Hypoxia-Triggered Nanoscale MetalâOrganic Frameworks for

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Hypoxia-triggered Nanoscale Metal-Organic Frameworks for Enhanced Anticancer Activity Ming Liu, Lei Wang, Xiaohua Zheng, Shi Liu, and Zhigang Xie ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b07570 • Publication Date (Web): 29 Jun 2018 Downloaded from http://pubs.acs.org on June 29, 2018

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ACS Applied Materials & Interfaces

Hypoxia-triggered Nanoscale Metal-Organic Frameworks for Enhanced Anticancer Activity

Ming Liu,†‡ Lei Wang,*† Xiaohua Zheng,†§ Shi Liu† and Zhigang Xie*†

†State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, P. R. China ‡The University of Chinese Academy of Sciences, Beijing 100049, P. R. China

§University of Science and Technology of China, Hefei 230026, P. R. China

*E-mail: [email protected] *E-mail: [email protected]

KEYWORDS ：Nanoscale Metal-Organic Frameworks, Photodynamic Therapy, Porphyrin, Tirapazamine, Combined Therapy

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ABSTRACT：： The oxygen dependent feature of most photosensitizers (PSs) and aggravated hypoxia tumor microenvironment seriously impede the photodynamic therapy (PDT) effectiveness. However, this undesirable impediment can be utilized to further trigger the activation of hypoxia-sensitive prodrugs. And a combined therapy can be realized through associating PDT with hypoxia-activated chemotherapy. Herein, a multifunctional Hf-porphyrin NMOF platform (Hf-TCPP), has been synthesized with high porphyrin loading capacity and well-ordered coordination array prevent porphyrin itself-quenching, thus greatly improve the generation efficiency of reactive oxygen species (ROS) which is helpful for PDT. Assynthesized Hf-TCPP nanoparticles possess more than 50 wt% of TCPP photosensitizers’ content, good crystallization, and large BET surface for further loading hypoxia-activated prodrug (tirapazamine (TPZ)) in a high loading content. Additionally, subsequent surface modification with a dopamine-derived polymer (DOPA-PIMA-mPEG) significantly improves their dispersibility and structural stability, as well as the controlled release kinetics of TPZ. Such nanoplatform can efficiently produce ROS for PDT upon irradiation, and also the depletion of the oxygen could further aggravate the hypoxic environment of tumors to induce the activation of TPZ for achieving an enhanced treatment efficacy. This work demonstrates the great advantages of NMOF-based platform in anti-tumor therapies for combined PDT with hypoxiaactivated chemotherapy.


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INTRODUCTION Photodynamic therapy (PDT), on the basis of irradiation of photosensitizers (PSs) to produce cytotoxic reactive oxygen species (ROS), is a noninvasive modality for treating various diseases that result in the killing or inactivation of pathological cells (e.g., infectious microorganisms or cancer cells).1-4 Under irradiation, the activated PSs may produce long-lived triplet electronic state, that would react with neighboring molecules via two distinct pathways, namely type I and type II photochemical reactions.2, 5-6 As for type I reaction, an oxygen-independent pathway, involves the electron transfer of triplet PSs to produce kinds of free oxygen radicals, including superoxide, hydroxyl radicals, and hydroperoxides.7 While the type II reaction is an oxygendependent pathway, in which the triplet PSs transfer energy (not electrons) to its neighboring molecular oxygen, leading to the produce of potent oxidizing agent singlet oxygen (1O2). Most of PSs for PDT undergo a type II reaction process. One unfavorable consequence for this process is the local hypoxia in tumor regions, greatly reducing the PDT efficiency.8-10 Several strategies have been developed to address this issue, including increasing the oxygen level in tumor region or decreasing the oxygen consumption of cancer cells.11-15 Notably, the adverse hypoxia situation already forms a new kind of target in cancer therapy. Among this, the usage of therapeutic targeted hypoxia-activated prodrugs has drawn great passions, which display a highly cytotoxicity to hypoxic cells but hardly any effect on normal cells and can be bio-reduced to cytotoxic radicals by some intracellular reductase enzymes under low-oxygen conditions.8, 16-21 Nanoscale metal-organic frameworks (NMOFs), consisted of metal ions or clusters with organic ligands in nanoscale regions, have been primarily adopted as promising platform to introduce active PSs molecules into the frameworks or pores of MOFs for applications in catalysis,22-23 optical devices,24-26 and biomedicine.27-30 Those introduced PSs as functional unit


