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Intelligent MnO 2 Nanosheets Anchored with Upconversion Nanoprobes for Concurrent pH-/H 2 O 2 -Responsive UCL Imaging and Oxygen-Elevated Synergetic Therapy

Wenpei Fan , Wenbo Bu , * Bo Shen , Qianjun He , Zhaowen Cui , Yanyan Liu , Xiangpeng Zheng , Kuaile Zhao , and Jianlin Shi *

Dr. W. Fan, Prof. W. Bu, Dr. Q. He, Dr. Z. Cui, Dr. Y. Liu, Prof. J. Shi State Key Laboratory of High Performance Ceramics and Superfi ne Microstructures Shanghai Institute of Ceramics Chinese Academy of Sciences Shanghai 200050 , P. R. China E-mail: wbbu@mail.sic.ac.cn; jlshi@mail.sic.ac.cn Prof. W. Bu Jiangsu Collaborative Innovation Center for Advanced Inorganic Functional Composites Nanjing Tech University Nanjing 210009 , P. R. China Dr. B. Shen Institute of Radiation Medicine Fudan University Shanghai 200032 , P. R. China Dr. X. Zheng Department of Radiation Oncology Shanghai Huadong Hospital Fudan University Shanghai 200040 , P. R. China Dr. K. Zhao Department of Radiology Shanghai Cancer Hospital Fudan University Shanghai 200032 , P. R. China

DOI: 10.1002/adma.201405141

temperature, redox, etc.) for on-demand imaging/therapy. [ 6 ] As we know, solid tumors demonstrate a unique metabolic profi le different from normal tissues. [ 7 ] On one hand, due to the upregulated glycolytic metabolism during tumorigenesis, solid tumors generate massive lactic acid, [ 8 ] which results in an acidic tumor microenvironment with a remarkable decrease in pH value. On the other hand, compared with normal cells, malignant cancerous cells produce excessive amounts of H 2 O 2 , [ 9 ] which causes a signifi cantly increased level of H 2 O 2 in the tumor microenvironment. So there exists an acidic and H 2 O 2 -rich microenvironment in solid tumors. Thanks to the pH-/redox-responsive properties, exfoliated manganese oxide (MnO 2 ) nanosheets can be reduced into Mn 2+ by acidic H 2 O 2 in solid tumors, which also generates a great deal of oxygen for signifi cantly improving the oxygen-dependent PDT/RT effects on hypoxic tumors. [ 10 ] Importantly, MnO 2 exhibits good bio-compatibility because manganese is a necessary nontoxic ele-ment involved in physiological metabolism. [ 11 ] Therefore, the exploration of MnO 2 -based 2D theranostic nanomaterials may give rise to the next generation of intelligent stimuli-responsive nanomedicine for future clinical applications.

Besides oxygen generation, by engineering MnO 2 nanosheets with imaging contrast agents, [ 12 ] stimuli (pH/H 2 O 2 )-responsive imaging can be also realized for enhanced diagnostic accuracy. As a typical noninvasive imaging probe, [ 13 ] upconversion nano-particles (UCNPs) demonstrate incomparable advantages (e.g., weak autofl uorescence, superior photostability, nonblinking, etc.) [ 14 ] and provide high-resolution upconversion luminescent (UCL) imaging guidance for the precisely positioned PDT/RT. [ 15 ] Moreover, the design of theranostic upconversion nano-probes (UCNP photosensitizers) can signifi cantly improve the PDT effects on deep-seated tumors due to the large penetration depth of NIR light, [ 16 ] and even elicit synergetic/superadditive PDT/RT effects upon coirradiation of NIR light/X-ray. Conse-quently, the integration of MnO 2 nanosheets and upconver-sion nanoprobes may be highly attractive to effi cient cancer theranostics.

In this study, we develop intelligent 2D theranostic nano-materials based on the MnO 2 nanosheets anchored with upconversion nanoprobes (UCSMs) for concurrent pH-/H 2 O 2 -responsive UCL imaging and oxygen-elevated synergetic radio/photodynamic therapy. On one hand, the quenched upconver-sion luminescence of UCSMs can be recovered/enhanced for diagnosis/monitoring through the decomposition of MnO 2 into Mn 2+ by acidic H 2 O 2 in solid tumors. On the other hand, the MnO 2 –H 2 O 2 redox reaction can generate massive oxygen in

As two representative noninvasive treatments, photodynamic therapy (PDT) and radiotherapy (RT) can force light/ionizing radiation precisely on targeted tumors to effi ciently induce cell death by generating a great deal of reactive oxygen spe-cies (ROS). [ 1 ] However, the oxygen-dependent-featured PDT/RT usually produces limited therapeutic effects on hypoxic solid tumors mainly because of the inadequate oxygen supply in tumor vascular systems. [ 2 ] Tumor oxygenation that aims at greatly increasing the oxygen concentrations in hypoxic regions should be an effective strategy to overcome hypoxia and sub-stantially enhance the PDT/RT effi cacy, [ 3 ] which highlights the urgency of developing intelligent theranostic nanomaterials that can “make” oxygen directly in solid tumors.

