Ï-Extended Benzoporphyrin-Based MetalâOrganic Framework for

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#-Extended Benzoporphyrin-Based Metal-Organic Framework for Inhibition of Tumor Metastasis Jin-Yue Zeng, Mei-Zhen Zou, Mingkang Zhang, Xiao-Shuang Wang, Xuan Zeng, Hengjiang Cong, and Xian-Zheng Zhang ACS Nano, Just Accepted Manuscript • Publication Date (Web): 27 Mar 2018 Downloaded from http://pubs.acs.org on March 27, 2018

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Π-Extended Benzoporphyrin-Based Metal-Organic Framework for Inhibition of Tumor Metastasis

Jin-Yue Zeng,† Mei-Zhen Zou,† Mingkang Zhang,† Xiao-Shuang Wang,† Xuan Zeng,† Hengjiang Cong† and Xian-Zheng Zhang†,*

†

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

* Corresponding author. Email: [email protected]

1

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ABSTRACT: We report on the benzoporphyrin-based metal−organic framework (TBP-MOF), with 10-connected Zr6 cluster and much improved photophysical properties over the traditional porphyrin-based MOFs. It was found that TBP-MOF exhibited red-shifted absorption bands and strong near-infrared luminescence for bioimaging, whereas the π-extended benzoporphyrin-based linkers of TBP-MOF facilitated 1O2 generation to enhance O2-dependent photodynamic therapy (PDT). It was demonstrated that poly(ethylene glycol)-modified nanoscale TBP-MOF (TBP-nMOF) can be used as an effective PDT agent under hypoxic tumor microenvironment. We also elucidated that the low O2-dependent PDT of TBP-nMOF in combination with αPD-1 checkpoint blockade therapy can not only suppress the growth of primary tumor, but also stimulate an antitumor immune response for inhibiting metastatic tumor growth. We believe this TBP-nMOF has great potential to serve as an efficient photosensitizer for PDT and cancer immunotherapy. KEYWORDS:

metal-organic

framework,

benzoporphyrin,

tumor

metastasis,

photodynamic therapy, immunotherapy

2


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Photodynamic therapy (PDT) is based on the accumulation of nontoxic photosensitizers (PSs) in target tissue, tissue oxygen and light to generate singlet oxygen to selectively damage target cells or tissue.1 The photodynamic activity of PSs for PDT mainly relies on their photochemical and pharmacological properties such as wavelength region of light absorption, quantum yield of photosensitization, molar absorption coefficient, the accumulation in tumor tissue and tissue oxygen concentration.2 It is well-known that the tumor hypoxia microenvironment, caused by distorted tumor blood vessels and the rapid proliferation of tumor cells, would lead to a negative effect on the formation of cytotoxic 1O2.3 The continued O2 consumption mediated by PSs would further aggravate the tumor hypoxia during PDT and greatly limit the O2-dependent PDT efficacy.4 To address these issues, it is critically important to develop the efficient photosensitizer with stronger light absorptions in the infrared region and effective 1

O2-generation under the native hypoxic microenvironment. Benzoporphyrin derivatives, a class of π-extended porphyrins, are promising PSs for

application in PDT.5 Due to the extension of their π-conjugation, benzoporphyrin derivatives exhibit red-shifted absorption bands (at λ > 650 nm), strong infrared luminescence and high chemical stability compared with the common porphyrins.6 With the development of efficient synthetic methods, benzoporphyrin derivatives are being investigated as a potential alternative to traditional porphyrins for PDT.7 Nevertheless, most of obtained benzoporphyrin derivatives are highly hydrophobic and suffer from defects such as lack of selectivity toward tumor cells, the propensity for self-aggregation 3


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and the limited solubility in solution.8 Although many nanomaterials have been involved as PS carriers, most of them usually exhibit limitations of imprecise loading, instability and leaching.9 Recently, porphyrin-based metal−organic frameworks (MOFs) were fabricated as PSs for PDT.10 These MOFs with crystalline structure and stable framework exhibit high PS loadings, the facile diffusion of 1O2 and avoidable self-quenching.11 Metastasis is the main reason for cancer mortality.12 However, PDT is mainly used for the treatment of superficial cancer in clinic due to the limited penetration of the light. It was reported that PDT can stimulate adaptive immune responses by releasing inflammatory mediators and cytokines into the tumor microenvironment.13 The antitumor immune response can be enhanced by targeting regulatory pathways in T cells.14 It has been well-studied that tumor cells themselves can induce T cell apoptosis through programmed cell death receptor 1 (PD-1)/programmed death 1 ligand (PD-L1) pathway for immune evasion.15 Recently, PD-1/PD-L1 checkpoint blockade strategies, which have demonstrated to be a promising tumor immunotherapy method, can be used to improve anti-tumor immunities by inhibiting cytotoxic T lymphocytes exhaustion.16 Therefore, the combination of checkpoint blockade with traditional therapy strategies, such as chemotherapy and PDT, could provide combined therapeutic benefits for the recurrence and metastasis of tumor.17 Here, benzoporphyrin-based metal-organic framework (TBP-MOF) was reported, which exhibited 10-connected Zr6 cluster and much improved photophysical properties due to the π-extended benzoporphyrin-based linkers. It was found that the 4


