Embedding Atomically Dispersed Manganese/Gadolinium Dual Sites in Oxygen Vacancy‐Enriched Biodegradable Bimetallic Silicate Nanoplatform for Potentiating Catalytic Therapy

Abstract Due to their atomically dispersed active centers, single‐atom nanozymes (SAzymes) have unparalleled advantages in cancer catalytic therapy. Here, loaded with chlorin e6 (Ce6), a hydrothermally mass‐produced bimetallic silicate‐based nanoplatforms with atomically dispersed manganese/gadolinium (Mn/Gd) dual sites and oxygen vacancies (OVs) (PMnSAGMSNs‐V@Ce6) is constructed for tumor glutathione (GSH)‐triggered chemodynamic therapy (CDT) and O2‐alleviated photodynamic therapy. The band gaps of silica are significantly reduced from 2.78 to 1.88 eV by doping with metal ions, which enables it to be excited by a 650 nm laser to produce electron‐hole pairs, thereby facilitating the generation of reactive oxygen species. The Gd sites can modulate the local electrons of the atom‐catalyzed Mn sites, which contribute to the generation of superoxide and hydroxyl radicals (•OH). Tumor GSH‐triggered Mn2+ release can convert endogenous H2O2 to •OH and realize GSH‐depletion‐enhanced CDT. Significantly, the hydrothermally generated OVs can not only capture Mn and Gd atoms to form atomic sites but also can elongate and weaken the O‐O bonds of H2O2, thereby improving the efficacy of Fenton reactions. The degraded Mn2+/Gd3+ ions can be used as tumor‐specific magnetic resonance imaging contrast agents. All the experimental results demonstrate the great potential of PMnSAGMSNs‐V@Ce6 as cancer theranostic agent.


Introduction
With the development of nanotechnology, nanozymes with a tumor microenvironment (TME)-specific catalytic nature have attracted considerable interest. [1]Therefore, reactive oxygen species (ROS)-mediated nanocatalytic strategies are considered promising cancer treatments. [2]Generally speaking, ROS mainly include singlet oxygen ( 1 O 2 ), hydroxyl radicals ( • OH), and superoxide anions radicals ( • O 2 − ), which are often employed to disrupt cell-adaptation mechanisms and induce cell death. [3]However, most reported nanozymes have fewer catalytic active sites and lower atomic utilization efficiency for enzyme-like catalytic process, severely limiting their catalytic activity. [4]Therefore, improving the atomic utilization of the nanozyme catalytic centers can be regarded as an effective strategy for enhancing the catalytic activity of nanozyme.
Recently, carbon-based singleatom nanozymes (SAzymes) by hightemperature pyrolysis method have been increasingly attracting attention in tumor catalytic therapy owing the adjustability of the coordination environment, well-defined electronic and geometric structures, maximum atomic utilization efficiency (100%), and unique quantum size effects. [5]Shi et al. reported a bioinspired hollow N-doped carbon sphere doped with a Cu-based SAzymes that can directly catalyze the decomposition of both oxygen (O 2 ) and H 2 O 2 to ROS to suppress tumor growth. [6]Meanwhile, SiO 2 was introduced into PCN-222 (Fe) to obtain high-loading single-atom catalysts to inhibit the agglomeration of Fe during pyrolysis. [7]However, most of the reported carbon-based SAzymes are challenging to degrade, metabolize, and remain in the body for a long time, which poses potential toxicity risks and greatly limits the use of SAzymes in cancer. [8]Therefore, the development of biodegradable SAzymes is necessary. [9]Compared to carbon-based supports, silicon-based supports are considered as ideal candidates owing to their unique structure, easy functionalization, and good biocompatibility.Benefiting from incorporation of transition metals (M = Mn, Fe, Cu, etc.) into the silsesquioxane framework (-Si-O-Si-), a metal silicate hybrid framework (-Si-O-M-) with additional functions, such as defect-engineered biodegradability and photosensitizing ability, can be used for photodynamic therapy (PDT) application.Due to their merits of variable composition, low toxicity, and low price, silicate-based materials have also been widely used in photocatalytic anticancer. [10]DT, which relies on external energy to convert O 2 into ROS, is one of the most optimum anticancer therapies because of its minimal side effects, good therapeutic effect, and strong spacetime specificity. [11]Due to its high singlet quantum yield, strong tissue penetration, high biocompatibility, and good PDT efficiency, Chlorin e6 (Ce6) has been widely employed for PDT. [12]owever, low ROS generation induced by hypoxia TME and quick energy attenuation often leads to limited PDT effects.To improve PDT outcomes, various nanozymes, including CaO 2 , MnO 2, and manganese silicate (MnSiO 3 ), have been used to relieve tumor hypoxia, thereby achieving O 2 self-sufficient PDT. [13]uang et al. developed novel zinc silicate photocatalyst using a low-temperature hydrothermal method, which endowed ZnSiO 4 with high photocatalytic activity. [14]Besides, Hyeon et al. designed a Ce6-loaded manganese ferrite nanoparticle-anchored mesoporous silica nanoparticles to enhance the therapeutic effects of PDT against hypoxic tumors. [15]1a,16] Dong et al. reported that an MnSiO 3 -supported CaO 2 nanoplatform achieved excellent CDT/PDT synergistic therapeutic effects. [1]The Fenton-like agent Mn 2+ released from MnSiO 3 can deplete ROS scavenger (GSH), further reducing ROS wastage and triggering GSH-depletion-enhanced CDT.Liu et al. successfully synthesized copper ferrite nanospheres as a nanodiagnostic and treatment platform for synergistic CDT/PDT therapy. [17]However, the narrow light response range and high recombination efficiency of photogenerated electron-hole pairs severely limit the practical application of silicates in photocatalytic anticancer applications. [18]efect engineering, especially that of oxygen vacancies (OVs), is considered an effective strategy for significantly boosting the photocatalytic activities of silicate nanomaterials. [19]OVs can serve as electron-or hole-trapping centers, significantly suppressing the recombination of photogenerated carriers. [20]In addition, OVs can broaden the light-absorption range of the catalysts.Li et al. reported Bi 2 O 3-x possessing a substantial absorption range, attributing to the OVs-induced local surface plasmon resonance effect. [21]Additionally, many local electrons near the OVs can be transferred to the anoxic surface.Not only that, some small molecules including O 2 and H 2 O 2 can be activated and dissociated, generating more active substances. [22]Wang et al. found that W 18 O 49 with enriched surface OVs increased the adsorption energy of H 2 O 2 , indicating that the O-O bond of H 2 O 2 was elongated and weakened owing to the stretching effect. [23]n light of these ideas, herein, coupled with Ce6, OVs-enriched PMn SA GMSNs-V@Ce6 with atomically dispersed Mn/Gd dual sites was designed as TME-responsive Fenton-like reagent and dual-photosensitizer for GSH-triggered CDT, O 2 self-sufficient PDT, and magnetic resonance imaging (MRI).PMn SA GMSNs-V continuously generated sufficient amounts of O 2 as a photodynamic substrate by the endogenous H 2 O 2 .Meanwhile, PMn SA GMSNs-V not only served as photosensitizer but also as a semiconductor.Upon 650 nm laser irradiation, PMn SA GMSNs-V produced 1 O 2 via energy transfer.Of course, PMn SA GMSNs-V can be excited by light to form photogenerated electrons and holes, thereby achieving multiple ROS.Subsequently, the photosensitizer molecule Ce6 was integrated into PMn SA GMSNs-V, and PMn SA GMSNs-V@Ce6 with dual photosensitizers displayed the high photodynamic efficiency and relieved hypoxia for enhanced PMn SA GMSNs-V@Ce6-mediated PDT.Significantly, atomic dispersion Mn active sites with 100% atomic utilization in PMn SA GMSNs-V@Ce6 can directly trigger multiple enzyme activities.Because the -Mn-O-bonds are sensitive to TME, Fenton-like Mn 2+ degraded from the PMn SA GMSNs-V@Ce6 may catalyze endogenous H 2 O 2 to produce toxic • OH, resulting in GSH-depletion-enhanced CDT.Particularly, the OVs in PMn SA GMSNs-V@Ce6 promote the decomposition of H 2 O 2 to achieve a large number of • OH radicals.TME-responsive T 1weighted MRI nature of released Mn 2+ /Gd 3+ and atomically dispersed Mn/Gd was employed to monitor the progress of multiple ROS-mediated cancer treatments.