The emerging 2D nanomaterials (e.g., graphene, MoS 2 , WS 2 , etc.) [ 4 ] have been widely explored/used in a variety of fi elds due to their unique physical/chemical properties. [ 5 ] Of par-ticular interest with 2D nanomaterials is their great promise to develop into the intelligent theranostic nanosystems that can respond sensitively to internal/external stimuli (e.g., pH,

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situ for signifi cantly improving the synergetic PDT/RT effects on solid tumors upon NIR/X-ray irradiation. To the best of our knowledge, this is the fi rst report of constructing intelligent 2D theranostic nanomaterials for simultaneous stimuli-responsive imaging and oxygen-elevated therapy, which may be extended to the effi cient treatment of various solid tumors.

A facile nanochemical protocol was established for synthe-sizing the intelligent 2D theranostic nanomaterials ( Figure 1 a). First, monodisperse UCNPs (NaYF 4 :Yb/Er/Tm) with uniform spherical morphology (Figure S1a, Supporting Information) were prepared by a typical thermal decomposition method. [ 17 ] Then, a uniform photosensitizer (SPCD)-incorporated dense silica shell was coated on the surface of UCNPs (Figures S1b, S2, Supporting Information), which gave rise to the thera-nostic upconversion nanoprobes (denoted as UCSs). During the dense silica coating process, TEOS acted as the reaction precursor and silica source, which would hydrolyze in the pres-ence of ammonia and condense to form the silica network. Moreover, SPCD molecules were simultaneously encapsulated into the silica network through the hydrolysis of TEOS and con-densation of silanol groups between SPCD and the silica shell, thus preventing the leakage of SPCD molecules (Figure S3, Supporting Information). [ 18 ] Herein, SPCD is chosen as the anti-tumor photosensitizer due to the optimal overlap between its absorption spectrum and the emission spectrum of UCNPs (Figure S4a, Supporting Information), which will facilitate the red luminescence energy transfer from UCNPs to SPCD (Figure S4b, Supporting Information) for the generation of sin-glet oxygen ( 1 O 2 ). Finally, amorphous MnO 2 nanosheets (as con-fi rmed by the diffused rings in the SAED pattern of Figure S1d in the Supporting Information) were wrapped out onto the surface of all UCSs (Figure S1c, Supporting Information) by the redox reaction between KMnO 4 and 2-( N morpholino) ethanesulfonic acid (MES). The XPS spectrum (Figure S5, Supporting Information) shows that the Mn 2p 3/2 and Mn

2p 1/2 peaks are centered at about 641.9 and 653.6 eV, respec-tively, with a spin-energy separation of 11.7 eV, which con-fi rms the Mn 4+ manganese species and formation of Mn(IV)O 2 nanosheets. [ 19 ] All the TEM/STEM images (Figures 1 b,c, S1c, Supporting Information), EDS spectrum (Figure S1e, Sup-porting Information), FTIR and UV–vis spectra (Figure S6, Supporting Information) demonstrate the successful formation of the UCSs-anchored MnO 2 nanosheet structure (denoted as UCSMs). During the KMnO 4 –MES reaction, the Mn atoms in the MnO 6 octahedron would bond to the O atoms in the silica shell of UCSs nanoparticles via intermolecular hydrogen or coordination bonding, [ 20a,b ] so the UCSs nanoparticles acted as anchor sites for the growth of MnO 2 nanosheets. Meanwhile, the electrostatic repulsion among the negatively charged silica shells would endow UCSs nanoparticles with high stability of dispersion in aqueous solutions and a uniform distribution of anchor sites [ 20c ] to bond onto MnO 2 nanosheets.

