MnO2-Disguised Upconversion Hybrid Nanocomposite: An Ideal

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MnO2-Disguised Upconversion Hybrid Nanocomposite: An Ideal Architecture for Tumor Microenvironment-Triggered UCL/MR Bioimaging and Enhanced Chemodynamic Therapy Binbin Ding, Shuai Shao, Fan Jiang, Peipei Dang, Chunqiang Sun, Shanshan Huang, Ping'an Ma, Dayong Jin, Abdulaziz A. Al Kheraif, and Jun Lin Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.9b00893 • Publication Date (Web): 20 Mar 2019 Downloaded from http://pubs.acs.org on March 20, 2019

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Chemistry of Materials

MnO2-Disguised Upconversion Hybrid Nanocomposite: An Ideal Architecture for Tumor Microenvironment-Triggered UCL/MR Bioimaging and Enhanced Chemodynamic Therapy Binbin Ding,†,‡ Shuai Shao,†,§ Fan Jiang,†,‡ Peipei Dang,†,‡ Chunqiang Sun,† Shanshan Huang,† Ping’an Ma,*,†,‡ Dayong Jin,⊥ Abdulaziz A. Al Kheraif‖ and Jun Lin*,†,‡ † State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China ‡ University of Science and Technology of China, Hefei 230026, P. R. China § Changchun University of Science and Technology, Changchun 130022, P. R. China

⊥Institute for Biomedical Materials and Devices, Faculty of Science, University of Technology, Sydney, NSW, 2007, Australia ‖Dental Health department College of Applied Medical Sciences, King Saud University, Riyadh, Saudi Arabia ABSTRACT: Upconversion nanoparticles (UCNPs) and MnO2 hybrid theranostic nanoplatform (UC-Mn) is highly desired, however, the rational design of such UC-Mn hybrid nanomaterials is still a great challenge. Herein, a simple and versatile strategy for in-situ growth of MnO2 on the surfaces of UCNPs was reported using a sacrificial template method to construct an ideal MnO2-disguised and tumor microenvironment (TME)-triggered architecture. Such sophisticated architecture not only achieves activatable magnetic resonance imaging (MRI) and restorable upconversion luminescence (UCL) imaging with over 100-fold enhancement of UCL in vivo, but also significantly improves the efficiency of chemodynamic therapy (CDT) by GSH depletion- and cisplatin activation-enhanced •OH generation simultaneously. Additionally, the synergetic effect of CDT and chemotherapy presents excellent therapeutic effect in vivo as compared to either CDT or chemotherapy alone. We believe that the ideal design of the MnO2-disguised upconversion hybrid nanocomposite will provide more revelations on the future research about nanoscale theranostic systems.

INTRODUCTION In solid tumors, the complicated tumor microenvironment (TME) characteristically displays mild acidity, high permeability, severe hypoxia, elevated glutathione (GSH) and H2O2 overproduction.1-3 The TME not only has a tremendous effect on tumor development and metastasis,4 but also furnishes the "gate" for selective tumor treatments.5, 6 Hence, designing nanoscale theranostic systems that are capable of being responsive to the inherent features of TME has been widely accepted to be a satisfactory and promising choice to realize tumor-specific treatment.7, 8 For example, many smart theranostic systems could produce stimulienhanced/activated diagnostic signals and release their cargos when they are entered into the tumor, so as to realize the tumor-specific imaging, enhanced therapeutic efficacy and reduced side effects.9-11 In recent years, lanthanide ion-doped upconversion nanoparticles (UCNPs) and MnO2 nanomaterials have been received increasing attention for cancer diagnosis and therapy.12-16 Upconversion materials are able to convert the low-energy photons into high-energy photons through a multiple-photon process, which gives UCNPs

unique upconversion luminescence (UCL) properties with superior photostability, low autofluorescence background and high tissue penetration depth.17-19 And MnO2 nanostructures as ideal TME-responsive and biodegradable agents not only achieve controlled drug deliveries, but also generate O2 for enhanced therapies and release Mn2+ at the same time that are able to dramatically improve T1-magnetic resonance imaging (MRI) contrast.10, 20-24 Particularly very recently, MnO2 was verified for chemodynamic therapy (CDT) by generating highly reactive hydroxyl radical (•OH) via a Fenton-like reaction.25 Hence, constructing UCNPs and MnO2 hybrid theranostic nanoplatform (UC-Mn) to integrate multifunction in one system is highly desired.2628

Unfortunately, the rational design of such TMEresponsive UC-Mn is still a great challenge. For example, the most common strategy is that KMnO4 was reduced by 2-(Nmorpholino)ethanesulfonic acid (MES) to form amorphous MnO2 nanosheets in situ.29, 30 However, this method is unmanageable and the obtained

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Scheme 1. Schematic illustration of UCMnPt for TME-triggered UCL/MR bioimaging and self-enhanced tumor therapy.