Results and Discussion
The as-obtained PMn SA GMSNs-V@Ce6 with atomically dispersed Mn and Gd sites was synthesized via simple hydrothermal strategy using SiO 2 nanospheres as template (Scheme 1).In this nanosystem, PMn SA GMSNs-V@Ce6 has some merits for potentiating cancer theranostics: i) PMn SA GMSNs-V can serve as semiconductor to generate photogenerated carriers to produce multiple ROS under 650 nm laser irradiation (charge separation).ii) PMn SA GMSNs-V@Ce6 continuously triggered catalase (CAT)-like activity to generate sufficient amounts of O 2 to continue PDT reaction.As a result, upon 650 nm laser irradiation, PMn SA GMSNs-V@Ce6 can directly convert O 2 into 1 O 2 , Scheme 1. Schematic illustration of the synthesis of PMn SA GMSNs-V@Ce6 and the theranostic mechanism of PMn SA GMSNs-V@Ce6 for O 2 selfsufficient PDT, tumor GSH-triggered CDT, and MRI under 650 nm laser irradiation.
exhibiting the hypoxia-relieved PDT (catalase-like activity).iii) Atomically dispersed Mn sites modulated by the Gd sites in PMn SA GMSNs-V@Ce6 could trigger efficient peroxidase (POD)like and oxidase (OXD)-like activities, endowing PMn SA GMSNs-V@Ce6 with high enzymatic activity.Meanwhile, the Mn 2+ release from the PMn SA GMSNs-V@Ce6 due to reduced GSH can react with endogenous H 2 O 2 to produce a large amount of • OH, resulting in GSH-depletion-enhanced CDT effects.Importantly, the large number of OVs in PMn SA GMSNs-V@Ce6 can elongate and weaken the O-O bond of H 2 O 2 , which favors the decomposition of H 2 O 2 to generate more • OH.Collectively, PMn SA GMSNs-V@Ce6 achieved high tumor inhibition based on the ROS-mediated catalytic therapy.Additionally, Mn 2+ and Gd 3+ can be used as MRI contrast agent.
The first step in fabricating SAzymes was successfully synthesizing monodisperse mesoporous silica nanoparticles (MSNs) via a microemulsion process.The images obtained using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) revealed that the average size of MSNs was ≈80 nm, as illustrated in Figure 1a Brunauer-Emmett-Teller (BET) fitting results, the MSNs possessed an average pore size of 3.4 nm and a specific surface area of 520.2 m 2 g −1 .The formation of crystalline phases for SiO 2 was studied using the powder X-ray diffraction (XRD) analysis.A prominent broad diffraction peak located at 20∼30°is observed in Figure S2, Supporting Information, which belongs to the typical diffraction peak of amorphous SiO 2 .When Mn ions were introduced into SiO 2 , silicate ions generated by MSNs in an alkaline environment can react with the manganese-ammonium complex ions (Mn(NH 3 ) 4 2+ ) to form MnSiO 3 during the hydrothermal process.In Figure S3a, Supporting Information, the SEM images showed that MnSiO 3 consists of uniform nanospheres with a mean size of ≈85 nm.The obtained MnSiO 3 had a rough surface formed by accumulation of some granular nanoparticles.The corresponding X-ray energy dispersive spectra (EDS) mapping of MnSiO 3 revealed that only C, Si, O, and Mn elements were detected (Figure S3b, Supporting Information).From the TEM images, it is clear that MnSiO 3 has a hollow structure, indi-cating more mesopores in MnSiO 3 (Figure S3c,d, Supporting Information).As the core of SiO 2 was constantly consumed, more basic MnSiO 3 was produced by Mn(NH 3 ) 4 2+ , forming a hollow shell.High-resolution transmission electron microscopy (HRTEM) shows a lattice spacing of 0.25 nm, corresponding to the typical lattice plane of MnSiO 3 . [24]The element percentages of MnSiO 3 were calculated to be C (41.39 wt.%), Si (14.67 wt.%), O (18.33 wt.%), and Mn (25.61 wt.%) (Figure S3e, Supporting Information).Moreover, the SEM, TEM images, and corresponding SEM-EDS mapping of Gd/SiO 2 are shown in Figure S4a-d XRD patterns of MnSiO 3 and Gd/SiO 2 are displayed in Figure S5, Supporting Information.13a] However, compared to standard Gd 2 O 3 pattern (JCPDS card 88-2165), Gd/SiO 2 showed poor crystallinity.This phenomenon can be explained by the fact that Mn ions can easily form Mn(NH 3 ) 4 2+ , indicating that Mn ions are more easily doped into SiO 2 than Gd ions.When Mn and Gd ions were simultaneously integrated into SiO 2 , atomically dispersed Mn/Gd dual sites embedded in the mesoporous Gd and Mn co-doped silicate nanospheres with OVs (Mn SA GMSNs-V), as shown in Figure 1c and the corresponding size of Mn SA GMSNs-V was about 85 nm.Subsequently, aberration-corrected high-angle annular dark-field scanning TEM (HAADF-STEM) was performed.In Figure 1d, many bright spots (circled in red) are attributed to the atomically dispersed Mn and Gd sites.The line-scanning intensity profiles indicated the existence of an atomic dispersion of Gd (slightly brighter Gd dots and darker Mn dots) in Figure S6a-b, Supporting Information.The corresponding atomic distance between Gd and Mn is ≈0.36 nm.Additionally, an obvious lattice fringe (0.25 nm) is observed, corresponding to the typical characteristic plane of MnSiO 3 .As shown in Figure S7, Supporting Information, a straightforward hydrothermal procedure may successfully manufacture Mn SA GMSNs-V (0.995 g) on a large scale in a 300 mL reactor.Furthermore, the corresponding XRD pattern of Mn SA GMSNs-V, which included distinctive peaks of both Gd 2 O 3 and MnSiO 3 , confirmed the successful preparation of Gd/Mn codoped silicate.In Figure 1e, the TEM-EDS mapping showed that Si, O, Mn, and Gd elements were evenly distributed throughout the Mn SA GMSNs-V structure.Notably, Si was concentrated on the shell of the nanospheres rather than in the middle, suggesting that the internal SiO 2 has been depleted.However, the morphology of Mn SA GMSNs-V differed from MnSiO 3 , which was attributed to the presence of internal Gd 2 O 3 .According to EDS results (Figure S8, Supporting Information), the element contents of Si, O, Mn, and Gd are 10.29 wt.%, 39.33 wt.%, 16.58 wt.% and 33.80 wt.%, respectively.
