Endogenous Catalytic Generation of O2 Bubbles ... - ACS Publications

Aug 10, 2017 - University of Chinese Academy of Sciences, Beijing 100049, People,s Republic ... Second Affiliated Hospital of Chongqing Medical Univer...
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Endogenous Catalytic Generation of O2 Bubbles for In Situ Ultrasound-Guided High Intensity Focused Ultrasound Ablation Tianzhi Liu,†,‡ Nan Zhang,§ Zhigang Wang,§ Meiying Wu,† Yu Chen,† Ming Ma,† Hangrong Chen,*,† and Jianlin Shi*,† †

State Key Laboratory of High Performance Ceramics and Superfine Microstructures, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, People’s Republic of China ‡ University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China § Second Affiliated Hospital of Chongqing Medical University, Chongqing 400016, People’s Republic of China S Supporting Information *

ABSTRACT: High intensity focused ultrasound (HIFU) surgery generally suffers from poor precision and low efficiency in clinical application, especially for cancer therapy. Herein, a multiscale hybrid catalytic nanoreactor (catalase@ MONs, abbreviated as C@M) has been developed as a tumorsensitive contrast and synergistic agent (C&SA) for ultrasound-guided HIFU cancer surgery, by integrating dendriticstructured mesoporous organosilica nanoparticles (MONs) and catalase immobilized in the large open pore channels of MONs. Such a hybrid nanoreactor exhibited sensitive catalytic activity toward H2O2, facilitating the continuous O2 gas generation in a relatively mild manner even if incubated with 10 μM H2O2, which finally led to enhanced ablation in the tissue-mimicking PAA gel model after HIFU exposure mainly resulting from intensified cavitation effect. The C@M nanoparticles could be accumulated within the H2O2-enriched tumor region through enhanced permeability and retention effect, enabling durable contrast enhancement of ultrasound imaging, and highly efficient tumor ablation under relatively low power of HIFU exposure in vivo. Very different from the traditional perfluorocarbon-based C&SA, such an on-demand catalytic nanoreactor could realize the accurate positioning of tumor without HIFU prestimulation and efficient HIFU ablation with a much safer power output, which is highly desired in clinical HIFU application. KEYWORDS: HIFU ablation, tumor microenvironment, hybrid nanostructure, catalytic nanoreactor, mesoporous organosilica nanoparticles

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liquid/solid core as contrast and synergistic agent (C&SA) for HIFU treatment.6−11 Theoretically, upon HIFU stimulation, gas bubbles will be generated from the temperature-sensitive compound inside the nanocarrier, dramatically changing the in situ acoustic environment of the tumor for enhanced ultrasound imaging and HIFU ablation. However, such gas-releasing strategy needs the exogenous HIFU prestimulation, wherein gas release and HIFU irradiation take place simultaneously without preimaging guidance. Therefore, in order to achieve higher efficiency and more safety for HIFU surgery, new gasreleasing strategies are highly desired to realize precise

maging-guided noninvasive tumor ablation (e.g., radio frequency ablation, microwave ablation, laser ablation, high intensity focused ultrasound ablation (HIFU)) has become a newly preferred treatment for certain cancers and patients due to its high accuracy, cost-efficiency, and faster recovery advantages.1,2 Owing to its high tissue-penetrating depth and real-time monitoring features, ultrasound-guided HIFU (US-HIFU) ablation has demonstrated its great potential and advantages among clinical cancer surgeries.3,4 Nevertheless, the current HIFU surgery still suffers from relatively low imaging resolution and therapeutic efficacy, which always takes hours of treating with repeated high-energy irradiation pulses, leaving normal tissue in danger along the ultrasound pathway.5 Nowadays, the fast development of nanomedicine and nanobiotechnology has refreshed the traditional US-HIFU surgery, by the design of a nanocarrier with phase-transition © 2017 American Chemical Society

Received: May 30, 2017 Accepted: August 10, 2017 Published: August 10, 2017 9093

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Scheme 1. (a) Synthetic Procedures for Thioether-Bridged MONs and Following Catalase Immobilization in MONs, (b) Multiscale Hybrid Nanostructure of Catalase@MONs, and (c) Ultrasound-Guided HIFU Therapy Mediated by Catalase@ MONs through Endogenous In Situ Catalytic Reaction in H2O2-Enriched Cancer Region