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tetrabenzoporphyrin-based linkers (TBP) of TBP-MOF are well-dispersed in the framework to avoid self-quenching of the excited states. It was also discovered that TBP-MOF shows high chemical stability, red-shifted absorption bands and strong infrared luminescence compared with the traditional porphyrin-based MOFs. Clearly, poly(ethylene glycol)-modified nanoscale TBP-MOF (TBP-nMOF) can be used as an effective PDT agent under hypoxic tumor microenvironment. TBP-nMOF-mediated PDT can not only induce the apoptosis and necrosis of 4T1 murine breast cancer cells, but also increase the presentation of tumor-activated antigens to T cells and stimulate the host immune system. It was further found that the low O2-dependent PDT of TBP-nMOF in combination with PD-L1 checkpoint blockade therapy can significantly inhibit the growth of the primary tumors and stimulate systematic immune response for inhibiting metastatic tumor growth (Scheme 1). RESULTS AND DISCUSSION Preparation and Characterization of TBP-MOF. In this investigation, we reported the rational design of the π-extended benzoporphyrin-based metal−organic framework. The crystallographic data and structural refinements for TBP-MOF (Cu) were summarized in Table S1-4 (Supporting Information, SI). Single-crystal X-ray diffraction studies revealed that TBP-MOF (Cu) was isoreticular with tetragonal topology and crystallized in space group I41/amd (Figure 1A and Figure S1-3). Different from the 12-connected Zr6 cluster showed in the UiO-66, ten edges of the Zr6 cluster were bridged by carboxylates from TBP (Cu) ligands and two edges were coordinated by two benzoic 5


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acid molecules in TBP-MOF (Cu) (Figure 1B). It was found that the framework of TBP-MOF (Cu) consists of 10-connected Zr6 clusters linked by the twisted planar TBP ligands giving rise to a 3-D framework (Figure 1C). In conventional Zr-MOFs, this secondary building unit differs from 6-connected and 8-connected Zr6 cluster in porphyrin-based MOFs.18-20 The diversity could be attributed to the stereo-hindrance effect and cyclic tension from tetrabenzoporphyrin-based linkers. Moreover, we also found that the 10-connected Zr6 cluster of TBP-MOF exhibited low symmetry and two types of coordinated TBP (Cu) could be differentiated into single or double carboxylate oxygens from TBP (Cu) ligands. The coordinated formula of Zr6 cluster in TBP-MOF (Cu) is assigned to be Zr6O4(OH)4(C6H5COO)2(1COO)4(2COO)6. The higher connectivity on Zr6 cluster resulted from the increased TBP-Zr ratio in the starting material. Thus, TBP-MOF with 10-connected Zr6 clusters could exhibit the higher photosensitizers loading efficiency. The 3D AFM images and topological height profiles for the morphology of TBP-MOF show the fluctuation changes of surface height at ~ 8 nm (Figure S4, 5). This was likely attributed to surface local defects of TBP-MOF crystal. To assess their crystallinity, all of samples were characterized by powder X-ray diffraction (PXRD) pattern (Figure 1D). It was found that the as-prepared TBP-MOF (Cu) was consistent with the simulated pattern from single crystal structure data, suggesting phase purity of TBP-MOF (Cu). Notably, PXRD pattern of TBP-MOF (free metal) also can match well with TBP-MOF (Cu), demonstrating the consistency of crystal structure (Figure 1E). It was further found that the activated TBP-MOF (Cu) remained the sharp 6


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peaks in their PXRD patterns, indicating the stability of the crystal structure (Figure S6). PXRD patterns also exhibited that TBP-MOF (Cu) remained the crystallinity in aqueous solutions with pH values in the range of 1−10, confirming the chemical stability of TBP-MOF (Cu) (Figure S7). Thermogravimetric (TG) analysis in the N2 stream showed that the framework of TBP-MOF (Cu) started to decompose around 400 °C. The weight loss of TBP-MOF (Cu) before 300 °C was attributed to coordinated hydroxide groups/water molecules and the isolated N,N-dimethylformamide (DMF) molecules (Figure S8). Furthermore, the Raman spectrums of TBP and TBP-MOF were obtained with exciting laser with wavelength of 514 nm (Figure S9). We cannot only investigate the modes associated with the carboxylate groups, but also the main differences between TBP and TBP-MOF can be observed. The porosity of TBP-MOF series was then investigated by nitrogen adsorption studies at 77 K. The N2 uptake and Brunauer-Emmett-Teller (BET) surface area of TBP-MOF (Cu) were determined to be 340 cm3 (STP) and 1124 m2 g-1, respectively. The N2 adsorption of TBP-MOF (Cu) showed a typical type-Ⅰ isotherm, which was fully consistent with the crystal structure (Figure 1F). TBP-MOF (free metal) also exhibited similar surface areas and N2 adsorption isotherms. Controlled Synthesis and Characterization of Nanoscale TBP-MOF. It was found that each linker of TBP-MOF was spatially isolated in the framework and its molecular property greatly could be preserved regardless of dimensions. As a result, TBP-MOF could be completely used as small molecules and their built-in properties could not be 7


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affected by particle size. We conceived whether this property could allow for the maximization of passive targeting through optimizing the size of TBP-MOF for PDT.21 Thus, we carried out the downsizing of TBP-MOF from millimeter scale to nanoscale by diluting the reaction system (Figure 2A, C). To preserve the phase purity of TBP-MOF, the stoichiometry between reactants was not changed, while aiming to produce more MOF

crystal

nucleus,

which

resulted

in

smaller

nanoparticles.

