In situ fabrication of MS@MnO2 hybrid as nanozymes for enhancing ROS-mediated breast cancer therapy

Xufeng Zhu a, Yanan Liu ab, Guanglong Yuan a, Xian Guo a, Jieqiong Cen a, Youcong Gong a, Jie Liu *a and Ye Gang *ac
aDepartment of Chemistry, College of Chemistry and Materials Science, Jinan University, Guangzhou 510632, People's Republic of China. E-mail: tliuliu@jnu.edu.cn
bCollege of Life Sciences, Shenzhen University, Shenzhen, Guangdong 518060, China
cDepartment of Gastroenterology, The First Affiliated Hospital of Jinan University, Guangzhou 510632, China

Received 22nd May 2020 , Accepted 25th September 2020

First published on 16th October 2020


Abstract

The reactive oxygen species (ROS)-mediated anti-cancer therapy that shows the advantages of tumor specificity, high curative effect, and less toxic side-effects has powerful potential for cancer treatment. However, hypoxia in the tumor microenvironment (TME) and low penetrability of photosensitizers further limit their clinical application. Here, we present a composite core–shell-structured nanozyme (MS-ICG@MnO2@PEG) that consists of a mesoporous silica nanoparticle (MS) core and a MnO2 shell loaded with the photosensitizer indocyanine green (ICG) and then coated with PEG as the photodynamic/chemodynamic therapeutic agent for the ROS-mediated cancer treatment. On the one hand, MS-ICG@MnO2@PEG catalyzes H2O2 to produce O2 for enhanced photodynamic therapy (PDT), and on the other hand, it consumes GSH to trigger a Fenton-like reaction that generates *OH, thus enhancing the chemodynamic therapy (CDT). At the cellular level, MS-ICG@MnO2@PEG nanozymes exhibit good biocompatibility and induce the production of ROS in 4T1 tumor cells. It disrupts the redox balance in tumor cells affecting the mitochondrial function, and specifically kills the tumor cells. In vivo, the MS-ICG@MnO2@PEG nanozymes selectively accumulate at tumor sites and inhibit tumor growth and metastasis in 4T1 tumor-bearing mice. Accordingly, this study shows that the core–shell nanozymes can serve as an effective platform for the ROS-mediated breast cancer treatment by enhancing the combination of PDT and CDT.


1. Introduction

ROS (including 1O2, *OH, and ˙O2)-mediated anti-cancer therapy has demonstrated strong potential in cancer treatment due to the advantages of tumor specificity, high curative effect, and less toxic side effects.1–3 Photodynamic therapy (PDT), as a clinical treatment method, adopts a certain wavelength of UV-vis/near-infrared light in combination with photosensitizers and oxygen to generate cytotoxic 1O2 to induce tumor cell apoptosis.4,5 Nevertheless, the hypoxic state of TME and the low penetration of photosensitizers further limit its clinical application.6,7 Therefore, there is an urgent need to conquer these disadvantages in order to achieve an effective therapeutic effect.8–10 In recent years, chemodynamic therapy (CDT) has attracted much attention due to its high efficiency and minimal side effects. The CDT converts endogenous H2O2 (100 μM ∼ 1 mM) into *OH through Fenton like or Fenton reaction, which is one of the most toxic ROS.11,12 However, simply regulating the ROS levels may not be sufficient to kill tumor cells with antioxidant capacity. Compared with normal cells, tumor cells are more dependent on the antioxidant system, and glutathione (GSH) is the most abundant and important non-enzymatic ROS scavenger in living cells.13–15 Notably, GSH level in cancer cells is approximately 4-fold than that in normal cells, and is highly susceptible to further oxidation induced by the exogenous ROS or compounds that inhibit the antioxidant system.16,17 Accordingly, the regulation of ROS levels in TME in combination with blocking the redox balance in tumor cells may be an effective way to treat breast cancer.

Various nanozymes that respond to TME and effectively regulate ROS levels have attracted widespread attention.18,19 Nanozymes are man-made enzymes based on nanomaterials, which hold the advantages of easy synthesis, low cost, and good catalytic stability.20 Nanozymes, such as platinum,21 Fe3O,22 CeO2,23 and MnO2,24–26 have been constructed and developed, which enhance the treatment of cancer and alleviate tumor hypoxia by catalyzing hydrogen peroxide to generate *OH or O2. The MnO2 nanozymes react with glutathione (GSH) to generate Mn2+, and then, Mn2+ catalyzes H2O2 to produce highly toxic OH in TME. For example, Chen's group developed the MnO2-based enhanced CDT nano-agents that exhibited Fenton-like Mn2+ transfer and GSH depletion characteristics.27 Furthermore, these MnO2 nanozymes could trigger the decomposition of H2O2 in TME into H2O and O2 to alleviate tumor hypoxia. The catalytic mechanism of the MnO2 nanozyme cascade could significantly enhance oxygen-dependent cancer therapies, such as radiation therapy and photodynamic therapy (PDT) (Scheme 1).


image file: d0nr03931d-s1.tif
Scheme 1 (a) Schematic representation of the synthesis of the MS-ICG@MnO2@PEG nanozyme, and (b) the process of enhancing PDT/CDT by a catalytic reaction and a Fenton-like reaction in vivo, achieving an increase in reactive oxygen species levels in tumor cells, thus inducing tumor cell apoptosis.

