Hypoxia-tropic nanozymes as oxygen generators for tumor-favoring theranostics

Journal Pre-proof Hypoxia-tropic nanozymes as oxygen generators for tumor-favoring theranostics Fangli Gao, Jin Wu, Heqi Gao, Xueyan Hu, Lihua Liu, Adam C. Midgley, Qiqi Liu, Zhiyuan Sun, Yijin Liu, Dan Ding, Yanming Wang, Deling Kong, Xinglu Huang PII:

S0142-9612(19)30734-3

DOI:

https://doi.org/10.1016/j.biomaterials.2019.119635

Reference:

JBMT 119635

To appear in:

Biomaterials

Received Date: 29 August 2019 Revised Date:

22 October 2019

Accepted Date: 16 November 2019

Please cite this article as: Gao F, Wu J, Gao H, Hu X, Liu L, Midgley AC, Liu Q, Sun Z, Liu Y, Ding D, Wang Y, Kong D, Huang X, Hypoxia-tropic nanozymes as oxygen generators for tumor-favoring theranostics, Biomaterials (2019), doi: https://doi.org/10.1016/j.biomaterials.2019.119635. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Hypoxia-tropic Nanozymes as Oxygen Generators for Tumor-favoring Theranostics

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Fangli Gao , Jin Wu , Heqi Gao , Xueyan Hu , Lihua Liu , Adam C. Midgley , Qiqi Liu , Zhiyuan Sun , a

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Yijin Liu , Dan Ding , Yanming Wang , Deling Kong * and Xinglu Huang

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Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, and State Key

Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin 300071, China. b

Department of Radiology, Tianjin First Central Hospital, Tianjin 300192, China.

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State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, Nankai University, Tianjin

300353, China. d

Joint Laboratory of Nanozymes, College of Life Sciences, Nankai University, Tianjin 300071, China.

*To whom correspondence may be addressed. Email: [email protected] or [email protected]

Abstract Oxygen deficiency is the main obstacle of hypoxia-related theranostics, thus this is a considerable amount of research focusing on the development of methods to supply oxygen by taking advantage of hypoxia-responsive properties of nanoparticles. However, strategies to properly penetrate hypoxic regions by the nanoparticles remains an unmet challenge. In this work, a biomimetic nanozyme capable of possessing catalase-like activity and the efficient direct penetration of hypoxic areas in tumor tissues was developed to supply oxygen based on catalytic tumor microenvironment-responsive reaction, providing substantial tumor hypoxia relief with nearly 3-fold reduction compared to untreated tumor tissues. To demonstrate the advantages of the nanozymes in overcoming hypoxia, a theranostic nanosystem model composed of the core/shell nanozymes and aggregation-induced emission (AIE) molecules was designed. The nanosystem was able to present multi-modal imaging of tumors and modulated the tumor microenvironment for improved photodynamic therapy (PDT) by cascade reactions of therapeutic effector molecules, thereby providing significantly enhanced therapeutic benefits in inhibiting tumor growth and lung metastasis of orthotopic breast cancer. This conceptual study showed the multifaceted features of biomimetic nanozymes as tumor therapeutics and

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demonstrated the encouraging potential for modulating hypoxia as an application for tumor theranostics. Keywords: hypoxia, nanozyme, biomimetic synthesis, aggregation-induced emission (AIE), theranostics

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Hypoxia, a characteristic feature of solid tumors and, is associated with cancer progression, angiogenesis, metabolism and metastasis [1, 2]. Accumulating evidence suggests that cancer cells in hypoxic regions have elevated resistance to treatments such as radiotherapy [3] and photodynamic therapy (PDT) [4] , due to the requirement of oxygen molecules in the process of cancer cell death. Thus, developing therapeutic strategies to alleviate tumor hypoxia has recently been considered an extremely valuable target in oncology. Recently, substantial efforts aimed at the modulation of hypoxia have incorporated multiple functionalities and moieties into nanoparticle design, such strategies include those which are oxygen-dependent [5-8], based on the stabilization of hypoxia-inducible factor-1α (Hif-1α) [9-11] or hypoxia-activated [12-14]. Among the strategies, MnO2-based nanoparticles have recently displayed promising potential as a unique type of theranostic agents due to their inherent mechanism of catalytic reaction, in response to the tumor hypoxic microenvironment 2+

[15-18]. The decomposed Mn

+

reacts with H within the tumor microenvironment, which also

enhances T1 MR imaging contrast. More importantly, MnO2 triggers the decomposition of acidic H2O2 +

2+

into oxygen and results in the alleviation of tumor hypoxia (MnO2 + 2H → Mn +

2+

+ 2H + H2O2 → Mn

+ H2O + 1/2O2; MnO2

+ 2H2O + O2). In other words, acid environment in tumor hypoxic areas is critical

for highly efficient decomposition of MnO2 nanoparticles. However, a fundamental issue is that the delivery efficacy of nanomedicine for tumor hypoxic cells is greatly limited owing to the presence of poor vascularization within tumor hypoxic areas, which in turn causes extremely low decomposition efficacy of intratumoral MnO2 nanoparticles. Until now, nanosystems capable of proper penetration into tumor hypoxic regions are lacking. As a promising human-derived nanocarrier, genetically engineered ferritin nanocages (FTn) have shown intrinsic hypoxia-tropic properties by efficient tissue penetration and active binding with hypoxic tumor cells [9, 19]. These intrinsic qualities are dependent on the receptor of FTn (TfR-1 in humans; TIM-2 in mice) [20, 21]. In other words, the intact binding domains of FTn on the nanocarrier surface is critical for targeting of hypoxia and subsequent actions. As such, developing FTn-based oxygen supply platforms that do not affect receptor-dependent hypoxia-tropic properties is challenging. Interestingly, the unique protein structure of FTn allows the diffusion of both metal ions and O2 (or H2O2) via shell channels [22-24]. Thus, the interior hollow 8 nm diameter cavity of FTn [25, 26] can be utilized as a natural nucleation nanoreactor due to abundant metal ion-bonding residues at the interior ferroxidase centers [27-30].