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could partly participate in the formation of MOF frameworks at a very high content, while this cannot be done with other nanoparticle material classes.31-32 Among them, Zr-porphyrin-based NMOFs have continually attracted much attention for their ease of synthesis, excellent thermal and chemical stabilities, and distinctive photophysical property.33-34 For biological application, these nanoscale forms could not only maintain the confinement effect of MOF frameworks to enlarge the intramolecular distance of porphyrin PSs and to prevent the self-quenching, but also produce the synergistic effect with metallic nodes, such as the so-called “heavy atom effect”, to facilitate the intersystem crossing (ISC) and improve the PDT efficiency.35-36 Besides, those nanosized carriers can also preferentially accumulate at the tumor area to increase the therapeutic concentration of PSs in the tumor cells.37-39 Great progresses of porphyrin-based NMOF have been achieved recently in combining photodynamic &radiation therapy or with chemotherapy,4042

but the multifunctional NMOF platform associating PDT with hypoxia-activated

chemotherapy is rare reported, which may benefit for the treatment and imaging of heterogeneity and diversity of tumors. Herein, a multifunctional Hf-porphyrin NMOF nanocarrier, named Hf-TCPP, have been designed and prepared to potentially application for drug delivery, CT imaging, and associated photodynamic and hypoxia-activated therapy (Scheme 1). Hf-TCPP nanoparticles with high TCPP loading content, good crystallization, and large BET surface have been firstly synthesized by using the modulator-assisted solvothermal approach in the reaction of HfCl4, Tetra (4carboxyphenyl) porphine (TCPP) and benzoic acid. And a hypoxia-activated prodrug tirapazamine (TPZ) has been selected and loaded into the Hf-TCPP to produce cytotoxic radical species (oxidizing hydroxyl radical and benzotriazinyl radical) via a single-electron reduction reaction under low-oxygen conditions.43-44 Additionally, another feature for MOF platform is the


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selective covalent attachment of polymers upon its external surface via post-synthetic modification.45 Based on the available coordination ability of modulator-occupied Hf metal nodes, the dopamine-derived polyethylene glycol (DOPA-PIMA-mPEG) have been introduced by chemical modification to improve the dispersibility, blood circulation time, and structural stability of drug carriers in biological media and also to control the drug-release.46-50 Hf metal could be utilized for CT imaging for its strong X-ray attenuation. Upon light irradiation, the enhanced intersystem crossing (ISC) of TCPP linkers can improve the efficiency of assynthesized TPZ/Hf-TCPP/PEG to produce more 1O2 for PDT and to induce the cell apoptosis. Meanwhile, since the PDT process would further deplete the oxygen level to aggravate the hypoxic environment of tumors, which could spontaneously induce the activation of TPZ from the NMOFs for bioreductive chemotherapy to achieve the enhanced synergistic antitumor activity.

RESULTS AND DISCUSSION Material Characterization Purple Hf-TCPP NMOFs have been synthesized by the modulator-assisted solvothermal method, and their size and morphology are further characterized to give spherical shapes with an average particle diameter of 55 nm (Figure 1A and B). Powder X-ray diffraction (PXRD) (Figure 1C) indicates that as-synthesized Hf-TCPP nanoparticles are highly crystalline. One of the advances for MOF nanoparticles in drug delivery system is their larger “void” volume and available biodegradation. However, the larger “void” volume and available biodegradation of NMOF could lead to some unwanted results, such as the burst release of loaded drugs and too fast


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structural decomposition during the drug delivery. To improve the stability and dispersibility of Hf-TCPP in various physiological media, DOPA-PIMA-mPEG with abundant coordination groups have been selected as a coating/functionalization agent for surface modification of HfTCPP. The abundant carboxyl and diphenol groups on the PIMA backbone could supply strong coordination ability to the Hf4+ ion on the outer surface of Hf-TCPP, and also the introducing PEG chains could guarantee the good dispersibility in aqueous media and better performance in the subsequent in vivo experiments. Introducing PEG does not affect the crystallinity, morphology and size of pristine Hf-TCPP nanoparticles (Figure 1C and Figure S1). And a new peak at 1050 cm-1 is appeared in the PEGylated Hf-TCPP/PEG as well, coming from the C-O-C stretching vibration of DOPA-PIMA-mPEG (Figure S2).51 Additionally, significant change of surface charges in ζ-potential measurements (Figure 1D) has been observed from 9.12 ± 0.52 mV of Hf-TCPP to -21.73 ± 0.86 mV of Hf-TCPP/PEG. The hydrodynamic diameter of HfTCPP/PEG measured by dynamic light scattering (DLS) slightly increased to 163 ± 5 nm from 149 ± 4 nm after DOPA-PIMA-mPEG modification (Figures S3). Importantly, Hf-TCPP/PEG also exhibits good structural stability (Figure S4) and well dispersibility in various physiological media (water, phosphate buffer saline (PBS) (pH = 7.4), Dulbecco’s modified eagle medium supplemented with 10% FBS (DMEM) and fetal bovine serum (FBS)) even up to 1 week (Figure 1E, the inserted picture), instead of rapid precipitates and structural decomposition are observed in the PBS solution of Hf-TCPP only within 2 days (Figure S5). Besides, the hydrodynamic diameter and PDI of Hf-TCPP/PEG in DMEM (Figure 1E) did not change significantly over time. All those above results could confirm the successful surface modification and superior structural stability and dispersibility of Hf-TCPP/PEG during long time incubation in various biological media, which is beneficial for application in cargo loading.