However, the long-term in vivo toxicity of nanozymes is still a major challenge that limits their clinical application.28,29 Therefore, it is important to rationally optimize the structure of synthetic nanozymes. Studies have found that the decomposition of MnO2 nanozymes into non-toxic and water-soluble Mn2+ can aid in quick excretion via the kidneys, causing less toxic- and side-effects in the body.30 In addition, a large number of research works have shown that MS have excellent biocompatibility and biodegradability, as well as high load capacity, and thus are widely used as drug/gene delivery systems.31–33 Moreover, the mesoporous structure does not prevent the entry and exit of substances (such as catalytic substrates and catalytic products), which may further improve the catalytic effectiveness. For example, Liu's group constructed a mesoporous MnO2 nanomaterial to catalyze endogenous H2O2 to overcome tumor hypoxia and enhance PDT in a multi-modal system for highly effective cancer treatment.34 Importantly, nanozyme-coated mesoporous materials are suitable for implementing multiple response mechanisms in the tumor microenvironment, thereby releasing chemotherapeutic drugs or other agents and fully exerting the catalytic activity.35,36

Here, we present a composite core–shell-structured nanozyme (MS-ICG@MnO2@PEG) that consists of mesoporous silica (MS) nanocore and a MnO2 shell loaded with the photosensitizer indocyanine green (ICG) and then coated with PEG coating as the photodynamic/chemodynamic therapeutic agent for ROS-mediated cancer treatment. On the one hand, the MnO2 shell can catalyze H2O2 to produce oxygen, which relieves tumor hypoxia and enhances ICG-mediated PDT. On the other hand, the MnO2 shell produces Mn2+ when glutathione is consumed, and further, Mn2+ generates ˙OH through a Fenton-like reaction, which destroys tumor cells. In addition, the consumption of GSH weakens the clearance of *OH and 1O2, thus synergistically enhancing the effect of PDT/CDT. This study shows that MSiO2-ICG@MnO2@PEG can be used as an effective platform for ROS-mediated breast cancer treatment by simultaneously enhancing the efficacy of the combination of PDT and CDT.

2. Experimental section

Cetyltrimethylammonium bromide (CTAB) was purchased from Aladdin Reagent (Shanghai) Co., Ltd. Indocyanine green (ICG), ethyl orthosilicate (TEOS), 2,2,6,6-tetramethyl-4-piperidone (TEMP), and 5,5-dimethyl-1-pyrrole porphyrin-N-oxide (DMPO) were obtained from Sigma. Polyacrylic acid (PAA), poly (allylamine hydrochloric acid) (PAH), 1,3-diphenylisobenzofuran (DPBF), methylene blue (MB), and reduced glutathione were bought from McLean. In addition, active oxygen detection kits, dimethyl sulfoxide (DMSO), and Aladdin Live/Dead Cell Staining Kit (Calcein-AM/PI) were acquired from Biyuntian. Unless specifically mentioned, all reagents and solvents were purchased commercially and used without further purification, and Ultrapure Milli-Q water (18.2 MW) was used in all experiments.

2.1 Synthesis of MSiO2

0.4 g of CTAB was added to a round-bottomed flask. To this, 200 mL of deionized water and a 2 mol L−1 NaOH solution were added and then stirred evenly to 80 °C. At the same time, 2 mL of TEOS was added and stirred for 2 h at a constant temperature. The white sample obtained after centrifugation was dispersed in 80 mL of ethanol. To this, 1.6 mL of concentrated hydrochloric acid was added, and the mixture was refluxed at 50 °C for 24 h. Subsequently, the product obtained after centrifugation was washed with ethanol. After washing the resulting MS twice with deionized water, ICG at a concentration of 20 μg mL−1 was added and stirred for 6 h in the dark. The sample was separated after centrifugation and unloaded ICG was washed with deionized water.

2.2 Synthesis of MS-ICG@MnO2 @PEG nanozymes

10 mg mL−1 of KMnO4 was dispersed in MS-ICG (v/v 1[thin space (1/6-em)]:[thin space (1/6-em)]5), and low-power ultrasound was used for 20 min to obtain MS-ICG@MnO2. The obtained MS-ICG@MnO2 at a concentration of 4 mg mL−1 was added to a 5 mg mL−1 PAH solution in the ratio of 1[thin space (1/6-em)]:[thin space (1/6-em)]2, stirred for 30 minutes and washed with deionized water three times. MS-ICG@MnO2 surface-modified with PAH was dispersed in a 5 mg mL−1 PAA solution, stirred for 30 minutes and washed three times with deionized water. Finally, 15 mg of EDC and 50 mg of PEG-NH2 were stirred for 12 h, then added to MS-ICG@MnO2 modified with PAA and PAH and washed three times with deionized water to obtain the MS-ICG@MnO2@PEG nanozyme.