Inductively coupled plasma (ICP) testing revealed the elemental content of Gd (34.90 wt.%) and Mn (17.00 wt.%) (Table S1) were consistent with EDS results.The surface elemental states and chemical compositions of the samples were examined using X-ray photoelectron spectroscopy (XPS).The full-scan spectra of Mn SA GMSNs-V further confirm the successful introduction of Gd and Mn (Figure S9, Supporting Information).The highresolution XPS spectrum of Gd 4d is shown in Figure 1f.With a spin-orbit splitting of 4.8 eV, the two prominent peaks at 142.4 and 147.2 eV are associated with the 4d 5/2 and 4d 3/2 energy levels of Gd, respectively. [25]These findings indicate that the main valence of Gd was 3+.Meanwhile, the peaks located at 653.3 and 641.4 eV are attributed to the 2 p 1/2 and 2 p 3/2 of Mn, respectively.For Mn 2p 3/2 , the peak at 641.4 eV was divided into the three main peaks located 640.8, 642.0, and 644.8 eV, which correspond to Mn 2+ (13.21%),13a] The possibility of redox interaction with intratumoral GSH is significantly increased by the high-valence Mn 3+ and Mn 4+ , thereby improving the PDT and Fenton-like effects (Figure 1g).Additionally, the high-resolution XPS spectrum of O 1s was fitted in Figure 1h.The peaks situated at 529.5, 531.2, and 533.2 eV were ascribed to lattice oxygen (O L ), OVs, and chemisorbed oxygen (O c ), respectively. [26] elucidate the chemical environment and coordination state of Mn and Gd species, Mn SA GMSNs-V were investigated using X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) spectroscopy.The Mn K-edge and Gd L 3 -edge for Mn SA GMSNs-V, as well as their respective reference samples, were studied using normalized XANES curves.Figure 2a demonstrates the near-edge absorption of Mn SA GMSNs-V, which is situated between Mn 2 O 3 and MnO 2 , indicating that Mn atoms carried positive charges with the oxidation state, and the corresponding valence of Mn element was between +3 and +4, which is in agreement with XPS results. [27]imilarly, the normalized XANES curves of the Gd L 3 -edge for Mn SA GMSNs-V are shown in Figure 2b.The absorption edge of the Mn SA GMSNs-V lies between those of Gd foil and Gd 2 O 3 , demonstrating that the valence of Gd was between +0 and +3.Notably, the Gd L 3 -edge absorption curve of Mn SA GMSNs-V has a positive tendency to move in the direction of higher energy, and the Mn K-edge absorption edge location was near to the lower energy direction, which supported the electron transfer from Gd to Mn in the XANES spectra. [28]These results further revealed that the local electrons of Mn sites could be modulated by the introduction of Gd sites, thereby achieving a rapid catalytic reaction.In addition, the Fourier transformed (FT) k 3 -weighted EX-AFS (without phase correction) of Mn space in Mn SA GMSNs-V were performed with reference samples (Figure 2c-d and Figure S10, Supporting Information).The FT-EXAFS spectrum of the Mn SA GMSNs-V displayed a predominant peak situated at 1.52 Å, corresponding to the Mn-O scattering path.There was no prominent contribution of metallic Mn-Mn, revealing the isolation of Mn single atoms throughout the Mn SA GMSNs-V samples, consistent with HAADF-STEM results.Another peak at 2.51 Å belonged to the Mn-O-Si coordination for Mn SA GMSNs-V, revealing the existence of MnSiO 3 , which follows the HAADF-STEM results.It is important to note that a weaker peak situated at 2.94 Å could be associated with Mn-O-Gd coordination.Furthermore, the fitting curve in k spaces (Figure S11, Supporting Information) was employed to investigate the structural parameters of reference samples and Mn SA GMSNs-V.
The corresponding curve-fitting results were in excellent agreement with the experimental data.Similarly, the k 3 -weighted FT-EXAFS was recorded at Gd L 3 -edge of the Mn SA GMSNs-V with Gd foil and Gd 2 O 3 (Figure 2e).The k 3 -weighted FT-EXAFS spectroscopy clarified the coordination surrounding of Gd.In Figure 2f, the two major peaks at about 1.88 and 3.29 Å for Mn SA GMSNs-V corresponded to the Gd-O and Gd-O-Gd coordination peak, respectively.Compared with Gd foil and Gd 2 O 3 (Figure S12, Supporting Information), the Gd-Gd coordination peak was not detected in Mn SA GMSNs-V, indicating that metallic Gd did not aggregate.Figure S13, Supporting Information, illustrates the results of the EXAFS curve fitting of the Mn SA GMSNs-V, Gd foil, and Gd 2 O 3 in k spaces to determine the coordination arrangement of Gd, which suggested the experimental data and the results of curve fitting were quite similar.Wavelet transform (WT) analysis with a higher resolution was used to determine the roles of different scattering paths.In Figure S14, Supporting Information, Mn foil has a predominant intensity maximum at 6.2 Å −1 , which was attributed to metallic Mn-Mn coordination.As displayed in Figure 2g-i, WT contour Normalized XANES of Mn K-edge spectra of Mn foil, MnO 2 Mn 2 O 3 , and Mn SA GMSNs-V and b) Gd L 3 -edge spectra of Gd foil, Gd 2 O 3 and Mn SA GMSNs-V.c) FT k 3 -weighted Mn K-edge EXAFS spectra of Mn foil, MnO 2 , Mn 2 O 3, and Mn SA GMSNs-V and d) corresponding FT-EXAFS fitting curves at R space of Mn.FT k 3 -weighted Gd L 3 -edge EXAFS spectra of Gd foil, Gd 2 O 3, and e) Mn SA GMSNs-V and f) corresponding FT-EXAFS fitting curves at R space of Gd. g-l) WT-EXAFS plots of Mn SA GMSNs-V and corresponding reference samples.plots of MnO 2 , Mn 2 O 3, and Mn SA GMSNs-V were obtained.For Mn SA GMSNs-V, three obvious intensity maximum at 4.4, 5.8, and 10.6 Å −1 was observed, corresponding to the Mn-O, Mn-O-Si, and Mn-O-Gd, respectively.Moreover, compared to the Gd foil and Gd 2 O 3 , the WT contour plots of Mn SA GMSNs-V showed the similar characteristic maximum intensity located at 4.06 and 6.7 Å −1 (Figure 2j,l) and no obvious Gd-Gd coordination peak was detected, indicating the existence of Gd 2 O 3 in Mn SA GMSNs-V.The Mn-O and Gd-O average coordination numbers were determined to be 3.7 and 5.5 based on the EXAFS fitting parameters, respectively, which revealed that Mn sites were bonded with four atoms in the structure of Mn SA GMSNs-V (Table S2).These data can further verify the successful doping of Gd and Mn, consistent with TEM mapping results.Taken all together, the coordination environment of the Mn element proves that some Mn atoms mainly exist in atomic dispersion in Mn SA GMSNs-V due Figure 3. a).N 2 adsorption-desorption isotherms of Mn SA GMSNs-V, b) UV-vis diffuse absorbance spectra of Mn SA GMSNs-V (insert: the corresponding plots of (h) n/2 for Mn SA GMSNs-V).c) Photocurrent transient responses, d) electrochemical impedance spectroscopy.e) The ESR spectra of pure SiO 2 and Mn SA GMSNs-V.The free energy diagrams of Mn SA GMSNs-V and f,g) Mn SA GMSNs during a catalytic process in an acidic environment.h) DOS of Mn SA GMSNs and i) Mn SA GMSNs-V.
to the capture of OVs, and Mn atoms do not agglomerate.However, according to the results of HAADF-STEM and EXAFS, Gd was primarily composed of Gd 2 O 3 and a small amount of atomic dispersion of Gd.
In Figure 3a, the N 2 adsorption-desorption isotherms exhibited an obvious hysteresis loop, indicating that Mn SA GMSNs-V possessed many abundant mesoporous structures. [24]The Brunauer-Emmett-Teller (BET) surface area and average pore size were calculated to be 432.3m 2 g −1 and 5.8 nm (Figure S15, Supporting Information), respectively.Compared to the pure SiO 2 , BET value of Mn SA GMSNs-V significantly decreased, which was attributed to the deduction of pore.Ultraviolet-visible diffuse reflectance spectra (UV-vis DRS) was employed to determine the light-absorption properties of the synthesized nanomaterials.The UV-vis DRS spectrum of pure SiO 2 is displayed in Figure S16a, and corresponding band gap was calculated to be 2.78 eV (Figure S16b) according to Kubelka-Munk Equation (1), [29] indicating lower light absorption of SiO 2 .