UC-Mn assemblies show irregular morphologies due to the rapid growth rate of MnO2, which easily leads to selfgrowth rather than growth onto the smooth surface of UCNPs. Another strategy is also obstreperous that UCNPs are anchored onto the prefabricated MnO2 nanosheets or honeycomb MnO2.27, 31 As a result, much efforts have been paid to pursue the core-shell or core–satellite UC-Mn with controllable structures by introducing silica or CaF2 as an interlayer.32, 33 While the efficiency of UCL decreases seriously after silica coating and CaF2 as an interlayer is restricted to cubic phase UCNPs. Consequently, there is an urgent need to exploit an ideal architecture that allows the UC-Mn hybrid nanomaterials to achieve harmonious integration. Herein, we report a simple and versatile strategy for insitu growth of MnO2 on the surfaces of UCNPs directly as an ideal TME-responsive theranostic nanoplatform. MnO2-coated NaYF4:30%Yb,0.5%Tm@NaYF4 upconversion hybrid nanocomposite (UCMn) was prepared using a sacrificial template method. In brief, carbons as structure-directing agents were firstly coated by the hydrothermal method, which then reacted with KMnO4 at room temperature to generate in-situ MnO2 onto the surface of UCNPs. By further anchoring cisplatinum pro-drug and polyethylene glycol (PEG) onto

the surface of UCMn (UCMnPt), the obtained ideal MnO2-disguised theranostic agents with fully TMEtriggered performances have following advantages: a) because of GSH-responsive degradative character of MnO2, MnO2 is capable of dissolving and then Mn2+/cisplatinum pro-drug both release; b) the released Mn2+ can achieve MRI and CDT by generating •OH via a Fentonlike reaction with endogenous H2O2; c) the depletion of GSH by MnO2 significantly enhances CDT efficiency due to the potent scavenging effect of GSH on •OH; d) the released cis-platinum pro-drug not only kills tumor cells by chemotherapy, but also enhances CDT efficiency by generating H2O2; e) UCL is effectively and maximally recovered after MnO2 dissolution. (Scheme 1) We believe that the ideal design of the MnO2-disguised upconversion hybrid nanocomposite will achieve harmonious integration of each component. RESULTS AND DISCUSSION Construction and composition of UCMn-Pt-PEG. Here, oleic acid (OA)-capped βNaYF4:30%Yb,0.5%Tm@β-NaYF4 core-shell UCNPs (UCNPs-OA) were prepared at first. The obvious changes of morphologies and sizes verified that the core-shell


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Figure 1. The characterization of UCMnPt. The transmission electron microscopy (TEM) images of βNaYF4:30%Yb,0.5%Tm core (A), β-NaYF4:30%Yb,0.5%Tm@β-NaYF4 core-shell UCNPs (B), UCNPs@C (C) and UCMn (D). The high resolution TEM images of β-NaYF4:30%Yb,0.5%Tm core (E), β-NaYF4:30%Yb,0.5%Tm@β-NaYF4 core-shell UCNPs (F), UCNPs@C (G) and UCMn (H). (I-L) The element mappings of UCMn. (M) XRD patterns and the standard diffraction peaks for carbons (JCPDS No. 41-1487) and β-NaYF4 (JCPDS No. 16-0334). A new peak at 26.4 degree in UCNPs@C was observed, which could be well indexed to the graphite (JCPDS No. 41-1487). (N) X-ray photoelectron spectroscopy (XPS) spectra. Two new peaks at 653.05 eV (Mn (IV) 2p1/2) and 641.45 eV (Mn (IV) 2p3/2) in UCMn were observed. (O) UV-Vis-NIR absorption spectra. The characteristic KMnO4 peaks located at 311, 525, and 545 nm. The broad absorption from visible to NIR region of UCNPs@C was observed owing to the highly graphitic components in the carbon shell. A new broad absorbance band around 340 nm appeared in UCMn, which could be resulted from the surface plasmon band of colloidal MnO2. (P) Fourier transform infrared spectroscopy (FTIR). After removal of oleate ligands, the stretching vibrations of methylene (CH2) in OA at 2925 and 2854 cm−1 as well as stretching vibrations of carboxylic group of OA at 1557 and 1466 cm−1 were absent. After carbons coating, the stretching bands at 2930 and 2857 cm−1 as well as 1241 and 2257 cm−1 referring to C-H and C=C in UCNPs@C were observed. UCNPs were successfully prepared as shown in Figure 1A, Figure 1B, Figure S1, Figure S2 and Figure S3. The

