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Engineering Magnetosomes for Ferroptosis/ Immunomodulation Synergism in Cancer Fan Zhang, Feng Li, Gui-Hong Lu, Weidong Nie, Lijun Zhang, Yanlin Lv, Weier Bao, Xiaoyong Gao, Wei Wei, Kanyi Pu, and Hai-Yan Xie ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.9b00892 • Publication Date (Web): 02 May 2019 Downloaded from http://pubs.acs.org on May 2, 2019

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Engineering Magnetosomes for Ferroptosis/Immunomodulation Synergism in Cancer Fan Zhang,† Feng Li,† Gui-Hong Lu,† Weidong Nie,† Lijun Zhang,‡, § Yanlin Lv,‡, Weier Bao,‡, § Xiaoyong Gao,‡, Wei Wei,‡, §, * Kanyi Pu,⊥ and Hai-Yan Xie†, * †School ‡State

of Life Science, Beijing Institute of Technology, Beijing, 100081, P. R. China

Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese

Academy of Sciences, Beijing, 100190, P. R. China §University ⊥School

of Chinese Academy of Sciences, Beijing, 100049, P. R. China

of Chemical and Biomedical Engineering, Nanyang Technological University, 637457,

Singapore

ABSTRACT: As traditional anticancer treatments fail to significantly improve the prognoses, exploration of therapeutic modalities is urgently needed. Herein, a biomimetic magnetosome is constructed to favor the ferroptosis/immunomodulation synergism in cancer. This magnetosome composes of Fe3O4 magnetic nanocluster (NC) as the core, and pre-engineered leukocyte membranes as the cloak, wherein TGF-β inhibitor (Ti) can be loaded inside the membrane and PD-1 antibody (Pa) can be anchored on the membrane surface. After intravenous injection, the membrane camouflage results in long circulation, and the NC core with magnetization and

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superparamagnetism enables magnetic targeting with magnetic resonance imaging (MRI) guidance. Once inside tumor, Pa and Ti cooperate to create an immunogenic microenvironment, which elevates H2O2 amount in polarized M1 macrophage and thus promotes the Fenton reaction with Fe ions released from NCs. The generated hydroxyl radicals (·OH) subsequently induce lethal ferroptosis to tumor cells, and the exposed tumor antigen in turn improves the microenvironment immunogenicity. The synergism of immunomodulation and ferroptosis in such a cyclical manner therefore leads to potent therapeutic effects with few abnormalities, which supports the engineered magnetosomes as a promising combination modality for anticancer therapy.

KEYWORDS: biomimetic magnetosomes, PD-1 antibody, TGF-β inhibitor, immunomodulation, ferroptosis, cancer therapy

Although chemotherapy remains the pillar in the fight against cancer in clinical settings, this treatment fails to significantly improve the prognoses. In this aspect, a wave of efforts has been spurred to explore anticancer modalities.1 Recently, ferroptosis has been discovered as a type of programmed cell death, which is significantly distinct from the apoptosis induced by chemotherapy.2-4 During this process, the Fenton reaction, typically initiated between ferric ions and intratumoral H2O2,5-7 efficiently generates potent reactive oxygen species (ROS),8 leading to the lethal lipid peroxidation.9-12 Such iron-dependent damage has thus inspired researchers to harness this function for killing tumor cells and shed light on anticancer therapy.13-15


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In addition to tumor cells, symbiotic stroma cells, such as tumor-associated macrophages and regulatory T (Treg) cells, inhabit tumor tissue as well.16 Owing to the vital role of these cells in tumor growth and metastasis, the regulation of their functions in the tumor microenvironment (TME) has also attracted booming interest.17,

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As the most famous strategy for TME

modulation, immune checkpoint therapy has provided a weapon,19, 20 winning the 2018 Nobel prize. Several checkpoint agents, such as the PD-1 antibody (Pa) Nivolumab®, have obtained FDA approval, and there is a high expectation that others in this class will also be approved within the next few years.21 Although promising, only a fraction of patients respond to this treatment in clinical settings. Given the dynamic and complicated nature of the TME, coordination with other agents that target important signals (such as transforming growth factorβ, TGF-β) involved in cancer is necessary.22 In this case, the response rate to TME immunomodulation can be increased with subsequent therapeutic benefit. Keeping these ideas in mind, we noticed the possible synergism of ferroptosis and immunomodulation in cancer, which has not yet been explored. On one hand, TME immunomodulation can induce macrophage polarization from M2 to M1, offering more intratumoral H2O2 for the Fenton reaction.23 On the other hand, ferroptosis in tumor cells can release tumor antigens and create an immunogenic TME, thus enhancing the response to immunomodulation. To achieve this synergism, elemental Fe, checkpoint antibody (Pa) and TGF-β inhibitor (Ti) SB-505124 hydrochloride should be utilized together. The way forward with this combination thus lies in the spatiotemporal cooperation of these agents in tumors. Unfortunately, their distinct stabilities and pharmacokinetics in vivo prevent them from accumulating at tumor sites in an identical manner. A possible way to solve this problem can be the construction of nanoparticles (NPs),24, 25 which can passively infiltrate tumor tissues through


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the enhanced permeability and retention (EPR) effect. However, the efficient loading of these agents, which have distinct molecule weights, charge and hydrophilicities, into a tiny NP platform seems difficult. To improve the delivery performance, functionalization should also be integrated for long-circulation capacities,26,

