pH-Responsive, Self-Sacrificial Nanotheranostic Agent for Potential In

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A pH-responsive, self-sacrificial nanotheranostic agent for potential in vivo and in vitro dual modal MRI/CT imaging, real-time and in-situ monitoring of cancer therapy Ludan Yue, Jinlong Wang, Zhichao Dai, Zunfu Hu, Xue Chen, Yafei Qi, Xiuwen Zheng, and Dexin Yu Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00562 • Publication Date (Web): 02 Jan 2017 Downloaded from http://pubs.acs.org on January 3, 2017

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Bioconjugate Chemistry

A pH-responsive, Self-sacrificial Nanotheranostic Agent for Potential in Vivo and in Vitro Dual Modal MRI/CT Imaging, Real-time and In-situ Monitoring of Cancer Therapy AUTHOR NAMES Ludan Yue,†,‡ Jinlong Wang,‡,§ Zhichao Dai,‡ Zunfu Hu,# Xue Chen, ‡ Yafei Qi, |Xiuwen Zheng,‡,* and Dexin Yu|,* AUTHOR ADDRESS †

College of Chemistry, Chemical Engineering & Materials Science, Shandong Normal

University, Jinan, Shandong, 250000, China, ‡College of Chemistry & Chemical Engineering, Linyi University, Linyi, Shandong, 276000, China,

§

College of Chemistry & Chemical

Engineering, Shandong University of Technology, Zibo, Shandong, 255000, China, #College of Chemistry & Molecular Engineering, Qindao University of Science & Technology, Qingdao, 266000, China and |Radiology Departments, Qilu Hospital of Shandong University, Jinan, Shandong, 250000, China. *

Address correspondence to Prof. Xiuwen Zheng

College of Chemistry & Chemical Engineering, Linyi University, Linyi, Shandong, 276000, China. Tel & Fax: +86 5398766600 E-mail: [email protected]; [email protected]

ACS Paragon Plus Environment


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KEYWORDS FePt· Theranostic· MRI/CT image-guided ·pH-responsive· ROS ABSTRACT Multifunctional nanotheranostic agents have been highly commended due to the application to realize image-guided cancer therapy. Herein, based on the chemically disordered face centered cubic (fcc) FePt nanoparticles (NPs) and graphene oxide(GO), we develop a pH-responsive FePt-based multifunctional theranostic agent for potential in vivo and in vitro dual modal MRI/CT imaging and in-situ cancer inhibition. The fcc-FePt will release highly active Fe ions due to the low pH in tumor cells, which would catalyze H2O2 decomposition into reactive oxygen species (ROS) within the cells and further induce cancer cell apoptosis. Conjugated with folic acid (FA), the iron platinum-dimercaptosuccinnic acid/PEGylated graphene oxide-folic acid (FePt-DMSA/GO-PEG-FA) composite nanoassemblies (FePt/GO CNs) could effectively target and show significant toxicity to FA receptor-positive tumor cells, but no obvious toxicity to FA receptor-negative normal cells, which was evaluated by WST-1 assay. The FePt-based multifunctional nanoparticles allow real-time monitoring of Fe release by T2-weighted MRI, and the selective contrast enhancement in CT could be estimated in vivo after injection. The results showed that FePt-based NPs displayed excellent biocompatibility and favorable MRI/CT imaging ability in vivo and in vitro. Meanwhile, the decomposition of FePt will dramatically decrease the T2-weighted MRI signal and increase the ROS signal, which enables real-time and in situ visualized monitoring of Fe release in tumor cells. And also, the self-sacrificial decomposition of fcc-FePt will be propitious to the self-clearance of the as-prepared FePt-based nanocomposite in vivo. Therefore, the FePt/GO CNs could serve as a potential multifunctional theranostic nanoplatform of MRI/CT imaging guided cancer diagnosis and therapy in clinic.


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INTRODUCTION Magnetic nanocomposites have drawn more attention due to the potential applications in biomedical diagnostics and therapies, such as magnetic resonance imaging (MRI) contrast agent1-4, computed tomography (CT)5 contrast agent, drug delivery6-8, biosensing2 and thermal therapy8, 9 in cancer diagnosis and treatment. Several magnetic nanoparticles including iron oxide (Fe3O4) NPs, metallic iron (Fe) NPs, and Fe-based alloy NPs, such as iron-cobalt (FeCo) and iron-palladium (FePd) NPs have been reported to possesses strong magnetic properties6, 10-13, but few magnetic nanomaterials were still being used for the treatment, such as ferumoxytol can be used as a medicine of anemia, and most magnetic nanomaterials were pulled from the market. Therefore, it is necessary to develop a kind of magnetic nanomaterials with high biological security and stabilized imaging function. Multi-modality imaging has been widely used in the diagnosis of tumors due to the capability of overcome the limitations of each single imaging modality, and improve the precision of diagnosis.14 Both CT and MRI are essential methods for clinical diagnosis. However, only few contrast agents could be employed in dual imaging contrast effect14. Several drawbacks hindered the further application of MRI as the sole methodology in the diagnosis of cancer, including the inability to assess lesion vascular system15-17. As another potent non-invasive imaging modality in clinical practice, it is difficult to diagnose the lesion region accurately by CT when the density of lesions similar with normal tissue, due to the contrast mechanism. Recently, some high performance MRI/CT contrast agents were developed in order to circumvent the drawbacks of the diagnosis using MRI or CT alone and improve the image contrast and sensitivity. For example, Liu and co-workers developed a PEGylated FePt@Fe2O3 core-shell nanoparticles as a promising multifunctional theranostic nanoplatform in imaging guided cancer therapy6. Chou et al. synthesized water-soluble, size-tunable superparamagnetic FePt nanoparticles and investigated their potential as a dual-modality contrast agent for MRI/CT10. Chen demonstrated that the engineered biocompatible FePt nanoparticles could be used for cellular