structure of UCNPs was further confirmed by the X-ray diffraction (XRD) patterns (Figure 1M) and selected area electron diffraction (SAED) pattern (Figure S4). The synthesis of ligand-free UCNPs with hydrochloric acid (HCl) treatments was proved by Fourier transform infrared spectroscopy (FTIR, Figure 1P). Then the thin carbons about 2.9 nm were coated by a hydrothermal method as presented in Figure 1C and Figure 1G. As shown in Figure 1M, a new peak at 26.4 degree in carbons-coated UCNPs (UCNPs@C) appeared, which could be well indexed to the graphite (JCPDS No. 411487).34 In addition, the broad absorption from visible to NIR region (Figure 1O) and the stretching bands of C-H and C=C of UCNPs@C (Figure 1P) were observed owing to the highly graphitic components in the carbon shell. All these data showed that UCNPs@C was synthesis. At last, UCMn was obtained using a sacrificial template

method. In brief, UCNPs@C was added into KMnO4 aqueous solution and reacted with KMnO4 at room temperature to generate MnO2 in situ. The equation was presented as following: 4MnO4- + 3C + H2O → 4MnO2 + CO32- + 2HCO3-. As shown in Figure S5, the solution of wine red was clearly observed, indicating the reaction happened. As exhibited in Figure 1D, Figure 1H, Figure S6 and Figure S7, the MnO2 layer about 5.1 nm as a uniform shell could be seen clearly thanks to carbons as the structuredirecting agents. The element mappings (Figure 1I-1L and Figure S8), X-ray photoelectron spectroscopy (XPS) spectra (Figure 1N) and the absorption spectra (Figure 1O) further proved the successful preparation of UCMn.



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Figure 2. GSH-responsive properties of UCMn. (A) Schematic illustration of GSH-responsive UCL of UCMn. (B) The absorption spectra of UCMn and luminescence spectrum of UCNPs. (C) The proposed energy transfer mechanism under 980 nm NIR laser irradiation in UCMn. (D) The UCL spectra of the obtained nanoparticles (inset: the photographs of the aqueous solution of UCNPs, UCNPs@C, UCMn or UCMn + GSH, respectively). (E) The absorption spectra of UCMn treated by GSH with different concentrations (inset: the photographs of UCMn treated by GSH with different concentrations). (F) The UCL spectra of UCMn treated by GSH with different concentrations (inset: the UCL photographs of UCMn with different treatments). (G) Cisplain and Mn2+ release profiles from UCMnPt nanoparticles treated by GSH with different concentrations. (H) The TEM images of UCMn treated by GSH with different concentrations. a) 0 mM of GSH; b) 0.4 mM of GSH and c) 4 mM of GSH. From the XPS high-resolution scans of Mn2p peaks of UCMn in Figure S9, two new peaks at 653.05 eV (Mn (IV) 2p1/2) and 641.45 eV (Mn (IV) 2p3/2) were observed. In addition, a new broad absorbance band around 340 nm appeared in UCMn, which could be resulted from the surface plasmon band of colloidal MnO2.20 All these factors indicated that this sacrificial template method is

very effective for the synthesis of MnO2-directly coated UCNPs with controllable morphologies. Next, the surface modification of UCMn with polyethyleneimine (PEI) and


