Bimetallic Oxide MnMoOX Nanorods for in Vivo Photoacoustic

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Bimetallic Oxide MnMoOX Nanorods for in vivo Photoacoustic Imaging of GSH and Tumor-Specific Photothermal Therapy Fei Gong, Liang Cheng, Nailin Yang, Qiutong Jin, Longlong Tian, Mengyun Wang, Yonggang Li, and Zhuang Liu Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.8b02933 • Publication Date (Web): 24 Aug 2018 Downloaded from http://pubs.acs.org on August 26, 2018

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Bimetallic Oxide MnMoOX Nanorods for in vivo Photoacoustic Imaging of GSH and Tumor-Specific Photothermal Therapy Fei Gong1, Liang Cheng*1, Nailin Yang1, Qiutong Jin1, Longlong Tian1, Mengyun Wang2, Yonggang Li2, and Zhuang Liu*1 1

Institute of Functional Nano & Soft Materials (FUNSOM), Jiangsu Key Laboratory for

Carbon-Based Functional Materials and Devices, Soochow University, Suzhou 215123, China. 2

Department of Radiology, the First Affiliated Hospital of Soochow University Suzhou, Jiangsu, 215006, China.

Corresponding Author: *E-mail: [email protected] (L. Cheng), Phone: +86-512-65882097 *E-mail: [email protected] (Z. Liu), Phone: +86-512-65882036

ABSTRACT: Accurate imaging of glutathione (GSH) in vivo is able to provide real-time visualization of physiological and pathological conditions. Herein, we successfully synthesize bimetallic oxide MnMoOX nanorods as an intelligent nanoprobe for in vivo GSH detection via photoacoustic (PA) imaging. The obtained MnMoOX nanoprobe with no near-infrared (NIR) absorption in the absence of GSH would exhibit strong GSH-responsive NIR absorbance, endowing PA imaging detection of GSH. Due to the up-regulated GSH concentration in the tumor microenvironment, our MnMoOX nanoprobe could be utilized for in vivo tumor-specific PA imaging. Moreover, MnMoOX nanorods with GSH-responsive NIR absorbance could also be employed to achieve tumor-specific photothermal therapy (PTT). Importantly, such MnMoOX nanorods show inherent biodegradability and could be rapidly cleared out from the body, minimizing their long-term body retention and potential toxicity. Our work presents a new type of GSH-responsive nanoprobe based on bimetallic oxide nano-structures, promising for tumor-specific imaging and therapy.

KEYWODS: GSH detection; MnMoOX nanorods; photoacoustic imaging; photothermal therapy; renal clearance 1

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Glutathione (GSH) is the most abundant endogenous antioxidant that plays a central role in maintaining the appropriate redox status of biological systems.1-4 However, aberrant generation of GSH would break the redox balance, which is associated with many diseases including cancer, aging, cardiovascular diseases, and other ailments.5-7 Thus, it is of significant importance to monitor the in vivo changes of GSH concentrations in real time. In particular, the concentration of GSH in cancer cells is much higher than that in the normal cells (up to 1000-folds),1, 8, 9 indicating that GSH may be a signal molecule to be utilized to improve the specificity of cancer imaging and therapy. Previously, many types of fluorescent probes based on the thiol-sensitive organic fluorophores have been employed to monitor the cellular GSH.8, 10, 11 Moreover, bioluminescence imaging for living cell GSH detection has also been developed by utilizing a combination of lanthanide-doped upconversion nanoparticles and manganese dioxide nanosheets.12 However, due to the shallow tissue-penetration depth and strong light scattering, both fluorescence and bioluminescence imaging modalities have limitations for in vivo GSH detection. Therefore, there is still much need to develop effective methods for in vivo GSH detection with high sensitivity and spatial resolution. Photoacoustic (PA) imaging is an emerging optical imaging technology that utilizes ultrasound signals generated by photothermal expansion of light-absorbing tissues or contrast probes under the laser irradiation.13-15 It can provide deeper tissue-penetration (up to 12 cm) and higher spatial resolution compared to the traditional optical imaging modalities.8, 13, 16-18 Up to now, various types of nanomaterials with near-infrared (NIR) absorption have been employed as probes for PA imaging.19-21 Recently, various smart nanoprobes with their NIR absorption responsive to some specific physiological signals such as pH, H2O2, and reactive oxygen or nitrogen species, have also received great interests.13,