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Additionally, Hf-TCPP NMOFs also possess a permanent porosity with specific surface areas of 386.2 m² g-1 and the main pore size distribution of 0.468 nm as determined by nitrogen adsorption-desorption isotherm (Figure 1F and Figure S6), which ensures for loading of cargos. A hypoxia-activated prodrug, TPZ, has been chosen as a model for “proof of principle”. For a typical experiment, Hf-TCPP solution (20 mg in 2 mL THF) firstly mixed with different amounts of TPZ and then stirred at 50 oC for 24 h to ensure fully loading. And then, 100 mg of DOPAPIMA-mPEG was introduced into this mixture with stir and went on for another 24 h at the same temperature to complete the PEGylation. The unloaded TPZ and free DOPA-PIMA-mPEG polymers were removed by centrifugal washing. The successful loading of TPZ on HfTCPP/PEG is evidenced by the UV-vis spectra of TPZ/Hf-TCPP/PEG (Figure 1G), in which the characteristic absorbance peak of TPZ at 265 nm is clearly identified. The increasing drug loading ratios and decreasing efficiency of Hf-TCPP have been observed after increasing the amount of TPZ in feed (Figure S7, S8 and Table S1). Considering the high hypoxia toxicity of TPZ, a moderate TPZ/sample ratio (1/5, W/W) has been used, which gives a loading efficiency and contents approximately 54.24% and 85.28 µg mg-1, respectively (Figure S7). And about 8.6 wt% of DOPA-PIMA-mPEG and 9.6 wt% of TPZ in final TPZ/Hf-TCPP/PEG are calculated from thermogravimetric analysis (TGA) (Figure 1H and Figure S9), which is consistent with the result of UV-vis analysis. To confirm the better controlled release of TPZ/Hf-TCPP/PEG, the drug release profiles have been examined by dispersing samples in PBS at 37 oC, and the release percentages of TPZ in the selected medium have been determined by UV. TPZ is released from the TPZ/Hf-TCPP/PEG with a relatively rapid release of ~ 60% within the first 10 h, and then exhibited a continuous slow-release profile (Figure 1I). But for TPZ/Hf-TCPP without PEGylation at the same condition, an obvious burst release behavior has been observed.


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Photostability, Singlet Oxygen and Hypoxia Generation Ability Before evaluating the singlet oxygen generation ability of TPZ/Hf-TCPP/PEG, we firstly investigated its photostability under a LED light irradiation (λ = 635 nm, 12 mW cm-2). UV spectra of TPZ/Hf-TCPP/PEG show negligible changes (Figure 2A), and the quantitative analysis of theirs absorbance intensities at 426 nm indicate that there is no significant photobleaching within 20 min and only less than 10% decay of all 60 min time of exposure (inserted picture of Figure 2A). The ROS generation ability of TPZ/Hf-TCPP/PEG upon light irradiation has been evaluated with 1, 3-diphenylisobenzofuran (DPBF), a probe which could irreversibly trap 1O2 to cause a decay of its absorbance intensity at about 410 nm. No any changes have been found in DPBF control group (Figure S10C). Both absorbance intensities of DPBF at 410 nm in TPZ/Hf-TCPP/PEG and Hf-TCPP/PEG dispersed in DMF solutions show a continuous decrease to almost 24% upon irradiation with an LED lamp (λ = 635 nm) within 9 min, indicating the good singlet oxygen generation ability and negligible effect of loaded TPZ to the singlet oxygen generation of Hf-TCPP/PEG (Figure 2B and Figure S10). Upon light irradiation, an obviously increasing red emission of oxygen sensor porphyrin-Pd52 in TPZ/Hf-TCPP/PEG and its Hf-TCPP/PEG control is detected, which means that the decreasing oxygen level causes by the gradual conversion of oxygen to ROS during PDT process (Figure 2C and Figure S11). Moreover, to further verify the PDT caused ROS generation and triggered hypoxia in tumor cells, human cervical cancer (HeLa) cells have been treated with TPZ/Hf-TCPP/PEG with irradiation or not, respectively. A Hypoxia/Oxidative Stress Detection Kit has been used for analyzing the stress levels of ROS and hypoxia. Cells treated with TPZ/HfTCPP/PEG upon irradiation (Figure 2D) exhibit obviously enhanced fluorescence intensity of ROS generation (green) and hypoxia production (red) when compared with negative controls