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Figure 3. The MB degradation by Mn2+ or UCMn-mediated Fenton-like reaction. (A) Schematic illustration of Mn2+ and UCMn-mediated Fenton-like reaction. (B) The absorption spectra of methylene blue (MB) treated with different solutions. (C) The MB degradation treated by H2O2 with different concentrations (0, 1, 2, 4, 8, 12 and 16 mM). (D) The MB degradation by Mn2+-mediated Fenton-like reaction in the presence of GSH with different concentrations (0, 1, 5 and 10 mM). (E) The MB degradation by UCMn-mediated Fenton-like reaction in the presence of GSH with different concentrations (0, 1, 5 and 10 mM). the following platinum (IV) pro-drugs (c,c,t[Pt(NH3)2Cl2(OOCCH2CH2COOH)2]) loading were performed, which was characterized by the thermogravimetric analysis (TGA, Figure S10), FTIR (Figure S11) and Zeta potentials (Figure S12). The Y, Mn and Pt contents of UCMnPt were determined by inductively coupled plasma mass spectrometry (ICP-MS), and the drug loading efficiency was about 3.6 %.

UCNPs (Figure 2B). As shown in Figure 2C, the proposed energy-transfer mechanism from βNaYF4:30%Yb,0.5%Tm@β-NaYF4 to MnO2 is presented.29 Hence, after MnO2 coating, the UCL quenched severely (Figure 2D). To verify whether UCL is restorable with GSH treatments, the absorption spectroscopy of UCMn with different concentrations of GSH was provided firstly. As exhibited in Figure 2E, the absorption values decreased over concentrations of GSH, indicating

GSH-responsive properties of UCMn. Next, the optical properties of UCMn were assessed. The absorption spectroscopy of UCMn showed an intense broad band centered at 340 nm, which overlaps with the emissions of



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Figure 4. In vitro experiments. (A) UCL images of HepG2 cells incubated with UCMn for 10 min, 1 h and 4 h at 37 °C. (B) The UCL spectrum of UCMn incubated by HepG2 cells with different treatments (L-BSO: GSH inhibitor; NAC: GSH promoter; UCMn incubated by HepG2 cells only; UCMn without treatments as a control). (C) The mass of platinum internalized in HepG2 cells after incubation with free DDP and UCMnPt for 10 min, 1 h and 4 h at 37 °C. (D) Relative cell viability of L929 cells incubated with Mn2+ or UCMn (Mn: 0, 6.25, 12.5, 25, 50 and 100 μM) for 24 h at 37 °C. (E) Intracellular •OH detections using DCFH-DA as a probe with different samples (no treatment as a control, Mn2+ or UCMn). (F) Viability of HepG2 cells after incubating with different concentrations of Mn2+, UCMn, DDP or UCMnPt for 24 h at 37 °C. (G) Intracellular GSH detections of HepG2 cells with various treatments (no treatment as a control, Mn2+, UCMn or UCMnPt). (H) Intracellular H2O2 detections of HepG2 cells (no treatment as a control, DDP, Mn2+, UCMn or UCMnPt) and L929 cells without treatment. (I) Poptosis induced by Mn2+, UCMn, DDP or UCMnPt in HepG2 cells. degradation of MnO2 (Figure S13). Then as expected, UCL gradually recovered with increasing amounts of GSH (Figure 2F and Figure S14). Remarkably, a 283-fold enhancement of upconversion emission at 363 nm was observed (Figure S15 and Figure S16). The magical luminescence recovery is attributed to the ideal architectonics of UCMn. TEM images further proved that MnO2 could decompose with the adding of GSH and the naked UCNPs were obtained (Figure 2H). For the GSHresponsive Mn2+ and cisplatin release test, GSH aqueous solution with different concentrations were added into

UCMnPt aqueous solution. After 10 minutes' standing, the supernatants were collected for the measure of cisplatin and Mn2+ contents by the inductively coupled plasmamass spectrometer (ICP-MS). As shown in Figure 2G, with increasing amounts of GSH, the cisplatin and Mn2+ showed similar release behaviors, indicating GSHresponsive Mn2+ and drug release. What’s more, the


