A Mn(III)-Sealed MetalâOrganic Framework Nanosystem for Redox

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A Mn(III)-Sealed Metal-Organic Framework Nanosystem for Redox-Unlocked Tumor Theranostics Shuang-Shuang Wan, Qian Cheng, Xuan Zeng, and Xian-Zheng Zhang ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.9b00300 • Publication Date (Web): 28 May 2019 Downloaded from http://pubs.acs.org on May 29, 2019

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A Mn(III)-Sealed Metal-Organic Framework Nanosystem for Redox-Unlocked Tumor Theranostics

Shuang-Shuang Wan, Qian Cheng, Xuan Zeng and Xian-Zheng Zhang*

Key Laboratory of Biomedical Polymers of Ministry of Education & Department of Chemistry, the Institute for Advanced Studies, Wuhan University, Wuhan 430072, P. R. China

* Corresponding author. E-mail: [email protected]

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ABSTRACT: Here, a Mn(III)-sealed metal-organic framework (MOF) nanosystem based on coordination between Mn(III) and porphyrin (TCPP) via a one-pot method was designed and constructed. Mn(III), as a sealer, not only quenched TCPP-based fluorescence but also inhibited reactive oxygen species (ROS) generation, which made MOFs an “inert” theranostic nanoparticle. Interestingly, upon endocytosis by tumor cells, MOFs were disintegrated into Mn(II) and free TCPP by intracellular glutathione (GSH) in tumor cells, owing to redox reaction between Mn(III) and GSH. This disintegration would lead to consumption of antioxidant GSH and activated Mn(II)based magnetic resonance imaging (MRI) as well as TCPP-based fluorescent imaging. More importantly, such a GSH-regulated TCPP release could implement controllable ROS generation under irradiation, which avoided side effects (inflammation and damage of normal tissues). As a consequence, after unlocking by GSH, Mn(III)-sealed MOFs could significantly improve therapeutic efficiency of photodynamic therapy (PDT) by combining controlled ROS generation and GSH depletion after precise dual tumor homing.

KEYWORDS: tumor theranostics, metal-organic framework, GSH depletion, controllable ROS generation, GSH-activated imaging


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In recent years, photodynamic therapy (PDT) as a minimally invasive therapeutic modality has been extensively studied for tumor treatment,1-3 which induces tumor cell death by transferring the energy of the photo-excited photosensitizer (PS) to oxygen to produce highly toxic reactive oxygen species (ROS).4 However, to date, further clinical application of PDT is still restricted by plenty of intractable problems.5 As a key element of PDT, the commonly used PSs possess poor tumor targeting and accumulation capability due to hydrophobic nature, complicated synthetic modification, low payloads, easy aggregation, etc.6,7 In addition, the high expression of glutathione (GSH) in tumor cells not only resists chemo-, radio- and photodynamic therapy but also serves as an antioxidant to scavenge cellular ROS,8,9 which is one of the most challenge for PDT application in vivo.10,11 More importantly, it is documented that excessive ROS is able to cause side effects, including phototoxicity on normal tissues and proinflammation at tumor region.12,13 Thus, an intelligent delivery system that simultaneously achieves effective PS-mediated ROS generation and elimination of intracellular GSH, is urgent to improve PDT at tumor site. Very recently, metal-organic framework nanoparticles (nMOFs) constructed by coordination of metal ion/clusters and organic bridging ligand, have drawn extensive attention

owing

to

high

drug

loading

ability,

intrinsic

biodegradability,

structural/compositional tunability, controlled size/shapes.14-18 Among which, porphyrin based nMOFs with high PS loading and facile diffusion of singlet oxygen (1O2) as well as avoidable self-quenching of porphyrinic fluorescence, are widely used for PDT of tumor.19-21 Although integrating metal ion/clusters function to maximize the


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antitumor efficiency of porphyrin based MOFs is a popular trend, the synthetic difficulty makes the utilization of metal ion/clusters in infancy.22 Moreover, as far as we know, there are rare reports about responsive nMOFs for specific and efficient tumor theranostics.23,24 Clearly, it is highly desired to exploit potential application of metal ion/clusters of porphyrin based nMOFs with intelligent response. Here, we designed and constructed a GSH-unlocked Mn(III)-sealed MOF nanosystem to achieve dual-modality guided monitored PDT by controllable ROS generation and GSH depletion. Considering oxidation and contrast effect of Mn element,25 Mn(III) was selected as metal nodes to coordinate with TCPP-based ligand for formation of Mn(III)-TCPP MOFs with a one-pot method. Mn(III) from MOFs was expected to lock theranostic function of MOFs due to quenched effect of Mn(III) on TCPP-based fluorescence and ROS generation. However, as depicted in Scheme 1, MOFs could react with intracellular GSH to produce glutathione disulfide (GSSG), Mn(II) and free TCPP after internalization by tumor cells. As a result, Mn(II)-based magnetic resonance imaging (MRI) and TCPP-based optical imaging (OI) would be activated. Simultaneously, controllable ROS generation with reduced side effects could be regulated by GSH-controlled TCPP release, and MOFs-medicated cellular GSH was consumed. Thus, the MOF nanoparticle is possible to achieve comprehensive suppression of tumors via synergistic GSH-controlled ROS generation and GSH depletion in tumor site.

