Manganese Silicate Nanospheres

5 days ago - Dong, Liu, Feng, Yang, Liu, Lai, Lu, Lovell, Chen, and Fang. 2019 19 (2), pp 997–1008. Abstract: Delivery of therapeutics into the soli...
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Biodegradable Biomimic Copper/Manganese Silicate Nanospheres for Chemodynamic/Photodynamic Synergistic Therapy with Simultaneous Glutathione Depletion and Hypoxia Relief Conghui Liu, Dongdong Wang, Shuyuan Zhang, Yaru Cheng, Fan Yang, Yi Xing, Tailin Xu, Haifeng Dong, and Xueji Zhang ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b09387 • Publication Date (Web): 22 Mar 2019 Downloaded from http://pubs.acs.org on March 22, 2019

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Biodegradable

Biomimic

Nanospheres Synergistic

for Therapy

Copper/Manganese

Silicate

Chemodynamic/Photodynamic with

Simultaneous

Glutathione

Depletion and Hypoxia Relief Conghui Liu, Yang,

Dongdong Wang,

†, ‡

Shuyuan Zhang, ‡ Yaru Cheng,

†, ‡

Fan

†, ‡

Yi Xing, †

†, ‡

†, ‡

Tailin Xu,

†, ‡

Haifeng Dong, *,

†, ‡

Xueji Zhang*,

†, ‡

Beijing Advanced Innovation Center for Materials Genome Engineering,

University of Science and Technology Beijing, Beijing 100083, P. R. China. ‡ Beijing

Key Laboratory for Bioengineering and Sensing Technology, Research

Center for Bioengineering and Sensing Technology, School of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing 100083, P. R. China. E-mail: [email protected], [email protected]

ABSTRACT: The integration of reactive oxygen species (ROS)-involved photodynamic therapy (PDT) and chemodynamic therapy (CDT) hold great promise for enhanced anticancer effect. Herein, we report a biodegradable cancer cell membrane-coated mesoporous copper/manganese silicate nanospheres (mCMSNs) with homotypic targeting ability to the cancer cell lines and enhanced ROS generation through singlet oxygen (1O2) production and glutathione (GSH)-activated Fenton reaction, showing excellent CDT/PDT 1

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synergistic therapeutic effect. We demonstrate that mCMSNs are able to relieve the tumor hypoxia microenvironment by catalytic decomposition of endogenous H2O2 to O2 and further react with O2 to produce toxic 1O2 with a 635 nm laser irradiation. GSH-triggered mCMSNs biodegradation can simultaneously generate Fenton-like Cu+ and Mn2+ ions and deplete GSH for efficient hydroxyl radical (·OH) production. The specific recognition and homotypic targeting ability to the cancer cells were also revealed. Notably, the relieving hypoxia and GSH depletion disrupt the tumor microenvironment (TME) and cellular antioxidant defense system (ADS), achieving exceptional cancer-targeting therapeutic effect in vitro and in vivo. The cancer cells growth were significantly inhibited. Moreover, the released Mn2+ can also act as advanced contrast agent for cancer magnetic resonance imaging (MRI). Thus, together with photosensitizers, Fenton agent provider and MRI contrast effect along with the modulating of the TME, allows mCMSNs to realize MRI-monitored enhanced CDT/PDT synergistic therapy. It provides a paradigm to rationally design TMEresponsive and ROS-involved therapeutic strategies based on a single polymetallic silicate nanomaterial with enhanced anticancer effect.

KEYWORDS: Fenton reaction, glutathione, chemodynamic therapy, cell membrane, photodynamic therapy

Emerging cancer treatments, such as photodynamic therapy (PDT) and chemodynamic therapy (CDT), utilize intracellular specific chemical reactions 2

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to generate cytotoxic reactive oxygen species (ROS), including singlet oxygen (1O2), super-oxide anions (∙O2-), hydroxyl radicals (∙OH), which can damage biomolecules and finally induce cellular apoptosis or necrosis once their concentration reach a certain level.1, 2 As external energy-triggered therapies, PDT converts oxygen (O2) to high-level ROS by light-activated photosensitizers with the advantages of slight pain, low side effects and minimal systemic toxicity compared to conventional chemotherapy and radiotherapy.3,

4

However, the

PDT therapeutic effects are limited by low ROS generation efficiency, which was resulted from the hypoxia tumor microenvironment (TME) and quick energy attenuation.5 Therefore, combination treatments including PDT/photothermal therapy (PTT),

6-9

PDT/chemotherapy

10, 11

and PDT/immunotherapy

12, 13

are

usually employed to overcome the insufficient tumor inhibition effect of monotherapy.14, 15 For example, the porphyrin-grafted and DOX-encapsulated lipid nanoparticle was a typical combination nanoplatform for synergistic chemo-photodynamic therapy, achieving significant suppression on tumor growth by initiating multiple antitumor mechanisms.10 Generally, CDT is dependent on an in situ Fenton reaction (Fenton-like) that generate oxidative ∙OH from hydrogen peroxide (H2O2) under the catalysis of ferrous ion (Fe2+) or other Fenton-like ions.16-27 CDT triggered by endogenous chemical energy in TME rather than external energy input effectively avoids the quick energy attenuation during the penetration to tumor tissue existed in PDT.28 Given the features including H2O2 overexpression (100 μM~1 mM), low 3

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catalase activity and mild acidity of TME,1, 29, 30 ∙OH is produced only in tumorspecific microenvironment but do little harm to normal tissues. However, the glutathione (GSH) related cellular antioxidant defense system (ADS) limits CDT efficacy21 and biodegradability of inorganic nanoparticles impedes its clinical translation.31,

