Erythrocyte Membrane Cloaked Metal–Organic Framework

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Erythrocyte Membrane Cloaked Metal-Organic Framework Nanoparticle as Biomimetic Nanoreactor for Starvation-Activated Colon Cancer Therapy Lu Zhang, Zhenzhen Wang, Yan Zhang, Fangfang Cao, Kai Dong, Jinsong Ren, and Xiaogang Qu ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b05200 • Publication Date (Web): 28 Sep 2018 Downloaded from http://pubs.acs.org on September 28, 2018

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Erythrocyte Membrane Cloaked Metal-Organic Framework Nanoparticle as Biomimetic Nanoreactor for Starvation-Activated Colon Cancer Therapy Lu Zhang,†,‡ Zhenzhen Wang,†,‡ Yan Zhang,†,‡ Fangfang Cao,†,‡ Kai Dong,† Jinsong Ren,*,† and Xiaogang Qu*,† †

State Key Laboratory of Rare Earth Resource Utilization and Laboratory of

Chemical Biology, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, PR China ‡

University of Chinese Academy of Sciences, Beijing 100039, PR China

ABSTRACT: Shutting down glucose supply by glucose oxidase (GOx) to starve tumors has been considered to be an attractive strategy in cancerous starvation therapy. Nevertheless, the in vivo applications of GOx-based starvation therapy are severely restricted by the poor GOx delivery efficiency and the self-limiting therapeutic effect. Herein, a biomimetic nanoreactor has been fabricated for starvation-activated cancer therapy by encapsulating GOx and prodrug tirapazamine (TPZ) in erythrocyte membrane cloaked metal-organic framework (MOF) nanoparticle (TGZ@eM). The fabricated TGZ@eM nanoreactor can assist the delivery of GOx to tumor cells and then exhaust endogenous glucose and O2 to starve

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tumors efficiently. Importantly, the resulting tumor hypoxia by GOx-based starvation therapy further initiate the activation of TPZ, which is released from the nanoreactor in acid lyso/endosomes environment, for enhanced colon cancer therapy. More importantly, by integrating the biomimetic surface modification, the immune escaping and prolonged blood circulation characteristics endow our nanoreactor dramatically improved cancer targeting ability. The in vitro and in vivo outcomes indicate our biomimetic nanoreactor exhibites the strong synergistic cascade effect for colon cancer therapy in an accurate and facile manner.

KEYWORDS: erythrocyte membrane, metal-organic framework, glucose oxidase, biomimetic nanoreactor, starvation-activated therapy Compared with normal tissue cells, tumor cells need a plenty of nutrients and energy to sustain their survival and growth on account of the disordered metabolic pathways.1,2 According to the Warburg effect, more than 50% of the cellular energy is generated by the low-efficient glycolytic pathway in tumor cells, which leads them to take in much more glucose than normal tissue cells.3 Once shutting down the glucose supply, the growth of tumor would be suppressed preferentially, thereby glucose-metabolic related cancer-starving therapy is increasingly considered to be a promising clinical translation.4-7 Up to now, some strategies have been put forward to starve tumors by consuming the glucose of cancer cells.8 Typically, utilizing the glucose oxidase (GOx) to catalyze glucose into hydrogen peroxide and gluconic acid in the presence of O2, which are based on the intratumor chemical reaction of glucose oxidation catalyzed by GOx, has been demonstrated as an effective strategy for

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fighting cancer.9-13 However, current GOx delivery strategies are primarily based on covalent conjugation or electrostatic interactions between nanocarriers and GOx.8,9 These approaches may suffer from enzyme leaching and aggregation, restricted mass transfer, low loading capacity, and loss of catalytic activity.14 Furthermore, passive immunological clearance and potential safe concerns seriously restricted the applicability of these nanocarriers.15,16 On the other hand, the consumption of intratumoral O2 during GOx-based starvation therapy would intensify the degree of the hypoxic microenvironment, which in turn make the therapeutic functions self-limiting.17 Therefore, it is highly desirable to explore an intelligent therapeutic system which not only could actively deliver GOx into tumor tissue with high enzymatic activity but also has the potential to overcome or even make use of the hypoxia dilemma for better therapeutic outcomes.

