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An Ultrasmall SnFe2O4 Nanozyme with Endogenous

Oxygen Generation and Glutathione Depletion for
Synergistic Cancer Therapy
Lili Feng, Bin Liu, Rui Xie, Dongdong Wang, Cheng Qian, Weiqiang Zhou, Jiawei Liu,
Deblin Jana, Piaoping Yang,* and Yanli Zhao*

photodynamic therapy (PDT) and photo-

The tumor microenvironment (TME) with the characteristics of severe hypoxia, thermal therapy (PTT), has aroused wide
overexpressed glutathione (GSH), and high levels of hydrogen peroxide (H2O2) attention on account of its unique advan-
dramatically limits the antitumor efficiency by monotherapy. Herein, a novel tages, such as deeper tissue penetration,
TME-modulated nanozyme employing tin ferrite (SnFe2O4, abbreviated as SFO) is negligible tissue injury, high efficiency,
and specificity for cancer treatment.[1–15]
presented for simultaneous photothermal therapy (PTT), photodynamic therapy
Phototherapy also has its inevitable imper-
(PDT), and chemodynamic therapy (CDT). The as-fabricated SFO nanozyme fection in spite of great progress achieved
demonstrates both catalase-like and GSH peroxidase-like activities. In the TME, in the fields of PDT and PTT. For example,
the activation of H2O2 leads to the generation of hydroxyl radicals (•OH) in situ for the hypoxia tumor microenvironment
CDT and the consumption of GSH to relieve antioxidant capability of the tumors. (TME) deteriorates the PDT efficiency and
Meanwhile, the nanozyme can catalyze H2O2 to generate oxygen to meliorate the the heat shock response weakens the PTT
effect.[16–26] As an emerging therapeutic
tumor hypoxia, which is beneficial to achieve better PDT. Furthermore, the SFO strategy, the fundamental mechanism of
nanozyme irradiated with 808 nm laser displays a prominent phototherapeutic chemodynamic therapy (CDT) involves the
effect on account of the enhanced photothermal conversion efficiency (η = 42.3%) generation of highly toxic hydroxyl radicals
and highly toxic free radical production performance. This “all in one” nanozyme (•OH) from endogenous hydrogen per-
integrated with multiple treatment modalities, computed tomography, and oxide (H2O2) catalyzed by transition metal,
metal, or peroxidase-like catalysts through
magnetic resonance imaging properties, and persistent modulation of TME
in situ treatment.[27–34] If the Fenton or
exhibits excellent tumor theranostic performance. This strategy may provide a Fenton-like reactions can simultaneously
new dimension for the design of other TME-based anticancer strategies. produce oxygen (O2) along with •OH,
the generated O2 may also relieve TME
1. Introduction hypoxia and enhance the therapeutic efficacy of PDT simulta-
neously.[35–37] Therefore, the combination of phototherapy with
Along with the rapid development of nanomedicine, near- other treatment modalities would provide a solution to solve the
infrared (NIR) light-activated phototherapy, representatively as low anticancer efficacy issue of monotherapy.
It has been confirmed that the malignant solid tumors
Dr. L. Feng, Dr. D. Wang, Dr. C. Qian, Dr. W. Zhou, Dr. J. Liu, D. Jana, possess immunosuppressive microenvironment, which
Prof. Y. Zhao is associated with the special TME with features of severe
Division of Chemistry and Biological Chemistry hypoxia, increased H2O2 level, and overexpressed glutathione
School of Physical and Mathematical Sciences (GSH).[38–44] The unique TME promotes the proliferation and
Nanyang Technological University
21 Nanyang Link, Singapore 637371, Singapore metabolism of tumor cells, resulting in the difficulty to cure
E-mail: [email protected] cancer thoroughly through the monotherapy model. There-
Dr. B. Liu, Prof. P. Yang fore, the synergistic therapy accompanied with TME regula-
Key Laboratory of Superlight Materials and Surface Technology tion is crucial for the elimination of malignant tumor. Lately,
Ministry of Education catalytic reactions based on multivalent metal ions (such as
College of Material Sciences and Chemical Engineering
Harbin Engineering University
Fe2+/Fe3+, Cu+/Cu2+, and Mn2+/Mn4+) in nanozymes, which
Harbin 150001, P. R. China imitate the functions of natural enzymes, have displayed a con-
E-mail: [email protected] spicuous effect in accommodating TME and then improving
Dr. R. Xie the treatment outcome.[45–48] For instance, Zhang et al. syn-
Department of Digestive Internal Medicine thesized MnFe2O4@MOF (MOF = metal-organic framework)
Harbin Medical University Cancer Hospital nanostructure with both catalase-like and GSH peroxidase-like
Harbin 150081, P. R. China
activities, consecutively generating O2 and depleting overex-
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/adfm.202006216.
pressed GSH to modulate TME for achieving high antitumor
effect.[49] Lin and co-workers prepared hollow mesoporous
DOI: 10.1002/adfm.202006216 Cu2MoS4 loaded glucose oxidase, which could effectively

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relieve tumor hypoxia and antioxidant capacity of tumor, region.[51–54] It can therefore be assumed that SFO could conduct
showing improved antitumor efficacy combined with the as a potential NIR light-triggered phototherapy agent. Herein,
photo­therapy upon 808 nm laser irradiation.[50] Consequently, we for the first time synthesized a novel SFO nanozyme by a
the rational design of persistent TME-regulating catalytic simple strategy to achieve an “all in one” function (computed
nanozymes is critical for ameliorating immunosuppressive tomography (CT) and magnetic resonance (MR) imaging, and
TME. However, these popular designs mainly focus on the magnetic targeting synergistic CDT/PTT/PDT). In this struc-
integration of several components to achieve the multifunction ture (Figure 1), the existence of Sn2+/Sn4+ and Fe2+/Fe3+ redox
by sophisticated procedures, which greatly hinder their clinical couples enable SFO to show excellent •OH production capacity
applications. Therefore, the development of single-entity nano- by Fenton-like reaction, simultaneously eliminating overex-
particles through a facile process to realize “all in one” func- pressed GSH in TME and reducing the antioxidant ability of
tion is extremely expected. the tumor via GSH peroxidase-like activity. Importantly, the
Bimetallic oxides, such as tin ferrite (SnFe2O4, denoted as SFO nanozyme can interact with endogenous H2O2 to produce
SFO), have drawn wide attention in the photocatalytic field O2, which could relieve the hypoxia of TME. Upon 808 nm light
based on the inherent existence of multivalent metal ions irradiation, the SFO nanozyme demonstrates significant tumor-
(Sn2+/Sn4+ and Fe2+/Fe3+) and significant absorption in the NIR killing capacity by phototherapy on account of its significantly

Figure 1. a) Schematic diagram of injectable “all in one” SFO for TME/NIR dual-responsive and imaging-guided synergetic CDT/PTT/PDT. b) Schematic
illustration of the synergistic therapy mechanism for SFO. NE: nanozyme.