Scanning electron microscope (SEM) and transmission electron microscopy (TEM) images of downsizing TBP-MOF clearly showed spherical morphology (Figure 2B, C). The size of such TBP-MOF nanoparticle with good monodispersity was measured to be ~ 50 nm (Figure 2C). PXRD data confirmed that the identity of TBP-nMOF nanoparticles was unchanged by size variation (Figure 2D). Moreover, the N2 uptake and BET surface area of TBP-nMOF were determined to be 303 cm3 (STP) and 1018 m2 g-1, indicating well maintained porosity compared to bulk crystalline TBP-MOF (Figure S10). To enhance the physiological stability, TBP-MOF nanoparticles were modified with poly(ethylene glycol) (PEG)-SH through surface coordination method. After modified with (PEG)-SH, the zeta potential of the TBP-nMOF reduced to ∼ -9.3 mV. Dynamic light scattering (DLS) measurements on TBP-nMOF gave an average diameter of 102.5 nm in dulbecco's modified eagle's medium (DMEM) with a polydispersity index of 0.067 (Figure S11). Furthermore, it was found that the size and zeta potential of TBP-nMOF showed no obvious changes in 72 h, indicating the stability at the physiological condition (Figure S12, 13). 8


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The improved photophysical properties of TBP-MOF were investigated by UV-vis absorption spectroscopy. It was found that TBP linkers exhibited a split Soret band at λmax 470 nm and three Q bands at 580, 646, and 692 nm. In contrast, TBP-nMOF showed slight red shifts for all of the Q bands, with peaks at 590, 655, and 705 nm (Figure 2E). Importantly, the lowest-energy Q band of TBP-nMOF was thus red-shifted by 50 nm relative to the traditional porphyrin MOF (Figure S14). We also found that TBP exhibited the fluorescence peaks at 705 and 780 nm, while the fluorescence emission of TBP-nMOF showed slight red shifts relative to that of TBP (Figure 2F). Notably, the lowest-energy emission of TBP-nMOF was red-shifted by ~100 nm compare to porphyrin MOF (Figure S15). Furthermore, it was confirmed that TBP-nMOF showed a relatively shorter fluorescence lifetime of 2.12 ns compared with TBP (3.16 ns) as determined because of enhanced intersystem crossing (ISC) upon coordination of the TBP ligands to Zr4+ ions through the carboxylate groups (Figure 2G). Therefore, TBP-nMOF could not only use as an efficient PDT photosensitizer, but also emit strong near-infrared fluorescence for bioimaging. Singlet Oxygen Generation of TBP-nMOF. Considering the improved photophysical properties of TBP-MOF, the

1

O2 generation of TBP-nMOF was investigated.

Anthracene-9,10-dipropionic acid (ADPA) was employed to determine to the 1O2 generation efficiencies of tetrakis (4-carboxyphenyl)porphyrin (TCPP), protoporphyrin (PpIX), porphyrin-based zirconium MOF (PCN-224), TBP and TBP-nMOF. The TBP-nMOF samples and controls were irradiated with 660-nm light-emitting diode (LED) 9


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and the adsorption decrease at 378 nm upon the reaction of ADPA with 1O2 was measured by UV-vis spectrophotometer. Notably, both TBP ligand and TBP-nMOF showed much higher 1O2 generation ability than the traditional PSs within the oxygen atmosphere (Figure 2H). Furthermore, the 1O2 generation efficiencies of TBP-nMOF were examined in different oxygen concentration. It was found that TBP-nMOF-induced 1O2 generation remained at high levels even under low oxygen concentration (Figure 2I). This result indicated that TBP-nMOF could serve as an efficient PDT photosensitizer within low oxygen concentration. We also further investigated the 1O2 generation efficiencies of PpIX, TCPP, TBP, porphyrin MOFs and TBP-nMOF (10 µM) under 660-nm LED irradiation by utilizing singlet oxygen sensor green (SOSG) as probe (Figure S16). SOSG could react with 1O2 to generate green fluorescence that could be quantified on a fluorimeter. Notably, TBP-nMOF is ∼8 times higher than porphyrin-based MOF (PCN-224) and ∼17 times as efficient as PpIX in generating 1O2. The Anti-Tumor Efficay of TBP-nMOF in Vitro. The hypoxic tumor microenvironment is the main cause of the limited PDT efficacy for therapy of solid tumors as oxygen plays an important role in the process of PDT. Given the efficient reactive oxygen species (ROS) production of TBP-nMOF at low oxygen concentration, we then investigated the therapeutic effects of TBP-nMOF in vitro by using the 4T1 cells. The intracellular distribution and uptake were confirmed by confocal laser scanning microscopy (CLSM) and flow cytometric measurement (Figure 3A and Figure S17). It was discovered that obvious red fluorescent signal from the endocytosis of the 10


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TBP-nMOF was observed through CLSM, indicating that TBP-nMOF was efficiently internalized within 4T1 cells. Furthermore, the intracellular ROS generation of TBP-nMOF was examined at different oxygen concentration by using DCFH as the ROS probe, which could emit green fluorescence in the existence of ROS via CLSM observation. As expected, TBP-nMOF exhibited significant DCF fluorescence against 4T1 cells in 21% O2 atmosphere, indicating efficient ROS production ability (Figure 3D). Importantly, after reduction of O2 atmosphere to 5%, the light-triggered ROS production of TBP-nMOF also was remarkably observed (Figure 3C). These results suggested that TBP-nMOF was an effective PDT agent under hypoxic tumor microenvironment. To investigate the PDT efficiency of TBP-nMOF, 4T1 cells were incubated with TBP-nMOF under 21% O2 and 5% O2 atmosphere for 6 h. The 4T1 cells then were irradiated with 660-nm LED (30 mW cm−2, 3 min). After 24 h, the cell viabilities were determined by the standard methyl thiazolyl tetrazolium (MTT) assay (Figure 3E, F). It was found that TBP-nMOF showed no obvious toxicity to 4T1 cells in dark. Notably, TBP-nMOF exhibited high phototoxicity to cells within 21% O2 and 5% O2 atmosphere. In contrast, although the PDT-mediated cell destruction by TBP-nMOF in 21% O2 atmosphere could be much high effective, the light-induced tumor cell-destruction efficiency of TBP-nMOF remained at relatively high levels even under the hypoxic condition. Meanwhile, we also found that TBP-nMOF showed enhanced phototoxicity to 4T1 cells compared to traditional porphyrin MOF (PCN-224) within 21% O2 and 5% O2 atmosphere (Figure S18, 19). To further evaluate the apoptosis stages of the 4T1 cells 11