RESULTS AND DISCUSSION


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Preparation and Characterization of Mn(III)-TCPP MOFs. The Mn(III)-TCPP nanoparticles were synthesized by simply stirring the mixture of Mn(OAc)3•2H2O and TCPP in a N,N-dimethylformamide (DMF) solution containing acetic acid (HAc) for 12 h. To characterize the structure of this synthetic nanoparticle, we synthesized a large crystal (Figure S1B) for single crystal diffraction analysis. The crystal structure of Mn(III)-TCPP MOF exhibited the tetragonal crystal system with I4/mmm space group and lattice parameter of a = 16.7860 Å, b = 16.7860 Å, c = 15.7415 Å (Table S1, Figure S2). Powder X-ray diffraction (PXRD) pattern of the nanoscale Mn(III)-TCPP MOFs showed the similarity compared with the single crystal, indicating that Mn(III)-TCPP MOFs with different sizes retain their crystal structure (Figure S3). By adjusting the volume of HAc/DMF solution, different sizes of fusiform-like nanoparticles were obtained (Figure S1A). Among which, in the optimized conditions (25 mL HAc/DMF solution), the nanoparticles possessed a suitable uniform size with monodisperse as measured by scanning electron microscopy (SEM, Figure 1A) and transmission electron microscopy (TEM, Figure 1B), approximately 170 nm in length and 50 nm in width. Besides, Mn(III)-TCPP nanoparticles had a thickness of about 100 nm by atomic force microscope (AFM) (Figure 1C). This demonstrated a spindle-shaped threedimensional structure of nanoparticles. Moreover, TEM mapping images (Figure 1D) and EDX result (Figure S4) showed a clear distribution of Mn, O and C elements, which confirmed that Mn element was involved in the formation of nanoparticles. This conclusion was further confirmed by X-ray photoelectron spectroscopy (XPS) (Figure S5). From the observation of PXRD patterns in Figure 1E, Mn(III)-TCPP nanoparticles


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presented a set of characteristic peaks that were different from ligand (TCPP and Mn(OAc)3) peaks, illustrating the crystal structure of the nanoparticles as presented lattice stripe in Figure 1B (insert). Moreover, the thermogravimetric analysis of Mn(III)-TCPP nanoparticles was carried out, indicating the molar ratio of Mn and TCPP (Figure 1F). In addition, a BET surface area of 169.69 cm2g-1 (pore volume 0.12 cm3g-1) for Mn(III)-TCPP nanoparticles was obtained by N2 sorption isotherm (Figure S6). Interestingly, Mn(III) from MOFs was found to only coordinate with the carboxyl group but not with center nitrogen atoms of TCPP, because MOFs had four Q bands peaks similar to that of TCPP in the UV-vis absorption spectrum instead of the two ones in the structure (TCPP(Mn)) coordinated by Mn center (Figure 1G).26 This conclusion could also be confirmed by the appearance of carboxylate peaks (1650 cm1

and 1400 cm-1) and the retention of amino peak (965 cm-1) of TCPP in infrared

spectroscopy (Figure S7). Such coordination mode would facilitate subsequent fluorescence recovery and ROS generation of TCPP, which would be quenched if TCPP center was coordinated by Mn as reported in the literature.27 Of special note, according to Mn 3s and 2p3/2 peak in XPS spectrum (Figure 1H and S8),28 we confirmed that Mn in coordination with organic ligands (TCPP), was trivalent valence. In addition, divalent and tetravalent coordination of Mn were ruled out because only Mn(III) without unpaired electrons was silent in electron spin-resonance (ESR) measurements (Figure 1I).29 Besides, we still assessed stability of MOFs for better biomedical applications. The time-dependent water stability of MOFs had been detected by DLS, TEM and XRD. The morphology (Figure S9) and crystal structure (Figure S10) as well


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as hydrodynamic size (Figure S11) of nanoparticles did not change too much with time, confirming good stability of MOFs in water. Moreover, such aqueous stability was also verified by socking MOFs in PBS and DMEM culture medium (Figure S12). Compared with MOFs coated with PEG, size and PDI of MOFs in buffer still remained stable (Figure S13), which demonstrated that MOFs without modification did not undergo obvious aggregation. GSH-Triggered Unlocking of MOFs. It was documented that high-valence Mn (Mn(III) or Mn(Ⅳ)) was easily converted to Mn(II) in the presence of reducing agents.30,31 As expected, the ESR signal of MOFs solution was changed from silence to a sextet characteristic peaks of Mn(II) after adding GSH (Figure 1I), which was well matched with that of Mn(OAc)3 solution treated with GSH (Figure S14). In addition, the morphology of Mn(III)-TCPP MOFs was seriously destroyed with the increase of GSH concentration (Figure 1J), and degradants did not form nanoparticles by coordination (Figure S15), which was well matched with increase of size in GSH solution (Figure S16). And these phenomena all indicated GSH concentration-related decomposed behavior of MOFs. Thus, we could draw a conclusion that MOFs could react with GSH to produce Mn(II), GSSG and TCPP, resulting in disintegration of MOFs as depicted in Figure 2A. To quantitatively illustrate the conclusion, the consumption of GSH was firstly detected with 5,5'-dithio-bis-2-(nitrobenzoic acid) (DNTB, Figure S17).32 As shown in Figure 2B, GSSG generation increased with the increase of GSH and MOFs concentration, confirming that GSH could be oxidized into GSSG in the presence of MOFs. On the other hand, disintegration of MOFs in solution