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enhanced photothermal conversion efficiency (PCE, ƞ = 42.3%) best-fitting curve of Sn 3d5/2 resulted in two separate peaks at
applicable for PTT and highly toxic •OH and superoxide anion 485.6 and 486.1 eV, which affirmed the coexistence of Sn2+ and
(•O2−) production capacity for PDT. A combined CT and MR Sn4+. Analogously, it was discovered that the Sn 3d3/2 signal can
imaging function was also realized for obtaining more accurate also be separated into two peaks having binding energy at 494.0
information and monitoring the treatment efficacy. Therefore, and 494.5 eV, which agreed with the binding energy of Sn2+ and
this “all in one” SFO nanozyme is highly potent for synergistic Sn4+, respectively. The ratio of Sn4+ and Sn2+ was estimated to be
therapy directed by multiple imaging modalities. approximately 0.92, and the presence of low valence Sn species
in SFO could be ascribed to the partial reduction of Sn4+ in the
reaction process. The existence of Fe2+/Fe3+ and Sn2+/Sn4+ redox
2. Results and Discussion couples presents a good prospect for Fenton-like, catalase-like,
and GSH peroxidase-like activities.
2.1. Nanostructure and Compositions of SFO Then, pS-PEG was adopted to encapsulate the surface of
SFO via non-covalent interactions for enhancing the solubility
To facilitate the clinical application of nanozymes, SFO was and biocompatibility. The stepwise surface modification pro-
synthesized by a simple hydrothermal method, and then cesses were confirmed by the Fourier transform infrared (FTIR)
functionalized by poly(styrene)-block-poly(ethylene glycol) spectroscopy (Figure S4, Supporting Information). In addition,
(pS-PEG) to enhance their hydrophilicity and biocompatibility the zeta potential of SFO changed from +3.59 to −5.24 mV after
(Figure 2a). As displayed by transmission electron microscopy the functionalization of pS-PEG (Figure S5a, Supporting Infor-
(TEM) images, the as-synthesized SFO demonstrated uniform mation). The modified SFO demonstrated excellent dispersity
dispersion with an average diameter of 9.3 nm (Figure 2c,d). and colloidal stability in various physiological solutions with
High-resolution TEM (HRTEM) indicated that SFO had a an average hydrodynamic size of about 13.5 nm (Figure S5b,
cube-shaped morphology (Figure 2e). Raman spectrum was fur- Supporting Information), and no apparent aggregation pheno­
ther provided to confirm the structure of SFO. The character- mena could be detected within 14 days of culture, identifying
istic peaks observed at 325, 482, and 672 cm−1 are corresponding that it could serve as a promising system for cancer treatment.
to the vibrations of SFO (Figure S1, Supporting Information). Moreover, the thermogravimetric analysis was introduced to
The crystalline spinel structure (Figure 2b) of SFO with a cubic analyze the modified SFO in the range of 30–800 °C, exhibiting
phase based on powder X-ray diffraction (PXRD) pattern was a loading amount of 4.35% (by wt.) for the pS-PEG component
presented (Figure 2j). More importantly, the lattice spacing of (Figure S6, Supporting Information).
0.258 nm is assigned to the (311) lattice plane of SFO (JCPDS
No. 11–0614). Such value is slightly larger than 0.252 nm of
Fe3O4 in the same direction, which is due to the substitution 2.2. In Vitro Synergistic Therapeutic Performance
of smaller Fe2+ by larger Sn2+ to fabricate the SFO lattice struc- of SFO Nanozyme
ture. In addition, the fast Fourier transform (FFT) of SFO was
achieved (inset in Figure 2e), which demonstrated four kinds The catalase-like capacity of SFO nanozyme to generate O2
of spots that can be assigned to the (220), (400), (222), and is crucial for the treatment of hypoxia tumor.[57,58] The homo­
(440) planes of SFO with good crystallization, in well consistent genous mixing of SFO nanozyme with H2O2 at lower initial
with the PXRD pattern and Raman spectrum. Furthermore, concentration (25 µM) under both pH values (7.4 and 6.0) indi-
the coexistence of Sn, Fe, and O characteristic signals was cated that O2 was effectively produced upon the SFO interac-
detected in the X-ray energy dispersive spectrum (EDS) of SFO, tion with H2O2, and a myriad of O2 bubbles can be observed
revealing that the ratio of Sn to Fe is approximately 1:2 within (Figure S7, Supporting Information). The concentration of O2
the nanostructure accuracy by quantitative analysis (Figure S2, continued to rise in the measured solution after the addition of
Supporting Information). The homogeneous distributions of SFO nanozyme and always remained at a high level for 20 min,
Sn, Fe, and O elements in SFO were also confirmed by the which was assigned to the presence of multivalent elements of
element mapping, indicating the successful synthesis of SFO Sn2+/Sn4+ and Fe2+/Fe3+ in SFO (Figure 3a). To further quan-
(Figure 2f–i). titatively assess the catalytic capacity, time-dependent H2O2
To further investigate the chemical state of SFO, the valence depletion assays were carried out by adding SFO nanozyme.
states of Sn, Fe, and O elements were carried out by X-ray photo­ As shown in Figure 3b, more than 50% of H2O2 was disinte-
electron spectroscopy (XPS). Figure S3, Supporting Informa- grated by SFO nanozyme with 12 min and almost completely
tion, shows the XPS survey spectrum of SFO, indicating the consumed within 3 h (Figure 3c and Figure S8, Supporting
presence of the Sn, Fe, and O elements simultaneously. On the Information). The above experimental results demonstrated
basis of XPS spectral analysis, Fe3+ and Fe2+ together with Sn4+ that SFO nanozyme had an excellent catalytic capacity. Fur-
and Sn2+ were detected in the structure of SFO.[55,56] Figure 2k thermore, its catalytic activity maintained constant after several
exhibits the high-resolution spectrum of Fe 2p, and the peak of cycles of H2O2 addition, confirming that SFO nanozyme pos-
Fe 2p3/2 at 709.7 eV belongs to the Fe2+ oxide. The oxidation state sessed significant catalytic persistence (Figure 3d).
of Fe3+ was manifested from the two peaks of Fe 2p3/2 and Fe The generation of •OH free radical via the Fenton reaction
2p1/2 located at the binding energy of 710.8 and 724.3 eV, respec- is the essential factor in CDT. 3,3’,5,5’-Tetramethyl benzidine
tively, with a separation value of about 13.5 eV between Fe 2p3/2 (TMB) was adopted to explore the •OH production capacity
and Fe 2p1/2. The high-resolution XPS spectrum of Sn 3d is pre- in a phosphate-buffered solution with the pH value of 6.0,
sented in Figure 2l, accompanied with the fitting curves. The where the characteristic absorption peak located at 653 nm is

