Tumor Microenvironment-Activated Ultrasensitive Nanoprobes for

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Biological and Medical Applications of Materials and Interfaces

Tumor Microenvironment-Activated Ultrasensitive Nanoprobes for Specific Detection of Intratumoral Glutathione by Ratiometric Photoacoustic Imaging Longguang Tang, Fei Yu, bowen tang, Zhen Yang, Wenpei Fan, Mingru Zhang, Zhantong Wang, Orit Jacobson, Zijian Zhou, Ling Li, Yijing Liu, Dale O Kiesewetter, Wei Tang, Liangcan He, Ying Ma, Gang Niu, Xianzhong Zhang, and Xiaoyuan Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b08100 • Publication Date (Web): 18 Jul 2019 Downloaded from pubs.acs.org on July 19, 2019

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ACS Applied Materials & Interfaces

Tumor Microenvironment-Activated Ultrasensitive Nanoprobes for Specific Detection of Intratumoral Glutathione by Ratiometric Photoacoustic Imaging Longguang Tang,†, ‡ Fei Yu,† Bowen Tang,§ Zhen Yang,‡ Wenpei Fan,*,‡ Mingru Zhang,‡ Zhantong Wang,‡ Orit Jacobson,‡ Zijian Zhou,‡ Ling Li, ‡ Yijing Liu,‡ Dale O. Kiesewetter,‡ Wei Tang,‡ Liangcan He,‡ Ying Ma,‡ Gang Niu,‡ Xianzhong Zhang,*,† Xiaoyuan Chen*, ‡ †State

Key Laboratory of Molecular Vaccinology and Molecular Diagnostics & Center for Molecular Imaging and Translational Medicine, School of Public Health, Xiamen University, Xiamen 361102, China ‡Laboratory

of Molecular Imaging and Nanomedicine, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD 20892, USA §Fujian

Provincial Key Laboratory of Innovative Drug Target Research, School of Pharmaceutical Science, Xiamen University, Xiamen 361102, China KEYWORDS: croconaine dye, polyoxometalate cluster, ratiometric photoacoustic imaging, glutathione, tumor microenvironment activation ABSTRACT: Glutathione (GSH), one of the most significant reducing species in vivo, plays important roles in a variety of diseases and cellular functions. Precise quantification of GSH via advanced non-invasive photoacoustic imaging (PAI) is of vital significance for the early diagnosis and prompt treatment of GSH-related deep-seated diseases, which stresses the need for customdesign of GSH-sensitive PAI probes with changeable NIR-absorption. In this work, a novel intelligent tumor microenvironmentactivated ratiometric PAI nanoprobe is first developed with the intention of specific ultrasensitive detection of intratumoral GSH by overcoming the limitations of previously reported fluorescent or PA imaging sensors. This special ratiometric PAI nanoprobe (CRPOM) is synthesized through the self-assembly of croconaine (CR) dye and molybdenum (Mo)-based polyoxometalate (POM) clusters with opposite NIR absorbance change in response to GSH. The resulting amplified ratiometric absorbance (Ab866/Ab700), the relatively low limit of detection (LOD) value (0.51 mM), and the unique acidity-activated self-aggregation contribute to the prolonged intratumoral retention and enhanced tumor accumulation of CR-POM for accurate quantification of intratumoral GSH (0.5-10 mM). Featuring the additional merit of 64Cu radiolabeling for whole-body PET imaging, the smartly designed CR-POM nanoprobe will open new horizons for real-time non-invasive monitoring of biodistribution and simultaneous accurate quantification of GSH levels especially in tumor and other GSH-related pathophysiological processes.

INTRODUCTION Glutathione (GSH), the most abundant small-molecularweight cysteine-containing thiol in cells (~0.5-10 mM), plays an essential role as a cellular antioxidant.1 The variations in GSH concentrations are usually associated with many diseases. For example, GSH deficiency results in strong oxidative stress, which will aggravate liver disease,2 Alzheimer’s disease,3 Parkinson’s disease,4 immune dysfunctions (AIDS),5 stroke,6 heart attack,7 and diabetes.8-9 On the contrary, the excess of GSH in cancer cells can confer resistance to radiotherapy/chemotherapy,10-11 and also promote tumor invasion/metastasis.12-14 Therefore, the detection and quantification of GSH levels are of significant importance for the early accurate diagnosis of GSH-related diseases. The leap-forward development of molecular imaging technology provides a convenient yet powerful tool for non-invasive realtime quantification of GSH levels in vivo. To date, enormous efforts have been made to design GSH-responsive fluorescent probes.15-18 However, the drawbacks of poor spatial resolution and shallow tissue penetration always impede the clinical

application of fluorescent imaging in deep tissue detection.19 A few GSH-activatable magnetic resonance imaging (MRI) nanoprobes for tumor cell imaging have also been developed,20 but a high-resolution MRI image takes a relatively long period of scanning, which fails to reflect the real-time GSH concentrations. In addition, most of the MRI/fluorescence imaging probes cannot distinguish GSH from other reducing agents, such as cysteine (Cys) and homocysteine (Hcy) which are abundant in vivo. Therefore, the development of a more precise imaging technique with deeper tissue penetration, enhanced sensitivity, and unique GSH specificity is required. Featuring the combination of optical excitation and ultrasonic detection, non-invasive photoacoustic imaging (PAI) has been developed to employ longitudinal experiments to obtain high-resolution deep tissue images in live animals by overcoming the optical diffusion limitation and achieving high optical resolution and deep imaging depth (up to 7 cm).19, 21-22 Although many ratiometric PAI probes have been synthesized for sensing ROS, pH, ClO-, and some other oxidants,23-25 very

