Nanoenzyme-Augmented Cancer Sonodynamic Therapy by Catalytic

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Nanoenzyme-Augmented Cancer Sonodynamic Therapy by Catalytic Tumor Oxygenation Piao Zhu, Yu Chen, and Jianlin Shi ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b00999 • Publication Date (Web): 03 Apr 2018 Downloaded from http://pubs.acs.org on April 3, 2018

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Nanoenzyme-Augmented Cancer Sonodynamic Therapy by Catalytic Tumor Oxygenation Piao Zhu,1,2 Yu Chen*1 and Jianlin Shi*1 1

State Key Lab of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, P. R. China 2

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

1

ACS Paragon Plus Environment

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Abstract: Ultrasound (US)-triggered sonodynamic therapy (SDT) can solve the critical issue of low tissue-penetrating depth of traditional photo-triggered therapies, but the SDT efficacy is still not satisfactorily high on combating cancer at current stage. Here we report on augmenting the SDT efficacy based on catalytic nanomedicine, which takes the efficient catalytic features of nanoenzymes to modulate the tumor microenvironment (TME). The multifunctional nanosonosensitizers have been successfully constructed by the integration of MnOx component with biocompatible/biodegradable hollow mesoporous organosilica nanoparticles (HMONs), followed by the conjugation with protoporphyrin (PpIX, as the sonosensitizer) and cyclic arginine-glycine-aspartic pentapeptide c(RGDyC) (as the targeting peptide). The MnOx component in composite nanosonosensitizer acts as the inorganic nanoenzyme for converting the tumor-overexpressed hydrogen peroxide (H2O2) molecules into oxygen and enhancing the tumor oxygen level subsequently, which has been demonstrated to facilitate the SDT-induced reactive oxygen species production and enhance the SDT efficacy subsequently. The targeted accumulation of these composite nanosonosensitizers efficiently suppressed the growth of U87 tumor xenograft on nude mice after the US-triggered SDT treatment. The high in vivo biocompatibility and easy excretion-out of these multifunctional nanosonosensitizers from the body have also been evaluated and demonstrated to guarantee their future clinical translation, and their TME-responsive T1-weighted magnetic resonance imaging capability provides the potential for therapeutic guidance and monitoring during SDT. Keywords: Sonodynamic therapy, Protoporphyrin, Nanoenzyme, Tumor microenvironment, Nanomedicine

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Though progresses have been achieved recently, the death rates of cancer are still increasing, causing cancer diseases one of the leading deaths nowadays.1 There is an urgent need for the development of minimally invasive or non-invasive therapeutic modalities to avoid the severe side effects and painful experience of traditional protocols, such as conventional surgical excision, radiation therapy and chemotherapy, which strongly depends on the tumor size, location, histologic type, etc.2, 3, 4, 5 The emerging external-triggered therapeutic modalities, such as light-triggered photodynamic therapy (PDT) and photothermal therapy (PTT), have been extensively explored for cancer therapy in the past decade, which, unfortunately, suffers from the intrinsically limited tissue-penetrating depth of light, leading to the non-effectiveness in the treatments against deep-seated tumor.6, 7, 8 Alternatively, ultrasound (US), as a mechanical wave, has been extensively explored for cancer diagnosis and therapy in clinics, such as US imaging and high-intensity focused US.9, 10, 11 Compared to light-triggered photosensitizers for PDT, US is also capable of triggering the sonosensitizers for the generation of reactive oxygen species (ROS) for cancer therapy, which is thereby termed as sonodynamic therapy (SDT).12,

13, 14

The low bioavailability and

chemical/biological instability of traditional organic sonosensitizer molecules, however, result in the low therapeutic efficacy of SDT and hinder its further clinical translation.15, 16,

17

Some

alternative inorganic nanoparticles such as TiO2, have been demonstrated to be effective in augmenting the SDT efficacy,18 but their hard biodegradation and unclear biosafety issues still remain unsolved.19 We have recently loaded organic sonosensitizer molecules into the mesopores for SDT-based cancer therapy.20 However, the low ROS-generation efficacy and the lack of targeting mechanisms remain critical issues in achieving desirable tumor-suppression effect and further high therapeutic outcome. After re-visiting the SDT mechanism, it has been found that the characteristic hallmarks in solid tumor, i.e., tumor microenvironment (TME), significantly influences the therapeutic 3


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efficacy of SDT. On the one hand, the insufficient oxygenation (hypoxia) in TME would lead to the low-production of singlet oxygen (1O2).20, 21, 22, 23, 24 On the other hand, the high concentration of glutathione (GSH) in TME consumes the as-produced 1O2 by SDT.25, 26, 27, 28 It is highly challenging to mitigate the influences of these two TME-associated factors on the SDT efficacy.29, 30, 31 Recent results have demonstrated that manganese oxide (MnO2) nanoparticles could efficiently modulate the TME oxygenation owing to their hydrogen peroxidase property and advantaged breaking-up behavior in either mildly acidic condition or in the presence of GSH. The related process can be expressed by the following reactions.28, 31, 32, 33 MnO2 + 2H+ → Mn2+ + H2O + 1/2O2↑ MnO2 + H2O2 + 2H+→ Mn2+ + 2H2O + O2↑ MnO2 + GSH → Mn2+ + GSSG MnO2 compound will decompose and generate oxygen in the reducing condition by GSH, which has been demonstrated to consume GSH and enhance the PDT efficacy.27, 28, 34 In addition, MnO2 nanoparticles have been demonstrated to act as the catalase-like nanoenzyme to react with overexpressed hydrogen peroxide (H2O2) in TME to produce oxygen molecules, which could overcome tumor hypoxia and enhance the therapeutic efficacy of radiation therapy 35, 36, 37 In order to modulate the hypoxia of TME and subsequently enhance the SDT efficacy on combating cancer, in this work, an intelligent nanoplatform has been constructed with excellent specificity to both endogenous TME and exogenous US irradiation. Among the large amount of silica-based delivery nanosysterms, hollow mesoporous organosilica nanoparticles (HMONs) have attracted great attention due to their well-designed structural features and ingenious adjustable composition characteristics as compared to inorganic mesoporous silica nanoparticles (MSNs). On one hand, the hollow nanostructure of HMONs can guarantee the high drug-loading capacity of the nanocarrier because such a hollow interior provides large reservoirs for the guest drug molecules. On the other hand, the organic-inorganic hybrid composition of HMONs brings 4


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the easy biodegradation feature of organic moieties within the hybrid framework, which can significantly accelerate the biodegradation of silica-based nanocarrier.38, 39, 40, 41, 42 Based on the strong oxidation potential of MnO4- and reduction potential of structure-directing organic agent CTAC,43, 44 MnOx nanoparticles were in-situ generated in the mesopore channels of HMONs by a simple redox reaction, which then acts as a nanoenzyme to catalyze the decomposition of over-expressed H2O2 in TME for oxygen production. Organic sonosensitizers (protoporphyrin, PpIX) were further loaded into the hollow interior and mesopores of HMONs-MnOx, followed by anchoring RGD (Arg-Gly-Asp) peptide onto the surface for active targeting delivery (PpIX@HMONs-MnOx-RGD, designated as PMR).45 The post-elevated oxygen level in TME could significantly enhance the SDT effect of as-designed PMR, which has been systematically evaluated and demonstrated both in vitro at cellular level and in vivo on tumor-bearing xenograft on nude mice.

Results and discussion Construction

and

Characterization

of

PMR

Nanosonosensitizers.

