Magnetic Targeting, Tumor Microenvironment-Responsive Intelligent

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Magnetic Targeting, Tumor Microenvironment Responsive Intelligent Nanocatalysts for Enhanced Tumor Ablation Lili Feng, Rui Xie, Chuanqing Wang, Shili Gai, Fei He, Dan Yang, Piaoping Yang, and Jun Lin ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b05042 • Publication Date (Web): 19 Oct 2018 Downloaded from http://pubs.acs.org on October 19, 2018

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Magnetic Targeting, Tumor Microenvironment Responsive Intelligent Nanocatalysts for Enhanced Tumor Ablation Lili Feng,†,‡,§ Rui Xie,#,§ Chuanqing Wang,† Shili Gai,†,* Fei He,† Dan Yang,† Piaoping Yang,†,* and Jun Lin‡,* †

Key Laboratory of Superlight Materials and Surface Technology, Ministry of Education,

Harbin Engineering University, Harbin 150001, P. R. China, ‡ State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130021, P. R. China, # Department of Digestive Internal Medicine & Photodynamic Therapy Center, Harbin Medical University Cancer Hosptial, Harbin, 150081, P. R. China

KEYWORDS: nanocatalysts, Fenton reaction, hydroxyl radicals, magnetic targeting, iron carbide

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ABSTRACT: Therapeutic nanosystems which can be triggered by the distinctive tumor microenvironment possess great selectivity and safety to treat cancers via in situ transformation of nontoxic prodrugs into toxic therapeutic agents. Here, we constructed an intelligent, magnetic targeting and tumor microenvironment responsive nanocatalysts that can acquire oxidation therapy of cancer via specific reaction at tumor site. The magnetic nanoparticle core of iron carbide-glucose oxidase (Fe5C2-GOD) achieved by physical absorption has a high enzyme payload and the manganese dioxide (MnO2) nanoshell as an intelligent “gatekeeper” shields GOD from premature leaking until reaching tumor tissue. Fe5C2-GOD@MnO2 nanocatalysts maintained inactive in normal cells upon systemic administration. On the contrary, after endocytosis by tumor cells, tumor acidic microenvironment induced decomposition of MnO2 nanoshell into Mn2+ and O2, meanwhile, released GOD. Mn2+ could serve as magnetic resonance imaging (MRI) contrast agent for real-time monitoring treatment process. Then the generated O2 and released GOD in nanocatalysts could effectively exhaust glucose in tumor cells, simultaneously, generate plenty of H2O2 which may accelerate the subsequent Fenton reaction catalyzed by Fe5C2 magnetic core in mild acidic tumor microenvironment. Finally, we demonstrated the tumor site-specific production of highly toxic hydroxyl radicals for enhanced anticancer therapeutic efficacy but minimized systemic toxicity in mice.


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Malignant tumor is one of the major diseases that seriously harm human health, its leading cause of death worldwide.1,2 As the most traditional therapeutic modality, chemotherapy suffers from toxic side effects due to the low bioavailability.3–8 Some other anticancer treatment modalities, for example radiation,9–12 photoacoustic,13–15 ultrasound16–18 or microwave19–22 therapies are able to locate the treatment sites via imaging-guidance, while these researches trigger limited lethality to tumor cells, serious injury to normal cells and/or unwanted cancer metastasis. Therefore, the ability to kill cancer cells, meanwhile, causing negligible toxicity to normal cells is of crucial importance and urgently needed. To acquire more efficient and tumor site-specific treatment, tumor microenvironment has been extensively explored lately, in which the tumor cells metabolize, physical environment and vascular formation are markedly different from those in normal tissue cells.23–25 Tumor acidic microenvironment-responsive drug release and diagnostic imaging have been widely investigated.26–31 However, the introduction of anticancer drug without tissue-specificity would still result in unwanted toxicity to normal cells. It is noteworthy that if the inherent substance in tumor cells is biocompatible and naturally non-toxic, while under the influence of intratumor-delivered non-toxic agents, it could be in situ converted into toxic anticancer substances in the tumor microenvironment, the cancer therapy with significantly enhanced therapeutic effect and negligible toxic side effects will be realized. Fenton reaction appears in almost all iron-related critical process.32–35 Some ferromagnetic nanoparticles (such as γ-Fe2O3, Fe3O4 and Fe5C2) could disintegrate H2O2 into non-toxic O2 and H2O in neutral pH condition. More significantly, they could efficaciously disassemble H2O2 into highly toxic hydroxyl radicals (•OH) in acidic condition.36–41 Therefore, magnetic nanoparticles are regarded as a kind of promising tumor targeting nanocatalytic enzyme due to the production of •OH which can cause apoptosis or necrosis of tumor cells under the acidic tumor


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microenvironment.42–46 Nevertheless, the level of intratumoral H2O2 is too low to produce large amount of •OH to achieve satisfactory catalytic treatment effect. Hence, it is necessary to propose a new method to increase the content of H2O2 in tumor cells. Recently, glucose oxidase (GOD), which is a natural aerobic dehydrogenase and can serve as a high efficiency H2O2-producing catalyst has received considerable attention, since it is being exploited to generate H2O2 in tumor site.47–51 Just recently, Shi and co-workers developed GOD and ultrasmall Fe3O4 loaded biodegradable silica nanoparticles to treat cancer through sequential catalytic reactions in tumor cells.52 Akiyoshi’s group demonstrated that enzyme-loaded therapeutic vesicular nanoreactors with intrinsically permeable membrane acquired increase oxidative stress and inhibition glutathione (GSH) for suppression tumor growth efficiently.53 However, the intrinsic substance glucose is accessible in vivo pervasively, resulting in no regulation of reactive oxygen species (ROS) production. On the other hand, as an aerobic enzyme, GOD cannot be effectively catalyzed to produce H2O2 in situ because of the hypoxic environment of tumor cells. In addition, these nanosystems are lack of tumor-targeting and tissue specificity, leading to inevitable damage to normal tissues. Therefore, it maintains a big challenge to synthesize tumor-targeting site-specific nanosystems with (i) “gatekeeper”, (ii) in situ generation of O2 effectively for subsequent catalytic reaction, (iii) and transformation of H2O2 into downstream highly toxic ROS to maximize therapeutic efficacy and minimize side effects. Herein, we presented an intelligent and sequentially functional theranostic nanoplatform for efficient tumor growth inhibition by tumor microenvironment responsive catalytic reactions with tumor specific targeting. We designed a manganese dioxide (MnO2)-encapsulated and GOD loaded magnetic iron carbide (Fe5C2) core-shell structured nanosystem (Fe5C2-GOD@MnO2)