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In this work, we prepared a hypoxia-tropic nanozyme as oxygen generator (OGzyme) by the biomimetic synthesis of MnO2 nanoparticles inside the hollow cavity of FTn (Scheme 1a). Since hypoxia plays major roles in tumor resistance to multiple treatments, we further explored the potential applications of OGzymes in tumor-favoring hypoxia-associated therapy. Recently, aggregationinduced emission (AIE) molecules were used as effective photosensitizers for PDT as a result of production of reactive oxygen species (ROS) as aggregation increased energy transference from singlet state to triplet state for minimizing loss of excited energy through non-radiative decay [31-33]. AIE embedded within a lipid bilayer to formed AIE-liposome complexes that could be efficiently delivered to tumor sites and reaggregated in situ as a result of liposome degradation, which proved an effective strategy for tumor PDT [34]. Therefore, we designed a reaction cascade theranostic nanosystem by integration of OGzymes with the PDT capabilities of AIE molecules. In our system, the AIE molecules and OGzymes were encapsulated into phospholipid bilayers and the inner cavity of liposome nanocarriers, respectively (Lipo-OGzyme-AIE). Under light irradiation, AIE molecules generated cytotoxic levels of ROS that also enabled imaging-guided PDT. At the same time, OGzymes provided adequate O2 for inherent conversion to ROS by taking advantage of the sensitive pH-/H2O2-responsive behaviors of MnO2. Furthermore, this secondary delivery system composed of liposome and FTn significantly improved the delivery efficacy of FTn in tumor by overcoming the short in vivo circulation time of FTn. After the degradation of liposomes, the OGzymes was released and further deeply penetrated into tumor tissues including but not limited in hypoxic areas. The generated O2 through MnO2 catalytic decomposition reactions not only relieved tumor hypoxia but also diffused 1

throughout the whole tumor tissues, providing the source of O2 inherent conversion of AIE molecules under irradiation. Therefore, this study demonstrated a conceptual theranostic nanosystem, which not only presented synergistic roles in overcoming biological barriers for improved delivery efficacy, but also enabled modulation of the tumor microenvironment for enhanced cancer theranostics by reaction cascades of therapeutic effector molecules (Scheme 1b).

RESULTS AND DISCUSSION Synthesis and characterization of OGzymes and Lipo-OGzyme-AIE The biomimetic synthesis of MnO2 into FTn utilized a one-step FTn-based biomineralization method, by taking advantage of the existing intrinsic properties of FTn for the capture and nucleation of metal

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ions. Several potential routes including eight 3-fold, twelve 2-fold, and twenty-four 1-fold channels, 2+

allowed the Fe

2+

entry from solution into inner cavity of FTn. Next, Fe

was located within the

ferroxidase centers, composed of A (Glu27, His65, Glu62) and B (Glu107 and Glu62) binding sites 2+

2+

[35]. Different from Fe , Mn 2+

wherein Mn

primarily binds with A sites in an active manner [36] (Figure 1a),

oxidizes and nucleates into MnO2 nanoparticles, when in H2O2-containing alkaline

solution. By observation of the resulting OGzyme via transmission electron microscopy (TEM), the images showed the hollow structure of FTn shells by protein negative staining (Figure 1b) and the uniform and monodispersed spherical morphology of MnO2 (Figure 1c). The diameter of FTn and MnO2 were determined to be approximately 12 nm and 5 nm, respectively, and it was implied that MnO2 particles located within the hollow cavity of FTn possessed diameters of approximately 8 nm. There were ~160 Mn atoms per FTn, as calculated by determining Mn content and protein concentration. X-ray photoelectron spectroscopy (XPS) of the OGzymes confirmed the chemical state of Mn by two characteristic peaks, displayed at 653.6 and 641.8 eV corresponding to the Mn (IV) 2p1/2 and Mn (IV) 2p3/2 spin–orbit peaks of MnO2, respectively (Figure S1a). In malignant tumors, cells generate excessive amounts of H2O2 due to imbalanced biochemical and metabolic reactions [37]. Meanwhile, upregulated glycolytic metabolism in hypoxic tissues produce excessive amounts of lactic acid, as a result of the acid-/redox-responsive tumor properties. 2+

Thus, MnO2 can theoretically be decomposed into Mn

by reaction with H2O2 in tumors [38, 39], along

with the release of O2 in situ to relieve tumor hypoxia. In other words, acidic H2O2 levels (i.e. hypoxic conditions) are boosted the generation of O2. We next explored whether the OGzymes possessed catalase-like activity, based on the capability to catalyze H2O2 into O2 through in vitro mimicry of the tumor tissue microenvironment. A high concentraiton of H2O2 (80 mM) was utilized to test visible O2 production of OGzymes (Figure 1d), which showed numerous O2 bubble production by OGzyme solution, but no obvious bubbles were observed in the control solution. Given the concentration of the endogenous H2O2 within tumor tissues ranges between 10-100 µM [40], we tested O2 generation by particles following triggered decomposition of H2O2 solution (100 µM). An oxygen probe was used to measure the dissolved O2. As expected, the rapid elevation of the dissolved O2 levels was observed when using OGzyme solution, whilst the production of O2 remained within a relatively stable level in PBS solution. Impressively, the catalase activity of the OGzymes at pH 6.5 was approximately 2-fold higher than that at pH 7.4, after the addition of H2O2 for 10 min, implying more efficent decomposition

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of MnO2 nanoparticles in tumor hypoxic microenvironment. The results demonstrated that the OGzymes not only possessed catalase-like activity, but also implied that they could have a higher energetic status within tumor hypoxia-like environments. In the following, the Lipo-OGzyme-AIE composed of liposome, AIE and the OGzyme was designed and prepared. The characterization and fluorescent properties of AIE molecules were shown in Figure S1b-d. Compared to the TEM images of liposomes alone (Figure 1e), the successful loading of the OGzymes into liposomes was observed (Figure 1f) with an encapsulation efficacy of ~30%, as fluorometrically determined by the amount of FTn within liposome. The ratio of FTn and AIE molecules loaded into liposome is ~1:480. Dynamic light scattering (DLS) revealed that the particles had a narrow size distribution range, and the mean diameter increased from 96.8 nm of Lipo-AIE to 122.5 nm of the Lipo-OGzyme-AIE (Figure 1g). The Lipo-OGzyme-AIE also showed catalase-like activity in a similar trend with that of the OGzymes, according to their catalytic decomposition of H2O2 1

into O2 (Figure 1h). Considering O2 is a central element to generate cytotoxic O2 during the process 1

of PDT, we next measured O2 production by particles within H2O2 solution using 9, 10-anthracenediylbis(methylene)dimalonic acid (ABDA) as an indicator under visible light irradiation (400-700nm) [41]. 1

By recording the absorbance value at 400 nm over time, we observed significant O2 generation in the 1

presence of particle, and O2 yield by Lipo-OGzyme-AIE is obviously higher than Lipo-AIE, at the same condition after irradiation (Figure 1i). These results implied that the OGzymes were able to produce O2 1

with the subsequent efficient O2 yield produced by AIE molecules, through a series of catalytic reaction cascades under conditions relevant to the tumor microenvironment.

