Glucose Oxidase-Instructed Traceable Self-Oxygenation/Hyperthermia Dually Enhanced Cancer Starvation Therapy.

Cancer theranostics based on glucose oxidase (GOx)-induced starvation therapy has got more and more attention in cancer management. Herein, GOx armed manganese dioxide nanosheets (denoted as MNS-GOx) were developed as cancer nanotheranostic agent for magnetic resonance (MR)/photoacoustic (PA) dual-modal imaging guided self-oxygenation/hyperthermia dually enhanced starvation cancer therapy. The manganese dioxide nanomaterials with different morphologies (such as nanoflowers, nanosheets and nanowires) were synthesized by a biomimetic approach using melanin as a biotemplate. Afterwards, the manganese dioxide nanosheets (MNS) with two sides and large surface area were selected as the vehicle to carry and deliver GOx. The as-prepared MNS-GOx can perform the circular reaction of glucose oxidation and H2O2 decomposition for enhanced starvation therapy. Moreover, the catalytic activity of GOx could be further improved by the hyperthermia of MNS-GOx upon near-infrared laser irradiation. Most intriguingly, MNS-GOx could achieve "turn-on" MR imaging and "turn-off" PA imaging simultaneously. The theranostic capability of MNS-GOx was evaluated on A375 tumor-bearing mice with all tumor elimination. Our findings integrated molecular imaging and starvation-based synergistic cancer therapy, which provided a new platform for cancer nanotheranostics. © The author(s).

Introduction

Glucose is an important source of energy in living organisms, the occurrence and development of many diseases are closely related to the glucose metabolism1-2. Utilization of glucose oxidation through the catalytic reaction of glucose oxidase (GOx) can cut off energy supply to inhibit the tumor growth3-5, which are known as GOx-based starvation therapy that has attracted more and more attention in cancer management in recent years4, 6-11. However, the starvation therapies based on GOx are restricted by the hypoxic state of tumor tissues, because glucose oxidation reactions need a large amount of oxygen (O2)12-13. Therefore, how to improve the O2 level of tumor tissues will directly decide the therapeutic effect of GOx-based starvation therapy.

The products of glucose oxidation are including gluconic acid and hydrogen peroxide (H2O2). Meanwhile, tumor microenvironments also contain a high content of H2O2. As we know, the decomposition of H2O2 can produce water and O2, thus promising the melioration of tumor hypoxia. Manganese oxide (MnO2) nanomaterials can catalyze decomposition reaction to produce O2 to modulate solid tumor hypoxia for effective starvation therapy14-18. Therefore, the combination of MnO2 and GOx will accelerate glucose consumption to improve the starvation therapeutic effect. Recently, MnO2-based theranostic agents have been widely explored14,19-23, such as urchin-shaped MnO2 nanoparticles17, 24-25, hollow MnO2 nanoplatform26-27, MnO2 nanosheets28-31 and Mn-based layered double hydroxide32 and so on. However, most of MnO2 nanomaterials need polyethylene glycol (PEG) modification to improve their biocompatibility and water solubility28. Melanin, an endogenous biomolecule, has been employed as coating materials to endow nanoplatforms with good biocompatibility, dispersibility, and strong near-infrared (NIR) absorption33-37. Moreover, melanin-based nanoplatforms have great potential in the biomedical applications of photoacoustic imaging (PAI) and photothermal therapy (PTT).

Herein, we used melanin as the biotemplate to directly synthesize MnO2 nanomaterials with different morphologies (such as nanoflowers, nanosheets and nanowires). The melanin coated MnO2 have good biocompatibility and strong NIR absorption. Afterwards, GOx armed manganese dioxide nanosheets (denoted as MNS-GOx) were explored for cancer nanotheranostics with following characteristics (Scheme 1): (i) the circular reaction of glucose oxidation and H2O2 decomposition; (ii) the enhancement of catalytic activity of GOx by the hyperthermia of MNS-GOx; (iii) activatable magnetic resonance (MR)/photoacoustic (PA) dual-modal imaging. The theranostic capability of MNS-GOx was carefully investigated both in vitro and in vivo. The as-prepared MNS-GOx has great potential in traceable cancer therapy, especially for MR/PA dual-modal imaging guided self-oxygenation/hyperthermia dually enhanced starvation cancer therapy.