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active targeting, selective release, and even

imaging guidance, which will further make the NP construction substantially more challenging. To this end, we herein engineered magnetosomes that could efficiently load Fe, Pa and Ti for high-performance ferroptosis/immunomodulation synergism in cancer (Scheme 1). In brief, Fe3O4 magnetic nanoclusters (NCs), with both satisfactory superparamagnetism and magnetic control, were synthesized as the core of the magnetosome, which could be utilized for Fe accommodation, magnetic resonance imaging (MRI) guides and magnetic targeting. Subsequently, these NCs were coated with a leukocyte membrane that was pre-engineered with azide (N3). Such camouflage not only prolonged the circulation time but also facilitated loading of hydrophobic Ti in the membrane and conjugation of dibenzocyclooctyne (DBCO)-modified Pa through mild and efficient click chemistry. After intravenous injection, the formed Pa-M/TiNCs were capable of accumulation at the tumor site, wherein Pa together with Ti created an immunogenic TME via increased rates of CD4+ T/Treg cells, CD8+ T/Treg cells, and M1/M2 macrophages. Meanwhile, the elevated H2O2 sourced from M1 polarization promoted the Fenton reaction with Fe ions, which were released from NCs in the slightly acidic TME. The generated hydroxyl radicals (·OH) induced lethal ferroptosis to tumor cells, and the exposed tumor antigen in turn improved the immunogenicity of the TME. Using seven different tumor models, we systematically demonstrated that such a synergism of ferroptosis/immunomodulation could result in potent anticancer therapeutic effects, showing great promise as a modality to fight cancer.


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Scheme

1.

Schematic

illustration

of

a

biomimetic

magnetosome

for

ferroptosis/

immunomodulation synergism in cancer.

Results Characterizations of engineered magnetosomes. Efficient delivery of Fe, Ti and Pa together into tumor tissue is a prerequisite for ferroptosis/immunomodulation synergism. Owing to the


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outstanding capability of being effectively directed to the tumor site by an external magnetic field, nanoparticles with high magnetization are very promising for tumor-targeted delivery. Meanwhile, the beneficial addition of high superparamagnetism will help us to noninvasively monitor the in vivo distribution via MRI guidance. However, these two contradictory properties seem difficult to integrate in traditional magnetic nanoparticles.28 To solve this problem, we developed a facile one-pot hydrothermal approach to synthesize uniform-sized NCs, which consisted of stacked ~10 nm Fe3O4 building units (Figure 1a). Such an architecture not only endowed the NCs with both superior magnetization and superparamagnetism but also accommodated an abundance of elemental Fe for the Fenton reaction. To prolong the circulation time in vivo, these NCs were further biomimetically camouflaged with a leukocyte membrane (M), which was pre-engineered with an azide group. Correspondingly, a membrane layer (Figure 1a) containing typical membrane protein components could be observed around the NC (Figure 1b and Figure S1, Supporting Information). The formed magnetosome thus facilitated Ti loading inside the membrane (verified by the UV absorption spectrum in Figure 1c). Moreover, Pa could also be decorated on the magnetosome surface through mild and efficient click chemistry (Figure S2, Supporting Information), which was demonstrated by the colocalization of Pa and M/Ti-NC in confocal laser scanning microscopy (CLSM) images (Figure 1d) and by the binding of secondary immune-antibody-linked Au nanoparticles (Au-Pa-M/Ti-NCs) in transmission electron microscopy (TEM) images (Figure 1e). The gradual increase in particle size and changed zeta potential also validated the successful Ti loading (Figure S3, Supporting Information), membrane camouflaging and Pa decoration, with amounts tuned up to 20 μg (Ti), 50 μg (M) and 10 μg (Pa) for every 100 μg NCs (Figure S4, Supporting Information).


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Figure 1. Characterization of Pa-M/Ti-NCs. a) TEM images (Scale bars: 50 nm). b) Western blot analysis of membrane proteins in (i) M-NCs, (ii) the leukocyte membrane and (iii) the whole cell. c) UV absorption spectra. d) CLSM images (Scale bar: 10 µm). e) TEM images of (i) Au-


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Pa-M/Ti-NCs and (ii) Au+M/Ti-NCs (Scale bar: 50 nm). f) The hydrodynamic sizes of Pa-M/TiNCs. Pa-M/Ti-NCs were stable in 10% FBS and PBS over two weeks, little change was found during storage. g) The magnetic hysteresis curve of Pa-M/Ti-NCs. h) CLSM images of FITClabeled Pa-M/Ti-NCs dispersed in water without (left) or with (right) a magnet. i) T2 relaxation rate (1/T2, s-1) as a function of Fe concentration (mM) for Pa-M/Ti-NCs. Inset: T2-weighted MRI image of Pa-M/Ti-NCs. j) Detection of released Fe ions in PBS. k) The hydroxyl radical generation induced by the released Fe3+/Fe2+ from Pa-M/Ti-NCs. All data represent the mean ± s.d. (n=3). For further utilization in vivo, the constructed Pa-M/Ti-NCs were systematically characterized. During two weeks of storage, little change was observed in the size of Pa-M/Ti-NCs (Figure 1f), showing good stability for intravenous (i.v.) injection (Figure S5, Supporting Information). Meanwhile, the Pa-M/Ti-NCs exhibited the required magnetic properties. On one hand, the high saturation magnetization reached up to 80 emu g-1 (Figure 1g), which enabled rapid enrichment in a microfluid by a commercial magnetic scaffold (Figure 1h). On the other hand, the 1/T2 signal intensity was proportional to the particle concentration (Figure 1i), indicating the capacity for MRI guidance. Considering another capacity of the NCs that served for Fe accommodation, we also estimated the Fe ion release profile in pH=7.4 and pH=6.5 buffers, which mimicked physiological and tumor media. As shown in Figure 1j, substantially more Fe ions were released at pH=6.5 than at pH=7.4, suggesting the significantly improved dissolution of the magnetosome core at tumor tissue. In this aspect, H2O2 could be well catalyzed to ·OH in the methylene blue bleaching experiment (Figure 1k). Such good Fenton reactivity and the aforementioned high loadings of Ti and Pa together shed light on the ferroptosis/immunomodulation synergism in vivo.