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based on the following considerations: (i) MnO2-encapsulated Fe5C2-GOD avoids premature drug leakage in normal cells; (ii) Fe5C2-GOD@MnO2 nanocatalysts which can localize accumulation into the tumor site under the driving of magnetic field will be monitored by magnetic resonance imaging (MRI); (iii) after endocytosis by tumor cells, the acidic microenvironment allows for decomposition of MnO2 shell, triggering the generation of O2 and release of GOD; (iv) acting as an enzyme catalyst, GOD can effectively catalyze glucose to generate H2O2 in tumor region under sufficient oxygen conditions. The elevated H2O2 can be further catalyzed by Fe5C2 nanoparticles via Fenton reaction to generate abundant highly toxic •OH, resulting in tumor cells apoptosis and death. Eventually, as-produced highly toxic •OH can effectively kill cancer cells and inhibit tumor growth via T1/T2-weighted dual-modal MRI-guided oxidation therapy

RESULTS AND DISCUSSION Anticancer Therapeutic Mechanism of Fe5C2-GOD@MnO2. The rational design and preparation of catalytic theranostic nanosystem are illustrated in Scheme 1a. GOD was integrated with magnetic Fe5C2 nanoparticles and encapsulated by pH-responsive MnO2 nanoshell, denoted as Fe5C2-GOD@MnO2. The significant therapeutic process is based on the specific tumor microenvironment, as presented in Scheme 1b.54,55 Primitively, Fe5C2-GOD@MnO2 could be transferred into tumor tissues via magnetic targeting and enhanced permeability and retention (EPR) effect. Due to the acidic characteristics of tumor microenvironment, MnO2 nanoshell can be decomposed into Mn2+ (T1 contrast agent) and O2 effectively, meanwhile, release trapped GOD for sequential biological-chemical catalytic reactions. The released GOD could exhaust glucose via enzyme-catalyzed biological reaction and generate a large amount of H2O2 in situ.


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Importantly, H2O2 produced in the above reaction could be further catalyzed by magnetic Fe5C2 nanoparticles (in Fe5C2-GOD@MnO2 nanocatalysts) to generate abundant highly toxic •OH, resulting in effectively cell apoptosis and necrosis. Hence, the designed Fe5C2-GOD@MnO2 nanocatalysts possess multiple functions, particularly according to their magnetic and catalytic properties, such as elevating the accumulative effect of Fe5C2-GOD@MnO2 in tumor tissue via magnetic targeting, exhausting glucose to starve the cancer cells, enhancing the H2O2 levels in tumor tissues for facilitating the Fenton reaction to generate a great deal of highly toxic •OH, dual-modal (T1/T2-weighted) MRI guided and monitored anticancer therapy. Synthesis and Characterization of Fe5C2-GOD@MnO2. Fe5C2 nanoparticles were fabricated by one-pot thermal decomposition method, and then modified with bovine serum albumin (BSA) to acquire excellent biocompatibility and solubility. GOD, a natural aerobic dehydrogenase was effectively loaded onto the surface of Fe5C2 nanoparticles by physical absorption. Afterwards, the MnO2 nanoshell layer which can be used as intelligent “gatekeeper” for pH-controlled release of GOD was introduced via reducing KMnO4 in the presence of cationic polyelectrolyte PAH, finally, resulting a multifunctional MnO2-encapsulated Fe5C2GOD therapeutic nanosystem (Fe5C2-GOD@MnO2). The representative transmission electron microscopy (TEM) image demonstrates that the as-prepared Fe5C2 nanoparticles possess a coreshell structure with uniform distribution and an average diameter of 13.5 nm, as shown in Figure 1a. The lattice spacing in the core is 0.205 nm indexed to the (510) plane of Fe5C2, nevertheless the shell structure emerges to be amorphous (Figure 1b). As presented in Figure 2a, x-ray diffraction (XRD) pattern indicates that the chemical structure is Fe5C2, which is matched well with the standard card (JCPDS No. 36-1248). For the XRD pattern of Fe5C2-GOD@MnO2 sample, the diffraction peaks of Fe5C2 and MnO2 could be detected simultaneously, which


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indicate that two phases coexist in the nanocatalysts. No apparent morphological and size changes were detected after modification with BSA and loading GOD, as shown in Figure S1a and Figure 1c. The energy-dispersive spectroscopy (EDS) spectrum in Figure S1b confirms that the Fe5C2-BSA nanoparticles are constituted of Fe, C, and O elements. However, the introduction of MnO2 shell layer results in a rough surface. Figure 1d presents the TEM image of Fe5C2-GOD@MnO2 with an overall size of 24.1 nm and the outer MnO2 shell can be clearly observed with the thickness of approximately 4.3 nm. Compared with the UV-vis absorption spectrum of Fe5C2 nanoparticles, the emergence of the typically characteristic peaks at 369 nm in the UV-vis absorption spectrum of Fe5C2-GOD@MnO2 is well consistent with that of MnO2, further confirming the successful introduction of MnO2 shell layer (Figure 2b). The dynamic light scattering (DLS) results exhibit the hydrodynamic sizes of Fe5C2-BSA, Fe5C2-GOD and Fe5C2-GOD@MnO2 achieved in aqueous solution are approximately 18.2, 24.4 and 37.8 nm, respectively (Figure S2a). In addition, Fe5C2-GOD@MnO2 nanoparticles are stable and dispersed well in different solutions with an average DLS diameter of ~37.2 nm (Figure S2b), the reason for the increase in diameter from DLS measurment may be on account of the BSA coating. As shown in Figure 2c, a series changes of zeta potential analysis support the successful coatings, which demonstrate a negative charge of Fe5C2-BSA (−13.8 mV) and a positive charge of Fe5C2-GOD@MnO2 (+8.9 mV). Figure 2d offers the experimental results of thermogravimetric analysis (TGA) curves of the Fe5C2@MnO2 and Fe5C2-GOD@MnO2 nanocatalysts in the range of 100 − 800 °C, suggesting a loading amount of 3.62 wt.% for the GOD. The size and morphology of the nanoparticles basically maintain constant after GOD loading in comparison with those without GOD. The nanoparticles without GOD loading are served as the control and designated as Fe5C2@MnO2 (Figure S3). X-ray photoelectron