Synergistic effects of secondary delivery system To understand the improved hypoxia-targeting ability, we investigated the synergistic roles derived from the secondary delivery system composed of the two nanocarriers (i.e. FTn and liposome). As a natural human-derived protein, FTn showed short half-life (~4 h) in blood circulation and was rapidly cleared from the body [21, 42]. Thus, the increased tumor uptake of FTn was expected by prolonging circulation time with liposomes. As such, an IVIS imaging system was used to visualize tumor sites and organ distribution of nanoparticles, following i.v. administration to mice bearing subcutaneous 4T1 tumors. Both Lipo-FTn-Cy 5 and FTn-Cy 5 showed obvious accumulation within tumor sites. In addition, the signal intensity within tumors of mice injected with Lipo-FTn-Cy 5 was approximately 3.3-

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fold higher than that of mice that received FTn-Cy 5 injection (Figure 2a and b). The limited tissue penetration of liposomes has been suggested to be due to “the binding site barrier effect” [43], as a result of their membrane fusion with adjacent tumor cells beyond blood vessels. Therefore, we next explored the penetration of FTn-Cy 5 loaded Lipo-AIE (Lipo-FTn-Cy 5-AIE ) throughout 3D multicellular tumor spheroids established using 4T1 cells. Multicellular tumor spheroids are wellestablished in vitro model for exploring the penetration of drug molecules and are characterized by the presence of hypoxic regions within the center of the spheroids [44]. Following incubation with LipoFTn-Cy 5-AIE, the 3D-reconstructed laser scanning confocal microscopy images of spheroids displayed that Lipo-AIE were primarily located near the periphery of tumor spheroids (Figure 2c). In contrast, FTn-Cy 5 were able to deeply penetrate throughout the entirety of the spheroids, including the hypoxic core. The quantification analysis also revealed the distribution of FTn within middle areas were significantly higher than that of liposomes, as determined by the mean signal intensity ratios between the center and peripheral regions (Figure 2d). To further understand the penetration ability in tumor microenvironment, we next studied in vivo tumor tissue distribution of Lipo-FTn-Cy 5-AIE following direct intratumoral injection. Figure 2e showed that the fluorescence signal from liposomes in cryo-section of 4T1 tumor tissue did not fully co-localize with that of FTn. In other words, the penetration distance of FTn showed differences to the liposomes within 4T1 subcutaneous tumors, showing nearly 2-fold greater distribution by FTn, when compared to the liposomes (Figure 2f and g). We also confirmed that FTn was able to deeply penetrate into tumor tissue, while liposome was mainly distributed in perivacular regions (Figure S2a). This phenomenon could be explained by the receptordependent transcytosis of FTn, an intrinsic property that allows for deeper penetration into tumor tissues, which has been demonstrated by our and others' works [20, 21]. Based on the above results, we further sought to investigate whether FTn, even though loaded into liposome, was still able to target tumor hypoxia cells following i.v. administration. We first studied the release of FTn from liposome by co-localization of FTn and liposome in the tumor tissue. The images revealed limited blood vessel distribution in hypoxic regions within the 4T1 subcutaneous tumor with vessel density less than one-third of that in normoxic tumor regions of the same tumor (Figure S2b). Next, tumor tissues were observed after immuno-staining for tumor hypoxia and for blood vessels. As expected, Cy 5-labeled FTn was preferentially found within hypoxic tissue (Figure 2h). These results implied that FTn released from liposome intratumorally and further penetrated into tumor hypoxic regions.

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Cellular uptake and cytotoxicity As mentioned above, our strategy based on in situ biomimetic synthesis of MnO2 nanoparticles inside FTn is critical for maintaining the intact binding domains of FTn on the surface. To confirm this point, we compared cell uptake of Cy 5 labeled FTn and Cy 5 labeled OGzymes at normoxic (21% O2) (Figure 3a) and hypoxic (1% O2) conditions (Figure 3b). Confocal images showed that both FTn and OGzymes distributed in cytoplasm of 4T1 cells regardless of oxygen conditions. Importantly, the fluorescence intensity of FTn in 4T1 cells was identical with that of OGzymes using flow cytometry analysis, demonstrating that MnO2 nanoparticles inside FTn do not affect the binding of FTn with its receptor. We next tested the cytotoxic effects of Lipo-OGzyme-AIE by irradiation-induced PDT. After incubation with 4T1 cells, we first checked whether Lipo-OGzyme-AIE were efficiently endocytosed. The confocal images showed strong fluorescent signal of AIE within the cytoplasm of cells (Figure 3c), which was further confirmed by using flow cytometry analysis. The efficient cellular uptake provided the basis for in situ cytotoxic ROS generation through reaction of endogenously produced H2O2 with the OGzymes. As such, dichlorofluorescein diacetate (DCFH-DA) was used to detect intracellular ROS 1

level. Figure 3d showed that the significant increased O2 intracellularly was observed after treatment 1