Metal organic frameworks (MOFs), fabricated from organic bridging ligands and metal ion/ion clusters, have recently attracted significant interest in biomedical fields, especially for enzyme and/or drug delivery.18-24 Owing to the large surface area, ultra-high porosity and tunable pore size, MOF is capable of exploiting the de novo approach to embed enzyme in its tight cavities with high loading efficacy.25-29 Moreover, such confinement encapsulation could significantly reduce the structural changes of enzymes, disperse their catalytic active sites, keep their enzymatic performance and prevent their leaching.30-34 Furthermore, the adjustable surface functionality and versatile structures of MOF endow it with multifunctionalities and stimuli-responsive payload controlled release.35-39 Recently, among various surface

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functionalization approaches, cell membrane coating has been used as a powerful method for enhancing the utility of nanoparticles.40-42 Taking advantages of cell-specific functionalities, nanoparticles coated with different cell membranes have been developed and studied extensively for biomedical applications.43-48 Especially, erythrocyte membrane coated nanoparticles have gained greater attention.49-51 This biomimetic strategy could “make up” nanoparticles with the biointerface of erythrocytes, which could reduce their elimination by the immune system, and then locate nanoparticles at the target tumor tissues by enhanced permeability and retention (EPR) effect.52 Thus, by taking advantage of these features, we envision that MOF and erythrocyte membrane could be combined to develop a biomimetic platform for maximizing the delivery of therapeutic agents with high activity to tumor tissues and realizing the effective cancer therapy.

Herein, we developed an erythrocyte membrane cloaked MOF-based biomimetic nanoreactor encapsulating GOx and prodrug for starvation-activated colon cancer therapy. As illustrated in Scheme 1, zeolitic imidazolate framework-8 (ZIF-8), which decomposed under acidic conditions but was stable under physiological conditions, was chosen as nanocarrier to encapsulate GOx and prodrug tirapazamine (TPZ). Then, the fabricated nanoparticles were further coated with erythrocyte membrane to obtain TPZ-GOx-ZIF-8@erythrocyte membrane (defined as TGZ@eM) nanoreactor. On the basis of biomimetic properties of erythrocyte membrane, TGZ@eM could effectively accumulate inside tumor tisssues with the immune escaping and prolonged blood circulation characteristics. The GOx in TGZ@eM nanoreactor could efficiently

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consume endogenous glucose and O2 to starve tumor cells. Meanwhile, the aggravated hypoxic microenvironment in tumors caused by the nanoreactor could transform prodrug TPZ, which were released from the nanoreactor in acid lyso/endosomes environment, into highly cytotoxic radicals for further inducing cell apoptosis. Taken together, the biomimetic nanoreactor not only could assist the delivery of GOx with high efficacy to specifically starve tumor cells, but also could utilize the aggravated hypoxic microenvironment caused by GOx-based therapy to initiate a starvation-activated cancer therapy. In this way, an augmentative synergistic efficacy between starvation therapy and valid prodrug therapy could be achieved by our biomimetic nanoreactor, which would effectively suppress the cancer growth in a more efficient and safer manner.

RESULTS AND DISCUSSION To confirm our protocol, ZIF-8 nanoparticles embedded with GOx and TPZ (denoted as TGZ) were constructed by a simple but effective one-pot encapsulation approach.26 The detailed synthetic process was clarified in the Supporting Information. GOx in ZIF-8, TPZ in ZIF-8 (denoted as GZ, TZ) and pure ZIF-8 (Figure S1) were also acquired with the same method. The scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images of the as-synthesized TGZ in Figure 1A verified its size distribution is uniform with a diameter of about 120 nm. In order to substantiate that GOx and TPZ were encapsulated into ZIF-8, we carried out the nitrogen adsorption-desorption isotherms assays. As illustrated in Figure S2, the BET surface areas of TGZ (506.4681 m2 g-1) was smaller than that of pure ZIF-8 crystals

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(980.4891 m2 g-1), which proved the presence of GOx and TPZ. Fourier transform infrared spectroscopy (FTIR) further demonstrated that both GOx and TPZ were successfully encapsulated into the ZIF-8 nanocarriers (Figure S3). Besides, the Zeta-potential measurements (Figure 1E) of ZIF-8 (+21.1 mV), GZ (-25.9 mV) and TGZ (-8.3 mV) also verified the successful encapsulation of GOx and TPZ into ZIF-8. Moreover, as shown in power X-ray diffraction (PXRD) graphs (Figure 1B), the crystal structure of TGZ was the same as that of the pure ZIF-8, indicating that the incorporation of GOx and TPZ had negligible influence on the crystallinity of ZIF-8 hosts. To ensure that GOx was indeed embedded in ZIF-8, TGZ was treated with surfactant to remove any surface absorbed GOx and then was examined by FTIR. As shown in Figure S4, the stretches characteristic of GOx at around 1640-1660 cm-1, corresponding to amide I, were primarily attributed to C=O stretching mode, which indicated the presence of GOx in TGZ. However, the samples prepared by washing the mixture of GOx and pre-formed TZ with surfactant did not show the stretches characteristic of GOx. These outcomes demonstrated that the mechanism of GOx encapsulation was not through the absorption or adsorption of the ZIF-8 pore network.53 In addition, the TEM images of TGZ and TZ after calcination could also be used as evidence to prove the encapsulation of GOx in TGZ (Figure S5). Small cavities were found in TEM image of the calcinated TGZ, which were not present in the calcinated TZ, demonstrating that GOx was indeed encapsulated into TGZ rather than adsorbed on the surface of the nanoparticles. The GOx loading efficiency (percentage of TGZ-encapsulated GOx over total GOx) calculated by BCA Protein