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imaging and in vivo MRI applications, which opens the way for several future applications of FePt NPs, including regenerative medicine and stem cell therapy in addition to enhanced MR diagnostic imaging2. All of above-mentioned studies indicate that the magnetic FePt-based nanoparticles have potential applications as multimodal imaging contrast agents in clinical settings. Various multifunctional nanomaterials are widely used in cancer treatment, either serving as drug carriers by loading therapeutic agents, or destructing tumors with their inherent properties usually under external physical stimuli.14 Fe-based nanoparticles were also found to effectively induce cancer cell apoptosis and further inhibit the growth of tumor. It is reported the FePt NPs could be heated up to couple of hundreds degrees within picoseconds under laser irradiation, which possess excellent photothermal efficiency for cancer therapy.8 FePt NPs was reported to release Fe in an acidic solution which could catalyze hydrogen peroxide (H2O2) decomposition into reactive oxygen species (ROS) intracellular that lead to rapid damage to oxidation and deterioration of cellular membranes9, 18, 19. Dhirendra9 and co-workers synthesized PEGylated mesoporous FePt-Fe3O4 composite nanoassemblies (CNAs) by a hydrothermal approach, and demonstrated that the presence of Fe allows the generation of reactive oxygen species (ROS) in the presence of hydrogen peroxide inside the cancer cells. The synergistic combination of chemotherapy (loaded DOX) and ROS is very efficient for killing cancer cells. Nanoparticles for biomedical applications must be highly chemical stable and relatively biocompatible, thus the biocompatibility of FePt NPs should be further improved.20 Graphene oxides (GO) possess a flexible size, excellent biocompatibility, abundant periphery carboxylic and hydroxylic groups on its surface, which endowed it the superior stability in aqueous solutions. Therefore, FePt NPs were loaded onto the PEGylated GO sheets to produce the multifunctional nanoparticle of FePt/GO CNs and use GO sheets as an ideal carrier21-23 for FePt NPs to improve the biocompatibility of it in physiological environment. However, in vivo study of FePt/GO CNs has never been reported before to our best knowledge.


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In the present work, we further optimize the synthesis of our previous reported24 FePt/GO-based nanocomposite via a sub-step method to improve the stability for in vitro and in vivo toxicity studies and MR/CT imaging. Briefly, the hydroxyl groups (OH) on GO sheets were converted to carboxylic acid (COOH) moieties to obtain GO-COOH. Inspired by the well-known serum stealth effect of PEG, 6-armed-PEG-NH2 was conjugated onto GO-COOH nanosheets via the amide linkage to fabricate a PEG-polymer–coated carrier, GO-PEG23, 25-27. FA was then modified as the targeting agent to enable the FePt/GO CNs entering the FA receptor-positive cells by the FA-receptor endocytosis mechanisms. FePt NPs modified by meso-2,3-Dimercaptosuccinnic acid (DMSA) were subsequently loaded on the GO sheets conjugated by 6-armed-PEG-NH2 and FA (GO-PEG-FA) which acted as a hydrophilic “stealth” carrier and renders the NPs stable at physiological environments. However, when the microenvironmental pH decreased down to 4.8, the active Fe ions released from FePt could react with H2O2 produced by the mitochondria, producing highly reactive ROS based on the Fenton reaction.24, 28, 29

As a result, the tumor cells would be damaged rapidly because of the high reactiveness for lipid

membrane oxidation by ROS. Next, we used FePt/GO CNs as T2-weighted imaging contrast agent for balb/c mice baring 4T1 tumor, observing MR significant signal attenuation effect and obvious inhibition effect in vivo. On the other hand, the decomposition of fcc-FePt will be propitious to the clearance of the as-prepared FePt-based nanocomposite in vivo26, 30, 31. This pH-responsive, self-sacrificial theranostic FePt/GO CNs show great promise in cancer diagnostics and therapy based on significant negative contrast in T2-weighted MRI and cellular membranes oxidation and deterioration damaged by ROS. This result may provide an insight for future clinical diagnostics and treatment by this new type of contrast agent in multimodal bio-imaging applications. RESULTS AND DISCUSSION The lipophilic FePt NPs were prepared according to a simple polyhydric alcohols method. As shown in Figure 1A, the FePt NPs were found to be monodisperse and uniform in size (3-4 nm in



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diameter). The lattice fringes of the FePt were clearly observed with adjacent fringe spacing of 0.224 nm, which were accordance to {111} lattice planes for face-centered cubic (fcc) phase of FePt nanoparticles (Figure 1A). Energy-dispersive X-ray spectrometric (EDS) analysis (Figure S1) confirms that the FePt consists of Fe and Pt at the ratio of 1:15, 32, 33, which is consistent with the results by the inductively coupled plasma mass spectroscopy (ICP-MS). For further biological applications, the nanoparticles were converted from lipophilic to hydrophilic by DMSA via ligand exchange reaction (Figure S2).