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Figure 2. Preparation and characterization of SFO. a) Schematic illustration for the synthetic process of SFO. b) Crystal structure of SFO (gray repre-
sents Sn atom, yellow represents Fe atom, and red represents O atom). c,d) TEM images of as-synthesized SFO at different magnifications. e) High-
resolution TEM image of as-synthesized SFO. Inset is the FFT pattern of SFO. f–i) Scanning TEM image and elemental mapping of Sn, Fe, and O
of SFO. j) PXRD pattern of SFO and corresponding standard card. k,l) XPS high-resolution spectra for Fe 2p and Sn 3d characteristic peaks of SFO.

triggered by generated •OH. As presented in Figure 2e, the effect produced from SFO nanozyme can distinctly enhance the
•OH production gradually ascended upon increasing the level efficiency of •OH production. The ionization process could be
of H2O2, indicating that the Fenton reaction occurred effec- facilitated by the temperature increase, which is beneficial to
tively. When exposed to 808 nm laser, the absorption intensity enhance the Fenton reaction rate. Furthermore, continuous flu-
of the detection probe was prominently higher than that in the orescence attenuation of 1,3-diphenylisobenzofuran (DPBF) was
absence of laser illumination, verifying that the hyperthermia detected when the SFO solution was exposed to 808 nm laser

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Figure 3. a) O2 generation in H2O2 solution added with PBS or SFO under different pH values (7.4 and 6.0). b) Decomposition of H2O2 with PBS or SFO
versus reaction time under different pH conditions. c) Corresponding absorption spectra of H2O2-Ti(SO4)2 solution in the presence of SFO at different
reaction times. d) Repetitive catalytic ability of SFO with repetitive addition of H2O2. e) H2O2 concentration-dependent and heat enhanced oxidation of
TMB due to generated •OH by SFO irradiated upon 808 nm laser. f) Irradiation time-dependent oxidation of DPBF, indicating ROS generation by SFO
with 808 nm laser illumination. g) Schematic illustration for the Fenton reaction of SFO responsible for the generation of •OH and the PTT-enhanced
Fenton reaction. h) Proposed mechanism of •OH/•O2− generation by SFO under 808 nm laser irradiation. i) ESR spectra of •OH/•O2− trapped by DMPO
under different conditions. j) GSH depletion by SFO with different concentrations characterized by the absorbance of 5,5’-dithiobis-(2-nitrobenzoic
acid) (DTNB). k,l) Time-dependent GSH depletion by SFO. The inset in (k) is a photograph of the solutions at different reaction times.

due to the production of •O2−, while the absorption intensity of electron trap site to restrain the electron-hole recombination,
DPBF showed no significant change upon 808 nm laser irra- leading to the more effective production of ROS (•OH and
diation without SFO nanozyme, demonstrating that SFO could •O2−) when exposed to the laser irradiation. As a consequence,
serve as a promising photosensitizer for 808 nm laser mediated SFO could be utilized as an efficient photosensitizer to gen-
PDT (Figure 3f and Figure S9, Supporting Information). All erate ROS for effective killing of tumor cells when exposed to
these experimental results revealed that SFO nanozyme as an 808 nm laser.
“all in one” theranostic agent possessed a remarkable applica- GSH is an abundance endogenous antioxidant, and it is
tion prospect for cooperative CDT, PTT, and PDT. capable of maintaining the redox balance of cells to inhibit the
Inspired by the efficient chemodynamic effect and O2 pro- cell destruction triggered by ROS. Hence, we then studied the
duction capacity of SFO nanozyme, electron spin resonance GSH depletion capacity of SFO nanozyme. Upon increasing
(ESR) spectrum was applied to analyze the production of reac- the SFO concentration and incubation time, the GSH deple-
tive oxygen species (ROS) by utilizing 5,5-dimethyl-1-pyrroline tion was enhanced obviously, due to the existence of multivalent
N-oxide (DMPO) as the •OH trapping agent. The characteristic Sn2+/Sn4+ and Fe2+/Fe3+ redox couple (Figure 3j–l). The special
peak intensity of SFO (pH = 6.0, 25 µM H2O2) under 808 nm performance of SFO nanozyme could prominently improve the
laser irradiation exhibited an apparent increase as compared PDT and CDT efficacy via destroying cellular antioxidant defense
with that without laser illumination, conforming that SFO system. XPS analysis indicated that the ratio of Sn2+/Sn4+ was
could indeed serve as a chemodynamic agent for CDT and the 0.92 in the pristine SFO and converted to approximately 1.15 after
hyperthermia effect could effectively enhance the •OH pro- reaction with GSH, verifying that a fraction of Sn4+ in SFO was
duction (Figure 3i). The SFO plus laser irradiation group also reduced to Sn2+ by GSH (Figure S10 and Table S1, Supporting
exhibited a typical 1:2:2:1 characteristic signal. As proposed in Information). The consumption of GSH by SFO would be ben-
Figure 3g,h, the unique nanostructure could elevate the sepa- eficial to retain a relatively high level of ROS inside tumor cells
ration efficiency of electron-hole pair and SFO may act as an in the photo/chemo-dynamic therapeutic process.