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Targeted delivery to tumor tissue. As the targeted delivery performance was highly correlated with the subsequent ferroptosis/immunomodulation synergism, we next focused our attention on the biodistribution behavior. To this end, we intravenously administered B16F10xenografted mice with different NC-based formulations (labeled with DIR dye) and comparatively investigated the time-elapsed fluorescent signals. As shown in Figure 2a, the signal of free NCs appeared in the tumor area until 6 h, and the intensity at 24 h remained unsatisfactory due to the rapid clearance in vivo. When the NCs were camouflaged with the leukocyte membrane (M-NCs), macrophage phagocytosis was significantly inhibited (Figure S6, Supporting Information). The resultant long circulation (Figure S7, Supporting Information) thus opened the time window to utilize the EPR effect, and we observed an accelerated and ameliorated tumor accumulation. Owing to the PD-1 receptor being highly expressed on stroma cells in the TME,29 the decoration of Pa on the magnetosome surface (Pa-M/Ti-NCs) bestowed the tumor-delivery performance. Upon a magnetic field (Pa-M/Ti-NCs(m)), the signal markedly and continuously increased with time, resulting in a 4-times enhancement over that of its counterpart in the case of pristine NC. Correspondingly, the fluorescence signal of Pa-M/TiNCs(m) in other organs significantly decreased (Figure 2b and Figure S8, Supporting Information), which would also help to reduce system toxicity. Considering the superparamagnetism of the NC core, we also validated the targeting performance by MRI (Figure 2c and d). Similarly, the T2 signal increased following the sequence of NCs, M-NCs, PaM/Ti-NCs, Pa-M/Ti-NCs(m). Such a distinction was further verified at the histological level. Compared with the sparse and scattered signal of NCs, the substantial signal of Pa-M/Ti-NCs(m) infiltrated throughout the tumor tissue (Figure 2e), which again demonstrated the success of utilizing engineered magnetosomes for tumor-targeted delivery.


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Figure 2. Biodistribution of different NC-based formulations. a) Fluorescence images of mice after intravenous (i.v.) injection. b) The fluorescence intensity of different formulations in organs. c) T2-MRI of mice before and after i.v. injection. d) The corresponding decline of the T2 value in T2-MRI after i.v. injection with different formulations. e) Corresponding nanoparticle distributions in tumor tissue (Scale bar: 50 µm). All data represent the mean ± s.d. (n = 6).


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Synergism of immunomodulation and ferroptosis. The superior delivery performance prompted us to investigate the outcomes in the TME. To this end, we harvested and analyzed the infiltrated stroma cells of B16F10-xenografted tumors after different treatments. As shown in Figure 3a, Pa-M/Ti-NCs (green) were bound to CD4+ T cells, CD8+ T cells, Treg cells and macrophages, which have been demonstrated to express PD-1 on the cell surface.30 In this case, the cooperated inhibition of PD-1 and TGF-β led to a significant improvement in the values of CD4+/Treg (Figure 3b), CD8+/Treg (Figure 3c), and M1/M2 (Figure 3d), which outperformed the counterparts from the case of utilizing a single immunomodulator or without a magnetic field. Such TME modulation was also verified by immunofluorescence staining. Compared to the results of the PBS group, the treatment with Pa-M/Ti-NCs yielded significantly more CD4+, CD8+, and M1 cells dominant in the TME. Meanwhile, another benefit of this inclination from immunosuppression to immunogenicity attracted our interest. The intratumoral polarization from M2 to M1 could produce substantially more hydrogen peroxide (Figure 3e), which has also been reported in inflammatory diseases.23 Together with the released Fe ions from the NC core, an abundance of ROS could thus be generated via the Fenton reaction (Figure 3f), which resulted in degradation of lipid molecules by the removal of electrons through oxidation.31-33 During this lipid peroxidation process, the depletion of the antioxidant glutathione compromised the lipid repair enzyme (glutathione peroxidase 4, GPX4)34 and metabolism enzyme (Fatty acid-CoA ligase 4, FACL4).35 Correspondingly, we observed the significant decrease of these two indicators’ expression by the Pa-M/Ti-NC group (Figure 3g), demonstrating potent ferroptosis. These performances were also found in 4T1-tumor model (Figure S9, Supporting Information).