favorable for the passive transport of macromolecules into the malignant tumor region via EPR (enhanced permeability and retention) effect.26,27 On the basis of our previous work,28 herein, we chose thioether-bridged dendritic MONs as carriers to immobilize catalase, obtaining a hybrid catalytic nanoreactor (catalase@MONs) for tumor-sensitive US-HIFU surgery owing to its controllable synthesis and potential biodegradability. The further modified micelle/precursor co-templating method endows MONs with a more open structure while maintaining its sub-50 nm size (Scheme 1a), which is comparable to the molecular dimension of catalase (9.0 nm × 6.0 nm × 2.0 nm).29 Thus, the subsequent catalase immobilization on MONs can be also regarded as a kind of nanoscale hybridization.30 Besides the molecular level of the hybrid framework of MONs, catalase@ MONs possess a multiscale hybrid structure (Scheme 1b). Such an elaborate hybrid nanoreactor is designed to serve as a tumorsensitive C&SA for US-HIFU ablation as it can realize H2O2triggered sustained O2 bubble release in the tumor region without HIFU prestimulation, enabling specific ultrasound imaging guidance and enhanced HIFU ablation at much reduced power output (Scheme 1c).

preimaging guidance and to further reduce the HIFU power output. As a characteristic feature of the malignant tumor,12 endogenous H2O2 could act as an effective internal stimulus for enhanced cancer therapy (e.g., photodynamic therapy and radiotherapy) based on O2-evolving catalytic reaction.13−16 In addition, the in vivo H2O2-responsive O2 gas-releasing phenomenon induced by catalytic nanoreactors has also been detected by enhanced ultrasound (US) and magnetic resonance (MR) imaging.17,18 Therefore, it is believed that such a H2O2enriched microenvironment of malignant tumors could also be exploited to realize the on-demand tumor-sensitive gas release for HIFU surgery, which could not only break the dependence on HIFU irradiation for gas generation but also enable the enhanced ultrasound preimaging for specific guidance of HIFU ablation. Mesoporous organosilica nanoparticles (MONs) with molecular hybrid framework have been extensively explored in nanotechnology, including ultrasound-based theranostic nanomedicine for cancer,19,20 due to their greatly improved biocompatibility and biodegradability compared to that of traditional mesoporous silica nanoparticles (MSNs) with a pure Si−O−Si framework.21,22 Moreover, MONs with dendrimerlike structure could serve as a carrier/support for the immobilization/encapsulation of biomacromolecules, such as proteins and enzymes due to their high surface area, large pore size/volume, and distinct pore morphology.23−25 However, it is still challenging to synthesize MONs having large pore size (≥5 nm) within small particle size (≤50 nm), which is considerably

RESULTS AND DISCUSSION The thioether-bridged dendritic mesoporous organosilica nanoparticles with a more open structure were synthesized with a fixed bis[3-(triethoxysilyl)propyl]tetrasulfide (BTES)/ tetraethyl orthosilicate (TEOS) mass ratio and reactant-mixing rate. Figure 1a−c shows typical transmission electron 9094

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Figure 1. (a−c) TEM images of MONs at different magnifications (inset: digital photo of MONs dispersed in DI water). (d) N2 adsorption− desorption isotherm and the corresponding pore size distribution of MONs (inset). (e) FTIR spectrum of MONs. (f) Raman spectra of BTES and MONs (inset: enlarged detail of f).

microscopy (TEM) images of the well-defined urchin-like MONs with ultralarge radial pores and an average particle diameter of 50 nm, which presents good colloidal dispersibility in DI water, as shown in the inset of Figure 1a. Scanning electron microscopy images in Figure S1 further demonstrate the uniform dendritic structure of MONs. The N2 sorption isotherm plot of as-synthesized MONs gives a type-IV character, indicating well-defined mesoporous structure of MONs with a large Brunauer−Emmett−Teller (BET) surface area of 547 m2 g−1 and a high pore volume of 1.80 cm3 g−1 (Figure 1d). The corresponding pore size distribution curve of MONs presents a peak value at around 7.4 nm, which accords well with the TEM images of MONs in Figure 1c. In addition to the characteristic peaks of silica (Si−O−Si, Si−OH), the appearance of adsorption peaks of C−C, C−H2 (1300−1500 cm−1) and S−S, S−C (520−720 cm−1) in the Fourier transform infrared (FTIR) spectrum indicates the formation of hybrid organic−inorganic framework of MONs (Figure 1e).31 Raman spectroscopy, as a sensitive tool for the detection of stretching vibrations of S−S and S−C bonds located at 438, 488, and 636 cm−1, also confirms the hybrid framework of MONs (Figure 1f).32 Obviously, the addition of BTES endows the MONs with a molecular hybrid framework. More importantly, compared to mesoporous silica nanoparticles (MSNs) with the same size (Figure S2), BTES plays a key role in the construction of the dendritic structure of MONs, which enables the subsequent catalase immobilization. In order to largely keep the molecular structure of catalase intact, physical adsorption was applied as the driving force for the successful immobilization on MONs by immersing the assynthesized MONs in PBS solution of catalase (pH 7.4).33,34