21-26

However, GSH-responsive nanoscale probes for in vivo GSH

detection via PA imaging have been rarely reported to our best knowledge. In this work, GSH-responsive bimetallic oxide MnMoOx nanorods were synthesized for in vivo GSH detection via PA imaging. Synthesized by the typical organic phase procedure, the obtained MnMoOX nanorods are then modified with polyethylene glycol (PEG). Without optical absorbance in the NIR window for the as-made MnMoOX-PEG, such nanoprobe would exhibit GSH-responsive strong NIR absorption, owing to the reduction of MoVI in the initial MnMoOx to MoV by GSH, which in the meanwhile triggers the transformation of nanorods to ultrasmall nanodots. Utilizing this behavior, PA imaging of GSH is realized with the MnMoOX-PEG nanoprobe. Due to the higher 2


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concentration of GSH in the tumor microenvironment (TME), PA imaging of tumors on mice is achieved by using this MnMoOX-PEG nanoprobe. Moreover, photothermal tumor ablation is also realized based on the strong GSH-activated NIR absorption of MnMoOX-PEG nanoprobe, which induces minimal non-specific heating to normal tissues. Notably, those MnMoOX-PEG nanorods could be degraded in vivo to endow rapid renal clearance. Our work thus presents a new type of GSH-responsive nanoprobe based on bimetallic oxide nanostructures, promising for tumor-selective imaging and therapy.

MnMoOX nanorods were prepared by a typical organic-phase synthesis procedure,27 in which Mo(CH2O)6 solution was firstly heated to 260 oC and then added with Mn(acac)3 precursor for 5 min reaction. The obtained MnMoOX nanorods were then modified by an amphiphilic polymer, poly(ethylene glycol)-grafted poly (maleic anhydridealt-1-octadecene) (C18PMH-PEG) to increase their water solubility (Scheme 1). Transmission electron microscopy (TEM) imaging showed the uniform morphology of the synthesized MnMoOX nanorods with an average length at ~40 nm and width at ~10 nm (Figure 1A). The high-resolution TEM (HRTEM) imaging (Figure 1A, inset) revealed the lattice spacing of the obtained nanostructure to be ~0.346 nm, which was attributed to (-2 2 0) lattice plane of the MnMoO4 structure (JCPDS No. 26-0575) (Figure 1D).28 Meanwhile, the elemental mapping further confirmed the co-existence of Mn, Mo, and O elements (Figure 1B and 1C). X-ray photoelectron spectroscopy (XPS) was also employed to analyze the metal valence states (Figure S1). The Mn 2p peaks at binding energies of 641.2 eV (2p3/2) and 653.4 eV (2p1/2) could be assigned to MnII (Figure 1E), while the Mo 3d peaks located at 235.4 eV (3d3/2), 232.3 eV (3d5/2) and 230.3 eV (3d5/2) could be ascribed to MoVI and MoV (with the ratio of ~3 : 1) (Figure 1F). Therefore, it is possible that the MoVI was partially reduced by the oleylamine in the reaction process. Moreover, according to ICP-OES (Inductively Coupled Plasma Optical Emission Spectrometry) and XPS analysis, the value of x in our MnMoOX nanorods was determined to be 3.875. To understand the formation mechanism of the MnMnOX nanorods, we have carefully investigated how the synthesis parameters such as the reaction time and the Mn : Mo ratio could affect the obtained nanostructures. Before the addition of Mn precursor (at 0 s), a large amount of MoOX nanodots were synthesized due to thermal decomposition of Mo(CH2O)6 (Figure 1G). After adding the Mn precursor, the morphology of the product was changed into nanoparticles (at 10s). 3