32

It is confirmed that Fenton or Fenton-like reactions may

simultaneously produce O2 along with ∙OH,22,

30

suggesting that CDT may

relieve oxygen deficiency in PDT and improve its therapeutic effect, while UV/vis or near-infrared laser irradiation could improve ∙OH generation efficiency in Fenton reaction to enhance antitumor effect of CDT.20,

33

For

example, UV/vis irradiation can improve ∙OH generation efficiency in photoFenton reaction by photoreduction of Fe3+ to regenerate the catalytic ions Fe2+, which avoids the termination of Fenton reaction.34, 35 Furthermore, combining NIR with upconversion nanoparticles (UCNPs) can convert NIR into UV/vis in photo-Fenton-involved CDT with reduced damage to normal tissues and the relative high penetration depth.36 Most of the previous research on CDT/PDT synergistic therapy were involved in complex incorporation or assembly of photosensitizers and Fenton-like agents to realize “all-in-one” multifunction, such as Cu2+–graphitic carbon nitride (g-C3N4) nanosheets,37 UCNPs@MnSiO3@g-C3N4 nanoplatforms,38 and mesoporous silica nanoparticles loaded with manganese ferrite nanoparticles and chlorin e6.30 Little efforts have been devoted to achieving synergistic CDT/PDT in single smart nanoagents. Recently, Liu et al. synthesized copper 4

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ferrite nanospheres as integrated theranostic nanoagents for CDT/PDT synergistic therapy, realizing photoenhanced CDT and improved PDT for effective tumor eradication.22 However, improved specificity, biocompatibility and targeting efficiency of theranostic CDT/PDT nanoagents are continuously required. Over the years, the cancer cell-membrane-coated technology is an emerging method to endow nanoparticles with improved biocompatibility and homotypic targeting ability by the source cancer cell lines since that the surface adhesion molecules of cancer cells endow them with the homologous adhesion capacity, leading to the formation of multicellular malignant cell aggregates.3942

It eliminates the complex preparation and the risk of the immune system

activation existed in traditional surface functionalization with polymers and targeting ligands.43, 44 Thus, rationally designing biomimic and biodegradable nanoplatforms integrated PDT and CDT hold great promise for efficient ROSmediated cancer treatments. Herein, we demonstrated a simple approach to prepare biodegradable mesoporous copper/manganese silicate nanospheres (CMSNs), and utilize them as TME-responsive Fenton-like agents and photosensitizers for magnetic resonance imaging (MRI)-guided CDT/PDT synergistic cancer therapy. The CMSNs were synthesized by a hydrothermal method using dendritic mesoporous silica nanoparticles (DPSNs) as self-sacrificing templates, and further camouflaged with MCF-7 cancer cell membranes to obtain the biomimetic mem@CMSNs (mCMSNs) (Scheme 1A). The copper silicate 5

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existed in the CMSNs acted as efficient photosensitizers of PDT, while Fentonlike Cu+ and Mn2+ released from the CMSNs by a redox reaction with GSH40-42 could catalyze endogenous H2O2 to generate toxic ·OH and simultaneously release O2 in CDT. This TME-mediated redox reaction simultaneously enabled hypoxia relief and GSH depletion, leading to enhanced CDT/PDT anticancer effects. In addition, longitudinal relaxivity (T1) of released Mn2+ allowed to monitor the progress of CDT/PDT with magnetic resonance imaging (MRI) (Scheme 1B, 1C).

RESULTS AND DISCUSSION The synthesized DPSNs with an average diameter of 130 nm (Figure S1A) were used as the template of CMSNs. During hydrothermal process, silicate ions released from DPSNs in the alkaline condition48-50 reacted with the copper/manganese–ammonium complex ions (Cu(NH3)42+ or Mn(NH3)42+)51, 52 to generate copper/manganese silicate, which continuously deposited on the surface of DPSNs until the DPSNs were completely consumed, finally leading to the formation of mesoporous CMSNs. The ammonium chloride (NH4Cl) added in the synthesis process was used to control the ammonia ionization rate and inhibit the hydroxide formation.53, 54 The morphology of the as-prepared CMSNs could be simply adjusted by the feeding ratios of copper and manganese chloride (Figure S1B-E, Table S1). The mesoporous silicate nanoparticles with relative smooth surface was selected to facilitate the cancer membrane coating. As shown in 6

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Figure 1A and 1B, the CMSNs had inner hollow cavities of ∼30 nm in diameter and mesoporous shells of ∼30 nm in thickness. It exhibited a specific surface area of 247.9 m2/g, pore volume of 0.684 cm3/g and average pore size of 5.16 nm (Figure 1C), and the large surface and mesoporous structure were useful for active molecular accessibility and substance transfer.55,

56

The X-ray

diffraction (XRD) pattern of CMSNs further confirmed the successful synthesis of copper/manganese silicate (CuMn6SiO12) along with characteristic peaks of both copper silicate and manganese silicate (Figure S2). X-ray photoelectron spectroscopy (XPS) analysis of CMSNs revealed that the Cu and Mn doping amount was nearly 13.4% and 14.3% (Figure S3), respectively. The highresolution XPS spectrum of Cu 2p and Mn 2p suggested the Cu existed primarily in the form of Cu2+ with shakup satellite peaks (Figure 1D), while Mn 2p3/2 was consisted of 68.6% Mn3+ (642 eV), 27.7% Mn4+ (644 eV) and 3.7% Mn2+ (641 eV) (Figure 1E and 1F).31, 38, 57 The high component of Cu2+, Mn3+ and Mn4+ provided great potential for redox reaction with GSH and improved PDT and Fenton-like effect. The outer cancer cell membrane lipid bilayer shell on the obtained mCMSNs was about 10 nm in thickness (Figure 1G and 1H). The increased hydrodynamic particle size (~25 nm) and the decreased surface zeta potential (~10 mV) of mCMSNs compared to CMSNs also confirmed the successful membrane coating (Figure 1I and 1J). Importantly, it was observed that the protein ingredients in mCMSNs were almost the same as those of MCF7 membranes analyzed by sodium dodecyl sulfate−polyacrylamide gel 7