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Figure 3. Pa-M/Ti-NC(m)-induced tumor immune microenvironment changes. a) CLSM images of bound Pa-M/Ti-NCs on cells separated from tumor tissue after i.v. injection (green: Pa-M/TiNCs, blue: nucleus, and red: cytoplasm membrane; scale bar: 10 µm). b), c), d) The ratios of


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CD4+ T to Treg cells, CD8+ T to Treg cells, and M1- to M2-associated macrophages as well as the corresponding immunofluorescence images (Scale bar: 100 µm). e) The production of H2O2 in the tumor tissues of different groups. f) CLSM images of hydroxyl radical generation (green) in tumor tissues (scale bar: 10 µm). g) Immunohistochemical analyses of FACL4 (red) and GPX4 (purple) in tumor tissues (Scale bars: 100 µm). All data represent the mean ± s.d. (n = 6). It is also worth mentioning the potential ferroptosis in tumor cells could in turn help immunomodulate the TME. Taking the M-NC group as an example, the values of CD4+/Treg cells, CD8+/Treg cells, and M1/M2 macrophages also achieved moderate increases in the absence of Pa and Ti immunomodulators (Figure 3b-d). A possible explanation could be the exposure to immunogenic antigens, which were released from the moderate ferroptosis induced by M-NC alone. With further loading of Pa and Ti, improved ferroptosis could be more helpful in creating a more immunogenic TME, which could continue to polarize M2 to M1 to offer more H2O2 for ferroptosis. Such a cyclic synergism undoubtedly maximized the cooperation between immunomodulation and ferroptosis, shedding light on subsequent therapeutic effects. Evaluation of therapeutic effects. Finally, we systematically evaluated the anticancer performance of the engineered magnetosomes. In the first model, B16F10-xenografted mice were utilized to study tumor inhibition after administration with different formulations (Figure 4a). Compared with the PBS group, intravenous injection with Ti, Pa or M-NC alone failed to yield a satisfactory tumor inhibition effect (Figure 4b). This was ameliorated when Ti was integrated into the magnetosomes (M/Ti-NCs). With Pa decoration (Pa-M/Ti-NCs), the antitumor efficacy was further enhanced. The application of a magnetic field at the tumor area led to the nearly complete suppression of tumor growth in the Pa-M/Ti-NC(m) group. Correspondingly, the expression of the cell proliferation indicator Ki 67 in tumor tissue (Figure


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4c) decreased in the following sequence: PBS, Ti, Pa, M-NCs, M/Ti-NCs, Pa-M/Ti-NCs, and PaM/Ti-NCs(m). As a result, most mice were tumor-free in the Pa-M/Ti-NC(m) group (Figure 4d), and all of them remained alive after 50 d (Figure S10, Supporting Information). Such a satisfactory therapeutic effect was achieved with few abnormalities in typical cytokines (Figure S11, Supporting Information), biochemical markers (Figure S12, Supporting Information), body weight, temperature (Figure S13, Supporting Information), and organ histology (Figure S14, Supporting Information), confirming the safe use of our engineered magnetosomes.

Figure 4. Pa-M/Ti-NC(m)-mediated inhibition of tumor growth in the B16F10 tumor model. a) Treatment schedule. b) Individual tumor growth kinetics in mice with different treatments. c)


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Immunohistochemical analysis of Ki 67 expression in tumor sections (Scale bar: 50 μm). d) Representative tumor photographs of different groups. To test the applicability to different tumors, we also investigated the therapeutic performance in 4T1 breast tumor models (Figure 5a). As shown in Figure 5b, the tumor growth rates again decreased in the order of PBS, Ti, Pa, M-NCs, M/Ti-NCs, Pa-M/Ti-NCs and PaM/Ti-NCs(m). Specifically, the Pa-M/Ti-NC(m) treatment achieved the most potent inhibition of tumor development, leading to a 100% survival rate after 50 d with few abnormalities (Figure S15, Figure S16 and Figure S17, Supporting Information). Considering the frequent distant metastasis to lung and bone for breast cancer in clinical settings, we also evaluated the antimetastatic performance. No sign of metastasis was observed in the mice treated with PaM/Ti-NCs(m), while the extent of metastatic foci in the lung and metastasis-induced erosion in the bone varied among other groups (Figure 5c, Figure S18 and Figure S19, Supporting Information). Additionally, to mimic a more malignant metastasis process, we intravenously injected 4T1 cells as a 4T1 xenograft model (Figure 5d). As these 4T1 cells had been engineered to express luciferase (Luc-4T1), we could monitor tumor development at both primary and metastasis sites through the bioluminescence signal (Figure 5e and f). As expected, only when Ti and Pa were simultaneously delivered by M-NCs could the metastasis signal disappear in the lung area. With further magnetic assistance, primary tumors were also completely suppressed in the Pa-M/Ti-NCs(m) group, again demonstrating the substantially superior therapeutic performance.


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Figure 5. Pa-M/Ti-NCs(m)-mediated inhibition of the growth and metastasis in 4T1 tumor model. a) Treatment schedule. b) Individual tumor growth kinetics of mice receiving different


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treatments. c) Representative computed tomography (CT) images of lung metastasis (blue) and spontaneous bone metastasis. d) Treatment schedule for the hematogenous metastasis model. e) Bioluminescence images of hematogenous metastasis in lungs with different treatments. Images are representative of six independent experiments. f) MFI statistics of primary tumors (down) and lung metastasis (up) in e). All data represent the mean ± s.d. (n=6). Owing to the tumor cells that remain after surgery, recurrence is another complicated problem in clinical settings.36 To this end, we established a tumor recurrence model by surgically resecting the majority of primary B16F10 tumors and evaluated the local tumor development after different treatments (Figure 6a). Compared with the results of the PBS group, neither Ti nor Pa alone succeeded in recurrence suppression, and the survival time remained unsatisfactory. On the contrary, almost no recurrence was found in the Pa-M/Ti-NC(m) group, and a 100% survival rate was achieved after 50 d (Figure 6b and c). Such an extraordinary result was further validated in the Luc-4T1 tumor recurrence model. Treatments with Ti or Pa alone only inhibited metastasis to lung and bone areas and failed to eliminate recurrence at primary sites. Once PaM/Ti-NCs(m) were administered after the surgery, the signals at both primary and metastasis sites disappeared (Figure 6d and e), again leading to a 100% survival rate during a long-term observation period (Figure 6f).