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Figure 4. a) UV–vis absorption spectra of dispersed SFO aqueous suspension at varied concentrations. b) Mass extinction coefficient of SFO at
808 nm. Normalized absorbance intensity at λ = 808 nm measured by the characteristic length of the cell (A/L) at varied concentrations (7.18, 15.6,
31.3, 62.5, 125, and 250 ppm). c) Schematic illustration of NIR laser triggered hyperthermia effect. d) Infrared thermal images of SFO aqueous solutions
with different concentrations (0, 62.5, 125, and 250 ppm) under 808 nm laser irradiation. e) Temperature change curves of SFO aqueous solutions with
different concentrations upon 808 nm laser irradiation. f) Heating and cooling curves of SFO aqueous solutions illuminated under 808 nm laser, and
linear time data obtained from the cooling period. g) Thermal stability of SFO (four cycles of laser on/off) under 808 nm laser illumination. h) UV–vis
absorbance spectra of SFO before and after laser radiation for varied time intervals. i) Hydrodynamic size distribution changes of SFO in the process
of 14-day incubation.

2.3. Photothermal Performance of SFO Nanozyme photothermal performance was investigated through recording
the temperature variations of SFO with varied concentrations
SFO nanozyme was expected to be NIR light-responsive. irradiated upon 808 nm laser (0.5 W cm−2), as exhibited in
Due to its small bandgap energy (1.38 eV), SFO may possess Figure 4c. An apparent concentration-dependent hyperthermia
unique advantages in PTT and enhance the generation of effect of SFO was obtained. The temperature increased with the
ROS irradiated with 808 nm laser for improving the syner- increasing sample concentration, in which the temperature rap-
gistic anticancer efficacy (Figure S11, Supporting Information). idly increased by 36 °C at the SFO concentration of 250 ppm,
UV–vis–NIR absorption spectrum of SFO exhibited a con- while the temperature of the control group only ascended by
centration-dependent NIR absorption band in the wavelength 1.3 °C (Figure 4d,e). The PCE of SFO was estimated to be 42.3%
range of 700–900 nm (Figure 4a). On account of the significant when exposed to 808 nm laser, which was higher than some
absorption, the extinction coefficient of SFO was determined to reported nanoagents including Cu9S5 nanocrystals, SiO2/MoS2
be 5.1 L g−1 cm−1 at 808 nm, which is higher than those reported nanoparticles, Fe(III)@WS2 nanocapsules, and niobium car-
photothermal nanomaterials (Figure 4b).[59–61] Subsequently, the bide (MXene), as demonstrated in Figure 4f.[60,62–64] Eventually,

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the photothermal stability of SFO was demonstrated by the 2.4. In Vitro Cytotoxicity
heating and cooling cycle curves (Figure 4g). The photothermal
conversion performance of SFO nanozyme showed negligible Before investigating in vitro synergistic anticancer effect, the
changes after several cycles, confirming its excellent thermo- blood compatibility of the SFO nanozyme was evaluated. The
stability. The absorption spectra at varied post-irradiation times hemolysis results indicated negligible hemolytic effect (less
demonstrated no obvious differences, further suggesting that than 2%) from SFO even when the concentration was as high
the laser irradiation did not give any apparent influence on as 250 ppm in phosphate-buffered saline (PBS), displaying
the photothermal efficiency of SFO (Figure 4h). The hydrody- its excellent hemocompatibility (Figure S12, Supporting
namic size of SFO was approximately 15.3 nm, which basically Information). The in vitro synergistic treatment efficiency of
remained unchanged during the 14-day incubation process in SFO was then assessed. As confirmed by the standard MTT
various physiological solutions (Figure 4i). The excellent PCE, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
thermal stability, and deep tissue penetration of NIR laser mide) assay, SFO demonstrated no significant toxicity to dif-
enabled SFO to be a promising photothermal agent for photo- ferent kinds of cell lines even when the concentration was
therapy of malignant tumor induced by NIR laser. as high as 250 ppm (Figure 5a and Figure S13, Supporting

Figure 5. a) Relative cell viabilities of 4T1 cells after incubation with SFO at different concentrations. b) Relative cell viabilities of 4T1 cells after different
treatments by MTT assay. c) Flow cytometry data to show PI-positive dead cells after different treatments. d) Quantitative analysis of GSH levels for
the treated groups in (g). e) Confocal images of 4T1 cells treated with FITC-modified SFO for different times (1 and 3 h) and stained with DAPI to label
cell nucleus. f) Confocal images of 4T1 cells stained with Calcein AM (green, live cells) and PI (red, dead cells) after different treatments (containing
control, Laser, SFO, SFO/Vc + L, SFO + L, and SFO + M + L). g) Confocal images of 4T1 cells stained by O2 indicator, for example, (Ru(dpp)3)Cl2, after
different treatments. h) Confocal images of 4T1 cells stained with Thiol Tracker Violet (GSH detection reagent) after incubation with various concentra-
tions of SFO. i) Confocal images of 4T1 cells stained with DCFH-DA after various treatments.