Silica-based

nanoplatforms have been extensively explored for drug delivery based on their specific mesoporous structure (e.g., large surface area, high pore volume and tunable pore size) and abundant surface chemistry.46, 47, 48 To further improve the biocompatibility and biodegradability of mesoporous silica, molecularly organic-inorganic hybrid HMONs were adopted to load MnOx components (as the catalase-like nanoenzyme) and subsequent protoporphyrin (PpIX, as the organic sonosensitizer) in the hollow interior and mesopore channels (Figure 1a). Typically, mesoporous silica nanoparticle was synthesized by sol-gel process, which acted as the hard template for the further coating of a mesoporous organosilica layer based on the “chemical homology” mechanism.38, 42, 49 The mesoporous silica nanoparticle was then removed according to our previously developed “structural difference-based selective etching” strategy.50 Potassium 5


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permanganate (KMnO4) is a strongly oxidative agent, which was further introduced to oxidize the hybridized organic components within the framework of HMONs, achieving the in-situ growth of MnOx components within the mesopores of HMONs (HMONs-MnOx). The surface of HMONs-MnOx was further modified with amino group via grafting (3-Aminopropyl) triethoxysilane (APTES) by reacting with silanol groups on HMONs (HMONs-MnOx-NH2) for the subsequent conjugation of PpIX (PpIX@HMONs-MnOx, abbreviated as PM). Finally PpIX@HMONs-MnOx was modified with RGD peptide via an intermediate connecting molecular bifunctional PEG derivative Maleimide PEG NHS Ester (designated as PpIX@HMONs-MnOx-RGD, abbreviated as PMR), which could specifically recognize and bind to the overexpressed integrin αvβ3 on the cancer-cell membrane.51 These PMR nanosonosensitizers could efficiently accumulate into the tumor tissue via both the passive targeting by enhanced permeability and retention (EPR) effect and positive RGD targeting (Figure 1b). Upon reaching the tumor tissue, the MnOx component acted as the catalase-like nanoenzyme to decompose H2O2 for the O2 production and alleviate the tumor hypoxia afterwards. The up-regulated oxygenation of tumor further augmented the SDT efficacy by producing more amounts of ROS such as singlet oxygen (1O2) upon external US irradiation. Especially, the integrated MnOx could act as the acidity-responsive contrast agents for T1-weighted MR imaging, providing the potential for therapeutic SDT guidance and monitoring.27, 31 The MSNs of around 70-80 nm in diameter and high dispersity (Figure 2a) was chosen as the hard template for coating a mesoporous organosilica layer with thioether-bridged hybrid composition (SiO2@MONs). These template-etched HMONs show the particle size of around 90 nm and maintains the high dispersity of initial MSNs (Figure 2b and c). Especially, the presence of well-defined mesopores can also be easily distinguished in the high-resolution SEM image (Figure 2b). The spherical morphology and well-designed hollow nanostructure were 6


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maintained after further stepwise MnOx integration, PpIX conjugation and RGD modification (Figure 2d, e and f). The initial organic-inorganic hybrid HMONs have a large surface area of 736.2 m2·g-1 and a high pore volume of 2.4 cm3·g-1. Comparatively, the as-obtained PMR after stepwise loading and modification shows much lowered surface area and pore volume of 97.6 m2·g-1 and 0.8 cm3·g-1, respectively (Figure 2g), which is due to the loading of multiple agents within the mesopores. This result also demonstrates that the mesopores can act as the reservoirs for the loading of multiple agents for varied theranostic purposes. The evolution of Zeta potential further confirms the successful synthesis in each preparing step (Figure 2h). Typically, the surface of HMONs was negatively charged due to the presence of large amounts of silanol groups, and the surface amino-group modification turned the negative Zeta potential into positive one. The subsequent PEGylation and RGD conjugation further decreased the surface potential of nanosonosensitizers. The hydrolyzed particle diameters of HMONs and PMR are 108.48 nm (PDI: 0.385) and 133.34 nm (PDI: 0.163) as determined by the dynamic light scattering technique (Figure 2i). This work adopts thioether-bridged organosilica (BTES) as the precursor to fabricate HMONs as the cargo carrier for further functionalization, which is based on the consideration that tetrasulfide bond (-S-S-S-S-) in thioether is physiologically active and responsive to the reducing microenvironment of tumor for easy biodegradation, making HMONs more promising as compared to traditional MSNs with pure -Si-O-Si- framework.38, 40, 49 After co-incubating with GSH (10 mM) for 24 h, PMR was found slightly biodegraded as compared to the pure SBF group with intact spheres. Especially, the co-incubation in GSH solution exhibited much higher biodegradation rate than the co-incubation in pure PBS in the following 15 days’ biodegradation evaluation (Figure S2). In addition, the accumulated percentage of Si component biodegradation in 10 mM GSH reached 70% (Figure S3), indicating the easy biodegradation of fabricated composite nanosysterm. The

13

C and

29

Si cross-polarization MAS (CPMAS) spectrum of 7


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HMONs shows the characteristic signal of silsesquioxane framework (Figure 3a and 3b). The 13

C NMR spectrum of HMONs shows prominent resonances at 12.3, 23.4, and 41.7ppm which

can be assigned to the 1C, 2C, and 3C carbon species of –Si-1CH22CH23CH2–S–S–S–S– 3

CH22CH21CH2–Si- within the framework of HMONs, respectively. Both Q and T silicon sites of

HMONs can be distinguished in

29

Si MAS solid-state NMR spectrum. In the right range, the

characteristic signal can be indexed to the resonances of Si (OSi)4 (4Q, δ = –110.3 ppm) and (OH)Si(OSi)3 (3Q, δ = –100.6 ppm), respectively, and the signals at –58.2 and–65.5 ppm can be assigned to the silicon resonances of 2T [(SiO)2(OH)SiC] and 3T [(SiO)3SiC], respectively.49, 52 The characteristic peaks of S-S and S-C (520–720 cm−1) bonds in the Fourier transform infrared (FTIR) spectrum (Figure S1) and the stretching vibrations of S-S and S-C (438, 487, and 633 cm−1) in the Raman spectrum (Figure 3c) further demonstrate the organic-inorganic hybrid compositions of HMONs.49 In addition to the specific organic-inorganic hybrid composition with bridged thioether groups within the framework, MnOx and PpIX were stepwise loaded into the interior and mesopores of HMONs to form PMR nanosonosensitizers (Figure 3d). MnOx-based nanoplatforms have been well demonstrated to catalyze the decomposition of H2O2 to generate oxygen, which can in situ elevate the oxygen level in TME.27,

28, 31, 34

To reveal the chemical status of MnOx,

HMONs-MnOx nanocomposites were analyzed by X-ray photoelectron spectroscopy (XPS). According to XPS simulation, the relative contents of Mn2+, Mn3+ and Mn4+ were determined to be 34.2%, 37.4% and 28.4%, respectively (Figure 3e). By quantifying the amount of MnO4- in the supernatant collected, the amounts of MnOx loaded in the nanoparticles was determined to be around 15%. Similarly, by comparing the UV-vis absorption spectra between PpIX@HMONs with HMONs, a prominent absorbance was distinguished at the site of 410 nm, which was indexed as the characteristic peak of PpIX (Figure 3f).20 The loading amount of PpIX was calculated to be 18% based on the typical Langer-Beer law (Figure S4). To further reveal the 8


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composition of PMR, the element mappings of O, Si, S, and Mn exhibit the high uniformity of each element distribution in the framework of PMR.