Scheme 1

Schematic illustration of MNS-GOx for magnetic resonance (MR)/photoacoustic (PA) dual-modal imaging guided self-oxygenation/hyperthermia dually enhanced starvation cancer therapy.

Experimental Section

Synthesis of MNS

MnO2 nanosheets (MNS) were synthesized by the biomimetic synthesis method using melanin as the biomimetic template. Typically, 0.4 mL melanin (2 mg/mL) and 3.2 mL ethanol were added into 48 mL deionized (DI) water. The pH value of the mixed solution was adjusted to 7.4~7.8. Then the solution was heated to 90 °C, 0.32 mL potassium permanganate (KMnO4) (0.05 M) was slowly added under vigorous stirring. The system was kept stirring for 30 min at 90 °C. The product was purified and concentrated by an ultrafilter (30 kDa).

Synthesis of MNS-GOx

GOx was conjugated on the surface of MNS through the 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC)/N-Hydroxysuccinimide (NHS) reaction between carboxyl groups of MNS and amine groups of GOx. In brief, 10 mg EDC and 5 mg NHS were added into 4 mL MNS (1 mg/mL). After 2 h, 40 µL GOx (2 mg/mL) was added and the mixture solution was stirred for another 8 h. The MNS-GOx was washed with DI water for several times and suspended in water for further use. In further experiments, the ratios of 200:1 and 1:1 of MNS: GOx were prepared for in vitro and in vivo experiments, respectively.

Characterization

The morphologies of MnO2 nanomaterials were observed by high-resolution field emission transmission electron microscope (TEM) (JEM-3200FS, JEOL, Japan). The thickness of MNS was measured by atom force microscope (AFM) (MultiMode 8, Bruker, Germany). X-ray diffraction (XRD) pattern of MNS was detected by X-ray diffractometer (D8 Advance, Bruker, Germany). Dynamic light scattering (DLS) and Zeta potential was measured on Zetasizer Nano-ZS90 (Malven, England). Fourier-transform infrared (FT-IR) spectra were collected on a FT-IR spectrometer (Spectrum Two, PerkinElmer, USA). UV/Vis absorption spectra were measured on a Cary 60 UV/Vis spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). The Mn element concentration of MNS was quantified by an inductively coupled plasma-atomic emission spectrometry (ICP-AES) (JY 2000-2, Horiba, France).

pH-Responsive Degradations of MNS and MNS-GOx

The pH-responsive degradations of MNS and MNS-GOx were monitored by UV/Vis spectrophotometer. The optical density (OD) at 400 nm of MNS (100 µg/mL) or MNS-GOx (100 µg/mL) in PBS solutions (5 mM glucose, pH 7.4) was measured over time. Additionally, 4 mL of MNS (100 µg/mL) or MNS-GOx (100 µg/mL) was loaded into a dialysis bag with the molecular weight cut-off of 3500 Da. Then the bag was immersed into 50 mL of buffer solutions with different pH values (5 mM glucose pH 5.0, 6.0, 7.4) under vigorous stirring at room temperature. At different time points, 2 mL of buffer solution was taken out and the released Mn2+ was detected by ICP-AES. Meanwhile, an isometric buffer solution was added to keep the total volume same.

Photothermal Performance of MNS

The aqueous solutions of MNS (0~200 µg/mL) were stored in Eppendorf tubes, and exposed to 808 nm laser at power density of 1 W/cm2 for 3 min. The photothermal stability of MNS (200 µg/mL) were irradiated upon different laser power density of 1 W/cm2 for four on/off cycles (on: 2 min, off: 6 min). Real-time thermal imaging was recorded by a SC300 infrared camera (FLIR, Arlington, VA) and quantified by FLIR Examiner software.

T1 Relaxivity of MNS and MNS-GOx

MNS and MNS-GOx with different Mn element concentration (0.2, 0.4, 0.6, 0.8, 1.0 mM) were dispersed in PBS solutions with different pH values (5, 6, 7.4) for 4 h. Then the T1 relaxivity was detected by a 3 T clinical MRI scanner (UMR 790, United-Imaging, Shanghai, China). An fast spin echo (FSE) sequence with 19 different values of T1 (TR (repetition time)=5000 ms; TE (ehco time) =100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900, 1000, 1200, 1300, 1500, 2000 ms) for T1 measurements. T1 relaxation times were calculated by fitting the signal intensities with increasing T1 to I(t)=I0[1-k•exp(-t/T1)] by using a nonlinear least-squares fit of the Levenberg-Marquardt algorithm.