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Figure 6. Pa-M/Ti-NCs(m)-induced suppression of the postoperative recurrence in B16F10 and 4T1 xenografts model. a) Treatment schedule. b) Individual tumor growth kinetics of primary tumors and recurrent tumors receiving different treatments in the B16F10 tumor model. c) The survival of the mice receiving different treatments in b. d) Bioluminescence images of primary tumors before or after tumor resection and recurrent tumors in mice receiving different


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treatments. Images are representative of six independent experiments (4T1 model). e) Individual 4T1 tumor growth kinetics of primary tumors and recurrent tumors receiving different treatments. f) The survival of the mice in e). Considering the immunogenic TME generated from the ferroptosis/immunomodulation synergism, we also verified the feasibility of utilizing our engineered magnetosomes to induce an immunological response and memory to improve cancer prognoses. To explore this, B16F10tumor-bearing mice with different administrations first received complete tumor resection, which was utilized to investigate the infiltrated DC cells. Four weeks later, mice were again inoculated with B16F10 cells, and the effect of immunological memory on the growth of this distant tumor was investigated (Figure 7a). As shown in Figure 7b and c, the Ti and Pa treatment only yielded moderate improvement in the mature DC amount and the cross-presentation level, while the PaM/Ti-NC(m) group presented substantial improvement. Similarly, the percent of effector memory T (TEM) cells in CD8+ T cells also increased in the following sequence: PBS, Ti, Pa, and Pa-M/Ti-NCs(m) (Figure 7d). As a result, we observed complete inhibition in the Pa-M/TiNC(m) group, while all mice in the other groups died within 3 weeks (Figure 7e and f). These distinct performances were also found for the Luc-4T1-xenografted mice (Figure S20 and Figure S21, Supporting Information). The Pa-M/Ti-NC(m) group still significantly outperformed its counterparts, and no sign of tumors was observed at the rechallenge site (Figure 7g, h, and i). All these results together demonstrated our engineered magnetosome could fulfil various clinical requirements to fight cancer.


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Figure 7. Pa-M/Ti-NCs(m)-induced long-term immune memory effects for tumor inhibition. a) Treatment schedule. b) Relative mature DCs in primary tumors with different treatments on the


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resection day. c) The flow cytometry analysis of the DC cross-presentation marker MHC-I in b). d) The percent of TEM cells in the spleens of the mice on the same day of rechallenging. e) Average tumor growth curves for the rechallenged distant tumors receiving different treatments. f) The tumor recurrence ratio of mice in b) after different administrations. g) Bioluminescence images of the primary tumors before or after tumor resection and the rechallenged tumors of mice receiving different treatments. Images are representative of six independent experiments. h) Average tumor growth curves of the rechallenged distant tumors receiving different treatments. i) The percentage of the tumor-free mice in h). All data represent the mean ± s.d. (n = 6). Conclusion In summary, we constructed magnetosomes using NCs as the core and pre-engineered leukocyte membranes as the cloak, wherein Ti could be largely loaded inside the membrane through hydrophobic interactions and Pa could be efficiently anchored on the membrane surface via a mild click reaction. Such a design incorporating recent nanoparticle technologies bestowed the magnetosomes with multiple features. After intravenous injection, the camouflage of the leukocyte membrane resulted in long circulation, and the superparamagnetism and magnetization of the NC core enabled subsequent magnetic targeting with MRI guidance. Once inside the TME, the Pa, Ti, and Fe cargo induced synergistic immunomodulation and ferroptosis in a cyclical manner. Consequently, potent therapeutic effects with few abnormalities were achieved in seven different tumor models. These satisfactory results, generated from the combination strategy, suggested our engineered magnetosomes could serve as a promising modality for safe and high-performance anticancer therapy.


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Experimental Section Reagents and material: TGF-β inhibitor (SB505124) was purchased from Selleck. Anti PD-1 monoclonal antibody was purchased from Biolegend. CD45 antibody was purchased from Santacruz. FITC-conjugated anti-mouse CD11c, APC/Cy7-conjugated anti-mouse CD3, eFlour 450-conjugated anti-mouse CD4, PE-conjugated anti-mouse CD8a, PE-conjugated anti-mouse CD40,