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spectroscopy (XPS) was applied to research the chemical state of Fe5C2-GOD@MnO2 nanocatalysts (Figure S4a-e). Firstly, the XPS survey spectrum exhibits C content of 46.67%, an O content of 36.72%, Fe content of 4.75% and Mn content of 11.89%, respectively. Secondly, the presence of two characteristic peaks at around 724.5 eV and 710.8 eV in the Fe 2p highresolution spectrum suggests the existence of Fe3O4, the other two peaks at 707.0 eV and 719.9 eV can be attributed to the Fe−C bond which are related with Fe5C2 nanoparticles, demonstrating the successful preparation of iron, the coexistence of a magnetic phase and carbide phase.56,57 Thirdly, the characteristic peak at 285 eV in the C 1s spectrum integrated with HRTEM image indicates the existence of amorphous carbon shell. Besides, the two peaks at 654.2 eV and 642.4 eV in the Mn 2p spectrum, which accorded with the Mn (IV) 2p2/3 and Mn (IV) 2p1/2 spin-orbit peaks of MnO2, respectively, further verifying that KMnO4 was converted into MnO2 through PAH. Fourier transform infrared spectroscopy (FTIR) also additionally affirms the successful loading of GOD on the surface of Fe5C2-BSA, as well as the further coating of MnO2 shell (Figure S4f). In addition, the coexistence of Fe, C, Mn, O and N signals is detected in the EDS spectrum of Fe5C2-GOD@MnO2, directly affirming the successful formation of multifunctional nanocatalysts combined with TEM image (Figure 1e). To confirm the MnO2 shell coating on Fe5C2, cross-sectional compositional line profiles of Fe5C2-GOD@MnO2 were measured and provided in Figure 1g. Obviously, the line profiles of Fe, C and Mn elements indicate the shell coating of MnO2, which indicates the successful preparation of core-shell structure. It is noteworthy that Fe5C2-GOD@MnO2 exhibits excellent magnetic performance in the presence of magnetic field, which makes it possible for magnetic targeting triggered cancer therapy (Figure 1f). The above experimental results confirm the successful loading of GOD and coating MnO2 shell layer simultaneously.


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In Vitro Catalytic Profiles of Fe5C2-GOD@MnO2. The catalytic treatment mechanism of Fe5C2-GOD@MnO2 nanocatalysts is illuminated in Scheme 1b. The MnO2 shell of nanocatalyst is initially decomposed into Mn2+ and O2 under acidic condition, and then β-D-glucose is catalyzed into H2O2 and β-D-glucono-1, 5-lactone via the packaged GOD. Whereafter, Fe5C2 nanoparticles facilitate the decomposition of H2O2 to produce highly toxic •OH in mildly acidic tumor microenvironment, while generate non-toxic O2 and H2O in neutral condition. To confirm the production of radical species, 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) as a nitrogen trap was utilized to capture free radicals with short-lived to generate comparatively long-lived radical-DMPO adducts. Electron spin resonance (ESR) spectroscopy was used for analyzed and confirmed these adducts. It is crucial to understand that the addition of glucose (10 mM) into Fe5C2-GOD@MnO2 aqueous solution under acidic pH environment (pH = 6.0) induces the production of plenty of •OH, which is verified by the characteristic peaks of •OH in ESR spectrum (Figure 3a). However, no significant signals are detected in ESR spectrum under the same experimental conditions without glucose addition. The comparison analysis indicates that the •OH radicals are effective generated by Fe5C2-GOD@MnO2 nanocatalysts under acidic condition in the presence of glucose. In order to quantitatively analyze the production of •OH, the 1,3-diphenylisobenzofuran (DPBF) was resorted to monitor the radical. The produced •OH will oxidize chromogenic DPBF to colorless DPBF cation-free radicals, which can lead to the reduction of its absorption intensity at the wavelength of 410 nm in UV-vis absorption spectrum. In Figure 3b-f, the absorption intensity of DPBF decreases apparently when Fe5C2-GOD@MnO2 solution containing glucose (10 mM) was mixed together under acidic environment (pH = 6.0) for 60 minutes. Comparatively, no significant change in absorption intensity can be detected in other control groups, including the groups in the absence of glucose under acidic/neutral


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condition and the groups in the presence of glucose under neutral condition. As a contrast, the absorption intensity of DPBF solution containing Fe5C2@MnO2 in the presence of glucose under neutral/acidic condition was also measured (Figure S5). No significant decreased can be detected in the absorption intensity of the DPBF mixture solution, which indicates that the GOD plays an important role in the sequential biological-chemical catalytic reactions. The above experimental results suggest the acidity-sensitive and glucose-activated generation of •OH under the catalysis by Fe5C2-GOD@MnO2. In Vitro Cytotoxicity Performance. The above in vitro survey exhibited the effective generation of •OH assisted by Fe5C2-GOD@MnO2 nanocatalysts under acidic condition in the prescence of glucose. To further explore the cytotoxicity profiles of Fe5C2-GOD@MnO2 nanocatalysts, HeLa cells were treated with Fe5C2-GOD@MnO2 at various concentrations (800, 400, 200, 100, 50, 25 and 12.5 μg/mL) in both neutral (pH = 7.4) and acidic (pH = 6.0) culture mediums for 24 h. The cytotoxicity was assessed by the standard methyl thiazolyl tetrazolium (MTT) assay. It could be observed that cell survival rates are mainly lay on the concentration of Fe5C2-GOD@MnO2 nanocatalysts and pH value of the culture medium (Figure S6a). In the cytotoxicity experiment, the Fe5C2-GOD@MnO2 nanocatalysts displayed 78.04%, 69.83%, 63.24%, 55.49%, 46.05%, 39.72% and 32.86% cell viabilities with the increase of sample concentration under acidic condition, while much higher cell survival rates of 98.26%, 96.45%, 97.95%, 96.87%, 89.55%, 94.96% and 95.15% could be detected at the corresponding concentration in neutral condition. Extraordinary, the cytotoxicity towards HeLa cells triggered by 800 μg/mL of Fe5C2-GOD@MnO2 could be rescued by adding L-ascorbic acid of different concentrations (a type of antioxidant agent, 50, 25, 12.5, and 6.25 μg/mL), hinting that the cytotoxicity of Fe5C2-GOD@MnO2 to cancer cells is caused by oxidative damage of •OH