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With the time increasing, the nanorods were formed and grew longer. The length-diameter (L/D) ratio was increased from 1.2 (at 10 s) to 4.4 (at 60 s) (Figure 1H). Furthermore, X-ray diffraction (XRD) spectra showed the intensities of the characteristic peaks of the MnMoOX nanorods increased significantly with the prolonging reaction time (Figure 1I). However, further prolonging the reaction time had little effect on the morphology of MnMoOX nanorods (Figure S2 and S3). Meanwhile, we also found that the morphology of MnMoOX nanorods could be tuned by the addition of Mn(acac)3 precursors. When the ratio of Mn : Mo increasing to 1 : 1, MoOX nanoparticles were changed into MnMoOX nanorods with uniform morphology, while further increase of this ratio would lead to disappearance of MnMoOX nanorods accompanied by the generation of MnO nanospheres (Figure S4 and S5). Based on the above results, we proposed a seed-mediated growth mechanism for the synthesis of MnMoOX nanorods as follows (Figure 2J).29, 30 Firstly, MoOX nanodots are quickly formed owing to the rapid thermal decomposition of Mo(CH2O)6. After the addition of the Mn precursor, MoOX nanodots would serve as seeds to react with the Mn precursor to generate MnMoOX nanoparticles immediately. With the prolonging of reaction time, the nanoparticles would grow longer to generate nanorods. Furthermore, we also successfully synthesized FeMoOX and CuMoOX nanostructures based on the same seed growth method (Figure S6). The obtained FeMoOX nanoflowers and CuMoOX nanoparticles showed uniform morphology, suggested that the seed growth method could be a simple and general method to prepare regular Mo-based bimetallic oxide (Figure S7). We chose MnMoOX nanorods prepared at the feeding Mn : Mo ratio of 1 : 1 (5 min of reaction time) for the following experiments. The as-prepared MnMoOX nanorods were then modified with C18PMH-PEG to increase their water solubility. The amount of PEG coated on the surface of MnMoOX nanorods was determined by thermogravimetric analysis (TGA) to be 47.9% (Figure S8). After PEGylation, MnMoOX-PEG nanorods showed excellent stability in the physiological solutions including RPMI 1640 cell culture medium, fetal bovine serum (FBS), and phosphate buffered saline (PBS) without any precipitation over a week (Figure S9), allowing their further application in biological systems. Moreover, with such biocompatible PEG coating, the synthesized MnMoOX-PEG showed negligible in vitro cell cytotoxicity (Figure S10). The polyvalent metallic elements (e.g. Fe, Cu, Ce, and Mo) that could be transformed into lower valence states in the presence of reductant from their high valence states are widely used in catalysis, 4


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energy, and medical applications.31-34 Due to the existence of Mo in the MnMoOX-PEG sample, we wondered whether our synthesized MnMoOX-PEG nanorods would show GSH redox-activation property. MnMoOX-PEG nanorods were incubated with different concentrations of GSH. Obviously, the color of the MnMoOX-PEG nanorods changed from colorless to blue, monotonically deepened with the increasing GSH concentration (Figure 2A and Figure 2B insert). The absorbance of MnMoOX-PEG solution at 830 nm increased in proportional to the concentrations of the added GSH in the range of 0.5~10 mM (Figure 2B). In contrast, no significant response was found after incubation of MnMoOX-PEG with a solution of 100 µM H2O2, an acidic buffer at pH = 5.4, the fresh mouse serum, fetal bovine serum, and 10 mM L-cysteine solution (Figure S11), suggesting that MnMoOX-PEG could serve as a highly specific GSH probe. Due to the strong absorbance at 830 nm, the GSH-activated MnMoOX-PEG could be used for PA imaging. As the concentration of GSH increased, the PA signal at 830 nm showed the significant enhancement (Figure 2C). Notably, obvious PA signals were still detected for the low concentration of GSH down to 0.5 mM, indicating the high sensitivity of this nanoprobe for GSH detection (Figure 2C insert). In the meanwhile, the same GSH-response phenomenon was also observed by changing the reaction time (Figure S12). Therefore, those MnMoOX-PEG nanorods can be used as a sensitive probe for the GSH detection. To clarify the detailed mechanism of GSH activated MnMoOX-PEG nanorods, XPS analysis was utilized to investigate the Mo valence states before and after incubation with GSH. XPS spectra of Mo 3d showed that there was 76.4% of MoIV and 23.6% MoV in the initial MnMoOX-PEG samples (Figure 2D). After being incubated with GSH for 4 h, the sample contained about 24.9% MoIV and 75.1% MoV (Figure 2E), indicating that most of MoIV was reduced to MoV under the GSH condition, a reason resulting the enhancement of NIR absorption. Moreover, XRD spectra revealed that the intensity of characteristic peaks decreased significantly after incubation of MnMoOX-PEG nanorods with GSH (Figure 2F). From the TEM images, ultra-small ones less than 5 nm instead of original nanorods were observed after incubating MnMoOX-PEG with GSH (Figure 2G and Figure S13). All these results indicated that most of MoIV was reduced into MoV by GSH in the MnMoOX-PEG sample, resulting in the color change from colorless to blue, as well as the degradation of MnMoOX-PEG nanorods into nanodots. Owing to the destruction of nanorods structure for the MnMoOX-PEG sample in the presence of 5