APC-conjugated

anti-mouse

CD80,

APC/Cy7-conjugated

anti-mouse

CD86,

PerCP/eFlour 710-conjugated anti-mouse MHC I, BV605-conjugated anti-mouse MHC II, eFlour 450-conjugated anti-mouse CD44, and PerCP Cy5.5-conjugated anti-mouse CD62L were purchased from eBioscience. Dulbecco’s modified eagle’s medium (DMEM), RPMI-1640 medium, and fetal bovine serum (FBS) were obtained from Gibco. Cell apoptosis kit with Annexin V Alexa Fluor® 488 & propidium iodide (PI) were purchased from Thermo Fisher Scientific. Rhodamine-phalloidin and Hoechst were purchased from Life technologies. NHSPEG5-DBCO (A134-10) was purchased from ClickChemistryTools. DID and DIR dyes were purchased from FANBO BIOCHEMICALS. The iron assay kit was purchased from Abcam. Other chemicals were purchased from Alfa Aesar, Co. or Sigma Aldrich. Synthesis and characterization of Fe3O4 magnetic nanoclusters (NCs): First, the NaOH/DEG stock solution was prepared by mixing 1 g of NaOH with diethylene glycol (DEG) (50 mL) under an Ar atmosphere and heated to 120 °C. When NaOH was dissolved, a paleyellow suspension solution was obtained. It was further heated for 1 h and then stored at 70 °C. After which, FeSO4·7H2O (0.128 g) was dissolved in a mixture of ethylene glycol (EG) and DEG (EG/DEG) (15 mL), and then polyethyleneimine (PEI) (0.06 g) was added and heated to 160 °C with vigorous stirring under an Ar atmosphere. Next, the mixture was quickly dropped into the NaOH/DEG stock solution. The reaction was heated and maintained at 220 °C for 1 h.


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After the solution was cooled to room temperature, the precipitate was collected by magnetic separation and washed with deionized water (dH2O). Cell cultures: The mouse leukocyte J774A.1 cell line, B16F10 cell line and 4T1 cell line were purchased from Peking Union Medical College Hospital (Beijing, China). Cells were cultured in media supplemented with 10% FBS and 1% penicillin-streptomycin at 37 °C with 5% CO2 and passaged at approximately 80% confluence. Animal use and care: BALB/c female mice (4 weeks, 16-18 g) and C57BL/6 female mice (4 weeks, 18-20 g) were obtained from Vital River laboratories (Beijing, China). The animals were maintained in accordance with the guidelines of Laboratory Animal Care, and the research protocol was approved by the Experimental Animal Ethics Committee in Beijing. Preparation of azide-modified leukocyte membrane fractions: Leukocyte membrane fractions were prepared according to the method we previously reported.37 Briefly, leukocytes (J774A.1) were cultured at 37 °C for 24 h. Then, the medium was replaced with fresh medium containing 0.1 mM azide-Cho. After 24 h incubation, the cells were washed with HEPES buffer solution (HBS) and resuspended in HBS supplemented with a protease inhibitor cocktail. After resuspension, the cells were degraded by IKA®T18 basic ULTRA-TURRAX (IKA, Germany) for different time intervals, and the cellular membrane fractions were purified by ultracentrifugation through a discontinuous sucrose density gradient. The gradient was divided into three different fractions, and the extracellular membrane biomarker CD45 and CD44 were analyzed by western blot. Modifying antibody with DBCO: A 50-fold molar excess of NHS-PEG5-DBCO linker was added to PD-1 antibody (Pa) solution and incubated at 4 °C overnight. Then, the sample was


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filtered using a centrifugal filter device (Amicon® Ultra-0.5, Millipore Co, Germany) at 7000 g for 50 min to remove the small molecules. The purified DBCO-PD-1 antibody (DBCO-Pa) was resuspended in phosphate buffered saline (PBS) and analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS, AXIMA-Performance MA, SHIMADZU, Japan). Loading TGF-β inhibitor (Ti) into leukocyte membrane fractions: Azide-modified leukocyte membrane fractions and TGF-β inhibitor (Ti) were mixed by ultrasonication, which resulted in the loading of Ti into the membrane fractions. The sample was filtered using a centrifugal filter device at 7000 g for 30 min to remove the redundant components. The Tiloaded membrane (M/Ti) was analyzed with UV spectrophotometry (U-3900 spectrophotometer, HITACHI, Japan). Membrane coating of NCs and decorating the particles with PD-1 antibody (Pa): The azide-modified leukocyte membrane fractions with or without Ti were mixed with positively charged NCs (diameter≈80 nm) in HEPES C, M/Ti-NCs or M-NCs and were formed through a porous membrane. Then, the DBCO-Pa was dissolved in PBS (pH 7.4) and left to react with M/Ti-NCs at 25 °C for 1 h. Finally, the Pa-decorated M/Ti-NCs (Pa-M/Ti-NCs) were washed and collected by magnetic separation. Characterization of Pa-M/Ti-NCs: The Pa-M/Ti-NCs were characterized by a Zeta Sizer (Nano series, Malvern, UK), H-7650 TEM (HITACHI, Japan), a physical property measurement system for magnetic properties (PPMS-9 Quantum Design), and a 7T Bruker pharmascan animal instrument (Bruker Optics, Tsukuba, Japan) for MRI. For fluorescence microscopy imaging, M/Ti-NCs and Pa were individually labeled with DID and fluorescence secondary antibody and