Catalytic Activity of GOx

30 µL of MNS-GOx (1 mg/mL) or GOx (1 mg/mL) was added into 1 mL of glucose solution (30 mM) under oxygen gas flow (5 mL/min). The glucose concentrations were immediately detected by Yuwell 590 glucometer at different time points (0, 2, 4, 6, 8 min). Moreover, the glucose solutions were heated up to 30, 40, 50 or 60 ºC. The kinetic parameters (Vmax) of GOx obtained from the Michaelis-Menten plots at different temperatures38-41. Meanwhile, the catalytic activity of GOx in MNS-GOx was investigated before and after laser irradiation (808 nm laser, 1 W/cm2 for 5 min).

In Vitro Synergistic Therapy

For in vitro synergistic therapy, four parallel groups were set: control, MNS + laser, MNS-GOx, and MNS-GOx + laser. A375 cells were seeded into a 96-well plate at 104/well and then cultured at 37 °C for 24 h. The cells were incubated with MNS or MNS-GOx (0~1 mM) for 4 h and then exposed to an 808 nm laser at 1 W/cm2 for 5 min. Then the old dulbecco's modified eagle medium (DMEM) media were removed and 5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in 100 μL DMEM media was added into each well. After co-incubation for another 4 h, the old DMEM media were replaced with 150 μL dimethylsulfoxide (DMSO) per well, and the absorbance at 490 nm was monitored by a microplate reader. The cytotoxicity was finally expressed as the viabilities of different-treated cells in contrast to the untreated control cells.

A375 cells were seeded into a 12-well plate at 7*104/well and then cultured at 37 °C for 24 h. MNS or MNS-GOx (1 mM, DMEM media) was added into each well and co-incubated for 4 h. Then the cells were exposed to an 808 nm laser at 1W/cm2 for 5 min. After co-incubation for another 24 h, the cells were co-stained by Calcein acetoxymethyl ester (calcein AM) and propidium iodide (PI), and then imaged using an Olympus FV1000 fluorescent microscope.

In Vivo MR/PA Dual-Modal Imaging

The tumor-bearing nude mice (∼20 g) were intratumoral injected with MNS or MNS-GOx (5 mg/kg). Magnetic resonance imaging (MRI) was performed by a UMR 790 3.0T (United-Imaging, Shanghai, China). T1-weighted images were acquired by FSE sequence at 0, 0.5, 1, 4, 8, 24 h and the parameters were as follows: TR=700 ms; TE=14.3 ms; Flip Angle=145 º; matrix size, 160 × 160; slice thickness, 1.5 mm. Signal intensities were measured in defined regions of interest (ROI) with software named Image J. The PA images were recorded by a Vevo LAZR2100 system (VisualSonics Inc. New York, NY) equipped with a 40 MHz, 256-element linear array transducer on tumors.

In Vivo Synergistic Therapy

A375 cells were implanted subcutaneously into nude mice (∼20 g). In vivo treatment was performed when the tumor reached 6 mm in average diameter (10 days after implant). The mice were divided into six groups: PBS as the control group; PBS with laser irradiation (PBS + laser); MNS; MNS-GOx; MNS with laser irradiation (MNS + laser); MNS-GOx with laser irradiation (MNS-GOx + laser). For laser irradiation groups, 808 nm laser with the power of 0.6 W/cm2 was used to irradiate tumor tissues for 5 min after intratumoral injection (dose: 5 mg/kg). For each group, the tumor volumes and body weight were measured every two days, and volume of tumors was calculated as (tumor length) × (tumor width/2)2. Relative tumor volume was calculated as V/V0 (V0 is the tumor volume when the treatment was initiated).

Hemolysis Assay

The red blood cells (RBCs) were isolated from serum by centrifugation of the mixture containing 0.5 mL blood sample and 1 mL PBS solution at 4500 rpm for 3.5 min, then washed the RBCs over five times and diluted the purified cells to 5 mL. Then 0.3 mL volume diluted RBCs suspension was added to quadruple volume of PBS solution with different concentrations of MNS (12.5 to 400 µg/mL). The mixtures were vortexed and kept to stand for 4 h at room temperature. Samples were then centrifuged to measure the absorbance of the supernatants at 541 nm by an UV-vis spectroscopy. RBCs treated with deionized water and PBS were set as positive and negative controls.