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Assay Kit was 92% (≈10 wt%), which was in accordance with thermo gravimetric analyzer (TGA) results (Figure S6, S7). Meanwhile, the amount of TPZ encapsulated by TGZ was about 13.2% as determined by UV-vis analysis (Figure S8). The relative low encapsulation efficiency in aqueous solution may attribute to the hydrophobic characteristic of small molecule TPZ.25,54

To fabricate a biomimetic nanoreactor, TGZ was further camouflaged with erythrocyte membrane vesicles by using previous method.55 As shown in Figure 1C and Figure S9, TGZ@eM showed a core-shell spherical structures compared to naked TGZ. The thickness of the homogeneous outer membrane shell was about 10 nm, which demonstrated the successful coating of erythrocyte membrane. Moreover, the results of dynamic light scattering (DLS) demonstrated the average hydrodynamic diameters of TGZ increased from 229 to 243 nm after the coating of erythrocyte membrane (Figure 1D). Simultaneously, the zeta potential of TGZ@eM was dramatically decreased to -29.6 mV (Figure 1E), which was approach to the value of erythrocyte membrane vesicles (-30.1 mV). These results reaffirmed the formation of a biomimetic surface. Subsequently, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to prove the existence of the membrane proteins and GOx in TGZ@eM (Figure 1F). The outcomes showed that the erythrocyte membrane proteins were reserved in abundance and the bands of GOx (about 70-120 kDa) were also observed in the SDS-PAGE of TGZ@eM, further indicating that GOx was successfully loaded into the erythrocyte membrane cloaked TGZ. In addition, TGZ@eM, ZIF-8@eM, GZ@eM and TZ@eM were also prepared

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to behave as controls in the same manner (Figure S1). By the way, though it was possible to fabricate GOx/TPZ@eM with the same procedures, the resulted products would not show the morphology and characteristic of nanoparticles, which was similar to the one of erythrocyte membrane (Figure S9A). In addition, regard to TGZ@eM, ZIF-8 as a skeleton to encapsulated GOx and TPZ could realize more abundant enrichment of the therapeutic reagents than the only membrane coated GOx and TPZ.

As the main intracellular energy source, glucose played a primary role in providing energy for tumor metabolisms. Specifically, tumor cells urgently developed the anaerobic glycolysis process with low ATP-production, which made them exceptionally dependent on the glucose nutrient.1 Based on this specific feature, our well-designed TGZ@eM nanoreactor has the potential to consume the glucose by the enzyme-catalyzed reaction to starve tumor cells specifically. As illustrated in Figure S10, TGZ@eM nanoreactor was capable of decreasing the glucose concentration effectively, and their catalytic ability was comparable to free GOx with the same concentration, suggesting that our designed nanoreactor could significantly reserve the enzymatic activity of GOx. In particular, TGZ@eM nanoreactor exhibited higher activity in a broadly acidic environment (4-6), whereas their catalytic activity was declined under the neutral and alkaline conditions (Figure 2A), inferring that our nanoreactor would have minor side effects on normal tissues. Aside from the consumption of glucose, the GOx-based bioreaction could in situ generate H2O2 molecules and gluconic acid in the presence of O2 (Figure 2B). Based on the

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peroxidase property of horseradish peroxidase (HRP), a colorimetric method was constructed to examine the production of hydrogen peroxide by utilizing HRP-catalyzed blue color reaction. As illustrated in Figure S11, after the addition of glucose and TGZ@eM in the meantime, the horseradish peroxidase (HRP) could effectively catalyze oxidation of TMB in the presence of the resulting hydrogen peroxide. Moreover, the generated H2O2 was increased in response to the elevated concentrations of glucose (Figure S12). With gluconic acid concentration increasing, a dramatic pH drop (from 7.68 to 3.82) was observed for TGZ@eM-catalyzed decomposition reaction of glucose (Figure S13), while the pH value was kept constant without the addition of glucose. Such property would induce the degradation of ZIF-8 hosts due to their pH-responsive behavior and be beneficial for subsequent payloads release. As shown in Figure 2C, upon exposure to lower pH environment (pH 5.0), the TGZ@eM nanoreactor displayed a burst release of drugs and more than 70 % of payload was released within 2 h. Comparatively, only 10 % drug was released at pH 7.4. Meanwhile, the change of O2 concentration during TGZ@eM-based catalytic reaction was also measured by a portable dissolved oxygen meter. As illustrated in Figure 2D, the O2 concentration decreased from 7.8 mg L-1 to 0.82 mg L-1 in 500 s. These outcomes strongly demonstrated that the catalytic ability of TGZ@eM could be used to deplete glucose for starving the cancer cells accompanying with the further anabatic hypoxia.

