Break-up of Two-Dimensional MnO 2 Nanosheets Promotes Ultrasensitive pH-Triggered Theranostics of Cancer

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Break-up of Two-Dimensional MnO 2 Nanosheets Promotes Ultrasensitive pH-Triggered Theranostics of Cancer

Yu Chen , Delai Ye , Meiying Wu , Hangrong Chen , Linlin Zhang , Jianlin Shi ,* and Lianzhou Wang *

simultaneously reduce the side-effects of anticancer agents to normal tissues and enhance their therapeutic effi ciency. [ 10 ] The introduction of contrast agents (CAs) for stimuli-enhanced diagnostic imaging [e.g., magnetic resonance imaging (MRI), ultrasound imaging, fl uorescence imaging, computed tomog-raphy imaging] can improve the imaging accuracy and sensi-tivity for the early diagnosis of cancer. [ 11 ] However, such nano-platforms with concurrent stimuli-responsive drug-releasing and diagnostic-imaging performances for cancer theranostics are still unavailable to our knowledge.

Herein, we report on the construction of an intelligent theranostic platform based on highly dispersed 2D MnO 2 nanosheets for concurrent ultrasensitive pH-responsive MRI and drug release/delivery. Manganese-based oxides have been demonstrated as alternative CAs for T 1 -weighted MRI (T 1 -MRI) to potentially substitute for clinical gadolinium (Gd)-based CAs due to their improved biocompatibility over cytotoxic Gd-based agents. [ 12 ] The US Food and Drug Administration (FDA) has warned that gadolinium is associated with nephrogenic sys-temic fi brosis with impaired kidney function. Manganese is a necessary element for physiological metabolism and in vivo biological systems can effi ciently control its homeostasis. [ 13 ] However, the imaging performances of Mn-based nanoparticles (typically r 1 < 0.5 mM −1 s −1 ) are considerably lower than those of commercial Gd-based CAs ( r 1 ≈ 3.4 mM −1 s −1 ). [ 12c–e ]

Tumorigenesis can generate a more acidic microenviron-ment in tumor tissues than in normal tissues due to the up-regulated glycolytic metabolism during tumorigenesis, which can generate lactic acid. [ 14 ] Here, elaborately constructed MnO 2 nanosheets with ultrasensitivity to the mild acidity to break-up, disintegrate, and release Mn( II ) ions; thus, the accessibility of paramagnetic centers [Mn( II )] to surrounding water mol-ecules can be substantially enhanced, which results in highly improved T 1 -MRI performances for tumor imaging and detec-tion ( Figure 1 ). [ 12b ] The extremely large surface area to mass ratio of MnO 2 nanosheets provides abundant anchoring points for anticancer agents to load. The break-up of drug-loaded MnO 2 nanosheets in a mild acidic environment can release the loaded cargos for internally pH-triggered drug release. The released Mn( II ) ions from the continuous break-up and disintegration of MnO 2 nanosheets can be easily metabolized by the kidneys, which allows circumvention of the intractable degradation issue of traditional inorganic nanosystems, such as well-known silica, gold, and carbon-based nanomaterials. [ 15 ] For practical clinical application, pH-responsive MRI will be critically important druing the diagnostic-imaging process to identify the cancer's location and evolution stage. In the thera-peutic process, the pH-responsive drug release, by breaking-up MnO 2 nanosheets, will be of signifi cant advantage. The toxic