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electrophoresis (SDS-PAGE) (Figure 1K), suggesting most of the cell membrane proteins were retained in mCMSNs.58,

59

The hydrodynamic size,

zeta potential and polymer dispersity index (PDI) of mCMSNs stored in 4 ℃ remained almost unchanged in 7 days, indicating the good stability of mCMSNs in solution (Figure S4). The catalytic effect of CMSNs on H2O2 decomposition was first demonstrated by bubbles generation in H2O2 solution (Figure S5) and rapid real-time O2 concentration increasing after addition of CMSNs into different concentrations of H2O2 solution (Figure 2A and S6), indicating that CMSNs could accelerate H2O2 decomposition to generate O2 and thus might relieve the hypoxia of tumors. In addition, the band gap of CMSNs was calculated to be 1.93 eV (Figure S7), suggesting that the CMSNs might be excited by a 635 nm laser (E=2.05 eV) to generate ROS for PDT.60 The 1O2 generation ability of CMSNs solution with or without H2O2 and CMSNs solution containing H2O2, GSH and inorganic ions were measured by 1,3-diphenylisobenzofuran (DPBF) degradation experiments. As shown in Figure 2B and S8A-C, the sharp downtrend of DPBF absorption at 420 nm for CMSNs solution irradiated with a 635 nm laser (0.6 W/cm-2) within 30 min compared to the control groups indicated the good CMSNs-mediated 1O2 production ability. Much more steep downtread was observed as the added H2O2 increased (Figure 2B and S9A-E), suggesting the 1O2 production ability was significantly enhanced due to more O2 generation by the Fenton reaction thus achieved improved PDT. It was 8

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further confirmed that the component of copper silicate rather than manganese silicate in CMSNs played the key role in 1O2 generation (Figure S8D and S8E). As for the CMSNs solution containing H2O2, GSH and inorganic ions (Figure S9), the degradation efficiency of DPBF decreased because reductive GSH could effectively eliminate reactive species including 1O2 generated in PDT though redox reaction.61, 62 The biodegradation possibility of CMSNs in mimic TME was directly observed by TEM analysis (Figure 2C). CMSNs remained largely intact in neutral condition (pH=7.4) in the absence of GSH, while most of CMSNs were significantly collapsed in acid condition (pH=6.5) after 24 h. Further simultaneously providing 10 mM GSH and acid condition, the framework of CMSNs were almost completely degraded. The Cu and Mn ions released from CMSNs treated with or without GSH at different pH was monitored by ICP-OES measurements (Figure 2D and 2E). As expected, the GSH enabled significantly induce CMSNs biodegradation to release Cu and Mn, while the mild acidic condition further enhanced the release. We also measured the elemental composition and valence of the reservation from CMSNs treated with different concentrations of GSH in acid condition (pH=6.5) for 24 h by XPS analysis to clarify the precise composition of synthesized CMSNs and reveal the reaction between CMSNs and GSH. As shown in Table S2 (obtained from Figure S10), the atomic and mass concentration percentages of Cu and Mn gradually decreased with the increasing concentration of GSH. Notably, the ratio of Mn3+ 9

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in Mn 2p3/2 of the reservation sharply decrease with the increasing GSH concentration. When the GSH concentration reached 10 mM, the Mn content could not be detected and the original Cu shakeup satellite peaks disappeared due to the efficient redox reaction between CMSNs and GSH (Table S2 and Figure S10D-I). These results suggested that the mild acidity (pH=6.5) and reductive

GSH

could

accelerate

the

biodegradation

of

CMSNs

to

simultaneously generate Fenton-like ions for CDT. The Fenton-like Cu+ in the presence of Cl- or HCO3- and Mn2+ with HCO3assistance showed good ·OH production capability in methylene blue (MB) degradation analysis, and the scavenging effect of GSH toward ∙OH was also oberved (Figure S11A-C), in agreement with provious reports.63, 64 The CMSNs displayed similar MB degradation capacity in the presence of Cl- and HCO3with a GSH-dependent pattern. As shown in Figure 2F and S11D, CMSNs could promote MB degradation by increasing·OH generation along with the increase of GSH from 0 to 1.0 mM, and finally obtained over 50% MB degradation even excessive GSH would scavenge OH in turn. The ESR spectra using 5,5dimethyl-1-pyrrolineN-oxide (DMPO) as ·OH trapping agent provided the similar results to the MB degradation analysis of CMSNs (Figure 2G). These results indicated the good ∙OH generation ability of CMSNs in the solution simultaneous containing H2O2, GSH, and HCO3- and the great potential of CMSN for enhanced CDT by GSH consumption. Addtionally, subsequent MRI capability of released Mn2+ ions from the CMSNs at different pH and GSH 10

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concentrations was further confirmed (Figure S12). It can be observed that the T1 signal derived from paramagnetic Mn centers showed enhanced brightness with the increasing Mn2+ concentration from 0 to 0.6 mM in phantom images of each group (Figure 2H). The initial r1 (longitudinal relaxivity coefficient) of CMSNs in neutral (pH=7.4) or mild acid (pH=6.5) condition were calculated to be low as 0.17 mM−1s−1 and 0.47 mM−1s−1 respectively, which may be caused by the inhibited chemical exchange between the isolated paramagnetic Mn centers in CMSNs and protons.21,