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containing GSH was also evaluated by analyzing degradants (TCPP and Mn ion). In Figure 2C, slow degradation (below 28%) of MOFs at 35 h implied the stability of MOFs in the HEPES buffer solution without GSH. However, once adding GSH, the released TCPP increased significantly over time and exhibited positive correlation with GSH concentration until no obvious change at high concentration (> 2.5 mM). Consistent with the release of TCPP, Mn ion from MOFs exhibited similar released trend (Figure S18). Motivated by the phenomenon, we further explored MRI contrast performance of MOFs. Considering MRI effect of Mn in nanoparticles was deteriorated due to inhibited chemical exchange between Mn and protons,33 T1 signal of MOFs before and after adding GSH (2.5 mM) was detected by MRI system. Compared with control, T1 relaxation rate (r1) had nearly 2.3-fold enhancement in the presence of GSH (Figure 2D), indicating the potential of MOFs as GSH-activated T1 contrast agent for cancer diagnosis. Change in Fluorescence and ROS Generation of MOFs after Unlocking. Next, we evaluated influence of GSH-triggered MOFs unlocking on fluorescence and ROS generation. Due to Förster resonance energy transfer (FRET) caused by overlay between the broad spectral absorption of Mn(III) and emission of TCPP, it was not surprising to observe obvious quenching fluorescence of MOFs compared with that of TCPP (Figure 2E). In contrast, fluorescence intensity of MOFs significantly enhanced with increased GSH concentration from 0 mM to 10 mM (Figure 2F), up to 18.2-fold improvement. Besides, we also assessed the fluorescence change over time at the optimized GSH concentration (2.5 mM). As exhibited in Figure S19, fluorescence of


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TCPP increased gradually until the peak at 200 S in the presence of GSH, indicating rapid and sensitive MOFs degradation by GSH. In contrast, an ignored fluorescence intensity in group without GSH further verified stability of MOFs. In addition to GSHresponsive fluorescence enhancement, it was more important to explore the effect of GSH-unlocking effect on ROS yield. As shown in Figure 2G, by comparing relative fluorescence intensity ((Ft-F0)/Ft) of SOSG (a ROS probe), it was worth noting a positive correlation between ROS yield and GSH concentration at the same irradiation time. This meant that we could control the amount of ROS by GSH-regulated TCPP release for effective PDT with little side effect. In the light of the scavenging effect of GSH as a reducing agent on ROS, the yield of ROS had decreased slightly with irradiation time when the GSH concentration exceeded 2.5 mM. Such phenomenon was also confirmed by a gradually decreasing SOSG fluorescence intensity from 2.5 mM GSH concentration to 10 mM in Figure 2H. Notably, SOSG fluorescence intensity still exhibited a 36 times stronger at excessive GSH (10 mM) than that without GSH, indicating an excellent ROS production ability of MOFs even in a strong antioxidant environment. In view of high expression of tumor cellular GSH (1-10 mM),34 we further compared ROS generation of MOFs and TCPP at 10 mM GSH. As shown in Figure 2I, MOFs-treated SOSG fluorescence intensity was nearly 2 times as high as that of TCPP after a short-time irradiation, demonstrating that MOFs was more advantageous for PDT in an GSH-maintained antioxidant system of tumor cells. In Vitro Disintegration of MOFs and Their Effect on Cellular Oxidative Stress. Subsequently, performance of MOFs in vitro was explored. As observed in Figure 3A,


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endocytic MOFs presented a gradually enhancement of TCPP fluorescence over time, which ascribed to cellular TCPP fluorescence recovery by GSH-triggered MOFs disintegration as proven in Figure 2F. Furthermore, the flow cytometry analysis (Figure 3B and 3C) verified that fluorescence had 6-fold (20 min post-culture) and 18-fold (40 min post-culture) stronger than that without post-culture. Such GSH-activated TCPP fluorescence was beneficial for monitoring disintegration and visualizing movement of MOFs in vitro. Accompany with the process, GSH of 4T1 cells would be consumed owing to redox reactions between MOFs and GSH. As expected, cellular GSH level downgraded to 40% after treatment with MOFs, while GSH content of Mn(OAc)2 and TCPP groups was up to 90% (Figure 3D), implying that Mn(III) from MOF indeed caused cellular GSH depletion. Then, MOFs-medicated change of cellular oxidative pressure was detected by flow cytometry and confocal laser scanning microscopy (CLSM) with DCFH-DA, a ROS probe which emitted green fluorescence after oxidized by ROS. Obviously, fluorescence signal of cells treated with MOFs was nearly 10 times as high as that of control group by flow analysis (Figure 3E and 3F), which was well matched with brighter green fluorescence in the MOFs group by observed in Figure 3G. This phenomenon ascribed to increased intracellular ROS levels indirectly induced by depletion of GSH detected in Figure 3D. What’s more, once irradiated with 660 nm laser, green fluorescence of MOFs by CLSM with ROS probes including ROSDCFHDA (Figure 3G) and SOSG (Figure S20), was further enhanced, approximately 12.6 times and 16.4 times than that of TCPP+ hv and MOFs group respectively. This indicated that MOFs-caused increase of intracellular oxidative stress was far higher