Two-dimensional (2D) nanomaterials have attracted much recent attention due to their distinct structure–property rela-tionships in optoelectronics, catalysis, separation, and energy-related applications, such as solar cells, supercapacitors, and lithium-ion batteries. [ 1 ] Especially, some families of 2D nano-materials have found biomedical applications in biological sensing, [ 2 ] drug delivery, [ 3 ] hyperthermia, [ 4 ] molecular imaging, [ 5 ] and tissue engineering. [ 6 ] The most explored graphene oxide (GO) and reduced GO (rGO) can transform absorbed near-infrared (NIR) laser into heat to initiate the hyperthermia of cancer cells. [ 7 ] The exceptionally large surface area to mass ratio of GO/rGO endows them with a high cargo-loading capacity for effi cient drug delivery. [ 8 ] Transition metal dichalcogenides, such as MoS 2 and WS 2 nanosheets, [ 4,9 ] were also demonstrated as effi cient hyperthermia agents for cancer eradication. Most of these reported 2D nanomaterials were explored as therapeutic agents for cancer therapy. The development of new 2D nano-material systems with concurrent diagnostic and therapeutic functions (designated as theranostic agents) can generate mul-tifunctional 2D nanosystems with unique fi nding, fi ghting, and following functions: the tumor tissues can be found by nanoparticle-based targeting/imaging, killed by loaded cargos or transformed energy, and followed by contrast-enhanced imaging, which is of great signifi cance for successful cancer treatment. [ 9 ]

It is highly desirable to develop intelligent multifunctional theranostic nanosystems that are responsive to internal or external triggers. The on-demand drug release triggered by intrinsic physiological-microenvironment changes (pH, redox, enzyme, heat, etc.) and/or external artifi cially introduced trig-gers (light, magnetic/electronic fi eld, ultrasound, etc.) can

Dr. Y. Chen, [+] Dr. D. Ye, [+] and Prof. Dr. L. Wang Nanomaterials Center, School of Chemical Engineering and Australian Institute for Bioengineering and Nanotechnology (AIBN) University of Queensland Queensland 4072 , Australia E-mail: [email protected] Dr. Y. Chen, Dr. M. Wu, Prof. Dr. H. Chen, L. Zhang, Prof. Dr. J. Shi State Key Laboratory of High Performance Ceramics and Superfi ne Microstructure Shanghai Institute of Ceramics Chinese Academy of Sciences Shanghai 200050 , P. R. China E-mail: [email protected][+]These authors contributed equally to this work.

DOI: 10.1002/adma.201402572

Adv. Mater. 2014, DOI: 10.1002/adma.201402572

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anticancer drugs can be released in the acidic microenviron-ment of tumor tissue, which simultaneously reduces the toxic side-effects to normal tissue and enhances their therapeutic performance.

The synthesis of exfoliated MnO 2 nanosheets was con-ducted according to a reported process with some modifi ca-tions (Figure 1 A). [ 16 ] Typically, layered Na–MnO 2 materials were initially prepared by co-precipitation between NaOH and Mn(NO 3 ) 2 in H 2 O 2 solution, followed by hydrothermal treat-ment in NaOH solution (2 M) at 150 °C for 24 h. A further ion-exchange procedure was applied to exchange Na + in Na–MnO 2 for H + to obtain H-MnO 2 by stirring in daily refreshed hydro-chloric acid solution (0.1 M) for a week at room temperature. The collected H-MnO 2 was exfoliated by stirring vigorously in tetrabutylammonium hydroxide [(C 4 H 9 ) 4 NOH, 0.24 M] solu-tion for 10 days at room temperature. After centrifugation at

10000 rpm, the suspension was collected to obtain exfoliated MnO 2 nanosheets highly dispersed in aqueous solution. For further PEGylation, the obtained MnO 2 nanosheets were redis-persed into amino-polyethylene glycol (PEG 5000 -NH 2 ) aqueous solution (1 mg mL –1 ) by ultrasound treatment for 4 h, which was repeated three times to guarantee effi cient PEGylation.

Exfoliated MnO 2 nanosheets exhibit the typical 2D sheet-like morphology ( Figure 2 ) with average lateral size of around 200 nm. Some variations in the contrast are attributed to the wrin-kling or folding of the nanosheets (Figure 2 a,b). [ 17,18 ] Detailed analysis based on Raman spectra (Figure S1, Supporting Infor-mation) and X-ray photoelectron spectroscopy (XPS, Figure S2) further confi rmed the features of MnO 2 nanosheets. The pre-pared MnO 2 nanosheets can be well dispersed in water for up to several months (Figure 2 d and S3) without apparent aggre-gation. However, the stability of MnO 2 nanosheets in saline



Figure 1. A) Schematic illustration of synthetic procedure for 2D PEG–MnO 2 nanosheets; B) theranostic function of PEG–MnO 2 nanosheets for intra-cellular pH-responsive drug delivery and T 1 -MRI. PEG=Poly(ethylene glycol).