24

After GSH (10 mM) addition, the r1 of

CMSNs in neutral condition (pH=7.4) increased to 5.01 mM−1s−1, and that in mild acid condition (pH=6.5) was up to 7.14 mM−1s−1 (Figure 2I), which suggested a significantly enhanced release of Mn2+ from CMSNs in acid and reductive conditions and confirmed the TME-responsive MRI contrast enhancement of CMSNs. The homotypic targeting ability of mCMSNs toward MCF-7 cells was investigated using NHDF normal cells and human non-small cell lung cancer cells (A549) as the controls (Figure 3A and S10). It was observed that mCMSNs preferentially accumulated in MCF-7 cells rather than NHDF cells and A549 cells. Similar results were also achieved by quantitative flow cytometry analysis shown in Figure 3B that MCF-7 cells exhibited approximated to 7-fold higher mean fluorescence intensity (MFI) than that in NHDF cells, indicating the highly specific self-recognition of mCMSNs toward MCF-7 cells owing to intercellular homologous binding capability endowed by membrane proteins on the outer 11

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MCF-7 cancer cell membrane.39-42, 65 The intracellular O2 generation capacity of mCMSNs was investigated by measuring the hypoxia level of cells after different treatments in hypoxic condition. Red Hypoxia Detection Reagent was used as hypoxia probe, which would emit fluorescent after degradation by nitroreductase presented in hypoxic cells. As shown in Figure 3C and the corresponding surface plot images, the intracellular red fluorescence intensity was significantly enhanced after hypoxia treatment compared with that of normoxia group, which confirmed the establishment of hypoxic condition. Further incubation with different treatment in hypoxia environment, the fluorescence intensity was observed to remain almost the same in PBS-treated group. On the contrary, the fluorescence intensity in mCMSNs-treated group obviously became weak, suggesting the hypoxia relief by mCMSNs due to O2 generation. The mCMSNs-mediated ROS production in MCF-7 cells was measured by the intracellular ROS probe 2, 7-dichlorofluorescin diacetate (DCFH-DA), which displayed green fluorescence after oxidized by ROS. As shown in Figure 3D and the corresponding surface plot images, the mCMSNstreated MCF-7 cells presented stronger fluorescence than that of the control group, whereas the green fluorescence was weak for the MCF-7 cells pretreated with the L-buthionine sulfoximine (L-BSO), an inhibitor of γglutamylcysteine synthetase which played the key role in intracellular GSH synthesis,66, 67 suggesting that it was the GSH triggered CMSNs-mediated ·OH generation, and the GSH depletion further enhanced ∙OH generation. The 12

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mCMSNs-treated MCF-7 cells with irradiation displayed the strongest green fluorescence owing to the enhanced ROS (∙OH and 1O2) generation through the combined CDT and PDT (Figure 3D and S11). Moreover, significant decrease of intracellular GSH/GSSH ratio compared to the control group was observed for MCF-7 cells incubated with mCMSNs after irradiation (Figure 3E), which was resulted from the synergistic CDT/PDT induced GSH depletion. The in vitro cytotoxicity of mCMSNs was further performed by cell counting kit 8 (CCK-8) assay. As shown in Figure 3F and S12, mCMSNs displayed enhanced dose- and time-dependent cytotoxicity to MCF-7 due to effective ·OH generation through Fenton-like reaction, whereas no obvious cytotoxicity to NHDF cells was observed when the concentration was lower than 50 μg/mL. It indicated mCMSNs could specifically kill cancer cells but cause negligible harm to normal tissues because the relative low cellular uptake of mCMSNs and low expression of GSH and H2O2 in normal cells. Then we evaluated the synergistic inhibition effect of mCMSNs-mediated CDT and PDT on MCF-7 cells (Figure 3G). The mCMSNs showed enhanced cytotoxicity than CMSNs attributed to the specific-targeting ability. Upon laser irradiation, the anticancer effect of mCMSNs was further enhanced and the cell viability decreased to less than 20% at the concentration of 50 μg/mL. Calcein-AM and propidium iodide (PI) co-staining fluorescence imaging (Figure 3H) and cell apoptosis assay by quantitative flow cytometry analysis (Figure S13) further confirmed the enhanced inhibition effect and significant cell apoptosis/death toward MCF-7 13

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cells received mCMSNs treatment and laser irradiation compared to control groups, whereas no obvious effect on NHDF cells received the same treatments. The good in vitro antitumor efficacy of mCMSNs encouraged us to further investigate the in vivo anticancer effect. The blood circulation and biodistribution in main organs of mCMSNs were first measured. As shown in Figure 4A, mCMSNs displayed a blood retention of 20.1 % ID/g, while only 2.3 % ID/g of bare CMSNs was found in blood circulation at 24 h after tail vein injection. The circulation half-life of the mCMSNs was calculated to be 7.07 h, which was much longer than that 3.21 h for the bare CMSNs attributed to the inherent properties of the cell membranes coating.65, 68, 69 The self-recognition ability of the cancer cell membranes also facilitated the tumor accumulation of mCMSNs, which was 2.85-fold higher than that of CMSNs (Figure 4B). Moreover, the whole tumor area became brighter after postinjection for 24 h (Figure 4C), demonstrating that the mCMSNs could be efficiently accumulated in tumor and gradually reduced to Mn2+ by GSH and the mCMSNs provided an efficient T1-weighted MRI contrast effect for tumor diagnosis. MCF-7 tumor-bearing nude mice (female, 4 weeks) were divided into 5 groups (n = 5) randomly to study the antitumor performance of different treatments. As shown in Figure 4E and 4F, neither PBS (pH=7.4, 10 mM) nor laser showed inhibition effect on tumor growth. As expected, obvious inhibition 14

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on tumor growth was observed in CMSNs- and mCMSNs-treated groups, and mCMSNs plus laser irradiation treatment exhibited the greatest suppression effect, which was resulted from the enhanced oxidative stress induced by the synergistic CDT/PDT treatment (Figure 4D). After mice were sacrificed, hematoxylin and eosin (H&E) staining of tumor tissues and main organs was further performed to confirm the anticancer effect and biocompatibility of different treatments. It could be observed that cancer cells of tumor tissues in synergistic therapy group underwent much severer apoptosis/death than that of other groups (Figure 4H). In addition, there were no histopathological abnormalities in main organs (Figure S14) and no obvious changes in body weights for all groups (Figure 4G). Therefore, the MRI monitored CDT/PDT synergistic therapy mediated by mCMSNs with advanced tumor inhibition effect and good biocompatibility was promising in anticancer treatments.