with Lipo-AIE under light irradiation, while the O2 level was further elevated in the presence of the OGzymes. The results were also confirmed by quantification analysis of the signal intensity of activated DCFH-DA probe inside cells using flow cytometry. Next, the PDT effect of the particles in vitro was further explored by evaluation of cell viability at normoxic (21% O2) and hypoxic conditions (1% O2). Once exposed to light irradiation, both Lipo-AIE and Lipo-OGzyme-AIE showed high cytotoxicity regardless of oxygen environment, while the OGzymes alone presented negligible cytotoxicity at equivalent dose to Lipo-OGzyme-AIE (Figure 3e). For the cells treated with Lipo-AIE, the cell killing effect under normoxia was better than that of hypoxic exposure, implying cell resistance to PDT under hypoxic environments. Furthermore, there were no significant differences between the cell viability of Lip-AIE and Lipo-OGzyme-AIE under normoxic condition. However, the cells treated with the OGzymes at hypoxic conditions showed apparent difference from those treated with Lipo-AIE alone, displaying an OGzyme concentration-dependent manner. Under hypoxic exposure, the cell viability when treated with Lipo-AIE was 27.2%, 29.3% and 22.8% compared to untreated cells, respectively. In contrast, the cell viability was decreased to 23.6 %, 7.7% and 4.1% at the presence of

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0.025, 0.05 and 0.1 mg/ml OGzymes, respectively. These results revealed the effective generation of intracellular cytotoxic ROS by Lipo-OGzyme-AIE, regardless of O2 environment.

In vivo tumor accumulation and hypoxia alleviation Following the in vitro evaluation, we thus sought to investigate the in vivo properties of the particles within animal tumor models. We first utilized in vivo fluorescence imaging to track the Lipo-FTn-AIE in 4T1 tumor-bearing mice following i.v. administration. The efficient accumulation of the particles was observed in tumor areas (arrow) over time (Figure 4a), indicating ~2-fold increases in AIE fluorescence signal intensity at 24 h, compared to 1 h post-injection (Figure 4b). Quantification analysis of the biodistribution within major organs and tumors isolated from 24 h post-injection demonstrated the high uptake of Lipo-AIE-FTn in tumor (Figure S3). The AIE fluorescence signal was further confirmed throughout the whole tumor by evaluating cryo-sections of tumor tissue, strong aggregation signals were especially evident within the tumor tissue interior (white arrow) (Figure 4c). The aggregation of AIE molecules occurred in tumor tissues since the molecules could remain in liposome and/or hydrophobic AIE molecules could easily aggregate in physiological microenvironment. As a secondary delivery system, we next sought to confirm that OGzymes also accumulated in tumor tissues. Since the accumulation of Lipo-OGzyme-AIE in the tumor was expected to be followed by 2+

H2O2-mediated decomposition of MnO2, we therefore monitored the yield of Mn within tumor tissues by using magnetic resonance (MR) imaging. The enhanced T1 MR imaging contrast of tumor at 24 h was observed compared to the tumor before injection (Figure S4). Quantification analysis also indicated that the T1 signal intensity at 24 h was significantly decreased by ~60%, compared to preinjected tumor. These results revealed efficient decomposition of MnO2 in tumors and high tumor accumulation of Lipo-OGzyme-AIE for MR imaging of tumor. To further understand the intratumoral behaviors of the systemically administrated particles, we assessed whether the particles efficiently alleviated hypoxic status. As such, an immunofluorescence assay was performed to study tumor slices obtained from 4T1 flank tumor. The cell nuclei, blood vessels and hypoxic areas were stained with DAPI (blue), anti-CD31 antibody (red), and antipimonidazole antibody (green), respectively. We found the untreated tissue contained abundant hypoxic aregions, accounting for 51.9±8.5% of the whole tumor tissue (Figure S5 and Figure 4d and e). Notably, the OGzymes substantially decreased the hypoxic areas, with nearly 3-fold decline in

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comparison with untreated tumor tissue. Moreover, liposome encapsulation promoted alleviation of the hypoxic status within tumor tissues most likely due to the enhanced tumor accumulation (Figure 2). A capacity for time-dependent hypoxia alleviation was also observed in tumor tissues by comparing different time points post i.v. injection of Lipo-OGzyme, with virtually negligible hypoxia within the whole tumor tissue at 24 h.

In vivo anti-tumor efficacy Next, we evaluated the in vivo efficacy of Lipo-OGzyme-AIE for enhanced PDT within 4T1 mouse tumor models. In this experiment, Balb/c mice bearing 4T1 subcutaneous tumors were randomly divided into five groups: PBS, OGzyme (10 mg/kg FTn), Lipo-AIE (5 mg/kg AIE), Lipo- OGzyme-AIE low (2.5 mg/kg AIE, 5 mg/kg FTn) and Lipo-OGzyme-AIE high (5 mg/kg AIE, 10 mg/kg FTn). Given the high -2

tumor accumulation at 24 h (Figure 4), the tumors were exposed to white light irradiation (0.35 W cm ) for 10 min 24 h after i.v. injection. The tumor sizes and body weights were measured every 2 days. As shown in Figure 5a, the OGzymes showed no statistical changes in tumor growth after 8 days posttreatment compared to untreated groups. For tumors in mice treated with Lipo-AIE, Lipo-OGzyme-AIE low or

Lipo-OGzyme-AIE high, tumor growth was clearly delayed. More importantly, the OGzymes

significantly improved therapeutic efficacy of Lipo-AIE including those in the low dose treatment group (half of AIE amount), demonstrating the remarkable synergistic effect of PDT within assistance from OGzyme relief of tumor hypoxia. Animal body weight remained unperturbed in all groups, suggesting no apparent systemic toxicity of the nanoformulations (Figure 5b). In addition, severely damaged tissues were observed by hematoxylin and eosin (H&E) staining of tumor slices in the Lipo-OGzymeAIE treated group, while the cells in other treatment groups largely or entirely remianed normal morphology and proliferation (Figure 5c). Immunohistochemistry experiments using TUNEL staining confirmed more positive signal in groups treated with Lipo-OGzyme-AIE than other groups, implying that the apoptosis of tumor cells was induced by the generation of ROS during PDT. Importantly, histopathological analysis revealed that no obvious systemic toxicity of OGzyme, Lipo-AIE and LipoOGzyme-AIE by evaluation of H&E staining images of major organ samples of animals for 14 days (Figure S6). These results clearly displayed the remarkable antitumor effect in vivo by taking advantage of the cascade reaction and synergistic delivery ability of the secondary delivery system

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designed. Undoubtedly, the OGzymes capable of prominent overcoming tumor hypoxia and effective self-generating O2 played criticle role in favoring anit-tumor ability.