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GSH, we thus suspect that Mn2+ would be released in the presence of GSH during this process. As Mn2+ would be an effective contrast agent in T1-weighted magnetic resonance (MR) imaging, different concentrations of MnMoOX-PEG samples were incubated with 10 mM GSH for 4 h and then imaged by a MR scanner. The T1-weighted MR images of MnMoOX-PEG exhibited a remarkable brightening effect and the r1 relaxivity of MnMoOX-PEG was significantly increased from 1.71 mM-1•S-1 in the absence of GSH to 6.94 mM-1•S-1 in the presence of GSH (Figure 2H and 2I). Such enhanced MR imaging is likely attributed to the Mn2+ released from MnMoOX nanorods. The above results indicated that MnMoOX-PEG nanorods after being reduced by GSH were destructed, likely resulting in polyoxometalate clusters (HX(MoVX)(MoVI1-X)O3) and hydrated manganese ion (Mn(H2O)62+).21 Encouraged by the above GSH-responsive properties of MnMoOX, we wondered whether our MnMoOX-PEG nanorods could be used for in vivo GSH detection. GSH is often found at higher concentrations in tumor cells (2~10 mM) than that in normal cells.35 Thus, a murine breast 4T1 tumor model was employed to demonstrate the potential of MnMoOX-PEG nanoprobes for GSH detection (Figure 3A and 3B). Mice were intratumorally (i.t.) injected with MnMoOX-PEG, which was also injected into the muscle as the control. Interestingly, rather strong PA signals were observed in the tumor after injected with MnMoOX-PEG for 2 h, whereas muscle showed much weaker PA signals (Figure 3B). In the following time, the PA signals showed gradual decrease, likely owing to the destruction of nanorods structure into ultra-small nanodots and metal ions in the presence of endogenous GSH, and then these ultra-small nanodots might be cleared out from the mouse body. These results demonstrated the possibility of using MnMoOX-PEG for tumor GSH detection by PA imaging. It has been reported that L-2-Oxothiazolidine-4-carboxylate (OTZ), a prodrug of cysteine, could stimulate the synthesis of intracellular GSH,36 while L-buthionine-sulfoximine (BSO), a drug that inhibits the activity of gamma-glutamylcysteine synthetase, could inhibit the synthesis of GSH inside cells.37 Therefore, OTZ and BSO were employed to modulate the GSH concentrations in the tumor (Figure 3A and 3B). OTZ or BSO was firstly i.t. injected to the mouse, then the mouse was i.t. injected with MnMoOX-PEG for PA imaging. Interestingly, it could be found that PA signals in OTZ-treated tumors were significantly enhanced, while BSO-treated tumors showed significantly reduced PA signals. Moreover, quantification analysis of PA signals for these groups further indicated 6