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(untreated cells or TPZ/Hf-TCPP/PEG alone), indicating the substantial ROS and induced hypoxia generation in the cells. These results demonstrate that TPZ/Hf-TCPP/PEG upon irradiation would produce ROS effectively and in turn the boost oxygen consumption could aggravate cellular hypoxia, which would lead to activate the anticancer activity of TPZ. Cellular Uptake and Cytotoxicity The cell uptake was evaluated by incubating HeLa cells with TPZ/Hf-TCPP/PEG (40 µg mL-1) for 6 h, next strong red emissions from TPZ/Hf-TCPP/PEG-itself were detected in cytoplasm under confocal laser scanning microscopy (CLSM), presented as Figure S12. Then quantitative analysis of Hf concentrations by ICP-MS shows that the internalization of TPZ/Hf-TCPP/PEG nanoparticles exhibits a time-dependent manner (Figure S13) and an amount of 463 ng/106 cells can be reached at an incubation time of 12 h. Those results indicated the effective internalization of TPZ/Hf-TCPP/PEG nanoparticles by cancer cells, which warrants their further biological application. To evaluate the synergetic PDT and hypoxia-activated chemotherapy for enhanced anticancer activity of as-synthesized TPZ/Hf-TCPP/PEG, the cytotoxicity against HeLa and mammary carcinoma (4T1) cells, were investigated by the standard methyl thiazoletetrazolium (MTT) cell viability assay. In light of the oxygen-dependent feature of PDT and hypoxia-activated TPZ, the cell experiments were carried out in two oxygen pressures, that is, 20% for normoxia and 2% for hypoxia. Under normoxia condition with or without light irradiation, the cells treated with free TPZ have a survival of more than 80% even at the highest concentration of 15 µg mL-1, implying that TPZ itself has little detrimental effects (Figure S14). On the contrary, TPZ itself exhibits the hypoxia-triggered chemotherapeutic effect and higher cell killing ability against HeLa (IC50 =


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1.94 µg mL-1) and 4T1 (IC50 = 2.41 µg mL-1) under hypoxia condition. We next studied the in vitro PDT performance of TPZ/Hf-TCPP/PEG and its Hf-TCPP/PEG control at normoxia and hypoxia conditions (Figure 3 A, B, and Figure S15). As expected, Hf-TCPP/PEG exhibits negligible dark cytotoxicity or oxygen content related cytotoxicity against two selected cell lines under both normoxia and hypoxia conditions, demonstrating its good biocompatibility. But once exposure at 635 nm light in the same experimental conditions, TPZ/Hf-TCPP/PEG and HfTCPP/PEG control show an oxygen pressure and sample concentration-dependent phototoxicity, which proves the PDT efficiency is significantly restrained under hypoxia. TPZ/Hf-TCPP/PEG gives a higher cytotoxicity than Hf-TCPP/PEG under normoxia or hypoxia, indicating an enhanced anticancer activity from the synergetic effects of PDT from the NMOF itself and hypoxia-activated TPZ. The cell death, apoptosis and necrosis with or without light irradiation, has been examined through a commercial Annexin V-FITC/PI apoptosis detection kit. As-mentioned synergistic apoptotic effects of TPZ/Hf-TCPP/PEG-triggered PDT with hypoxia-responsive TPZ in flow cytometry (Figure 3C). Very close cell viabilities have been observed in treatments with PBS or free TPZ alone whether under light irradiation or not, proving that the light irradiation alone or TPZ-itself have little phototoxicity or chemotherapeutic cytotoxicity. However, significant apoptosis and necrosis cells are appeared in incubation with TPZ/Hf-TCPP/PEG and HfTCPP/PEG. Remarkably, TPZ/Hf-TCPP/PEG sample results in apparently enhanced dead cells ratio of 32.8% than that of its control Hf-TCPP/PEG (23.3%) and also the lowest viability of 59.2%. Combining with MTT results and flow cytometric analyses, we could conclude that the rapid oxygen depletion during the photochemical reaction for PDT further results in intracellular


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hypoxia and then induces TPZ producing toxic oxidizing radical species to realize excellent synergistic therapeutic effect. In vivo CT imaging and Therapeutic efficacy X-ray computed tomography (CT) is common in biological field for its high spatial resolution, deep tissue penetration, and 3D visibility.53-54 The Hf-TCPP/PEG is also anticipated to be candidate for CT imaging agent for strong X-ray attenuation of Hf.55-56 The CT signals and attenuation values of Hf-TCPP/PEG solutions tend to be a concentration-dependent behavior, when scanned by with concentrations ranged from 0.63 to 25 mg mL-1 at 120 kVp (Figure 4A). A maximum value of 264 Hounsfield units (HU) and brighter CT image are observed at sample concentration of 25 mg mL-1. HU values of Hf-TCPP/PEG are proportional to their concentrations, and the simulated line slope is about 10.2 HU L g-1, which is higher enough and available for the application in CT imaging.57-58 To further validate the ability of in vivo imaging, 4T1 tumor-bearing mouse have been intratumorally injected with Hf-TCPP/PEG (20 mg mL-1, 50 µL). Compared with the tumor site before injection (47 HU), an obvious enhanced contrast with the CT signal value of 571 HU in the tumor site has been detected after injection, and also is shown in its corresponding three-dimensional (3D) reconstructed CT images (Figure 4B). These results demonstrate that Hf-TCPP/PEG can be utilized as efficient contrast-enhancing medium for in vivo CT imaging. Before evaluating the anticancer efficacy of TPZ/Hf-TCPP/PEG in vivo, their blood circulation, biodistribution, and fate after administration were systematically investigated through monitoring the concentration of Hf ions by ICP-MS. In blood circulation study (Figure S16A), the drug concentration decreased gradually over time and has a half-life of ~2.65 h. The