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Information). The cell survival rate of 4T1 cells descended as SFO concentration, the intracellular GSH level decreased fur-
the SFO concentration increased, which could be ascribed ther. The experimental results indicate that SFO as an efficient
to a small quantity of •OH generation based on Fenton-like nanozyme possesses the capability to deplete GSH, beneficial to
reaction in tumor cells. Inspired by the hyperthermia effect PDT/CDT-induced cancer cell killing effect. The production of
of SFO upon 808 nm laser irradiation, the single PTT could intracellular ROS was further investigated via a chemical probe
be understood by introducing Vitamin C (Vc) as a ROS scav- 2’,7’-dichlorofluorescin diacetate (DCFH-DA), which could
enger. SFO-based PTT triggered relatively high cell mortality be oxidized by generated ROS with green fluorescence emis-
than single CDT due to insufficient H2O2 content in tumor sion. 4T1 cancer cells incubated with SFO exhibited weak green
cells. In comparison with the group of SFO exposed to fluorescence due to the generation of a small amount of •OH
808 nm laser, nearly all 4T1 cells were destroyed after being in tumor cells by Fenton-like reaction. The group of SFO/Vc
irradiated with 808 nm laser under the guidance of magnetic plus 808 nm laser irradiation showed almost no fluorescence.
field owing to the magnetic targeting synergistic CDT/PTT/ After the 4T1 cells were incubated with SFO plus 808 nm laser
PDT (Figure 5b). irradiation, the level of ROS generation in 4T1 cells apparently
Then, the cellular uptake capacity of SFO on 4T1 tumor cells was increased, which was attributed to the hyperthermia-enhanced
researched. The green fluorescence signal from fluorescein iso- CDT and PDT (Figure 5i). Significantly, 4T1 cells incubated
thiocyanate (FITC)-labeled SFO and the blue fluorescence signal with SFO in the presence of laser and magnetic field demon-
derived from 4’,6-diamidino-2-phenylindole (DAPI)-labeled cell strated the strongest green fluorescence, as a plenty of ROS was
nucleus demonstrated effective overlay, certifying that SFO could produced inside the cells (Figure S15, Supporting Information).
accumulate effectively in tumor cells via endocytosis (Figure 5e). These results indicate that SFO is able to improve hypoxia in
To further determine the in vitro tumor cell killing effect of SFO, the TME and plays an important role in acquiring high photo/
Calcein AM, and propidium iodide (PI) co-staining assay was per- chemo-dynamic treatment efficiency.
formed (Figure 5f). The 4T1 tumor cells irradiated with 808 nm
laser alone demonstrated no significant decrease in cell viability,
while SFO or SFO/Vc plus 808 nm laser irradiation exhibited a 2.5. Imaging Performance
portion of 4T1 cell death due to the single CDT or synergistic effect
of CDT and PTT. Significantly, almost all cancer cells were dead The guidance by bioimaging technique would be beneficial to
in the group of SFO plus 808 nm laser irradiation in the guidance achieve accurate laser irradiation concentrating on the tumor
of magnetic field based on the magnetic targeting synergistic site in the course of treatment, to avoid damaging the sur-
therapeutic effect, and thus strong red fluorescence from PI was rounding normal tissues. Hence, it is crucial to monitor the
observed. Strong red fluorescence was detected under both hypoxia time-dependent tumor accumulation of nanozyme to confirm
and normoxia conditions, indicating that the ROS generation effi- the optimal treatment time for laser irradiation. Primarily, the
ciency of SFO was unaffected by the hypoxia due to the production blood circulation half-life of SFO was calculated to be 2.52 h
of endogenous O2 (Figure S14, Supporting Information). The flow by the two-compartment model (Figure S16, Supporting Infor-
cytometry results also confirmed that a large number of cells were mation). The relatively long circulation time would profit the
effectively killed by SFO plus laser irradiation under the presence accumulation of nanozyme within the tumor region. To acquire
of magnetic field (Figure 5c). These results illustrate that SFO pos- above-mentioned objective, SFO was injected into tumor-
sesses low biotoxicity without laser irradiation, while having high bearing mice intravenously, and in vivo hyperthermia effect of
photo/chemo-dynamic toxicity against tumor cells with laser irra- SFO was monitored using an infrared thermal imager at spe-
diation and magnetic field. cific time points. As shown in Figure 6a,b, the temperature of
We also researched the intracellular O2 production for tumor region treated with SFO obviously increased by approxi-
improving the tumor hypoxia. [Ru(dpp)3]Cl2 as an intracellular mately 36.3 °C within 5 min upon exposure to 808 nm laser in
O2 level indicator was adopted to investigate the endogenous the presence of magnetic field, which not only destroyed the
O2 generation (Figure 5g). 4T1 cells incubated with SFO under malignant cells, but also facilitated the photothermal enhanced
hypoxic condition demonstrated weaker fluorescence as com- CDT. The result was also much better than that without mag-
pared with the control group under normoxic condition, veri- netic field. As a control, the temperature of tumor region with
fying the production of intracellular O2 from SFO (250 ppm) only 808 nm laser irradiation demonstrated a finite increase
via catalytic reaction. Upon increasing the SFO concentrations, under the same conditions. These results once again confirmed
even weaker red fluorescence signal was detected because cata- that SFO possesses superior photothermal effect and magnetic
lase-like SFO could interact with overexpressed H2O2 in cancer targeting capability during the bioimaging process, endowing it
cells to generate a certain amount of O2, suggesting that the with highly efficient and safe tumor ablation ability.
intracellular O2 production was concentration-dependent and Taking similar atomic numbers of Sn and iodine into con-
therefore the photo/chemo-dynamic therapeutic effect could sideration, we studied the in vitro and in vivo CT imaging capa-
be significantly enhanced. In order to verify the peroxidase-like bility of SFO. Hounsfield unit (HU) value increased linearly
capability of SFO to consume overexpressed GSH inside tumor as increasing the concentration of SFO, since the attenuation
cells, the Thiol Tracker Violet staining assay was performed coefficient of Sn (0.61 cm2 kg−1 at 140 keV) was similar to that
(Figure 5d,h). In comparison with the control group under nor- of iodine (Figure 6c).[65] For in vivo CT imaging capacity, SFO
moxic condition, 4T1 cells incubated with SFO under hypoxic was administrated into tumor-bearing mice intravenously.
condition demonstrated weaker green fluorescence on account As shown in Figure 6d,e, the CT images of mice after the
of the GSH consumption inside the cells. As increasing the SFO treatment demonstrated significant signal enhancement

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Figure 6. a) Time-dependent in vivo photothermal imaging of 4T1 tumor-bearing mice after intravenous administration with saline and SFO with or
without magnetic field upon 808 nm laser irradiation. b) Quantification of temperature changes from the tumor region based on the images in panel
(a). c) In vitro CT images of SFO aqueous solutions with different concentrations. The CT value of as-prepared samples is in accordance with the
concentrations of SFO. d) In vivo CT images of tumor-bearing mice: pre-injection and post-injection via intravenous manner. e) Corresponding cross-
sectional CT line profiles before and after injection of SFO, corresponding to (d). f) T2 relaxation rates and MR images of SFO aqueous solutions with
different concentrations. g) MR imaging of mice before and after 24 h intravenous injection with SFO (10 mg kg−1). h) Quantification of MR signal
intensity from the tumor sites. (***p < 0.001, **p < 0.01, or *p < 0.05). i) Biodistribution of SFO in mice at different injection days based on ICP-MS
measured Fe levels. j) Fe content in urine and feces at different days after SFO injection. k) Schematic illustration of the body’s rapid clearance of SFO.