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that the levels of GSH with the order of OTZ-treated tumor > untreated tumor > BSO-treated tumor ≈ muscle (Figure 3C and Figure S14). To further study the potential of MnMoOX-PEG for the tumor GSH detection, mice bearing 4T1 tumors were intravenously (i.v.) injected with MnMoOX-PEG (Figure 3D, Figure S15 & S16). Interestingly, the PA signals at 830 nm obviously increased over time, owing to the time-dependent tumor accumulation of MnMoOX-PEG nanorods and their subsequent reaction with GSH within the tumor (Figure 3E). In addition to PA imaging, T1-weighted MR imaging was also carried for tumor-bearing mice after i.v. injection of MnMoOX-PEG nanorods. An obvious brightening effect could be observed in the tumor region after i.v. injection with MnMoOX-PEG, suggesting that GSH-responsive MnMoOX-PEG could serve as a promising contrast agent for MR imaging (Figure 3F and 3G). All above results verified that our GSH-responsive MnMoOX-PEG nanoprobe could be used as an intelligent nanoagent for PA/MR dual-modal imaging. Considering that the NIR absorption of MnMoOX-PEG could be significantly enhanced in the presence of GSH, we proposed that this nanoprobe may also be employed as a GSH-activated photothermal agent for cancer therapy. After incubation with GSH, MnMoOX-PEG showed obviously increased photothermal effect (Figure S17-S20). Then, mice bearing 4T1 tumor were intratumorally (i.t.) and intramuscularly (i.m.) injected with MnMoOX-PEG on both sides of each mouse. At 2 h post injection, mice were exposed to the 808 nm laser irradiation. The temperature of the tumor site rapidly increased to 54 oC, while the muscle injected with MnMoOX-PEG showed no significant temperature change (Figure 4A and 4B), demonstrating that our MnMoOX-PEG nanorods could act as a tumor-specific photothermal agent, which is activated within the tumor but not in normal tissues. Next, we carried out a more careful in vivo photothermal therapy study by i.v. injection of MnMoOX-PEG into 4T1-tumor-bearing mice (Figure 4C and Figure S21). The tumor temperature of mice injected with MnMoOX-PEG sharply increased to 52 oC while the control group showed no significant change, indicating that such GSH-responsive MnMoOX-PEG nanorods could be employed as an efficient tumor-specific photothermal agent. Thus, the in vivo PTT performance of MnMoOX-PEG nanorods was evaluated by 4T1-tumor-bearing mice (Figure S22). The tumors on mice treated with MnMoOX-PEG plus laser irradiation were completely eliminated, without recurrence (Figure 4D), demonstrating the excellent therapeutic efficacy of MnMoOX-PEG nanorods. 7


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More importantly, the mice in the treated group (MnMoOX-PEG + Laser) showed significantly 100% survival within 30 days, in marked contrast to the other three groups (Figure 4E). To further verify the antitumor efficacies, hematoxylin and eosin (H&E) staining of tumor slices were carried out. Severe damages of tumor cells could be observed for the MnMoOX-PEG + laser group, while other three groups possessed negligible cell damage (Figure 4F). Furthermore, the body weight of the treated mice remained almost invariability (Figure S23). These results together demonstrated that this MnMoOX-PEG nanoprobe could be served as an efficient GSH-activated PPT agent. Then, we wondered whether MnMoOX-PEG could be cleared out from the mouse body. Mice were intravenously injected with MnMoOX-PEG, and then sacrificed after 1, 7, 14, and 30 days, with their main organs harvested for biodistribution study. It was found that MnMoOX-PEG nanorods were mainly accumulated in the liver and spleen at 1 day post injection (p.i.), and quickly decreased in the following time points (Figure 4G). After 30 days, the Mo concentrations in the liver and spleen were decreased to 1.16 ± 0.08 %ID/g and 1.32 ± 0.22 %ID/g, respectively, suggesting that the majority of MnMoOX-PEG was decomposed and cleared out from the mouse body. Notably, high levels of Mo could be detected in the urine of mice after injection of MnMoOX-PEG, indicating that those nanorods within the mouse body would be gradually degraded into ultra-small polyoxometalate clusters (HX(MoVX)(MoVI1-X)O3) and manganese ions to allow efficient renal excretion (Figure 4H). H&E staining also evidenced that MnMoOX-PEG showed no noticeable toxicity to the mice (Figure S24). Considering the efficient clearance and the little retention of Mo in the body after 30 days, the synthesized MnMoOX-PEG nanoprobe would not cause significant long-term toxicity. In summary, an intelligent bimetallic oxide MnMoOX nanoprobe has been successfully synthesized for in vivo GSH detection via PA imaging. The obtained MnMoOX nanoprobe is highly sensitive to GSH, with the detection sensitivity down to the micromole level. After the GSH activation, the nanoprobe exhibited strong NIR absorption due to the reduction of MoVI to MoV. Considering the up-regulated GSH concentrations in the tumor microenvironment than other normal tissues, the MnMoOX-PEG nanoprobe could be employed for in vivo PA imaging of tumors. Furthermore, GSH-responsive MnMoOX-PEG with high NIR absorbance after activation by GSH could also allow tumor-specific photothermal therapy. Meanwhile, the MnMoOX-PEG nanorods could be rapidly cleared out from the body due to their inherent biodegradability. Therefore, our work presents a new type of GSH-responsive nanoprobe based on bimetallic oxide nanostructures, 8