2.3 Characterization

The as-prepared nanozymes were characterized by using various methods. The size distribution and zeta potential of the nanozymes were measured by dynamic light scattering (DLS) using a ZEN3600 Zetasizer Nano-ZS (Malvern Instruments Ltd, USA). The nanomaterial solution was dispersed onto a holed carbon film on copper grids to obtain the micrographs on a Hitachi H-7650 TEM (Hitachi Ltd, Japan) operating at an accelerating voltage of 80 kV. The SEM-EDX analysis performed on an EX-250 system (Japan H-field Co., Ltd) was used to check the elemental composition of MS-ICG@MnO2@PEG. The morphology and roughness of the nanozyme were identified by bio-atomic force microscopy (JPK Instruments, Germany). The crystalline nature of the nanozyme was analyzed using a Gemini S Ultra (Oxford Diffraction Ltd, UK) with copper K(alpha) radiation.

2.4 Evaluation of the ROS properties of the MS-ICG@MnO2@PEG nanozyme

MS-ICG@MnO2@PEG (25 mM) was mixed with different concentrations of GSH, and the amount of Mn2+ produced was measured. The relative content of ICG released was detected at different time periods. MS-ICG@MnO2@PEG was reacted with 100 μM H2O2 at different pH conditions for 6 min, and the O2 content was measured under constant temperature and humidity.

2.5 Detection of 1O2

The MS-ICG@MnO2@PEG nanozyme was mixed with 0.0002 M DPBF. After adding H2O2 at a final concentration of 100 μM for 3 minutes, the solution was irradiated using an 808 nm near-infrared light source at 0.4 W cm−2 for 9 min, and the fluorescence spectrum of DPBF was detected using a fluorescence spectrophotometer. For the measurement of 1O2, 40 μL of 2.0 M TEMP was mixed with an equal volume of 10 μg mL−1 MS-ICG@MnO2@PEG and H2O2 to achieve a final concentration of 100 μM. After 3 min of reaction, the mixture was irradiated with an 808 nm near-infrared light source at a power density of 0.4 W cm−2.

2.6 Detection of the content of *OH

For the detection of *OH, the MS-ICG@MnO2@PEG nanozyme was reacted with GSH at different concentrations for 15 min and centrifuged. 5 μg mL−1 MB and 100 μM H2O2 were added to the supernatant. After the reaction was complete, a UV-vis spectrophotometer was used for detection. For the measurement of *OH, the MS-ICG@MnO2@PEG nanozyme was reacted with 4 M GSH for 15 min and centrifuged. 10 μL of the supernatant was mixed with 40 μL of 2.0 M DMPO aqueous solution and 10 μL of 100 μM H2O2 after 10 min.

2.7 Detection of intracellular ROS level

4T1 cells were seeded on a confocal dish. Serum-free medium containing 12.5 μg mL−1 of different drugs was used instead of a complete medium and incubated for 24 h. For the PDT treatment group, incubation was performed for 6 h with an 808 nm near-infrared light source at a power density of 0.4 W cm−2 for 9 min. Finally, all the drug treatment groups were tested for reactive oxygen levels in the cells using a reactive oxygen detection kit according to the method provided by the manufacturer.

2.8 Detection of GSH level

The GSH level was monitored in 4T1 cells after incubation with different nano-agents for 24 h. GSH depletion was measured using Ellman's reagent.

2.9 Inhibitory effect of the MS-ICG@MnO2@PEG nanozyme on 4T1 cells

4T1 cells were inoculated on a confocal plate with the drugs from different experimental groups for 24 h. The cells were then treated according to the experimental method provided by the kit manufacturer and stained for live/dead cells and examined by CLSM (LSM 880, Carl Zeiss).

2.10 Construction and treatment of 4T1 tumor-bearing mouse model

All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Jinan University and approved by the Animal Ethics Committee of Jinan University. 4-Week-old female BALB/c nude mice purchased from the Guangdong Medical Experimental Animal Center to construct a 4T1 tumor-bearing mouse model after one week of observation. The mice were randomly divided into two batches: subcutaneous tumors and lymphatic metastases. Each batch was randomly divided into 5 groups: saline treatment group, ICG + NIR treatment group, MS-ICG@PEG treatment group, MS@MnO2@PEG treatment group, and MS-ICG@MnO2@PEG nanozyme + NIR treatment group. 200 μL of 4T1 cells were subcutaneously inoculated into the right armpit of each nude mouse (the number of cells was about 5 × 106). White spots appeared at the inoculation site two days later, and small solid tumors appeared one week later, indicating that a mouse tumor model was established. When the tumor volume grew to about 60 mm3, drug treatment was started according to different groups.