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few has been reported to detect reductants, especially GSH,26 presumably attributed to the difficulty in the custom-design of probes with changeable NIR-absorption under reductive activation.27 Herein, we report a novel ratiometric GSH-specific PAI nanoprobe synthesized through the self-assembly of croconaine (CR) dye and molybdenum (Mo)-based polyoxometalate (POM) cluster into uniform nanoparticles (CR-POM). In particular, for the first time, we find that the CR dye can be reduced specifically by GSH (rather than by Cys or Hcy), showing the distinct GSH concentrationdependent decrease in the absorbance at 700 nm. In contrast, the Mo-based POM clusters can be reduced by GSH to increase their absorbance at 866 nm due to the GSH-activated Mo(VI) to Mo(V) conversion.28-30 Thus, the PA signal ratio of CR-POM at these two wavelengths (PA866/PA700) is much higher than most existing ratiometric PAI probes. More importantly, the acidic tumor microenvironment (TME) can drive the self-assembly and aggregation of CR-POM through protonation-induced hydrogen bonding of POM clusters, which favors its prolonged intratumoral retention for enhanced tumor accumulation (Figure 1).31-32 Such a unique hybrid nanoprobe will serve as an unprecedented ratiometric PAI paradigm for both tumor-specific delivery and ultrasensitive detection of GSH, so as to achieve highly accurate detection and quantification of intratumoral GSH levels. It is worth mentioning that the CR dye can be radiolabeled with 64Cu through its binding with the carbonyl oxygens of the dye, which also enables quantitative pharmaco-imaging to monitor the whole-body distribution of CR-POM nanoprobes in vivo by PET imaging. RESULTS AND DISCUSSION The CR dye was synthesized through a three-step reaction (Figure 2a). First, compound 1 was obtained by nucleophilic substitution reaction between thiophene-2-thiol and amino groups. Then compound 2 was synthesized by condensation reaction of 2 equivalent of compound 1 with croconic acid, and the final product CR was obtained by ester hydrolysis. Chemical analyses of the intermediates and the final product CR were presented in Figures S1-S4. CR could specifically react with GSH, leading to decreased NIR absorption, which drew our strong attention to explore the possible mechanism behind the GSH-activated absorption change of CR. It has been reported that the sulfhydryl of GSH can react with some dyes through a nucleophilic substitution by attacking the carbon-carbon double bond.33-36 Furthermore, CR dyes are electrophiles because of their electron-deficient central fivemembered ring, which is highly vulnerable to nucleophilic attack by thiols and leads to the abrupt decrease of NIR absorbance. Therefore, it is expected to form the structure illustrated in Figure 2b after mixing CR and GSH in PBS, in which the carbon-carbon double bond near the thiophene is attacked by the mercapto group of GSH. The molecular weight was analyzed by LC-MS before and after adding GSH. As shown in Figure 2c, the target mass of CR-GSH was found to be the same as that of the supposed structure. To further confirm its structure, NMR was conducted to show the chemical shift of CR with and without GSH. When 1 equiv. of GSH was added into the DMSO-d6 or D2O solution of CR, the chemical shift of CR was changed a lot and the intensity was

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also decreased in 5 min. The peaks of CR even disappeared when 4 equiv. of GSH was added into the CR-D2O solution (Figure S5 and Figure S6). However, no reaction was found when excess of Cysteine (Cys) or Homocysteine (Hcy) was added into the D2O solution of CR (Figure S7a and S7b), which was also confirmed by testing their absorbance (Figure 2d). This unexpected is against the previous studies of squarylium dyes37 or other croconaine dyes38 that are more sensitive to Cys or Hcy than to GSH. Featuring the unique GSH concentration-dependent NIR absorption (Figure 2e), CR is indeed a promising dye to detect GSH specifically. To gain deep insights into the mechanism of the high selectivity and photophysical changes of CR towards GSH binding, we performed quantum chemical calculations of ground and excited electronic states of CR, CR-GSH, CRHcy, and CR-Cys using density functional theory (DFT) and time-dependent DFT (TDDFT) with the B3LYP hybrid exchange-correlation functional and the 6-31g(d) basis set. Although GSH, Cys and Hcy have the same reactive fragment R-S-H, their different 3D conformations with CR may determine their different tendencies to react with CR. As shown in Figure 3a, the 3D structure of CR-GSH is very similar in both non-covalent binding (before the reaction) and covalent binding (after the reaction). The H-bonds between the carboxylic acid groups may contribute to the mercapto nucleophilic substitution between GSH and CR as they exist in both states. However, even though the R-S-H from Hcy and Cys can keep an active state with the distance less than 5 Å, it may be difficult for them to keep the active state because the carboxylic acid groups between them and CR are more amenable to form H-bonds, which will inhibit the reaction of mercapto nucleophilic substitution since the S atom will be far away (> 5 Å) from the reaction site on CR due to the small size of Hcy and Cys. In line with the experimentally determined results (Figure 3b) that the absorption of CR-GSH in NIR region was dramatically reduced, the computationally predicted vertical transition wavelengths for CR and CR-GSH are around 700 nm (CR) or exhibit a marked blueshift to 400 nm (CR-GSH). (Figure 3c and Table S1). In contrast to CR that showed a decrease of absorption after reaction with GSH, the Mo-based POM clusters displayed an increase of absorption owing to the GSH-induced reduction of Mo(VI) into Mo(V) (Figure 4a). The increased reduction of POM promoted the delocalized electron density and occupied cation sites of Mo(V) through reversible and multi-step electron exchange, which meanwhile led to the enhancement of electron relaxation polarization and NIR absorption.28 The NIR absorbance of POM at 866 nm showed a GSH concentration-dependent increase behavior (Figure 4b), contrasting with GSH concentration-dependent NIR absorbance decrease of CR. Therefore, a ratiometric PA imaging probe could be obtained by the combination of CR and POM. Three types of POM clusters at varied reduction states were prepared by adding different concentrations of Lascorbic acid during the synthesis, which were named as R0POM (with no reduction), RmPOM (at medium reduction level) and RhPOM (at high reduction level) (Figure S8). Thanks to the metal-carboxyl coordination chemistry, the two carboxyl groups in the CR dye could link with POM clusters to form hybrid CR-POM nanoprobes through the selfassembly of POM and CR in water. Corresponding to the three types of POM clusters at varied reduction levels, three