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concentrations of Hf ions in the major organs and tumor were determined as well (Figure S16B). High levels accumulations of TPZ/Hf-TCPP/PEG particles locate in liver and spleen, but a still relatively high tumor accumulation of them could be seen after 12h p.i. In vivo anticancer efficacy of TPZ/Hf-TCPP/PEG has been further evaluated by using the 4T1 subcutaneous xenograft murine models via tail vein administration (5.0 mg mL-1, 200 µL). Forty tumor-bearing mice have been randomly assigned into eight groups and then treated with kinds of protocols: 1) PBS, 2) PBS + light, 3) TPZ, 4) TPZ + light, 5) Hf-TCPP/PEG, 6) Hf-TCPP/PEG + light, 7) TPZ/Hf-TCPP/PEG, and 8) TPZ/Hf-TCPP/PEG + light. A rapid tumor growth and no notable difference has been detected in the control group regardless of light irradiation or not, and the tumor volume increases nearly 6-fold on day 14 compared to that of original (Figure 5A and B). Meanwhile, the mice treated with TPZ alone or only under irradiation show marginally inhibited efficacy towards tumor growth. PDT performance of Hf-TCPP/PEG upon irradiation gives moderate tumor inhibition capability, as evidenced by the partly controlled tumor growth. Noticeably, the significant inhibition of tumor has been achieved in mice treated with TPZ/ HfTCPP/PEG and light irradiation at 635 nm (Figure 5B and D). Combining with the in vitro date of TPZ/Hf-TCPP/PEG, such in vivo result manifests the great superiority of associating the oxygen consumption of PDT and hypoxia activated TPZ for reinforcement of anticancer efficacy. Hematoxylin and eosin (H&E) also present markedly dead cells without nuclei in mice treated with TPZ/Hf-TCPP/PEG under light irradiation, further demonstrating the synergistic effects of this combined PDT and hypoxia-induced chemotherapy (Figure 5E). Furthermore, the biosafety was judged by changes of body weight, hematology and serum biochemistry tests, and histological section. The body weight of all groups changed slightly by any treatment, indicating the good biocompatibility of TPZ/Hf-TCPP/PEG (Figure 5C).


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Negligible fluctuations of vital blood parameters during all selected time intervals further indicate the good hemocompatibility of as-synthesized TPZ/Hf-TCPP/PEG sample (Figure S17A). Major blood biochemistry parameters were in the normal ranges, and no hepatic or kidney dysfunction were found after TPZ/Hf-TCPP/PEG administration (Figure S17B). Moreover, there are no significant pathological abnormalities of H&E of control group (Figure S18). These results revealed that TPZ/Hf-TCPP/PEG shows no distinct toxicity and owns great potential for in vivo cancer therapy.

Conclusions In conclusion, Hf-TCPP nanoparticles with spherical shapes and average size of 55 nm have been synthesized by modulator-assisted solvothermal method. The porous nature of Hf-TCPP ensures its high TPZ loading of 85.28 µg mg-1. Further surface PEGylation with DOPA-PIMAmPEG significantly enhances its dispersibility and structural stability in various tested physiological media. Meanwhile, the PEGylation significantly controls the release rate of TPZ within TPZ/Hf/TCPP/PEG. Under light irradiation, TPZ/Hf/TCPP/PEG is able to efficiently produce ROS and induce the activation of TPZ, generation great cytotoxicity against both HeLa and 4T1 cells. In vivo results also validate the remarkable antitumor efficacy can be achieved by this synergistic treatment in 4T1 tumor model. In addition, all CT imaging tests prove that such NMOF has good imaging ability of Hf4+ metal nodes. We believe this multifunctional NMOF could reverse the adverse situation of PDT, realizing enhanced anticancer ability through synergetic photodynamic and hypoxia-response therapy. This work also highlights the great potential of porous materials in drug delivery, imaging and the treatment of heterogeneous


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tumors. For the further pre-clinical translation of NMOF, there has a long way to undergo. Besides the green synthesis, controllable particle size or shapes, and necessarily surface modification of NMOFs, many endeavors should be done in a more profound level to fully understand the structure-biological property relations, such as activating immune system during PDT treatment,59-60 and also to evaluate the absorption, distribution, metabolism, and excretion mechanism of NMOFs after in vivo administration.28-29 Finally, closely multidisciplinary teamwork among the chemistry, materials, and medicine etc. al should be developed urgently.