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promising for applications in cancer imaging and therapy. Moreover, as GSH is able eliminate cytotoxic reactive oxygen species generated during certain types of cancer therapies,38, 39 in future studies, we will investigate whether MnMoOX-PEG could serve as the sensitizer to enhance photodynamic therapy or sonodynamic therapy, whose performances are related to the GSH levels in the tumor.

ASSOCIATED CONTENT Supporting Information. Detailed synthesis and characterization, experimental procedures, supplementary figure S1~S24. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (L. Cheng), Phone: +86-512-65882097 *E-mail: [email protected] (Z. Liu), Phone: +86-512-65882036 The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENT This

article

was

partially

supported

by

the

National Research

Programs

of

China

(2016YFA0201200), the National Natural Science Foundation of China (51525203, 51761145041, 51572180), Collaborative Innovation Center of Suzhou Nano Science and Technology, a Jiangsu Natural Science Fund for Distinguished Young Scholars (BK20130005, BK20170063), and a Project Funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions.

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(28) Peng, C.; Gao, L.; Yang, S.; Sun, J. Chem. Commun. 2008, 43, 5601-5603. (29) Luo, M.; Ruditskiy, A.; Peng, H. C.; Tao, J.; Figueroa-Cosme, L.; He, Z.; Xia, Y. Adv. Funct. Mater. 2016, 26, 1209-1216. (30) Chen, L.; Huang, B.; Qiu, X.; Wang, X.; Luque, R.; Li, Y. Chem. Sci. 2015, 7, 228-233. (31) Gong, F.; Luo, L.; Yao, Y.; Dai, D.; Lu, W.; Chen, W. Chem. Eng. J. 2016, 304, 440-447. (32) Zou, J.; Ma, J.; Chen, L.; Li, X.; Guan, Y.; Xie, P.; Pan, C. Environ. Sci. Technol. 2013, 47, 11685-11691. (33) Mitra, R. N.; Gao, R.; Zheng, M.; Wu, M. J.; Voinov, M. A.; Smirnov, A. I.; Smirnova, T. I.; Wang, K.; Chavala, S.; Han, Z. ACS Nano 2017, 11, 4669-4685. (34) Ni, D.; Jiang, D.; Valdovinos, H. F.; Ehlerding, E. B.; Yu, B.; Barnhart, T. E.; Huang, P.; Cai, W. Nano Lett. 2017, 17, 3282-3289. (35) Anderson, E. D.; Gorka, A. P.; Schnermann, M. J. Nat. Commun. 2016, 7, 13378. (36) Choi, J.; Park, K. H.; Kim, S. Z.; Shin, J. H.; Jang, S. I. Molecules 2013, 18, 3467-3478. (37) Trachootham, D.; Alexandre, J.; Huang, P. Nat. Rev. Drug Discov. 2009, 8, 579-591. (38) Zhang, W.; Lu, J.; Gao, X.; Li, P.; Zhang, W.; Ma, Y.; Wang, H.; Tang, B. Angew. Chem. 2018, 130, 4985-4990. (39) Fan, H.; Yan, G.; Zhao, Z.; Hu, X.; Zhang, W.; Liu, H.; Fu, X.; Fu, T.; Zhang, X. B.; Tan, W. Angew. Chem. 2016, 55, 5477-5482.