The band gaps of Gd/SiO 2 (2.63 eV) (Figure S16c,d, Supporting Information) and MnSiO 3 (2.12eV) (Figure S16e,f, Supporting Information) are lower than those of pure SiO 2 .However, as Gd and Mn were co-doped into SiO 2 , the band gap of Mn SA GMSNs-V (1.88 eV) is shown in Figure 3b, indicating that Mn SA GMSNs-V showed a broad absorption range and could be stimulated using a 650 nm laser (E = 1.91 eV) for PDT.
Separation efficiency of charge carriers was further revealed by photoluminescence (PL) emission spectroscopy and photoelectrochemical characterizations.Generally, a lower emission intensity is caused by a lower efficiency radiative recombination. [30]igure S17, Supporting Information, shows the separation efficiency of charge carriers for MnSiO 3 , Mn SA GMSNs-V, and Mn SA GMSNs-V@Ce6.Obviously, MnSiO 3 showed the strongest fluorescence signal among all materials, revealing that MnSiO 3 has the highest recombination rate of carriers.For Mn SA GMSNs-V, the lower fluorescence intensity was observed due to the doping of Gd element.By contrast, Mn SA GMSNs-V@Ce6 exhibited the lowest fluorescence intensity due to the quenching of molecule aggregation.
Moreover, the charge transfer and separation efficiencies over MnSiO 3 and Mn SA GMSNs-V@Ce6 were investigated.A weaker intensity is consistent with lower separation efficiency.As shown in Figure 3c, Mn SA GMSNs-V displayed the highest photocurrent value than MnSiO 3 , indicating that more photogenerated electrons were generated under light irradiation.Electrochemical impedance spectroscopy (EIS) was conducted to study the charge-transfer performance (Figure 3d).The semicircle of pure SiO 2 is the largest, indicating that its resistance is the largest.When Mn ions were doped into SiO 2 , MnSiO 3 exhibited the smaller semicircle than SiO 2 due to its lower resistance.Among these samples, Mn SA GMSNs-V had the lowest semicircle, indicating Mn SA GMSNs-V possessed the lowest resistance and highest efficiency of electron-hole transfer by the introduction of Gd sites, which can accelerate the catalytic reaction. [31]The time-resolved transient PL (TRPL) decays of pure SiO 2 and Mn SA GMSNs-V were fitted to reveal the specific charge carrier dynamics (Figure S18, Supporting Information).The average lifetimes of pure SiO 2 and Mn SA GMSNs-V were calculated to be 5.27 and 4.73 ns, respectively.19a] Furthermore, linear sweep voltammetry (LSV) was employed to study the adsorption capacity of different materials, including SiO 2 , MnSiO 3 , and Mn SA GMSNs-V for H 2 O 2 substrate (Figure S19, Supporting Information).In the presence of H 2 O 2 , pure SiO 2 displayed poor current density under weakly acidic conditions.Compared to SiO 2 , an obvious current density for MnSiO 3 was observed, indicating that MnSiO 3 showed better affinity under the same conditions.To our delight, Mn SA GMSNs-V possessed the highest current density along with doping of Gd and Mn, suggesting Mn SA GMSNs-V exhibited the best affinity for H 2 O 2 .Electron spin resonance (ESR) can be regarded as powerful evidence to verify the presence of OVs in Mn SA GMSNs-V.As shown in Figure 3e and Figure S20, Supporting Information, pure SiO 2 showed a weaker Lorentz line due to the presence of a small amount of unpaired electrons, suggesting the existence of OVs.However, the SiO 2 after hydrothermal treatment showed a slightly stronger signal.Compared to pure SiO 2 and SiO 2 after hydrothermal treatment, a stronger electron paramagnetic resonance signal for Mn SA GMSNs-V (g = 2.001) was observed, indicating the number of unpaired electrons was markedly increased.This phenomenon reveals that many unpaired electron-localized OVs can anchor redundant Mn atoms that are not converted into MnSiO 3 .That is, the Mn atoms can occupy OVs sites. [32]Vs with many local electrons can activate or dissolve some small molecules, such as O 2 and H 2 O 2 , and thus positively affect the catalytic process.Subsequently, density functional theory (DFT) computations were performed to determine the critical ef-fects of OVs.The adsorption capacity of H 2 O 2 for Mn SA GMSNs-V without or with OVs at different active sites was studied.As shown in Figure S21a-d, Supporting Information, compared to the Gd sites without OVs (E ads = −0.639eV), lower adsorption energy (E ads = −1.163eV) was calculated for Gd sites with OVs.However, when Mn was used as the active site, Mn sites with OVs had lower adsorption energy (E ads = −1.832eV) than Mn sites without OVs (E ads = −1.054eV), which verified that OVs favored the adsorption for H 2 O 2 .In addition, the adsorption energy of Mn sites with OVs was lower than Gd sites with OVs, indicating that the adsorption process was more likely to occur at the Mn sites.
The existence of OVs might elongate and weak the O-O bond of H 2 O 2 , which may accelerate H 2 O 2 breakdown.Importantly, the free energy diagrams of Mn SA GMSNs-V and Mn SA GMSNs were simulated during weaker acidic conditions (Figure 3f,g).The simulated catalytic mechanism of Mn SA GMSNs-V under acidic conditions could be divided into the following processes.Initially, activated *H 2 O 2 may be produced by simply absorbing H 2 O 2 molecules onto the Mn sites of Mn SA GMSNs-V.Compared to Mn SA GMSNs (ΔG = −0.39135 eV), the lower free energy for Mn SA GMSNs-V (ΔG = −0.93542 eV) was observed, indicating Mn SA GMSNs-V was easy to combine with H 2 O 2 molecules.Secondly, hydroxyl groups (OH*) bonded to the Mn sites, and reactive • OH were generated by uniformly cleaving the activated H 2 O 2 molecules.Then, • OH could be generated under the stage.However, compared to the Mn SA GMSNs, the free energy barrier in the rate-determining step of the • OH formation for Mn SA GMSNs-V was much negative (−1.367 versus −0.613 eV), indicating Mn SA GMSNs-V is more beneficial for the formation of • OH because of the existence of OVs.
In addition, the density of states (DOS) for Mn SA GMSNs and Mn SA GMSNs-V was employed to elucidate the catalytic reaction mechanisms.As illustrated in Figure 3h-i, the strongest interactions between Mn SA GMSNs-V and H 2 O 2 were inferred by the larger DOS around the Fermi level, and several novel hybridized electronic states were detected for Mn SA GMSNs-V.Subsequently, titanium oxide sulfate (TiOSO 4 ) was used to detect changes in the concentration of H 2 O 2 solution treated with PMn SA GMSNs-V at different times.In Figure S22, Supporting Information, the UV-Vis absorption peak of TiOSO 4 solution gradually decreased, indicating that H 2 O 2 was consumed along with the extension of time.To verify CAT-like activity of Mn SA GMSNs-V, their catalytic activity of Mn SA GMSNs-V on H 2 O 2 decomposition was investigated.The corresponding curves of O 2 concentration change in the different solution were also obtained.As shown in Figure S23, Supporting Information, when H 2 O 2 was added into Mn SA GMSNs-V, an obvious real-time increase in O 2 concentration was observed than SiO 2 and MnSiO 3 .Almost no oxygen bubbles were observed in the pure SiO 2 and H 2 O 2 solution (Video S1).Compared to pure SiO 2 and H 2 O 2 solution, a small amount of oxygen bubbles were observed in pure MnSiO 3 solution.When Mn SA GMSNs-V was added into H 2 O 2 solution, an obvious generation of oxygen bubbles was observed owing to the introduction of Gd sites, indicating that Mn SA GMSNs-V can facilitate the breakdown of H 2 O 2 into O 2 , confirming the CAT-like activity of Mn SA GMSNs-V (2): (2) UV-vis absorption spectra of SiO 2 , Mn SA GMSNs-V, Ce6, Mn SA GMSNs-V@Ce6, MB degradation by • OH generated by different concentrations of GSH-treated b) Mn SA GMSNs-V@Ce6 (1 mg mL −1 ) and c) H 2 O 2 (8 mM).d) Time-dependent absorption spectra and e) the corresponding retention rate of DPBF in the presence of Mn SA GMSNs-V and Mn SA GMSNs-V@Ce6 under 650 nm laser irradiation.GSH depleting abilities using DTNB as f) the trapping agent of sulphydryl (-SH) in GSH.ESR spectra for detection of 1 O 2 , • O 2 − and • OH versus magnetic field in different conditions for Mn SA GMSNs-V and Mn SA GMSNs-V@Ce6 (g-i).