In Vitro Catalase-Like Nanoenzyme Activity and ROS-Generating Efficacy of PMR upon US Irradiation. The MnOx component within the mesopores of PMR exhibits catalase-like activity on the decomposition of H2O2 to generate O2. Its catalytic activity is also strongly dependent on the pH value, and the mild acidity could promote such a catalytic reaction. It is considered that the TME is also mildly acidic, therefore such a nanoenzyme-catalyzed reaction is highly beneficial for tumor-specific therapy because the large amount of oxygen (O2) production would effectively mitigate the tumor hypoxia and provide the oxygen source for the ROS production (Figure 4a). It has been found that the O2 production is strongly dependent on the Mn concentrations in PMR where the higher Mn concentration leads to the production of more oxygen molecules (Figure 4b). Importantly, the mild acidity causes the substantially enhanced oxygen production rate in the presence of PMR as the nanoenzyme (Figure 4c). It should be noted that the mild acidity could also induce the dissolution of MnOx component in PMR, which results in the slow release of manganese ions as confirmed by previous reports

35, 36, 37, 53, 54, 55, 56

and our observations (Figure S5). Such a phenomenon is beneficial for the T1-weighted MR imaging because the released manganese ions would create the maximum interaction opportunity to access to the surrounding water molecules, therefore the MRI performance could be substantially enhanced afterwards.44, 57, 58 The nanoenzyme-catalyzed O2 generation could provide the oxygen source for SDT-induced ROS

production.

To

verify

the

exact

reactive

oxygen

species

generated

by

nanosonosensitizers-assisted SDT process, the electron spin resonance (ESR) spectra were acquired

after

US

irradiation

under

different

conditions

(Figure

4d).

2,

2,

6,

6-tetramethylpiperidine (TEMP) is a typical spin-trapping agent for 1O2, which yields TEMPO (2, 9


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2, 6, 6-tetramethyl-1-piperidinyloxyl) free radical, and causes the ESR signal to split into a characteristic 1:1:1 triple signal.59,

60, 61

Though all setting groups clearly showed the

characteristic signal of 1O2, higher signal intensity was achieved in PpIX@HMONs groups compared with the non-materials group. Much stronger signal appeared at prolonged time duration or increased power density of the US irradiation (Figure 4d). In addition, a weak signal intensity of 1O2 was observed in the PpIX@HMONs + TEMP group even no ultrasonic treatment was adopted, due to the fact that PpIX is also a photosensitizer and the whole experimental process would be unavoidably exposed to light, as a result the weak 1O2 signal was observed due to this photodynamic effect. With the prolonging of ultrasonic-treatment duration, the absorbance intensity of 1, 3-diphenylisobenzofuran (DPBF), a typical 1O2 analytical reagent, decreased significantly, demonstrating that DPBF was oxidized by the generated 1O2. In addition, a photoluminescence measurement was conducted to detect the intensity of a Singlet Oxygen Sensor Green (SOSG) probe, which is highly specific to 1O2. Under harsher chemical environment in the presence of acidic H2O2, the PM group exposure to US irradiation showed a largely enhanced 1O2 production efficiency as compared to PpIX@HMONs group without the integrated MnOx component, which was contributed by the MnOx-catalyzed H2O2 decomposition to produce O2 as the oxygen source of SDT, as well as the MnOx-induced GSH oxidation to protect 1O2.62

In Vitro Nanoenzyme-Augmented SDT Efficacy against Cancer Cells. The PMR nanosonosensitizers could selectively bind to the overexpressed integrin αvβ3 on the surface of U87 cancer cells by the surface-conjugated c(RGDyC) peptide,63 which then enter the cancer cells via the typical endocytosis process (Figure 6a). The US-triggered SDT process triggers the 1

O2 generation to kill the cancer cells, which is enhanced by the MnOx-catalyzed O2 production.

Initially, confocal laser scanning microscopy (CLSM) and flow cytometry analysis were 10


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conducted to observe the intracellular uptake of PMR because the red fluorescence of PpIX in PMR could be used to track the nanosonosensitizers inside the cancer cells. It was found that the red fluorescence intensified at the extended co-incubation durations (from 4 h to 8 h) of PMR with U87 cancer cells (Figure 5a). Importantly, the targeted intracellular uptake of PMR was demonstrated owing to the presence of RGD peptide, a binding agent to cancer-cell membrane (U87 cancer-cell line) specifically overexpressed with integrin αvβ3, making PMR easier to enter the U87 cancer cells. The intensity of intracellular red fluorescence of PM-endocytosed cancer cells is much weaker as compared to that in the PMR group (Figure 5a), demonstrating the high targeting efficacy. Compared with PpIX loaded within HMONs, the naked drug PpIX is too small to keep retained in cancer cells, leading to a much weakened red fluorescence intensity (Figure S6). The further cytometry analysis is also in consistence with the CLSM observation where the PMR group has much higher intracellular red-fluorescence intensity (Figure 5b). To verify the in vitro mechanism of nanoenzyme-augmented SDT effect against cancer cells at cellular level, a ROS probe, 2′-7′-dichlorofluorescein diacetate (DCFH-DA) was used to detect the intracellular 1O2 levels by CLSM observation, which was based on the green fluorescence from 2, 7-dichlorofluorescein (DCF) converted from DCFH-DA in the presence of 1O2. As expected, upon US irradiation, PMR + US group exhibits stronger green fluorescence than that of PR + US group (Figure 6b, S7), which is in accordance with the result of SOSG intensity in the TME-modulated solution resulted from the enhanced 1O2 generation. This effect is due to the additional oxygen produced from MnOx nanoenzyme-catalyzed H2O2 decomposition, as well as the down-regulation of GSH to protect the generated 1O2 by the reaction between GSH and MnOx component. The weak green fluorescence was also observed in the PMR group without US irradiation because of the photosensitive effect of PpIX, which is in consistence with in vitro ESR characterization. For comparison, the non-treated group and US only group show weak green fluorescence. 11


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To

evaluate

the

therapeutic

efficacy

of

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nanoenzyme-augmented

SDT

by

PMR

nanosonosensitizers, both CLSM observation and flow cytometry analysis were used (Figure 6b). It has been found that the introduction of PMR combined with US irradiation significantly induces the cancer-cell death, and the therapeutic trend is in consistence with the 1O2 production efficacy. Furthermore, the quantitative therapeutic efficacy was evaluated by the standard cell-counting kit 8 (CCK-8) assay (Figure 6c). After co-incubation with U87 cancer cell for 24 h, the cell viability still maintain above 80% even at the PMR concentration reaching 50 µg·mL-1. Especially, these PMR nanoparticles also show the low cytotoxicity to the normal cells such as human umbilical vein endothelial cells (HUVECs) (Figure S8). Upon exposure to US irradiation, the cell viability sharply decreases. Especially, such a SDT-induced therapeutic effect is PMR concentration-

(Figure

6d),

US

power

density-

(Figure

6e) and

US

irradiation

duration-dependent (Figure 6f).