MARERIALS AND EXPERIMENTs Materials. Hafnium (IV) tetrachloride and dopamine hydrochloride were purchased from Acros Organics. TCPP was prepared in our lab.60 TPZ, Benzoic acid (BA), poly (ethylene glycol) methyl ether amine (Mn 550), and DPBF were obtained from Sigma-Aldrich. Poly(isobutylenealt-maleic anhydride) (PIMA) (Mw 6000) was obtained from Heowns Biochemical Technology. Characterization. Hydrodynamic size and zeta potential of the NMOFs were measured by Malvern Zeta Sizer-Nano ZS90 instrument. UV-vis and Fluorescence spectrum were monitored on SHIMADZU UV-2450 spectrometer and PerkinElmer LS55 luminescence spectrometer, respectively. FTIR were recorded on Nicolet Impact 410 infrared spectrometer. TEM and SEM images were obtained by electron microscope JEOL JEM-1011 and FEI/Philips XL-30, respectively. PXRD was collected by Bruker D8 diffractometer. TGA was carried out via a NetzchSta 449c with a heating rate of 10 oC min-1 under air atmosphere. Micromeritics ASAP 2010 analyzer was used to study the nitrogen adsorption isotherm. The content of Hf4+ has been quantified by ICP-MS.


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Synthesis of Hf-TCPP NMOFs. Syntheses of Hf-TCPP NMOFs were performed by solvothermal method. Briefly, HfCl4 (75 mg, 0.23 mmol), TCPP (6.5 mg, 8.22 × 10-3 mmol) and BA (200 mg, 1.64 mmol) were mixed into a separate vial with 10 mL of N, Ndimethylformamide (DMF). After ultrasonication for 5 min, the reaction mixture was placed in an 120 oC oven for 24 h. The dark purple product was gathered by centrifugation and then washed three times with DMF, Methanol (MeOH) and Tetrahydrofuran (THF), respectively. The obtained NMOFs were dispersed in THF for further use. Synthesis of DOPA-PIMA-mPEG. DOPA-PIMA-mPEG was synthesized according to a published paper with some modification.46 Equimolar amounts of H2N-PEG-OMe and dopamine were adopted in the synthesis process. Synthesis of Hf-TCPP/PEG or TPZ/Hf-TCPP/PEG. For the entrapment of TPZ inside the Hf-TCPP NMOF, suspension contained 20 mg of NMOF was blended with 1 mL of TPZ solution (4 mg mL-1). Then 100 mg of DOPA-PIMA-mPEG in 2 mL of THF was introduced for another 24 h. The unloaded TPZ in washing solutions was quantified according to a calibration curve for TPZ in MeOH. The TPZ encapsulation efficiency was figured out based on the ratio of the drug amount loaded into Hf-TCPP to that of added. The Hf-TCPP/PEG was prepared in the same way without the TPZ loading. In vitro TPZ Release from TPZ/Hf-TCPP or TPZ/Hf-TCPP/PEG. Firstly, 6 mg of dried TPZ/Hf-TCPP or TPZ/Hf-TCPP/PEG was immersed into 2 mL of PBS with shaking at 100 rpm under 37 oC. At each predetermined time, 1 mL of supernatant was extracted and displaced with fresh medium after centrifugation. Finally, the concentration of TPZ released has been determined by UV. The same experimental processes have been repeated in triplicate.


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Photostability and Singlet Oxygen 1O2 Generation. For the photostability test, the TPZ/HfTCPP/PEG was dispersed in deionized water and irradiated with a LED light (λ = 635 nm, 12 mW cm-2) for 60 min. Their UV-vis spectra were monitored every ten minutes. DPBF was adopted to study the singlet oxygen generation potential and its UV-vis absorbance was monitored over time. For example, Hf-TCPP/PEG or TPZ/Hf-TCPP/PEG was dispersed in DMF (10 µg mL-1, 2 mL) and then the solution was blended with DPBF solution (4 mM, 20 µL) before irradiation. And the photostability of DPBF control was investigated at the same method. Oxygen Level Assay. Porphyrin-Pd was used to monitor the change of oxygen level before and after the PDT process, whose fluorescence would be strongly quenched by oxygen molecules. Typically, 50 µL of DMF solution of Porphyrin-Pd (1.3 × 10-3 M) was added to 3 mL of Hf-TCPP/PEG or TPZ/Hf-TCPP/PEG (10 µg mL-1) aqueous solution and bubbled with O2 for 5 min. The fluorescence intensity of Porphyrin-Pd (λex = 420 nm) at 675 nm was recorded before and after 20 min irradiation. Cell Culture. HeLa and 4T1 cells were cultured at 37 oC under 5% (v/v) CO2 in DMEM medium (GIBCO) supplemented with 10% FBS (GIBCO) and 1% penicillin-streptomycin. Cell Uptake by CLSM and ICP-MS. To examine the uptake by CLSM, HeLa cells were seeded on a sterile cover slip for adhering to. After 24h, the cells were treated with TPZ/HfTCPP/PEG (40 µg mL-1) for another 4 h. Cells were washed and fixed with 4% of formaldehyde before being observed with CLSM (Zeiss LSM 780). The red emission from TPZ/Hf-TCPP/PEG was collected within the range 600-700 nm (λex = 405 nm). For ICP-MS assay, the cells were firstly incubated with TPZ/Hf-TCPP/PEG. Next the cells were collected, counted with a