CONCLUSIONS In conclusion, we successfully fabricated biodegradable mesoporous copper/ manganese silicate nanospheres (CMSNs) with biomimetic MCF-7 cancer cell membrane coating (mCMSNs) for synergistic CDT/PDT anticancer treatment. The resulting mCMSNs was first comprehensively characterized. We demonstrated GSH could trigger biodegradation of mCMSNs to release Fenton-like Cu+ and Mn2+ that catalyzed H2O2 to produce ∙OH for GSHdepletion enhanced CDT and simultaneously generate O2 to potentially relieve the hypoxia of tumors. Under a 635 nm laser-irradiation, the mCMSNs could 15

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also act as photosensitizers to generate 1O2 for hypoxia-relieved improved PDT. The cancer membrane coating endowed the mCMSNs with good tumor targeting ability both in vitro and in vivo. The target-cell-specific GSH depletionenhanced CDT and hypoxia-relieved PDT by mCMSNs impaired the antioxidant defense of cancer cells and made the cells more sensitive to ROS, resulted in excellent inhibition effect on tumors. In addition, the released Mn2+ from mCMSNs in the redox reaction with GSH exhibited high T1 relaxivity, which could be used as MRI contrast agents to monitor the synergistic CDT/PDT treatment. This work demonstrated a single polymetallic silicate nanomaterial with multifunction including hypoxia-relieved photodynamic effect and TME responsive chemodynamic activity and MRI imaging, providing a promising candidate for cancer theranostics.

MATERIALS AND METHODS Materials: All of the chemical reagents were used without further purification. Hexadecyltrimethylammonium

p-toluenesulfonate

(CTA·Tos),

1-Butyl-3-

methylimidazolium Trifluoromethanesulfonate ([BMIM] OTF), triethylamine (TEA), tetraethoxysilicon (TEOS), copper(II) nitrate trihydrate (CuNO3∙3H2O), manganese(II) chloride tetrahydrate (MnCl2∙4H2O), 3-(4, 5-dimethyl-2thiazolyl)-2,

5-diphenyl-2-H-

tetrazoliumbromide

(MTT)

and

Tris(hydroxymethyl)methyl aminomethane (Tris) were purchased from SigmaAldrich (China). Ammonium chloride (NH4Cl) and ammonium hydroxide (NH3∙H2O) were obtained from Sinopharm Chemical Reagent Co., Ltd (Beijing, 16

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China). PBS (pH 7.4, 10 mM), fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), Opti-MEM, trypsin-EDTA and penicillin-streptomycin were purchased from Gibco Life Technologies (AG, Switzerland). 4% paraformaldehyde fix solution was obtained from Beyotime. Instruments: The morphologies of nanoparticles were obtained by transmission electron microscopy (TEM, HT7700, Hitachi, Japan) and scanning electron microscope (SEM, JSM7610F, Hitachi, Tokyo, Japan). The particle size and zeta potential were performed by Zetasizer Nano ZS90 system (Malvern, UK). Nitrogen absorption−desorption isotherm and porosity was measured by a surface area analyzer (QuadraSorb SI 2000-08, Quantachrome Instruments). XRD was measured with a Bruker D8 ADVANCE X. X-ray photoelectron spectroscopy (XPS) spectra were obtained using Thermo Scientific ESCALAB 250 XI. Real-time measurement of O2 concentration in solution was performed by portable dissolved oxygen meter (JPBJ-608, Rex, INESA Scientific Instrument). The UV−visible absorption was obtained by a UV-1800 spectrophotometer (Shimadzu, Japan). The cell fluorescence imaging experiments were carried out using confocal laser scanning fluorescence microscope (CLSM, FV1200, Olympus, Japan). Cell apoptosis and death was analyzed by flow cytometry (Beckman Coulter, CytoFLEX). The concentration of released Cu and Mn of CMSNs and the concentration of Cu and Mn in blood samples and tissues were measured by inductively coupled plasma-optical emission spectrometry (ICP-OES, 5110DVD, Agilent). ∙OH was quantified by 17

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an ESR spectrometer (JEOL FA-200). Flow cytometry (Beckman Coulter, CytoFLEX) was used to analyze the target ability of mCMSNs. The MRI in vitro and in vivo was conducted by an animal MRI scanner (BioSpec70/20USR, Bruker, Germany). Synthesis

of

Dendritic

Mesoporous

Silica

Nanoparticles

(DPSNs).