In Vivo Up-Regulation of Tumor Oxygenation and MR Imaging Capability of MR. The MnOx component in nanosonosensitizers is effective for decomposing H2O2 to produce oxygen molecules (Figure 7a). Based on the H2O2 overexpression of tumor, H2O2 decomposion is expected to increase the oxygen level in TME. To demonstrate this assumption, photoacoustic (PA) imaging was adopted to in-situ characterize the changes of oxygen levels in vivo because PA imaging is a typical method for real-time mapping the oxygenation status in vivo based on the different absorbance spectra between oxygenated and deoxygenated hemoglobin.64 The tumor-bearing mice were subjected to PA imaging at the wavelengths 850 and 750 nm after the intravenous injection of nanosonosensitizers.65 An obviously enhanced PA signal was observed after the nanosonosensitizer administration (Figure 7b), and the intra-tumor oxyhemoglobin saturation shows the substantial increase from 4.8% to 18.7% (Figure S10b), indicating the effectiveness of MnOx component in the nanosonosensitizers in TME oxygenation. 12


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To further verify whether the hypoxia could be alleviated by the injection of the nanosonosensitizers or not, U87 tumor sections were ex vivo subjected to HypoxyprobeTM immunofluorescence stain, an immunohistochemistry marker to directly detect the hypoxia area of tumor, 4 h after intravenous injection.66 No differences were observed between control and PR group without MnOx integration, while significant suppressions in tumor hypoxia could be distinguished in MR and PMR groups in comparison to those without MnOx (Figure 7d). This result is also in consistence with the hypoxia-inducible factor 1 (HIF-1α) expression level as analyzed by western blot technique (Figure 7c). The HIF-1α is a main regulator of the transcriptional response to acute and chronic hypoxia.32, 67, 68. As expected, the HIF-1α/β-actin ratio of groups containing MnOx was decreased by almost 1.5 times as compared to the groups without MnOx (Figure S10a). In terms of protein expression, MnOx induced an essential change of the TME, rather than a transient tumor oxygenation.69 To evaluate the potential T1-weighted MR imaging capability of MnOx component in nanosonosensitizer, HMONs-MnOx dispersions with gradiently increased concentrations of Mn2+ (0.01 mM, 0.02 mM, 0.04 mM, 0.08 mM, 0.15 mM,0.30 mM and 0.60 mM) and pretreated under different concentrations of GSH and different pH at room temperature were scanned in the clinical MR scanner. The relaxation rate (r1 value) of HMONs-MnOx at pH 7.4 without GSH was measured to be only 0.33 mM-1s-1, and it increased to 3.02 mM-1s-1 and 3.39 mM-1s-1 under the GSH concentrations of 5 mM and 10 mM, respectively (Figure 8a). Especially, after soaking of HMONs-MnOx in acid solutions without GSH addition, the r1 value increased to 4.25 mM-1s-1 and 4.51 mM-1s-1 in the solutions of lowered pH values of 6.0 and 5.0, respectively (Figure 8c). Such a more than ten times enhancement in T1-weighted MR relaxation rate is attributed to the Mn2+ released from MnOx component under the reducing or acidic condition, as can be visualized by the weakening brown color (Figure S9), which results in more exposed paramagnetic centers accessible to water molecules.70 The in vitro T1-weighted MR imaging 13


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reveals significant brightening effects with the increases of Mn concentration (Figure 8b, 8d) and GSH concentration (Figure 8b), as well as with the decrease of pH (Figure 8d). Therefore, these MnOx-HMONs nanocomposites could act as TME-responsive contrast agents for T1-weighted MR imaging (Figure 8g). U87 tumor-bearing mice were used to conduct T1-weighted in vivo MRI by intravenous injection of MR contrast agent. A significant time-dependent brightening effect was observed in the T1-weighted MR images at the tumor site after the intravenous administration of MR (Figure 8e). Compared with non-tumor tissue area, the quantitative MRI signal intensities of tumor-bearing group show a substantial enhancement (Figure 8f). Both the passive targeting by EPR effect and the positive targeting of conjugated RGD peptide contribute to the high accumulation of MR into tumor, followed by the release of Mn2+ from MR in the specific TME (reducing microenvironment and mild acidity), which maximizes the interaction between paramagnetic Mn ions and water molecules and enhances the MRI contrast subsequently.

In vivo biocompatibility and excretion assay of PMR. Furthermore, in vivo biocompatibility assays of PMR nanosonosensitizers were comprehensively conducted on healthy female Kunming mice. All the blood indexes, including alanine aminotransferase (ALT, 41.63 ± 13.91 U/L), aspartate aminotransferase (AST, 82.80 ± 39.85 U/L), alkaline phosphatase (ALP, 154.00 ± 32.00 U/L), blood urea nitrogen (BUN, 4.55 ± 1.35 mmol/L), creatinine (CREA, 31.82 ± 17.68 µmol/L), red blood count (RBC, 8.78 ± 1.12 1012/L), white blood count (WBC, 8.77 ± 2.95 109/L), hemoglobin (HGB, 145.50 ± 20.50 g/L), mean corpuscular hemoglobin concentration (MCHC, 295.20 ± 26.50 g/L), red cell distribution width (RDW-SD, 16.62 ± 1.09 fL), mean corpuscular hemoglobin (MCH, 16.59 ± 1.09 pg), mean corpuscular volume (MCV, 56.39 ± 3.75 fL), and blood routine levels of hematocrit (HCT, 49.43 ± 6.70 %) of PMR-administrated group, show no significant differences as compared to the control group (Figure 9) and reference values 14


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of 9-weeks aged Kunming mice, except for CREA that many be related to the feeding forage. In addition, no significant body-weight losses were found after intravenous injections of PMR at the Si doses of 5, 10 and 20 mg·kg-1 for one-month feeding. Hence, the PMR administration shows no negative impact on the hematochemistry and physiochemistry of mice in the time frame. In addition, the stable serum ALT, AST, and BUN indexes also indicate that PMR has no significant renal and hepatic toxicities.71 The histological sections of heart, liver, spleen, lung and kidney from the mice sacrificed 30 days post-injection of PMR also reveal no detectable signs of damage or inflammatory lesions from hematoxylin-eosin (H&E)-stained organ slices, indicating the high histocompatibility of PMR nanosonosensitizers (Figure 10a). To quantify the excretion of PMR out of the body, female Kunming mice were intravenously injected with PMR, and the urine and feces were collected for analysis after the injection for prolonged durations. After administration for 48 h, a large amount of Si component was detected to be excreted out of mice (Figure 10b). The accumulated Mn content in urine was higher than that in feces, which might be attributed to the Mn ion release from MnOx in the nanosonosensitizers (Figure 10c). The easy excretion of PMR nanosonosensitizers can be the result of facile biodegradation of these composite nanosystems.

In vivo nanoenzyme-augmented SDT against tumor-bearing xenograft on nude mice by PMR nanosonosensitizers. The high in vitro therapeutic efficacy of PMR-enhanced SDT indicates the potential effectiveness for in vivo applications. To demonstrate this assumption, U87 tumor-bearing xenograft was established for evaluating the in vivo therapeutic efficacy of PMR nanosonosensitizers. Initially, the organ biodistributions were assessed. After the intravenous injections of PMR into tumor-bearing mice for 4 h, 24 h and 48 h, the major organs and tumor tissue were collected for quantitative analyses by ICP-OES to determining the Si contents in each organ and tumor. It has been found that PMR could efficiently accumulate into 15


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tumor with high targeting efficiency of 5.7 % at 4h, which increased to 10.6 % at 48h. (Figure 11a). Both the passive targeting via EPR effect and positive targeting by surface-conjugated RGD contribute to the high targeting efficacy. The blood-circulation half-time was calculated to be 1.1 h (Figure 11b), which indicates the easy elimination of the PMR from the central chamber, such as liver and kidney. By fitting with the double chamber model, the eliminating rate constant of the first 1 h was calculated to be -0.39 µg·mL−1·h-1, almost 39 times higher than that in the following 23 h (Figure 11c). Furthermore, five groups were set to conduct the SDT-based therapeutic evaluation, including control group (non-treated), PMR group (intravenous injection with PMR only), US group (US irradiation only), PR + US (intravenous injection of PR followed by US irradiation) group and PMR + US (intravenous injection of PR followed by US irradiation) group. Based on the results of metabolism, biodistribution and blood circulation half-time, the US irradiation in US group, PR+US group and PMR+US group was repeated at the time point of 1 day, 3 day and 5 day (Figure 11d-f). The body weight and the tumor-growth curves were recorded every two days. During the whole SDT process, no significant abnormal body weight changes were observed (Figure 11e), further indirectly indicating the high therapeutic biosafety of PMR nanosonosensitizers. After US irradiation, even though the tumor growth in the US only group was slightly suppressed, the tumor still kept quick growing. Comparatively, the tumor volumes in PR + US group and PMR + US group show significant suppression effects in the first two weeks as compared to control group and PMR only group (Figure 11d). Fifteen days later, the tumors in PR + US group show significant reoccurrence at the original position. At the end of the whole therapeutic evaluation, the tumor inhibition rate of PMR + US group was as high as 96%, much higher than those of PR + US group and US only group, respectively. The digital photos of tumor sites were in accordance with the tumor-volume change curve (Figure 11g). In comparison, the tumor volume of the control group and PMR group kept growing continuously 16