Figure 1. Advanced chemical properties and structural analysis of FePt/GO CNs. (A) TEM and HRTEM images of FePt NPs. The lattice fringes of the FePt with adjacent fringe spacing of 0.224 nm corresponding to lattice planes for fcc-FePt. (B) TEM image of FePt/GO CNs. (C) The magnetic properties of FePt/GO CNs, showing the superparamagnetism of the FePt-DMSA/GO-PEG. (D) The T2-weighted MR signals of FePt/GO CNs with Fe concentration of 20, 40, 60, 80, 100 µg/ml. (E) The linear correlation of the T2 relaxation rates (1/T2) against the iron concentration.


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Carboxylated GO was obtained by converting -OH to -COOH on GO sheets for further modification (Figure S5). The 6-armed-PEG-NH2 and FA were conjugated onto GO-COOH nanosheets via the amide linkage for targeted MR imaging of cancer cells overexpressing high-affinity FA-receptor. Thus, the surface chemistry could be easily tuned for biological stealth and targeting functions. FePt/GO CNs were prepared by loading FePt-DMSA onto the PEG-GO sheets following by modified FA (Figure 1B and Figure S3). UV-Vis and fluorescence spectra are used to illustrate the structure of FePt/GO CNs, in order to demonstrate the conjugation of FA onto the FePt/GO (Figure S4 and FigureS5). The magnetic properties of FePt/GO were measured at room temperature (298 K) (Figure 1C). The obtained saturation magnetization value of the FePt/GO CNs (Ms =4.10 emu/g) is comparable with the reported values in the literature34, which suggests its potential application as an MRI contrast agent in cancer diagnosis. The T2 weighted MR image for FePt-based NPs was obtained by a 3.0 T MR imager at various Fe concentrations. Figure 1D shows the T2 weighted images of FePt/GO CNs in the Fe concentration range of 0 - 0.596 mmol/L in deionized water. A significant concentration-dependent inverse MR image contrast was observed. Figure 1E shows the linear correlation of the T2 relaxation rates (1/T2) against the Fe concentration. The calculated relaxation rate (r2) value was 12.425 mM-1·s-1, indicating that the FePt/GO CNs could be used as a good MRI contrast agent.



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Figure 2. (A) Bright field image and fluorescence image (B) of MCF-7 cells and equal L02 cells cultured with FePt-DMSA/GO-PEG-FA-FITC NPs for 6h. (C) The merged images of A and B. White arrow points to the L02 cells while the red arrow points to the MCF-7 cells. (D) The cell viability of MCF-7 cells, HeLa cells, HepG2 cells, L02 cells and BRL 3A cells after cultured with different concentration of FePt/GO CNs for 10 hours.

As is known to all, the FA receptor were overexpressed on the cytomembrane of malignant tumor cells and low-expressed on normal cells, so that the result indicated that the FA could enable the FePt/GO CNs enter the FA receptor overexpressed tumor cells. To evaluate the feasibility of FePt/GO CNs for tracking FA receptor-positive tumor cells, the CNs cultured tumor cells and normal cells were prepared. After the malignant MCF-7 cells and normal L02 cells were co-cultured for 6h with fluorescein isothiocyanate (FITC) modified FePt/GO CNs (FePt-DMSA/GO-PEG-FA-FITC), the fluorescence images of FePt-based NPs were obtained. As shown in Figure 2, strong green fluorescence signal was observed in the MCF-7 cells (Figure. 2B), while no fluorescence could be


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found in L02 cells (Figure. 2 C and 2D). These results demonstrate that the FA receptor-mediated nanoparticles could effectively target tumor cells due to the over-expressed of FA receptor compared to the normal cells. And also, cancer cells can be easily visually identified from the co-cultured normal cells due to a distinct difference in fluorescence, which provides a facile route for early detection of tumor35. Furthermore, a standard flow cytometry assay was conducted with the FR-positive MCF-7 cells and FR-negative L02 cells after cultured by FePt-DMSA/GO-PEG-FA-FITC NPs for 6 hours, respectively. From the Figure S6, it could find that very weak fluorescence in L02 cells while strong fluorescence in MCF-7 cells were observed, indicating that almost no NPs enter into the L02 cells but large amounts of the FePt-DMSA/GO-PEG-FA-FITC NPs entered into the MCF-7 cells. The results could demonstrate that the FePt-DMSA/GO-PEG-FA-FITC NPs could target the FR-positive cells rather than the FR-negative cells. To estimate the influence of FePt/GO CNs on cell proliferation and viability of different cell lines, the WST-1 assay was conducted. Three malignant cell lines, human breast cancer cells (MCF-7), human cervical carcinoma cancer cells (HeLa) and human hepatocellular carcinoma cells (HepG2), and two normal cell lines rat hepatocyte cells (BRL 3A) and human hepatocyte L02 cells (L02) were incubated with FePt/GO CNs for 10 hours over the same range of dosages from 0 to 100 µg/ml and the cell viabilities were recorded. As shown in Figure 2A, the survival rate of MCF-7cells, HeLa cells, and HepG2 cells decreased significantly with the increase of Fe concentration while the survival rate of L02 and BRL 3A show no obvious change even at a high Fe concentration of 100 µg/mL. These results indicated that the FePt/GO CNs showed significant cytotoxicity to FA receptor-positive tumor cells (MCF-7, HeLa, HepG2), while negligible cytotoxicity to normal cells (BRL 3A and L02). In addition, the half maximal inhibitory concentration (IC50) of FePt/GO CNs against MCF-7, HeLa cells and HepG2 cells were calculated to be 40, 52, 47 µg/mL, respectively. Furthermore, a competitive inhibition experiment with FR-positive cells pre-incubated with excessive FA were conducted to evaluated the combination of FA and FA receptor. The MCF-7 cells were treated with excessive FA. And then, the FePt-DMSA/GO-PEG-FA CNs were added to treat the pre-treated cells with different