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hemocytomter, and lysed by concentrated nitric acid for ICP-MS analysis. The cellular uptake amounts of Hf were measured by ICP-MS and normalized with cell numbers. ROS and Hypoxia Analysis. ROS-ID® from Enzo Life Sciences was utilized to detect the levels of Hypoxia/ROS inside cells. HeLa cells with TPZ/Hf-TCPP/PEG (40 µg mL-1) were incubated in 96-well plates for 6 h. After irradiation in the hypoxia/oxidative stress detection mix medium, PBS washed cells were imaged by CLSM (for fluorescence signal of hypoxia (λex = 420 nm, λem = 600-700 nm) and ROS (λex = 488 nm, λem = 500-530 nm)). The other control groups were as follows: untreated samples, TPZ/Hf-TCPP/PEG without irradiation, and the positive groups (ROS-induced and hypoxia-induced, respectively). Cytotoxicity and Apoptosis Evaluation. HeLa and 4T1 cells were used to assess in vitro anticancer effect, respectively. Different pO2 conditions were generated by placing cultured cells in an incubator, with the partial pressure of CO2 maintained at 5% (the partial pressure of O2 maintained at 21% for normoxia and 2% for hypoxia). After incubation under normoxic, cells subsequently treated with various samples at a series concentrations in hypoxic (2%) or normoxia (21%) for 12 h before 30 min irradiation. Cell viability was evaluated by the MTT assay after being incubated for an additional 24 h. The apoptosis and necrosis were tested using Annexin V-FITC/PI Apoptosis Detection Kit. Briefly, HeLa cells were dealt with DMEM, TPZ, Hf-TCPP/PEG and TPZ/Hf-TCPP/PEG for 12 h followed by 30 min irradiation or not and incubated for an additional 24 h. The detection was carried out and processed using FlowJo. Animal Model. Balb/c mice (female, 4-6 weeks) were bought from the Animal Center of Jilin University and handled under the protocol established by Jilin University Studies Committee.


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The subcutaneous tumor models were initiated by injection of 5 × 106 4T1 cells into the right side of each mouse near the armpit. In vitro and in vivo X-ray CT Imaging. Hf-TCPP/PEG aqueous dispersions with different concentrations (0.63, 3.13, 6.25, 10, 12.5, 25 mg mL-1), were used to assess the CT contrast potential in vitro. As for in vivo experiment, tumor-bearing Balb/c mouse was intratumorally injected with Hf-TCPP/PEG (20 mg mL-1, 50 µL). All the CT images were taken by Activion 16 isx-031CT, TOSHIBA. In vivo Pharmacokinetics and Biodistribution. Tumor free mice were injected with TPZ/HfTCPP/PEG (50 mg kg-1) via the tail vain. Then blood samples were taken at each predetermined time points and lysed with concentrated HNO3 before ICP-MS analysis. The major organs and tissue samples were excised at each selected interval. Subsequently, the collected samples were weighted and digested with concentrated HNO3 at 150 oC for 2 h, and the content of Hf ions was quantified by ICP-MS. All the experiments were done in quadruplicate. Blood Biochemistry Assay. The mice were injected with TPZ/Hf-TCPP/PEG or PBS, respectively. At each predetermined time, the mice were sacrificed to harvest their blood samples for biochemistry studies. The serum biochemistry data were measured by the automatic biochemical analyzer (Mindray, BS-220), and complete blood samples were analyzed by an automatic blood cell analyzer (ABX MICROS 60). In vivo Therapeutic Efficacy. Experiments were carried out when tumors volume to ~100 mm3. And the mice were randomly assigned into eight treatment groups. Each mouse was given an intravenous injection (200 µL) of one of the following: (Ⅰ) PBS alone; (Ⅱ) PBS + light; (Ⅲ) TPZ alone (0.19 mg mL-1); (Ⅳ) TPZ (0.19 mg mL-1) + light; (Ⅴ) Hf-TCPP/PEG (5.0 mg mL-1)