2.11 Metabolic distribution of MS-ICG@MnO2@PEG nanozyme in 4T1 tumor-bearing mice

After ICG and the MS-ICG@MnO2@PEG nanozyme were injected through the tail vein, photos were taken using a live small animal fluorescence imaging system at 1 h, 6 h, 12 h, 24 h, and 48 h, respectively. Under the same conditions, ICG and the MS-ICG@MnO2@PEG nanozyme injected through the tail vein at a dose of 20 μL 5 mg kg−1 were metabolized by the 4T1 tumor-bearing mice for 12 h. Then the mice were euthanized, and the heart, liver, spleen, lung and kidney were dissected out and imaged using a fluorescence imaging system to observe the metabolism of the drug in the internal organs.

2.12 Analysis of in vivo treatment effects

Different drugs were injected into the tail vein at a dose of 40 μL 5 mg kg−1. For the PDT treatment group, 9 minutes after the tail vein injection, the tumor site of the mice was irradiated with a near-infrared light source at a power density of 0.4 W cm−2 for 12 min. All drug treatment groups were injected every three days for a treatment cycle of 15 days. For lymphatic tumor metastasis mice, the treatment cycle was 30 days. During the treatment period, the tumor volume was measured using Vernier calipers every three days, and changes in the bodyweight of nude mice were recorded. The tumor volume was calculated as V = (length × width2)/2. After treatment, the nude mice were euthanized. The intact subcutaneous tumor was peeled off, weighed and photographed. The tumor tissue, heart, liver, spleen, lung and kidney of the nude mice were removed, washed three times with physiological saline, fixed with 10% paraformaldehyde and subjected to histopathological analysis. Tumor sections were prepared and stained with the TUNEL kit according to the method provided by the manufacturer. The HE and Ki67 immunohistochemical staining samples and CD31 and HIF-α immunofluorescence staining sections were observed under a fluorescence microscope.

2.13 Statistical analysis

Statistical analysis was conducted using Origin Pro 8.5 and GraphPad Prism 5.0 software. All data are presented as mean ± standard deviation (SD). Statistical analysis was performed using the Student t-test or one-way analysis of variance (ANOVA). Significant differences were considered as *p < 0.05, **p < 0.01, and ***p < 0.001.

3. Results and discussion

3.1 Preparation and characterization of the MS-ICG@MnO2@PEG nanozyme

We constructed an MS-ICG@MnO2@PEG nanozyme that contained a MnO2 shell wrapped around a mesoporous silica nanocore (MS)39(Fig. 1a). As shown in Fig. 1b, the mesoporous silica particles had a spherical morphology with a clear pore size distribution on the surface. The nanoparticle was ∼54 nm in size and showed good dispersibility. The photosensitizer ICG was loaded in MS by physical adsorption. Transmission electron microscope (TEM) images showed that loading ICG did not change the morphology and size of MS (Fig. 1c). The loading efficiency of ICG was 25.6%, as determined by UV spectroscopy (Fig. S5). In order to obtain MS-ICG-MnO2, MnO2 was grown on the MS surface by in situ crystallization. TEM imaging showed that there was a shell made of MnO2 on the surface of MS, and the morphology changed significantly (Fig. 1d). On testing the hydrated particle size of the MS-related nano-formulations, the results showed that the size of MS-ICG-MnO2 was ∼70 nm, which proved that the MnO2 shell was successfully coated on MS (Fig. S2). Studies have shown that the core–shell structure can improve catalytic activity dramatically.9,23,24 The elemental mapping images (Fig. 1e) of MS-ICG-MnO2 displayed the presence of Si, Mn, O, and C elements clearly. The Si element (green) had the highest proportion, and its distribution was relatively concentrated. The Mn (red) and O (yellow) elements were distributed on the outer surface and throughout the entire nanozyme, which was in line with our prediction. The C element (blue) may be from the copper net. Energy-dispersive spectroscopy (EDS) line scan element analysis was further performed (Fig. S3). X-ray photoelectron spectroscopy (XPS) was used to investigate the composition and valence states of MS-ICG@MnO2@PEG (Fig. 1f). The peaks with binding energies at 654.2 and 642.4 eV belonged to Mn(IV) 2p1/2 and Mn(IV) 2p3/2, respectively. These results revealed the successful formation of the MnO2 shell. The UV-vis spectrum (Fig. 1g) showed that the characteristic absorption peak of ICG in MS-ICG was around 780 nm, indicating that ICG was successfully loaded on MS. Meanwhile, MS-ICG-MnO2 showed a new absorption band near 300–400 nm, which may be due to the plasmon band on the surface of MnO2, indicating that MnO2 was successfully coated on the surface of MS-ICG. The near-infrared spectrum (Fig. 1h) showed a C–O–C characteristic absorption peak of PEG at 1100 cm−1. In addition, the zeta potential data of the MS-functionalized products showed that the sequential modification of ICG, MnO2, and PEG caused the zeta potential of MS to change (Fig. S4). All results demonstrated the successful synthesis of the MS-ICG-MnO2@PEG nanozyme (Fig. S1).
image file: d0nr03931d-f1.tif
Fig. 1 Synthesis and characterization of the MS-ICG@MnO2@PEG nanozyme. (a) Synthetic process of the MS-ICG@MnO2@PEG nanozyme; (b) TEM image of MS, (c) MS-ICG, and (d) MS-ICG@MnO2; (e) elemental spectrum of MS-ICG@MnO2; (f) XPS spectra of MS-ICG, MS-ICG@MnO2 and MS-ICG@MnO2@PEG and (g) high-resolution Mn2p XPS spectra of MS-ICG@MnO2@PEG;UV-vis spectrum and (h) infrared spectra of the MS-ICG@MnO2@PEG nanozyme.