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during the whole duration, indicating the high efficacy of PMR-enhanced SDT on tumor growth suppression. To further clarify the mechanism of the efficient suppression on the tumor-growth by PMR nanosonosensitizers, tumor sections from mice sacrificed at the end point of the therapeutic evaluation were stained with H&E, TUNEL, and Ki-67 antibody. Typically, both H&E staining and TUNEL staining are used to show the pathological changes in tumor tissues, while Ki-67 staining is for cellular proliferation in tumor sections. In the PR + US and PMR + US groups, only large amounts of cell necrosis are shown in the H&E and TUNEL staining, and the PMR + US group exhibits much stronger cell damages. On the contrary, there is no significant cell necrosis in the control group and PMR only group. The cells show much suppressed proliferation in PMR + US group as shown in the Ki67 staining as compared to other groups (Figure 11h), further indicating the high SDT efficacy as assisted by targeted PMR nanosonosensitizers.

Conclusions In summary, we have successfully developed a well-designed nanosonosensitizer for enhancing US-triggered SDT efficacy by nanoenzyme-based modulation of tumor oxygenation, which has been achieved by integrating MnOx component and sonosensitizer molecules into biocompatible/biodegradable mesoporous organosilica-based hollow nanoplatforms (HMONs in this work). The MnOx component as the nanoenzyme catalyzes the decomposition of tumor-overexpressed hydrogen peroxide (H2O2) to produce oxygen and alleviate the tumor hypoxia subsequently, which provides abundant oxygen sources for SDT-induced ROS production and significantly enhances the SDT efficacy. Especially, the surface RGD peptide conjugation targetedly enhances the substantial accumulation of nanosonosensitizers into tumor tissue. Extensive in vitro and in vivo (U87 tumor xenograft on nude mice) evaluations have demonstrated the high therapeutic efficacy of the targeted nanosonosensitizers for killing the 17


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cancer cells and suppressing the tumor growth. The high in vivo biocompatibility and easy excretion out of the body of these multifunctional nanosonosensitizers have also been demonstrated to guarantee their further clinical translation. This work not only enhance the SDT efficacy by modulating the specific features of TME to a great degree, but also provides an efficient therapeutic modality on combating cancer by US irradiation without the tissue-penetration limitation, which are suffered critically by traditional photo-triggered therapeutics, such as PDT and PTT.

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Methods Synthesis of Molecularly Organic-Inorganic Hybrid HMONs. For the synthesis of hollow mesoporous organosilica nanoparticles (HMONs), a facile “chemical homology” approach was used. Briefly, CTAC (Sigma-Aldrich) aqueous solution (5 g, 10 wt%) and TEA (Sinopharm Chemical Reagent Co.) aqueous solution (0.2 g, 10 wt%) were premixed and stirred at room temperature for 20 min, followed by the addition of TEOS (Sinopharm Chemical Reagent Co., 0.375 mL) dropwise after transferring to a water bath at 90 ℃. The mesoporous silica nanoparticles (MSNs) were formed after hydrolysis/condensation reaction for 1 h. Then, the core-shell MSNs@MONs nanoparticles were prepared by adding a pre-mixed silicon sources, TEOS (0.25 mL) and BTES (Aladdin Biochemical Technology Co., Ltd., 0.5 mL), to the reaction solution for another 4 h reaction. After washing with ethanol for three times, the product was then dispersed into water (4 mL). Part of the solution (0.8 mL) was taken out to be dispersed into water (21 mL), together with ammonia solution (0.4 mL), to etch the MSNs core. The process was lasted for 3 h at 95 °C, and then the white product was collected by centrifugation and washing with water to remove the remnants. To remove the structure-directing agent CTAC, the products were extracted in a mixture of methanol (400 mL) and NaCl (Sinopharm Chemical Reagent Co.., 5 g) for 12 h at 78 °C for three times. Finally, the collected HMONs were centrifuged and washed with ethanol for three times.

MnOx Integration into Mesopores of HMONs. Oxidation/reduction (O/R) reaction between MnO4− and organic moiety within HMONs framework in aqueous solution was in situ triggered to form manganese oxide (MnOx) nanoparticles within mesopores. Briefly, HMONs (100 mg) without extraction were dispersed into deionized water (10 mL) and then KMnO4 (Sinopharm Chemical Reagent Co.) aqueous solution (10 mL, 0.1 M) was added dropwise into HMONs dispersion under the magnetic stirring at room temperature. Followed by transferring the mixture 19


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into a water bath under the magnetic stirring for another 4 h at 40℃ away from light, the production was collected by centrifugation and washed with plenty of water to remove residual MnO4− ions. The final product was extracted by a mixture of methanol and NaCl as mentioned above.

Loading Protoporphyrin into Hollow Interior and Mesopores of MnOx-HMONs. Before loading protoporphyrin (PpIX, Sigma-Aldrich) into the hollow interior and mesopores of HMONs or HMONs-MnOx, the collected HMONs or HMONs-MnOx were firstly dispersed into ethanol (100 mL), followed by adding APTES (Sigma-Aldrich, 100 µL). After water bathing for 6 h at 80℃, the obtained amino-grafted HMONs-NH2 or HMONs-MnOx-NH2 were transferred into MES solution (120 mL, 0.1 mol·L-1, pH = 5). Then, EDC (Sigma-Aldrich, 35.2 mg) and NHS (Sigma-Aldrich, 6.8 mg) were added to activate amidation reaction between amino group of mesoporous nanoparticles and the carboxyl group of PpIX (10 mg), followed by stirring at room temperature for 12 h. The production PpIX@ HMONs or PpIX@HMONs-MnOx (PM) was collected by centrifuging and washing with ethanol.

Synthesis of PEG or RGD Peptide Modified Composite Nanosonosensitizers. For anchoring PEG molecules onto the surface of composite nanosonosensitizers, Methoxy PEG silane (Jenkem Technology Co., Ltd., 25 mg) was dissolved into their ethanol solution (100 mL), which was then refluxed at 80℃ for 12 h. For RGD peptide conjugation, the terminal N-hydroxysuccinimide (NHS) of bifunctional PEG derivative Maleimide PEG NHS Ester (NHS-PEG2000-MAL, Jenkem Technology Co., Ltd.) was used to obtain amino group-modified nanosonosensitizer in PBS under magnetic stirring for 24 h at room temperature. Then, the obtained HMONs-PEG2000-MAL were incorporated ultrafiltration membranes (cut off = 4000 20


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kDa) to remove the residual reactants for several times, which was further re-dispersed into PBS. The c (RGDyC) (Chinese Peptide Company) was added into the dispersion under the magnetic stirring for 24 h at room temperature to trigger the reaction between the thiol group in cysteine and

Maleimide

of

MONs-PEG2000-MAL,

by

which

PpIX@HMONs-RGD

(PR),

HMONs-MnOx-RGD (MR) or PpIX@HMONs-MnOx-RGD (PMR) were synthesized.