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then imaged by a confocal laser scanning microscope (Leica, USA). For TEM imaging, the secondary immune-antibody-linked Au nanoparticles (≈10 nm) were left to incubate with PaM/Ti-NCs before TEM imaging. To detect the magnetic manipulation, the FITC-labeled PaM/Ti-NCs were dispersed in water without (left) or with (right) a magnet in a microfluid and then imaged by a laser scanning confocal imaging system (FV3000, Olympus). To monitor the stability, Pa-M/Ti-NCs were dispersed in PBS or FBS (10%), and then, the zeta potential and diameter were measured every day over the following two weeks. Fe ion releasing: The Fe iron released from Pa-M/Ti-NCs was measured for 20 h with an iron assay kit and an automatic microplate reader at different time intervals. The concentration of Fe ions in the solution was calculated according to the absorbance at 593 nm. In vitro hydroxyl radical detection: The generation of hydroxyl radicals (·OH) induced by Fe3+ or Fe2+ ions released from Pa-M/Ti-NCs were evaluated by a methylene blue (MB) bleaching assay. The ·OH was selectively trapped by MB at first, and then, the absorption intensity at 664 nm at each time point was measured. The bleaching of MB, due to the presence of ·OH in the sample, was indicated by a discoloration from dark blue to almost white. In vivo fluorescence and magnetic resonance imaging (MRI): A subcutaneous transplantable melanoma model was established by inoculating B16F10 cells (5×105 cells) in the right flank. Then, the tumor-bearing mice were individually injected (i.v.) with different M-NCbased formulations labeled with DIR as NCs, M-NCs, Pa-M/Ti-NCs or Pa-M/Ti-NCs with a magnetic field in the tumor area (Pa-M/Ti-NCs(m)). Finally, the mice were scanned using an In Vivo Imaging System (FX Pro, Kodak, Japan) at different time intervals. For resected organ imaging, the animals were euthanized, and then, the tumors and organs were excised and imaged.


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The distribution of M-NC-based formulations in vivo was also detected by an In Vivo MR Imaging System (BioSpec 70/20 USR, BRUKER). Flow cytometry assay of cells in tumor tissues: Tumor tissues were harvested and digested into single-cell suspensions. The cells were collected and dispersed in 1 mL of PBS after red blood cell lysis. The tumor-associated macrophages (M1, M2), CD4+ T cells, CD8+ T cells and Foxp3+ regulatory T cells (Treg cells) were stained with Cy5-conjugated anti-ly6c, BV510conjugated anti-CD206, eFlour 450-conjugated anti-mouse CD4, PE-conjugated anti-mouse CD8a and FITC- conjugated anti-mouse Foxp3, respectively, and then analyzed by flow cytometry. In vivo tumor inhibition study: The B16F10 tumor-bearing C57BL/6 or 4T1 tumor-bearing BALB/c mice with an initial tumor size of 80-100 mm3 were randomized into seven groups (n=6), and then individually administered with (i) PBS, (ii) Ti, (iii) Pa, (iv) M-NCs, (v) M/TiNCs, (vi) Pa-M/Ti-NCs or (vii) Pa-M/Ti-NCs(m) via intravenous injection twice a week at a Ti dose of 1.0 mg kg-1 and a Pa dose of 0.5 mg kg-1. The body weight and tumor volume of each mouse were measured daily during the whole period of treatment. The estimated tumor volume was calculated by using the formula volume (mm3) = length×width2/2, and the experimental endpoint was defined as either death or a tumor size greater than 1000 mm3. The lung and hind leg of each mouse in 4T1 tumor-bearing BALB/c mice were imaged by computed tomography (CT) to study the metastasis originated from the primary tumor. In vivo anti-metastasis effect: A subcutaneous transplantable model was established by inoculating luciferase-expressing 4T1 (Luc-4T1) cells (5×105 cells) into BALB/c mice. The mice were then randomly divided into 7 groups and individually treated with (i) PBS, (ii) Ti, (iii) Pa,


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(iv) M-NCs, (v) M/Ti-NCs, (vi) Pa-M/Ti-NCs or (vii) Pa-M/Ti-NCs(m) on days 7, 10, 13 and day 16. On day 10, the mice were injected (i.v.) with 100 μL of PBS (pH 7.2) containing 3×105 luciferase-expressing 4T1 cells. The primary tumor and the metastasis in lungs were imaged by an IVIS Spectrum on day 20 (PerkinElmer, USA) after intraperitoneal injection with D-Luciferin sodium salt at a dose of 3 mg/mouse. Post-operation recurrence inhibition effect: C57BL/6 mice were subcutaneously inoculated with 5×105 B16F10 cells and randomly divided into 4 groups on -10 d. On day 0, the majority of tumors were surgically resected when the tumor volume reached 500 mm3. On days 3, 6, 9 and 12, the mice were individually treated with (i) PBS, (ii) Ti, (iii) Pa or (iv) Pa-M/Ti-NCs(m) at a TGF-β inhibitor dose of 1.0 mg kg-1 and a PD-1 antibody dose of 0.5 mg kg-1. The recurrent tumor size and survival were measured every two days, and the experimental endpoint was defined as either death or a tumor size greater than 1000 mm3. The post operation recurrence inhibition effect was also studied by using Luc-4T1 tumor model. The primary and distant tumors were imaged by IVIS Spectrum (PerkinElmer, USA) after intraperitoneally injection with D-Luciferin sodium salt at a dose of 3 mg/mouse. Long-term immune memory effect: C57BL/6 mice were inoculated subcutaneously with 5×105 B16F10 cells on day -45 and randomly divided into 4 groups. After inoculation, they were individually treated with (i) PBS, (ii) Ti, (iii) Pa or (iv) Pa-M/Ti-NCs(m) at a Ti dose of 1.0 mg kg-1 and a Pa dose of 0.5 mg kg-1 on days -35, -32 and -29, and then, the primary tumors were surgically resected on day -28. Four weeks later, these mice were rechallenged with B16F10 cells in the right flank. The distant tumor size and survival were measured every two days, and the experimental endpoint was defined as either death or a tumor size greater than 1000 mm3. The Luc-4T1 tumor-bearing BALB/c mice were also treated as the B16F10 tumor model to study