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(Figure S6b). The in vitro therapeutic effect of Fe5C2-GOD@MnO2 nanocatalysts with various concentrations under the influence of magnetic field was also investigated (Figure 4a). HeLa cells incubated with Fe5C2@MnO2 show negligible cell toxicity, indicating the excellent biocompatibility of our nanomaterials. Simple oxidative damage induced by Fe5C2-GOD@MnO2 (t-test, P=1.5×10−3 versus control) is not as effective as the magnetic targeted therapy group. It is also indicated that cancer cells treated with Fe5C2-GOD@MnO2 in the case of magnetic targeting (t-test, P=2.1×10−3 versus control) exhibit significantly reduced cell viabilities, which can be attributed to the improved material ingestion and production of a large amount of •OH. The in vitro cytotoxicity of Fe5C2@MnO2 and GOD@MnO2 nanocatalysts for different culture mediums are also tested for cancer cells (Figure S6c and d). No significantly decrease in cell survive rate is detected, suggesting that the cytotoxicity of Fe5C2-GOD@MnO2 is resulted from the synergistic effect of GOD and Fe5C2 instead of GOD or Fe5C2 alone. Likewise, the acquired hemolysis ratio is much less than 1% at the maximum Fe5C2-GOD@MnO2 concentration (800 μg/mL), indicating that the synthesized nanoparticles have good biocompatibility (Figure S7). To observe the distribution of living cells and dead cells more intuitively, HeLa cells were treated with different samples for 6 h, and then stained with calcein-AM and propidium iodide (PI) simultaneously. The dead and viable cells were stained with red and green fluorescence, respectively. The images could be detected by using confocal laser scanning microscopy (CLSM), as shown in Figure 4b. For control groups, HeLa cells treated with culture medium and Fe5C2@MnO2 groups exhibit no significant cell damage. The largest number of dead cells is detected when treated with Fe5C2-GOD@MnO2. Significantly, almost all the HeLa cells incubated with Fe5C2-GOD@MnO2 in the case of magnetic field are dead. The percentage of PIpositive cells is about 95% at Fe3+-equivalent concentration of 400 μg/mL, suggesting relatively


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high efficient cell apoptosis (Figure S8). These results are consistent with the cytotoxicity experiment, which is considered to be induced by the highly toxic •OH in situ produced by means of Fe5C2-GOD@MnO2 nanocatalysts with magnetic targeting. The magnetic property of Fe5C2-GOD@MnO2 could be utilized to magnetically promote drug uptake. HeLa cells were treated with Fe5C2-GOD@MnO2 nanocatalysts with or without a magnetic field for different times. Then, the images were acquired via CLSM. In Figure S9a and b, Fe5C2-GOD@MnO2 nanocatalysts could efficiently enter cancer cells as affirmed by the presence of intracellular green fluorescence rooted from FITC of FITC conjugated Fe5C2GOD@MnO2 nanocatalysts, and the green fluorescence intensity increases gradually with the extension of incubation time. HeLa cells treated with Fe5C2-GOD@MnO2 nanocatalysts for 3 h in the presence of magnetic field display significantly enhanced cellular uptake, in contrast to that without magnetic field. In addition, the cellular uptake process of Fe5C2-GOD@MnO2 was also measured by different methods containing inductively coupled plasma mass spectrometry (ICP-MS) and flow cytometry (FCM). ICP-MS was utilized to determine the level of iron in the cell lysis solution. The iron content internalized in cells from Fe5C2-GOD@MnO2 ascends with the incubation time and reaches the maximum at 6 h, and then declines at 9 and 12 h incubation time points (Figure S10a). The cellular uptake of Fe5C2-GOD@MnO2 was further quantified with FCM by evaluating the fluorescent intensity of FITC, which derived from the FITC modified Fe5C2-GOD@MnO2 nanocatalysts incubated with HeLa cells. The efficiency of cell phagocytosis is enhanced with incubation time, which is accordance with the above experimental results (Figure S10b). Bio-TEM, which is a significant approach to verify cellular uptake of Fe5C2-GOD@MnO2 nanocatalysts was carried out after HeLa cells were treated with Fe5C2GOD@MnO2 for 6 h. As displayed in Figure S10c and d, it is clear that the Fe5C2-GOD@MnO2


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nanocatalysts could be validly internalized by HeLa cells. The above experimental results indicate that the nanoparticles can be ingested by cells. To understand the intracellular mechanism of Fe5C2-GOD@MnO2 as therapeutic agents, the generation of intracellular ROS was quantified by 2’-7’-dichlorofuorescin diacetate (DCFH-DA), which could be converted from non-fluorescent into fluorescent 2’-7’-dichlorofuorescin (DCF) when oxidized by ROS. In Figure 4c, no apparent green fluorescence can be detected in control group and Fe5C2@MnO2 treated cancer cells, suggesting insignificant ROS production by Fe5C2@MnO2. Notably, Fe5C2-GOD@MnO2 with magnetic targeting triggers a large number of intracellular ROS generation as exhibited by the strong green fluorescence emission of DCF, which presents the quantitative assessment on the ROS production capacity of the nanocatalysts. Similarly, HeLa cells treated with Fe5C2-GOD@MnO2 gives a weak fluorescence compare to the presence of magnetic field, indicating that much less ROS is generated without magnetic targeting, because fewer nanocatalysts are ingested by cells. The relative fluorescence intensity was also determined to more intuitive comparison of the generated ROS (Figure S11). Furthermore, the relative intracellular fluorescence intensity was also measured through FCM which allows comparison of ROS generation after HeLa cells incubated with different samples (Figure 4d). No apparent ROS generated for the HeLa cells incubated with Fe5C2@MnO2, and the fluorescence intensity of DCF exhibits significant increase in Fe5C2-GOD@MnO2 treated HeLa cells. While HeLa cells incubated with Fe5C2-GOD@MnO2 under the driving of magnetic field have higher green fluorescence compared with that without magnetic targeting group. Integrated with ESR experimental result, it can be concluded that Fe5C2-GOD@MnO2 nanocatalysts could be effectively swallowed by tumor cells with magnetic targeting, which


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produce •OH under tumor acidic microenvironment in the presence of H2O2 to induce the toxicity effect and acquire excellent therapeutic efficacy afterward. Biodegradation Behavior of Fe5C2-GOD@MnO2 Nanocatalysts. The biodegradation and biocompatibility are crucial factors in determining the further possibility clinical application of as-prepared nanocatalysts. As a result, the biodegradation behavior of Fe5C2-GOD@MnO2 was monitored in simulated body fluid mediums with various pH values (pH = 7.4 and 6.0), which were utilized to imitate the neutral environment of normal cells and mildly acidic microenvironment of tumor cells (Figure 5a). The Mn-based biodegradation product concentration was carried out by ICP-MS measurement. Comparatively, Fe5C2-GOD@MnO2 nanocatalysts exhibit the rapid biodegradation rate under acidic medium than neutral one during the 72 h degradation process, indicating that Fe5C2-GOD@MnO2 could propose more permanent treatment performance in the tumor acidic microenvironment (Figure 5b). In addition, the biodegradation morphology and structure variation of Fe5C2-GOD@MnO2 were further researched and observed via bio-TEM straightly in HeLa cancer cells. The bio-TEM images of HeLa cells treated with Fe5C2-GOD@MnO2 nanocatalysts for different times are presented in Figure 5c-f. The Fe5C2-GOD@MnO2 could be endocytosed by cancer cells and appropriate structural changes could be detected in the first 24 h of intracellular biodegradation. The morphology of the nanoparticles is gradually destroyed with the extension of the incubation time. No intact nanoparticles structure and almost completely damaged structure could be observed in the tumor cells until the incubation period of 48 h. The bio-TEM detection results are in agreement with the degradation experiments in phosphate buffered solution (PBS) solution with different pH values (Figure 5b). So I draw the conclusion that the microenvironment with tumor