Monodispersed DPSNs with the diameter of 130 nm were synthesized according to previous reports.48, 49 Briefly, CTA·Tos (0.96 g), TEA (0.105 g) and [BMIM] OTF (10 mg) were first mixed in 50 mL of water, then the mixture was stirred at 80 °C for 1 h. Next, 7.8 mL of TEOS was quickly added into the mixture. After stirring at 80 °C for another 2 h, the mixture was purified by washing and centrifugation with ethanol and water. Then, template extraction was employed to remove the CTA·Tos from as-prepared DPSNs. After addition of ethanolic HCl (concentrated HCl (15 mL) in ethanol (100 mL)) into 0.1 g of the as-prepared DPSNs, the suspension was sonicated for 2 h and then stirred at 70 °C for 24 h. This extraction procedure was repeated three times. Finally, the precipitate was collected by washing with ethanol and drying in a vacuum oven at 60 °C for 24 h. Synthesis of Mesoporous Copper and Manganese Sillicate Nanospheres (CMSNs).52,

54

CMSNs were synthesized by hydrothermal treatment and the

DPSNs were used as self-sacrificing templates. Briefly, CuNO3∙3H2O (0.4 mmol) and NH4Cl (10 mmol) were first mixed in 20 mL water, then 1 mL of 18

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NH3∙H2O (28%) was added dropwise into the mixture. The obtained mixture of copper-ammonia coordination complex ions was solution A. DPSNs (50 mg) and MnCl2∙4H2O (0.4 mmol) were mixed in 20 mL water to obtain solution B. Solution A and B were mixed and stirred for 30 min, then transferred into a Teflon-lined autoclave (50 mL) and maintained at 180 °C for 24 h. The precipitates were collected, washed with water until the pH was 7, and finally dried in a vacuum oven at 60 °C. Preparation of Cancer Cell Membrane. The MCF-7 cell membrane was obtained first by Membrane Protein Extraction Kit. Briefly, the collected 5*107 MCF-7 cells were dispersed in 1 mL of membrane protein extraction buffer solution A pre-added 1mM PMSF and placed in an ice bath for 10−15 min. The resulting MCF-7 cell suspension was frozen at -80 ℃ and then thawed at room temperature. This freezing-thawing cycle was repeated for 3 times, followed by centrifugation at 3500 rpm for 15 min at 4 °C. The obtained supernatant was further centrifuged at 14000 rpm for 30 min at 4 °C. Finally, the precipitate was preserved and stored at -80 ℃. Preparation of Cancer Cell Membrane-coated CMSNs (mCMSNs).44 The mCMSNs were constructed using Avanti mini extruder. Briefly, the human breast adenocarcinoma MCF-7 cell membrane solids and the CMSNs were dissolved in water at the concentration of 2 mg/mL and sonicated for 15 min respectively. Then, 0.5 mL of MCF-7 cell membrane (2 mg/mL) was extruded 19

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at least 10 times by Avanti mini extruder using polycarbonate porous membrane (400 nm). Next, 0.5 mL of CMSNs (2 mg/mL) was added into the pretreated MCF-7 cell membrane, and the mixture was extruded at least 10 times using polycarbonate porous membrane (200 nm). Finally, the mixture was centrifugated at 6000 rpm for 10 min at 4 °C and washed with water for 3 times to remove excess free membrane. Extracellular Real-time O2 Concentration Measurement. 3 mg CMSNs and 90 μL of H2O2 (1 M) were added to 30 mL water in turn under vigorous stirring, and the O2 concentration of solution was monitored by a portable dissolved oxygen meter in real time. Extracellular 1O2 Generation Measurement. DPBF solution (20 μL, 10 mM in DMSO) was added to the sample solution (250 μg/mL, 2 mL) under irradiation (635 nm, 0.6 W/cm2) for 30 min. Finally, the absorbance change of DPBF at 420 nm was recorded at appointed time. Extracellular Degradation of CMSNs. Typically, 200 μg CMSNs were respectively added into 1 mL PBS solution (10 mM, pH=7.4 or pH=6.5) without or with GSH (10.0 mM) to investigate the influence of pH and GSH on CMSNs biodegradation. The testing solution was put into a shaker at 37 °C. At given time point, the concentration of released Cu and Mn were measured by ICPOES and the morphology change of CMSNs was observed by TEM.

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Extracellular Chemodynamic Activity of CMSNs. CMSNs (100 μL, 2 mg/mL), NaCl (200 μL, 50 mM) and different concentrations of GSH (0, 0.5, 1.0 and 10 mM) were added in 25 mM NaHCO3/5% CO2 buffer solution (total volume: 800 μL). Then the mixed solution was shaken for 15 min at 37 °C. After centrifugation, MB (100 μL, 100 μg/mL) and H2O2 (100 μL, 80 mM) were added to the supernatant followed by incubation at 37 °C for 30 min. Finally, the absorbance change of MB at 664 nm was recorded. Extracellular MRI Imaging Property. 100 μL mCMSNs ([Mn]: 6 mM) was added into 900 μL PBS solution (10 mM, pH=7.4 or pH=6.5) contaning 10 mM NaCl and 25 mM NaHCO3/5% CO2 without or with GSH (10.0 mM) and shaken at 37 °C for 1 h. After centrifugation, MRI images and the T1 relaxation time of diluted supernatant (Mn concentration: 0, 0.05, 0.1, 0.2, 0.4 and 0.6 mM) were measured by MRI system (BioSpec70/20USR, Bruker, Germany) at 7.0 T with a gradient echo sequence (TR = 299 ms and TE = 6.01 ms). Cell Culture. MCF-7, A549 and NHDF cells were incubated in DMEM medium containing 10% FBS and 1% antibiotics (penicillin−streptomycin, 10000 U/mL) at 37 °C under 5% CO2. Cellular Uptake of mCMSNs. MCF-7, A549 and NHDF cells were seeded in culture dishes respectively and incubated for 24 h. OPTI-MEM solution containing mCMSNs (50 μg/mL) was stained with green fluorescent probe of cell membrane (DiO) for 15 min and washed with PBS (10 mM, pH=7.4) for 21