In order to further verify CAT enzyme activity of the PMn SA GMSNs-V@Ce6, a hypoxia-inducible factor-1 (HIF-1) protein was employed to assess the degree of cell hypoxia.Compared with control group, western blot analysis of PMn SA GMSNs-V@Ce6 showed that the signal intensity of HIF-1 decreased (Figure S24, Supporting Information).These results can suggest that PMn SA GMSNs-V@Ce6 could alleviate hypoxia via O 2 generation following cellular uptake.
To confirm that Ce6 was successfully loaded in Mn SA GMSNs-V, the UV-vis absorption spectra of all prepared nanozymes were investigated.As shown in Figure 4a, Mn SA GMSNs-V exhibited the wide absorption than pure SiO 2 in the visible light range.For Ce6, two absorption peaks were observed at 402 and 655 nm, which correspond to the typical peaks of Ce6.Moreover, Mn SA GMSNs-V@Ce6 demonstrated characteristic peaks similar to those of Ce6 at 422 nm and 672 nm, indicating the successful loading of Ce6. [12]Notably, an obvious red-shift for Mn SA GMSNs-V@Ce6 was observed due to the insertion of metal ion into the framework of Ce6. [33]Besides, the loading amount of Ce6 was calculated based on the UV-Vis absorption spectra of Ce6 at varying concentrations (Figure S25a) and the standard curve for Ce6 (Figure S25b, Supporting Information).As a result, Ce6 loading efficiency in the Mn SA GMSNs-V@Ce6 was calculated to be 24.7 wt.% (Figure S25c, Supporting Information).Subsequently, the response capacity of Mn SA GMSNs-V to different concentrations of GSH and H 2 O 2 was investigated.As displayed in Figure 4b, under mild acid conditions, the absorbance value of MB obviously decreased at 664 nm as the GSH concentration increased from 0 to 10 mM, demonstrating a concentration-dependent relationship between the absorbance and GSH concentrations.These results further revealed that the weakly acid environment (pH 6.5) and reduced GSH promoted rapid biodegradation of Mn SA GMSNs-V to coincidentally trigger a Fenton-like reaction, which was attributed to the response of Mn-O bonds. [27]Similarly, the absorbance of MB also decreased as the concentration of H 2 O 2 increased due to more ROS generation (Figure 4c).
Moreover, 1,3-diphenylisobenzofuran (DPBF), with a maximum absorbance peak located at 420 nm, was employed to verify the generation of 1 O 2 . [34]Figure S26, Supporting Information, shows absorption spectrum of DPBF at different times in the presence of Mn SA GMSNs-V by 650 nm laser irradiation, indicating Mn SA GMSNs-V can act as photosensitizer (3).When Ce6 was integrated into Mn SA GMSNs-V, a significant decrease in the absorption spectra of DPBF over Mn SA GMSNs-V@Ce6 was observed (Figure 4d).Compared to the Mn SA GMSNs-V, Mn SA GMSNs-V@Ce6 displayed lower absorbance in DPBF absorption spectra (Figure 4e), which was attributed to the synergistic effects of Mn SA GMSNs-V and Ce6 (4).
Meanwhile, 650 nm laser illumination (0.6 W cm −2 ), Mn SA GMSNs-V and Mn SA GMSNs-V@Ce6 for ROS generation were investigated using singlet oxygen sensor green (SOSG) as a detector.2b] Figure S27, Supporting Information, showed that there was no detectable fluorescence emission signal from H 2 O 2 , suggesting H 2 O 2 basically does not generate 1 O 2 .For Mn SA GMSNs-V, lower emission intensity was observed, confirming that Mn SA GMSNs-V can serve as a photosensitizer, thereby transforming triplet oxygen ( 3 O 2 ) to 1 O 2. Furthermore, Mn SA GMSNs-V@Ce6 displayed the strongest fluorescence intensity, indicating excellent performance toward the 1 O 2 generation due to the synergistic effects of Mn SA GMSNs-V and Ce6.
Additionally, 5, 5′-dithio-bis (2-nitrobenzoic acid) (DTNB) was employed to evaluate the depletion ability of GSH.It is reported that DTNB can react with GSH to produce yellow chromogenic products with a prominent absorbance peak located at 412 nm. [35]or pure DTNB, there was no detectable absorption signal at 412 nm in the UV-vis spectra (Figure 4f).Interestingly, an obvious absorption peak was detected, which was attributed to the color reaction of GSH and DTNB.When Mn SA GMSNs-V was added to the GSH solution, a lower absorbance was observed in the UV absorption spectra, implying Mn SA GMSNs-V can consume GSH.The GSH degradation rate in HeLa cells treated with varying concentrations of PMn SA GMSNs-V is shown in Figure S28, Supporting Information.The intracellular GSH levels showed a concentration-dependent decline after treatment with PMn SA GMSNs-V.As a result, GSH-responsive behavior can effectively prevent the ROS loss, thereby realizing GSHdepletion-enhanced CDT.
The low-temperature ESR spectra were collected using 2,2,6,6-tetramethylpiperidine (TEMP) and 5,5-dimethyl-1pyrroline N-oxide (DMPO) as spin-trapping agents to identify ROS production. [36]As shown in Figure 4g, both Mn SA GMSNs-V and Mn SA GMSNs-V@Ce6 presented a straight line in the presence of only pure H 2 O 2 , indicating 1 O 2 cannot be pro-duced.However, when Mn SA GMSNs-V were illuminated by a 650 nm laser (5 min), there was a significant signal for 1 O 2 with a strength ratio of 1:1:1, revealing Mn SA GMSNs-V can be regarded as photosensitizer, which is following with SOSG results. [37]Stronger signals were observed as the illumination time increased (10 min).Compared to the Mn SA GMSNs-V, Mn SA GMSNs-V@Ce6 displayed strongest 1 O 2 signals under the same time conditions, which was attributed to the synergistic effects of Mn SA GMSNs-V and Ce6.The Mott-Schottky (MS) plots were used to examine the conduction band (CB) of Mn SA GMSNs-V to explore the ROS-generating process better.Figure S29, Supporting Information, shows that the slope of the MS plot was unequivocally positive, indicating that Mn SA GMSNs-V was n-type semiconductors. [38]As a matter of fact, the location of the CB is close to the flat potential of the n-type semiconductors. [39]It was discovered that the flat potential of Mn SA GMSNs-V was −0.75 eV (vs.Ag/AgCl at pH 7).The calculated value for the matching CB of Mn SA GMSNs-V was about −0.55 eV (vs.Normal Hydrogen Electrode (NHE)) based on Equation (5).Meanwhile, the VB of Mn SA GMSNs-V was evaluated by XPS valence band spectrum (Figure S30), consistent with MS results.Based on Equation ( 6), the VB of Mn SA GMSNs-V was calculated to be +1.33 eV.
When the Mn SA GMSNs-V was illuminated by 650 nm laser (> 1.88 eV), electrons originating from the VB of Mn SA GMSNs-V were able to transition to the corresponding CB to generate electron-hole pairs (7).The VB energy of Mn SA GMSNs-V (+1.33 eV) was lower than that of E  ( • OH/H 2 O) (+1.99 eV), indicating • OH could not be produced in this manner. [40]Nevertheless, the CB potential (−0.55 eV) of Mn SA GMSNs-V is more negative than that of E  (O 2 / • O 2 − ) (−0.33 eV), suggesting the formation of an active species ( • O 2 − ) might be formed. [41]In addition, the ESR spectra (Figure 4h) further reveal the generation of • O 2 − , which was attributed to the fact that the photogenerated electrons were further captured by O 2 (8).