3.2 Study of MS-ICG-MnO2@PEG nanozyme-catalyzed ROS production in vitro

Glutathione, the main scavenger of reactive oxygen species, in cancer cells is four times more abundant than in normal cells.16 MnO2 can convert reduced glutathione into its oxidized form, degrade it to Mn2+, and catalyze H2O2 to produce cytotoxic *OH (Fig. 2a). Because of the glutathione consumption capacity of MnO2, the color of MS@MnO2@PEG turned lighter as the GSH concentration increased while using different concentrations of GSH and MS@MnO2@PEG, and Mn2+ produced in the process was also concentration-dependent. Liquid chromatography and mass spectrometry also showed that reduced GSH was converted to oxidized glutathione (GSSG) after MS@MnO2@PEG was added (Fig. S6a and b). The MnO2 shell disintegrated itself when GSH was consumed. With the release of MSG-loaded ICG, it was possible to achieve tumor environment-responsive release of drugs. As shown in Fig. 2c, the release rate of ICG was related to the concentration of GSH; that is, the release rate of ICG increased with an increase in GSH concentration. MnO2 is a known H2O2 catalyst, which can catalyze the production of oxygen in the presence of H2O2. This system used its oxygen generation ability to make up for photodynamic oxygen dependence (Fig. 2b). As shown in Fig. 2d, MS@MnO2@PEG could catalyze the production of oxygen depending on the pH value. Compared with the acidic environment, a neutral environment showed a stronger ability to generate oxygen, which may be because the acidic environment inhibits H2O2 decomposition and oxygen generation. After loading ICG, the system could improve the oxygen dependence of ICG and thus enhance PDT. As shown in Fig. 2e, MS-ICG-MnO2@PEG produced a higher concentration of 1O2. Compared with ICG alone, the MS-ICG-MnO2@PEG nanozyme was less affected by oxygen when it produced 1O2, and ICG could also produce oxygen under simulated hypoxic conditions in vitro, which may be due to the small amount of oxygen dissolved in water (Fig. 2f). The reaction of GSH with MnO2 was accompanied by the production of Mn2+, and Mn2+ catalyzed the formation of *OH from H2O2 through the Fenton-like reaction. As shown in Fig. 2g, the MS-ICG-MnO2@PEG nanozyme reacted with H2O2 to produce more OH after the GSH reaction. We used MB as the indicator of *OH to detect the production of OH in the system. As shown in Fig. 2h and i, the production of OH was dependent on GSH concentration, and an increase in GSH concentration caused more production of OH, which in turn increased the consumption of hydroxyl radicals by MB. Compared with Mn2+ alone, the MS-ICG-MnO2@PEG nanozyme had a stronger ability to catalyze the production of and *OH, thereby degrading MB.
image file: d0nr03931d-f2.tif
Fig. 2 Study of MS-ICG-MnO2@PEG nanozyme-catalyzed ROS production in vitro. (a) Mechanism of in vitro ROS production by the MS-ICG-MnO2@PEG nanozyme; (b) the release of Mn2+ accompanying the reaction of MS@MnO2@PEG with different concentrations of GSH; (c) ICG release rate after the MS-ICG@MnO2@PEG nanozyme reacts with different concentrations of GSH; (d) oxygen release rate of MS@MnO2@PEG at different pH; (e) the ability of ICG and MS-ICG-MnO2@PEG nanozyme to react with H2O2 and generate 1O2 determined under the radiation of NIR light at 808 nm; (f) under hypoxic and normoxic conditions, the ability of the MS-ICG-MnO2@PEG nanozyme and ICG to react with H2O2 and generate 1O2; (g) after reacting with GSH, the ability of the MS-ICG-MnO2@PEG nanozyme to react with H2O2 and generate ˙OH; (h) the effect of MS@MnO2@PEG treated with different concentrations of GSH on the degradation of MB by catalyzing H2O2, control group: MB alone; (i) comparison of the catalytic ability of MS@MnO2@PEG and Mn2+ toward H2O2 in the presence of 10 mM GSH.