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after various treatments, the cells were examined by flow cytometry using annexin V−FITC and PI (Figure S20). It was demonstrated that TBP-nMOF-mediated PDT significantly induced the apoptosis and necrosis of 4T1 cells in 5% O2 atmosphere under 660-nm LED irradiation (30 mW cm−2, 3 min). Therefore, different from conventional PSs, TBP-nMOF was not only effective under normoxic conditions, but also could use as an effective PDT photosensitizer even within the hypoxic environment. The Biodistribution and Antitumor Efficay of TBP-nMOF in Vivo. To evidence the feasibility of TBP-nMOF in vivo, 4T1 tumor xenograft in Balb/c mice model was established for evaluating the biodistribution of TBP-nMOF on a small animal imaging system. The fluorescence imaging was measured after intravenous injection of TBP-nMOF. It was found that the fluorescence signal gradually increased in the tumor tissue and came to a head at 6 h post-injection (Figure 4A, C). The tumor and main organs were harvested at 6 h after the injection of TBP-nMOF for imaging, indicating that TBP-nMOF effectively accumulated in tumor tissues (Figure 4B, D). Meanwhile, we also found that the fluorescence in bladder was strong, suggesting that TBP-nMOF possess good biological compatibility and can be effectively metabolized in vivo. To quantitatively assess the biodistribution of TBP-nMOF, the amounts of Zr in major organs and tumor tissues were tested by ICP-MS, indicating Zr element mainly accumulated in tumor tissue and liver (Figure 4E). The pharmacodynamic study demonstrated that TBP-nMOF possessed relatively long circulation time in the blood (Figure S21, 22). These results suggested that TBP-nMOF has distinct advantages for 12


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bioimaging. First, TBP-nMOF exhibit red-shifted absorption bands and strong infrared luminescence compared with traditional porphyrin MOF, which can effectively avoid the interference of biological background fluorescence (Figure S23). Second, TBP-nMOF with the size of ~50 nm is appropriate for efficient tumor tissues accumulation by the enhanced permeability and retention (EPR) effect. Third, TBP-nMOF with good stability and hydrophilicity can effectively evade RES recognition and elimination at physiological conditions.22 Considering the low dependent ability of TBP-nMOF to oxygen for PDT, we investigated the ability of low O2-dependent PDT to induce apoptosis and necrosis in vivo. The 4T1 tumor-bearing mice were randomly divided into five groups (6 mice per group). The mice were treated with TBP-nMOF solution by intravenous method and then the tumors were irradiated with 660-nm laser after 6 h. The tumor volumes and mice body weights were recorded in following 2 weeks (Figure 4F, G). At 15 day, the tumors of all mice were weight and collected. It was found that traditional porphyrin MOF with 660-nm laser irradiation partially inhibited the tumor growth compared with PBS group (Figure S24). Notably, TBP-nMOF with 660-nm laser irradiation, the tumors exhibited the smallest volumes and slowest growth speed at the end of treatment (Figure 4H and Figure S25). Furthermore, hematoxylin and eosin (H&E) staining also displayed that most of tumor cells became necrotic and apoptotic in the group of TBP-nMOF with 660-nm laser irradiation (Figure S26A). Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) of PDT group showed significant immunofluorescence, 13


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which was attributed to PDT of TBP-nMOF stimulated immune responses by releasing inflammatory mediators and cytokines into the tumor microenvironment (Figure S26B). H&E staining images of major organs from PDT group suggested that TBP-nMOF induced negligible toxic side effects to mice (Figure S27). The Antitumor Effect of Synergistic TBP-nMOF-Mediated PDT and αPD-1 in Vivo. Although TBP-nMOF-mediated PDT could significantly inhibit the primary tumor growth, PDT alone could not eliminate completely the 4T1 tumors and prevent tumor recurrence. PDT can stimulate antitumor immunity and cause the activation of cytotoxic T cells and their infiltration to the tumors, which may increase immunogenicity and improve the presentation of tumor-derived antigens to T cells.23 It has been well-studied that tumor cells themselves can induce T cell apoptosis through PD-1/PD-L1 pathway for immune evasion.16 Encouraged by the immune responses that induced by low O2-dependent TBP-nMOF-mediated PDT, we thus hypothesized that such a therapy strategy might have any synergistic effect with PD-1/PD-L1 checkpoint blockade. To evaluate the anti-tumor effect of synergistic PDT and αPD-1 in vivo, the therapeutic study was carried out by using a 4T1 tumor xenograft in Balb/c mice model (Figure 5A). As shown in Figure 5B, PBS or TBP-nMOF exhibited no obvious effect on the tumor growth, whereas αPD-1 or PDT alone partially inhibited the tumor growth. Notably, combined PDT and αPD-1 almost eliminated the 4T1 tumors without resulting in body weight loss, suggesting PD-L1 blockade significantly enhanced PDT-mediated tumor immunotherapy (Figure 5C, D and Figure S28). Furthermore, TUNEL and H&E staining of the tumor 14