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concentration of Fe for 10 h. As shown in Figure S7, no distinct influence on the cells apoptosis were found, indicating that the pre-added FA could combine with the FA-receptor and decrease the binding of FePt-DMSA/GO-PEG-FA CNs.

Figure 3. (A)The cellular viability of MCF-7 cells after incubated by FePt/GO CNs for 2, 4, 6, 10, 24 hours at the Fe concentration of 40 µg/ml. (B) The enhanced fluorescence of DCF was visualized through the bright green fluorescent image of the cells after incubation for 6 h, indicating the formation of ROS. (C) The DCFA fluorescence intensity of intracellular ROS stained by DCFH-DA for 2, 4, 6, 10 and 24 h, it shows that FePt/GO CNs entered into cells and induced the intracellular ROS. (D) The MR imaging of MCF-7 cells cultured with FePt/GO CNs in DMEM for 2, 4, 6, 10 and 24h. (E) The DCF fluorescence intensity and T2-wighted MR signals of MCF-7 cells after cultured with FePt/GO CNs in DMEM for 2, 4, 6, 10 and 24h.


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To check if the cytotoxicity changes with time, we have also carried out the WST-1 assay using MCF-7 cells as the model. In this case, the cells were treated with FePt/GO CNs at the Fe concentrations of IC50 under different incubation times (2h, 4h, 6h, 10h, and 24h). As shown in Figure.3A the cell viability decreased dramatically with time as expected, indicating that the cytotoxicity increased with the time. This result is consistent with the mechanism that Fe could be released from FePt since the slightly acidic microenvironment (pH=4.8) in tumor cells (Figure S8 and S9), which is favorable for Fe acidic stimuli triggered release in cancer cells6. The released active Fe ions will further catalyze intracellular hydrogen peroxide (H2O2) transformed into reactive oxygen species (ROS) 36, which could damage the cellular membranes, thus lead to rapid lipid oxidation, DNA and protein damage, and eventually cell death18 due to their strong oxidation reaction (Scheme 1). In the following work, a commercially available fluorescence probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) was used for the detection of ROS in the cancer cells. As shown in Figure 2B, after the FePt NPs loaded MCF-7 cells were further co-incubated with DCFH-DA, obvious green fluorescence signals were observed. What’s more, the fluorescence intensity of DCF (λex/λem 480/520 nm) was collected after reacted with ROS at different incubation time (2, 4, 6, 10 and 24h, respectively) by FePt/GO CNs. To further demonstrate that the generation mechanism of ROS, control experiments, MCF-7 cells previously treated with H2O2 for half an hour or not were incubated with GO-FA nanosheets, were conducted under the same conditions. As shown in Figure 3C , Figure S10 and Figure 3D, the fluorescence intensity of DCF (reaction product of DCFH-DA with ROS) at 520 nm was enhanced gradually with the increased incubation time by FePt/GO CNs, which implies that the release of Fe in the acidic stimuli leaded to the generation of ROS. Meanwhile, the T2-weighted MRI signals showed a decreasing trend after incubated with FePt/GO CNs for different time (Figure 3D), which is accordance to the fact recognized in the previous reports. On the other hand, it should be emphasized that the new nanoparticle FePt/GO CNs could be treated as a self-decomposed agent, which would get rid of the potential threat caused by the accumulation of nanomaterials.



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Scheme 1. The pH-responsive theranostic probe and its active Fe release process intracellular. FePt/GO CNs enter the tumor cells via the folate receptor-mediated endocytosis, subsequently Fe released from FePt due to the acidic stimuli in tumor cells, and the released Fe will catalyze H2O2 into ROS, which could damage the cellular membranes, eventually lead to cell death.