3.3 In vitro antitumor effect and absorption of the MS-ICG@MnO2@PEG nanozyme

The effective uptake of nanozymes by tumor cells directly affects the outcome. Laser confocal microscopy and flow cytometry were used to study the uptake of MS-ICG-MnO2@PEG nanozyme by 4T1 breast cancer cells. As shown in Fig. 3a, laser confocal imaging data showed that compared with ICG, the uptake of the MS-ICG-MnO2@PEG nanozyme by 4T1 cells was better (Fig. 3b–d). This may be due to the rough surface of MS-ICG-MnO2@PEG.38 On comparing the uptake of MS-ICG-MnO2@PEG nanozyme by 4T1 at different temperatures, we found that compared to 4 °C, the uptake efficiency of the MS-ICG-MnO2@PEG nanozyme at 37 °C was more than double. The cell uptake pathway of MS-ICG-MnO2@PEG nanozyme may be energy-dependent endocytosis.37 The MS-ICG-MnO2@PEG nanozyme showed the ability to release drugs in response to TME and was able to use the tumor microenvironment to specifically suppress tumor cells. This inspired us to further explore its effects and mechanisms at the cellular level. We found that the MS-ICG-MnO2@PEG nanozyme did not affect the proliferation of SHY5Y cells and RAW 264.7 macrophages (Fig. S7a and b). This shows that the nanozyme had good biocompatibility. In order to further investigate the enhanced CDT/PDT therapeutic effect, we used 4T1 cancer cells as a model to examine the anticancer effect of the MS-ICG-MnO2@PEG nanozyme under normoxic and hypoxic environments to observe the contrast. Upon 808 nm laser irradiation, MS-ICG-MnO2@PEG nanozyme treatment showed greatly enhanced cytotoxicity than both MS@MnO2@PEG and MS-ICG-MnO2@PEG in the normoxic environment (Fig. 3e), which was consistent with the calcein-AM and propidium iodide (PI) co-staining results that most of the cancer cells were killed when treated with MS-ICG-MnO2@PEG nanozymes plus irradiation (Fig. 5a). Notably, the hypoxic condition had an obvious influence on the cell-killing ability of MS@MnO2@PEG and MS-ICG-MnO2@PEG, whereas that of the MS-ICG-MnO2@PEG nanozyme-treated group remained almost the same both in normoxic and hypoxic conditions owing to the O2-self-supplying property of MnO2 that catalyzes endogenous hydrogen peroxide (Fig. 3f).
image file: d0nr03931d-f3.tif
Fig. 3 In vitro antitumor effect and absorption of MS-ICG@MnO2@PEG nanozymes. (a), (b) and (c) uptake by 4T1 cells after incubation with ICG and the MS-ICG@MnO2@PEG nanozyme for 24 h. Scale bar = 20 μm. (d) The uptake of the MS-ICG@MnO2@PEG nanozyme by 4T1 cells under different temperatures. Cell viability after incubation with different concentrations of the MS-ICG@MnO2@PEG nanozyme and 4T1 cells under (e) normoxia and (f) hypoxia. The data represent the mean of n (n = 3) determinations, and the error bars represent the standard deviation of the mean.

image file: d0nr03931d-f4.tif
Fig. 4 Modulation of intracellular reactive oxygen species and GSH levels by the MS-ICG-MnO2@PEG nanozyme. (a) Reactive oxygen species levels and (b) quantitative statistics in cells of different treatment groups. (c) The relative content of glutathione in cells of different treatment groups.

image file: d0nr03931d-f5.tif
Fig. 5 Inhibitory effect of the MS-ICG-MnO2@PEG nanozyme on 4T1 cells and its mechanism. (a) After different treatments, live/death staining of 4T1 cells. (b) Flow cytometry analysis of the effect of different treatment groups on the apoptosis of 4T1 cells. (c) Flow cytometry analysis of the effects of different treatment groups on the mitochondrial membrane potential of 4T1 cells.

3.4 Regulation of reactive oxygen species in 4T1 cells

The MS-ICG-MnO2@PEG nanozyme could enhance PDT in vitro and enhance CDT by consuming glutathione. Therefore, the effect of the MS-ICG-MnO2@PEG nanozyme on the intracellular reactive oxygen species was further investigated. As shown in Fig. 4a and b, compared with PDT and CDT alone, the MS-ICG-MnO2@PEG nanozymes could enhance the PDT/CDT combination and significantly increase the level of reactive oxygen species in 4T1 cells. In in vitro experiments, MnO2 down-regulated GSH levels in the cells. The relative GSH content in the cells after different treatment showed that the MS-ICG@MnO2@PEG nanozyme could also down-regulate the GSH levels in 4T1 cells. PDT-treated cells showed a relative increase in GSH levels, which may be due to the intracellular redox stress caused by PDT. The MS-ICG-MnO2@PEG nanozyme showed a stronger GSH depletion effect than MS-MnO2@PEG, and it is possible that the MS-ICG-MnO2@PEG nanozyme caused disturbances in the redox cycle in 4T1 cells (Fig. 4c). These results indicate that the MS-ICG-MnO2@PEG nanozyme could enhance CDT/PDT by consuming GSH and up-regulate toxic ROS levels in tumor cells.