To visualize the cellular uptake behaviors of the well-designed nanoreactor in tumor cells and their immune evading ability, Rhodamine B (denoted as Rhm B) with

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high fluorescence brightness was specifically chosen as TPZ substitute in our work with encapsulation efficiency at 13.3% (Figure S14). As illustrated in Figure 3A, both Rhm B-GOx-ZIF and Rhm B-GOx-ZIF@eM treatments exhibited significant red fluorescence in CT26 cells, confirming that ZIF-8 could function as a high-performance nanocarrier to improve the cellular uptake of GOx and the erythrocyte membrane coating negligibly impaired the internalization of nanoreactor by tumor cells. Moreover, the quantitative flow cytometry analysis had shown the similar outcomes (Figure 3C, D). Subsequently, the immune evading ability of Rhm B-GOx-ZIF@eM was evaluated through antiphagocytosis against RAW264.7 murine macrophages, in which Rhm B-GOx-ZIF without erythrocyte membrane coating was used for comparison. As illustrated in Figure 3B, a bright red fluorescence was detected for RAW264.7 cells with the treatment of Rhm B-GOx-ZIF nanoparticles. Conversely, only a dim red fluorescence was detected in RAW264.7 cells after treating with Rhm B-GOx-ZIF@eM. Moreover, the quantitative analysis by flow cytometry indicated that the fluorescence intensity in RAW264.7 cells incubated with Rhm B-GOx-ZIF was approximatively 2.9-folds higher than that of Rhm B-GOx-ZIF@eM (Figure 3E, F), vividly indicating a good immune escape ability of Rhm B-GOx-ZIF@eM. The above outcomes demonstrated that the erythrocyte membrane cloaking endowed the nanoreactor with the ability of escaping from the systemic clearance of immune system, which might result in the prolonged blood-circulations spans.

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Having demonstrated the exciting cellular uptake and immune evading ability of the nanoreactor, co-localization studies were conducted to track their subcellular distributions inside CT26 cells. The red fluorescence of Rhm B-GOx-ZIF@eM was found to match well with LysoTracker Green after 4 h of incubation, indicating that our nanoreactor was trapped in lysosome via endocytosis pathway (Figure S15). Better yet, the acid environment of lyso/endosomes in CT26 cells would fabricate intracellular drug release. As expected, the intracellular fluorescence intensity of Rhm B was significantly increased with prolonged incubation time (Figure S16).

To evaluate the tumoricidal potential of TGZ@eM nanoreactor, their in vitro cytotoxicity was implemented first according to the standard methyl thiazolyl tetrazolium (MTT) assay. The results (Figure 4A) revealed that ZIF-8@eM exhibited low cytotoxicity against CT26 cells because of the good biocompatibility of ZIF-8 and erythrocyte membrane. GZ@eM treatment resulted in lower cell viability compared to control groups. By consuming glucose within the tumor in a way of glucose-metabolic reaction, we strategically starved the tumors as well as produced a high concentration of H2O2, which elevated redox stress inside the tumor for a much stronger anticancer effect. However, on account of that the continuous O2 consumption during starvation therapy would create a hypoxic environment and make the therapeutic efficacy self-limiting, it failed to inhibit cancer cell proliferation completely. To demonstrate this, the HIF-1α immunostaining assay was carried out. As illustrated in Figure S18, strong green signals could be legibly observed only in the nanocomposites contained GOx component with high HIF-1α expression,

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suggesting that GOx-based starvation therapy was sufficient to generate hypoxia. Such revellent hypoxia could activate TPZ to produce toxic oxidizing radical species which

could

cause

cytotoxic

double-strand

breaks

in

DNA

through

a

topoisomerase-II-dependent process.56 In this way, an excellent anticancer effect was achieved (Figure 4A). As a comparison, the therapeutic efficiency of various nanocomplexes by incubating with CT26 cells in either normoxia or hypoxia conditions was also examined. As illustrated in Figure S17, TZ@eM exhibited enhanced cytotoxicity due to the activation of TPZ in the oxygen-deficient conditions. Simultaneously, the efficacy of GZ@eM was obviously restricted under the hypoxia conditions. However, TGZ@eM displayed comparable outcomes in the both oxygen-deficient and oxygen-sufficient microenvironment which was benefited by the sequential therapeutic mode. Besides, to further accurately simulate the heterogeneity of tumor microenvironment with hypoxic region,57 we then fabricated CT26 multicellular tumor spheroids (MCTS) to investigate the effect of GOx in therapeutic groups on inducing hypoxia via utilizing hypoxia inducible factor (HIF)-1α staining assay. The confocal laser scanning microscopic (CLSM) images exhibited obvious green fluorescence in the internal and edge of MCTS after treatments of GZ@eM and TGZ@eM due to the continuous O2 consumption by GOx (Figure 4B and Figure S18B). Subsequently, the cell cytotoxicity of nanoreactor was further tested with LIVE/DEAD kit against both MCTS and the traditional monolayer cell cultures and verified that TGZ@eM treatment offered the most effective cancer cell killing ability (Figure 4C, D), which was consistent with MTT results.

