleate: 0.681 g of zinc chloride (5 mmol) and 4.567 g of sodium oleate (15 mmol) were dissolved in a mixture of 15 mL of distilled water, 20 mL of ethanol, and 30 mL of hexane. The solution was heated to 60 °C and maintained for 3 h with stirring. After the reaction, the upper layer containing zinc oleate was washed three times with distilled water. The resulting zinc oleate as white powder was attained after natural volatilization of hexane on an open dish. The two oleate complexes were stored for further use. Synthesis of Fe3O4 and ZnxFe3-xO4 Octapods with Different Zn Doping Ratios. A one-pot method was used to produce ZnxFe3-xO4 octapods (x = 0.44 for example). 0.903 g (1 mmol) of iron oleate (dark red-brown waxy solid, stored at least for two weeks in the dish) and 0.108 g (0.172 mmol) zinc oleate were dissolved in 12 mL 1-octadecene, with the addition of 0.189 mL (0.586 mmol) of oleic acid. The solution was first heated in vacuum at 100 C for 30 min to remove other low boiling point impurities in air and solvent, then heated to 350 C rapidly and refluxed at that temperature for 2 h in a N2 atmosphere. After the solution was cooled down to room temperature, the products were separated by centrifugation and dispersed in hexane. After three times wash with ethanol, the NPs were dispersed in hexane again for further use. ZnxFe3-xO4 octapods with other doping ratios were synthesized in the same way by adjusting the ratio of ferric oleate and zinc oleate. The preparation of Fe3O4 octapods is similar to this procedure except using zinc oleate as a precursor. Preparation of Sodium Citrate Coated Fe3O4 and ZnxFe3-xO4 Octapods. 100 mg of sodium citrate in 4 mL of distilled water and 50 mg of the as-prepared NPs in 4 mL of hexane were mixed with the adding of 6 mL of acetone. The resulting mixture was stirred for 60 °C to undergo a ligand exchange process. Because the chelation capability of citrate is stronger than that of oleic oleate, Fe3O4 and ZnxFe3-xO4 octapods were transferred to water with oleate replaced by citrate gradually. NPs in the bottom layer were collected by centrifugation, dispersed in water, washed three times with acetone, and finally dispersed again in water for further use. 12 ACS Paragon Plus Environment

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Cytotoxicity Evaluation. 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) assays was used to evaluate the cytotoxicity of citrate-coated Fe3O4 (x = 0) and ZnxFe3-xO4 (x = 0.21, 0.44, 0.76, and 0.99) octapods with SMMC-7721 cells. Cells were firstly seeded into a 96-well plate in RPMI 1640/DMEM at a density of 1 × 104 cells per well and incubated under 5% CO2 at 37 °C overnight. Then the cells were incubated with citrate-coated Fe3O4 and ZnxFe3-xO4 octapods for 24 h at different [Fe] or [Zn + Fe] concentrations (0.469, 0.938, 1.875, 3.75, 7.5, 15, 30, 60, and 120 μg/mL). After adding 100 μL of fresh media containing 0.5 μg/mL MTT to each well, the cells were further incubated at 37 °C for 4 h. OD492 value (Abs.) of each well was acquired on a MultiSkan FC microplate reader immediately and the cell viability was calculated accordingly. In Vitro MRI Measurements. The T1/T2 relaxivities and MRI phantom images at 1.5 T were conducted on an HT-MICNMR-60 system. T1- and T2-weighted phantom images were acquired with a spin-echo (SE) sequence: TR/TE = 100/8.3 ms (T1), TR/TE = 5000/37 ms (T2), matrices = 128 × 128, thickness = 0.8 mm, slice = 1. On a Varian 7 T micro MRI system, the relaxivities and MRI phantom images were recorded using a T2-weighted fast spin-echo multi-slice sequence (fSEMS): TR/TE = 2500/40 ms, FOV = 80 × 80 mm, thickness = 2 mm, slice = 1. In Vivo Liver MR Imaging. All animal experiments in this paper were conducted according to the protocol approved by the Animal Protection and Utilization Commission of Xiamen University. In vivo T2-weighted MR imaging of liver was performed with BALB/c mice as a model on a Varian 7 T micro MRI system. The images of the liver in the transverse and coronal planes were attained after intravenous injection of Fe3O4 (x = 0) and ZnxFe3-xO4 (x = 0.21, 0.44, 0.76, and 0.99) octapods at a dose of 1 mg [Fe]/kg or [Zn + Fe]/kg (n = 3/group). The parameters of fSEMS sequence were TR/TE = 2500/32.8 ms, FOV = 40 × 40 mm (transverse plane) and FOV = 40 × 80 mm (coronal plane), thickness = 1 mm, and averages = 4. The MR images were acquired at 0, 0.5, 1, 2, and 4 h after injection. Signal-to-noise ratio (SNR) was calculated by this equation: SNRliver = SIliver/SDnoise, where SI represents signal intensity and SD represents standard deviation. The SNR changes (∆SNR) is define as ∆SNR = |SNRpost - SNRpre|/SNRpre. Hepatic Tumors MR Imaging. The MR images of hepatic tumors in the sagittal plane were obtained at 0.5, 1, 2 and 4 h after intravenous injection of Feraheme (2 mg [Fe]/kg) and ZnxFe3-xO4 (x = 0.44) octapods at a dose of 1 mg [Zn + Fe]/kg, 0.5 mg [Zn + Fe]/kg and 0.2 mg [Zn + Fe]/kg, 13 ACS Paragon Plus Environment


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respectively (n = 3/group). The parameters of imaging were TR/TE = 2500/40 ms, thickness = 1.5 mm, and slice = 8. The contrast-to-noise ratio (CNR) changes of tumor is defined as CNR = (SNRtumor – SNRliver) /SNRtumor. Statistical Analysis. Statistical differences were evaluated with Student’s t tests. All data are presented as mean ± standard deviation.