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Figure 5. In vivo imaging and therapy. (A) MRI of a tumor-bearing mouse with intratumor injection of UCMnPt. The mouse was imaged on a 1.2 T MRI Scanner (Shanghai, China) at various time periods (0, 5, 10, 30 and 60 min). The tumor site is labeled with red arrows. (B) T1-weighted MRI of UCMn aqueous solution at different Mn concentrations with or without excessive GSH treatment, and T1 transverse relaxation rate as a function of Mn concentration. (C) UCL images of a tumor-bearing mouse with intratumor injection of UCMnPt. The mouse was imaged on an in vivo Maestro whole-body imaging system equipped with an external 980 nm laser as the excitation source at various time periods (10, 30 and 60 min). (D) The total signal intensity of UCL and MR for different time. The body weight (E), relative tumor volume (F), the tumor photographs (G) and representative photographs of nude mice (H) in different groups. release of Mn2+ further demonstrated that MnO2 is capable of dissolving by reacting with GSH. The MB degradation by Mn2+ or UCMn-mediated Fenton-like reaction. Recently, CDT utilizing iron-initiated Fenton chemistry to kill tumor cells by converting endogenous H2O2 into •OH, has been receiving considerable attention.35-39 And Mn2+-mediated Fenton-like reaction was also reported to achieve CDT by generating •OH like Fe2+.25 Here, to verify the •OH generation, methylene blue (MB), a dye that can be degraded by •OH,25 was chosen as an indicator of •OH generation as exhibited in Figure 3B. Unlike iron-initiated Fenton reaction, bicarbonate (HCO3-) is necessary for Mn2+-mediated Fenton-like reaction (Figure 3B). At the same time, it is easy to understand that the •OH generation by Mn2+-mediated Fenton-like reaction is H2O2 concentration-dependent (Figure 3C). With increasing amounts of H2O2, the absorbance of MB decreased, indicating more •OH generation. It is worth noting that

intracellular GSH as the scavenging agent of •OH greatly restricts CDT efficacy. As shown in Figure 3D, with the adding of GSH, the degradation of MB by Mn2+-mediated Fenton-like reaction was tremendously limited. On the contrary, MnO2 could effectively remit this scavenging effect owing to GSH depletion properties (Figure 3E and Figure S18). Therefore, compared with Mn2+, MnO2 could dramatically enhance generating of •OH in the presence of GSH (Figure 3A). In vitro UCL imaging and the cellular uptake. The cellular uptake in HepG2 cells were examined using UCL imaging and ICP-MS. As presented in Figure 4A, the typical UCL of Tm3+ was observed and increased over time, indicating the efficient uptake of UCMn. Afterward, in order to prove the restorable UCL of UCMn was GSH-responsive, HepG2 cells were treated with L-buthionine sulfoximine (LBSO, a GSH inhibitor) and N-acetyl-L-cysteine (NAC, a GSH promoter), respectively. As shown in Figure 4B, compared