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is relatively low. Thus, the surface of MnO 2 nanosheets was further modifi ed with PEG 5000 -NH 2 through Mn–N coordinate bonding to improve their stability in physiological conditions. The PEGylated MnO 2 nanosheets (designated as PEG–MnO 2 ) kept the sheet-based morphology (Figure 2 c) and high disper-sity (Figure 2 e) in saline after the surface modifi cation.

The break-up of PEG-MnO 2 nanosheets in a mild acidic environment could be clearly observed by the color change of their solution from brown to colorless, while no color change occurred under neutral conditions (Figure S4). The relevance of the break-up process and corresponding enhanced T 1 -MRI could be directly demonstrated by dynamic in situ clinical MRI (3T) scanning. PEG-MnO 2 nanosheets were encapsulated within a dialysis bag (molecular weight cut-off: 5000 Da), which was then immersed into the buffer solutions at pH values of 4.6 ( Figure 3 a) and 7.4 (Figure 3 b). These two pH values were chosen to imitate the normal blood circulation environment and acidic microenvironment in tumor tissues. The released Mn( II ) could penetrate the dialysis bag and enter the buffer solution while the initial PEG–MnO 2 nanosheets were too big to pass out of the dialysis bag. Accompanying the fast break-up of MnO 2 nanosheets and the release of Mn( II ) in an acidic environment, a small positive-enhanced T 1 -MRI region could be found in the initial stage (30 min), while substantial posi-tive T 1 -MRI signal intensity enhancement could be directly observed in the whole buffer solution within 60 min. Com-paratively, soaking PEG-MnO 2 nanosheets in the neutral buffer solution showed no signifi cant signal changes. Quantitative signal intensity after 60 min soaking in acidic buffer solution showed a nearly 3.6-fold increase in signal over that of the neu-tral buffer solution (Figure 3 c).

The relaxation rate ( r 1 value) of initial PEG-MnO 2 nanosheets was very low at 0.007 mM −1 s −1 . Such a low r 1 value is attributed to the high valence ( IV ) of manganese and shielded paramag-netic centers inaccessible to water molecules. [ 19 ] Importantly, the r 1 value was substantially increased from the initial value of 0.007 to 3.4 and 4.0 mM −1 s −1 after soaking in the acidic buffer solution at pH 6.0 and 4.6 for 2 h, respectively (Figure S5). Such a pH-responsiveness results in a 571-fold magnitude increase of the r 1 value after changing the pH value from 7.4 to 4.6. This r 1 value is also comparable to those of commercial Gd-based CAs ( r 1 ≈ 3.4 mM −1 s −1 ) and nearly eightfold magni-tude increase over those of MnO x NPs with varied particle sizes (typically smaller than 0.5 mM −1 s −1 ). [ 12d ] Such an ultrasensitive T 1 -MRI performance is believed applicable for the nanosheets to function as CAs for in vivo pH-responsive T 1 -MRI. The PEG-MnO 2 nanosheets also exhibited a redox-responsive T 1 -MRI performance subject to the reduction of Mn( IV ) into Mn( II ) and subsequent disintegration of the nanosheet (Figure S6, S7, and related discussion).