To further investigate the therapeutic effect of the FePt/GO CNs, we conducted studies on balb/c mice with tumor in conformity with national guidelines and with approval of the regional ethics committee for animal experiments. Twelve balb/c mice bearing 4T1 tumor at the right leg were randomly divided into 3 groups and used for the treatment when the tumor volume reached ~100 mm3. They were given intratumoral injections every 5 days of the following agents: 0.1 mL, 0.9% of PBS


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solution as negative control (group A), 0.1 mL of FePt-DMSA-FA at the Fe concentration of 200µg/ml (group B), and 0.1 mL of FePt-DMSA/GO-PEG-FA (FePt/GO CNs) at the Fe concentration of 200µg/ml (group C). On the 21st day, tumors were excised for further analysis. The sizes of tumors were measured every three days after treatment and plotted as a function of time in Figure 4A. In the control group A, tumors showed rapid growth. And, the mice treated with FePt-DMSA-FA (group B) exhibited lower growth than that in group A, indicating the added FePt-DMSA-FA have inhibition effect in a certain extent to 4T1 tumor. While for the treatment with FePt/GO CNs in group C, the tumor growth shows obvious inhibition effect compared to group A and B. Figure 4B displays the typical images for the excised tumor after the treatment at 21st day, showing the significant difference in the size. This could also be verified by the T2-weighted MR imaging (Figure 4C and Figure S11). In this work, neither death nor significant body weight drop was observed in all groups during the treatments, implying that the toxic side effects to other organs are negligible. To further explore the side effects of the as-prepared FePt/GO CNs to other organs including heart, liver, spleen, lung and kidney from the balb/c mice treated with FePt/GO CNs or not were harvested and analyzed via hematoxylin and eosin (H&E) staining (Figure 4D). It shows that all of the organs have well-organized cellular structure without obvious abnormality. No appreciable damage, lesion, or inflammation was observed, confirming that the FePt/GO CNs have no obvious side effects to other organs6, 37 and potentially high clinical safety.



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Figure 4. In vivo evaluation of therapeutic effect. (A) Comparative therapeutic-efficacy study in vivo animal model after the 20 days treatment with: 0.1 mL, 0.9% of saline solution as negative control (group A), 0.1 mL of FePt-DMSA-FA at the Fe concentration of 200µg/ml (group B), 0.1 mL of FePt-DMSA/GO-PEG-FA NPs (FePt/GO CNs) at the Fe concentration of 200µg/ml (group C). (B)Excised tumors from mice of group A, B and C.(C) MRI image of mice after treated with 0.1 mL, 0.9% of saline solution as negative control (group A), 0.1 mL of FePt-DMSA-FA at the Fe concentration of 200µg/ml (group B), 0.1 mL of FePt-DMSA/GO-PEG-FA (FePt/GO CNs) at the Fe concentration of 200µg/ml (group C) for 20 days(injected every 5 days).(D)The H&E assay images of tumor tissue after 20 days treatment from the group A(above) and C(below).


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Based on the outstanding R2 value and T2-wighted MRI ability in cell, the possibility of using the FePt/GO CNs as a contrast agent was further explored in vivo. In this experiment, balb/c mice bearing 4T1 tumors were scanned by a 3.0 T MR scanner before and after intratumorally injection of FePt/GO CNs at different time including 0 h, 1 h, 2 h, 24 h and 48 h. The results were showed in Figure 5 and quantitated in Figure S12.

Figure 5. MR images of mice after injection in 48 hours. The results indicated that the MR signal was slowly decreased with the time, indicating the decomposing of fcc-FePt.

T2-weighted MR images of the tumor region treated by FePt/GO CNs showed that the MR signal slowly decreased with the time and exhibited remarkable T2 contrast from the surrounding normal tissue. Compared to the image before injection, the injection site became significantly darker, indicating the FePt/GO CNs could be used as excellent contrast agent. Subsequently, the dark area began to expand, and the whole tumor region turned dark within 2 hours after the injection of NPs, indicating the injected FePt/GO CNs was circulating in vivo. Then the FePt/GO CNs penetrated into the tumor tissue followed by cell internalized with the time. Finally, the tumor became brighter at elongated time in 48h, which improves that the as-synthesized FePt/GO CNs could real-time monitor Fe releasing by activatable T2 imaging in vivo. Comprehensive analyze with the Figure 3D, one could



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find that FePt/GO CNs significantly decomposed in tumor cells within 24 h, and show almost no signal at 48 h in vivo, indicating the FePt/GO CNs have self-decomposed function in a certain extent in vitro and in vivo. All the above-mentioned results could further indicate that the FePt/GO CNs can induce tumor cells apoptosis and inhibit tumor growth, and meanwhile, the process can be monitored in real-time via MRI imaging.

Figure 6. (A) The CT imaging capability of FePt/GO CNs and the HU value of FePt/GO CNs increased by Pt concentration (0.17, 0.35, 0.70, 1.39, 2.79 mg/ml). (B) The 3D reconstructed CT images and the 2D coronal CT image(C) after FePt/GO CNs injection.