with UCMn incubated by HepG2 cells alone, the declining UCL treated with L-BSO and enhanced UCL treated with NAC were observed, respectively. These data powerfully confirm that UCL of UCMn is restorable and GSHresponsive. To further evaluate the endocytosis of UCMnPt, HepG2 cells were incubated with UCMnPt or cisplatin [cisdiammine-dichloroplatinum (II), DDP] for 10 min, 1 h and 4 h, respectively. As shown in Figure 4C, compared with DDP, UCMnPt exhibited better time-dependent uptake thanks to the special endocytosis of nanoparticles. All these data suggested the excellent abilities for the cellular uptake. Intracellular •OH detection and toxicity assay. The generation of intracellular •OH, the most harmful reactive oxygen species (ROS) caused by the Mn2+-mediated Fentonlike reaction, was confirmed by 2,7dichlorodihydrofluorescein (DCFH-DA) as a •OH probe, which can be rapidly oxidized to 2’,7’-dichlorofluorescein (DCF) with green fluorescence by the generated •OH.40 As shown in Figure 4E and Figure S19, compared with the control group with no treatments, the medium green fluorescence signals in HepG2 cells incubated with Mn2+ were observed clearly, indicating the •OH generation. When the HepG2 cells were treated with UCMn, the stronger green fluorescence was presented, suggesting more •OH generation thanks to the GSH depletion properties of MnO2. Subsequently, the in vitro therapy effect with different treatments was evaluated by the standard methyl thiazolyl tetrazolium (MTT) assay. Briefly, HepG2 cells were incubated with various concentrations of Mn2+, UCMn, DDP or UCMnPt (0, 6.25, 12.5, 25, 50 and 100 μM of Mn; 0, 3.125, 6.25, 12.5, 25 and 50 μM of Pt) at 37 °C for 24 h. As expected, UCMn-exposed HepG2 cells presented significantly greater inhibition ratio relative to MnCl2, which is consistent with the results of •OH detection. It was also found that UCMnPt with CDT and chemotherapy simultaneously showed the highest anticancer efficacy compared with UCMn and DDP alone. (Figure 4F) Especially, a flow-cytometry apoptosis assay based on the typical AnnexinV-FITC and PI costaining protocol was performed to further verify the synergistic effect of CDT and chemotherapy. The percentages of cell apoptosis treated with Mn2+, UCMn, DDP and UCMnPt were 18.7%, 28.9%, 47.7% and 55.4%, respectively (Figure 4I), which was consistent with the above MTT results. Intracellular GSH and H2O2 detections. Next, to further explain the GSH depletion capabilities of MnO2 shell, in vitro GSH detection was carried out. Simply, HepG2 cells were treated with (a) untreated as a control; (b) Mn2+ (50 μM of Mn), (c) UCMn (50 μM of Mn) and (d) UCMnPt (50 μM of Mn) for 6 h, respectively. The amount of GSH was detected using GSH assay kits (Beijing Solarbio Science & Technology Co., Ltd, BC1175) according to the manufacturer’s instructions. As can be seen in Figure 4G and Figure S20, the group incubated with Mn2+ like the control group showed no obvious changes of GSH content. In contrast, the administration of UCMn and UCMnPt both could dramatically consume intracellular GSH, which was attributed to the reaction of GSH and MnO2 shell. It is well known that CDT is able to convert endogenous H2O2 into •OH, which causes the depletion of intracellular H2O2. Similarly, for in vitro H2O2 detection, HepG2 cells were treated with (a) untreated, (b) Mn2+ (50 μM of Mn), (c)

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UCMn (50 μM of Mn) and (d) UCMnPt (50 μM of Mn), respectively. The amount of H2O2 was detected using H2O2 assay kits (Beyotime Biotechnology, S0038) according to the manufacturer’s instructions. As exhibited in Figure 4H and Figure S21, compared with the control group, a remarkable decrease of H2O2 was observed in the groups treated with Mn2+, UCMn and UCMnPt attributed to Mn2+-mediated Fenton-like reaction. It has been widely reported that DDP has the ability to activate nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) and efficiently convert O2 to O2•−, which is further dismutated by superoxide dismutase (SOD) enzyme to form H2O2, causing an upsurge in cellular H2O2.41, 42 To prove this effect, the H2O2 content incubated with DDP was also detected and a slight increase of H2O2 content was observed. These results verify that cisplatin not only achieves chemotherapy, but also is responsible for activation-enhanced and H2O2-dependent CDT. And the amount of H2O2 in L929 (mouse fibroblast cell line) cells with no treatments was also detected. More interestingly, compared with the HepG2 cells, the amount of H2O2 in L929 cells was observably low. These data explain that the Mn2+ and UCMn possess low cytotoxicity and good biocompatibility on L929 cells (Figure 4D) while high cytotoxicity on HepG2 cells at the same conditions because of the different H2O2 content. It is very inspiring for pursuing the tumor-specific and enhanced therapy as well as reduced side effects on normal cells. In vivo UCL/MR imaging. It is widely accepted that in vivo bio-imaging is essential for the accurate cancer diagnosis and therapy.43, 44 Particularly, the TME-responsive and specific imaging is highly desired. Here, the release Mn2+ from MnO2 shell by reacting with GSH are able to dramatically improve T1-MRI contrast. For the GSH-responsive MRI test, 0.5 mL of GSH aqueous solution (10 mM) was added into 0.5 mL of UCMn aqueous solution with different concentrations (0, 0.063, 0.125, 0.25, 0.5 and 1 mM of Mn) (Figure S22). After 10 minutes' standing, the obtained solution was directly for the tests. The UCMn aqueous solutions with different concentrations treated without GSH were as controls. As shown in Figure 5B, T1 value with GSH treatment was up to 8.65 mM-1S-1 while without GSH treatment was only 0.63 mM-1S-1, suggesting GSH-mediated activatable MR imaging. For in vivo MRI, H22 tumor-bearing Balb/c mice with intratumor injection of UCMnPt were imaged on a 1.2 T MRI Scanner (Shanghai, China) at various time periods (0, 5, 10, 30 and 60 min). The relative intensity of T1 weighted imaging was determined from black and white images using densitometry scans to obtain quantitative data (Image J). Figure 5A and Figure 5D showed that about triple increases of relative intensity was observed from 0 min of injection to 60 min, indicating GSH-mediated activatable MR imaging in vivo. Similarly, for in vivo UCL imaging, H22 tumor-bearing Balb/c mice with intratumor injection of UCMnPt, were imaged on an in vivo Maestro whole-body imaging system equipped with an external 980 nm laser as the excitation source at various time periods (10, 30 and 60 min). The total signal intensities of UCL were also recorded. As presented in Figure 5C and Figure 5D, over 100-fold enhancement of UCL was appeared from 10 min of injection to 60 min. The inconceivable luminescence recovery is attributed to the precise synthesis and ideal architectonics of UCMn. All these results demonstrated that our obtained UCMnPt as an ideal