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alone; (Ⅵ) Hf-TCPP/PEG (5.0 mg mL-1) + light; (Ⅶ) TPZ/Hf-TCPP/PEG (5.0 mg mL-1) alone; or (Ⅷ) TPZ/Hf-TCPP/PEG (5.0 mg mL-1) + light. Each mouse in group (Ⅱ), (Ⅳ), (Ⅵ), (Ⅷ) was anesthetized and then irradiated with light (635 nm, 0.1 W cm-2) for 30 min at the tumor site, 12h post injection. Following treatment, the body weight of mice and tumor volume evolution were monitored every two days. The tumor sizes were calculated as (length × width2)/2. Relative tumor volumes were normalized to its initial size prior to treatment. 14 days later, all the mice were executed and excised tumors were photographed and weighted. Besides, the tumor tissues and other major organs were dissected to prepare paraffin section for H&E staining to monitor the histological changes. Statistical analysis. All results are presented as means ± SD from at least three parallel tests. The statistics were done with SPSS Statistics 17.0 software by the two-tailed t test (*p < 0.05, **p < 0.01 and ***p < 0.001).


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ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Additional information for the structural and morphological characterizations, drug delivery, biostability, cytotoxicity, and bio-safety of Hf-TCPP/PEG and TPZ/Hf-TCPP/PEG.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] *E-mail: [email protected]

Notes The authors declare no competing financial interest. ACKNOWLEDGMENT Financial support was kindly provided by the National Nature Science Foundation of China (Project No. 21771174).

ABBREVIATIONS NMOFs

Nanoscale metal-organic frameworks

PDT

Photodynamic therapy

TCPP

Tetrakis (4-carboxyphenyl) porphyrin

TPZ

Tirapazamine


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ISC

Intersystem crossing

ROS

Reactive oxygen species

PIMA

Poly(isobutylene-alt-maleic anhydride)

PSs

Photosensitizers

Hf-TCPP

Hf-porphyrin NMOF

Hf-TCPP/PEG

PEGylated Hf-TCPP NMOF

DOPA-PIMA-mPEG

Dopamine-derived polyethylene glycol

THF

Tetrahydrofuran

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Scheme 1. (A) Schematic synthesis route of TPZ/Hf-TCPP/PEG. (B) The in vivo synergetic photodynamic and hypoxia-activated therapy of TPZ/Hf-TCPP/PEG.


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Figure 1. Characterizations and TPZ release of as-synthesized samples. (A) TEM and (B) SEM image of Hf-TCPP. The inserted picture, statistics of particle size from SEM data. (C) PXRD patterns of Hf-TCPP, Hf-TCPP/PEG, and TPZ/Hf-TCPP/PEG. (D) The Zeta-potential of HfTCPP, Hf-TCPP/PEG, and Hf-TCPP/PEG dispersed in distilled water. (E) The changes of the average diameter and PDI of Hf-TCPP/PEG in DMEM over different time monitored by DLS. The inserted picture, photograph of Hf-TCPP/PEG dispersed in water, PBS (pH = 7.4), DMEM and FBS after one week (5 mg mL-1). (F) Nitrogen adsorption isotherm of Hf-TCPP at 77 K. (G) The representative UV−vis absorbance spectra of TPZ, Hf-TCPP, Hf-TCPP/PEG, and TPZ/HfTCPP/PEG dispersed in water at room temperature. (H) The TGA curves of Hf-TCPP, HfTCPP/PEG, and TPZ/Hf-TCPP/PEG under air atmosphere. (I) TPZ release from TPZ/Hf-TCPP and TPZ/Hf-TCPP/PEG in PBS (pH = 7.4).


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Figure 2. The photostability and singlet oxygen generation ability of TPZ/Hf-TCPP/PEG. (A) The changes of UV-vis absorbance spectra of TPZ/Hf-TCPP/PEG dispersed in water under light irradiation; the insert image is its corresponding quantitative result. (B) Comparison of the decay rates of DPBF only and in the presence of Hf-TCPP/PEG or TPZ/Hf-TCPP/PEG under light irradiation. (C) The fluorescence intensity variation of Porphyrin-Pd before and after irradiation with adding Hf-TCPP/PEG and TPZ/Hf-TCPP/PEG samples. (D) Intracellular fluorescence detection of HeLa cells with ROS/hypoxia detection probes in different treatments by CLSM. Scale bar = 50 µm.


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Figure 3. In vitro cytotoxicity of TPZ/Hf-TCPP/PEG against (A) HeLa and (B) 4T1 cells. Cells were cultured in hypoxic (2%) or normoxia (21%) atmosphere with or without light irradiation for 30 min (635 nm, 12 mW cm-2), *p

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