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We then embarked on evaluating the possible death mechanism of CT26 cells induced by TGZ@eM nanoreactor employing annexin V-fluorescein isothiocyanate (Annexin V-FITC) and propidium iodide (PI) staining assay. Necrosis cells, late apoptosis cells, vital cells and early apoptosis cells were represented by Q1, Q2, Q3 and Q4, respectively. The proportion of apoptosis cells was 9.6%, 15.1%, 22.2% and 35.3% after treatment with ZIF-8@eM, TZ@eM, GZ@eM and TGZ@eM, respectively (Figure 4E). The increased apoptosis-inducing potential of TGZ@eM was due to the fact that GOx-based starvation therapy could result in strong enough hypoxic environments to activate the intracellular TPZ to synergistically realize enhanced apoptosis of cancer cells. Furthermore, no severe necrosis was shown for our designed nanoreactor, which minimized inflammation and damage to surrounding cells. Taking together, these in vitro results clearly demonstrated the sequentially activated therapeutic superiority of TGZ@eM compared to the single starvation therapy or prodrug therapy.

In order to investigate whether erythrocyte membrane coating could improve the blood circulating capacity of TGZ, the pharmacokinetics of TGZ@eM was studied by using CT26 tumor-bearing mice (n = 3) as the model. The blood circulation half-life (Figure 5A) of TGZ@eM (t1/2=4.7) was about 2 times longer than that of bare TGZ (t1/2=2.4). Such enhanced blood circulation capacity could be attributed to the fact that erythrocyte membrane cloaking rendered the TGZ “stealthy” to the host immune system on account of the enriched membrane proteins and antigens. At 24 h post injection, all the mice were euthanized and their major organs and tumors were

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collected for biodistribution analysis. Owing to the clearance of reticuloendothelial system (RES), the TGZ nanoparticles were primarily accumulated in liver, spleen and lung (Figure 5B). In stark contrast, TGZ@eM exhibited lower distribution in liver, spleen and lung, further indicating that erythrocyte membrane cloaking made the nanoreactor stealthy to RES. More importantly, the accumulation of TGZ@eM in tumor was almost 2 times than that of TGZ, which could be due to the prominent long circulation capacity and enhanced EPR effect after erythrocyte membrane coating.

In order to identify the stability of TGZ@eM in the blood, 1 mL blood was incubated with TGZ@eM (200 uL, 1 mg mL-1) for 2 h, 8 h, 16 h and 24 h, respectively. After that, the pH value of blood was tested and the supernatants were collected to identify the released amount of Zn2+ by using ICP-MS. The pH value (Figure S19) was nearly constant after incubating with TGZ@eM (200 uL, 1 mg mL-1). In addition, little amount of Zn2+ was tested (Figure S20). These indicated that TGZ@eM exhibited good stable in blood. After that, healthy female Balb/c mice were administrated with phosphate buffered saline (200 uL) and TGZ@eM (10 mg kg-1, 200 uL) intravenously to investigate the in vivo biocompatibility. We studied the blood biochemical levels and hematological parameters of the mice at 1, 7 and 28 days post injection. As illustrated in Figure 5D, the level of white blood cell, alburnin and creatinine improved slightly within 24 h, it demonstrated a little effect on innate immune system and the functions of liver and kidney. In contrast, no evident difference on blood indexes (red blood cells, white blood cell, mean corpuscular hemoglobin, hemoglobin, hematocrit value, and mean corpuscular volume), kidney

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functions (creatinine and blood urea nitrogen) and liver status (alburnin, aspartate transaminase, globulin, alanine aminotransferase and total protein) was observed at 7 and 28 days post injection, indicating that there was no significant toxicity during the evaluation period. Subsequently, we studied the long-term biodistribution evaluation at 3 and 7 days post injection of TGZ@eM (Figure S21). The results indicated the nanoreactor could be cleared out from the body for further reduced burden of these organs, which would be the reason for good long-term biocompatibility of TGZ@eM. Furthermore, the blood glucose level of mice after the injection with TGZ@eM (i.v.) was also recorded within 8 h. As illustrated in Figure 5C, the value of blood glucose exhibited a moderate and transient decrease at 30 min post injection, however, which spontaneously recovered in 1 h. Meanwhile, there was no pathoglycemia during the evaluation period. These primarily but comprehensive in vivo results indicated that TGZ@eM possessed good biocompatibility, which provided a chance for in vivo therapeutic applications.