The immobilized amount of catalase was determined by measuring the specific absorbance of catalase at 405 nm in the supernatant after centrifugation based on Beer−Lambert Law. Figure S3a shows the UV−vis absorption spectra of catalase at different concentrations, and the corresponding calibration curve is presented in Figure S3b. As shown in Figure 2a, MONs achieved the saturation catalase loading within only 1 h with a high capacity of 375 mg/g, which is obviously higher than MSNs with the same particle size (Figure 2b). As it is reported that the pore size has a significant influence on enzyme immobilization,35 it has been proven that catalase can be confined in a nanospace of about 7 nm in diameter in mesoporous silica by a direct visualization technique.36 The higher loading amount of catalase for MONs is mainly attributed to its larger open pore structure compared to that of MSNs with the same particle size. Furthermore, compared to naked MONs, the decreased BET surface area and pore volume of catalase@MONs confirm the presence of catalase within the open pores of MONs (Figure S4). It is worth noting that the average hydrated diameter size of MONs increased slightly from 135.5 nm (PDI = 0.073) to 145.9 nm (PDI = 0.180) after the catalase immobilization (Figure 2c), demonstrating that part of the immobilized catalase is distributed around the surface of MONs. Moreover, catalase@MONs show lower ζpotential compared to MONs (Figure 2c, inset), demonstrating a higher colloidal stability with a deeper yellow color appearance when dispersed in PBS owing to the catalase loading, as shown in Figure 2d. Owing to the immobilized catalase around the outer surface of MONs, catalase@MONs exhibit further reduced cytotoxicity and hemolysis compared to naked MONs (Figure S5), 9095

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Figure 2. (a) Absorption spectra of the supernatant after MONs have been immersed in catalase solution for varied time periods. (b) Absorption spectra of the supernatant after the catalase immobilization in MSNs and MONs under the same conditions. (c) Dynamic light scattering and ζ-potential results of MONs and catalase@MONs. (d) Digital photos of MONs and catalase@MONs dispersed in PBS. (e) In vitro B-mode ultrasound imaging of free catalase and catalase@MONs under different H2O2 concentrations. (f) Catalytic reaction kinetics of free catalase and catalase@MONs toward H2O2 decomposition.

decompose H2O2, free catalase could remarkably accelerate the formation of oxygen bubbles in a rapid manner even under 10 μM H2O2. Comparatively, catalase@MONs exhibit a lasting and much more moderate mode for this catalytic process, facilitating the sustained release of oxygen gas bubbles, which induces continuous magnification of echo signal of ultrasonography. Figure 2f further compares the different behaviors of catalytic reaction kinetics between catalase@MONs and the free catalase. It is clear that the free catalase group reveals a H2O2 concentration-dependent reaction rate, which dramatically decreases as the catalytic process proceeds.39 However, the catalase@MONs group presents a much slower but stable selfdecomposition rate of H2O2. Furthermore, the corresponding enzymatic activities of the two groups can be calculated based on their dynamic curves. Though the catalase activity after immobilization is 625 units/mg, accounting for only 20.8% of the corresponding free catalase activity, probably due to the conformation changes of the immobilized catalase,40 the catalase@MONs could exhibit catalytic stability and O2generating durability much higher than that of free catalase. In other words, thanks to its moderate and stable performance of catalytic decomposition for H2O2, this hybrid nanoreactor (catalase@MONs) enables the continuous generation of O2 bubbles, resulting in a sustained signal enhancement of ultrasound imaging, which is highly desired in the clinical theranostics of cancer.

possessing good biocompatibility with great potential feasibility for in vivo application without need of further surface modification. Since catalase and MONs are both negatively charged in PBS solution (pH 7.4), hydrogen bonding between the carboxylic and amino groups of the catalase and surface silanols of MONs is considered to be the main driving force for the catalase immobilization.37 Then, in vitro catalase release test was conducted to further evaluate the stability of this hybrid composite (catalase@MONs). Notably, only 25% of the immobilized catalase was released after 72 h in PBS solution. Similarly, catalase@MONs show no significant catalase release even under the high ionic strength condition (PBS + 0.1 M KCl) and cell culture medium (+fetal bovine serum) (Figure S6).38 Such a strong enzyme-support attachment enables catalase@MONs to be a relatively stable hybrid composite in physiological environment without serious catalase leakage problems. Therefore, catalase@MONs were regarded as a whole to measure the catalytic performance for decomposing H2O2 into oxygen bubbles. As expected, substantial gas bubbles were released upon co-incubation of catalase@MONs with 50 and 10 μM H2O2 (Figure S7). Furthermore, the in vitro ultrasound imaging was carried out to visually observe the O2-generating catalytic process of free catalase and catalase@MONs under different H2O2 concentrations. As evidenced by the B-mode ultrasound real-time imaging in Figure 2e, catalase@MONs display completely different oxygen-evolving behavior compared to the free catalase. As a specific biocatalyst to 9096