Inhibition of lung metastasis in orthotopic breast cancer Lung metastasis is a common feature of breast cancer in clinic because the cancer cells can break away from the primary tumor and travel to distant lung. Inspired by the above results, we next explored whether the nanoparticles inhibited cancer metastasis using mouse models of established 4T1 orthotopic breast cancer with lung metastasis mice. To track in vivo tumor growth and metastasis, the primary breast cancer and metastatic lung cancer were simultaneously monitored by imaging firefly luciferase overexpression in 4T1 cells. Figure 5d exhibited that the signal intensity of luminescence imaged in the fat pad location were equal amongst all groups before treatment. Then, the primary tumors in situ were performed with PDT following i.v. administration of particles for 24 h. At 14 days, mice treated with PBS, OGzymes or Lipo-AIE showed obvious tumor growth, as evidenced by remarkedly increased signal at the implanted sites, whereas Lipo-OGzyme-AIE treatment displayed a restricted increase in signal. Subsequenly, the major organs were isolated from the above mice for imaging the biodistribution of breast cancer metastasis. We found the luciferase signal was observed in the lungs only (Figure S7). Notably, compared to other groups, the lung signal when treated with Lipo-OGzyme-AIE was negligible under the same setting parameters (Figure 5e). Quantification analysis demonstrated Lipo-AIE significantly inhibited the lung metastasis of 4T1 orthotopic breast cancer, along with a 14.5% metastasis compared to PBS group. The metastasis was further decreased to ~5% in the presence of OGzymes. The significant inhibited lung metastasis could be explained by the delayed growth and development of primary breast cancer after PDT treatment.

CONCLUSION In conclusion, we introduced a multifaceted nanozyme based on a naturally occurring human protein shell and biomimetic one-step synthesis of a MnO2 core, which were capable of actively penetrating into hypoxic tumors by overcoming intratumoral barriers and provided highly efficient generation of an oxygen supply through catalytic reactions, in response to the tumor microenvironment. Based on the powerful properties of OGzymes, we further designed a secondary delivery system, showing, i) improved tumor delivery efficacy of the OGzymes and deep penetration into hypoxic tumor tissue; ii)

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highly tumor hypoxia alleviation and enhanced production of cytotoxic singlet oxygen based on a cascade of converting events; iii) multimodal imaging of tumors for imaging-guided therapy; iv) efficient tumor cell killing, especially for boosted cytotoxic effect under hypoxic condition. As a result of these benefits, this delivery nanosystem offers impressive in vivo antitumor therapeutic outcomes and effective lung metastasis inhibition in models of orthotopic breast cancer, followed a single treatment. Beyond the designed system shown here, we believe OGzymes also have potential for combinational therapy with other PDT molecules and other therapeutic modes associated with modulation of hypoxia.

Methods Preparation of FTn. Human ferritin heavy chain was prepared following our recent reports [9, 45]. Briefly, a plasmid encoding human ferritin heavy chain was constructed by typical molecular cloning and the resulting plasmid vector was transformed into E.coli BL 21. To obtain FTn, the protein production was induced by IPTG, and the resulting protein solution was subsequently purified to obtain the final protein solution by heating at 60 °C and gel-filtration chromatography (GFC) with a Superose 6 column (GE Healthcare), respectively. The cage-like nanostructure of FTn was characterized by HITACHI HT7700 EXALENS transmission electron microscopy (TEM) after negative staining with 1% uranyl acetate. FTn concentration was determined by standard BCA protein assay kit (Thermo Fisher Scientific). Conjugation FTn with Cy 5 (FTn-Cy 5). For the Cy 5 labeling with FTn, 30 molar equivalents of Cy 5 NHS ester (Ex 650 nm, Em 670 nm) were reacted with FTn in the pH 8-9 buffer at room temperature for at least 4 hours. The mixture was purified using a PD-10 desalting column. Finally, the resulting FTn-Cy 5 was determined to be ~7 per FTn by measuring the amount of the labeled Cy 5 and FTn, respectively. Biomimetic synthesis of the OGzymes. The MnO2 core was prepared into FTn by following the modified method of iron oxide biomineralization [46]. Specifically, a degassed solution (8.0 mL of 100 mM NaCl) was added to a jacketed reaction vessel under N2 condition, followed by addition of FTn (2.0 mg, 3.9 nmol) in 100 mM NaCl to the vessel. The temperature of the vessel was kept at 65 °C using water bath. The pH was titrated to 8.5 using 50 mM NaOH (INESA, pH SJ-4F). Mn (II) was added (12.5 mM MnCl2) to attain a theoretical loading factor of 4000 Mn per protein cage. Stoichiometric equivalents (H2O2:Mn, 1:2) of freshly prepared degassed H2O2 (4.17 mM) was also