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cells can cause the fracture of Mn–O bonds, and then the shell biodegradation can be realized, and eager to be excreted out of in vivo after therapeutic functioning. Pharmacokinetics

and

Biocompatibility

of

Fe5C2-GOD@MnO2

Nanocatalysts.

Investigation of systemic toxicity and clearance of nanotheranostic agents are crucial for possible biomedical application. To research whether Fe5C2-GOD@MnO2 would cause any side effect, the bio-distribution of the nanocatalysts in major organs versus various injection time intervals were initially researched (Figure 6a). The mice administrated with Fe5C2-GOD@MnO2 via intravenously were scarified at various time points, and then their main organs and tumor were collected for bio-distribution detection. The Fe contents in solubilized tissues were measured via ICP-MS. It can be observed that Fe5C2-GOD@MnO2 nanocatalysts are primary distributed in spleen and liver on account of the capture by the reticuloendothelial system. The Fe levels are found to be 16.25% of the injected dose per gram tissue (%ID/g) in the tumor at 8 h postinjection, 18.31% at 24 h post-injection and eventually 7.50% at 7 days post-injection. The nanocatalysts can effectively accumulate in the tumor site attributed to EPR effect. A circulating half-life of 3.23 h in blood stream is received via blood circulation experiment (Figure 6b). The eliminating rate constants of Fe5C2-GOD@MnO2 nanocatalysts are calculated to be -0.1319 μg/mL per hour in the first stage, and decrease to -0.028 μg/mL per hour in the second stage within a varying time period of 6.429 h (Figure 6c). The obvious volume percentage of distribution of Fe5C2-GOD@MnO2 distribution demonstrates the raising distribution kinetics in the blood throughout the body (Figure 6d). The above experimental results show a time dependent clearance and metabolized effect mainly by the liver and kidney, which is apparently conducive to eliminate potential long-term toxicity.


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In addition, the examination of the standard biochemical index was carried out on tumorbearing mice intravenous injection of saline (control group) and Fe5C2-GOD@MnO2. The body weights of the tumor-bearing mice were measured during 14 days of treatment, which exhibited that the injection of Fe5C2-GOD@MnO2 nanocatalysts has no discernible effect on the mice growth (Figure S12a). The levels of blood urea nitrogen (BUN) and creatinine (CREA) in Fe5C2GOD@MnO2 injected group demonstrate no apparent changes compared with the control group, suggesting that all the mice have normal kidney function, as exhibited in Figure S12b and c. Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) levels remain normal for Fe5C2-GOD@MnO2 nanocatalysts injected group, illuminating the healthy liver function (Figure S12d). No significant variations on blood indicators containing red blood cells, mean corpuscular hemoglobin, mean corpuscular volume, hemoglobin, and hematocrit could be detected, confirming the homogeneous biochemical status and no apparent infections or effect on physiological regulation of immune throughout the evaluation process (Figure S12e-i). Finally, the main organs including heart, liver, spleen, lung and kidney were achieved for H&E staining (Figure S13). No obvious pathological abnormalities are detected in main organs from representative mice injected with Fe5C2-GOD@MnO2 within 14 days of treatment, suggesting the excellent histocompatibility of Fe5C2-GOD@MnO2 nanocatalysts. Based on the above biosafety experimental results, it may be reasonable to infer that Fe5C2GOD@MnO2 nanocatalysts have high biocompatibility for possible in vivo anticancer therapeutic applications. MRI Performance Measurements. Magnetization measurement shows that Fe5C2 and Fe5C2-GOD@MnO2 possess superparamagnetic feature with a saturation magnetization (Ms) of 76 emu g–1 and 54 emu g–1 at room temperature, respectively (Figure S14). As known, MnO2 can


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be stable in neutral pH condition, while it would be disassembled into Mn2+ and generated O2 in acidic condition, and Mn2+ with five unpaired 3d electrons is an excellent T1 MRI contrast agent. In addition, due to the presence of Fe5C2 magnetic core-shell structured nanoparticles, the Fe5C2GOD@MnO2 exhibits strong superparamagnetic characteristics, as confirmed by field-dependent magnetization measurement. The synthesized nanoparticles could be served as T1/T2-weighted dual-modal MRI contrast agent in the tumor acidic microenvironment. Then, in vitro T1/T2weighted MR images of Fe5C2-GOD@MnO2 nanocatalysts with different Fe concentrations after incubation with various pH values buffered solution (pH = 6.0 and 7.4) for 1 h were carried out. An apparent concentration dependent brightening effect is detected in T1-weighted MR images of Fe5C2-GOD@MnO2 nanocatalysts at pH 6.0, while the Fe5C2-GOD@MnO2 nanocatalysts show a rather weak signal at pH 7.4 (Figure 7a). The Fe5C2-GOD@MnO2 nanocatalysts demonstrate a very low longitudinal relaxation (r1) value of 2.787 mM−1 s−1 at pH 7.4, which should be assigned to the high valence (IV) of manganese and isolated paramagnetic centers unapproachable to water molecules. Significantly, the r1 value recorded at pH = 6.0 augment apparently from the initial value to 24.639 mM−1 s−1, due to the disintegration of MnO2 into paramagnetic Mn2+. In the meantime, in vitro T2-weighted MR images of Fe5C2-GOD@MnO2 nanocatalysts were also detected, implying a concentration-dependent darkening effect in both neutral and acidic conditions. High transverse relaxation (r2) values of 35.740 and 79.555 mM−1 s−1 are achieved, respectively (Figure 7b). Hence, Fe5C2-GOD@MnO2 nanocatalysts could serve as pH-responsive dual-modal MRI contrast agent, which is specifically available for imaging in consideration of the acidic tumor microenvironment. Based on the in vitro data analysis, it is indicated that the higher concentration of the nanoparticles accumulate in the tumor site, the better imaging effect will be achieved. Benefiting