Characterization. Transmission electron microscopy (TEM) images were acquired on a JEM-2100F electron microscope operated at 200 kV. Scanning electron microscopy (SEM) images and element mapping were obtained on a field emission Magellan 400 microscope (FEI Company). UV-vis-NIR absorption spectra were conducted on a UV-3101PC Shimadzu spectroscope with QS-grade quartz cuvettes at room temperature. The specific surface area and pore

size

of

the

composite

nanosonosensitizers

were

determined

by

a

nitrogen

adsorption-technique at 77 K on a Micrometitics Tristar 3000 system. X-ray photoelectron spectroscopy (XPS) spectrum was recorded by ESCAlab250 (Thermal Scientific). Dynamic light scattering (DLS) and zeta potential measurements were conducted on a Zetasizer Nanoseries (Nano ZS90, Malvern Instrument Ltd.). The quantitative analysis of each element content in the composite nanosonosensitizers was obtained by inductively coupled plasma atomic emission spectrometry (ICP-AES, Agilent Technologies, US). The confocal laser scanning microscopy (CLSM) images were acquired on FV1000 (Olympus Company, Japan). Flow cytometry analysis for cell apoptosis and the cellular uptake of the nanosonosensitizers were conducted by BD LSRFortessa. The monitoring of the intratumoral oxygen saturation (sO2) was performed by using a Vevo LAZR PA Imaging System (VisualSonics Company, Canada). The ESR characterization was performed on a Bruker EMX electron paramagnetic resonance spectrometer. Raman spectrum was operated on a DXR Raman microscope (Thermal Scientific, USA) with a 532 nm excitation length. All the experiments involving US treatment were conducted using an 21


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US transducer (Chattanooga Co., US).

Degradation Evaluation of PMR in SBF Solution. Typically, in order to determine the time-dependent structural evolution and the accumulated Si release of PMR during the biodegradation process, PMR nanoparticles were added into simulated body fluid (SBF) without or with GSH (5 mM and 10 mM) at 37 °C under slow stirring, respectively. The partially biodegraded PMR was collected by centrifugation, and directly transferred for TEM observation and ICP-OES test at the given time.

In Vitro Detection of O2 Generation. The generated O2 was detected by a JPBJ-609L portable dissolved oxygen meter according to operation instructions in a 40 mL glass bottle under the magnetic stirring. In brief, several varied conditions were chosen for the experiment, including different Mn concentrations (0, 10, 20, 40 µg·mL-1), pH value (pH = 6.5, 7.4) and H2O2 amounts (0, 100 µM), which were conducted by dispersing HMONs-MnOx into H2O2 aqueous solutions.

Qualitative and Quantitative 1O2 Detection. By using the trapping agent TEMP (Dojindo Molecular Technologies), the 1O2 generation by US-activated PpIX@HMONs was detected. For comparison, TEMP + US group, TEMP + PpIX@HMONs (500 µg·mL-1) group, TEMP + PpIX@HMONs + US (with different US irradiation parameters) group were tested by electron paramagnetic resonance spectrometer. In addition, PpIX@HMONs dispersed in N, N-dimethylformamide (DMF, Sigma-Aldrich) (100 µg·mL-1) was employed to determine the quantitative 1O2 generation trend with time followed by adding DPBF (Sigma-Aldrich, 40 µL, 8 mM). The mixture’s absorbance at the wavelength of 410 nm was detected after US irradiation (1.0 MHz, 1.5 W·cm-2, 50% duty cycle, 1 min) every 1 min on a UV-vis spectroscope. Finally, a Singlet Oxygen Sensor Green (SOSG) 22


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probe, highly specific to 1O2, was used for detecting 1O2 from PM under harsher chemical formulation simulated TME in the presence of acidic H2O2 (100 µM) and GSH (10 mM, Sigma-Aldrich).

62, 72

After US irradiation (1.0 MHz, 1.5 W·cm-2, 50% duty cycle, 1 min), the

mixture was tested by a photoluminescence measurement every one min by a fluorescence spectrophotometer (HORIBA FluoroMAX-4, France) using excitation/emission of 488/525 nm for solutions containing: 10 µM SOSG reagent and 100 µg·mL-1 PpIX@HMONs or PM in 10 mL PBS (pH =6.5).

In Vitro and In Vivo MR Imaging. The in vitro T1-weighted signals of HMONs-MnOx at different concentrations treated with different concentration of GSH and different pH values were measured. The prepared HMONs-MnOx nanocomposites were dispersed into different buffer solutions ([GSH] = 0, 5, 10 mM and pH = 7.4, 6.0, 5.0), which were further shaken at a speed of 120 rpm for 1 h at 37 °C. Then, the HMONs-MnOx buffer solutions were taken out and diluted to corresponding concentrations with xanthan gum buffer solution for in vitro MRI test in 2 mL Eppendorf tubes. The in vivo tumor model was established by subcutaneously implanting U87 glioma cells purchased from Shanghai Cell Bank, Chinese Academy of Sciences (CAS) into the tested mice. The nude mice were intravenous injected with MR physiological saline solutions at the Mn dose of 0.08 mg·kg−1 when the tumor volume reached around 200 mm3, and the in vivo T1-weighted MR imaging of U87 tumor-bearing mice was acquired at tumor site and non-tumor tissue area after intravenous injection at pre-determined time intervals. The animal procedures were in agreement with the guidelines for the Animal Care Ethics Commission of Shanghai Tenth People’s Hospital, School of Medicine of Tongji University. The in vitro and in vivo MRI experiment was carried out on a Signa HDXT 3.0 T equipment (GE Medical System, Fudan University Cancer Hospital). The experimental parameters for 23


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T1-weighted fast-recovery spin−echo sequence were set as follows: Slice = 3 mm, Space = 0.5 mm, TR = 1000, 2000, 3000, and 4000, Phase fov = 0.8, Freq × Phase = 384 × 256, Fov = 20, Nex = 2, ETL = 2.

Intracellular Endocytosis by CLSM Observation and Flow Cytometry Analysis. Typically, U87 cells were seeded into the CLSM-specific dish (35 mm × 10 mm, Corning Inc., New York) at a density of 1 × 105 cells/dish and they were adhered to the wall of the dish after incubating for 24 h. Then, the culture media containing PpIX, or PM, or PMR (1 mL, at the equivalent PpIX dose of 10 µg·mL-1 in MEM,) was cultured for 4 h and 8 h. For locating the cell, 100 µL DAPI (Beyotime Biotechnology) was added into the dish to stain the cell nuclei for 15 min followed by observation with CLSM. For flow cytometry analysis, U87 cells were seeded into 6-well microplates at a density of 1 × 105 cells/plate and they were adhered to the wall of the dish overnight. Then, the culture media were replaced by the gradient concentration of PM or PMR (1 mL, at the equivalent PpIX dose of 2.5, 5, 10, 20 µg·mL-1 in MEM) and then were cultured for 4 h. The cellular uptake of nanosonosensitizer was acquired by a flow cytometry equipment.

In Vitro 1O2 Generation at Intracellular Level. Five groups (non-treated cells as control, PMR, US, PR + US, PMR + US) were set to compare the 1O2 generation capability in vitro. Similar to intracellular endocytosis as observed by CLSM, cells were co-cultured with different nanoagents for 4 h. Then, the cells treated with DCFH-DA (Beyotime Biotechnology, 100 µL, 1 µL/9 µL in DMEM) were irradiated by US (1.0 MHz, 1.5 W·cm-2, 50% duty cycle, 1 min) and incubated for another 1 h. Finally, the cells were washed with PBS for three times and observed by CLSM.