To evaluate whether such an ultrasensitive pH-responsive-ness is suitable for in vivo tumor imaging, a nude mice 4T1 cancer xenograft was established for the in vivo assessment. PEG–MnO 2 saline solution was directly injected into the tumor tissues and normal subcutaneous tissues for comparison. Figure 4 a shows that PEG–MnO 2 does not lead to positive con-trast-enhancement in T 1 -MRI image at the initial stage (OFF state) due to its low imaging performance (within 20 min) for T 1 -MRI. However, clear positive signal-enhancement around the injection points within tumor tissues could be observed over time (ON state). Such an interesting positive T 1 -MRI signal enhancement could be attributed to the break-up of PEG–MnO 2



Figure 2. a) Bright-fi eld and b) dark-fi eld TEM images of MnO 2 nanosheets; c) TEM image of PEG-MnO 2 nanosheets; d) digital picture of MnO 2 nanosheets dispersed in water; e) photos of MnO 2 and PEG-MnO 2 nanosheets dispersed in saline.


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nanosheets that leads to quick Mn( II ) release in the mildly acidic microenvironment of the tumor region, which enables the Mn( II ) to act as a highly effi cient T 1 -MRI CAs. Quantita-tive measurement shows that the T 1 -MRI signal intensity increases 2.3-fold on break-up of the PEG–MnO 2 nanosheets (Figure 4 c). In coronal T 1 -MRI images, the tumor tissue at the injection site exhibits similar time-dependent positive T 1 -MRI signal enhancement while the subcutaneous injection position shows no signifi cant signal enhancement throughout the whole evaluation process (Figure 4 b), which was further illustrated by the quantitative measurements of T 1 -MRI signal intensities

(Figure 4 d). This interesting phenomenon not only gives direct evidence that the tumor tissues are more acidic than the normal tissues, but also gives the fi rst evidence that such pH variations can be utilized for ultrasensitive pH-responsive MRI simply by choosing adequate compositions of nano-CAs, in addition to those based on complicated design/synthesis of pH-responsive chemical bonds. [ 11b , 11c , 20 ]

The large surface area to mass ratio of exfoliated MnO 2 nanosheets provides a large number of anchoring points for drug molecules, which means that MnO 2 nanosheets could func-tion as carriers for drug delivery. It is anticipated that the loaded



Figure 3. In vitro dynamic measurement of T 1 -MRI of PEG–MnO 2 in either mildly acidic environment (pH = 4.6, a) or neutral conditions (pH = 7.4, b); c) the T 1 -MRI signal intensities of PEG-MnO 2 aqueous solutions under pH 7.4 and 4.6 for prolonged periods. The T 1 -MRI images were obtained every 3 min after soaking PEG-MnO 2 nanosheets in the buffer solution at various pH values.


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cargos can be released very fast in an acidic microenviron-ment, based on the break-up of MnO 2 nanosheets ( Figure 5 a). This MnO 2 nanosheet-based intelligent nanosystem gives the fi rst example of inorganic nano drug delivery systems (DDSs) for carrier-disintegration-based pH-responsive drug release, which means that such a controlled release is no longer an organic-DDS-specifi c characteristic. Doxorubicin (Dox), a typ-ical broad-spectrum anticancer drug, was loaded onto the sur-face of MnO 2 nanosheets via electrostatic interaction and Mn–N coordinate bonds. The initial zeta potential of the suspension of MnO 2 nanosheets was –40.6 mV, but it became positive (+22.3 mV) after Dox loading due to the positive nature of Dox molecules absorbed onto the surface of the negatively charged MnO 2 nanosheets (Figure 5 b). In addition, the Mn atoms in the sheets could form special coordinate bonds with the N atoms in Dox molecules. [ 21 ] Such two factors, i.e., electrostatic interaction and coordinate bonding, endow the MnO 2 nanosheets with a high Dox-loading capacity of 450 mg g –1 .