The outstanding in vitro antitumor activity and biocompatibility of TGZ@eM would signify their possibly high in vivo therapeutic efficiency. Prior to investigating the therapeutic capability of our nanoreactor, we initially studied the hypoxia status of tumors after different treatments by HIF-1α staining assay. As shown in Figure 6A, HIF-1α positive signals were significantly proved for tumor slices after injection of TGZ@eM and GZ@eM intravenously, confirming that the tumor hypoxia status were significantly enhanced, which was beneficial for activating TGZ as well as indicating the consumption of glucose in tumor tissues. Furthermore, the relevant up-regulated

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expression of VEGF was found in tumor tissues treated with the therapeutic groups contained GOx component compared to other groups, further confirming the revellent hypoxia production. Inspired by this feature, we subsequently investigated whether the oxygen deprivation effects of GOx had the capacity to ignite the therapeutic effect of TPZ and suppress tumor growth in vivo. After the tumor size reached about ~ 100 mm3, CT26 tumor-bearing mice were divided into five groups (n =5) at random and intravenously injected with saline (control, 200 uL), ZIF-8@eM (10 mg kg-1, 200 uL), GZ@eM (starvation therapy, 10 mg kg-1, 200 uL), TZ@eM (prodrug therapy, 10 mg kg-1, 200 uL) and TGZ@eM (synergistic therapy, 10 mg kg-1, 200 uL), respectively. The body weights of mice were not significantly affected by various treatments compared to control group during 13 days of the therapeutic period (Figure 6B), implying low toxicity of these erythrocyte membrane cloaked nanocomposites. On tumor suppression assessments, therapeutic groups presented different suppression effects. Specifically, the suppression rates were calculated according to the variations of the relative tumor volume (Figure 6C). For GZ@eM group, the tumor growth inhibition (TGI) rate was limited to 63.5% due to the intrinsic hypoxic microenvironment of tumors and self-activated hypoxia. Treatment of TZ@eM also showed moderate anticancer capability (77.2%). This was attributed to that the inner hypoxia of tumor could activate TPZ to generate toxic oxidizing radical species for further inducing the apoptosis of cancer cells. However, owing to sufficient oxygen supply of the tumor cells nearby tumor vasculatures, only using the hypoxia-activated prodrugs could not provide complete antitumor effects. Comparatively, when

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TGZ@eM nanoreactor was administrated intravenously, the TGI rate was improved to 97.6%, conferring the most satisfactory therapeutic outcomes. The digital photographs of tumors also showed that the tumor sizes in TGZ@eM group were much smaller than that of other groups (Figure 6D). Moreover, the tumor mass from each group was removed and the results indicated that the tumors treated with TGM@eM nanoreactor was almost eliminated (Figure 6E), further validating their outstanding therapeutic outcome. Such satisfactory antitumor efficacy could be attributed to the fact that GOx-based starvation therapy could dramatically intensify the degree of tumor hypoxia to activate TPZ into toxic radicals, through which a strong synergistic effect was realized.

To further study the mechanism of synergistic therapy, the apoptosis levels of tumors and histological damages were investigated concretely by using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and hematoxylin and eosin (H&E) staining assay, respectively, at 24 h after different treatments. As illustrated in Figure 6F, severe apoptosis and morphology change were shown after tumors treated with TGZ@eM nanoreactor, while only moderate levels of damages were examined for those mice treated with TZ@eM or GZ@eM, further demonstrating the synergistic effects. These results demonstrated that our designed nanoreactor not only could greatly enlarge the accumulation of GOx in tumors with high catalytic efficacy, but also overcome the hypoxic dilemma of the starvation therapy. Moreover, H&E sections and digital photographs of main organs (heart, liver, spleen, lung, and kidney) demonstrated no obvious lesions or abnormalities as

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compared with the untreated group during the whole therapeutic period (Figure S22, S23), further confirming the high biocompatibility of erythrocyte membrane camouflaged nanoreactor.

CONCLUSIONS In summary, an erythrocyte membrane cloaked MOF-based biomimetic nanoreactor was prepared for highly precise starvation-activated colon cancer therapy. Through the membrane cloaking approach, the merits of erythrocyte membrane were grafted to the nanoreactor, which endowed it with the longer retention time and the ability of enhanced immune escaping. And then the nanoreactor could be preferentially accumulated in tumor post tail vein injection. The nanoreactor could not only sustain the high GOx catalytic activity to deprive a large amount of endogenous glucose and O2 but also induce a sufficiently strong hypoxic microenvironment to activate the prodrug TPZ, through which the high-effective synergistic tumor therapy was achieved. Taken together, the biomimetic nanoreactor fabricated here represented a promising strategy for colon cancer therapy, which would be highly beneficial to design more intelligent nanoplatforms for further clinical applications.