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Dual-modality contrast agent is expected to be of great theranostic importance to diagnose tumor in clinical application.38 Therefore, the CT imaging capability of FePt/GO CNs was evaluated and shown in Figure 6A. Compared to the clinically used iodine-based CT contrast agents, FePt/GO CNs may have shown more significant CT contrast ability. The in vitro X-ray attenuation (CT) potency of these FePt/GO CNs with various concentrations of Pt was examined. A significant increase of the Hounsfield Unit (HU) value was achieved in a concentration dependent manner. These results demonstrated the significantly enhanced CT contrast imaging of FePt/GO CNs, which was due to the high X-ray absorption coefficient of Pt contained in FePt/GO CNs. Figure 6B and C provides the 3D reconstructed CT images after FePt/GO CNs injection. Contrast enhancement of tumor could be seen from Figure 6C after intratumoral injection, while the whole bone structure could be clearly observed in the image. No other organs could be seen in the same image, which demonstrated that the FePt/GO CNs were relatively stable with no metastasis to other organs as expected, indicating that the FePt/GO CNs have the potential as a successful CT imaging contrast agent. CONCLUSION In this work, we reported a pH-responsive, self-sacrificial nanotheranostic agent for potential in vivo and in vitro multi-modal imaging, real-time and in-situ monitoring of cancer therapy. FePt nanoparticles were synthesized with an average diameter of 3~4nm in size and loaded onto the PEGylated GO sheets. The FePt/GO CNs could actively target FA-receptor positive tumor cells. The cytotoxicity of FePt-based nanocomposite, evaluated by WST-1 assay, showed significant toxicity toward MCF-7, HeLa and HepG2 cells, and the corresponding half maximal inhibitory concentration (IC50) is ca. 40, 52 and 47 µg/ml, respectively. In vitro and in vivo MRI and CT studies demonstrated that these NPs rendered superior contrast enhancement for both CT and MR imaging, indicating the FePt/GO CNs nanoparticles could be used as multi-mode detection probe in early diagnosis of cancer. The excessive ROS generated from H2O2, which was catalyzed by the active Fe ions released from FePt resulted in the rapid damage of the cells. Furthermore, ROS signal was firstly related with the



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MRI signal to synergistic characterize the Fe releasing in tumor cells. Besides, Fe releasing from FePt indicated that FePt/GO CNs could be treated as a self-sacrificial agent. The therapy applicability of the probe on tumor and tumor cells indicated that FePt/GO CNs could be treated as a promising theranostic agent for the inhibition of cancer. In conclusion, the MR/CT imaging-guided FePt/GO CNs nanoparticles may hold great potential for further cancer diagnosis and therapy in clinic.

MATERIALS AND METHODS Chemicals and reagents All chemicals and solvents used were of analytical grade. Iron acetylacetonate (Fe(acac)3, 98%) and Pt(II) acetylacetonate (Pt(acac)2, 98%) was purchased from Beijing HWRK Chem. Graphite powder was purchased from Aladdin Reagent Co. Ltd. 1-Octadecene (ODE, 90%) was purchased from Aladdin. Oleylamine (OAm, > 70%) was purchased from Energy Chemical. Oleic acid (OA) and DMSA were purchased from TCI. 1-Ethyl-3-carbodiimide hydrochloride (EDC, 97%) was purchased from Damas-Beta. N-hydroxysuccinimide (NHS, 99%) was purchased from Xiya Reagent. 6-armed-PEG-NH2 was purchased from Yare Bio. FA was purchased from TCI. Anhydrous ethanol, hexane, dimethyl sulfoxide (DMSO) and toluene (99%) were used as received.

Preparation of carboxylated graphene oxide 1g graphene was added into 23 ml concentrated H2SO4 and stirred for 10 mins in 0 ℃ water bath. 3g KMnO4 was added and the temperature was kept below of 20 ℃, the mixture was kept stirring until the solution turned yellow green, and then stirring for another 2h at 35 ℃. Subsequently, 46 ml of triple-distilled water was added and stirred for 15 mins, another 140ml of triple-distilled water was added after that, followed by stopped heating and cooled down to room temperature. 2 ml 30% H2O2 was added before the mixture was centrifuged. The precipitation was washed to neutral with water. The solid was exfoliated at 400 w for 30 mins. After that, the mixture was centrifuged and the supernatant was retained. The graphene oxide was obtained in the solution.


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5 g sodium hydroxide and 5 g sodium chloroacetate was added into 20ml water, and the mixture was stirred until it dissolved. 50mg GO was added and ultrasound for 3 h. the mixture was centrifuged and washed to adjust the pH to 7, carboxylated graphene oxide was obtained after dialysis for 2~3 days. Graphene oxide modified by PEG 70 mg EDC, 42.39 mg NHS, and 20 mg 6-armed-PEG-NH2

(21, 25, 39)

were added to 4ml

carboxylated graphene oxide aqueous of concentration of 1mg/ml. Mixed and vigorously stirred for 48 hours. Then centrifuged at a high speed of 12000rpm, the supernatant was collected, after dialyzing(40) (MWCO: 5000) against triple-distilled water and freeze-drying, the product GO-PEG-NH2 was obtained as a brown fluffy powder. Synthesis of FePt-DMSA 0.5mmol Pt(acac)2，1mmol Fe(acac)3 and 3mmol 1,2-hexadecanediol was added into 30 mL dioctyl ether in a 200ml three-necked flask, Nitrogen was bubbled into the three-necked flask for 20 min to exclude oxygen in it. Raise the temperature to 100 ℃, and kept for 20min, 0.17ml oleylamine and 0.16ml oleic acid was added under a nitrogen atmosphere, raise the temperature to 295 ℃ rapidly, then cooled to room temperature before heated for 60 min. 30ml ethanol and 70ml of n-hexane was added into the flask, the mixture was centrifuged at the speed of 11,000 rpm. The precipitate was washed and centrifuged for three times. The remained product was FePt. 30mg FePt and 50 mg DMSA was added into 2ml DMSO before shaking at room temperature for 1 h, the mixture was centrifuged for 10 minutes, the precipitate was washed with anhydrous ethanol before the FePt-DMSA was obtained. Load monodispersive FePt nanoparticles on GO sheets 7.5 mg FePt-DMSA was dispersed in 10ml of ethanol, was added into the mixture of 10 mg GO-PEG-NH2 and 10ml DMF (dimethylformamide) solution, sonication for 3 hours. 10ml of ethanol was added, centrifuged at 12000 rpm for 15 min, the precipitate was washed by 10ml of ethanol and centrifuged at 12000 rpm for 15 min, the product FePt-DMSA/GO-PEG-NH2 was obtain.