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imaging agent could be appropriate for activatable MR and restorable UCL bioimaging. Anti-tumor efficacy. The satisfactory TME-responsive properties encouraged us to further study the antitumor performance of UCMnPt in vivo. HepG2 tumor-bearing nude mice with an average tumor volume of 80 mm3 were randomized into five groups (n=5, each group) and were treated by tail intravenous injection with (1) normal saline; (2) Mn2+ (0.1 mL, 4 mM); (3) UCMn (0.1 mL, 4 mM); (4) DDP (0.1 mL, 2 mM); (5) UCMnPt (0.1 mL, 4 mM of Mn and 2 mM of Pt), respectively. The tumors of mice treated with normal saline and Mn2+ were found to grow very quickly, suggesting that they both had poor antitumor efficacy (Figure 5F, 5G and 5H). Both UCMn and DDP could suppress the tumor growth moderately due to the single CDT and chemotherapy. In contrast, the administration of UCMnPt achieved effective tumor growth inhibition thanks to the combined effect of CDT and chemotherapy, indicating remarkable improved therapeutic efficacy among all groups without causing any obvious body weight change (Figure 5E). To evaluate the therapeutic safety after various treatments, hematoxylin and eosin (H&E) staining of the major organs (heart, liver, spleen, lung, and kidney) was carried out. No appreciable organ damage was caused in therapeutic groups (Figure S23), suggesting the relatively high therapeutic biosafety.

CONCLUSIONS In conclusion, a simple and versatile strategy for in-situ growth of MnO2 on the surfaces of NaYF4:30%Yb,0.5%Tm@NaYF4 UCNPs was reported using a sacrificial template method to construct an ideal MnO2disguised and TME-triggered architecture. Such architecture not only achieves activatable MR and restorable UCL bioimaging, but also significantly improves the efficiency of CDT by GSH depletion- and cisplatin activation-enhanced •OH generation. Additionally, the synergetic effect of CDT and chemotherapy presents excellent therapeutic effect in vivo as compared to either CDT or chemotherapy alone. We believe that the ideal design of the MnO2-disguised upconversion hybrid nanocomposite will provide more revelations on the future research about nanoscale theranostic systems.

ASSOCIATED CONTENT Supporting Information. Experimental section, TEM images, size distribution, SAED pattern, element mappings, XPS high-resolution scans, TGA, FTIR, Zeta potential, UCL spectra, intracellular •OH, GSH and H2O2 detections and H&E-stained images.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] (P. Ma). * E-mail: [email protected] (J. Lin).

Author Contributions All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This project is financially supported by the National Natural Science Foundation of China (Grant Nos. NSFC 51720105015, 51672269, 51772124, 51872282 and 51572257), Science and Technology Development Planning Project of Jilin Province (Grant Nos. 20170101188JC and 20180520163JH), Youth Innovation Promotion Association of CAS (Grant No. 2017273), Overseas, Hong Kong & Macao Scholars Collaborated Researching Fund (Grant No. 21728101), CASCroucher Funding Scheme for Joint Laboratories (Grant No. CAS18204) and the Distinguished Scientist Fellowship Program of King Saud University. All animals in this study were handled according to a protocol approved by the Institutional Animal Care and Use Committee of Jilin University.

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MnO2-Disguised Upconversion Hybrid Nanocomposite: An Ideal

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