Ex vivo Histological Staining

Tumor tissues were collected from tumor-bearing mice in different groups at day 16 and sectioned into slices for hematoxylin and eosin (H&E) staining. For the toxicity evaluation of MNS or MNS-GOx, mice with intratumoral injection of MNS or MNS-GOx (dose: 5 mg/kg) were sacrificed at day 30 (3 mice per group). Then tumor tissues and major organs (including heart, liver, spleen, lung, kidney and tumor.) were collected, fixed in 10% neutral buffered formalin, embedded in paraffin, cut into 4 µm sections and stained with H&E. Finally, the images of these histological tissue sections were obtained by a BX41 bright field microscopy (Olympus).

Statistical Analysis

Data were presented as mean ± standard deviation (SD). Statistical differences among experimental groups were analyzed using one-way analysis of variance (ANOVA) followed by two-tailed Student's t test. P < 0.05 was considered as statistically significant.

Results and Discussions

Preparation and Characterization of MNS and MNS-GOx

MNS were prepared by one-step reduction of KMnO4 using melanin as a template under neutral conditions, and then modified with GOx by a cross-linker, as shown in Figure . Due to an abundant of catechol groups of melanin, it can anchor on the surface of metals oxides42. Therefore, the morphologies of MnO2 were strongly depended on the solubility of melanin under different reaction conditions. The as-prepared MnO2 nanomaterials were nanoflowers (Figure at acidic condition (pH < 7), ultrathin nanosheets (Figure at neutral condition (pH 7) and nanowires (Figure at alkaline condition (pH 10). The nanoflowers can be formed when the decomposition rate of KMnO4 was faster in acidic condition and the nanowires were probably formed when the solubility of melanin increased in alkaline solution43. The MNS with two sides and large surface area was chosen as the vehicle to carry and deliver GOx. As shown in , the hydrodynamic diameter of MNS was about ~70 nm measured by DLS. High resolution TEM image of MNS indicated that the lattice fringe spacing of MNS were 0.232 and 0.288 nm (, which can be attributed to (311) and (411) according to the XRD pattern (. EDS spectrum confirmed the high concentration of manganese and oxygen elements in MNS, which is further supported by element mapping images in . As shown in , the thickness of MNS was ~ 2 nm, which indicated the MNS with ultrathin sheet structure.

Melanin as the biotemplate plays an important role in regulating the morphology of MnO2 nanomaterials during the synthesis process. It can be adsorbed on the surface of MNS through the abundant catechol groups of melanin42, 44. As shown in Figure , UV-vis-NIR absorption spectrum of MNS contained the broad absorbance of melanin35 and the characteristic peak of MnO2 (~400 nm)44. The absorbance at 808 nm of MNS was positively correlated with MnO2 concentration, and the typical equation was Y = 0.003X + 0.0102 (R2 = 0.9736) (. The photothermal effect of MNS exhibited a MNS concentration-dependent under 808 nm laser irradiation (. After 4 cycles of laser irradiation, MNS still remained its excellent photothermal conversion property, suggested that MNS has a great photostability (. The melanin modified MnO2 had a large number of carboxyls for further surface modification. GOx can be covalently conjugated onto the surface of MNS by EDC/NHS reaction. FT-IR spectrum of MNS-GOx showed a new peak at about 1650 cm-1, indicating that GOx was successfully grafted onto the surface of MNS (Figure . The zeta potentials of MNS and MNS-GOx were -24.6 and -16.7 mV, respectively (Figure . These results indicated GOx was successfully loaded by MNS.