The acidity-induced break-up of MnO 2 nanosheets could pro-mote the fast release of loaded cargos in a mildly acidic environ-ment. To validate this idea, three typical pH microenvironments were established to imitate normal tissues (pH = 7.4) and tumor acidic conditions (pH = 6.0 and 4.6). As envisaged (Figure 5 c), Dox release in neutral buffer solution is quite low with only a 24.8 % released in 5 h. Comparatively, the release behavior is signifi cantly accelerated in mild acidic buffer solutions with 58.9 % (pH = 6.0) and 94.3 % (pH = 4.6) released in 5 h. Such an ultrasensitive pH-responsive drug release is very favorable

for cancer chemotherapy because the Dox release from MnO 2 nanosheets is rather low when it is circulating within the neu-tral blood vessels. When Dox-loaded MnO 2 nanosheets accu-mulate within tumor tissues, the acidic environment of tumors substantially promotes the break-up of the carrier and corre-sponding fast release of the loaded therapeutic agents, which can signifi cantly mitigate the side-effects of therapeutic drugs. The acidity-induced break-up of MnO 2 nanosheets and the Dox release take place simultaneously, which means that the Dox-release process could be monitored in situ by using MRI based on the leakage of Mn( II ) from the carrier. The preliminary results show that the T 1 relaxation time of 5 h in acidic envi-ronments (pH = 6.0 and 4.6) is signifi cantly shorter than that under neutral conditions (Figure 5 d), which indicates that fur-ther potential observation and determination of pH-responsive drug release by using T 1 -MRI during chemotherapy is possible.

The capability of intracellular drug delivery and cor-responding therapeutic effi ciency via Dox-loaded MnO 2 nanosheets was further systematically evaluated in Dox-resistant MCF-7/ADR cancer cells. The time-dependent intracellular Dox delivery and release were observed by using confocal laser scanning microscopy (CLSM, Figure 6 a). The red fl uorescent intensity of Dox became much stronger after 12 h co-incuba-tion (Figure 6 a 3 ) than that at the initial 4 h (Figure 6 a 1 ) and 8 h (Figure 6 a 2 ) co-incubation time-points, which indicates that Dox–MnO 2 can be effi ciently endocytosized into cancer cells and exhibits time-dependent release behavior that is probably trig-gered by the intracellular acidic environment within endosomes



Figure 4. a) Axial and b) coronal T 1 -MRI of 4T1 tumor-bearing nude mice before (a 1 ,b 1 ) and after (a 2 –a 9 and b 2 –b 9 ) administration of PEG–MnO 2 nanosheets within tumor and normal subcutaneous tissue. T 1 -MRI images were acquired every 10 min. Quantitative T 1 -MRI signal intensity before and after the administration of PEG–MnO 2 nanosheets c) axial tumor region and d) coronal tumor region and normal subcutaneous tissue.


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and lysosomes (ca. 5.0–5.5). [ 22 ] Single-cell CLSM images (Figure 6 a 4 –a 6 and S8) and 3D fl uorescence reconstruction (Figure 6 a 7 –a 9 ) further indicat that Dox molecules were present within the cancer cells, and that large amounts of them entered the nuclei. Substantially increased cell uptake of Dox could be

realized when using MnO 2 nanosheets compared to free Dox, as determined by quantitative fl uorescent measurement based on fl ow cytometry (FCM, Figure 6 b) under the different co-incu-bation concentrations and durations. This result means that MnO 2 nanosheet-mediated intracellular Dox delivery can result

Figure 5. a) Schematic illustration of pH-triggered Dox-release by breaking MnO 2 nanosheets up; b) zeta potential of MnO 2 nanosheets and Dox-loaded MnO 2 nanosheets; c) the release percentage of Dox from Dox-loaded MnO 2 nanosheets in different buffer solutions with various pH values (pH = 7.4, 6.0, and 4.6); d) T 1 values of the releasing medium after 300 min of release. Top MRI-T 1 images from left to right: water, buffer solution at pH 7.4, 6.0, and 4.6; **P < 0.01.