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tissue showed that the combination of PDT and αPD-1 induced the highest apoptotic ability among all of the treatment groups, which was attributed to both PDT and PD-L1-blockade triggered antitumor immune response and recruited tumor infiltrating T cells (CD8+ and CD4+) (Figure 5E, F). The proliferation ability of the CD8+ T cells was examined through analyzing tumor infiltrating lymphocytes (TILs) stained with anti-CD3-FITC, anti-CD4-PE and anti-CD8a-APC antibodies by flow cytometry (Figure 5G). The tumor infiltrating CD8+ T cells in the TBP-nMOF+light+αPD-1 group showed the highest proliferation activity, indicating that TBP-nMOF-mediated PDT significantly promoted the proliferation of CD8+ T cells (Figure 5H). Similar result was demonstrated in the main immune organ of spleen (Figure S29). We further investigated the influence of αPD-1 and PDT on the secretion of predominant cytokine in CD8+ T cells of tumor tissue. It was found that PDT significantly increased the secretion of interferon gamma (IFN-γ) and tumor necrosis factor (TNF-α) dual-positive tumor infiltrating CD8+ T cells (Figure 5I, J). These results also further demonstrated that PDT and PD-L1 blockade synergistically activated the CD8+ T cells. To confirm the activation of protective immune effect, the anti-tumor effect of combined PDT and αPD-1 was further tested by using a lung metastatic 4T1 tumor-bearing Balb/c mice model.24 All of the mice groups were intravenously rechallenged with luciferase-4T1 cells (luc-4T1) and monitored for the growth of lung metastatic tumor by bioluminescent imaging (Figure 6A and Figure S30). Obvious lung metastasis was observed in all the mice of the control groups (Figure 6B). In contrast, the 15


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combination of PDT and αPD-1 was much more efficient than single PDT in preventing tumor recurrence and dramatically inhibited lung metastasis of 4T1 tumor in a period of 22 days, indicating that a PD-L1 blockade significantly improved the long-lasting immune

response.

The

inhibition

effect

of

lung

metastasis

of

combined

TBP-nMOF-mediated PDT and αPD-1 was further demonstrated by H&E staining of the lungs. It was found that control groups showed obvious metastatic lesions (Figure 6C). These

results

indicated

that

the

combination

of

PD-L1

blockade

and

TBP-nMOF-mediated PDT was a potential therapy for metastatic 4T1 tumor establishment in lung. In this study, we confirmed that TBP-nMOF-mediated PDT could be enhanced by αPD-1-induced immunotherapy. Although many efforts have been devoted for PD-L1/PD-1 checkpoint blockade with anti-PD-L1 or anti-PD-1 monoclonal antibodies in past years, our strategy of combining PD-L1 silencing with PDT have distinct advantages. TBP-nMOF-mediated PDT can induce significant apoptosis and necrosis of the tumor cells in hypoxia microenvironment and active the systemic immune response (Figure 6D). PDT-mediated immune response can further be improved by stimulating chemokine expression via silencing of PD-L1. Therefore, PDT-induced tumor infiltration T cells and PD-L1 blockade can synergistically enhance the therapy effect and inhibit the metastasis of tumor. Moreover, TBP-nMOF-mediated PDT can also be carried out under the guidance of near-infrared fluorescence imaging to prevent nonspecific phototoxicity. CONCLUSIONS 16


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In summary, we developed a benzoporphyrin-based metal−organic framework, TBP-MOF, which exhibits enhanced stability and much improved photophysical properties. It was found that the π-extended benzoporphyrin-based linkers of TBP-MOF show efficient 1O2 generation over the traditional porphyrin-based MOFs. We confirmed that TBP-nMOF could serve as an effective PDT agent even within the hypoxic environment. Moreover, TBP-nMOF also shows red-shifted absorption bands and strong near-infrared luminescence, which could be used for near-infrared fluorescence imaging of tumor. It was further demonstrated that TBP-nMOF-mediated PDT induces adaptive immune response, triggering the secretion of inflammatory cytokines (IFN-γ, TNF-α) and recruiting tumor-infiltrating T cells (CD4+, CD8+). Moreover, PDT-induced host antitumor immune response could be improved by αPD-1 checkpoint blockade therapy. As a result, TBP-nMOF-mediated PDT in combination with αPD-1 checkpoint blockade therapy can not only suppress the growth of the primary tumors, but also inhibit the metastasis of tumors. METHODS Preparation of TBP-MOF(free metal). TBP (5 mg, 0.005 mmol), ZrCl4 (15 mg, 0.05 mmol), benzoic acid (400 mg) and acetic acid (0.05 mL) in 1 mL of DMF were ultrasonically dissolved in a Pyrex vial. The mixture was heated to 140 °C in oven for 96 h. After cooling down to room temperature, dark purple cubic crystals were harvested. As-prepared TBP-MOF were washed with DMF five times and thrice with dichloromethane, and then immersed in dichloromethane for over 48 h. After the removal 17


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of dichloromethane, the samples were further activated under vacuum for 6 h at 40 °C (Yield: 2.4 mg, 34.3%). Preparation of TBP-MOF(Cu). Cu-TBP (5 mg, 0.005 mmol), ZrCl4 (15 mg, 0.05 mmol), benzoic acid (400 mg) and acetic acid (0.05 mL) in 1 mL of DMF were ultrasonically dissolved in a Pyrex vial. The mixture was heated to 140 °C in oven for 72 h. After cooling down to room temperature, dark purple cubic crystals were harvested. As-prepared TBP-MOF(Cu) were washed with DMF five times and thrice with dichloromethane, and then immersed in dichloromethane for over 48 h. After the removal of dichloromethane, the samples were further activated under vacuum for 6 h at 40 °C (Yield: 3.0 mg, 42.9%). Preparation of TBP-nMOF. TBP (50 mg,0.05 mmol), ZrOCl2·8H2O (150 mg, 0.465 mmol), and benzoic acid (1.4 g) were dissolved in 100 mL of DMF in a 250 mL round-bottom flask and the mixture was stirred (500 rpm) at 100 °C for 4 h. After the reaction was done, TBP-MOF were collected by centrifugation (12000 rpm, 30 min) followed by washing with fresh DMF for 3 times. The as-obtain TBP-MOF were further dispersed in 20 mL of Milli-Q water. 20 mg of poly(ethylene glycol) (PEG)-SH were added under gently stirring and left to react for 12 h. After that, excess PEG-SH molecules were removed by repeated centrifugation (11000 rpm, 30 min). The resulting TBP-nMOF was suspended in water for further characterization and analysis. Generation and Detection of 1O2. Anthracene-9,10-dipropionic acid (ADPA) was employed to determine to the 1O2 generation efficiencies of PpIX (5 µg mL−1), TCPP (5 18