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the long-term immune memory effect. The primary and distant tumors were imaged by IVIS Spectrum (PerkinElmer, USA) after intraperitoneally injection with D-Luciferin sodium salt at a dose of 3 mg/mouse. Dendritic cell (DC) maturation and cross-presentation in tumors: Tumor tissues were harvested from the mice in different treatment groups and triturated into a single-cell suspension. Then, the cells were stained with FITC-conjugated anti-mouse CD11c, APC/Cy7-conjugated anti-mouse CD86 and BV605-conjugated anti-mouse MHC II for the analysis of DC maturation; cells were also stained with FITC-conjugated anti-mouse CD11c and PerCP/eFlour 710conjugated anti-mouse MHC I for analysis of DC cross-presentation. Memory T cells assay: The splenocytes were collected and stained with anti-CD3-PerCPCy5.5, anti-CD8-PE, anti-CD44-FITC and anti-CD62L-APC antibodies. After being washed, the cells were assessed by flow cytometry to detect effector memory T cells (TEM, CD3+ CD8+ CD62L- CD44+). Immunohistochemical section evaluations: The mice were sacrificed, and the tumors were harvested, sliced and stained. For evaluating ferroptosis in tumor tissues, the glutathione peroxidase 4 (GPX4) and lipoxygenase enzyme (FACL4) proteins were labeled with an immunofluorescence stain. The following antibodies were used to analyze tumor-associated macrophages and T cells in tumors: anti-CD206, anti-Ly6C, anti-CD4, anti-CD8 and anti-Foxp3. Ki 67 detection through immunohistochemical staining was used to measure tumor proliferation. Safety Estimation: To evaluate the safety of different M-NC-based formulations in vivo, body weight changes were recorded. For humane reasons, animals were sacrificed when the implanted tumor volume reached 1000 mm3. The tissue sections of the hearts, livers, spleen, lung, and


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kidneys were stained with H&E (hematoxylin/eosin) and analyzed. The serum levels of urea nitrogen (BUN), lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) were determined spectrophotometrically using an automated analyzer (Hitachi-917, Hitachi Ltd., Tokyo, Japan). Statistical analysis: All the data were presented as mean ± s.d.. Unpaired student’s t-test (twotailed) was used for comparison between two groups. Statistical significance was set at ***p < 0.01. ASSOCIATED CONTENT Supporting Information. Additional experimental figures including magnetosome characterizations, stealth ability, in vivo pharmacokinetic curves, biodistribution of Pa-M/Ti-NCs (m), Pa-M/Ti-NC(m)-induced tumor immune microenvironment changes and ferroptosis, therapeutic results, cytokine secretion, lung metastasis, safe estimation and Pa-M/Ti-NC(m)-induced long-term immune memory effects of 4T1 tumor model. 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] ORCID Hai-yan Xie: 0000-0002-6330-7929 Wei Wei: 0000-0002-6244-3187


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Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (No. 81571813, No. 21874011 and No. 21622608) and National Key R&D Program of China (2017YFA0207900). REFERENCES (1) Liu, J.; Chen, Q.; Feng, L.; Liu, Z. Nanomedicine for Tumor Microenvironment Modulation and Cancer Treatment Enhancement. Nano Today 2018, 21, 55-73. (2) Zhang, C.; Bu, W.; Ni, D.; Zhang, S.; Li, Q.; Yao, Z.; Zhang, J.; Yao, H.; Wang, Z.; Shi, J. Synthesis of Iron Nanometallic Glasses and Their Application in Cancer Therapy by a Localized Fenton Reaction. Angew. Chem. Int. Ed. 2016, 55, 2101-2106. (3) Bogdan, A. R.; Miyazawa, M.; Hashimoto, K.; Tsuji, Y. Regulators of Iron Homeostasis: New Players in Metabolism, Cell Death, and Disease. Trends Biochem. Sci. 2016, 41, 274-286. (4) Cao, Z.; Zhang, L.; Liang, K.; Cheong, S.; Boyer, C.; Gooding, J. J.; Chen, Y.; Gu, Z. Biodegradable 2D Fe-Al Hydroxide for Nanocatalytic Tumor-Dynamic Therapy with Tumor Specificity. Adv. Sci. (Weinh) 2018, 5, 1801155. (5) Feng, W.; Han, X.; Wang, R.; Gao, X.; Hu, P.; Yue, W.; Chen, Y.; Shi, J. NanocatalystsAugmented and Photothermal-Enhanced Tumor-Specific Sequential Nanocatalytic Therapy in both NIR-I and NIR-II Biowindows. Adv. Mater. 2019, 31, 1805919.


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Engineering Magnetosomes for Ferroptosis/Immunomodulation Synergism in Cancer Fan Zhang,† Feng Li,† Gui-Hong Lu,† Weidong Nie,† Lijun Zhang,‡, § Yanlin Lv,‡, Weier Bao,‡, § Xiaoyong Gao,‡, Wei Wei,‡, §, * Kanyi Pu,⊥ and Hai-Yan Xie†, *

A feature-packed biomimetic magnetosome was constructed. A Fe3O4 magnetic nanocluster (NC) core, PD-1 antibody (Pa) and TGF-β inhibitor (Ti) can spatiotemporally cooperate with each other in tumors for ferroptosis/immunomodulation synergism in cancer.


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Immunomodulation

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