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than that of a single one, demonstrating superiority of MOFs+ hv as a synergistic treatment with simultaneous ROS production and GSH consumption. Besides, to demonstrate the controllable ROS production via GSH-regulated TCPP release in vitro, we still conducted an experiment by comparing MOFs-treated cells with different expression of GSH including tumor (4T1) and normal cells (3T3, Figure S21). The Assessment of MOFs-Induced Cytotoxicity. On the basis of results above, MOFs-induced cytotoxicity was assessed against various cell lines. In Figure 4A, difference cell lines exhibited obvious differences in cell viability, which attributed to the difference in tolerance and sensitivity of various cells to GSH depletion. Of special note, tumor cells (CT26, 4T1 and B16) with MOFs treatment were found a low viability in comparison with normal cells (3T3), indicating tumor-specific killing effect of MOFs while avoiding toxicity to normal cells. Furthermore, to demonstrate PDT enhanced by GSH depletion, we evaluated the cell viability by introducing a GSH synthesis inhibitor, L-buthionine sulfoximine (L-BSO). As expected, at a non-toxic concentration of LBSO (Figure S22), cells pre-treated L-BSO caused a higher cytotoxicity than that of without L-BSO (Figure 4B). Furthermore, anti-tumor effect of MOFs with light was evaluated in vitro. Among all groups treated with different length of particles (Figure S23), MOFs nanoparticles with 170 nm had the best therapeutic effect on cancer cells, demonstrating that particles of this length were best suited for cancer treatment. Besides, as shown in Figure 4C, cells treated with Mn(OAc)2 had a negligible cytotoxicity compared to an obvious decreased viability in MOFs group, which confirmed MOFsinduced toxicity originated from GSH consumption rather than Mn(II)-medicated


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Fenton-like reaction. Inspiringly, when cells were subjected with 660 nm irradiation for 8 min, 4T1 cells proliferation was significantly inhibited and cell viability was only 11.2% at a low concentration of MOFs. Such excellent therapeutic effect was attributed to more severe oxidative stress from TCPP-based ROS generation and Mn(III)-based GSH depletion. In addition, live/dead cell staining assay with Calcein AM and PI was further performed to illustrate the killing ability of tumor cells by MOFs. Matched with results of MTT assay above, MOFs with light presented the highest percentage of dead cells (red fluorescence) of all, nearly 100% (Figure 4D). Furthermore, the lethal mechanism of MOFs was studied by flow cytometry. Obviously, all groups excepted for control were observed cell migration from Q4 (live cell) to Q2 (early apoptosis) and Q3 (late apoptosis) (Figure 4E), suggesting apoptosis-activated cell death pathway. More importantly, percentage of apoptosis cells (Figure S24) in MOFs+ hv group reached 40.1%, obviously higher than that of group treated with MOFs (10.84%) or TCPP+ hv (19.8%). Together, a conclusion was drawn that synergistic therapy with MOFs significantly enhanced apoptosis-induced cell death by combining controlled ROS production and GSH elimination to increase intracellular oxidative stress, which laid a good foundation for treatment in vivo. Imaging Effect of MOFs in Vivo. After that, imaging effect in response to GSH of tumor region was studied by intratumorally injecting with MOFs. The fluorescence of tumor site obviously increased from 0 min to 30 min (Figure 5A), implying GSHunlocked near-infrared fluorescence for optical imaging owing to TCPP gradual release from MOFs by GSH in vivo. Similarly, MRI performance of MOFs in vivo also


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descripted GSH-enhanced Mn-based T1 contrast signal over time after intratumoral injection within 30 min (Figure 5B). More importantly, enhancement of tumoral OI and MRI signal could not only achieve accurate diagnosis but also monitor MOF disintegration for efficient treatment in vivo. Further, accumulation of MOFs in targeting site was explored by intravenous injection. As shown in Figure 5C, a gradual bright tumor region over time suggested enhanced T1 contrast signal and time-related tumor accumulation of MOFs. This accumulation behavior was also confirmed by observing the fluorescence of tumor from progressive increase to slow decrease over time in intravenous OI (Figure 5D). From which, maximum enrichment time was found at 12 h after post-injection, which provided us an optimal irradiation time with 660 nm laser for subsequent efficient PDT. Besides, the strong fluorescence after 12 h implied long-term selective retention of MOFs in tumor sites, which was also reflected in fluorescence signal and the corresponding mean fluorescence intensity (MFI) value (Figure 5E) as well as autofluorescence (Figure S25) of tumor ex vivo at 36 h postinjection. In addition, as shown in Figure 5F, mice treated with MOFs had a higher TCPP remaining levels in blood than that of TCPP, indicating the formulation of nanoparticles could effectively avoid being cleared by protein during blood circulation. Collectively, in comparison with the formulation of a free photosensitizer (TCPP), MOFs showed a greater advantage to target and accumulate as well as retention in tumor sites due to good solubility and optimized size favorable for enhanced permeation and retention (EPR) effect and difficult clearance in pharmacokinetic. The Evaluation of Biosafety and Therapeutic Efficiency. As a fresh synthesized