Furthermore, the ESR spectra revealed that pure Mn SA GMSNs-V had no • O 2 − without 650 nm laser irradiation.In contrast, when Mn SA GMSNs-V were excited by 650 nm laser irradiation, a distinguishable characteristic peak that has an intensity ratio of 1:1:1:1 was detected, suggesting the presence of • O 2 − .Importantly, Mn SA GMSNs-V possessed the strongest • O 2 − signal after 10 min of illumination, revealing more electron-hole pairs could induce and generate more ROS.Remarkably, the typical ESR signal of • O 2 − were detected not only under 650 nm laser irradiation but also non-light conditions (only in the presence of H 2 O 2 ) (Figure S31, Supporting Information), indicating the existence of atomically dispersed Mn sites in the Mn SA GMSNs-V, thereby achieving excellent OXD-like activity.Similarly, a free-radical trapping experiment further verified the presence of Mn single atom (Figure S32, Supporting Information).Moreover, the production of • OH was confirmed by ESR assays.As shown in Figure 4i, pure H 2 O 2 presented a straight line and hardly produced • OH.After GSH and Mn SA GMSNs-V were introduced in H 2 O 2 solution (5 min), the ESR spectrum exhibited a characteristic 1:2:2:1 • OH signal peaks due to the generation of • OH.Notably, more • OH was detected with increased reaction time (10 min).More importantly, the signal of • OH for Mn SA GMSNs-V was also detected only in the presence of H 2 O 2 (Figure S33, Supporting Information).This phenomenon can be attributed to the atomically dispersed Mn in the Mn SA GMSNs-V, thereby inducing more • OH generation.Besides, compared to the absence of GSH, Mn SA GMSNs-V showed enhanced • OH signal in the presence of GSH due to the release more Fenton-like ions.
Mn SA GMSNs-V were endowed with degradable property due to the response of Mn-O bonds in Mn SA GMSNs-V.At first, the accumulated releasing of Mn and Gd ions from MnSiO 3 and Gd/SiO 2 was evaluated.As show in Figure S34, Supporting Information, the accumulated releasing rate of Mn from pure MnSiO 3 was faster, indicated that MnSiO 3 exhibited an obvious biodegradation ability compared to that of Gd/SiO 2 .Subsequently, the biodegradation behavior of Mn SA GMSNs-V in the simulated TME was directly observed by SEM and TEM analysis.The pictures of Mn SA GMSNs-V treated with different solutions for 24 h were shown in Figure S35a-b, Supporting Information.It was obvious that reductive GSH and acid conditions have a significant effect, thereby accelerating the biodegradation behavior of Mn SA GMSNs-V to achieve the enhanced CDT (9).
As shown in Figure S36a, Supporting Information, Mn SA GMSNs-V had a completely spherical nanostructure under neutral condition (pH 7.4) without GSH, whereas the majority of Mn SA GMSNs-V significantly collapsed under acidic conditions (pH 6.5) and in the presence of GSH (10 mM) after 24 h.Moreover, ICP-OES was used to analyze the Mn and Gd ions liberated from Mn SA GMSNs-V after treatment with or without GSH under various pH conditions (Figure S36b,c, Supporting Information).As anticipated, under the mildly acidic conditions, reduced GSH levels greatly stimulated Mn SA GMSNs-V biodegradation to release Mn and Gd.In the simulated environment, the cumulative degradation rate of Mn was much higher than that of Gd, because the Mn-O bond was more sensitive to the tumor environment.Due to the cleavage of the Mn-O bond, the release of Mn ions originating from the Mn SA GMSNs-V resulted in a large number of defects in the skeleton under acidic conditions and GSH, which could further induce the break of the Gd-O bonds.
Benefiting from the biodegradation of Mn and Gd, the MRI capabilities of Mn SA GMSNs-V were examined under simulated normal and tumor conditions.As shown in Figure S36d, Supporting Information, the T 1 signal exhibited increasing brightness of phantom picture in all groups when Mn 2+ and Gd 3+ concentration were increased from 0 to 1.6 mM.The enhancements in brightness with reductive GSH (10 mM) and weakly acidic con-ditions (pH 6.5) exhibited an extraordinarily higher degradation rate than that in the neutral (pH 7.4) and the absence of GSH conditions (0 mM).The corresponding initial longitudinal relaxation rate (r 1 ) of Mn SA GMSNs-V in normal condition or weakly acid condition without or with GSH was evaluated to be 1.14 and 6.11 mM −1 s −1 , respectively (Figure S36e, Supporting Information), indicating the remarkably improved release of Mn 2+ and Gd 3+ from Mn SA GMSNs-V under acid reduced environment, thereby confirming the enhancement of TME-responsive MRI of Mn SA GMSNs-V (10).
PMn SA GMSNs − V + GSH∕H + → Gd 3+ + Mn 2+ + GSSG (10)   Biological transmission electron microscopy (bio-TEM) was used to observe intracellular biodegradation of PMn SA GMSNs-V.When the incubation time of PMn SA GMSNs-V was only 0.5 h, in addition to the observation that part of the shell remained almost intact, apparent shell collapse and degradation fragments (red circle) were also observed (Figure S36f, Supporting Information).In addition, a stronger T 1 -weighted MRI signal was observed in the tumor-bearing mouse treated with PMn SA GMSNs-V injection than in control mice (Figure S36g, Supporting Information).As displayed in Figure S37, Supporting Information, the intensity of the MRI signal first increased and then decreased.At an injection time of 1 h, the intensity of MRI signal reached its maximum value.Notably, a much brighter tumor area (red circle) was observed after injection, indicating PMn SA GMSNs-V accumulated in the tumor area and gradually released Mn 2+ and Gd 3+ via GSH, thereby providing an effective T 1 -weighted MRI contrast for diagnosing tumors.
To improve the biocompatibility of Mn SA GMSNs-V@Ce6, HS-PEG was employed to synthesize PMn SA GMSNs-V@Ce6 (Figure S38).The peaks located at 1072 and 2979 cm −1 were observed for PMn SA GMSNs-V@Ce6 and were ascribed to the stretching vibration of C-H and -CH 2 -O-, indicating the successful modification of Mn SA GMSNs-V@Ce6.The cellular phagocytosis behavior of PMn SA GMSNs-V decorated by FITC was evaluated by the HeLa cells.As shown in Figure S39, Supporting Information, cell phagocytosis results for PMn SA GMSNs-V at different times were collected by the confocal laser scanning microscopy (CLSM).In the overlay image, the nuclei and cytoplasm of HeLa cells were labeled with DAPI (blue fluorescent) and FITC (green fluorescent), respectively, indicating that more PMn SA GMSNs-V were taken up by the HeLa cells over time.Subsequently, the in vitro cytotoxicity of PMn SA GMSNs-V and PMn SA GMSNs-V@Ce6 at the cellular level were studied by 4,5-dimethylthiazol2-yl-2,5-diphenyl tetrazolium bromide (MTT) method.The cell viabilities of L929 were calculated to be above 95% in each group when the concentration of PMn SA GMSNs-V and PMn SA GMSNs-V@Ce6 increased from 0 to 400 μg mL −1 (Figure S40, Supporting Information).These results revealed that PMn SA GMSNs-V and PMn SA GMSNs-V@Ce6 were nontoxic to normal cells and had good biocompatibility.The cytotoxicity of PMn SA GMSNs-V@Ce6 in the HeLa cells was investigated based on the synergy of CDT and PDT (Figure 5a).The survival rate of HeLa cells in each group irradiated with 650 nm laser irradiation was as high as 96%, revealing that laser irradiation by 650 nm was non-toxic to HeLa cells.Nevertheless, the viability of HeLa cells incubated with PMn SA GMSNs-V decreased with in- Viabilities of HeLa cells in the control group, treated with 650 nm, PMn SA GMSNs-V, PMn SA GMSNs-V plus 650 nm, PMn SA GMSNs-V@Ce6 and PMn SA GMSNs-V@Ce6 plus 650 nm (0.6 W cm −2 , 10 min).b) Intracellular ROS detection using DCFH-DA probe and c) CLSM images dyed with AM and PI of HeLa cells treated with PMn SA GMSNs-V, PMn SA GMSNs-V plus 650 nm, PMn SA GMSNs-V@Ce6 and PMn SA GMSNs-V@Ce6 plus 650 nm.Apoptosis of HeLa cells detected by flow-cytometry in the groups of control, 650, PMn SA GMSNs-V, PMn SA GMSNs-V@Ce6, PMn SA GMSNs-V plus 650, and d) PMn SA GMSNs-V@Ce6 plus 650.CLSM images of HeLa cells stained by JC-1 after incubation with PMn SA GMSNs-V, PMn SA GMSNs-V@Ce6, PMn SA GMSNs-V plus 650 nm, and e) PMn SA GMSNs-V@Ce6 plus 650 nm (error bars denote the standard deviation (n = 3, mean ± SD).