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from the excellent magnetic performance of Fe5C2-GOD@MnO2 nanocatalysts, in vivo T1/T2weighted MRI was further evaluated. Here, Fe5C2-GOD@MnO2 nanocatalysts were administrated into the U14 tumor-bearing mice intravenously. The T1-weighted MRI signal intensity at the tumor site exhibits the obvious brightening effect at 24 h post-injection, indicating high tumor accumulation of Fe5C2-GOD@MnO2 nanocatalysts via the EPR effect, meanwhile the gradual disintegration of MnO2 shell into Mn2+ in the mildly acidic tumor environment. Meanwhile, the as-obtained T2-weighted MRI demonstrates that a rather strong darkening effect is also observed at the tumor region after 24 h injection with Fe5C2GOD@MnO2 nanocatalysts (Figure 7c). Furthermore, magnetic targeting was also used to direct magnetic nanoparticles to the tumor site. In comparison with that without a magnetic field, an obviously enhanced MRI effect is detected in the targeted treatment group, indicating a much higher level of Fe5C2-GOD@MnO2 nanocatalysts accumulated in the tumor site via both magnetic targeting and EPR effect (Figure 7d). Based on the above experimental results, it can be concluded that Fe5C2-GOD@MnO2 could be served as a promising therapeutic drug for T1/T2weighted dual-modal MRI-guided anticancer treatment. In Vivo Tumor Catalytic Therapeutic of Fe5C2-GOD@MnO2 Nanocatalysts. The astonishing in vitro catalytic therapeutic effect and excellent biocompatibility/biodegradability of Fe5C2-GOD@MnO2 nanocatalysts encouraged us to further explore their potentially in vivo therapy. The satisfactory tumor growth inhibition effect is attributed to the highly toxic •OH generated by the tumor microenvironment responsive biodegradation and sequential biological/chemical-catalytic reactions via Fe5C2-GOD@MnO2 (Scheme 1b). In the process of reactions, the Fe5C2-GOD@MnO2 can substantially consume the glucose nutrients and generate abundant of H2O2 synchronously for subsequent chemo-catalytic Fenton reaction via magnetic


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Fe5C2 nanoparticles in the tumor acidic microenvironment. The generated toxic •OH could effectively kill tumor cells by means of a mitochondria-mediated apoptosis pathway. In order to affirm the feasibility in tumor suppression with Fe5C2-GOD@MnO2 nanocatalysts, the intravenous injection was administrated for the U14 cancer cells subcutaneously implanted tumors on Kunming mice. The mice were stochastically separated into 4 groups when the tumor volume increased to 60 mm3: saline (group 1), Fe5C2@MnO2 (group 2), Fe5C2-GOD@MnO2 (group 3), Fe5C2-GOD@MnO2 + M (group 4). The injected Fe dose in 100 μL of saline was 10 mg/kg mice body weight. A magnet was bound to the tumor site for magnetic targeting. As demonstrated in the tumor growth profiles, Fe5C2-GOD@MnO2 with magnetic targeting group (t-test, P=2.1×10−5 versus Fe5C2-GOD@MnO2 group) displayed significant tumor growth inhibition and complete tumor ablation after 14 days treatment (Figure 8a). In sharp contrast, in saline and Fe5C2@MnO2 groups, no apparent tumor growth inhibition could be detected and the tumor volume increased to over 8-fold than the original one. Fe5C2-GOD@MnO2 group provided apparently better antitumor efficacy as the sample was accumulated in the tumor location via EPR effect and generated toxic •OH in situ. During 14 days of the treatment, the mean body weights of mice in control and all treatment groups demonstrated no obvious variations (Figure 8b), suggesting that no apparent systemic toxicity has been caused by the administration of Fe5C2-GOD@MnO2 in the anticancer treatment process. Specifically speaking, in accordance with the transformation of the relative tumor volume, the tumor growth inhibition rates have been researched. Mice receiving Fe5C2-GOD@MnO2 under magnetic field exhibited the significant therapeutic effect with tumor growth inhibition rate up to 92%. For other control groups, the tumor-bearing mice group treated with Fe5C2-GOD@MnO2 demonstrated about 63% growth inhibition. However, no significant tumor growth inhibition efficiency can be detected in


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Fe5C2@MnO2 treatment group, indicating the excellent biocompatibility of as-obtained nanocatalysts. After the therapeutic period, the representative tumors from tumor-bearing mice in different treatment groups were dissected and compared in Figure 8c and S15, which more intuitive confirmed that tumor growth could be effectively inhibited after the intravenously injection of Fe5C2-GOD@MnO2 with magnetic targeting at the current dosage. In addition, the haematoxylin and eosin (H&E) staining images of tumor sections from various treatment groups are provided in Figure 8d. The tumor cells in untreated and other control groups primarily maintained their normal morphology with distinctive membrane and nuclear structures, while cancer cells were seriously damaged in the group administrating Fe5C2-GOD@MnO2 with magnetic targeting. The above results are agreement with the tumor growth inhibition rate, further verifying the superior treatment effect by Fe5C2-GOD@MnO2. In addition, H&E staining experiments of the major organs with the Fe5C2-GOD@MnO2 nanocatalysts treatment exhibit no apparent pathological changes (Figure S16). Therefore, the above experimental results exhibited that the Fe5C2-GOD@MnO2 can reduce the side effect of anticancer drugs to normal tissues and promote the therapeutic efficiency. In order to further affirm and popularize our experimental results, the in vivo therapeutic efficacy of Fe5C2-GOD@MnO2 was also executed on another tumor xenograft (4T1 mammary orthotopic tumor model and subcutaneous tumor model, respectively) according to a similar protocol in parallel with the intravenous assessment on U14 cervical subcutaneous cancer model. No apparent body weight changes of mice in control and all the treatment groups within 14 days of the treatment period, suggesting that no obvious toxicity has been triggered by the administration of Fe5C2-GOD@MnO2 intravenously (Figure S17a, d). On tumor inhibition assessments, both 4T1 mammary orthotopic and subcutaneous tumor models exhibit the crucial