MATERIALS AND METHODS Reagents. Glucose oxidase (GOx), tirapazamine (TPZ), and 2-Methylimidazole were purchased from Sigma Aldrich and used as received. Zinc nitrate hexahydrate (Zn(NO3)2•6H2O, ≥ 99%) was provided by Aladdin-Reagent Co. Ltd. (China). All

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other chemicals were of analytically pure and used directly. Ultrapure water (18.2 MΩ; Millipore Co., USA) was to prepare all buffers and used in all experiments. Measurements and characterizations. SEM images were recorded using a Hitachi S-4800 FE-SEM. FTIR analysis was performed on a Bruker Vertex 70 FITR Spectrometer. TEM images were obtained with a FEI TECNAI G2 20 high-resolution transmission electron microscope. Ultraviolet-visible (UV-vis) spectra were obtained with a JASCO-V550 spectrometer. TGA experiments were carried out on a PerkinElmer Pyris Diamond TG/DTA analyzer. The zeta potential of the nanoparticles in water was obtained on a Zetasizer 3000HS analyzer. N2 adsorption-desorption isotherms were recorded using a Micromeritics ASAP 2020 automated sorption analyzer. X-ray measurements were performed on a Bruker D8 FOCUS Powder X-ray Diffractometer. The amount of Zn ions in the tissues of the mice was quantified by inductively coupled plasma-atomic emission spectrometry (ICP-AES, X Series 2, Thermo Scientific USA). The flow cytometry data were recorded using BD LSRFortessaTM Cell Analyzer.

The synthesis of TPZ-GOx-ZIF. According to the previous report with slight change, TPZ-GOx-ZIF nanoparticles were prepared successfully. Specifically, Zn (NO3)2•6H2O (50 mg), GOx (10 mg), TPZ (1 mg) and 2-methylimidazole (0.97 g) were dissolved in 5 mL of ultrapure water at room temperature. Under stirring, the solution became orange turbid quickly. After 10 min, the nanocrystals were collected by centrifugation and washed for three times. In the end, the samples dried at 40 °C in

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a vacuum oven. As controls, GOx-ZIF, TPZ-ZIF, pure ZIF-8 and Rhm B-GOx-ZIF were synthesized with the same method.

The loading efficiency of GOx and TPZ. The loading amount of GOx was determined via BCA Protein Assay. BCA reagents were obtained from Beyotime, and the protocol was provided with the supplier. At the meanwhile, the payload of TPZ was obtained by using the standard calibration curve based on the UV-vis absorption intensity at 470 nm of the difference between the supernatant collected after centrifugation and initial solution. Then, the calculation of the TPZ loading efficiency was as following formula:

(MI - MR) × 100 / MI

MI referred to the initial mass of TPZ and MR referred to the residual mass of TPZ after loading.

Preparation of erythrocyte membrane obtained vesicles. The whole blood from female Balb/c mice was centrifuged at 3000 rpm for 5 min to remove the plasma. Afterwards, erythrocytes were washed with PBS for several times and then hemolysis in water for 1 h at 4 °C. Subsequently, the erythrocyte membrane was collected by centrifugation for 10 min at 12000 rpm and washed with water for several times until the supernatant became colorless. After that the collected erythrocyte ghosts were sonicated in a capped glass vial for 5 min. The resulting vesicles were subsequently extruded serially through 400-nm and then 200-nm polycarbonate porous membranes

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using an Avanti mini extruder (Avanti Polar Lipids). Then the vesicles which dispersed in water stored at - 80 °C for further use.

Preparation and Characterizations of TGZ@eM. Erythrocyte membrane obtained vesicles were blended with 1 mg of TGZ nanoparticles, and then the mixture was sonicated for 60 s. Afterwards, the above solution was extruded more than 10 times via a 200-nm polycarbonate porous membrane. The extra erythrocyte membrane was eliminated by centrifugation, and TGZ@eM nanoreactor was dissolved in water for subsequent use. Zeta potential values of nanoparticles were measured with DLS. For membrane protein analysis, proteins on TGZ@eM and erythrocyte membrane were dissolved via sonication in 5% Triton, quantified using BCA assay and then analyzed through sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE).

Statistical analysis. In this article, all data were presented as mean result ± standard deviation (SD). The statistical analysis was performed by using Origin 8.0 software. All figures illustrated were obtained from several independent experiments with similar results. Asterisks indicate significant differences (*P < 0.05, **P < 0.01, ***P < 0.001).

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Scheme 1. Schematic illustration of A) the preparation of TGZ@eM nanoreactor and B)

an

erythrocyte

membrane

cloaked

MOF

biomimetic

starvation-activated colon cancer therapy.