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different kinds of hybrid CR-POM nanoprobes (CR-R0POM, CR-RmPOM and CR-RhPOM) were prepared to compare their responsiveness to GSH reduction. X-ray photoelectron spectroscopy (XPS) spectra showed that part of Mo in these nanoprobes was reduced to different levels from VI to V oxidation state (Figure S9). The energy dispersive X-ray spectroscopy (EDS) spectrum further validated the successful synthesis of hybrid CR-POM nanoprobes (Figure S10). All of the three spherical CR-POM nanoprobes demonstrated high dispersity at PBS buffer (pH 7.4) with narrow hydrated size distribution centered at 100 nm (shown by transmission electron microscopy (TEM) and dynamic light scattering (DLS) results in Figure 5a-c, Figure S11). Moreover, the hydrated sizes of CR-POM nanoprobes exhibited little change during 30 days of storage in PBS (pH = 7.4) (Figure S12). The stability of CR-POM nanoprobes in vitro was further tested by inductively coupled plasma-optical emission spectrometry (ICP-OES) analysis of potentially leaked Mo element (Figure S13), which showed that the degradation percentages of CR-R0POM, CR-RmPOM and CR-RhPOM were less than 5% in 48 h. After verifying the high dispersity/stability of the three kinds of hybrid CR-POM nanoprobes, their sensitivities to GSH were tested in PBS buffer (pH = 7.4) with varied concentrations of GSH. The colors of the three samples became darker blue with the increasing GSH concentrations. Especially, CR-RhPOM showed the greatest change and darkest blue color (Figure S14). For the absorption spectra of the three kinds of hybrid nanoprobes (Figure 5d-f), the absorption peak of CR at 700 nm decreased gradually by adding increasing concentration of GSH, while the absorption peak of POM at 866 nm showed a positive correlation with the GSH concentration. Significant absorption enhancement at 866 nm was observed for the group of CR-RhPOM, which was in sharp contrast to the inapparent absorption change of CRR0POM at 866 nm. The ratiometric spectral changes of these nanoprobes towards GSH could be quantified by the ratio of absorbance at 866 nm to that at 700 nm (Ab866/Ab700). Among these three nanoprobes, CR-RhPOM showed the best liner correlation between the ratiometric absorbance (Ab866/Ab700) and GSH concentration with the highest slope and r2 value (Figure 5g-i). The limit of detection (LOD) values of CRR0POM, CR-RmPOM, and CR-RhPOM to GSH concentrations were calculated to be 1.584 mM, 0.441 mM and 0.314 mM, respectively. The calculation method for LOD was according to the following formula: LOD = 3SD/K. (SD: standard deviation of the response, K: the slope of the calibration curve). Importantly, the LOD value of CR-RhPOM was low enough to detect the GSH concentration in cells (0.5-10 mM). Moreover, the fast reaction (~ 10 min) between GSH and the CR-RhPOM nanoprobe might contribute significantly to the ultrasensitive GSH detection in vitro/in vivo (Figure S15). Therefore, CR-RhPOM was chosen as the best nanoprobe to quantify GSH in the following studies. As shown in Figure 6a, after incubation with increasing concentrations of GSH, CR-RhPOM demonstrated gradual attenuation of PA signal at 700 nm but obvious enhancement of PA signal at 866 nm. A good linear correlation was formed between the PA signal ratio (PA866/PA700) and the GSH concentration with the low LOD value of 0.512 mM and the assay linear range up to 14 mM, which was calculated according to the linear regression curve in Figure 6b (ymax is

about 1.5 according to Figure 5f), revealing the capability of CR-RhPOM for GSH quantification. To make sure whether the acid-activated aggregation will affect the photoacoustic ratio of CR-RhPOM, the GSH response of CR-RhPOM was tested in the PBS solution. The linear correlation changed very little, and the difference was negligible (Figure S16). To further evaluate its specificity and selectivity towards GSH, 14 kinds of other amino acids (10 mM) and H2S (50 µM) were incubated with CR-RhPOM to observe the changes in absorbances and PAI signals (Figure 6c-f, Figure S17). Almost all the other amino acids and H2S did not change the absorption intensity or PA signal of CR-RhPOM, except for slight changes by Cys and HCy. This was because POM could be partially reduced by Cys and Hcy, leading to an overall increase in the absorption intensity of CR-RhPOM. However, CR could not react with these amino acids, so the absorption intensity of CR remained unchanged. Therefore, the final ratio of absorption intensity at 866 nm to 700 nm would change very little, indicating that CR-RhPOM was also insensitive to Cys and HCy. All the above results testified that the CRRhPOM nanoprobe could serve as a colorimetric and photoacoustic chemodosimeter for the ratiometric detection of GSH with high sensitivity and selectivity. Furthermore, the low cytotoxicity and high biocompatibility of CR-RhPOM (Figure S18) guaranteed its application for the in vivo ratiometric PAI of GSH. Encouraged by the exciting results of ratiometric PA imaging of GSH in vitro, the potential of CR-RhPOM as a ratiometric PA probe for real-time monitoring of GSH in vivo was first tested in normal mice. The mice were subcutaneously injected with 50 µL of CR-RhPOM (100 µg mL-1), and then the PA signals were acquired at 700 and 866 nm with or without subcutaneous pre-injection of GSH (10 mM) (Figure S19a). Free dye CR and RhPOM were also evaluated as control groups. The mice injected with CR-RhPOM but without pre-injection of GSH, exhibited strong PA700 signal and weak PA866 signal with a PA866/PA700 ratio value of only 0.33. For the mice pre-injected with GSH, the PA700 signal drastically decreased and the PA866 signal remarkably increased with a PA866/PA700 ratio up to 1.2 (Figure S19b). However, despite pre-injection of GSH, the mice injected with CR or RhPOM, showed either a decrease or an increase for both PA700 and PA866 signals, respectively (Figure S20), resulting in almost no change for the ratio value of PA866/PA700. Although the CR-RhPOM nanoprobe demonstrated high performance in ratiometric PAI of GSH in subcutaneous normal mice model, its ability to monitor the GSH concentration within tumor still remain to be eluidated. Thanks to the unique advantage of Mo-based POM in acidityactivated aggregation through protonation-induced hydrogen bonding,28-29 the high density of surface-decorated POM could drive the self-assembly of the CR-RhPOM nanoprobes into much larger aggregates in the acidic tumor microenvironment, which contributed greatly to their prolonged intratumoral retention and enhanced tumor accumulation. As shown in Figure 7a, CR-RhPOM exhibited uniform and monodisperse spherical morphology with an average diameter of ~100 nm at pH 7.4. Interestingly, when incubation in mildly acidic condition (pH = 6.5), the CR-RhPOM nanoprobes were found to self-assemble into larger particles with a diameter of ~935 nm (Figure 7b), which could be increased to micrometers