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three times. Then, the cells were treated with pre-stained mCMSNs solution and incubated for 4 h. Finally, the cells were stained with Hochest 33342 (1 μL) for 15 min, washed with PBS (10 mM, pH=7.4) and observed by CLSM. For flow cytometry analysis, the cells treated with pre-stained mCMSNs were analyzed after collection. Intracellular Hypoxia Relief. The hypoxic environment was built by incubation adherent cells in hypoxia chamber with 1% O2, 5% CO2, and 94% N2 gas for 4 h. First, MCF-7 cells were seeded in culture dishes (1 × 105 cells/mL) and then divided into four groups with different treatments. (1) Normoxia group. The cells were incubated in normoxia. (2) Hypoxia group. The adherent cells were incubated in hypoxia chamber for 4 h. (3) Hypoxia + PBS group. The adherent cells were incubated in hypoxia chamber for 4 h and then treated with OPTIMEM medium containing 5% PBS (10 mM, pH=7.4) for 4 h in hypoxia chamber. (4) Hypoxia + mCMSNs group. The adherent cells were incubated in hypoxia chamber for 4 h and then treated with OPTI-MEM medium containing 5% mCMSNs (1 mg/mL in PBS) for 4 h in hypoxia chamber. Then, the cells were washed with PBS (10 mM, pH=7.4) for three times and stained with Hypoxia detection probe (0.5 μL, 1 mM) (Hypoxia Detection Kit, Enzo) for 30 min. After that, the cells were washed with PBS (10 mM, pH=7.4) for three times and observed by CLSM.

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Intracellular ROS Measurement. MCF-7 cells and NHDF cells were seeded in plates (1 × 105 cells per mL) respectively for 12 h. Then, the cells were treated with OPTI-MEM solution containing PBS (10 mM, pH=7.4) or mCMSNs (50 μg/mL) for 4 h. The MCF-7 cells pre-treated with L-BSO (100 μM) for 4 h and then treated with OPTI-MEM solution containing mCMSNs (50 μg/mL) for 4 h were also prepared. Then the cells were washed with PBS (10 mM, pH=7.4) and exposed to a 635 nm laser (0. 6 W/cm2) for 10 min. Finally, the cells were incubated with 1 μL DCFH-DA (10 mM) for 15 min, washed with PBS (10 mM, pH=7.4) and observed by CLSM. The intracellular 1O2 was detected by Singlet Oxygen Sensor Green (SOSG) reagent with the same steps. Intracellular GSH Measurement. The Intracellular GSH was measured by the GSH and GSSG Assay Kit (Beyotime). MCF-7 cells were seeded in 6-well culture dishes (105 cells/well) and treated with PBS (10 mM, pH=7.4), CMSNs (50 μg/mL) or mCMSNs (50 μg/mL). After incubating for 4 h, the cells were washed with PBS (10 mM, pH=7.4) for 3 times, exposed to 10 min irradiation (635 nm, 0.6 W/cm2) and further incubated for 12 h. Then, the cells were collected with centrifugation and the supernatant was discarded. The cell precipitates were re-suspended in protein remover M (10 mg/30 μL), subjected to 3 cycles of freezing-thawing, and then centrifugated at 1000 g for 10 min at 4 °C. The supernatant was reserved for GSH and GSSH assay according to the manufacturer’s protocol.

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Cytotoxicity Measurement. MCF-7 and NHDF cells were seeded in 96-well plates (104 cells per well) respectively and incubated for 12 h. Then, the cells were treated with OPTI-MEM solution containing CMSNs or mCMSNs at desired concentrations for 4 h. After replaced with fresh culture media, the cells were further incubated for 24 h. Finally, 10 μL CCK-8 solution was added to each well and incubated for another 4 h. The absorbance at 450 nm of cells in each wells were measured by microplate reader. Intracellular CDT/PDT Performance. MCF-7 cells and NHDF cells were seeded in plates (2 × 105 cells/mL) respectively for 12 h and then treated with OPTIMEM containing PBS, CMSNs (50 mg/mL) or mCMSNs (50 mg/mL). After 4 h, the cells were replaced with a fresh DMEM medium, exposed to 10 min irradiation (635 nm, 0.6 W/cm2) and further incubated for 12 h. For cell living/death evaluation, Calcein-AM (1 μL) and PI (1 μL) were added to the medium and incubated for 15 min. Then, the cells were washed with PBS (pH= 7.4, 10 mM) for three times and imaged by CLSM. For cell apoptosis/death evaluation, cells were co-stained with Annexin V-FITC (1 μL) and PI (1 μL) for 15 min, washed with PBS (pH= 7.4, 10 mM) for three times and analyzed by flow cytometer. Tumor Models. MCF-7 cancer-bearing female Balb/c mice (4 weeks) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and used under protocols approved by the Department of Laboratory Animal 24

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Science of Peking University Health Science Center. 2×106 MCF-7 cells in 200 μL of PBS (pH = 7.4, 10 mM) were subcutaneously injected to the right axilla of each mouse to build the MCF-7 tumor model. When the tumor sizes reached about 50 mm3, the mice were randomly divided into five groups for in vivo experiments (5 mice in each group) and intravenously injected with different formulations every third day for four doses: 1. PBS (10 mM, pH=7.4, 200 μL); 2. 635 nm; 3. CMSNs (2 mg/mL, 200 μL); 4. mCMSNs (2 mg/mL, 200 μL); 5. mCMSNs (2 mg/mL, 200 μL) + 635 nm. For group 2 and 5, the tumors were irradiated with a 635 nm laser (0.6 W/cm2) for 15 min (1 min break after 3 min irradiation). The tumor size and body weight of each mouse were recorded every other day for 2 weeks. The tumor size was calculated as follows: V = width2 × length/2. After 14 days, the cancer tissues and main organs were collected from the sacrificed mice for histological analysis.