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Scheme 1. Schematic illustration of the preparation and the GSH-responsive tumor theranostic application of MnMoOX-PEG nanorods

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Figure 1. Synthesis and characterization of MnMoOX nanorods. (A&B) The TEM image (A) and EDX elemental mapping (B) of as-synthesized MnMoOX nanorods. (C&D) EDS spectrum (C) and XRD spectrum (D) of as-synthesized MnMoOX nanorods. (E&F) XPS spectra of Mn 2p (E) and Mo 3d (F) peaks of the synthesized MnMoOX nanorods. (G-I) TEM images (G), L/D ratios (H), and XRD spectra (I) of MnMoOX nanorods prepared after different periods of reaction time (0, 10, 30, 50, and 60s). (J) Scheme of the mechanism showing the synthesis of MnMoOX nanorods via the seed-mediated growth. 13


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Figure 2. GSH-responsive MnMoOX-PEG nanorods. (A&B) UV-vis absorption spectra of MnMoOX-PEG after incubated with different concentrations of GSH. Inset in (B) is a photo of the above solutions. (C) PA signal intensities of MnMoOX-PEG incubated with various GSH concentrations. The insets are PA images of the above solutions. (D&E) Mo 3d peaks in XPS spectra of MnMoOX-PEG before (D) and after (E) incubation with GSH. (F&G) XRD spectra (F) and TEM 14


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images (G) of MnMoOX-PEG incubated with 10 mM GSH. (H&I) T1 relaxation rates and T1-weighted MR imaging of MnMoOX-PEG solutions with different concentrations before and after incubated with 10 mM GSH.

Figure 3. PA imaging for in vivo GSH detection with MnMoOX-PEG. (A) Schematic illustration showing PA imaging of OTZ-treated and BSO-treated tumors, with increased and decreased GSH concentrations, respectively, by using MnMoOX-PEG as the nanoprobe (i.t. injection). OTZ or BSO was firstly i.t. injected to each mouse to adjust the tumor GSH concentration. After 6 h, the mice were i.t. injected with MnMoOX-PEG for PA imaging. (B) In vivo PA imaging of muscle, untreated-tumor and tumor at 6 h post injection of OTZ or BSO with intratumor injection of MnMoOX-PEG. (C) PA signals at 830 nm based on PA imaging data in (B). (D) Schematic illustration showing the detection of tumor GSH by intravenous injection with MnMoOX-PEG. (E) In vivo PA imaging of tumors after i.v. injected with MnMoOX-PEG. (F) T1-weighted MR imaging of 4T1 tumor bearing mice before and 24 h after i.v. injected with MnMoOX-PEG. (G) The quantification of T1 MR signals from the tumors pre-inject and post injection of MnMoOX-PEG (***p < 0.001, **p < 0.01, or *p < 0.05). . 15


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Figure 4. In vivo PTT with MnMoOX-PEG. (A&B) IR thermal images and temperature change curves of muscle and tumor after i.t. injection with MnMoOX-PEG. (C) IR thermal images of 4T1 tumor-bear mice after i.v. injection with MnMoOX-PEG (Dose: 20 mg/kg; Laser: 0.8W/cm2 for 10 min). (D) Tumor growth curves of mice after various treatments. (E) Survival curves of mice after various treatments. (F) H&E stained tumor slices collected from different treatment groups. (G) Biodistribution of MnMoOX-PEG after intravenous injection in mice at different days. The Mo contents were measured by ICP-OES. (H) The detected Mo mass in urine and feces at different time points after intravenous injection of MnMoOX-PEG (dose 20 mg/kg) (***p < 0.001, **p < 0.01, or *p < 0.05).

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TOC Figure

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Bimetallic Oxide MnMoOX Nanorods for in Vivo Photoacoustic

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