with further acidification to pH 5.5 (Figure 7c). Accordingly, the hydrodynamic particle sizes of CR-RhPOM nanoprobes were increased from 93.1 ± 5.6 nm at pH = 7.4 to 945.7 ± 20.6 nm at pH = 6.5 and 1967.1 ± 50 nm at pH = 5.5 (Figure 7d), which further confirmed their pH-responsive self-assembly behavior. The feature of CR-RhPOM in acidity-activated selfaggregation was beneficial to enhance its tumor accumulation (Figure 7e), which was verified by positron emission tomography (PET) imaging. As croconaine dyes were able to bind with divalent metal ions at the carbonyl oxygens of the central five-membered ring, CR-RhPOM could chelate 64Cu with a high radiolabeling yield of more than 90%, as examined by instant thin layer chromatography (iTLC) analyses (Figure S18a). The 64Cu-CR-RhPOM showed very high stability after 48 h of incubation in PBS and mouse serum (Figure S21), indicating its suitability for in vivo PET imaging with negligible 64Cu leakage. The decay-corrected PET images of 64Cu-CR-R POM displayed a high tumor-to-normal contrast in h U87MG tumor-bearing mice (Figure 7f). The tumor uptake of 64Cu-CR-R POM reached a peak with 8.6 ± 0.68 %ID/g at 8 h h postinjection (p.i.), much higher than that of free dye 64Cu-CR (4.35 ± 0.42 %ID/g) at 2 h p.i. (Figure 7g). Moreover, the biodistribution data acquired by gamma counting (Figure 7h) also demonstrated that the tumor uptake of 64Cu-CR-RhPOM was 3.4-fold higher than that of 64Cu-CR, which should be attributed to the acidic TME that activated the POM-driven aggregation of CR-RhPOM nanoprobes for enhanced tumor accumulation. The enhanced tumor accumulation of CR-RhPOM via acidic TME-activated self-assembly featured its application in tumor-specific PAI of GSH. The PA signals were simultaneously recorded at 700 nm (rainbow) and 866 nm (red) in the U87MG xenograft tumor model after intravenous injection of CR-RhPOM. The PA signal at 700 nm exhibited a slight increase over time upon the injection of CR-RhPOM and reached a peak at 8 h, while that at 866 nm presented a fast increase over time from 0 to 8 h and reached the maximum at 8 h p.i. (Figure 8a). The difference of PA signal changes at 700 nm and 866 nm might be attributed to their different “accumulation–attenuation equilibrium” mechanisms: the relatively weak PA signal intensity at 700 nm possibly resulted from the opposite interaction between GSH reductioninduced PA signal decrease of CR at 700 nm and acidic TMEtriggered tumor accumulation of CR-RhPOM; However, the strong PA signal intensity at 866 nm should be ascribed to synergistic effect of both the absorption intensification of RhPOM at 866 nm activated by the high concentration of intratumoral GSH and enhanced tumor accumulation of CRRhPOM. Based on the different behavior of these two channels, the ratiometric PA signal (PA866/PA700) was shown to increase gradually over time and reached the maximum (1.33 ± 0.12) at 8 h p.i. (Figure 8c). For comparison, the above PA imaging studies were also performed on mice, which were treated with an inhibitor of GSH synthesis, Lbuthionine-S, R-sulfoximine (BSO), to deplete intratumoral GSH. As a result, the PA signals at these two wavelengths demonstrated an opposite trend to the group without GSH inhibitor, which meant that the PA signal at 700 nm showed a strong increase while that at 866 nm exhibited a weak increase (Figure 8b). As such, the ratiometric PA signal (PA866/PA700) was much weaker than that of the untreated group, which

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reached a peak at only 0.68 ± 0.08 (Figure 8c). According to the linear correlation between the GSH concentration and the ratiometric PA value (PA866/PA700), the GSH concentration at U87MG tumor sites was calculated to be about 8.6 mM, whereas it was decreased to be about 2.8 mM when the mice was treated with a GSH inhibitor (Figure 8d). To verify the accuracy of the quantification of intratumoral GSH by ratiometric PA imaging, a GSH colorimetric detection kit was employed to quantitatively measure the GSH concentration ex vivo within tumor (Figure 8d). The results showed that the estimated GSH level from the ratiometric PA imaging in vivo correlated well with that measured by the GSH detection kit ex vivo, which confirmed that the ratiometric PA imaging of the well-designed CR-RhPOM could succeed in sensitive detection of intratumural GSH. CONCLUSION In summary, we have rationally designed a novel PAI organic-inorganic hybrid nanoprobe for ratiometric detection of GSH with high specificity and sensitivity. The ratiometric PAI nanoprobe was established on the opposite NIR absorbance changes of CR dye and POM in response to GSH, which showed enhanced tumor accumulation for tumorspecific real-time ultrasensitive monitoring of GSH in vivo, due to the substantially increased EPR effect by the aciditydriven self-assembly and aggregation of CR-POM nanoprobes in situ. More importantly, the ratio between the two wavelengths (PA866/PA700) was much higher than most existing ratiometric probes, since one of the PA signal value (PA700 of CR) was decreased and the other one (PA866 of POM) was increased in presence of GSH, leading to improved sensitivity. Furthermore, the LOD (0.51 mM) and maximum detection value (about 14 mM) cover exactly the range of the GSH concentration in vivo (0.5-10 mM), which was highly competent for non-invasive quantification of GSH in vivo. The collective merits of ratiometric PAI of GSH and acidityactivated self-aggregation also feature the extensive application of CR-POM nanoprobes for non-invasive accurate diagnosis of other GSH-related biological processes and diseases. EXPERIMENTAL SECTION Materials. All chemicals were purchased from Alfa Aesar, Thermo Scientific, Fischer Scientific, or Sigma-Aldrich and used without further purification. Ultrapure water was prepared using a Milli-Q Plus System. Synthesis of methyl 1-(thiophen-2-yl)piperidine-4carboxylate (1). Methyl isonipecotate (1.07 g, 7.5 mmol, 1.5 equiv) and thiophene-2-thiol (0.58 g, 5 mmol, 1 equiv) were dissolved in toluene (10 mL) and refluxed under argon atmosphere for overnight. Then the toluene was evaporated under vacuum and the solid was purified by column chromatography on silica gel using hexane and ethyl acetate as the eluent, affording the product as white solid with a yield of about 78%. 1H NMR (300 MHz, CDCl3): δ (ppm) 6.83-6.80 (q, J = 6 Hz, 1H), 6.64 (q, J = 6 Hz, 1H), 6.17-6.16 (q, J = 3 Hz, 1H), 3.59-3.53 (m, 2H), 2.92-2.83 (m, 2H), 2.52-2.42 (m, 1H), 2.10-1.87 (m, 4H).13C NMR (75 MHz, CDCl3) δ 175.06, 159.46, 126.14, 112.43, 105.69, 51.85, 51.57, 40.48, 27.68 ppm. ESI-MS m/z: 225.14 (M)+. Synthesis of (Z)-2-(5-(4-acetoxypiperidin-1-yl)-thiophen2-yl)-5-(5-(4-(methoxycarbonyl)-piperidin-1-ium-1ylidene)-thiophen-2(5H)-ylidene)-3,4-dioxocyclopent-1-en-