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µg mL−1), PCN-224 (containing 5 µg mL−1 of TCPP), TBP (5 µg mL−1) and TBP-nMOF (containing 5 µg mL−1 of TBP). 80 µL ADPA (1 mg/mL) was mixed with 3 mL of TBP-nMOF or control samples. Furthermore, the

1

O2 generation efficiencies of

TBP-nMOF were measured at different oxygen concentration. The samples were irradiated with 660-nm LED (30 mW cm-2), and the decrease in optical densities was recorded by UV-vis spectrophotometer. The 1O2 generation efficiencies of PpIX, TCPP, TBP, porphyrin MOFs and TBP-nMOF (10 µM) under 660-nm LED irradiation were further investigated by using singlet oxygen sensor green (SOSG) as probe. SOSG can react with 1O2 to generate green fluorescence that can be quantified on a fluorimeter (λex = 504 nm, λem = 525 nm). The 1O2 generation curve was fitted with an exponential function: IF = A (1-e-kt), where t is irradiation time and IF represents fluorescence intensity, while A and k are fitting parameters. Generation and Detection of Intracellular ROS. The intracellular ROS were measured via CLSM using DCFH-DA as the ROS indicator. 4T1 cells were incubated with TBP-nMOF (20 µg mL−1) for 6 h under different oxygen atmosphere. Then, the medium was removed and DCFH-DA was added (final concentration 1 × 10−6 M) and incubated for 30 min under different oxygen atmosphere. Thereafter, the cells were further irradiated with a 660-nm LED irradiation (30 mW cm−2, 3 min), respectively. In Vitro MTT Cytotoxicity Assay. The in vitro cytotoxicity against 4T1 cells were investigated by using MTT assay. The 4T1 cells were seeded onto the 96-well plates and incubated in medium (100 µL) at 37 °C for 24 h. Then the original medium was replaced 19


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with fresh medium (100 µL) containing different concentrations of TBP-nMOF (3.2, 4.7, 7.0, 10.5, 15.8 and 23.7 ug mL-1). After incubation for 6 h under different oxygen atmosphere, the cells were irradiated with a 660-nm LED irradiation (30 mW cm−2, 3 min). For the control groups, the cells were cultured in dark all the time. Then the cells were cultured for another 24 h and 20 µL of MTT (5 mg mL−1 in PBS) was added and further incubated for 4 h. Thereafter, the supernatants were carefully removed, and 150 µL of DMSO was added into each well. After shaking for several minutes, the optical density (OD) was recorded at 570 nm on a microplate reader (BIO-RAD, Model 550, USA). The relative cell viability was calculated by the following equation: cell viability (%) = OD (sample) × 100/OD (control), where OD (control) was obtained in the absence of samples and OD (sample) was obtained in the presence of samples. Cell Apoptosis Analysis. The cell apoptosis analysis against 4T1 cells was performed by utilizing a flow cytometric assay of Annexin V-FITC and PI co-staining. 4T1 cells were seeded in 6-well plates and were treated with TBP-nMOF (20 µg mL−1). After incubation for 6 h under different oxygen atmosphere, the cells were irradiated with a 660-nm LED irradiation (30 mW cm−2, 3 min). Then the cells were cultured for another 24 h. 4T1 cells were stained with Annexin V-FITC/PI for 20 min. Subsequently, the cells were collected for flow cytometry measurement. Fluorescence Imaging and Pharmacokinetic Studies in Vivo. When the tumors grew to ~100 mm3 in volume, 150 µL of TBP-nMOF (600 µg mL−1 containing TBP: 4.0 mg kg−1) were injected into the mice by intravenous method. Thereafter, the mice were 20


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anesthetized and imaged in a small animal imaging system (PE Spectrum & Quantum FX) at 2, 4, 6, 8, 10, 12 and 24 h post-injection. The excitation wavelength was 675 nm and the fluorescence emission at 740 nm was collected. For the tissue distribution study of the composites, the mice were sacrificed at 6 h post-injection, and major organs were collected for ex vivo imaging. To quantitatively assess the biodistribution of the TBP-nMOF, major organs (heart, liver, spleen, lung and kidney) and the tumor tissues were obtained and digested to determine the amounts of Zr by ICP-MS measures at 6 h post-injection. For pharmacokinetic studies, 150 µL of TBP-nMOF (600 µg mL−1 containing TBP: 4.0 mg kg−1) were injected into the mice by intravenous method. The blood samples were taken at the 0.5, 1, 1.5, 2, 3, 5, 8, and 12 h time points after post-treatment. The samples were measured in a small animal imaging system (Ex = 675 and Em = 740 nm). In Vivo Antitumor Study. All animal experiments were carried out according to the guidelines of laboratory animals, which were defined by the Wuhan University Center for Animal Experiment/A3-Lab. Animals and Tumor Model: the female BALB/c mice (5-week old) were bought from Wuhan University Animal Biosafty Level III Lab and used for animal experiments. The tumor-bearing mice were obtained by injecting 4T1 cells (1 × 106 cells) into subcutaneous of female mice on the right back of hind leg region. When the tumors reached the size of ~100 mm3 in volume, the mice were randomly divided into 4 groups (6 mice per group). Then, tumor-bearing mice were treated with 1) PBS, 2) PBS+Light, 3) Porphyrin MOF+Light, 4) TBP-nMOF and 5) TBP-nMOF+Light. 21