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Conjugated FA and FITC on FePt/GO surface 10mg FePt/GO-PEG-NH2 was added into 10mL 0.01 M phosphate-buffered saline (PBS, pH 7.4), 5 mg N-(3-Dimethylaminopropyl-N’-ethylcarbodiimide) hydrochloride (EDC) and 5 mg NHS was added to the solution followed by sonication for an additional 30 min to activated the carboxyl group of FePt-DMSA/GO-PEG-NH2. 5mg FA, 10mg EDC, 10mg NHS was added into 5ml 0.01 mM PBS (pH=7.4), sonication for 15min to mix them followed by a 10 h shock at room temperature, the mixture was washed for 3 times to obtain the FePt-DMSA/ GO-PEG-FA. 5mg FA, 5mg FITC dissolved in 0.5ml DMSO, 20mg EDC, 20mg NHS was added into 5ml 0.01 mM PBS (pH=7.4), sonication for 15min to mix them followed by a 10 h shock at room temperature, the mixture was washed for 3 times to obtain the FePt-DMSA/GO-PEG-FA-FITC. Characterization GO, GO-PEG-NH2 ， FePt, FePt-DMSA and FePt/GO CNs were characterized by JEOL JEM-2100 transmission electron microscopy transmission electron microscopy (TEM) at 200 kV equipped with an energy dispersive spectrometry (EDS). GO sheets were imaged with atomic force microscopy (AFM) on a silicon substrate. The ultraviolet spectra were recorded from a cary 4600 spectrometer, which scanned over the range of 200~700 nm. The Fluorescence spectra were recorded from a Hitachi F-4500 spectrometer, which scanned over the range of 200~650 nm. The images of Fluorescence were confirmed by Fluorescence Imager (Olympus). The concentration of Fe was measured by ICP-MS (iCAP Q, Thermo Fisher) method. Magnetic measurement was carried out on a Magnetic Property Measurement System (MPMS SQUID VSM, Quantum Design, USA) at 298K. The T2 relaxivity was calculated by a linear fit of the inverse T2 (1/T2) relaxation time as a function of the Fe concentration. MR imaging were acquired under a 3.0 T clinical MRI scanner (GE Signa HDx 3.0T MRI, USA) equipped with a special coil designed for small animal imaging. T2-weighted images were acquired using the following parameters: repetition time (TR) 3000 ms; (echo time) TE 6ms; slice thickness 2.0 mm; slice spacing, 1.0mm; imaging matrix, 256× 192; NEX 2, field of vision (FOV) 7.0


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cm × 7.0cm. The images were reconstructed by a GE Advantage Workstation AW4.4 (GE Medical Systems). A GE Light Speed VCT clinical imaging system (GE Medical Systems) was used for CT scanning with the following parameters: beam collimation 64 ×0.625 mm, able speed 27 mm per rotation, beam pitch 1.25, gantry rotation time 1.0 s, and the images were reconstructed by a GE Advantage Workstation AW4.6 (GE Medical Systems). Cell culture. MCF-7 (human breast cancer cell line), HeLa (human cervical carcinoma cancer cell line), and BRL 3A (rat normal hepatocyte cell line) were provided by the Cell Bank of Chinese Academy of Sciences. L02 (human hepatocyte L02 cell line) were obtained from the American Type Culture Collection. HepG2 (human hepatocellular carcinoma cell line) were purchased from the Type Culture Collection of the Chinese Academy of Science. No cross-contamination of other human cells was observed. All cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 4.5 g/L D-Glucose, 110 mg/L Sodium Pyruvine, 100 U·ml-1 penicillin and 1 mg·ml-1 streptomycin. The cultures were maintained at 37 ℃ under a humidified atmosphere containing 5% CO2. Prussian blue staining MCF-7 cells were cultivated in DMEM medium with 10% fetal bovine serum, 1% penicillin and streptomycin, and maintained at 37 ℃ under a humidified atmosphere containing 5% CO2 for 24 hours. Phosphate buffered saline (PBS, 0.1 M, pH =7.4) was used to wash the cells twice before and after that incubated with FePt/GO CNs for 3 hours. Cells were fixed by 4% paraformaldehyde for 10 min at room temperature and incubated with 1% potassium ferrocyanide (K4Fe(CN)6·3H2O) and 2% HCl for 30 min (K4Fe(CN)6·3H2O: HCl = 1:1 by volume), and then added with 1% neutral red to stained nucleus. All Prussian blue stained cells were examined under a digital microscope (Leica QWin). Stability in PBS