Due to the large overlap between the emission spectrum of UCSs and the absorption spectrum of MnO 2 (Figure S7b, Sup-porting Information), the upconversion luminescence (UCL) should be signifi cantly absorbed/quenched by MnO 2 . With the increasing amount of KMnO 4 to react with MES, more and more MnO 2 nanosheets are produced (as confi rmed by the UCSMs’ color change from blue to black in Figure S7a in the Supporting Information), thus leading to the gradually decreased UCL intensity (Figure S7c, Supporting Information). According to the MnO 2 –H 2 O 2 reaction equation (Figure S8a, Supporting Information), MnO 2 only serves as a catalyst to pro-mote the disproportionation reaction of H 2 O 2 for producing O 2 and H 2 O in neutral solutions of H 2 O 2 (pH = 7.4). [ 21 ] However, in acid solutions of H 2 O 2 (pH = 5.5), MnO 2 is reduced into Mn 2+ while H 2 O 2 is oxidized to oxygen (Figure S8b, Supporting Infor-mation). [ 22 ] Furthermore, with the increasing concentrations of H 2 O 2 (0–50 × 10 −3 M ) added to react with MnO 2 of UCSMs, more and more gas bubbles are clearly observed (Figure S9a,

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Figure 1. a) Schematic illumination of the construction of intelligent MnO 2 nanosheets anchored with upconversion nanoprobes (UCSMs). b) TEM images of UCSMs. c) STEM image and the corresponding element mapping of UCSMs: c 1 ) Si; c 2 ) O; c 3 ) Mn; c 4 ) F.

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Supporting Information), thus confi rming the generation of much more dissolved O 2 (as evidenced by the quantitative dis-solved O 2 concentrations in Figure S9b in the Supporting Infor-mation). Moreover, the MnO 2 –H 2 O 2 redox reaction also leads to the increased UCL intensity of UCSMs upon NIR excitation ( Figure 2 a). In neutral solutions of H 2 O 2 , as MnO 2 is only a cat-alyst but not reduced itself (Figures 2 b, S8a, Supporting Infor-mation), the UCL signal is hardly recovered (Figure 2 c) owing to the quenching by the black MnO 2 nanosheets. However, in acid solutions of H 2 O 2 , MnO 2 nanosheets can be quickly reduced and decomposed into colorless Mn 2+ (Figures 2 b, S8b, Sup-porting Information), thus resulting in the remarkable enhance-ment in UCL emission intensity (Figure 2 c) and the complete recovery of originally quenched UCL signal (Figure S10, Supporting Information). Consequently, our designed UCSMs may be developed as an effective biosensor to quantitatively detect H 2 O 2 by examining the UCL emission intensity as well as the generated oxygen concentration.

In addition, the 1 O 2 generation of UCSMs upon NIR irradia-tion was tested by using 1,3-diphenylisobenzofuran (DPBF) as an indicator, which could react with 1 O 2 and cause a decrease in the absorption intensity of DPBF band centered at 400 nm. As seen from Figure 2 d, little 1 O 2 is produced (Figure S11a, Sup-porting Information) due to the UCL quenching by MnO 2 . After addition of H 2 O 2 , the signifi cantly decreased DPBF absorp-tion intensity demonstrates much enhanced 1 O 2 production (Figure S11b, Supporting Information) mainly owing to the O 2

generation by the MnO 2 -catalyzed disproportionation reaction of H 2 O 2 . More importantly, when the pH decreases from 7.4 to 5.5, the quenched red luminescence of UCNP core could be recovered and transferred to SPCD due to the reduction of MnO 2 by acidic H 2 O 2 , which leads to the further enhancement of 1 O 2 generation (Figure S11c, Supporting Information) as well as the greatly increased PDT effi cacy.

It has been reported that the hypoxic cells in solid tumors produce elevated concentrations of endogenous acidic H 2 O 2 , [ 23 ] which can react with MnO 2 of UCSMs to generate O 2 in situ for enhancing the synergetic PDT/RT effi cacy upon NIR/X-ray irradiation by generating massive ROS ( Figure 3 a). Herein, hypoxic murine breast cancer (denoted as hc-4T 1 ) cells were cultured in a hypoxic incubator under mixed atmos-phere of 2% O 2 , 5% CO 2 , and 93% N 2 . As shown in Figure 3 c, hc-4T 1 cells still demonstrate over 90% viability after incu-bation with up to 200 µg mL −1 UCSMs for 24 h, which con-fi rms the relative low cytotoxicity of UCSMs. The gradually increased UCL intensity of UCSMs after reaction with endog-enous acidic H 2 O 2 can be used to observe the cellular uptake of UCSMs. As shown in Figures 3 b and S12 (Supporting Information), after incubation with hc-4T 1 cells for 1 h, UCSMs show negligible UCL signal (Figures 3 b 1 , S12a,b, Supporting Information) probably due to the little cellular uptake and luminescence quenching by MnO 2 . With the increasing incubation duration up to 8 h (Figures 3 b 2 , S12c,d, Supporting Information) and 20 h (Figures 3 b 3 , S12e,f,

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Figure 2. a) Schematic illustration of the decomposition of MnO 2 nanosheets arising from the redox reaction between UCSMs and acidic H 2 O 2 , which leads to the enhanced UCL imaging for diagnosis/monitoring as well as the massive oxygen generation for improving the synergetic PDT/RT effects. Comparison of the b) UV–vis absorption intensity, c) UCL emission intensity, and d) 1 O 2 production among UCSMs, UCSMs after reaction with H 2 O 2 in neutral solutions (pH = 7.4), and UCSMs after reaction with H 2 O 2 in acid solutions (pH = 5.5).