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The mice were performed with a dose of 100 µL and then the laser irradiation was implemented after 6 h. Tumor size and mice weight were measured every day. Tumor size was recorded by a caliper and tumor volume was defined as: V = W2 × L/2, where W and L were the shortest and longest diameters of tumors, respectively. On day 15, all mice were killed and then the tumors were excised and weighed. Simultaneously, the main organs (heart, liver, spleen, lung and kidney) were also obtained and utilized for histology analysis. The Antitumor Efficacy of Combined PDT and Immunotherapy. To evaluate the antitumor efficacy of combined TBP-nMOF-mediated PDT and αPD-1 in vivo, 4T1 tumor xenograft in Balb/c mice model was established by subcutaneous injecting 1×106 4T1 cells. When the tumors reached the size of ~100 mm3 in volume, the mice were randomly divided into 5 groups (7 mice per group): 1) PBS, 2) PBS+light+αPD-1, 3) TBP-nMOF+αPD-1, 4) TBP-nMOF+light and 5) TBP-nMOF+light+αPD-1. The mice were performed with 100 µL of TBP-nMOF or PBS. Then, the 4T1 tumor-bearing Balb/c mice were irradiated with 660-nm laser after 6 h (300 mW·cm−2, 4 min). Furthermore, the mice were injected with αPD-1 antibody (25µg per mouse) and then repeated every other day for three dose in all. Tumor sizes and mice body weights were measured every day during the animal studies. Tumor size was recorded by a caliper and tumor volume was defined as: V = W2 × L/2, where W and L were the shortest and longest diameters of tumors, respectively. Relative tumor volume was calculated as V/V0 (V0 represents the tumor volume at the first treatment). On day 23, all mice were killed and then the tumors 22


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were excised and weighed. Simultaneously, the main organs (heart, liver, spleen, lung and kidney) were obtained and utilized for histology analysis. Immune Response of Combined PDT and αPD-1. To evaluate the immune response of combined TBP-nMOF-mediated PDT and αPD-1, cytokine secretion and tumor infiltrating lymphocytes (TILs) were detected using the 4T1 tumor xenograft in Balb/c mice model. When the tumors reached the size of ~100 mm3 in volume, the mice were randomly divided into 5 groups (7 mice per group): 1) PBS, 2) PBS+light+αPD-1, 3) TBP-nMOF+αPD-1, 4) TBP-nMOF+light and 5) TBP-nMOF+light+αPD-1. The mice were performed with 100 µL of TBP-nMOF or PBS. Then, the 4T1 tumor-bearing Balb/c mice were irradiated with 660-nm laser after 6 h (300 mW·cm−2, 4 min). Furthermore, the mice were injected with PD-1 antibody (25µg per mouse) and then repeated every other day for three dose in all. On day 12, the peripheral blood was isolated for following analysis. The production of tumor necrosis factor (TNF-α, 4A Biotech Co., Ltd) and interferon gamma (IFN-γ, 4A Biotech Co., Ltd) were analyzed by enzyme-linked immunosorbent assay according to manufacturer’s protocol. Meanwhile, to make single cell suspension, the tumor tissues were harvested and digested by 2% FBS RPMI 1640 medium. The suspension was filtered through 75 µm filters and washed with cold PBS twice after the red blood cells were removed by using ACK Lysis Buffer. Then the TILs were stained with anti-CD3-FITC, anti-CD4-PE, anti-CD8a-APC antibodies (all from biolegend) after the Fc block of the samples.

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Antimetastasis Effect of Combined TBP-nMOF-Mediated PDT and αPD-1. To further evaluate the immune response of combined TBP-nMOF-mediated PDT and αPD-1, a lung metastatic 4T1 tumor-bearing Balb/c mice model was established. When the tumors reached the size of ~100 mm3 in volume, the mice were randomly divided into 5 groups (7 mice per group): 1) PBS, 2) PBS+light+αPD-1, 3) TBP-nMOF+αPD-1, 4) TBP-nMOF+light and 5) TBP-nMOF+light+αPD-1. The mice were performed with 100 µL of TBP-nMOF or PBS. Then, the 4T1 tumor-bearing Balb/c mice were irradiated with 660-nm laser after 6 h (300 mW·cm−2, 4 min). Furthermore, the mice were injected with PD-1 antibody (25µg per mouse) and then repeated every other day for three dose in all. On day 6, 2×105 luc-4T1 cells were injected. On day 23, each mouse was intraperitoneal injected with PBS solution containing 3 mg of D-Luciferin potassium salt. Then, the mice were sacrificed and the lung tissues were harvested for bioluminescence image and H&E staining. ASSOCIATED CONTENT Supporting Information Available: The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org. Experimental details, materials and measurements, synthesis of ligands, TGA analysis of TBP-MOF, additional UV−vis absorbance spectrum, fluorescence spectrum, PXRD patterns, size and zeta potential, intracellular uptake, flow cytometry analysis, pharmacodynamic study, histology analysis, bioluminescence images, control experiments and crystallographic data (PDF). 24


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AUTHOR INFORMATION Corresponding Author: *E-mail: [email protected] Acknowledgments J.-Y. Zeng and M.-Z. Zou contributed equally to this work. This work was supported by the National Natural Science Foundation of China (51690152, 21721005 and 51533006). The single crystal XRD was performed using beamline BL17B in Shanghai Synchrotron Radiation (SSRF). We thank Xiaohui Xu, Qi Liu (Wuhan University) for their invaluable help and discussion. Crystallographic data for the reported crystal structure was deposited at the Cambridge Crystallographic Data Centre through www.ccdc.cam.ac.uk with the code 1819983.