creasing concentrations of PMn SA GMSNs-V from 0 to 400 μg mL −1 , suggesting that a large amount of • OH was produced by high-efficiency Mn single atomic active centers.Similarly, when Ce6 was integrated into PMn SA GMSNs-V, the cells treated with PMn SA GMSNs-V@Ce6 without laser irradiation exhibited a survival rate equivalent to that of PMn SA GMSNs-V.When the concentration of PMn SA GMSNs-V and PMn SA GMSNs-V@Ce6 reached a maximum of 400 μg mL −1 , the survival rates of HeLa cells were ≈46.1 and 45.9% in the existence of PMn SA GMSNs-V and PMn SA GMSNs-V@Ce6 alone, respectively.Then, when HeLa cells were exposed to 650 nm light illumination, it was obvious that the viabilities of cells incubated with PMn SA GMSNs-V decreased to ≈36.8%, which was attributed to the increased ROS generation by inducing efficient separation of photogenerated electron-holes.More importantly, the viability of HeLa cells decreased obviously to about 13.8% after treatment with PMn SA GMSNs-V@Ce6 plus 650 nm light illumination (0.6 W cm −2 ) due to the additive effects of numerous ROS.
1c] To further investigate the reaction mechanism by which PMn SA GMSNs-V@Ce6 kill cancer cells, an intracellular 2,7-dichlorohydrofluorescein diacetate (DCFH-DA) probe was used to study the production of ROS under different conditions based on the oxidized DCFH with green-fluorescence signal. [42]As shown in Figure S41, Supporting Information, no obvious green fluorescence signals were detected in the groups of control and 650 nm, suggesting that no ROS can be generated in the aforesaid treatment.Because of the active sites of MnSiO 3 and atomic dispersed Mn in PMn SA GMSNs-V, HeLa cells incubated with PMn SA GMSNs-V exhibited green fluorescence (Figure 5b).For PMn SA GMSNs-V@Ce6, HeLa cells incubated with PMn SA GMSNs-V without 650 nm laser irradiation showed similar the fluorescence intensity similar to that of PMn SA GMSNs-V.However, a stronger green fluorescence was observed in HeLa cells after treatment with PMn SA GMSNs-V plus 650 nm, which was attributed to the multiple ROS generation originating from synergistic effects of the efficient separation of photogenerated electrons-holes and atomic dispersed Mn sites.In addition, HeLa cells incubated with PMn SA GMSNs-V@Ce6 with 650 nm laser irradiation presented strongest green fluorescence in all groups when Ce6 was introduced to PMn SA GMSNs-V, following ESR results.Under the 650 nm irradiation, PMn SA GMSNs-V acted as a photosensitizer, and Ce6 were excited simultaneously to generate 1 O 2 .Meanwhile, PMn SA GMSNs-V can not only trigger CAT effect to achieve the decomposition of H 2 O 2 and efficiently convert H 2 O 2 to O 2 and H 2 O due to the introduction of Gd sites but also generate many electron-hole pairs.On the one hand, the generation of O 2 can further alleviate hypoxia in tumors.On the other hand, O 2 will also serve as a substrate for PDT, thereby efficiently achieving 1 O 2 conversion.Then, photogenerated electrons in the CB of PMn SA GMSNs-V can capture O 2 to further produce • O 2 − .2b] Benefiting from atomic dispersed Mn sites in PMn SA GMSNs-V, PMn SA GMSNs-V can generate • OH and • O 2 − by POD-like and OXD-like activities, respectively.
Subsequently, in response to TME (GSH, weakly acidic conditions), the PMn SA GMSNs-V degraded and released Mn 2+ to trigger a Fenton-like chemical reaction to produce a large amount of • OH.Because of the production of multiple ROS, the cancer cell-killing efficiency of PMn SA GMSNs-V@Ce6 based on the synergy of CDT and PDT was investigated using calceinacetoxymethyl ester (calcein-AM) (green signals for representing living cells) and propidium iodide (PI) (red signals for representing dead cells), respectively. [43]As shown in Figure S42, most HeLa cells presented green regions in the groups of control and 650 nm, indicating most of the HeLa cells maintained normal physiological activity.In contrast, the ratio of red regions was detected in PMn SA GMSNs-V and PMn SA GMSNs-V@Ce6 groups because of the Mn single atomic active centers and GSH depletion-induced CDT reaction (Figure 5c).Upon 650 nm laser irradiation, the HeLa cells treated with PMn SA GMSNs-V showed an increased ratio of red cells.For PMn SA GMSNs-V@Ce6 plus 650 nm laser irradiation, the whole regions demonstrated the strongest red fluorescence compared to other groups, indicating PMn SA GMSNs-V@Ce6 plus 650 nm possessed the highest killing ability for the HeLa cells owing to the synergistic results between PDT and CDT.Moreover, the effect of HeLa cells apoptosis was revealed by flow cytometry (Figure 5d and Figure S43).The corresponding proportions of tumor cells apoptosis for PMn SA GMSNs-V, PMn SA GMSNs-V@Ce6, PMn SA GMSNs-V plus 650, and PMn SA GMSNs-V@Ce6 plus 650 were evaluated to be 47.99, 49.25, 59.48 and 80.15%, respectively.Compared with other groups, the HeLa cells treated with PMn SA GMSNs-V@Ce6 plus 650 nanozymes had severe mortality, consistent with MTT results. [44]any studies have shown that apoptosis is closely associated with mitochondrial destruction.Subsequently, the difference in the potential of mitochondrial membrane was detected by 1,1′,3,3′-tetraethyl-imidacarbocyanine (JC-1) staining.Notably, JC-1 mainly displayed red aggregation fluorescence in healthy mitochondria and green monomer fluorescence in unhealthy mitochondria. [45]For control and 650 nm groups, the HeLa cells treated with by JC-1 demonstrated the strongest red fluorescence in whole regions, suggesting no obvious changes in mitochondrial membrane potential (Figure S44, Supporting Information).However, for PMn SA GMSNs-V and PMn SA GMSNs-V@Ce6 without 650 nm laser irradiation, the strong fluorescence intensity ratio of green to red was observed in HeLa cells (Figure 5e).Importantly, the group treated with PMn SA GMSNs-V@Ce6 plus 650 nm laser presented strong green monomer fluorescence, suggesting that the mitochondria of most HeLa cells were destroyed.Besides, the intratumoral GSH levels were further detected using glutathione detection reagent.As shown in Figure S45, Supporting Information, CLSM images display the strongest green fluorescence, indicating highest GSH levels in the groups of control and laser irradiation.Obviously, lower fluorescence signals were observed for PMn SA GMSNs-V and PMn SA GMSNs-V@Ce6 without laser irradiation.More importantly, CLSM images of PMn SA GMSNs-V and PMn SA GMSNs-V@Ce6 with 650 nm laser irradiation showed a sharp decrease in green fluorescence intensity, which was attributed to the GSH depletion.The HeLa cells treated with PMn SA GMSNs-V@Ce6 and laser irradiation exhibited the lowest fluorescence intensity, indicating that the ROS consumed large amounts of GSH produced.In fact, CDT-induced GSH depletion can prevent the loss of ROS, thereby achieving synergy between PDT and CDT.