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In Vitro Cytotoxicity against HUVECs and U87 cancer cell. Human umbilical vein endothelial cells (HUVECs) and U87 human glioblastoma cells were seeded (3×103 cells in 100 µL of corresponding culture medium per well) in 96-well microplates, and allowed to adhere overnight. Then, the culture media were replaced by 100 µL fresh media containing PM or PMR with gradient concentrations of PpIX for a further 24-h incubation. After washing with PBS (Shanghai Ruicheng BioTech Co., Ltd.) for three times, CCK-8 (Shanghai Ruicheng BioTech Co., Ltd., 100 µL, 10%) was added into each well. A microplate reader (Bio-TekELx800, USA) was used to determine the cytotoxicity according to the absorbance at the wavelength of 450 nm after 90 min co-incubation. In Vivo Sonotoxicity against Cancer Cell. To investigate the sonotoxicity of PMR under different ultrasonic parameters, the U87 cell viabilities were acquired similar to the protocol as mentioned above after US irradiation (1.0 MHz, 1.5 W·cm-2, 50% duty cycle, 1 min) and further incubation for 3 h. For the sonotoxicity evaluation in different experimental groups, the procedure was similar to the 1O2 generation by CLSM observation as mentioned above except the substitution of DCFH-DA with Calcein-AM/PI (Dojindo Molecular Technologies) and Annexin V-FITC/PI (Dojindo Molecular Technologies), followed by the measure with CLSM and BD LSRFortessa, respectively.

In Vivo Biocompatibility Assay. PMR dispersed in saline was intravenously injected into the healthy female Kunming mice (n = 6 in each group) at the different Si doses (0, 5, 10, and 20 mg·kg-1). After a whole month’s recording of the body weight of mice every two days, they were sacrificed and their organs (heart, liver, spleen, lung, and kidney) and blood samples were collected for evaluation, including hematoxylin and eosin (H&E) staining and various blood-indexes monitoring.

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Measuring Intratumoral Blood Oxygen Saturation by PA Imaging. Mice bearing U87 tumors were intravenously injected with MR saline solution (100 µL, [Mn] = 1 mg·kg-1) when the tumor volume reached approximately 200 mm3. In vivo PA images of estimated real-time oxygen saturation were collected pre- and 10 minutes post-injection using the Oxyhem mode (with excitation wavelengths of 750 and 850 nm).

Western Blot Analysis and Immunofluorescent Assay of the Hypoxia. U87 cells and mice bearing U87 tumors were divided into four groups (non-treated cells or mice as control, PR, MR, PMR) to assess the function of nanosonosensitizer as hypoxia mediator. After co-incubation with nanosonosensitizer ([Si] =20 µg·mL-1 in MEM) for 4 h, cells were washed with PBS for three times, and then collected by trypsinization and centrifugation for evaluating HIF-1α expression. As for HypoxyprobeTM immunofluorescence stain, mice bearing U87 tumors were sacrificed and the tumors were taken out, embedded in paraffin and sectioned after intravenous injection with the nanosonosensitizers (100µL, [Si]= 10 mg·kg-1) for 4 h.

Evaluation of Blood Circulation Time of PMR in Male Kunming Mice. PMR (100 µL, [Si] = 4 mg·mL-1) in saline solution was intravenously injected into the female Kunming mice (n = 3), and the blood (15 µL) was taken out at different time intervals (2 min, 5 min, 8 min, 10min, 0.25 h, 0.5h, 1 h, 2 h, 4 h, 8 h and 24 h), which was then melt by chloroazotic acid. The Si content in blood was determined by ICP-OES.

Bio-distribution of PMR in Tumor Tissue. The female BALB/c nude mice (n = 5) were intravenously injected with PMR (100 µL, [Si] = 4 mg·mL-1) when the tumor volume reached around 100 mm3. After the injection for 4 h, 24 h and 48 h, the mice were sacrificed, and the 26


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contents of Si in their main organs (heart, liver, spleen, lung and kidney) and tumor were tested by ICP-OES after melting the organs and tumors with chloroazotic acid.

Evaluation of PMR Excretion in Urine and Feces. The female Kunming mice (n=5) were administrated with PMR (100 µL, [Si] = 20 mg·kg-1) to investigate the metabolism process in vivo. The Si and Mn contents of the urine and feces collected at different time intervals (2 h, 6 h, 12 h, 24 h, 36 h and 48 h) were measured by ICP-OES after chloroazotic acid treatment.

In Vivo Evaluation of Enhanced SDT Efficacy by PMR Nanosonosensitizers. Initially, the tumor model was established by subcutaneously injecting female BALB/c nude mice with U87 cells. The mice were divided into five groups (the non-treated mice as control, PMR, US, PR + US and PMR + US, n = 7 in each group), when the tumor volume reached around 80 mm3. Then, the mice in each group were administrated with nanosonosensitizers intravenously at the PpIX dose of 5 mg·kg-1 (100 µL) except for the non-treated group. In US irradiated groups, the mice were treated by the US irradiation (1.0 MHz, 1.5 W·cm-2, 50% duty cycle, 3 min) 3 h ，3 d and 5 d after the injection. The tumor volume was recorded every two days on the basis of a standard protocol (V= (ab2)/2, where a and b refer to the largest length and width of tumor, respectively), as well as the photography of tumor was recorded accordingly. Finally, the mice were sacrificed and the tumor were dissected and conduct hematoxylin-eosin staining (H&E), TdT-mediated dUTP Nick-End Labeling (TUNEL), and Ki-67 antibody staining for histological analysis at the end of the therapeutic process.

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Figure 1. Schematic illustration of the construction of PMR nanosonosensitizers and their MRI-guided and catalytic oxygen generation-enhanced SDT against cancer. (a) The detailed steps of the fabrication of PMR nanosonosensitizers. MnOx were in-situ introduced into the hollow interior and mesopores of HMONs via a simple redox reaction, followed by PpIX anchoring into the hollow interior and mesopores of HMONs-MnOx. The targeting peptide RGD (Arg-Gly-Asp)

was further

nanosonosensitizers.

(b)

modified

Schematic

onto

the

illustration

surface of

to

targeted

construct the accumulation

targeting of

PMR

nanosonosensitizers into tumor for in vivo tumor microenvironment-responsive MR imaging and nanoenzyme-enhanced SDT.

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Figure 2. PMR characterizations. (a) TEM image of mesoporous SiO2 nanoparticles as hard template. (b) SEM and (c) TEM images of hybrid HMONs. (d, e) STEM images of PMR at different magnifications under secondary-electron imaging mode and (f) dark-field mode. (g) N2 adsorption-desorption isotherms of HMONs and PMR. The inset shows their corresponding BET specific surface area and pore volume. (h) A series of changes of Zeta potential of samples obtained from each synthetic step. (i) Particle-size distributions of HMONs and PMR.

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Figure 3. Composition characterization of PMR. Solid-state (a)

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13

C and (b)

29

Si CP/MAS

NMR spectra of organic-inorganic hybridized HMONs. (c) Raman spectra of HMONs. (d) Schematic illustration of the microstructure and composition of PMR. (e) XPS spectrum of HMONs-MnOx. (f) UV-vis absorption spectra of HMONs and PpIX@HMONs. (g) Elemental mappings of O, Si, S and Mn of PMR.