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Scheme 1. Schematic illustration of TBP-nMOF for inhibition of tumor metastasis. (A) A scheme indicating the fabrication process of TBP-nMOF. (B) Schematic presentation of TBP-nMOF-mediated photodynamic cancer immunotherapy. TBP-nMOF-mediated PDT induces adaptive antitumor immune response, recruiting tumor-infiltrating T cells for inhibition of tumor metastasis.

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Figure 1. Illustration and characterization of TBP-MOF structure. (A) Crystal structure and underlying network topology of TBP-MOF (Cu). (B) The 10-connected Zr6 in TBP-MOF (The carbon atoms from benzoic acid, single-coordinated carboxylate oxygen of TBP and double-coordinated carboxylate oxygen of TBP are shown in purple, yellow and light black, respectively). (C) Schematic representation of TBP-MOF framework, in which 10-connected Zr6 cluster and TBP ligand are simplified as blue nodes and red plane, respectively. (D) PXRD patterns for simulated and experimental TBP-MOF (Cu); and (E) as-synthesized TBP-MOF (Cu) and TBP-MOF (free metal). (F) N2 adsorption isotherm of TBP-MOF series at 77 K, 1 atm.

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Figure 2. Controlled synthesis and characterization of TBP-nMOF. (A) Microscope image of the crystal of TBP-MOF. (B) SEM image of TBP-nMOF. (C) TEM image of TBP-nMOF. (D) PXRD patterns for simulated and experimental TBP-nMOF samples. (E) UV−vis spectra of TBP and TBP-nMOF. Inset shows expanded views of the Q-band regions. (F) Fluorescence spectra of TBP and TBP-nMOF. (G) Time-resolved fluorescence decay traces of TBP and TBP-nMOF. (H) Singlet oxygen generation by PpIX, TCPP, PCN-224, TBP, and TBP-nMOF with a 660-nm LED irradiation (30 mW/cm2). (I) Singlet oxygen generation of TBP-nMOF at different oxygen concentration.

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Figure 3. The antitumor efficay of TBP-nMOF in vitro. (A) CLSM images of 4T1 cells after treatment with TBP-nMOF and stained with Hochest 33342. (B) Intracellular ROS detection by CLSM after 4T1 cells were incubated with TBP-nMOF and DCFH-DA, and (C) TBP-nMOF and DCFH-DA in 5% O2 under light irradiation, and (D) TBP-nMOF and DCFH-DA in 21% O2 under light irradiation (30 mW cm−2, 3 min). B1–D1) bright fields; B2–D2) DCFH fluorescence (green) depicted intracellular ROS; B3–D3) overlay images. (E) The dark and light toxicities of TBP-nMOF against 4T1 cells by MTT assay under 21% O2, and (F) 5% O2 atmosphere.

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Figure 4. The biodistribution and antitumor efficay of TBP-nMOF in vivo. (A) The in vivo fluorescence images of 4T1 tumor-bearing mice at various time intervals (black circle: tumor tissues; purple pane: bladder). (B) The ex vivo tissue fluorescence imaging of the mice after 6 h post-injection. (C) Semiquantitative mean fluorescence intensity (MFI) analysis at the tumor site and bladder at various time intervals after intravenous injection. (D) The corresponding MFI values of mice tissues. (E) The quantitative analysis on zirconium element by ICP-MS in main organs and tumor tissue after 6 h post-injection. (F) The tumor volume and (G) body weight changes in 15 days after various treatments, ***P < 0.001. (H) The average tumor weight after various treatments for 15 days, ***P < 0.001.

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Figure 5. The antitumor effect of synergistic PDT and αPD-1 in vivo. (A) Schematic illustration of the therapy process of combined PDT and αPD-1. (B) The relative tumor volume and (C) body weight changes in 22 days after various treatments, and (D) the average tumor weight after various treatments for 22 days, p values were calculated by a 35


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Student’s t test (**P < 0.01, ***P < 0.001). (E) H&E staining and (F) TUNEL immunofluorescence staining of the sacrificed tumor tissues (E1-F1: PBS; E2-F2: PBS+Light+αPD-1; E3-F3: TBP-nMOF+αPD-1; E4-F4: TBP-nMOF+Light; E5-F5: TBP-nMOF+Light+αPD-1). (G) Representative flow cytometry data of cytotoxic T lymphocytes (CTL) infiltration in tumors (Q1: CD8+; Q3: CD4+; Q2: double negative T cells; Q4: double positive T cells). CD8+ cells were defined as CTLs. (H) The percentage of CD8+ cells. (I) The production of IFN-γ and (J) TNF-α in sera of mice determined on the 12th day post various treatments.

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Figure 6. The inhibition of 4T1 tumors metastasis. (A) Bioluminescence imaging of the representative mice. (B) Bioluminescence images of the lung metastatic sites of the Luc-4T1 tumors. (C) H&E staining of the lung metastatic sites of the Luc-4T1 tumors (A1-C1: PBS; A2-C2: PBS+Light+αPD-1; A3-C3: TBP-nMOF+αPD-1; A4-C4: TBP-nMOF+Light; A5-C5: TBP-nMOF+Light+αPD-1). (D) The proposed mechanism of antitumor immune responses induced by combined PDT and αPD-1.

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