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material, it was necessary to assess biosafety and biocompatibility for biomedical application. The main parameters of blood biochemistry and routine displayed in Figure 5G and Table S2, similar to that of the blank group, indicted that the material did not produce systemic toxicity to mice even at high doses. In addition, hepatic metabolism of MOFs (Figure S26) and hemolysis test (Figure S27) also implied biocompatibility and biosafety of MOFs. Motivated by the above proof in vivo, therapeutic effect was further assessed on the 4T1 tumor-bearing mice. As observed in Figure 5H, the strong green fluorescence of DCFH in tumor tissues verified MOFs-medicated increased oxidation pressure in vivo by depleting GSH. This could also explain why tumor treated with MOFs+ hv had stronger DCFH fluorescence than that of TCPP+ hv. Subsequently, there was no significant change in the weight of the mice during 14-day treatment in Figure 6A, further suggesting a negligible toxic side effect induced by MOFs on treated mice. More importantly, as shown in Figure 6B, tumor-bearing mice treated with MOFs had a significant inhibition of tumor growth due to Mn(III)-induced oxidative damage. Once irradiated with 660 nm laser, tumor volume in MOFs+ hv group was comprehensively suppressed, which fully demonstrated the dual role (TCPP-based ROS generation and Mn(III)-based GSH elimination) played by MOFs in achieving tumor ablation in vivo. Therefore, on the day of treatment termination (on the 14th day), the weight of tumor tissues (Figure 6C) obtained by anatomy in MOFs+ hv group was the lightest of all, only 0.377 g. And the photograph of tumor (Figure 6D) ex vivo also indicated the smallest size after treatment with MOFs+ hv. Further histological analysis by H&E staining implied that the fewest cancer cells were found in tumor tissues treated


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with MOFs+ hv (Figure 6E) and a negligible side effect on main organ (Figure S28). And immunofluorescence results also verified the most apoptosis cells (Tunel) and the weakest cell proliferation (Ki67) signal in MOF+ hv group. Together, all the above phenomena indicated MOFs integrated controlled ROS generation and GSH depletion, could achieve in vivo comprehensive tumor suppression via synergistic treatment while avoiding side effects on normal tissues. CONCLUSIONS In summary, a Mn(III)-sealed MOF nanosystem was successfully constructed by a one-pot method. In vitro and in vivo experimental results indicated that these MOFs could undergo a reductive disintegration by overexpressed GSH in tumor cells, producing GSSG, Mn(II) and free TCPP. During which, intracellular GSH was consumed, Mn(II)-based MRI and TCPP-based OI were activated. Moreover, GSH could also effectively control ROS generation by regulating TCPP release from MOFs, which could avoid side effect by excessive ROS. By the aid of precisely activatable tumor homing with MRI and OI, MOF nanoparticle with controllable ROS generation and GSH depletion presented comprehensive tumor inhibition by significantly enhanced therapeutic effect of PDT.


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MATERIALS AND METHODS Materials. Dimethyl formamide (DMF) and acetic acid (HAc) were purchased from Shanghai Reagent Chemical Co. (China). Mn(OAc)3·2H2O was obtained from Bide Pharmatech Ltd. 4,4,4,4-(Porphine-5,10,15,20-tetrayl)tetrakis(benzoic acid) (H2TCPP) was obtained from Innochem (China). Glutathione (GSH) was from Macklin (China) and 5,5’-Dithiobis(2-nitrobenzoic acid) (DTNB, 98%) was purchased from Aladdin Reagent Co. Ltd.(China) and singlet oxygen sensor green (SOSG) was purchased from Invitrogen (USA). 2’,7’-dichlorofluorescin diacetate (DCFH-DA), Hoechst 33342, 3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium-bromide (MTT) and Propidium Iodide (PI) were provided by Beyotime Institute of Biotechnology (China). Annexin VFITC/PI apoptosis and necrosis detection kit was obtained from Yeasen, Shanghai, China. Roswell Park Memorial Institute (RPMI) 1640 medium, penicillin-streptomycin, trypsin and fetal bovine serum (FBS) were purchased Gibco Invitrogen Corp (USA). All of the other reagents and solvents were of analytical grade and used as received. Instruments. The UV-vis absorbance spectra were detected using a UV-vis spectrophotometer (Lambda Bio40, Perkin-Elmer) and the fluorescence of all sample were detected by LS55 luminescence spectrometer. Thermogravimetric property (TGA) was detected via a Pyris1 thermo gravimetric analyzer (Perkin-Elmer). The 660 nm lasers (Beijing Laserwave Optoelectronics) were used to the photodynamic therapy (PDT) study. The morphology of nanoparticles was observed by transmission electron microscopy (TEM) (JEM-2100 microscope) and field emission scanning electron microscope (SEM) (Zeiss SIGMA). Small animal imaging was performed on a small animal imaging system (PE Spectrum & Quantum FX). The hydrodynamic size was measured by dynamic light scattering (DLS) on a PSS Z3000 instrument. Confocal laser scanning microscopy (CLSM) images were collected by an UltraVIEW VoX