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Figure 4. In vitro evaluation on the O2 and 1O2 generation. (a) Scheme of MnOx as the catalase-like nanoenzyme for the O2 production and further 1O2 generation upon US irradiation. (b, c) O2 generation by tuning the concentrations of Mn (0-40 µg·mL-1) and H2O2 (0, 100 µM), as well as pH values (6.5, 7.4). (d) ESR spectra of 1O2 trapped by TEMP in PpIX@HMONs dispersions after US irradiation under different conditions, including varied US power densities and US irradiation durations. (e) UV-vis absorption spectra of the 1, 3-diphenylisobenzofuran (DPBF) in the presence of PpIX@HMONs upon US irradiation for prolonged durations. (f) Fluorescence changes of SOSG (λex = 488 nm, λem = 525 nm) in the presence of PpIX@HMONs or PM dispersion after exposure to US irradiation for prolonged durations pretreated by TME simulated solution ([GSH] = 10mM, [H2O2] = 100 µM, pH = 6.5).

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Figure 5. Intracellular endocytosis of nanosonosensitizers with or without RGD-targeting modification. (a) CLSM analysis of U87 cellular uptakes of different agents, including PpIX (naked drug), PM (no RGD peptide conjugation), PMR (with RGD peptide conjugation) at the PpIX concentration of 10 µg·mL-1, at varied time points. Scale bar = 40 µm. (b) Flow cytometry analysis of cellular uptake of different agents with or without targeting modification at different concentrations (from left-to-right, the equivalent PpIX concentration: 2.5, 5, 10 and 20 µg·mL-1). The inset histogram (the first figure) shows the statistical result of flow cytometry assay. All the scale bars are 40 µm.

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Figure 6. In vitro nanoenzyme-augmented SDT efficacy against U87 cancer cells. (a) Schematic illustration of augmented SDT process as-assisted by PMR nanosonosensitizers at cellular level. (b) CLSM observation and flow cytometry analysis of cancer cells after various treatments at the PpIX concentration of 10 µg·mL-1, including control group, PMR group, US group, PR + US group and PMR + US group. The scale bar is 40 µm (DCFH-DA) and 100 µm (Calcein-AM & PI) (c) Cell viabilities of U87 cancer cells after treated with PM and PMR at varied concentrations of PpIX for a further 24-h incubation. (d-e) Cell viabilities of U87 cells after exposure to US irradiation under different conditions, including (d) different PMR concentrations, (e) elevated US power-densities and (f) prolonged US irradiation durations.

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Figure 7. In vivo up-regulation of oxygenation in U87 tumor model on nude mice. (a) Schematic illustration of alleviating the tumor hypoxia and enhancing the oxygenation by MnOx component-catalyzed decomposition of H2O2 to produce O2 in tumor tissue. (b) Tumor oxygenation status as determined by in vivo photoacoustic (PA) imaging ([Mn] = 1 mg·kg-1). (c) HIF-1α expression levels ([Si] = 20 µg·mL-1) in tumor after different treatments, including control group, PR group, MR group and PMR group. (d) ex vivo immunofluorescence staining of the tumor tissue ([Si] = 10 mg·kg-1). All the scale bars are 40 µm.

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Figure 8. In vitro and in vivo contrast-enhanced T1-weighted MR imaging by adopting multifunctional MR nanocomposites as the contrast agents. (a) 1/T1 versus Mn2+ concentration and (b) corresponding in vitro MR imaging for HMONs-MnOx in buffer solution (pH = 7.4) at different GSH concentrations ([GSH] = 0, 5.0 and 10 mM) after soaking for 1 h. (c) 1/T1 versus Mn2+ concentration and (d) corresponding in vitro MR imaging for HMONs-MnOx in buffer solution ([GSH] = 0 mM) at different pH values (pH = 5.0, 6.0 and 7.4) after soaking for 1 h. (e) T1-weighted MR imaging of U87 tumor-bearing mice and (f) corresponding quantitative MRI-signal intensity of tumor after intravenous administrations of MR at a certain time interval. (g) Schematic illustration of the dissolution of MnOx from MR nanosonosensitizers under the mildly acidic or reducing TME for contrast-enhanced T1-weighted MR imaging.

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Figure 9. In vivo body-weight curve and hematological index. The changes of body weight of Kunming mice with time during one-month feeding and the hematological assays of mice 30 days post-treated with different doses of PMR (5 mg·kg-1, 10 mg·kg-1 and 20 mg·kg-1).

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Figure 10. In vivo histocompatibility and excretion of PMR. (a) Histological sections of heart, liver, spleen, lung and kidney from the mice sacrificed 30 days post-injection at different Si doses of PMR (5 mg·kg-1, 10 mg·kg-1 and 20 mg·kg-1) after stained by hematoxylin-eosin staining (H&E). All the scale bars are 200 µm. Accumulated (b) Mn and (c) Si contents in urine and feces of female Kunming mice after the intravenous administration of PMR for 2 h, 4 h, 12 h, 24 h, 36 h and 48 h.

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Figure 11. In vivo evaluations on the nanoenzyme-augmented SDT against U87 tumor by PMR nanosonosensitizers. (a) In vivo Si biodistributions after the intravenous injections of PMR for 4 h, 24 h, and 48 h. (b) Blood circulation curve of Si concentration after the intravenous administration of PMR. The blood-circulation half-time was calculated to be 1.1 h. (c) Double chamber model of eliminating PMR according to the blood-circulation curve based on the relationship between concentration and time. (d) Time-dependent tumor-growth and (e) time-dependent body-weight curves of mice in each experiment group, including control group, PMR group, US group, PR + US group and PMR + US group (*p < 0.05, **p < 0.01, ***p < 0.001). (f) In vivo therapeutic protocol of SDT on U87 tumor-bearing mice. (g) Digital images of tumors from each group at the ends of the treatments. (h) H&E staining, TUNEL staining, and

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Antigen Ki-67 immunofluorescence staining in tumor sections from each experiment group. All the scale bars are 200 µm.

ASSOCIATED CONTENT Supporting Information Available: Additional figures, table, and results as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] (J.L. Shi), [email protected] (Y. Chen).

Author contributions Y.C. and J.L.S. designated the idea of the present work and supervised the project and commented on the project. P.Z. synthesized and characterized the nanoenzyme composites, performed in vitro and in vivo experiment and analyzed the data. P.Z. wrote the initial manuscript draft and J.L.S. finalized it . All the authors contributed to the discussion during the whole project.

ACKNOWLEDGMENT We greatly acknowledge the financial support from the National Key R&D Program of China (Grant No. 2016YFA0203700), National Nature Science Foundation of China (Grant No. 51722211 and 51672303), Natural Science Foundation of Shanghai (Grant No. 13ZR1463500) 39


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ACS Nano

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Here we report on augmenting the SDT efficacy based on catalytic nanomedicine, which takes the outstanding catalytic features of nanoenzymes to modulate the tumor microenvironment (TME). The multifunctional nanosonosensitizers have been successfully constructed by the integration of MnOx component with biocompatible/biodegradable hollow mesoporous organosilica nanoparticles (HMONs), followed by the conjugation with protoporphyrin (PpIX, as the sonosensitizer) and cyclic arginine-glycine-aspartic pentapeptide c(RGDyC) (as the targeting peptide). The MnOx component in composite nanosonosensitizer acts as the inorganic nanoenzyme for converting the tumor-overexpressed hydrogen peroxide (H2O2) molecules into oxygen and enhancing the tumor oxygen level subsequently, which has been demonstrated to facilitate the SDT-induced reactive oxygen species production and enhance the SDT efficacy subsequently.

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Nanoenzyme-Augmented Cancer Sonodynamic Therapy by Catalytic

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