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tumor growth inhibition (Figure S17b, e). The Fe5C2-GOD@MnO2 alone could partially suppress the tumor growth with current dose. Strikingly, in comparison with the Fe5C2GOD@MnO2 alone, the Fe5C2-GOD@MnO2 nanocatalysts in the presence of magnetic field treatment group exhibited prominently greater tumor growth suppression, revealing an enhanced toxic •OH generated under magnetic field. More precisely, based on the changes of relative tumor volume, the tumor inhibition rates have been presented to be 61.32% and 90.47% respectively by the intravenous administration of Fe5C2-GOD@MnO2 in the absence and presence of magnetic field in 4T1 mammary orthotopic tumor model, meanwhile 59.78% and 91.02% at the corresponding experimental conditions in 4T1 mammary subcutaneous tumor model. After the treatment sessions ended, the representative tumors of mice in each group are collected and compared on the 14th day. The relative tumor volume (Figure S17c, f) and digital photos of mice (Figure S17g, h) also demonstrated that the tumor volume of mice within Fe5C2GOD@MnO2 in the presence of magnetic field treatment group was smaller than other groups. Furthermore, the haematoxylin and eosin (H&E) staining of tumor tissue sections from different groups were presented, as demonstrated in Figure S17g, h. In comparison to the control group, the tumor cells with the Fe5C2-GOD@MnO2 treatment under the guidance of magnetic field exhibit remarkable damage. The Fe5C2-GOD@MnO2 alone demonstrated slight destruction of tumor cells. Besides, no significant pathological changes of the major organs from all experimental groups could be observed by H&E stained organ slices, indicating the excellent biocompatibility of Fe5C2-GOD@MnO2 in the course of treatment (Figure S18 and S19). The above experimental results showed that 4T1 mammary tumor growth could be effectively inhibited after the intravenous injection of Fe5C2-GOD@MnO2 with magnetic targeting both orthotopic tumor model and subcutaneous tumor model.


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CONCLUSIONS In brief, we put forward an original MnO2-encapsulated and GOD-loaded Fe5C2 (Fe5C2GOD@MnO2) multifunctional nanocatalysts that can enhance sample accumulation at the tumor site by magnetic field mediated-orientation and real-time monitoring of therapeutic effects via T1/T2-weighted MRI. Under the stimulation of the tumor acidic microenvironment, the nanocatalysts can be decomposed to generate Mn2+ and O2 in situ, and further release the encapsulated GOD. The released GOD could effectively exhaust glucose in cancer cells and generate a plenty of H2O2 for further Fenton catalyst reaction induced by Fe5C2 magnetic core in mild acidic tumor microenvironment, leading to efficient production of toxic •OH to kill cancer cells. As expected, the in vivo treatment performance of the Fe5C2-GOD@MnO2 nanocatalysts demonstrates highly tumor inhibition effect. This research designs a special nanotheranostic system which can be transferred into the tumor tissue effectively via magnetic targeting and induce continuous catalyst reactions in response to the mild acidic tumor microenvironment to inhibition tumor growth, providing a promising strategy for effective cancer treatment simultaneously with improved tumor specificity and minimized side effects to normal tissues.

EXPERIMENTAL SECTION Materials and Reagents. All of the chemicals reagents mentioned in this article were used without further purification. All agents were purchased from Sigma-Aldrich other than pentacarbonyl iron (Fe(CO)5) and glucose oxidase (GOD) were acquired from Aladdin Chemical Reagent.


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Synthesis of Fe5C2 Nanoparticles. According to the previous literature, Fe5C2 were synthesized by thermal decomposition method.58 Typically, 0.05 g of CTAB was added into a mixed solution including oleylamine (10 mL) and 1-octadecene (10 mL) in a container, and then heated to 110 °C in vacuum to eliminate the impurities. The above mixture was heated to 120 °C under nitrogen protection for 30 min, then 0.55 mL of Fe(CO)5 was injected at 140 °C. The above solution heated to 185 °C maintain for 10 min, subsequently continued to heat to 320 °C last for 10 min. The product was obtained via centrifugation and dissolved in cyclohexane. Fabrication of Fe5C2-BSA Nanoparticles. Fe5C2-BSA was prepared on the basis of reported literature with a simple revision.59 The as-obtained nanoparticles were scattered in moderation cyclohexane. Afterwards, a solution of dimethylsulfoxide (DMSO, 3 mL) and dopamine (DOPA, 30 mg) was mixed with the above nanoparticles at 70 °C under magnetic stirring for 1 h. The Fe5C2 modified with dopamine were collected and added into BSA aqueous solution with rigorously stirring overnight. The sample (Fe5C2-BSA nanoparticles) was achieved by centrifugation and rinsed several times. Synthesis of Fe5C2-GOD@MnO2 Nanocatalysts. The Fe5C2-BSA were dissolved in sodium acetate buffer (pH = 5.2) involving 10 mg GOD. After stirring for 12 h, the nanoparticles could be acquired by centrifugation, and then dispersed in KMnO4 solution (0.42 mg/mL). Magnetic stirring for 10 min, PAH (1 mL, 50 mg/mL) was appended into the above solution tardily. The final sample Fe5C2-GOD@MnO2 nanocatalysts were achieved for further characterization. Fe5C2@MnO2 nanocatalysts are obtained using the same method. Characterization. Transmission electron microscope (TEM) was acquired for morphology and nanoparticles size on electron microscope operated. Malvern Zetasizer Nanoseries (Nano ZS90) was utilized to measure zeta potential and particle size for different samples. The UV-vis


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absorbance spectra of the samples were carrried out using UV-1601 spectrophotometer. ESR spectrum of Fe5C2-GOD@MnO2 was achieved by Bruker spectrometer. The T1/T2-weighted dual-modal MRI was conducted on a 1.2 T MR scanner. In Vitro Degradation Experiment. Fe5C2-GOD@MnO2 with the concentration of 400 μg/mL were dispersed into PBS solution with various pH values (pH = 7.4 and 6.0). The in vitro degradation experiment was performed at 37 °C by the sufficient stirring. Cultivate for different time intervals, inductively coupled plasma mass spectrometry (ICP-MS) was applied to detect the content of Mn in the aqueous solution. Cellular Uptake. Cell phagocytosis was assessed via confocal laser scanning microscopy (CLSM), ICP-MS and flow cytometry, respectively. Hela cells were planted in six-well plates and incubated grew against the wall. The cells were incubated with Fe5C2-GOD@MnO2 (400 μg/mL) with various times (0.5, 1, 3, 6, 9 and 12 h). The uninternalized nanocatalysts are washed several times with PBS and dissolved through the cell lysate. ICP-MS was utilized to measure the iron content in the aqueous solution. The FITC labeled nanoparticles were acquired according to the previous literature with a slightly modification. Simply, FITC (2 mg/mL) and APTES (50 μL/mL) were dissolved in 5 mL of ethanol with tightly stirring, APTES-FITC was achieved after several hours. Then, 10 μL of above solution was mixed with Fe5C2-GOD@MnO2 nanocatalysts for 6 h, FITC modified Fe5C2GOD@MnO2 nanocatalysts were acquired. Hela cells were planted in a 6-well plate and cultured to achieve attached cells. Subsequently, 1 mL FITC modified Fe5C2-GOD@MnO2 (400 μg/mL) was incubated with cells for different times (0.5, 1, 3 h) with or without a magnet, respectively. A magnet (magnet magnetic force = 0.2 T) was placed at the bottom of the petri dish for 30 min, and then removed the magnet and continued to culture for another 2.5 h. Afterwards, the