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(PerkinElmer) confocal laser scanning microscope (CLSM, Nikon C1-Si TE2000, Japan). Intracellular fluorescence change was analyzed by flow cytometry (BD FACSAria™ III, USA) Synthesis of Mn(III)-TCPP MOF Nanoparticles. 10 mL freshly prepared Mn(OAc)3·2H2O (DMF, 10.4 mM) was added into different volume (20 mL, 25 mL, 27 mL, 30 mL, 35 mL and 40 mL) of HAc/DMF (V/V=1/4) solution. After stirring for 5 min, 10 mL H2TCPP (DMF, 1.3 mM) was slowly added into the solution above and the color of the solution turned purple gradually. The reaction was allowed to proceed at room temperature for 12 h and then the purple particles were collected by centrifugation (11 000 rpm, 15 min), following by washing with DMF for 3 times. The Detection of GSH Depletion. The depletion of GSH was detected by Ellman’s reagents. In detail, 5 L DTNB (DMSO, 100 mM) was added into 995 μL GSH solution (0, 0.625 mM, 1.25 mM, 2.5 mM, 5 mM and 10 mM). After being mixed 2 min, the absorbance of the solutions at 405 nm was measured using a microplate reader (BIORAD, Model 550, USA). According to the absorption value, the standard curve of AGSH could be obtained. At the same time, the standard curve of the MOFs at 420 nm was obtained by measuring the absorbance of the nanoparticle solution. Different concentrations of nanoparticle solutions were added in a certain concentration of GSH (C0), and then 5 μL DTNB (100 mM, DMSO) was added to 995 μL mixture. After standing for 10 min, the absorbance (Ax) of mixture was measured. The following formula was used to calculate the consumption of GSH (C): C = (Ax – AMOFs) / (AGSH – AMOFs) × C0 The Disintegration of MOFs in Response to GSH. GSH-responsive MOFs disintegration was estimated by testing TCPP and Mn release. 500 μL MOFs solution (1 mg/mL) was put in dialysis bags (MWCO: 3500 Da), which was immersed in 10 mL


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HEPES buffer containing different GSH concentration (0, 1.25 mM, 2.5 mM, 5 mM and 10 mM) in a horizontal laboratory shake (37 ℃, 100 rpm). At given intervals, 1 mL buffer was taken out and supplemented with 1 mL fresh one. Finally, released TCPP and Mn concentration in dialysate were detected by UV-vis spectrophotometer and ICP-MS (IRIS Intrepid II XSP), respectively. Fluorescence Change of MOFs. 100 μL MOFs were dispersed in 1 mL different concentrations of GSH solutions (0, 0.625 mM, 1.25 mM, 2.5 mM, 5 mM and 10 mM). After being placed at 37 ℃ for 20 min, the fluorescence of solutions were measured. (λex = 415 nm, λem = 630 nm). The same method was used to detect fluorescence change of MOFs in the absence or presence of GSH (0 or 2.5 mM) at different reaction times (0, 0.5 min, 1 min, 2 min, 10 min and 20 min). ROS Detection. Singlet oxygen sensor green (SOSG) agent as a probe was used to monitor reactive oxygen generation. 10 μL SOSG (500 μM, DMSO) was added to 990 μL MOFs solution containing 10 ug MOFs. After irradiated (660 nm, 0.03 W/cm2) for different time (10 s, 20 s, 30 s, 40 s, 60 s, 90 s, 120 s, 150 s, 180 s), fluorescence of the solution at 530 nm was measured. The ROS generation ability was defined as (Ft – F0)/F0, where F0 refers to the initial fluorescence intensity. GSH Depletion in Vitro. According to the method in the previous literature 35 with some modifications. 4T1 cells were seeded in six-well plate and cultured for 24 h. Then cells were incubated with different samples (PBS, Mn(OAc)2, TCPP, MOFs) for 4 h. Subsequently, the medium was removed and cells were washed with PBS for 3 times. 80 μL of Triton-X-100 lysis buffer (0.4%) was added for lysing cells obtained. Afterwards, the lysates were centrifuged with 6000 r/min for 5 min, and 50 μL supernatant was mixed with DTNB (200 μL, 0.5 mM). In the end, the absorbance (405 nm) of the solutions was measured by a microplate reader.