pH-Responsive Degradation of MNS-GOx

MnO2 nanomaterials can be decomposed into Mn2+ in the tumor microenvironment for MRI and alleviating the hypoxia of tumor24, 28, 45-46. As shown in Figure , the OD at 400 nm of MNS-GOx decreased from 0.70 to 0.17 within 8 h in 5 mM glucose solution, while that of MNS kept stable at about 0.84. Because MNS-GOx can catalyze glucose to produce gluconic acid and H2O2, which promoted the degradation of MNS. The color of MNS-GOx solution was changed from brown to colorless over time (. Moreover, the quantified concentration of Mn2+ released from MNS or MNS-GOx was further verified the degradation of MNS. As shown in Figure , the Mn2+ released from MNS-GOx can reach 89% at pH 5, but it was only 56.3% of MNS at the same condition. The concentration of released Mn2+ will directly affect the following longitudinal relaxivity (r1). Because the released Mn2+ had five unpaired electrons, Mn-based contrast agents have been widely explored as T1 contrast agents. Based on the profile of 1/T1 vs Mn element concentration of MNS (Figure and MNS-GOx (Figure , the r1 MNS increased from 0.084 to 7.0 mM-1s-1 from pH 7.4 to 5.0, while r1 MNS-GOx increased from 3.3 to 22.8 mM-1s-1(Figure . The r1 MNS enhancement is due to the pH-responsive degradation of MNS, while the r1 MNS-GOx enhancement is due to pH/glucose dual-responsive degradation of MNS, which accelerated the release of Mn2+. In order to observe the degradation process of MNS-GOx, their morphology change was recorded by TEM imaging. As shown in Figure , when MNS-GOx was incubated with 5 mM glucose, most of MNS was decomposed at 17 min, and no MNS can be found at 30 min. These results suggested MNS-GOx exhibited pH/glucose dual-responsive performance.

In vitro Synergistic Therapy

MnO2 nanomaterials can catalyze the decomposition reaction of H2O2 to produce O2, which can promote the reaction of glucose oxidation16. As shown in Figure , the glucose oxidation rate of GOx and MNS-GOx were 2.69 and 3.43 mM/min, respectively. The catalytic activity enhancement of GOx by MNS can be attributed to the self-oxygenation of MNS-GOx during the glucose oxidation reaction that is an O2-depandent reaction. Afterwards, we investigated the catalytic activity of GOx at different temperature. The glucose concentrations were recorded by a glucometer, and extra O2 was supplied into glucose solution for sufficient O2 during the reaction. The results shown that Vmax of MNS-GOx increased from 2.69✕103 M/min at 30 °C to 3.43✕103 M/min at 50 °C (Figure . It was consistent with previous reports that the maximum activity of GOx at 50 °C 40-41, 47-48. Encouraged by the catalytic activity enhancement of GOx at high temperature, in vitro experiments were conducted on A375 cells for hyperthermia-enhanced synergistic therapy. MNS or MNS-GOx was incubated with A375 cells for 24 h, and then irradiation with or without 808 nm laser (1 W/cm2, 5 min). As shown in Figure , cells incubated with MNS kept their viability over 81%. Cell viabilities of MNS + laser, MNS-GOx and MNS-GOx + Laser groups showed concentration-dependent therapeutic effect. Importantly, MNS-GOx + laser group exhibited better therapeutic effect than that of MNS-GOx only. The synergistic therapeutic effect was further evidenced by live/dead cell staining (Figure . No dead cells were found in control group (Figure . For MNS + laser and MNS-GOx + laser groups, most of cells were dead in laser spots (Figure . These results indicated the catalytic activity of GOx could be further improved by the hyperthermia of MNS-GOx upon 808 nm laser irradiation. Additionally, we assessed the catalytic activity of GOx before and after laser irradiation. As shown in , the glucose reaction rates of MNS-GOx kept similar, which suggested the laser irradiation (808 nm, 1 W/cm2, 5 min) can preserve the catalytic activity of GOx.