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added as an oxidant. The Mn (II) and H2O2 solutions were added simultaneously at a constant rate of 31.3µL /min (100 Mn/(protein/min)) using a peristaltic pump (Longer Pump, BT100-1F). The constant pH was dynamically monitored using 50 mM NaOH. After the completion of the reaction, 200 µL of 300 mM sodium citrate was added to chelate free Mn. The resulting solution was further purified using PD-10 desalting column (GE Healthcare). The morphology of the samples was characterized by transmission electron microscope (TEM) following with or without the negative staining of the specimen with 1% uranyl acetate. The content of Mn was quantified by ICP (Thermo). The concentration was determined by quantification FTn protein concentration. Preparation of AIE loaded Liposome. To obtain AIE loaded liposome, the mixture of 1,2-dioleoyl-snglycero-3-phosphocholine (DOPC), cholesterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N[methoxy(polyethylene glycol)-2000] (Ammonium Salt) (DSPE-PEG2k), and AIE molecules at a molar ratio of 12:6:0.8:1 was dissolved in chloroform and then dried under a rotary evaporator. Afterward, the dried lipid film was hydrated with PBS, followed by extrusion through 400 nm, 200 nm and 100 nm polycarbonate filters for 20 times. The resulting Lipo-AIE was condensed with an Amico filter device with a molecule weight cutoff of 30 kDa (Millipore) for further use. Loading OGzymes into Lipo-AIE. The resulting lipid film of Lipo-AIE was fully hydrated with 1 mL solution for 1 hour at 60°C containing FTn-MnO 2 at a concentration of 2 mg/mL. Then the mixture was subjected to five freeze-thaw cycles (5 min in liquid nitrogen and 5 min at 60°C) to make sure the OGzymes loaded into Lipo-AIE. After removing the free FTn-MnO2 by gradient centrifugation, the obtained pellet was resuspended with PBS. The resulting solution was extruded through 400 nm, 200 nm and 100 nm polycarbonate filters for further use. The encapsulation efficacy of the OGzymes was fluorometrically determined by using Cy 5-labeling FTn loaded liposome as a model system. Measurement of dissolved oxygen concentration. Oxygen generation was determined in an anaerobic chamber with an oxygen electrode (Dissolved Oxygen Meter, WLDO-300). Briefly, the stock samples including the OGzymes and Lipo-OGzyme-AIE, were diluted into different pH of PBS buffer (pH=7.4 and 6.5), respectively. The solution was fully removed O2 with N2 for 30 min. Then, 100 µM H2O2 was added into the solution and the dissolved O2 level was recorded at the indicated time points by the electrode probe. ROS detection. ROS production was determined by using 9,10-anthracenediyl-bis(methylene) dimalonic acid (ABDA) as an indicator. Briefly, 5 µL ABDA in DMSO solution (10 mM) was added into

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the Lipo-AIE and Lipo-OGzyme-AIE solution (AIE equivalent of 5.9 mM) containing 100 µM H2O2 at pH=6.5, respectively. Subsequently, the absorption spectra of the solution after O2 removal upon white -2

light irradiation (0.15 W cm ) were recorded every 30 seconds at 400 nm. The descending absorbance value at 400 nm after designated irradiation time intervals was used to determine the ROS generation. The produced ROS efficacy was analyzed with lnA0/A: A0 is the initial absorbance, while A represents the final absorbance after irradiation. Cell culture. 4T1 Cells were cultured in RPMI 1640 medium (Gibco), supplemented with 10% fetal bovine serum (BI, Biological Industries) and 1% Penicillin-Streptomycin. For normoxic and hypoxic cell culture, cells were incubated in a humidified incubator (37 °C, 5% CO 2) with normal (21% O2) and hypoxic (1% O2) conditions, respectively. Penetration through 4T1 cell spheroids. The 4T1-based multicellular spheroids were prepared as described elsewhere with some modifications [47]. Briefly, 50 µL RPMI 1640 medium containing 1% 4

(w/v) agarose was added and solidified in each well of 96-well microplates. Then, 1× 10 cell/well 4T1 cells were seeded and cultured for 4−5 days to obtain 3D multicellular tumor spheroids with diameter of 500−600 µm. The spheroids were carefully transferred to glass-bottom dishes, where FTn-Cy 5 loaded Lipo-AIE were added and incubated for 6 h. Finally, cell spheroids were then observed under LSM 710 Meta laser confocal microscope (Carl Zeiss) and reconstructed using ZEN microscopy software (Carl Zeiss). The penetration ability of FTn and liposome were determined by evaluating the intensity distribution profile using a custom-made formula A0/A1: A0 is the mean intensity of the area distant from center within 50 µm; A1 defines the mean intensity of the area from outside to inside 50 µm. The image-based quantification analyses of signal intensity were performed by Image J software (NIH). Cell uptake. To evaluate cellular uptake of Cy 5 labeled FTn (FTn-Cy 5) and OGzymes (OGzyme-Cy 4

5) in different O2 conditions, 4T1 cells were seeded into glass-bottom dishes at 5×10 cells and cultured at 21% O2 and 1% O2 for 48 h, respectively. To compare the uptake, fresh medium (without FBS) containing FTn-Cy 5 (20 µg/mL FTn) and OGzyme-Cy 5 (Cy 5 equivalent) was added and continued to culture for 2 h in different O2 conditions, respectively. The cells were washed with PBS three times and fixed with 4% paraformaldehyde solution for 20 min, followed by staining for 15 min with DAPI. The distribution of the particles in cells was observed by using Zeiss confocal microscopy. The signal intensity of the particles in cells was quantified by flow cytometry analysis (FACS Calibur).

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For cell uptake of Lipo-OGzyme-AIE, Lipo-OGzyme-AIE (20 µg/mL FTn) was added to incubate for 6 h. The cells were evaluated at the excited wavelength of ~488 nm by confocal microscopy and flow cytometry. Intracellular ROS detection. Dichlorofluorescein diacetate (DCFH-DA) was used to detect intracellular ROS level. The cells were incubated with particles for 6 h. After washing, the DCFH-DA (10 µM) was added and incubated for 20 min. The cells were washed with PBS three times and stained for 20 min with Hoechst 33258. Before observation with confocal microscopy, the cells were exposed with white light irradiation (400-700 nm) for 2 min. For flow cytometry experiments, the cells were further collected and analyzed after the above procedures. Cytotoxicity. Cytotoxicity was assessed with a standard MTT method following PDT procedures. 3

Briefly, 4T1 cells were seeded into 96-well plates (5× 10 per well). After adherent to the plate, the cells were incubated with the OGzymes, Lipo-AIE or Lipo-OGzyme-AIE at the equivalent FTn concentration of 0.025, 0.05, 0.1 mg/mL. The cells were continued to culture in the normoxic (21%) and hypoxic (1%) conditions for overnight. Next, each well of the 96-well plates was exposed to white -2

light irradiation (0.15 W cm ) for 10 min. After another 12 h incubation, cytotoxicity was assessed with a standard MTT method. Animals and tumor model. Female BALB/c mice (6-8 weeks old) were purchased from SPF (Beijing) Biotechnology Co. Ltd. All Animals were handled in accordance with governmental and international 6

guidelines on animal experimentation. For subcutaneous 4T1 tumor model, 1×10 4T1 cells were subcutaneously injected into right shoulder of the mice. The tumor growth was monitored every day. 2

Tumor volume was calculated as (tumor length)×(tumor width) /2. Intratumoral penetration. Tumor-bearing mice were administrated with 20 µL FTn-Cy 5 loaded LipoAIE (FTn equivalent of 2.8 mg/kg) by intratumor injection using an automatic syringe at a constant rate of 1.33 µL/min. Animals were sacrificed 2 h after administration, and the tumors were harvested and frozen in OCT. Then, the whole tumor tissues were cryosectioned at a thickness of 10 µm (Leica Biosystems), and a series of sections were imaged using confocal microscopy. The background signal of the tissues was offset by using an untreated tumor tissue. The maximum coverage area of fluorescence signal in tumor tissues were chosen as representative images for determining the penetration distance. The image-based quantification analyses were performed by Image J software.