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Intracellular ROS Detection. The intracellular ROS was measured via a confocal laser scanning microscope and flow cytometry, and DCFH-DA was served as the ROS indicator. 4T1 cells were seeded in small dishes and cultured for 24 h. Then, cells were incubated with different samples (an equivalent TCPP amount of 20 mg/L) for 4 h. After washed with PBS, DCFH-DA solution (1 ×10-6 M) was added and incubated with cells for 15 min. When subjected with irradiation (660 nm, 0.03 W/cm2) for 3 min, cells were observed by CLSM or collected and analyzed by flow cytometry. In Vitro MTT Assay. The 4T1 cells were cultured in 96-well plates and incubated for 24 h. After that, gradient concentrations of Mn(OAc)2, MOFs, and TCPP were added and incubated with cells for another 4 h. After cells were accepted with irradiation (660 nm, 0.03 W/cm2) for 8 min and then cultured for 24 h, 20 μL of MTT (5 mg/mL) was added. 4 h later, the supernatant was removed and 150 μL DMSO was added. The optical density (OD) at 570 nm was recorded by a microplate reader. The cell viability was calculated according to the formula: cell viability (%) = OD (sample) / OD (control) ×100. The same method was employed to evaluate MOFs-induced cytotoxicity against 3T3, CT26 and B16 cells under dark condition. Live/Dead Cell Staining Assay. 4T1 cells were seeded in 6-well plates and cultured for 24 h. Subsequently, previous medium was replaced with fresh one containing MOFs or TCPP (an equivalent TCPP amount of 20 mg/L) for 4 h co-culture. After that, for light groups, the cells were irradiated with 660 nm laser (0.03 W/cm2) for 8 min. 4 h later, cells were stained with 5 μL Calcein AM (4 ×10-6 M) and 10 μL PI (4 ×10-6 M) for 30 min and then observed by fluorescence inverted microscope (Olympus UHGLGPS, China). Cell Apoptosis and Necrosis Assay. The apoptosis and necrosis assay of 4T1 cells was performed by flow cytometry. The cells were seeded in 6-well plates and cultured


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for 24 h. Then, cells were incubated with MOFs or TCPP (an equivalent TCPP amount of 20 mg/L) for 4 h. After irradiation (660 nm, 0.03 W/cm2) for 8 min, cells were cultured for another 4 h. Finally, cells were collected and stained with PI and AnnexinV-FITC for 15 min, following by flow cytometry analysis. In Vivo Fluorescence Imaging. All live animals experiments were conducted according to the Institutional Animal Care and Use Committee of the Animal Experiment Center of Wuhan University (Wuhan, China) and the Regulations for the Administration of Affairs Concerning Experimental Animals. Animal model was built by subcutaneous injection of 100 μL 4T1 cells (1 ×106 cells) in the right hind leg of BALB/c mice (6-week old). Tumor-bearing mice used for imaging had a tumor volume of up to 200 mm3. For GSH-activated fluorescence of MOFs in tumor, the 50 μL MOFs (TCPP equiv. 2.5 mg/kg) solution was administrated by intratumoral injection. After that, fluorescence of tumor was observed at several specific time points by using IVIS imaging systems. To study samples target and enrichment in tumor site, 100 μL MOFs or TCPP solution (TCPP equiv. 5 mg/kg) were intravenously injected into mice. Then mice were imaged at several specific time. 36 h later, the tumor and major organs collected from the sacrificed mice were imaged by IVIS imaging systems. T1-Weighted MRI Imaging. In solution, different concentrations of MOFs (Mn equiv. 5 mM, 2.5 mM, 1.25 mM, 0.625 mM, 0) were dispersed in 2.5 mM GSH solution or not for activated MRI by a Bruker BioSpec 7T/20 cm system (Bruker, Ettlingen, Germany). For GSH-enhanced MRI, 50 μL MOFs solution was intratumorally injected into 4T1 tumor-bearing mice. At 10 min, 20 min, 30 min post-injection, the T1-weight contrast signals were recorded using MRI system. Besides, 200 μL MOFs solution were intravenously injected into 4T1 tumor-bearing mice (Mn equiv. 1.3 mg/kg), and then T1-weight contrast was imaged at preset time (4 h, 8 h, 12 h) after injection.


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In Vivo Antitumor Study. After tumor volume of tumor-bearing mice reached about 100 mm3, the mice were randomly divided into 6 groups (8 mice per group), and intravenously injected with PBS (group 1 and 2), TCPP (group 3 and 4) and MOFs (group 5 and 6) (TCPP equiv. 5 mg/kg). 12 h later, 8 min of laser irradiation (660 nm, 0.22 W/cm2) was performed on mice from groups 1, 3, and 5. The tumor size and weight were measured every day. Tumor volume (V) was calculated according to the formula: V = W2 ×L/2. (W and L were the shortest and longest diameters of tumors). After 14day treatment, tumor ex vivo was weighted and photographed, following by collecting major organs and tumors for further histological analysis. Evaluation of Intratumoral Oxidative Stress. A DCFH-DA solution containing MOFs or TCPP was directly injected into tumors. Afterwards, 5 min of irradiation (660 nm, 0.22 W/cm2) was performed on mice from some groups. Subsequently, obtained tumors tissues from each group were collected and cryo-sectioned onto slide for selffluorescence of tumor. Hemolysis Test. The whole blood from BALB/c mice was centrifuged to obtain red blood cells. Subsequently, the blood cells were incubated with different concentrations MOFs at 37°C for 30 min. After that, all mixture was centrifuged to examine the hemolysis, where the RBC in PBS and distilled water without adding NP served as negative and positive control, respectively. The corresponding concentrations of MOFs solution were used as another control. Blood Biochemistry and Routine Examination. BALB/c mice were intravenously administrated with MOFs solution (TCPP equiv. 5 mg/kg). After 48 h post-injection, the blood samples were collected from treated-mice and analyzed using blood biochemistry analyzer (MNCHIP POINTCARE) and auto hematology analyzer (MC6200VET).