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nanoreactor

for

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Figure 1. A) TEM (inset) and SEM images of TGZ. B) The XRD pattern of TGZ and ZIF-8. C) TEM image of TGZ@eM. Inset: high magnifcation image of TGZ@eM, negatively staining with uranyl acetate. D) The DLS results of TGZ and TGZ@eM. E) Surface potential of ZIF-8, GZ, TGZ, TGZ@eM and erythrocyte membrane. F) Protein analysis of erythrocyte membrane, TGZ@eM, and GOx by using SDS-PAGE. Samples were stained with Coomassie Brilliant.

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Figure 2. A) Comparison of the catalytic activity of TGZ@eM nanoreactor under different pH values. The error bar is the standard deviation from the mean (n = 3). B) The detection of product gluconic acid after TGZ@eM and glucose were incubated in 0.5 mM pH 5.0 PBS for 30 min. C) TPZ release from TGZ@eM at pH 5.0 and 7.4 in PBS. The error bar is the standard deviation from the mean (n = 3). D) The O2 concentration variations of TGZ@eM solution upon the addition of glucose.

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Figure 3. The fluorescence micrographs of cellular uptakes against Rhm B-GOx-ZIF@eM and Rhm B-GOx-ZIF of A) CT26 cells and B) macrophages. The red fluorescence was associated with released Rhm B; blue fluorescence was expressed by Hoechst. The scale bar was 20 µm. Flow cytometry analysis of Rhm B-GOx-ZIF (yellow) and Rhm B-GOx-ZIF@eM (blue) against CT26 cells C) and macrophages E), respectively. Cells with no treatment were determined to be the control (pink). Relevant mean fluorescence intensity (MFI) values of CT26 cells D) and macrophages F) were analyzed by flow cytometry. 1 represented Rhm B-GOx-ZIF@eM and 2 represented Rhm B-GOx-ZIF. The error bar is the standard deviation from the mean (n = 3). Asterisks indicate significant differences (*P < 0.05, **P < 0.01, ***P < 0.001).

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Figure 4. A) CT26 cells viability after incubation with different concentrations of nanoparticles for 24 h. B) HIF-1α staining of MCTS in different groups, blue fluorescence of Hoechst 33342, green immunofluorescence staining of HIF-1α antibody. C) The CLSM images of MCTS incubated with c1) PBS, c2) ZIF-8@eM, c3) GZ@eM, c4) TZ@eM, and c5) TGZ@eM and D) Micrographs of CT26 cells after treatment with d1) PBS, d2) ZIF-8@eM, d3) GZ@eM, d4) TZ@eM, and d5) TGZ@eM; Dead cells were stained red with PI; viable cells were stained green with calcein AM. All the images in B), C) were obtained under magnification of 4 and the scale bar of D) is 50 µm. E) Flow cytogram representing apoptosis assay based on Annexin V-FITC and PI staining of CT26 cells after treatment with different

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therapeutic groups for 8 h. Asterisks indicate significant differences (*P < 0.05, **P < 0.01, ***P < 0.001).

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Figure 5. A) In vivo pharmacokinetic curves during 24 h after intravenously injected with TGZ@eM or TGZ. B) Biodistribution of TGZ@eM or TGZ at 24 h after the injection. C) Blood glucose level of mice in time of day. Mice (n = 3) were injected with TGZ@eM (i.v.) at 09:00 (a.m.). D) The blood biochemical levels and hematological parameters of the mice after treatment with TGZ@eM for 1, 7 and 28 days. Asterisks indicate significant differences (*P < 0.05, **P < 0.01, ***P < 0.001).

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Figure 6. A) VEGF and HIF-1α staining of tumor tissues obtained from mice treated with various groups. The scale bar is 50 µm. B) Relative mice body weight after treated with various groups. C) Relative tumor volume after treated with various groups. D) Photographs of the CT26 tumor-bearing mice before treatment and on day 13 after different treatments. E) Photographs of the tumor dissection. F) Fluorescence microscopy photographs of TUNEL and H&E stained tumor slices 24 h after the first treatment with different groups (i.v.). The scale bar is 50 µm. Asterisks indicate significant differences (*P < 0.05, **P < 0.01, ***P < 0.001).

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ASSOCIATED CONTENT

Supplementary materials contain twenty-three supplementary figures. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] *E-mail: [email protected] ACKNOWLEDGMENT Financial support was provided by the National Natural Science Foundation of China (Grants 21871249, 21820102009, 21533008, 21673223, 21431007, 21601175), the Key Program of Frontier of Sciences (CAS QYZDJ-SSW-SLH052) and the Jilin Province Science and Technology Development Plan Project (Grant Nos. 20160520129JH and 20170101184JC).

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