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Accumulation in tumor hypoxic areas. 4T1 tumor-bearing mice were administered with FTn-Cy 5 and FTn-Cy 5 loaded Lipo-AIE (FTn equivalent of 28 mg/kg) by tail vein injection, respectively. After 24 h, major organs and tumors were harvested and imaged by the Xenogen IVIS Luminar Imaging System (IVIS Lumina II Xenogen). The tumor tissues were collected for cryosection (Leica Biosystems). The hypoxic area of the tumor tissues was immunostained using a Hypoxyprobe-1 kit TM

(Hypoxyprobe ). The immunostaining was performed according to the standard procedure. Specifically, pimonidazole HCl (60 mg/kg) was given to the mice 1.5 h prior to sacrifice. The hypoxic regions of the tumor were stained using FITC-conjugated antibody in the kit. To co-stain with blood vessels, the tissues were simultaneously incubated with anti-pimonidazole antibody using PEconjugated anti-CD31 antibody (Biolegend). The images of the tissues were acquired using an LSM 710 Meta confocal microscope. In vivo and ex vivo imaging. For tracking distribution of the nanoparticles in vivo, 4T1 tumor bearing mice were administered with FTn loaded Lipo-AIE via tail vein injection (AIE equivalent of 10 mg/kg). At the indicated time points, the mice were imaged by an in vivo imaging system (IVIS Lumina II Xenogen). The optimal imaging parameters of the particles were determined based on the specific signal intensity in mouse body obtained from the imaging system. For analyzing the biodistribution, the mice were sacrificed and major organs including heart, liver, spleen, lung, kidney, muscle and tumor were collected for imaging. The accumulation and distribution of the particles were semi-quantified by using the Living Image 2.50 software. To further confirm the positive signal in tumor tissues, the cryosection of tumor tissues were observed with an excitation wavelength of 488 nm by confocal microscope. 3

Hypoxia alleviation in tumor tissue. When subcutaneous tumors reached ~100 mm , the mice were administrated with PBS, OGzymes, Lipo-OGZymes at the FTn dose of 10 mg/kg via tail vein injection. At the specific time points, the pimonidazole HCl (60 mg/kg) was given to the animals 1.5 h prior to sacrifice. The tumor tissues were immunostained with Hypoxyprobe following the procedures mentioned above. The acquired images were analyzed via Image J. The proportion of hypoxic regions in the tumor tissues was evaluated by following the formula AH/AT: AH represents the area of hypoxic regions; AT is the area of whole tumor tissues. In vivo photodynamic therapy and tissue evaluation. In this experiment, Balb/c mice bearing 4T1 subcutaneous tumors were randomly divided into five groups: PBS, OGzyme (10 mg/kg FTn), Lipo-

16

AIE (5 mg/kg AIE), Lipo-OGzyme-AIE low (2.5 mg/kg AIE, 5 mg/kg FTn) and Lipo-OGzyme-AIE high (5 3

mg/kg AIE, 10 mg/kg FTn). When tumor volumes reached ~100 mm , the mice were systemically administrated to allow the nanoparticles accumulation into tumors. After 24 h administration, the -2

tumors of the mice were exposed to white light irradiation (0.35 W cm ) for 10 min. The tumor sizes and body weight were recorded every other day until scarifice. For H&E staining analysis, after 14 days post-treatment, H&E staining of the tumor tissues was performed following standard procedures. Briefly, the tumors were isolated to fix in 4% paraformaldehyde in PBS, followed by embedding into paraffin. The paraffin-embedded tumor samples were sectioned into slices with 5 µm thickness. The slices were then stained with hematoxylin and eosin (H&E) for histopathological examination. For TUNEL staining, different from the above studies, the tumors were isolated 24 h post-treatment while not 14 days. The paraffin-embedded tumor slices were prepared and then TUNEL staining was performed according to the manufacturer instruction of Colorimetric TUNEL Apoptosis Assay Kit (Beyotime Biotechnology). Lung metastasis model. 4T1 orthotopic breast cancer xenografts were established according to a previous reported method [48]. Mouse nipples served as positional cues for the fat pad location. Female BALB/c mice were anesthetized and a small incision between the fourth nipple and the midline 6

was made. Subsequently, 1×10 4T1 cells overexpressed firefly luciferase were injected into the mammary fat pad of mice, followed by suture of the wound. Solid tumors were allowed for growth for 7 days, the mice were randomly assigned and imaged using IVIS Lumina II Xenogen after tail vein injected with D-Luciferin for 10 minutes. The mice were treated following the above imaging procedures. Then, the mice were administrated for 24 h, to allow the nanoparticles accumulation into tumors following i.v. injection. The tumors of the mice were exposed to white light irradiation (0.35 W -2

cm ) for 10 min. Two weeks later, the mice were imaged again after injection of D-Luciferin for 10min and then sacrificed. Major organs were harvested and imaged. The signal intensity of lung was quantified and analyzed by Living image software. Statistical analysis. The Student's unpaired t-test was used for statistical analysis between two groups of independent data. If multiple comparisons were involved, one-way or two-way analysis of variance was used as needed. Differences were considered statistically significant at p < 0.05. Results were shown as the mean±SEM.

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Acknowledgements This work was supported by the National Natural Science Foundation of China (31870999), the Natural Science Foundation of Tianjin (18JCYBJC40900), National Thousand Young Talents Program, Nankai University Hundred Young Academic Leaders Program, and “the Fundamental Research Funds for the Central Universities”, Nankai University (63191120).