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uninternalized nanocatalysts were rinsed away with PBS and stained with DAPI. Finally, the images of cells were observed using a Leica TCS SP8 instrument. In vitro Cell Viability Assay. For in vitro cytotoxicity assays, Hela cells were planted and incubated overnight to allow the attachment of cells. Subsequently, Fe5C2-GOD@MnO2 (100 μL) with a serial concentration of 12.5, 25, 50, 100, 200, 400 and 800 μg/mL were added into the 96well plate and treated for 12 h. The pH value was changed to 6.0 via adding HCl. Then 20 μL of MTT solution was added into each well. Continued to cultivate for another 4 h, DMSO (150 μL) was added and the absorbance at 490 nm was acquired to calculate cell survival rate. In addition, the in vitro cell survival rate of GOD@MnO2 and Fe5C2@MnO2 to L929 cells was also estimated via standard MTT assay. The concentration of nanoparticles is 12.5, 25, 50, 100, 200, 400 and 800 μg/mL. Hemolysis Assay. Blood red cells were obtained by centrifugation and washed with PBS, then diluted with a proper amount of PBS. Next, blood samples (0.3 mL) were added into Fe5C2GOD@MnO2 solution with different sample concentrations (12.5-800 μg/mL). PBS and deionized water set as negative control and positive control, respectively. After being stationary for two hours, centrifuge was performed to the samples, the supernatant was obtained to access their absorbance and calculated the hemolysis ratio. Detection of ROS. Hela cells were planted and incubated make the cells adhered to the wall. 1 mL of high glucose culture medium containing Fe5C2@MnO2 and Fe5C2-GOD@MnO2 were added. Then, the solution was removed and washed with PBS several times. Subsequently, 100 mM non-fluorescent 2’,7’-dichlorofluorescin diacetate as a fluorescent probe was added and incubated for 20 min to detect ROS, generating fluorescent product DCF, which could be observed via CLSM.


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T1/T2-weighted Dual-Modal Magnetic Resonance Imaging. The in vitro MRI performance was executed in a 1.2 T MRI magnet. Fe5C2-GOD@MnO2 nanocatalysts solution with different Fe concentrations at pH 7.4 and 6.0 were prepared (determined by ICP-MS measurement), respectively. T2 signal intensity versus repetition time (RT) at different sample concentrations was measured. Finally, r2 relaxivity value was assessed by the fitted curve of 1/T2 relaxation time (s−1) in accordance with Fe concentrations. In vivo T2-weighted MRI experiments were acquired by injecting Fe5C2-GOD@MnO2 nanocatalysts into the mice via intravenously (10 mg Fe/kg) on 1.2T clinical MRI equipment. After 24 h injection, the mice in control group and intravenous injection of Fe5C2-GOD@MnO2 nanocatalysts were scanned. A magnet (magnet magnetic force = 0.2 T) was bound to the tumor site for 1 h to research magnetic targeting, no magnetic field was used as a control. Furthermore, in vitro and in vivo T1-weighted MRI was also carried using the similitude experiment method. In Vivo Anticancer Treatment Performance. Female Kumming mice (18-22 g) were purchased from the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Harbin, China). The Kumming mice were split up (n = 5) into four groups. The mice were subcutaneously transplated with U14 cancer cells at the left armpit. The treatment began when the tumors were grow to about 60 mm3. The first group were injected saline (control group), the Fe5C2@MnO2 nanocatalysts (group two) and Fe5C2-GOD@MnO2 (group three and four) were also injected into the mice via intravenously. In group four, the magnet (M) was placed at the tumor site to improve Fe5C2-GOD@MnO2 nanocatalysts accumulation in tumors further understand the magnetic targeting anticancer treatment. A magnet (magnet magnetic force = 0.2 T) with 2 cm in length and 1 cm in width was bound on the tumor site for 1 h to guide the accumulation of nanocatalysts at the tumor sites. The injected Fe (Fe5C2-GOD@MnO2


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nanocatalysts) dose in 100 μL of saline was 10 mg/kg body weight. The relative tumor volume was enumerated according to Vr = V/V0 × 100% (V0: tumor volume on the first day). The mice body weights and tumor volume were sueveyed to access the treatment effect. To further confirm the in vivo anti-cancer treatment effect, BALB/c nude mice were divided (n = 5) into groups randomly. They were orthotopically and subcutaneously transplanted with 4T1 mammary cancer cells (2 × 105 cells each mouse) in the mammary fat pad and at the right back, respectively. Treatment was initiated when the tumor volume reached approximately 60 mm3 according to a similar protocol in parallel with the intravenous assessment on U14 cervical subcutaneous cancer model. Histological Staining. 14 days after treatment, the representative tumor-bearing mice from different treatment groups were sacrificed. Then, tumors and main organs were collected for histopathological analysis. The histological changes were analyzed using CLSM. Evaluation of Blood Biochemical Index. The blood biochemical indicator was measured to evaluate the cumulative effect of Fe5C2-GOD@MnO2 nanocatalysts. The tumor-bearing mice were divided into 2 groups (5 per group). The mice were intravenous administrated Fe5C2GOD@MnO2 nanocatalysts (100 μL, 10 mg Fe/kg) via and the control group injected the same dose of saline. After 14 days of treatment period, the blood sample was obtained by pulling out the eyeball from representative mice and analyzed by the Hematology analyzer. In Vivo Pharmacokinetic Evaluation. All mice were treated with Fe5C2-GOD@MnO2 nanocatalysts (100 μL, 10 mg Fe/kg) intravenously. At 1, 8, 24, 48 h and 7 days post-injection, the mice from different groups were euthanized. Main organs and tumors were collected, weighed and dissolved with HNO3 and H2O2 mixed solution. The biodistribution in different tissues were estimated as Fe percentage of tissues by ICP-MS.


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Statistics Analysis. The quantitative data analysis were manifested as mean ± S.D. More than three samples of statistical significance were collected. The student’s test was adopted to execute statistical analyses. The statistical significance was speculated at a value of *p

Magnetic Targeting, Tumor Microenvironment-Responsive Intelligent

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