3.5 Apoptotic effect on 4T1 tumor cells and its mechanism

Excessive ROS in tumor cells can cause oxidative damage to lipids, proteins, and DNA, which in turn can induce tumor cell death.40 The MS-ICG-MnO2@PEG nanozyme could up-regulate ROS levels in tumor cells and disrupt the redox cycle to achieve an enhanced PDT/CDT treatment effect. As shown in Fig. 5a, live/dead cell staining showed that the MS-ICG-MnO2@PEG nanozyme had stronger cell lethality than CDT and PDT alone. The flow cytometry analysis further demonstrated that the MS-ICG-MnO2@PEG nanozyme could enhance PDT/CDT and significantly induce apoptosis in 4T1 cells (Fig. 5b). In addition, the mechanism by which the MS-ICG-MnO2@PEG nanozyme induced tumor cell death was studied. Flow cytometry was used to analyze the changes in the mitochondrial membrane potential of the cells in different treatment groups. Compared with PDT and CDT alone, the MS-ICG-MnO2@PEG nanozyme caused a significant depolarization effect on the mitochondrial membrane potential (Fig. 5c). These results indicated that the MS-ICG-MnO2@PEG nanozyme induced tumor cell apoptosis by affecting mitochondrial oxidative stress in tumor cells.

3.6 In vivo distribution

Based on the near-infrared imaging characteristics of ICG, 4T1 tumor-bearing mice were constructed to study the biodistribution of the nanozyme in vivo. Using a specific time point to detect the nanozyme in the body, it was intuitively found that compared with free ICG, the nanozyme selectively accumulate at the tumor site due to the EPR effect, and a strong signal appeared at the tumor site after 12 h of administration (Fig. 6a and b). ICG was substantially or completely metabolized at 12 h, while MS-ICG-MnO2@PEG exhibited longer intratumor retention time than free ICG. At 1 h and 6 h, the distribution of MS-ICG-MnO2@PEG in organs other than the tumor sites may be due to ICG leakage caused by the incomplete coating of the MnO2 shell. After 24 h of systemic metabolism, fluorescence imaging analysis of mouse internal organs showed that MS-ICG-MnO2@PEG specifically accumulated at the tumor sites and also showed slight distribution in other metabolic organs, such as the kidney, heart, and spleen (Fig. 6c and d).
image file: d0nr03931d-f6.tif
Fig. 6 The metabolic effects of the MS-ICG-MnO2@PEG nanozyme in vivo. (a) Fluorescence imaging and (b) the corresponding quantitative statistics of the MS-ICG-MnO2@PEG nanozyme and ICG administered by tail vein injection in 4T1 tumor-bearing mice at different times. (c) Fluorescence imaging and (d) corresponding quantitative analysis of the visceral distribution of the MS-ICG-MnO2@PEG nanozyme and ICG 12 h after tail vein injection in 4T1 tumor-bearing mice. The data represent the mean of n (n = 3) determinations and the error bars represent the standard deviation of the mean, and the differences between the mean values of each level group were statistically significant, as determined by one-way ANOVA (**p < 0.01, ***p < 0.001).