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at the tumor site as compared with the case before the treat- illustration of the tumor treatment process. The mice admin-
ment. The HU value of the tumor enhanced apparently from istrated with saline or NIR laser irradiation alone displayed no
19.4 ± 2.3 HU to 348.1 ± 1.5 HU after the administration, apparent inhibitory effect on the tumor growth. The mice treated
indicating that SFO could be a potent contrast agent for CT with SFO alone or SFO/Vc plus NIR laser irradiation exhibited
imaging. Based on the inherent magnetic performance of SFO partial tumor growth suppression due to the CDT or synergistic
(Figure S17, Supporting Information), we investigated its poten- CDT and PTT effect. Excellent antitumor efficiency of the mice
tial capability as a T2-weighted MR imaging contrast agent. As treated with SFO upon NIR laser irradiation was observed, which
presented in Figure 6f, in vitro T2-weighted MR images of SFO should be attributed to the synergistic therapeutic effect. As
demonstrated an apparent darkening effect upon increasing the expected, significant tumor growth inhibition and elimination
sample concentration, and the transverse relaxivity (r2) value were achieved in mice treated with SFO plus NIR laser irradia-
was calculated to be 228.90 mM−1 s−1, which was significantly tion under the assistance of magnetic field (Figure 7b). Subse-
higher than that of commercial contrast agents, including quently, the toxicity of such nanozyme was also evaluated. No
Feridex (120 mM−1 s−1), Resovist (186 mM−1 s−1), and Combidex evident decline in the body weights of the mice in each group
(65 mM−1 s−1). Encouraged by the imaging potency of SFO in over the duration of treatments revealed negligible toxicity of
vitro, we further assessed the MR imaging effect in vivo by SFO (Figure 7c). After 14 days of treatment period, the mice were
using 4T1 tumor-bearing mice, as revealed in Figure 6g,h. In euthanized and the tumors from various treatment groups were
this case, SFO was administrated into mice intravenously and collected. The difference in the tumor mass was consistent with
imaged at 24 h post-injection. The MR imaging signal intensity the relative tumor volume growth curves (Figure 7d). The photo-
of tumor was prominently improved (1.2-fold) than that of pre- graphs of excised tumors from representative mice in each group
injection, suggesting that SFO could accumulate effectively at were also recorded, as presented in Figure 7e. The treatment effi-
the tumor site. cacy acquired by SFO was superb, indicating an enhanced syner-
The magnetic targeting ability and tumor accumulation gistic effect by modulating the TME.
effect of SFO were also verified by inductively coupled plasma Synergistic therapeutic efficacy was further assessed by
mass spectrometry (ICP-MS). The biodistribution of SFO in hematoxylin and eosin (H&E) staining of tumor slices after
major organs and tumors after 24 h injection intravenously with different treatments (Figure 7f). The tumors of harvested mice
or without magnetic field was investigated. Compared to the administrated with SFO nanozyme exposed to 808 nm laser
absence of a magnetic field, SFO showed efficient accumulate under the assistance of magnetic field were absolutely eradi-
at the tumor site with the efficiency of about 7.83% within cated at the end of the treatment session. Apparent tumor
24 h, which verified its magnetic targeting capacity in vivo tissue shrinkage and damage were evidenced by cell necrosis
(Figure S18, Supporting Information). Based on the ICP-MS and apoptosis, while the mice treated with other groups dis-
measurement of the Fe level in the tumor site at different played only light damage or less necrotic areas. These results
injection time points, an effective tumor accumulation of the confirmed that SFO was a magnetic targeting nanosystem
nanozyme followed by a clearance process can be speculated. with highly efficient antitumor capacity for 4T1 tumor-bearing
The effective accumulation of the nanozyme in the tumor site mouse model. Afterward, we investigated the body clear-
can be assigned to the enhanced permeability and retention ance behavior of SFO nanozyme. According to the Fe content
effect and magnetic targeting ability (Figure S19, Supporting determined by ICP-MS, a biodistribution profile versus injec-
Information). Hence, SFO could serve as a promising CT/MR tion time was presented (Figure 6i and Figure S20, Supporting
imaging contrast agent to precisely diagnose the lesion area Information). Prominent retention of SFO was chiefly detected
and monitor the treatment process with accurate information. in the liver and spleen of mice receiving SFO after 24 h admin-
istration. Upon prolonging the treatment time, the Fe levels in
these major organs drastically decreased, confirming the clear-
2.6. In Vivo Synergistic Therapeutics ance of SFO nanozyme. At the 14th day, the retention of the Fe
level declined to be only <0.9% ID g−1, indicating that SFO was
Based on the remarkable cooperative treatment efficacy of SFO excreted from the mouse body. The levels of Fe in the mouse
nanozyme in vitro, we further investigated its in vivo anticancer urine and feces were further evaluated. High contents of Fe
effect using a 4T1 orthotopic tumor-bearing mouse model. When were observed in the urine samples from mice at varied time
the tumor volume increased to about 60 mm3, the mice were points post injection of SFO, implying the elimination of SFO
separated into six groups randomly (five mice per group) for dif- via the renal filtration pathway (Figure 6j,k). The body elimina-
ferent treatments: 1) administrated saline (control), 2) injected tion of Fe minimizes the concern regarding the long-term bio-
saline and irradiated with 808 nm laser, 3) received SFO injec- toxicity of SFO.
tion, 4) received SFO/Vc administration with 808 nm laser illu- The in vivo blood biochemistry assay, complete blood sample
mination, 5) injected SFO upon NIR laser irradiation for 5 min, analysis, and histology evaluation were comprehensively per-
and 6) administrated SFO under the magnetic field with 808 nm formed to research the long-term biotoxicity of SFO, and SFO
laser illumination for 5 min. The mice were administrated with demonstrated no apparent systemic toxicity to the treated mice
100 µL of saline (groups 1 and 2) or SFO (10 mg kg−1, groups 3, at the used dose (Figure S21, Supporting Information). A neg-
5, and 6) or SFO (10 mg kg−1)/Vc (25 µmol kg−1) (group 4) intra- ligible difference in major liver and kidney function indicators,
venously and irradiated upon 808 nm laser (0.5 W cm−2, 5 min) indicating alanine aminotransferase (ALT), alkaline phosphatase
after 24 administrations (groups 2, 4, 5, and 6), and the treat- (ALP), aspartate aminotransferase (AST), and blood urea nitrogen
ments were repeated three times. Figure 7a shows the schematic (BUN), between the treatment and control groups, was observed,