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in much higher drug accumulation within the cancer cells than with free anticancer agents. To assess the therapeutic effi -ciency, the cell death mechanisms of MCF-7/ADR cells treated with free Dox and Dox–MnO 2 at different concentrations were assessed by typical FCM and fl uorescence-activated cell-sorting (FACS) protocols. Free Dox shows very little infl uence on the apoptosis and necrosis of MCF-7/ADR cells as indicated by limited small numbers of apoptotic cells (Figure 6 c–e), which can be attributed to the Dox-resistant nature of MCF-7/ADR cells. [ 22 ] Comparatively, Dox-loaded MnO 2 nanosheets induce the remarkable apoptosis of MCF-7/ADR cells (Figure 6 f–h). The quantitative therapeutic effi ciency was obtained by using a typical MTT assay, which shows that signifi cant cell death can be achieved when Dox is delivered by MnO 2 nanosheets (Figure 6 i,j). For example, the therapeutic effi ciencies of Dox-MnO 2 are 49.5 % (24 h) and 59.6 % (48 h) at a Dox concentra-tion of 30 µg mL –1 , much higher than those of free Dox (28.2 % for 24 h and 26.5 % for 48 h). Such a restored sensitivity of Dox-resistance cancer cells to Dox can be attributed to the high accu-mulation of Dox within cancer cells, pH-responsive intracellular drug release, and apoptotic acceleration mediated by MnO 2 nanosheets (Figure 1 B). The multidrug effl ux pump effect of MCF-7/ADR cells by over-expressed P-glycoprotein (P-gp) can pump free Dox from the cytoplasm out of the cancer cells, [ 23 ] but such a P-gp-induced effl ux process can be bypassed when using MnO 2 nanosheets due to the signifi cantly larger sizes of 2D MnO 2 nanosheets than the free Dox molecules. [ 24 ]

In summary, a new concept of concurrent ultrasensitive pH-triggered T 1 -weighted MRI and anticancer-drug releasing was

successfully demonstrated based on chemically exfoliated 2D MnO 2 nanosheets for intelligent cancer diagnosis and therapy (theranostics). The fast break-up of 2D MnO 2 nanosheets in a mildly acidic environment can substantially enhance the in vitro and in vivo T 1 -MRI performance based on the acidic microenvironment of tumor tissues. Such pH-activatable breaking-up behavior can promote the fast release of loaded anticancer drugs for on-demand drug release and circumvent the multidrug resistance of cancer cells. This proof-of-concept design of MnO 2 -nanosheet-based theranostics with dual pH-responsive functions (MRI and drug release) provides an effi -cient and cost-effective new protocol to realize successful thera-nostics of cancer in an intelligent and on-demand manner, which may pave the ways towards the creation of new nanosys-tems for biomedical applications based on 2D multifunctional nanomaterials.

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

Acknowledgments Y. Chen and D. Ye contributed equally to this work. We acknowledge fi nancial support from Australian Research Council through its DP and Future Fellowship scheme. The National Nature Science Foundation of China (Grant No. 51302293, 51132009), Shanghai Rising-Star

Figure 6. a) CLSM images of MCF-7/ADR cancer cells after co-incubation with Dox-loaded MnO 2 nanosheets for 4 (a 1 ), 8 (a 2 ), and 12 h (a 3 ); a 4 –a 6 : CLSM images of single cells after 4 h co-incubation with Dox-MnO 2 (a 4 : nuclei, a 5 : Dox, a 6 : merged image); a 7 –a 9 : 3D fl uorescence reconstruction images corresponding to a 4 -a 6, ; b) fl uorescent intensities of MCF-7/ADR cells after co-incubation with free Dox and Dox-MnO 2 for various durations and concentrations, as determined by FCM; cell death mechanisms of MCF-7/ADR cells after co-incubation with c–e) free Dox and f–h) Dox-MnO 2 at different concentrations (c,f: 5, d,g: 10, e,h: 20 µg mL –1 ) by FCM and FACS; cell viabilities of MCF-7/ADR after co-incubation with free Dox and Dox-MnO 2 for i) 24 and j) 48 h, *P < 0.05.


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Program (14QA1404100), Natural Science Foundation of Shanghai (13ZR1463500) and Foundation for Youth Scholar of State Key Laboratory of High Performance Ceramics and Superfi ne Microstructure (Grant No. SKL201203).

Received: June 11, 2014 Revised: July 3, 2014

Published online:

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