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1-olate (2). A solution of compound 1 (225 mg, 1 mmol, 2 equiv) and croconic acid (71 mg, 0.5 mmol, 1 equiv) in the mixture of n-butanol and toluene (10 mL, 1:1) was stirred at 120 oC for 2 h. After cooling to room temperature, the mixture was filtered and washed with methanol to afford the product as black solid (230 mg, 0.415 mmol, 83%). 1H NMR (300 MHz, DMSO-d6): δ (ppm) 8.53 (s, 2H), 7.04 (s, 2H), 4.01 (d, J = 12 Hz, 4H), 3.64 (s, 6H), 3.53 (t, J = 12 Hz, 4H), 3.36 (s, 1H), 2.79 (t, J = 9 Hz, 2H), 2.06 (d, J = 12 Hz, 4H), 1.81-1.70 (m, 4H), 1.23 (s, 2H). Synthesis of (Z)-5-(5-(4-carboxypiperidin-1-ium-1ylidene)thiophen-2(5H)-ylidene)-2-(5-(4-carboxypiperidin 1-yl)thiophen-2-yl)-3,4-dioxocyclopent-1-en-1-olate (3, CR). A mixture of compound 2 (200 mg, 0.36 mmol, 1 equiv), 1Methylimidazole Hydrobromide (176 mg, 1.08 mmol, 3 equiv) and methanesulfonic acid (103 mg, 1.08 mmol, 3 equiv) was magnetically stirred and heated to 120 oC for 2 h. After the completion of the reaction, the product was purified by semipreparative HPLC to afford CR as black powder (171 mg, 90% yield). 1H NMR (300 MHz, DMSO-d6): δ (ppm) 12.48 (s, 2H), 8.53 (s, 2H), 7.04 (s, 2H), 3.99 (s, 4H), 3.52 (s, 4H), 2.67 (s, 2H), 2.04 (d, J = 9 Hz, 4H), 1.73 (q, J = 21 Hz, 4H), 1.23 (s, 1H). MS (ESI): m/z = 528.389 [M + H]+. Quantum chemistry. All density functional theory (DFT) and time-dependent-DFT (TD-DFT) calculations of CR, CRGSH, CR-Hcy, and CR-Cys were performed using Guassian 09. The structures of CR, CR-GSH, CR-Hcy, and CR-Cys were generated with Schrödinger Maestro (Schrödinger Release 2016-2: Maestro, Schrödinger, LLC, New York NY, 2016) and the geometry of the ground states was optimized using a tight self-consistent field convergence criterion of 10-8 hartree. All DFT and TD-DFT calculations were carried out with the hybrid B3LYP functional, the 6-31g (d) basis set, and an ultrafine accuracy level and integration grid with maximum grid density. For the calculation of the electronic spectra, the excitation energies and oscillator strengths for excited states were computed using linear response TD-DFT with the Tamm-Dancoff approximation (TDA) for all geometry optimized CR, GSH, and CR-GSH. Preparation of POM Clusters at Varied Levels of Reduction. The POM clusters were synthesized by a facile one-pot method. 1.17 mmol of NaH2PO4•12H2O dissolved in 5 mL of ultrapure water was rapidly added into a 10 mL solution of 2 mmol of (NH4)6Mo7O24·4H2O, and stirred at room temperature. Afterwards, to obtain the reduced POM at varied reduction levels, which were named as R0POM, RmPOM and RhPOM, 2 mL solution of L-ascorbic acid at different concentrations of 0, 150 and 600 mg/mL was dropwise added into the solution under stirring, respectively. After stirring for another 15 min, the resulting POM clusters were precipitated with 50 mL of ethanol, separated by centrifugation, washed with water and ethanol for three times, and finally dried in a lyophilizer. Preparation of Hybrid Croconaine Dye-POM NPs (CRR0POM, CR-RmPOM, and CR-RhPOM). Generally, CR (10 mg) was dissolved in DMSO (1 mL), and the three different kinds of POM solutions were prepared by dissolving 50 mg of POM in 10 mL of deionized water. The CR solution was slowly dropped into the POM solution under sonication and the mixture was dialyzed (MWCO: 8kDa) against PBS (pH 7.4) under bubbling of nitrogen flow to remove excess free