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Figure 7. In vivo synergistic anticancer efficacy with SFO. a) Schematic illustration of the in vivo anticancer treatment procedure on tumor-bearing
mice. b,c) Relative tumor volume growth curves and body weight changes of mice after various treatments during the therapy (***p < 0.001, **p < 0.01,
or *p < 0.05). d) Average weights of tumors collected from different treatment groups. e) Representative photographs of excised tumors and cor-
responding mice collected from different treatment groups. f) H&E staining of tumor sections from the tumor-bearing mice in different treatment
groups. The yellow and red arrows represent fibroblast and inflammatory cells. Scale bar: 100 µm.

confirming the compatible hepatic and kidney safety of SFO 3. Conclusions

(Figure S21a–d, Supporting Information). For the blood routine
analysis, indexes containing white blood cell (WBC), red blood Nanozyme has aroused an extensive research interest, since
cells (RBC), hemoglobin (HGB), hematocrit (HCT), mean cor- nanomaterials with intrinsic enzyme-like activities exhibit
puscular volume (MCV), mean corpuscular hemoglobin (MCH), superior therapeutic efficacy in the cancer treatment. In this
mean corpuscular hemoglobin concentration (MCHC), and work, we have for the first time constructed a multifunctional
platelets (PLT) were recorded (Figure S21e–l, Supporting Infor- nanozyme, that is, ultrasmall SFO modified with pS-PEG,
mation). What is gratifying is that all the measured indicators which presented magnetic targeting and imaging-guided syn-
are within the normal ranges, again indicating negligible blood ergistic CDT/PDT/PTT of tumor. SFO as an H2O2-responsive
toxicity of SFO at the current treatment dose in vivo. Ultimately, nanozyme exhibits catalase-like and GSH peroxidase-like activi-
the histological analysis was performed by H&E staining of the ties, achieving continuous production of O2 and depletion of
main organs after various treatments. No evident tissue necrosis overexpressed GSH to relieve the antioxidant ability of the
or inflammatory lesion was inspected in the main organs from tumor. In addition, SFO possessing multivalent elements dis-
all the treatment groups, revealing that the SFO nanozyme had plays a potent •OH production capacity by Fenton-like reaction
excellent histocompatibility (Figure S22, Supporting Informa- to facilitate CDT. Significantly, the SFO nanozyme has demon-
tion). All these experimental results fully confirmed that SFO has strated remarkable phototherapeutic effect with excellent PCE
no significant systemic toxicity, showing highly promising appli- (42.3%) and free radical (•OH and •O2−) production capacity
cation potential as a safe and efficient theranostic nanozyme. when exposed to 808 nm laser. Such design strategy has led to