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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Table showing crystal structure information, the main hematological and biochemical parameters. Figures including SEM images, EDS spectrum, XPS spectrum, FTIR spectrum, DLS data, ESR spectrum, UV-vis absorption spectrum, the released percentage of Mn, fluorescence spectrum, cell viability, statistical analysis data, self-fluorescence of tumor tissues, hemolysis assay, and H&E staining images. Crystallographic data for the reported crystal structure was deposited

at

the

Cambridge

Crystallographic

Data

Centre

through

www.ccdc.cam.ac.uk with the code 1917238. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]. Author Contributions S. S. W. and Q. C. contributed equally to this work. ORCID Xian-Zheng Zhang: 0000-0001-6242-6005

ACKNOWLEDGMENT The authors gratefully acknowledge the funding provided by the National Natural Science Foundation of China (51833007, 51873162 and 51690152).


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Scheme 1. Schematic illustration of endocytosis Mn(III)-sealed MOF nanosystem for MRI- and OI-guided PDT by controlled ROS generation and GSH depletion after unlocked by overexpressed GSH in tumor cells.


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Figure 1. Characterization of Mn(III)-TCPP MOFs. (A) SEM, (B) TEM and highresolution TEM (insert), (C) AFM images and (D) TEM mapping of MOFs. (E) XRD patterns. (F) Thermogravimetric analysis. (G) UV-vis spectrum of TCPP, MOFs and TCPP(Mn). (H) XPS spectrum of Mn 3s. (I) ESR spectrum of Mn element. (J) SEM images of MOFs with different concentration of GSH.


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Figure 2. (A) The mechanism of MOFs unlocking triggered by GSH. (B) The detection of GSH consumption with DNTB. (C) GSH-responsive TCPP release. (D) T1-weighted imaging of MOFs with or without GSH (2.5 mM). (E) UV-vis spectrum of Mn(OAc)3 and fluorescence spectrum of MOFs and TCPP. And fluorescent images (insert). (F) MOFs fluorescence changes with GSH concentration. (G) The detection of ROS generation with SOSG. (H) The fluorescence intensity of SOSG after MOFs irradiated with 660 nm in high concentration GSH solution. (I) Different sampled-induced ROS generation at 10 mM GSH. ***P < 0.001


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Figure 3. (A) Fluorescence changes of intracellular MOFs by CLSM over time after 4 h incubation. (B) Fluorescence signal of intracellular MOFs at 0 min (red), 20 min (orange), and 40 min (green) after 4 h incubation by flow analysis and (C) the corresponding statistical analysis results. (D) Intracellular GSH levels after treatment with different samples. (E) Intracellular ROS detection after treated with PBS + hv (red), Mn(OAc)2 + hv (blue), TCPP + hv (orange), MOFs (green) and MOFs + hv (dark green) and (F) the corresponding statistical analysis results. (G) The detection of cellular ROS


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generation by CLSM. Scale bar: 20 μm.


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Figure 4. (A) MOFs-induced cell viability against various cells lines. (B) The cell viability after incubated with TCPP+ hv and TCPP+ L-BSO+ hv. (C) Cell viability with different treatment. (D) The detection of live/dead cells after various treatments. Live and dead cells were stained with Calcein-AM (green) and PI (red), respectively. Scale bar: 100 μm. (E) Cell death mechanism assessment with Annexin V-FITC/PI by flow cytometry. ***P < 0.001, **P < 0.01.


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Figure 5. (A) Fluorescence and (B) T1 contrast signal in tumor sites by intratumoral injection within 30 minutes. (C) In vivo MRI signal after intravenously injected with MOFs. (D) Fluorescence imaging of mice over time by intravenous injection and tissues imaging images at 36 h post-injection. (E) The MFI values of mice tissues after 36 h post-injection. (F) The pharmacokinetics of MOFs and TCPP in blood circulation. (G) Heatmap of hematological and biochemical analysis. Data was processed by normalizing the values of blank. (H) The oxidation pressure of tumor tissues with DCFH-DA (green).


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Figure 6. (A) The body weight and (B) tumor volume of mice after various treatments during 14-day treatment. (C) Weight and (D) photographs of tumor tissues ex vivo. (E) H&E staining, Tunel (green) and Ki67 (green) immuno-fluorescence of tumor after 14day treatment. ***P < 0.001.


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