dye and POM (1-2 nm). The final hybrid CR-POM NPs were dissolved in PBS and filtered through a 0.22 μm filter for further experiments. The POM loading content of CR-POM was calculated by ICP-OES, which was about 35%. Cytotoxicity Test. Cells were seeded in 96 well plates (1000 cells in 100 µL per well) for 24 h, and then the three different materials (CR, RhPOM, and CR-RhPOM) at varied concentrations (0, 5, 10, 25, 50, and 100 µg/mL) were added into the media. Cells were incubated for another 24 h then followed by the addition of MTT (20 µL, 5 mg/mL) for 4 h of incubation. The precipitate was dissolved by adding 150 µL of DMSO and the absorbance at 490 nm was measured with a microplate reader. The cell proliferation was expressed as the percentage of untreated control cells. Radiolabeling and in Vitro Stability Studies. 100 μL of CR-RhPOM (1 mg/mL in water) was added into a solution of 64CuCl (222 MBq) diluted with 2 mL of 0.1 M sodium acetate 2 buffer (pH 5.5). The mixture was shaken at 50 °C for 15 min and the labeling conversion was tested by iTLC; the complexed product was purified by passing through a PD-10 column using PBS as the eluent. The in vitro stability study was conducted by incubating the 64Cu-CR-RhPOM in PBS and mouse serum at 37 °C for 48 h and then evaluated by iTLC. In Vivo PET Imaging. All animal experiments were conducted under the protocol approved by the National Institutes of Health Clinical Center Animal Care and Use Committee (NIH CC/ACUC). U87MG cells (2×106) were subcutaneously injected into the right back flank of mice (athymic nude, 5 weeks old) to establish the tumor model. When the tumor size reached ∼100 mm3, the mice were intravenously injected with around 100 µCi of 64Cu-CRRhPOM and then scanned on a micro PET (Siemens Inveon) scanner at different time points. The 3-dimensional region of interests (ROIs) were drawn on the tumor area in decaycorrected PET images to calculate the tumor uptake. In Vitro and in Vivo PA Imaging. For the in vitro experiments, the CR-RhPOM solutions treated with different concentrations of GSH or 10 mM of different amino acids were filled into polyethylene 50 capillaries. The tubes mentioned above were placed in a container filled with 1% of agarose gel to perform the PA imaging. For in vivo imaging, U87MG tumor-bearing mice were treated with different samples (CR, RhPOM, CR-RhPOM and CR-RhPOM + BSO) solutions in PBS (150 µL, 50 µg/mL, CR content) by intravenous injection. PA signals were monitored at different post-injection time points at 700 and 866 nm by Visual Sonic Vevo 2100 LAZR system. Average PA intensities were acquired from the region of interests (ROIs). Statistical Analyses. Data were reported as mean values ± SE. We compared groups by the Student’s t test. The significance level was defined as P < 0.05.

ASSOCIATED CONTENT Supporting Information The 1H NMR, 13C NMR, and GC-MS spectrum, the XPS, EDS, and DLS characterization, the stability and cell viability of probe, the in vivo PA imaging. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author



*[email protected]; [email protected]; [email protected].

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

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was funded by the National Science Foundation of China (51602203, 81601489), and the Intramural Research Program (IRP), National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH). Longguang Tang was partially funded by the China Scholarship Council (CSC).

REFERENCES (1) Meister, A.; Anderson, M. E., Glutathione. Annu. Rev. Biochem. 1983, 52, 711. (2) Yuan, L.; Kaplowitz, N., Glutathione in Liver Diseases and Hepatotoxicity. Mol. Aspects Med. 2009, 30, 29. (3) Pocernich, C. B.; Butterfield, D. A., Elevation of Glutathione as a Therapeutic Strategy in Alzheimer Disease. Biochim. Biophys. Acta 2012, 1822, 625. (4) Smeyne, M.; Smeyne, R. J., Glutathione Metabolism and Parkinson's Disease. Free Radic. Biol. Med. 2013, 62, 13. (5) Herzenberg, L. A.; De Rosa, S. C.; Dubs, J. G.; Roederer, M.; Anderson, M. T.; Ela, S. W.; Deresinski, S. C.; Herzenberg, L. A., Glutathione Deficiency is Associated with Impaired Survival in HIV Disease. Proc. Natl. Acad. Sci. U. S. A. 1997, 94, 1967. (6) Zimmermann, C.; Winnefeld, K.; Streck, S.; Roskos, M.; Haberl, R. L., Antioxidant Status in Acute Stroke Patients and Patients at Stroke risk. Eur. Neurol. 2004, 51, 157. (7) Shimizu, H.; Kiyohara, Y.; Kato, I.; Kitazono, T.; Tanizaki, Y.; Kubo, M.; Ueno, H.; Ibayashi, S.; Fujishima, M.; Iida, M., Relationship between Plasma Glutathione Levels and Cardiovascular Disease in a Defined Population: the Hisayama Study. Stroke 2004, 35, 2072. (8) Sekhar, R. V.; McKay, S. V.; Patel, S. G.; Guthikonda, A. P.; Reddy, V. T.; Balasubramanyam, A.; Jahoor, F., Glutathione Synthesis is Diminished in Patients with Uncontrolled Diabetes and Restored by Dietary Supplementation with Cysteine and Glycine. Diabetes Care 2011, 34, 162. (9) Townsend, D. M.; Tew, K. D.; Tapiero, H., The Importance of Glutathione in Human Disease. Biomed. Pharmacother. 2003, 57, 145. (10) Vidyasagar, M. S.; Kodali, M.; Prakash Saxena, P.; Upadhya, D.; Murali Krishna, C.; Vadhiraja, B. M.; Fernandes, D. J.; Bola Sadashiva, S. R., Predictive and Prognostic Significance of Glutathione Levels and DNA Damage in Cervix Cancer Patients Undergoing Radiotherapy. Int. J. Radiat. Oncol. Biol. Phys. 2010, 78, 343. (11) Traverso, N.; Ricciarelli, R.; Nitti, M.; Marengo, B.; Furfaro, A. L.; Pronzato, M. A.; Marinari, U. M.; Domenicotti, C., Role of Glutathione in Cancer Progression and Chemoresistance. Oxid. Med. Cell. Longev. 2013, 2013, 972913. (12) Balendiran, G. K.; Dabur, R.; Fraser, D., The Role of Glutathione in Cancer. Cell Biochem. Funct. 2004, 22, 343. (13) Estrela, J. M.; Ortega, A.; Obrador, E., Glutathione in Cancer Biology and Therapy. Crit. Rev. Clin. Lab. Sci. 2006, 43, 143. (14) Ishimoto, T.; Nagano, O.; Yae, T.; Tamada, M.; Motohara, T.; Oshima, H.; Oshima, M.; Ikeda, T.; Asaba, R.; Yagi, H.; Masuko, T.; Shimizu, T.; Ishikawa, T.; Kai, K.; Takahashi, E.; Imamura, Y.; Baba, Y.; Ohmura, M.; Suematsu, M.; Baba, H.; Saya, H., CD44 Variant Regulates Redox Status in Cancer Cells by Stabilizing the xCT