In Vivo Dual-Modal Imaging

MNS-GOx was composed of Mn element and melanin, which can be used as contrast agents for MRI and PAI, respectively. In vivo MR/PA dual-modal imaging was performed on the A375 tumor-bearing mice. MNS or MNS-GOx was intratumoral injected into A375 tumor-bearing mice at 5 mg/kg dose. Then the treated mice were scanned on 3T clinic MRI scanner. Both MNS and MNS-GOx displayed an obvious T1-weighted enhancement in tumor tissues (Figure . Compared with MNS treated mice, mice in MNS-GOx group exhibited stronger contrast effect at each scan time point. Because MNS-GOx are pH/glucose dual-responsive, it can release more Mn2+ in tumor microenvironment than MNS, and the concentration of released Mn2+ will directly affect the longitudinal relaxivity and MRI contrast effect. So MNS-GOx group exhibited stronger contrast effect compared with MNS group. On the contrary to the increase of MR signal, PA signal of gradually decreased after injection over time (Figure . As shown in Figure , the quantified MR signal/noise ratio (SNR) of tumors treated with MNS-GOx gradually increased to the peak value of 2.67 at 8 h post-injection, while the SNR of tumors treated with MNS was only increased to 2.0 at 24 h post-injection. As shown in Figure , the quantified PA signals of tumors treated with MNS or MNS-GOx gradually decreased over time. These results suggested MNS-GOx could achieve “turn-on” MR imaging and “turn-off” PA imaging simultaneously. MRI can offer high resolution contrast for soft tissue without tissue depth limits, but restricted by its poor sensitivity and time-consuming. PAI is convenient and time-saving possesses high optical imaging contrast, but limited imaging depth. So the dual-model imaging can integrate different information to provide accurate diagnosis. A “turn on” MRI can light the deep tissues for precision cancer diagnosis, and the “turn off” PAI can monitor the treatment progress for individual treatment.

Based on in vitro synergistic PTT/starvation therapy effect, the in vivo synergistic therapy was conducted on A375 tumor-bearing mice. Mice were randomly divided into six groups: PBS, laser only, MNS, MNS+laser, MNS-GOx and MNS-GOx+laser. For all groups, the intratumoral injection dose was 5 mg/kg. For laser groups, 808 nm laser (0.6 W/cm2, 5 min) was immediately irradiated on A375 tumor tissues after injection. The tumor volumes were measured every 2 days during 16 days. As shown in Figure , the tumors of PBS and PBS + laser groups grew very fast, while MNS-GOx treated group shown great suppression effect compared with MNS group. Interestingly, the tumors of MNS-GOx + laser group were completely eliminated; while MNS + laser group began to regrow at 10 days after treatment.

On the one hand, the increase of tumor local temperature upon laser irradiation can improve the catalytic activity of GOx, thus consuming more intratumoral glucose(∼50 °C, ), on the other hand, MNS-GOx can perform the circular reaction of glucose oxidation and H2O2 decomposition for enhanced starvation therapy. The self-oxygenation/hyperthermia dually enhanced starvation cancer therapy shown higher tumor suppression effect than any single treatment. Furthermore, the survival rate of mice in MNS-GOx group was greatly prolonged (Figure . All treated mice had no obvious body weight change during different treatments (Figure . The photographs of A375 tumor-bearing mice further evidenced the synergistic PTT/starvation effect (Figure . Hematoxylin and eosin (H&E) staining images of tumor sections after treatments also indicated an obviously membrane fragmentation or shrinkage of nuclei in MNS + laser group and MNS-GOx + laser group (Figure . But no significant damage or inflammation from H&E stained images of main organs (heart, liver, spleen, lung, kidney), negligible change of blood biochemistry results, and the hemolysis rates of virous concentrations (15.5-400 µg/mL) of MNS were lower than 5.5% (. These findings indicated that MNS-GOx exhibited a great potential in hyperthermia enhanced starvation synergistic therapy.

Conclusions

In summary, three different morphologies of MnO2 nanomaterials were successfully synthesized by a biomimetic approach using melanin as a biotemplate at different pH conditions. Afterwards, GOx loaded MnO2 nanosheets (MNS) was explored as cancer nanotheranostics, especially for self-oxygenation/hyperthermia dually enhanced starvation therapy. The as-prepared MNS can decompose H2O2 to supply O2 for the GOx catalyzed glucose oxidation reaction, thus promising the circular reaction of H2O2 decomposition and glucose oxidation. Moreover, the catalytic activity of GOx could be further improved by the hyperthermia of MNS-GOx upon NIR laser irradiation. Most intriguingly, MNS-GOx could achieve “turn-on” MR imaging and “turn-off” PA imaging simultaneously. The theranostic capability of MNS-GOx was evaluated both in vitro and in vivo. Finally, the as-prepared MNS-GOx exhibited pH/glucose dual-responsive performance, activatable MR/PA dual-modal imaging, and hyperthermia enhanced starvation synergistic cancer therapy, which provided a new nanoplatform for cancer nanotheranostics.

Supplementary figures.

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