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Supporting Information), much stronger yellow luminescence can be detected surrounding the nucleus, which means that more and more UCSMs have been uptaken into the cytoplasm of hc-4T 1 cells and their UCL intensity is signifi cantly dequenched/enhanced owing to the decomposition of MnO 2 nanosheets.

Meanwhile, the redox reaction between UCSMs and endoge-nous acidic H 2 O 2 also increases the dissolved O 2 concentration, which can enhance the PDT and RT effi cacy in killing hc-4T 1 cells. As shown in Figure 3 e, both X-ray and NIR irradiation show negligible infl uence on the viability of hc-4T 1 cells, which indicates that hypoxic tumor cells are insensitive to NIR and X-ray irradiation. After addition of UCSMs, the PDT (UCSMs + NIR) and RT (UCSMs + RT) effects are both signifi cantly improved owing to the O 2 generation (Figure 3 d), thus causing more hc-4T 1 cells’ death. Furthermore, upon coirradiation of NIR and X-ray, the treatment of UCSMs + RT + NIR can result in the lowest cell viability as well as the most signifi cant cells’ apoptosis/necrosis (Figure S13, Supporting Information), which should be attributed to the synergetic PDT/RT effects that achieve much higher anticancer effi cacy.

Encouraged by the above in vitro imaging/therapy results, the in vivo oxygenation experiment was also performed. Herein, photoacoustic (PA) imaging was used to evaluate the vascular saturated O 2 (sO 2 ) within 4T 1 solid tumors by measuring oxygen-ated and deoxygenated hemoglobin at different wavelengths of 850 and 750 nm, respectively. As shown in Figure 4 a, compared with the saline as control, the injection of UCSMs can not only signifi cantly increase the signal intensity of oxygenated/deoxy-genated hemoglobin (Figure S14, Supporting Information),

but also enhance tumor sO 2 by about 7% (Figure 4 b). Mean-while, the tumor injected with UCSMs also demonstrates the remarkable down-expression of HIF-1α (Figure 4 c 2 ), thus indi-cating the decreased hypoxia and increased oxygenation. Mean-while, the recovered/enhanced UCL signal of UCSMs can be used for luminescent imaging of solid tumors. As shown in Figure S15 (Supporting Information), original UCSMs emit negligible luminescence, so the tumor shows little UCL signal in 1 h postinjection (Figure 4 d 7 ) owing to the nonreduction of MnO 2 . However, in 12 h of the MnO 2 –H 2 O 2 redox reaction in vivo, most black UCSMs (Figure 4 d 1 ) turn into blue UCSs (Figure 4 d 2 ), which can emit strong UCL signal for high-reso-lution luminescent imaging of solid tumor (Figure 4 d 8 ). These results indicate that UCSMs can achieve concurrent pH-/H 2 O 2 -responsive UCL imaging and oxygen generation in vivo.

Then, we conducted the in vivo therapy experiments on 4T 1 solid tumor-bearing mice. Animal procedures were in agree-ment with the guidelines of the Regional Ethics Committee for Animal Experiments. As shown in Figure 4 e, both NIR irradia-tion and UCSMs show little inhibitory effects on the growth of 4T 1 solid tumors, which demonstrates the negligible thermal damage of NIR and little toxicity of UCSMs. Only high-energy X-ray irradiation also demonstrates limited inhibitory effects due to the resistance of hypoxic tumor regions to RT. Interest-ingly, both the treatments of UCSMs + NIR and UCSMs + RT demonstrate signifi cant tumor growth inhibition attributable to the tumor oxygenation as well as enhanced UCL intensity by the reduction of black UCSMs into blue UCSs (Figure S16, Supporting Information), which can cause large-scale apoptosis/

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Figure 3. a) Schematic illustration of the in vitro pH-/H 2 O 2 -responsive UCL imaging and oxygen-elevated PDT/RT of hc-4T 1 cells by using UCSMs. b) Confocal laser scanning microscopy (CLSM) images of hc-4T 1 cells after incubation with UCSMs for b 1 ) 1 h, b 2 ) 8 h, and b 3 ) 20 h, respectively. The nucleus is stained with DAPI to emit blue luminescence while the UCNP core of UCSMs could emit yellow luminescence (merging of green and red luminescence) upon NIR excitation. c) In vitro toxicity evaluation of hc-4T 1 cells after incubation with varied concentrations of UCSMs for 24 h. d) In vitro evaluation of O 2 generation by incubating UCSMs with hc-4T 1 cells. e) In vitro evaluation of oxygen-elevated treatment of hc-4T 1 cells by UCSMs upon NIR/X-ray irradiation.