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efficient tumor cell death in vitro and complete ablation of the was stained with DAPI for 10 min. Then, these cells were rinsed with
tumor in vivo through synergistic PDT/PTT and hyperthermia- PBS several times and imaged by confocal laser scanning microscopy
enhanced CDT. Therefore, this theranostic nanozyme with (CLSM).
In Vitro Synergistic Therapeutic Efficacy: 4T1 cancer cells were cultivated
the regulation of TME will significantly promote accurate and into 96-well plates and then treated with PBS, SFO, SFO/Vc plus laser
highly efficient tumor treatment for future clinical translation. irradiation, SFO plus laser irradiation, and SFO in the presence of
Future major research effects will be investigating the possibili- magnetic field plus laser irradiation (125 ppm of SFO nanozyme) at
ties of SFO nanozyme mediated anti-angiogenesis in the tumor 37 °C. After that, the cells were irradiated with 808 nm laser at the power
tissues and tumor immunity. density of 0.5 W cm−2 for 5 min. The relative cell viability after synergistic
treatment was determined by performing the standard MTT assay. To
observe the cell viability using CLSM, Calcein AM, and PI solutions were
utilized to distinguish live and dead cells, respectively.
Intracellular Free Radical Production: DCFH-DA ROS assay kit was
4. Experimental Section utilized to monitor the intracellular ROS generation. 4T1 cells were
Synthesis of SFO: Solvothermal method was adopted to synthesize implanted into 6-well plates and grew adherently to the wall. Fresh
the magnetic SnFe2O4 nanoparticles. Firstly, SnCl2 • 2H2O (0.017 M) and culture medium containing SFO nanozyme (125 ppm) was added, which
FeCl2 • 4H2O (0.034 M) were dispersed in ethylene glycol (25 mL) under was further cultured for 6 h. After washing three times with PBS, the
magnetic stirring to obtain a mixture solution. Then, NH4OH solution cells were irradiated upon 808 nm laser (0.5 W cm−2) for 5 min. Then,
(5 mL) was added and adjusted with NaOH to make sure that the pH of DCFH-DA was added to culture with 4T1 cells for another 30 min. The
the solution was around 10. Soon afterward, the solution was transferred generation of free radicals was observed by CLSM.
into autoclave and heated to 200 °C for 15 h. After cooling down to room Intracellular GSH Consumption: To determine intracellular GSH level
temperature, the samples were obtained by centrifugation, and rinsed for 4T1 cells after different treatments, 4T1 cells were seeded in 6-well
with ethanol and deionized water alternately several times, which is plates for 24 h, and then 4T1 cells were incubated with SFO nanozyme
abbreviated as SFO. at varied concentrations (250, 125, 62.5, and 0 ppm). Subsequently, cells
Surface Modification of pS-PEG on SFO: SFO (10 mg) was dispersed were treated with Thiol Tracker Violet (20 µM) for 30 min. All the images
in deionized water (1 mL). After adding pS-PEG (1 mg), the aqueous were acquired by CLSM.
solution was stirred at 4 °C for 2 h. Then, the product of SFO nanozyme Hemolysis Assay: Blood samples were rinsed with PBS three times and
was achieved by centrifugation, and washed with deionized water three diluted with PBS (15 mL). Afterward, blood samples (0.3 mL) were mixed
times to remove excess pS-PEG. with PBS (1.2 mL, negative control), deionized water (1.2 mL, positive
H2O2 Consumption: Based on the principle of forming orange control), or SFO nanozyme with various concentrations (0–250 ppm).
complex between H2O2 and titanium ions (Ti4+) in acidic medium, the After incubation for 2 h, the samples were collected by centrifugation.
titanium sulfate (Ti(SO4)2) spectrophotometric method was utilized The supernatants were obtained, and their absorbance was measured by
to investigate the H2O2 consumption.[24] Solution A: SFO nanozyme a UV–vis spectrometer to calculate the hemolysis ratios.
(2 mg) was dispersed into H2O2 (1 × 10−3 M, 5 mL) and stirred at room In Vitro and In Vivo MR Imaging Performance: The in vitro and in vivo
temperature. Solution B: Ti(SO4)2 (24%, 665 µL) and H2SO4 (4.165 mL) MR imaging was performed on a 3.0 T clinical MR imaging instrument.
were diluted with water (25 mL). Afterward, solution A (100 µL) was To evaluate the in vitro imaging performance, SFO nanozyme at varied
mixed with solution B (200 µL) every 30 min. SFO nanozyme was Fe concentrations (0, 0.05, 0.1, 0.2, 0.4, and 0.8 mm) was dissolved in
separated by centrifugation, and the remaining H2O2 was determined dibasic sodium phosphate-citric acid buffer solution (1 mL, pH = 7.4)
by the absorbance at the wavelength of 405 nm via UV–vis absorption containing a certain amount of agarose gel, and placed in centrifuge
spectra. The continuous catalytic effect was evaluated by repetitive tubes (1.5 mL) for MR image scanning. T2-weighted MR images were
addition of H2O2 (1 × 10−3 M, 5 mL) to the SFO nanozyme solution, and obtained to calculate the relaxation rate r2. For in vivo MR imaging
the content of residual H2O2 was monitored. evaluation, SFO nanozyme was administrated into the tumor-bearing
GSH Consumption: To confirm the concentration-dependent GSH mice intravenously, and the imaging performance was monitored before
consumption, SFO nanozyme (475 µL, 250, 125, 62.5, 31.3, and 0 ppm) and after 24 injections.
was treated with GSH (25 µL, 1 × 10−3 M) and DTNB (5 µL, 10 mg mL−1) Measurement of Blood Circulation Half-Life and Biodistribution: The
in PBS at 37 °C for 1 h. Similarly, to investigate time-dependent GSH tumor-bearing mice were administrated with SFO nanozyme saline
consumption, SFO nanozyme (475 µL, 125 ppm) was co-incubated solution (100 µL, 10 mg kg−1) intravenously. Soon afterward, blood
with GSH (25 µL, 1 × 10−3 M) and DTNB (5 µL, 10 mg mL−1) in PBS sample (10 µL) was collected at various time points (10 min, 30 min, 1 h,
at varied time (0, 1, 3, 6, 12, and 24 h). Next, the mixture solution was 2 h, 4 h, 8 h, 12h, and 24 h) and diluted into 1 mL by physiological saline
centrifugated and the supernatant was required for the GSH depletion solution with ethylenediaminetetraacetic acid disodium salt (10 mm)
measurement by UV–vis spectroscopy. as the anticoagulant. The Fe concentration was determined by ICP-
Cytotoxicity Measurement of SFO Nanozyme: Cytotoxicity evaluation MS. The blood circulation half-life was analyzed and calculated based
of SFO nanozyme was performed on L929 fibroblast cells, normal on the two-component pharmacokinetic model. At the 1st d, 7th d, and
human embryonic kidney cells (HEK 293), human mesenchymal stem 14th d post-injection, mice were sacrificed. Main organs (heart, liver,
cells (hMSCs), KB cells, and 4T1 mouse breast tumor cells, respectively. spleen, lung, and kidney) were dissected, weighed, and digested for
These cells were implanted into 96-well microplates and permitted to biodistribution analysis via ICP-MS.
adhere overnight. Subsequently, the culture medium was substituted In Vivo Anticancer Performance of Synergetic Therapeutics: Animal
by fresh culture medium including SFO nanozyme at diluted sample experiments were performed according to the guidelines of the National
concentrations of 0, 7.8, 15.6, 31.25, 62.5, 125, and 250 ppm, respectively. Regulation of China for Care and Use of Laboratory Animals. The in vivo
After the coincubation for 24 h, the culture medium was substituted by animal experiments were approved by the Ethics Committee of Harbin
MTT (20 µg mL−1) culture solution and incubation for 4 h. Ultimately, Medical University Cancer Hospital. Female Balb/c nude mice aged
dimethyl sulfoxide (150 µL) was added to each well. Cell viability was 4–5 weeks old were purchased from Beijing Weitong Lihua, Experimental
calculated by measuring the absorbance at λ = 490 nm to the control via Animal Technology Co., Ltd. To obtain the 4T1 orthotopic breast cancer
a microplate reader. SFO nanozyme did not inhibit the cell growth in the model, the mice were inoculated orthotopically in the mammary fat
range of concentrations used. pad with 4T1 breast tumor cells (1 × 105 cell per site) in PBS (100 µL of
Cellular Uptake: 4T1 cells were plated in a 35 mm confocal dish dose for each mouse). Anticancer treatment performance of synergetic
(5.0 × 105 cells each well) and incubated with FITC-conjugated SFO PDT/PTT/CDT after intravenous injection was performed when the
nanozyme (125 ppm) at 37 °C for various time intervals. The cell nucleus tumor volume reached about 60 mm3. 4T1 tumor-bearing nude mice

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