3.7 In vivo antitumor effect

The MS-ICG-MnO2@PEG nanozyme could induce 4T1 cell apoptosis in vitro. To analyse if it has the antitumor ability in vivo, we divided the 4T1 tumor-bearing model mice into 5 groups and treated them with PBS, ICG + NIR, MS-ICG@PEG, MS@MnO2@PEG, and MS-ICG-MnO2@PEG nanozymes + NIR. When the tumor volume increased to about 60 mm3, the tail vein injection of drugs was performed. Based on the results of fluorescence imaging, 12 h after the injection of the drug, near-infrared light was used for irradiation, and the injection was performed every three days up to 15 d (Fig. 7a). The results of the relative tumor volume during the treatment and the tumor weight after treatment showed that, compared with the CDT and PDT alone treatment groups, the MS-ICG-MnO2@PEG nanozyme showed an excellent antitumor effect in vivo. Compared with other treatment groups, the MS-ICG-MnO2@PEG nanozyme could significantly suppress the voluminous growth of tumors and produced the lightest weighing tumor after treatment (Fig. 7b and d). As shown in Fig. 7e, the picture of the solid tumor after treatment further illustrated the excellent anti-cancer effect of the MS-ICG-MnO2@PEG nanozyme. Based on the results of H&E staining and TUNEL staining of the tumor, it was found that the MS-ICG-MnO2@PEG nanozyme could significantly induce apoptosis in tumorous tissues, while a lesser extent of apoptosis was observed in the PDT and CDT alone groups (Fig. 7f). As shown in Fig. 7c, the weight change in the mice from each treatment group showed that the treatment method did not cause side effects on the mouse's health. These results indicate that the MS-ICG-MnO2@PEG nanozyme was more effective against tumors by enhancing PDT/CDT.
image file: d0nr03931d-f7.tif
Fig. 7 The therapeutic effect of the MS-ICG@MnO2@PEG nanozyme on 4T1 tumor-bearing model mice. (a) Schematic diagram of the treatment process of tumor-bearing model mice with the MS-ICG@MnO2@PEG nanozyme. (b) Statistics of relative tumor volume changes in mice from different treatment groups every three days. (c) The body weight changes in the mice from the different treatment groups during the course of treatment. (d) Tumor weights in mice from different treatment groups after treatment. (e) Photographs of the solid tumors subcutaneously stripped from mice in different treatment groups. (f) H&E and TUNEL staining of the tumor tissues of mice from different treatment groups, scale bar = 200 μm. (g) Ki-67, CD-31 and HIF-α staining of mouse orthotopic tumor tissues, and HE of liver and lung.

Since 4T1 cells can spontaneously metastasize to the lung and liver, the effect of the MS-ICG-MnO2@PEG nanozyme on the lymphatic metastasis of 4T1 tumors was studied using tissue sections. As shown in Fig. 7g, the HE sections of the liver and lungs of mice showed that the MS-ICG-MnO2@PEG nanozyme treatment alleviated the metastasis of 4T1 cells in mice compared with the CDT and PDT treatments alone. Through the immunohistochemistry and immunofluorescence sections of the mouse tumorous tissues, we found that the number of Ki-67-positive cells in the tumorous tissue of the MS-ICG-MnO2@PEG nanozyme treatment group was reduced relative to the PDT and CDT alone treatment groups, indicating that the MS-ICG-MnO2@PEG nanozyme could significantly inhibit tumorous cell proliferation. The immunofluorescence analysis of CD31 and HIF-α in tumor tissues showed that, compared with the other treatment groups, the MS-ICG-MnO2@PEG nanozyme treatment group had reduced vascularity and hypoxia-inducible factor expression. This shows that the MS-ICG-MnO2@PEG nanozyme could improve the hypoxic environment in tumors and inhibit blood vessel growth. It is known that tumor metastasis and blood vessel growth are related to the tumor microenvironment. The upregulation of ROS in tumors further affects tumor metastasis. Therefore, it is speculated that the MS-ICG-MnO2@PEG nanozyme might alleviate tumor metastasis in vivo by improving the tumor microenvironment and damaging and inhibiting vascular growth.41–43 The in vivo safety of a nanozyme is very important. The organs (heart, liver, spleen, lung, and kidney) of mice were evaluated for safety by H&E. As shown in Fig. S8, the organs of the mice related to the different treatment groups were fresh and free of edema, necrosis, and inflammation. It shows that within a certain dose range, the MS-ICG-MnO2@PEG nanozyme did not cause toxic side effects on other internal organs during metabolism in vivo. This could be because most likely MnO2 and MS in the nanozyme were easily degraded in the acidic environment of the tumor and eventually excreted through the kidney without accumulation in the body.

4. Conclusions

In summary, we successfully constructed a GSH/H2O2 dual-response core–shell MS@MnO2 nanozyme to enhance ROS-mediated cancer treatment. Studies show that the core–shell MS@MnO2 nanozyme does not only respond to GSH to release the loaded photosensitizer ICG, but also degrades to form Mn2+ when GSH is converted to GSSG, catalyzing H2O2 to produce cytotoxic OH. At the same time, MnO2 catalyzes endogenous H2O2 to achieve in situ oxygen supply and enhances ICG-mediated PDT. At the cellular level, the core–shell nanozyme shows good biocompatibility and induces the production of ROS in 4T1 tumor cells. It disrupts the redox balance in tumor cells, affects mitochondrial function, and specifically kills tumor cells. In vivo, the core–shell nanozyme selectively accumulates at the tumor sites and inhibits tumor growth and metastasis in 4T1 tumor-bearing mice. Therefore, this study shows that the core–shell nanozyme can serve as an effective platform for ROS-mediated breast cancer treatment by enhancing the efficacy of the combination of PDT and CDT.

Ethical statement

All the animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Jinan University and approved by the Animal Ethics Committee of Jinan University.

Conflicts of interest

The authors declare no competing financial interests.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (81803027, 21877051), and the Planned Item of Science and Technology of Guangdong Province (2016A020217011).

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Footnote

Electronic supplementary information (ESI) available. See DOI: 10.1039/d0nr03931d

This journal is © The Royal Society of Chemistry 2020