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necrosis of tumor cells via the enhanced PDT and RT (as shown by the H & E and TUNEL images in Figure 4 g 1–7 ,h 1–7 ). Furthermore, by the combination of PDT/RT upon NIR/X-ray irra-diation, UCSMs + RT + NIR produces greatly promoted synergetic PDT/RT effects on 4T 1 solid tumors, thus leading to the much higher anti-tumor effi cacy by remarkably regressing the solid tumor volumes (Figure 4 i), as clearly seen from the digital photos (Figure S17, Supporting Information). Meanwhile, UCSMs + RT + NIR also results in the most remarkable down-regulation of HIF-1α and VEGF (Figure S18, Supporting Information) among these treatments, which confi rms the remarkable advantages of the oxygen-elevated synergetic PDT/RT in inhibiting the tumor angiogenesis and suppressing hypoxia. Finally, all the mice dem-onstrate negligible weight fl uctuations (Figure 4 f), thus confi rming little adverse effects of these treatments on the mice’s health.

Besides, the corresponding in vivo UCL imaging-guided therapy experiment by intravenous injection of UCSMs was also performed. First, negligible abnormality in the major organs of mice is observed (Figure S19, Supporting Information), which confi rms the good biocompatibility of UCSMs in vivo. Second, strong UCL signal is observed in the 4T 1 solid tumor (Figure S20, Supporting Information), which indicates that UCSMs can suc-cessfully accumulate in tumors. Third, the mice treated with UCSMs + RT +NIR not only demonstrate signifi cant tumor growth delay ( Figure 5 a–c, Figure S21, Supporting Information) and remarkable tumor cells’ apoptosis/necrosis (Figure 5 d 1,2 ) via oxygen-elevated synergetic PDT/RT, but also lead to the decreased hypoxia and circumvention of tumor angiogen-esis/metastasis via the down-regulation of HIF-1α and VEGF (Figure 5 d 3,4 ). However, due to the limited doses of UCSMs delivered into solid tumors by intravenous injection, its treat-ment effi ciency is still much lower than that by intratumoral injection. [ 24 ] Therefore, during the next stage, more effective strategies (e.g., proper surface modifi cation, attachment of targeting ligands, etc.) would be adopted to enhance the tumor accumulation for further improving the synergetic PDT/RT effects via the systemic administration of UCSMs.

In summary, we introduce nanotechnology into the design of intelligent MnO 2 nanosheets anchored with upconversion nanoprobes (UCSMs) for the concurrent stimuli-responsive imaging/therapy of solid tumors by overcoming hypoxia. On the basis of the redox activity of MnO 2 toward acidic H 2 O 2 , oxygen generation and upconversion luminescent imaging can be simultaneously achieved, which allows the simulta-neous diagnosis and positioned treatment of solid tumors via the oxygen-elevated synergetic radio/photodynamic therapy under the guidance of high-resolution UCL imaging. The cor-responding in vitro and in vivo results undoubtedly confi rm the unique advantages of UCSMs in inducing hypoxic cell death and inhibiting solid tumor growth. Furthermore, UCSMs can be hopefully developed as biosensors for H 2 O 2 as well as extended to treat various kinds of solid tumors, which may fi nally realize our “one drug fi ts all” dream.

Supporting Information Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements This work was fi nancially supported by the National Natural Science Foundation of China (Grant Nos. 51372260, 51132009, 21172043, 51102259, 81472794, 51402338) and the Shanghai Yangfan Program (Grant No. 14YF1406400). The authors thank Prof. Fuyou Li, Dr. Wei Yuan from Fudan University for the help in the UCL imaging in vivo. The authors thank Prof. Gang Liu, Dr. Xiaoyong Wang, Ms. Qiaoli Peng from Xiamen University for the help in the PA imaging in vivo. They also thank Prof. Fangfang Xu from Shanghai Institute of Ceramics, Chinese Academy of Sciences for the help in the SAED and EDS characterizations.

Received: November 10, 2014 Revised: May 8, 2015

Published online: June 8, 2015

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