In Vivo Biodistribution. MCF-7 cancer-bearing mice were intravenously injected with CMSNs or mCMSNs (2 mg/mL, 200 μL). At indicated time points (1, 2, 4, 8, 12, and 24 h) after intravenous injection, 50 μL blood was extracted from the tail of each mouse and weighed every time. After intravenous injection for 24 h, the mice were sacrificed to collect the liver, spleen, kidney, heart, lung and tumor. Then the Mn amount of the collected blood and tissue samples were measured by ICP-OES.

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In Vivo MRI. The tumor-bearing mice were intravenously injected with mCMSNs (2 mg/mL, 200 μL). T1-weighted MRI images were obtained after intravenous injection at appointed time by an animal MRI scanner (BioSpec70/20USR, Bruker, Germany) at 7.0 T with a gradient echo sequence (TR = 300.0 ms, TE = 5.48 ms).

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: TEM images, XRD and XPS spectra, DLS results, zeta potential, depletion of DPBF, MB degradation, CLSM images of cell uptake of mCMSNs in A549 cells, CCK-8 assay, flow cytometry results, and H&E-stained images of major organs (PDF)

AUTHOR INFORMATION Corresponding Authors *E-mail (H. Dong): [email protected]. *E-mail (X. Zhang): [email protected]. ORCID Haifeng Dong: 0000-0002-6907-6578 Xueji Zhang: 0000-0002-0035-3821

ACKNOWLEDGEMENTS

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The work was supported by National Natural Science Foundation of China (21874008, 21475008), Special Foundation for State Major Research Program of China (Grant Nos. 2016YFC0106602, 2016YFC0106601); the Open Research Fund Program of Beijing Key Lab of Plant Resource Research and Development, Beijing Technology and Business University (PRRD-2016-YB2); the Fundamental Research Funds for the Central Universities (Grant No. FRFBD-17-016A) and Beijing Municipal Science and Technology Commission (Grant No. z131102002813058).

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Scheme 1. (A) Fabration procedure for mCMSNs and (B, C) the scheme of therapeutic mechanism of mCMSNs for PDT under laser, and GSH-triggered CDT and MRI.

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Figure 1. (A) TEM image, (B) SEM image, (C) nitrogen absorption−desorption isotherm with corresponding pore-size distribution of CMSNs. The highresolution (D) Cu 2p and (E) Mn 2p XPS spectra of CMSNs. (F) The valence state fraction of Mn in the CMSNs. (G) TEM image of mCMSNs. (H) TEM image of mCMSNs stained with uranyl acetate. (I) DLS characterization and (J) surface zeta potential of CMSNs and mCMSNs. (K) SDS-PAGE protein analysis: L1: marker, L2: MCF-7 cell membrane, L3: CMSNs, L4: mCMSNs.

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Figure 2. (A) O2 generation of CMSNs (100 μg/mL) with H2O2 (3 mM). (B) Timedependent degradation of DPBF caused by 1O2 with or without irradiation. ([CMSNs] =250 μg/mL, [H2O2] =8 mM. Laser: 635 nm, 0.6 W/cm2, 30 min). (C) TEM images of CMSNs (200 μg/mL) after different treatments for various periods of time. Scale bar: 100 nm. Accumulated releasing (D) Mn and (E) Cu elements from CMSNs (200 μg/mL) with different treatments. (F) MB degradation by ∙OH generated by different concentration of GSH-treated CMSNs (200 μg/mL) plus H2O2 (8 mM). (G) ESR spectra of different reaction systems with DMPO as the spin trap. (H) In vitro MRI of CMSNs with different treatments and (I) the corresponding r1 value.

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Figure 3. (A) CLSM images of MCF-7 cells and NHDF cells after treatment with prestained mCMSNs (50 μg/mL) for 4 h and stained with Hochest 33342. Scale bar: 100 μm. (B) Flow cytometry analysis of MCF-7 cells and NHDF cells without any treatments or treated with mCMSNs (50 μg/mL) for 4 h and their corresponding MFI. CLSM images of (C) hypoxia level and (D) ROS production in MCF-7 cells and corresponding surface plot images, respectively. Scale bar: 100 μm. (E) Intracellular GSH/GSSH ratios of MCF-7 cells with different treatments. (F) Cell viability of MCF-7 cells and NHDF cells treated with different concentrations of mCMSNs by CCK-8 assay (**p < 0.01, two-tailed t test). (G) The effect of CMSNs or mCMSNs concentrations and 635 nm-laser irradiation (0.6 W cm−2, 10 min) on cell viability of MCF-7 cells by CCK-8 assay (*p < 0.05, **p < 0.01 and ***p < 0.001, two-tailed t test). (H) CLSM images of Calcein-AM- and propidium iodide (PI)-costained MCF-7 cells with different treatments. Scale bar: 200 μm.

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Figure 4. (A) Blood circulation and (B) biodistribution of CMSNs and mCMSNs over a span of 24 h after intravenous injection into MCF-7 tumor-bearing mice (**p < 0.01, two-tailed t test). (C) In vivo MRI of MCF-7 tumor-bearing mice before and after intravenous injection of mCMSNs at 0, 4 and 24 h. (D) DCFHstaining of tumor tissues from different groups at 24 h post-injection. Scale bar: 200 μm. (E) Representative photos of the mice with different treatments for 1 day, 7 days and 14 days, respectively, and representative tumor tissues collected from different groups at 14 days. (F) The relative cancer volume and (G) body weight changes of MCF-7 tumor-bearing mice after various treatments in 14 days (**p < 0.01 and ***p < 0.001, two-tailed t test). (H) H&E-stained slices of tumor tissues from different groups collected at 14 days. Scale bar: 100 μm. 43

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