Benefiting from the satisfactory results on a cellular level, the in vivo antitumor experiments for PMn SA GMSNs-V@Ce6 were systematically investigated in a U14 tumor-bearing mouse model (Figure 6a).First, based on the different treatment conditions, the tumor-bearing animals were randomly divided into six groups including control, 650 nm laser irradiation only (650 nm), PMn SA GMSNs-V, PMn SA GMSNs-V@Ce6, PMn SA GMSNs-V plus 650 nm and PMn SA GMSNs-V@Ce6 plus 650 nm to evaluate the antitumor effects.In Figure 6b, photographs of tumors dissected from the different treatments are collected.Compared to control and 650 nm groups, PMn SA GMSNs-V and PMn SA GMSNs-V@Ce6 exhibited a low tumor suppression effect due to the atomically dispersed Mn only.However, when PMn SA GMSNs-V@Ce6 was illuminated at 650 nm, PMn SA GMSNs-V@Ce6 plus 650 nm demonstrated the highest effect of inhibiting tumor growth due to the synergistic effects of PDT by dual photosensitizers, CDT by GSH-depleted and atomically dispersed Mn active center.Additionally, differences in the body weight and tumor volume were recorded to assess the therapeutic effect during 14 days of treatment (Figure 6c).In comparison with the groups of control and 650 nm, the weight of animals in the other groups was also normal and slightly increased over time, suggesting these treatments basically had no harmful effects on the health of mice.Subsequently, the relative tumor volumes of mouse was recorded for 14 days (Figure 6d).Compared with the control and 650 nm groups, limited suppression of tumor growth was observed in PMn SA GMSNs-V and PMn SA GMSNs-V@Ce6 groups.As expected, for the group of PMn SA GMSNs-V@Ce6 plus 650 nm laser irradiation, much more obvious tumor inhibitory effects were observed, which was attributed to multiple ROS generation.Besides, the hematoxylin and eosin (H&E) stained images of main organs (Figure S46, Supporting Information), including heart, liver, spleen, lung, and kidney, showed no obvious damage or inflammation, indicating the excellent histocompatibility and biosafety of PMn SA GMSNs-V@Ce6.Subsequently, the H&E stained images of representative tumor issues from different groups were also examined to verify the destruction of tumor cells.As shown in Figure 6e, PMn SA GMSNs-V@Ce6 plus 650 nm group exhibited a remarkable decrease in the proportion of blue-stained nuclei, verifing that PMn SA GMSNs-V@Ce6 plus 650 nm treatment displayed the maximum destruction level to tumor cells.Besides, Ki67 staining and terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end labeling (TUNEL) were used to evaluate the tumor apoptosis and proliferation.Compared with other groups, TUNEL and Ki67 stained tumor tissue slices showed that many of tumor cells were necrotic and the proliferation of tumor cells was reduced in the PMn SA GMSNs-V@Ce6 plus 650 nm group.To evaluate the biosafety of PMn SA GMSNs-V@Ce6, the corresponding the Mn and Si contents from tumor tissues and the main organs at various time point after injection were detected by biodistribution in vivo (Figure S47 and Figure S48, Supporting Information).Owing to the capture of blood circulation and reticuloendothelial system, the injected PMn SA GMSNs-V@Ce6 nanozymes were mainly located in the liver and tumor, suggesting that nanozymes possessed efficient excretion capacity and that the toxicity of PMn SA GMSNs-V@Ce6 to mice during treatment was negligible.These results indicated PMn SA GMSNs-V@Ce6 could act as an efficient antitumor agent with high biosafety because of the multiple ROS generation in acidic tumor microenvironment.

Conclusion
In summary, a mass-produced SAzymes synthetic strategy based on OVs-immobilization by a simple hydrothermal process was developed to prepare TME-activable PMn SA GMSNs-V@Ce6 with atomically dispersed Mn/Gd dual sites for synergistic CDT/PDT.OVs induced by co-doping of Gd and Mn ions into SiO 2 can capture unbound metal ions to form atomically dispersed Mn and Gd, as verified by the XANES and HAADF-STEM.The local electrons of atom-catalyzed Mn active sites were modulated by the introduction of Gd sites, thereby achieving effective CAT-like-, OXD-like-and POD-like-enzyme activities.Systemic DFT calculation results revealed that the O-O bond of endogenous H 2 O 2 could be elongated and weakened due to the presence of OVs, thereby promoting the decomposition of endogenous H 2 O 2 .Importantly, PMn SA GMSNs-V@Ce6 can serve as dual photosensitizers to generate 1 O 2 upon 650 nm light excitation.The PMn SA GMSNs-V-triggered CAT effect is beneficial for O 2 generation and favorable of PDT.In addition, laserirradiated PMn SA GMSNs-V can generate several electron-hole pairs to facilitate the generation of • O 2 − .Significantly, intratumoral GSH triggered the biodegradation of PMn SA GMSNs-V to liberate Fenton-like Mn 2+ that reacted with H 2 O 2 to produce • OH for self-enhanced CDT.The in vivo experiments results revealed that PMn SA GMSNs-V@Ce6 had the highest anticancer efficacy due to effective GSH depletion and multiple ROS.Interestingly, tumor GSH-induced release of Mn 2+ and Gd 3+ exhibited ideal T 1 relaxivity for tumor-enhanced MRI.Our work provides a novel strategy to encapsulate atomically dispersed Mn/Gd dual sites by OVs-anchored in a biodegradable nano-diagnostic platform with dual photosensitizers to achieve synergistic effects of GSHdepletion enhanced CDT and hypoxia-improved PDT.
Figure 1.a).SEM image of MSNs, b) TEM images of MSNsand c) Mn SA GMSNs-V, d) HAADF-STEM image of Mn SA GMSNs-V, e) corresponding TEM-EDS mapping, and EDS of Mn SA GMSNs-V.The high-resolution XPS spectra of Mn SA GMSNs-V for f) Gd 4d, g) Mn 2p, and h) O 1s.

Figure 2
Figure 2. a).Normalized XANES of Mn K-edge spectra of Mn foil, MnO 2 Mn 2 O 3 , and Mn SA GMSNs-V and b) Gd L 3 -edge spectra of Gd foil, Gd 2 O 3 and Mn SA GMSNs-V.c) FT k 3 -weighted Mn K-edge EXAFS spectra of Mn foil, MnO 2 , Mn 2 O 3, and Mn SA GMSNs-V and d) corresponding FT-EXAFS fitting curves at R space of Mn.FT k 3 -weighted Gd L 3 -edge EXAFS spectra of Gd foil, Gd 2 O 3, and e) Mn SA GMSNs-V and f) corresponding FT-EXAFS fitting curves at R space of Gd. g-l) WT-EXAFS plots of Mn SA GMSNs-V and corresponding reference samples.

Figure 4 .
Figure 4. a).UV-vis absorption spectra of SiO 2 , Mn SA GMSNs-V, Ce6, Mn SA GMSNs-V@Ce6, MB degradation by • OH generated by different concentrations of GSH-treated b) Mn SA GMSNs-V@Ce6 (1 mg mL −1 ) and c) H 2 O 2 (8 mM).d) Time-dependent absorption spectra and e) the corresponding retention rate of DPBF in the presence of Mn SA GMSNs-V and Mn SA GMSNs-V@Ce6 under 650 nm laser irradiation.GSH depleting abilities using DTNB as f) the trapping agent of sulphydryl (-SH) in GSH.ESR spectra for detection of 1 O 2 , • O 2 − and • OH versus magnetic field in different conditions for Mn SA GMSNs-V and Mn SA GMSNs-V@Ce6 (g-i).