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1 ml FePt/GO CNs (1 ml, 100 ug/ml Fe) in a dialysis bag dialyzed in PBS (0.01M, pH=4.8). The concentration of Fe in water was tested by ICP-MS after dialyzed for 0-50 h. Relaxivity measurements and CT imaging capability In a typical relaxivity measurement, FePt/GO CNs were dissolved in 1 ml deionized water at an Fe concentration of 0, 20, 40, 60, 80, 100µg/ml and diluted to 3 ml. The samples were transferred to PE tubes, and T2 relaxation time was determined under a 3.0 T clinical MRI scanner. For CT imaging capability study, the as-synthesized FePt/GO CNs were diluted in distilled water at the Fe concentration of 50, 100, 200, 400, 800µg/mL（The corresponding concentrations of Pt were 0.17, 0.35, 0.70, 1.39, 2.79 mg/ml). Samples were transferred to PE tubes. CT imaging ability of the FePt/GO CNs was determined by using the GE LightSpeed VCT clinical imaging system. In vitro MRI imaging For a typical MRI measure, MCF-7 cells were incubated with FePt/GO CNs dissolved in DMEM with the Fe concentrations of 40 µg/ml at 37°C for 2, 4, 6, 10, and 24 hours, respectively. After that, the cells were washed with phosphate buffered saline (PBS, 0.1 M, pH =7.4) three times. Subsequently, the cells were dispersed and suspended in 1mL of agarose gel (0.5%). The samples were then quickly transferred to a PE tube. MRI imaging was performed by using the 3.0 T whole-body MR scanner. Characterization of reactive oxygen species (ROS) For characterization of reactive oxygen species (ROS), 105 of MCF-7 cells per well were seeded in 24-well assay plates and cultured in DMEM (Dulbecco’s Modified Eagle Medium) supplemented with 10% FBS (fetal bovine serum), 1% penicillin and streptomycin. FePt/GO CNs (the equivalent Fe concentration is 40 µg/ml) was added and incubated for 2, 4, 6, 10 and 24 h. The cells were washed three times with PBS, before 1 ml fresh medium without FBS was added. 1 ul DCFH-DA was added into the medium followed by 1 h culture. Then the cells were trypsinized, resuspended, and washed with water by centrifugation at 2000 rpm, and then dispersed in 2 ml water. Finally, the fluorescence


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signals of cells (λex/λem 488/520 nm) were recorded at the same temperature on a fluorescence spectrophotometer. The fluorescence image was acquired by an Olympus digital camera. In vivo therapy and imaging study Balb/c mice were obtained from Jinan Peng Yue Biological Technology Co Ltd and used under protocols approved by Qilu Hospital Laboratory Animal Center. The 4T1 tumor model was generated by subcutaneous injection of 2 × 105 cells in 100 µl PBS into the right leg of female Balb/c mice. The mice were used for our experiments when the tumor volume reached ~100 mm3. These mice were maintained on a folate-free diet for 2 weeks before tumor inoculation. For in vivo study, twelve balb/c mice bearing 4T1 tumor at the right leg were randomly divided into 3 groups and used for treatment when the tumor volume reached ~100 mm3. They were given intratumoral injections every 5 days as following agent: 0.1 mL, 0.9% of saline solution as negative control (group A), 0.1 mL of FePt at the Fe concentration of 200 µg/ml as contrast (group B), 0.1 mL of FePt/GO CNs at the Fe concentration of 200 µg/ml (group C). On the 21st day, tumors were excised for further measurement. The dimension of each tumor was measured every three days using a caliper, and the tumor volumes were calculated based on the following formula: V=

ab 2 2

In this equation, V, a and b are the volume (mm3), length (mm) and width (mm) of tumor, respectively. The relative tumor volume was calculated as V/V0, where V0 is the tumor volume at initiation of the treatment. For folate-targeting in vivo MR imaging, female balb/c mice bearing KB tumors were intravenously injected with FePt/GO CNs (0.1 ml, 200 µg/ml of Fe) and imaged under the 3.0 T clinical MR scanner. For in vivo CT imaging, female balb/c mice bearing KB tumors were intravenously injected with FePt/GO CNs (0.1 ml, 1 mg/ml of Fe) and imaged under an IVIS Lumina XR system. Haematoxylin and eosin (H&E) staining



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Mice treated for 20 days with FePt/GO CNs or PBS were sacrificed at the 21st day. Major organs including liver, spleen, kidney, heart, and lung were harvested, fixed in 4% neutral-buffered formalin, processed routinely into paraffin, stained with hematoxylin and eosin (H&E), and then examined under a digital microscope (Olympus BX51). ACKNOWLEDGMENT This work is financially supported by Natural Science Foundation of China (21375057, 21675073) and Shandong Province Natural Science Foundation (No.ZR2016BB05, ZR2012HM012). SUPPORTING INFORMATION AVAILABLE EDS information of FePt NPs, image of MCF-7 cells stained by Prussian Blue, characterization of the GO sheets by AFM, ultraviolet absorption spectra and fluorescence spectrum of FePt/GO CNs, and evaluation of the cellular viability of MCF-7 cells after incubation. This material is available free of charge via the internet at http://pubs.acs.org.


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pH-Responsive, Self-Sacrificial Nanotheranostic Agent for Potential In

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