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ASSOCIATED CONTENT The Supporting Information is available free of charge on the ACS Publications website. TEM images, XPS spectra, DLS analysis, T1 and T2 relaxivity measurements, characterization of Feraheme, cytotoxicity test, in vivo T2 MRI, uptake in the liver and tumor, measurements of tumors’ sizes, quantification of MR signal-to-noise and contrast-to-noise ratios.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Author Contributions (L.Y., C. S., and H.L.) These authors contributed equally to this work. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (21771148, 21602186, 21521004, and 81430041), IRT_17R66, Natural Science Foundation of Fujian Province of China (2018J01011), and Fundamental Research Funds for the Central Universities (20720180033, 20720170088, and 20720170020).



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Figure 1. TEM images of Fe3O4 and ZnxFe3-xO4 octapods with different zinc doping ratios. (a) Fe3O4 (x = 0), (b) ZnxFe3-xO4 (x = 0.21), (c) ZnxFe3-xO4 (x = 0.76), and (d) ZnxFe3-xO4 (x = 0.99). Scale bar, 100 nm.


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Figure 2. Characterization of ZnxFe3-xO4 (x = 0.44) octapods. (a) A typical TEM image, (b) a typical HRTEM image, (c) selected area electron diffraction (SAED) patterns, (d) energy-dispersive X-ray (EDX) elemental line scanning analysis (inset, STEM-HAADF image), and (e) EDX mapping images of octapods. The lattice distance in HRTEM was an average of at least two hundred octapods via Image J analysis.



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Figure 3. Structure characterization and magnetic properties of Fe3O4 and ZnxFe3-xO4 octapods with various zinc doping ratios. (a) X-ray powder diffraction (XRD) patterns of Fe3O4 and ZnxFe3-xO4 octapods. (b) X-ray photoelectron spectroscopy (XPS) spectra of ZnxFe3-xO4 (x = 0.44) octapods. (c) Field-dependent magnetization (M–H) curves at 300 K and (d) saturated magnetizations (Ms) of Fe3O4 and ZnxFe3-xO4 octapods.


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Figure 4. Stability, cytotoxicity and biocompatibility evaluations of ZnxFe3-xO4 (x = 0.44) octapods after modifications with sodium citrate. (a) Size analysis by dynamic light scattering (DLS) measurements. (b) The viabilities of SMMC-7721 cells incubated with ZnxFe3-xO4 octapods at different concentrations for 24 h (n = 5/group). (c) H&E staining of different organs (heart, liver, spleen, lung and kidney) after injection. BALB/c mice were sacrificed two weeks after caudal venous injection of ZnxFe3-xO4 octapods at a dose of 2 mg [Zn + Fe]/kg.



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Figure 5. In vitro T2 contrast performance of Fe3O4 and ZnxFe3-xO4 octapods with different zinc doping levels at 1.5 and 7.0 T, including analysis of relaxation rate R2 (1/T2) measured on (a) 1.5 and (d) 7.0 T scanners, T2 relaxivities at (b) 1.5 and (e) 7.0 T, and T2-weighted phantom imaging at (c) 1.5 and (f) 7.0 T. r2 values were calculated from the slopes of the best-fit lines for experimental data.


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Figure 6. In vivo T2-weighted MRI of mouse liver at 7.0 T. (a) T2-weighted MR images of liver in the transverse plane before and after intravenous injection of Fe3O4 (x = 0) and ZnxFe3-xO4 (x = 0.21, 0.44, 0.76, and 0.99) octapods at a dose of 1 mg [Fe]/kg or 1 mg [Zn + Fe]/kg to mouse body weight (n = 3/group). (b) Quantitative analysis of signal-to-noise ratio (SNR) changes in the liver region at different time points (0.5, 1, 2, 4 h) after administration (* p < 0.05, ** p < 0.01, n = 3/group, compared with the control group, x = 0). (c) The liver uptake of total metal ions after injection via ICP-MS analysis. Contributions from endogenous iron were subtracted.



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Figure 7. Hypersensitive contrast-enhanced MRI of orthotopic and metastatic hepatic tumors. (a) In vivo T2-weighted MR images of orthotopic liver tumors (in red circles) of BALB/c mice in the sagittal plane at 7.0 T. The mice were inoculated with an injection of 3-5  105 H22 cells in the liver. Two weeks later, the images were acquired at 0, 0.5, 1, 2, and 4 h after intravenous injection of ZnxFe3-xO4 (x = 0.44) octapods (1.0 mg [Zn + Fe]/kg, 0.5 mg [Zn + Fe]/kg, and 0.2 mg [Zn + Fe]/kg) or Feraheme (2.0 mg [Fe]/kg) (n = 3/group). (b) Corresponding quantitative contrast-to-noise ratio (CNR) changes of tumors, CNR = (SNRtumor – SNRliver)/SNRtumor (* p < 0.05, ** p < 0.01, n = 3/group, compared with the control group, Feraheme). (c) In vivo T2-weighted MR images of metastatic hepatic carcinomas in the sagittal plane before and after intravenous injection of ZnxFe3-xO4 (x = 0.44) octapods at 1 h with a dose of 0.5 mg [Zn + Fe]/kg and 0.2 mg [Zn + Fe]/kg (n = 3/group). The orthotopic tumors and the metastatic tumors are indicated by white arrows and red arrows, respectively.


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Sensitive Contrast-Enhanced MR Imaging of Orthotopic and Metastatic

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