Page 6 of 15

Subunit of System xc(-) and Thereby Promotes Tumor Growth. Cancer Cell 2011, 19, 387. (15) Chen, X.; Zhou, Y.; Peng, X.; Yoon, J., Fluorescent and Colorimetric Probes for Detection of Thiols. Chem. Soc. Rev. 2010, 39, 2120. (16) Niu, L. Y.; Guan, Y. S.; Chen, Y. Z.; Wu, L. Z.; Tung, C. H.; Yang, Q. Z., BODIPY-based Ratiometric Fluorescent Sensor for Highly Selective Detection of Glutathione over Cysteine and Homocysteine. J. Am. Chem. Soc. 2012, 134, 18928. (17) Lim, S. Y.; Hong, K. H.; Kim, D. I.; Kwon, H.; Kim, H. J., Tunable Heptamethine-azo Dye Conjugate as an NIR Fluorescent Probe for the Selective Detection of Mitochondrial Glutathione over Cysteine and Homocysteine. J. Am. Chem. Soc. 2014, 136, 7018. (18) Yin, J.; Kwon, Y.; Kim, D.; Lee, D.; Kim, G.; Hu, Y.; Ryu, J.-H.; Yoon, J., Cyanine-Based Fluorescent Probe for Highly Selective Detection of Glutathione in Cell Cultures and Live Mouse Tissues. J. Am. Chem. Soc. 2014, 136, 5351. (19) Wang, L. V.; Hu, S., Photoacoustic Tomography: in Vivo Imaging from Organelles to Organs. Science 2012, 335, 1458. (20) Zhao, Z.; Fan, H.; Zhou, G.; Bai, H.; Liang, H.; Wang, R.; Zhang, X.; Tan, W., Activatable Fluorescence/MRI Bimodal Platform for Tumor Cell Imaging via MnO2 Nanosheet-aptamer Nanoprobe. J. Am. Chem. Soc. 2014, 136, 11220. (21) Ntziachristos, V.; Razansky, D., Molecular Imaging by Means of Multispectral Optoacoustic Tomography (MSOT). Chem. Rev. 2010, 110, 2783. (22) Jiang, Y.; Upputuri, P. K.; Xie, C.; Lyu, Y.; Zhang, L.; Xiong, Q., Broadband Absorbing Semiconducting Polymer Nanoparticles for Photoacoustic Imaging in Second Near-Infrared Window. Nano Lett. 2017, 17, 4964. (23) Pu, K.; Shuhendler, A. J.; Jokerst, J. V.; Mei, J.; Gambhir, S. S.; Bao, Z.; Rao, J., Semiconducting Polymer Nanoparticles as Photoacoustic Molecular Imaging Probes in Living Mice. Nat. Nanotechnol. 2014, 9, 233. (24) Zhang, J.; Zhen, X.; Upputuri, P. K.; Pramanik, M.; Chen, P.; Pu, K., Activatable Photoacoustic Nanoprobes for In Vivo Ratiometric Imaging of Peroxynitrite. Adv. Mater. 2017, 29, 1604764. (25) Chen, Q.; Liang, C.; Sun, X.; Chen, J.; Yang, Z.; Zhao, H.; Feng, L.; Liu, Z., H2O2-Responsive Liposomal Nanoprobe for Photoacoustic Inflammation Imaging and Tumor Theranostics via In Vivo Chromogenic Assay. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, 5343. (26) Gong, F.; Cheng, L.; Yang, N.; Jin, Q.; Tian, L.; Wang, M.; Li, Y.; Liu, Z., Bimetallic Oxide MnMoOX Nanorods for in Vivo Photoacoustic Imaging of GSH and Tumor-Specific Photothermal Therapy. Nano Lett. 2018, 18, 6037. (27) Yin, C.; Tang, Y.; Li, X.; Yang, Z.; Li, J.; Li, X.; Huang, W.; Fan, Q., A Single Composition Architecture-Based Nanoprobe for Ratiometric Photoacoustic Imaging of Glutathione (GSH) in Living Mice. Small 2018, 14, 1703400. (28) Zhang, C.; Bu, W.; Ni, D.; Zuo, C.; Cheng, C.; Li, Q.; Zhang, L.; Wang, Z.; Shi, J., A Polyoxometalate Cluster Paradigm with SelfAdaptive Electronic Structure for Acidity/Reducibility-Specific Photothermal Conversion. J. Am. Chem. Soc. 2016, 138, 8156. (29) Ni, D.; Jiang, D.; Valdovinos, H. F.; Ehlerding, E. B.; Yu, B.; Barnhart, T. E.; Huang, P.; Cai, W., Bioresponsive Polyoxometalate Cluster for Redox-Activated Photoacoustic Imaging-Guided Photothermal Cancer Therapy. Nano Lett. 2017, 17, 3282. (30) Ni, D.; Jiang, D.; Kutyreff, C. J.; Lai, J.; Yan, Y.; Barnhart, T. E.; Yu, B.; Im, H.-J.; Kang, L.; Cho, S. Y.; Liu, Z.; Huang, P.; Engle, J. W.; Cai, W., Molybdenum-based Nanoclusters Act as Antioxidants and Ameliorate Acute Kidney Injury in Mice. Nat. Commun. 2018, 9, 5421. (31) Yang, Z.; Fan, W.; Tang, W.; Shen, Z.; Dai, Y.; Song, J.; Wang, Z.; Liu, Y.; Lin, L.; Shan, L.; Liu, Y.; Jacobson, O.; Rong, P.; Wang, W.; Chen, X., Near-Infrared Semiconducting Polymer Brush and pH/GSH-Responsive Polyoxometalate Cluster Hybrid Platform for Enhanced Tumor-Specific Phototheranostics. Angew. Chem. Int. Ed. Engl. 2018, 57, 14101. (32) Tang, W.; Fan, W.; Wang, Z.; Zhang, W.; Zhou, S.; Liu, Y.; Yang, Z.; Shao, E.; Zhang, G.; Jacobson, O.; Shan, L.; Tian, R.;