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Scheme 1. Schematic illustration of the preparation of nanoparticles and cascade reactions induced by the nanozymes. a) Structures of the OGzymes and Lipo-OGzyme-AIE. b) Proposed action mechanism of the nanoparticles in tumors. Efficient tumor uptake of the OGzymes was achieved by encapsulation into liposome. Subsequently, the OGzymes further penetrated hypoxic tumor tissues, in addition to normoxic tumor areas. The OGzymes of possessing catalase-like activity intratumorally generated oxygen based on catalytic reaction responsive to tumor microenvironment, especially for hypoxia. Inspired by the properties of the OGzymes, a theranostic nanosystem composed of aggregation-induced emission (AIE) molecules and liposome was prepared, providing multi-modal imaging of tumor and modulation of tumor microenvironment for improved photodynamic therapy (PDT) by cascade reactions of therapeutic effector molecules.

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Figure 1. Characterization of the OGzymes and liposome encapsulation. a) Model of protein crystal 2+

structure of FTn. Enlarged frame is the interaction model between Mn and amino acid residues (Glu27, His65, Glu62) of single FTn subunit. b) TEM image of cage-like structures of the OGzymes following negative staining with 1% uranyl acetate. Scale bar=20 nm. c) TEM image of MnO2 core of the OGzymes without negative staining. Scale bar=20 nm. d) O2 generation ability of the OGzymes based on catalase activity. Left, production of a large amount of O2 bubbles in the presence of 80 mM H2O2 substrate. Right, dynamic changes of dissolved O2 level at pH 6.5 and pH 7.4 in the presence of 100 µM H2O2 substrate, respectively. e) TEM image of Lipo-OGzyme-AIE after negative staining. Scale bar=50 nm. f) TEM image of Lipo-OGzyme-AIE without negative staining. Scale bar=50 nm. g) Size distribution of Lipo-AIE and Lipo-OGzyme-AIE by DLS characterization. h) Dynamic changes of dissolved O2 level at different pH in the presence of 100 µM H2O2 solution. i) Decomposition rates of ABDA of Lipo-AIE, Lipo-OGzyme-AIE under white light irradiation. A0 and A are ABDA absorbance at 400nm before and after irradiation, respectively.

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Figure 2. Advantages of secondary delivery system in tumor uptake and intratumoral tissue penetration. a) Improved tumor accumulation of FTn after loading into liposome following intravenous injection for 24 h (n=3 per group). b) Quantification analysis of biodistribution of FTn-Cy 5 and LipoFTn-Cy 5 in different organs, respectively. c) Penetration of FTn-Cy 5 loaded Lipo-AIE through a 4T1 tumor spheroid model. Scale bar = 100 µm. d) Image-based quantification analysis of penetration of FTn-Cy 5 loaded Lipo-AIE in spheroids by using a custom-made formula A0/A1, where A0 is the mean intensity of the area distant from center within 50 µm; A1 defines the mean intensity of the area from outside to inside within 50 µm. Scale bar = 100 µm. e) Distribution of FTn-Cy 5 loaded Lipo-AIE in the 4T1 tumor tissues following intratumoral injection (n=3 per group). Image-based quantification analysis of f) distribution area and g) penetration distance of intratumorally administrated FTn-Cy 5 loaded Lipo-AIE, respectively. h) Colocalization of i.v. administrated Lipo-FTn-Cy 5 (red) with hypoxic areas (blue). Blood vessels were stained with anti-CD31 antibody (green). Scale bar = 100 µm.

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Figure 3. Cellular uptake and PDT-mediated tumor cell killing of the particles in vitro. The difference of cell uptake between Cy 5 labeled FTn and Cy 5 labeled OGzymes in a) normoxic (21% O2) and b) hypoxic (1% O2) conditions were analyzed by using (left) confocal imaging and (right) flow cytometry. Scale bar = 10 µm. c) Confocal imaging and flow cytometry analysis of cellular uptake of LipoOGzyme-AIE after incubation with 4T1 cells for 6 h. Scale bar = 20 µm. d) Confocal imaging and flow 1 cytometry evaluation of intracellular O2 level using DCFH-DA sensor. Scale bar = 5 µm. e) Cell viability of 4T1 cells treated with different concentration of OGzyme, Lipo-AIE and Lipo-OGzyme-AIE -2 under normoxic and hypoxic conditions following with white light irradiation at 0.15 W cm . The relative activity of untreated cells was set to be 100%. The Lipo-AIE concentration was determined by the equivalent OGzyme of Lipo-OGzyme-AIE.

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Figure 4. In vivo tumor accumulation and hypoxia alleviation. a) In vivo fluorescence imaging of 4T1 tumor (white arrow) bearing BALB/c mice over time following i.v. administration of Lipo-FTn-AIE. b) Image-based quantification analysis of mean intensity of Lipo-AIE in tumor and muscle tissues at different time points. c) Distribution of AIE molecules in tumor tissues from a). Aggregation-induced emission (white arrow) was observed by confocal imaging of cryosection of tumor tissues. d) Representative confocal imaging of hypoxia alleviation in tumor slices after immunostaining. Following treatment with the particles, the tumor tissues were stained with anti-pimonidazole antibody (green) and anti-CD31 antibody (red), respectively. Nuclei was stained with DAPI (blue). n=3 per group. Scale bar=100 µm. e) The relative hypoxia positive areas in tumor tissues after treatments.

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Figure 5. In vivo anti-tumor activity. a) Tumor growth curves (normalized to tumor volume at the start of treatment) of different groups of 4T1 tumor-bearing mice after various treatments indicated (n=8 per group). * p<0.05, **p<0.01. b) Body weight changes of the mice with different treatments. c) H&E (upper) and TUNEL (lower) staining of tumor slices collected from mice following different treatments indicated. Scale bar is 100 µm. d) Bioluminescence signal of the mice over time through IVIS imaging. The orthotopic breast cancer model was established with luciferase overexpressed 4T1 cells inoculated into fat pad location (n=5 per group). e) IVIS imaging (upper) and image-based quantification analysis (lower) of metastatic breast cancer in the lung14 days after treatments. **p<0.01.

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Graphical abstract

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