Page 7 of 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Cheng, S.; Lin, L.; Dai, Y.; Shen, Z.; Niu, G.; Xie, J.; Chen, X., Acidity/Reducibility Dual-Responsive Hollow Mesoporous Organosilica Nanoplatforms for Tumor-Specific Self-Assembly and Synergistic Therapy. ACS Nano 2018, 12, 12269. (33) Yin, C.; Huo, F.; Zhang, J.; Martinez-Manez, R.; Yang, Y.; Lv, H.; Li, S., Thiol-addition Reactions and Their Applications in Thiol Recognition. Chem. Soc. Rev. 2013, 42, 6032. (34) Umezawa, K.; Yoshida, M.; Kamiya, M.; Yamasoba, T.; Urano, Y., Rational Design of Reversible Fluorescent Probes for Live-cell Imaging and Quantification of Fast Glutathione Dynamics. Nat. Chem. 2017, 9, 279. (35) Sreejith, S.; Divya, K. P.; Ajayaghosh, A., A Near-infrared Squaraine Dye as a Latent Ratiometric Fluorophore for the Detection of Aminothiol Content in Blood Plasma. Angew. Chem. Int. Ed. Engl. 2008, 47, 7883.

(36) Hewage, H. S.; Anslyn, E. V., Pattern-based Recognition of Thiols and Metals Using a Single Squaraine Indicator. J. Am. Chem. Soc. 2009, 131, 13099. (37) Shin, I. S.; Gwon, S. Y.; Kim, S. H., Chromogenic Sensing of Biological Thiols Using Squarylium Dye. Spectrochim. Acta. A. Mol. Biomol. Spectrosc. 2014, 120, 642. (38) Zhang, X.; Chao, L.; Cheng, X.; Wang, X.; Zhang, B., A Near-Infrared

Croconium

Dye-based

Colorimetric

Chemodosimeter for Biological Thiols and Cyanide Anion. Sens.

Actuators B Chem. 2008, 129,152.



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Figures and Figure Captions

Figure 1. Schematic illustration of the CR-POM nanoprobe for specific ratiometric PA imaging of intratumoral GSH. CR-POM is first synthesized through the self-assembly of CR dye and POM clusters. CR-POM exhibits two typical absorbance peaks at 866 nm (red) and 700 nm (blue) to afford two PA signals with a small PA866/PA700 ratio. The acidic TME further triggers the conversion of CR-POM from small nanoparticles (105 nm) into large particles (1-2 µm), which contributes greatly to enhancing its selective retention at tumor sites and amplifying its PA signals. In the presence of GSH, the PA866 signal becomes stronger while PA700 signal gets weaker, which results in a large PA866/PA700 ratio.


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Figure 2. (a) Chemical structures and synthetic scheme for CR. Reagents and conditions: (i) Toluene, reflux, 4 h, 78%; (ii) Toluene/nButanol, croconic acid, reflux, 5 h, 83%; (iii) 1-Methylimidazole hydrobromide, methanesulfonic acid, 120 ℃, 2 h, 90%. (b) Proposed reaction between CR and GSH. (c) LC-MS spectrometry of CR before (top) or after (bottom) adding GSH. (d) UV-Vis absorption spectra of CR (25 μg/mL) after incubation with 10 mM of Cys, Hcy and GSH in 1×PBS (pH = 7.4) for 5 min. (e) UV-Vis absorption spectra of CR (25 μg/mL) upon addition of GSH.



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Figure 3. (a) Geometry optimized 3D structures of CR before and after reaction with GSH, Cys and Hcy. (b) Computed absorbance spectra of CR and CR-GSH obtained by TD-DFT TDA.


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Figure 4. (a) Schematic illustration of POM clusters with conversion of oxidation states of Mo(VI) to Mo(V) upon GSH reduction. (b) UV-Vis absorption spectra of RmPOM clusters dispersed in deionized water before and after incubation with various concentrations of GSH.

Figure 5. Three kinds of CR-POM nanoprobes and their different responsive activities towards GSH. (a-c) TEM iamges of (a) CR-R0POM, (b) CR-RmPOM, and (c) CR-RhPOM. (d-e) UV-Vis absorption spectra of (d) CR-R0POM, (e) CR-RmPOM, and (f) CR-RhPOM after reacting with varied concentrations of GSH. (g-i) Ratiometric absorbance ratios (Ab866/Ab700) of (g) CR-R0POM, (h) CR-RmPOM, and (i) CR-RhPOM as a function of GSH concentration.



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Figure 6. (a) PA imaging of the CR-RhPOM nanoprobe at wavelengths of 700 nm and 866 nm after reaction with varied concentrations of GSH. (b) Ratiometric PA signals (PA866/PA700) of CR-RhPOM as a function of GSH concentration. The red line represents linear fitting. (c) UV-vis absorption spectra of CR-RhPOM (1 µM) in the absence (control) or presence of different amino acid (10 mM) in PBS. (d) Quantification of the absorbance ratio (Ab866/Ab700) of CR-RhPOM in the absence or presence of different analytes (10 mM). (e) PA imaging of CR-RhPOM at wavelengths of 700 nm and 866 nm in the absence (control) or presence of Hcy, Cys, or GSH. (f) The ratiometric PA signals of CR-RhPOM (1 µM) in the absence (control) or presence of different amino acid (10 mM). The error bars represent the standard deviation of three separate measurements.


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Figure 7. (a-c) TEM images of CR-RhPOM nanoprobes at pH = 7.4 (a), pH = 6.5 (b), and pH = 5.5 (c). (d) DLS measurements of CRRhPOM nanoprobes with successive acidifications from pH = 7.4 to pH = 6.5 and to pH = 5.5. (e) Schematic diagram of the self-assembly and absorbance change behavior of CR-RhPOM in response to the intratumoral acidity and reducibility in vivo. (f) Representative PET images of U87MG tumor-bearing mice at different time points post-injection of 64Cu-CR and 64Cu-CR-RhPOM. (g) The corresponding tumor uptake (white cycle in f) quantified from decay-corrected PET images in (f). n = 5. (h) Biodistribution of 64Cu-CR and 64Cu-CRRhPOM determined by gamma counting at 24 h post injection. n = 5, **p

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