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Figure 3. (a) In vitro ultrasound images of the transparent phantom before and after the injections of saline, H2O2, C@M, and C@M+H2O2, followed by HIFU exposure at different irradiation powers. (b) Corresponding mean gray values of the ultrasound images of different treatment groups. (c) Digital photos of the transparent phantom after the injections of saline, H2O2, C@M, and C@M+H2O2, followed by HIFU exposures at different irradiation powers. (d) Corresponding ablated volumes after the injections and HIFU exposures of different groups (n = 3); *** represent the significant differences in ablated volume by comparing the C@M+H2O2 group with the control groups (saline, H2O2, C@M) at P ≤ 0.001 respectively. (e) Schematic diagram of the assessment process of the H2O2-triggered synergistic effect of catalase@MONs using the tissue-mimicking phantom.

(Figure 3a). The corresponding quantitative data in Figure 3b clearly show the significantly increased gray value after injection of C@M+H2O2 upon HIFU exposure. Comparatively, the other groups (saline, H2O2, C@M) exhibit limited increase of the mean gray value even after HIFU irradiation at 100 W (Figure 3b). In addition, the digital photos of the gel in Figure 3c give a visual display of the treatment effects by HIFU irradiation, wherein C@M+H2O2 presents the most significant color change in the focal point after HIFU irradiation with different power outputs, demonstrating the valid coagulative damage in the gel. The corresponding ablated volumes in the transparent phantom of each group were calculated by using the following equation:

To further evaluate the synergistic effect of catalase@MONs for HIFU ablation, a kind of polyacrylamide (PAA) gel containing egg white was constructed as a model of an optical transparent tissue-mimicking phantom to visualize the therapeutic outcomes of HIFU in vitro.41−43 Meanwhile, ultrasound imaging was applied during the whole theranostic process to record the change of gray value at specific regions. Four group samples of saline, H2O2 (50 μM), catalase@MONs (1 mg/mL in saline, abbreviated as C@M), and catalase@MONs+H2O2 (1 mg/mL in 50 μM H2O2, abbreviated as C@M+H2O2) were separately injected into the transparent phantom with the same dose and then exposed to the HIFU irradiations at 70 and 100 W for 10 s. As expected, a remarkable contrast enhancement can be found in the transparent phantom injected with C@M +H2O2 due to the catalytic generation of O2 bubbles. More importantly, the contrast enhancement is further magnified after HIFU exposure for the C@M+H2O2 group, whereas the other groups (saline, H2O2, C@M) show no apparent changes of the acoustic environment at the same HIFU irradiation dose

V = π × L × W2 /6

(1)

where L is the length of the coagulated lesion, W is the width of the coagulated lesion, and the results are summarized in the Figure 3d. Interestingly, compared to the saline group, both 9097

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Figure 4. (a) In vivo ultrasound imaging of MB231 xenograft tumors in nude mice before and after intravenous injections of C@M at different time intervals. (b) In vivo ultrasound images of MB231 xenograft tumors in nude mice before/after intravenous injections of saline, catalase, MONs, and C@M, followed by HIFU exposure at 80 W for 8 s. (c) Corresponding mean gray value of the ultrasound images in (b). (d) TTC staining results and the corresponding ablated volumes of MB231 tumors in nude mice treated with saline, catalase, MONs, and C@M, followed by HIFU exposure at 80 W for 8 s; *** represents the significant differences in ablated volume by comparing the C@M treated group with the control groups (saline, catalase, MONs) at P ≤ 0.001. (e) Histological assessments from MB231 xenograft tumors borne in mice treated with saline, catalase, MONs, and C@M, followed by HIFU exposure at 80 W for 8 s.

can only be detected right after the intratumor injection of C@ M (Figure S9). No contrast enhancement can be found without H2O2 in vitro for C@M (Figure S10). Thus, it is believed that the endogenous catalytic generation of O2 bubbles from the intratumoral H2O2 is the main cause of enhanced ultrasound imaging of the three tumor models. Moreover, the in vivo ultrasound imaging was evaluated at different time intervals on nude mice bearing MB231 xenograft tumors after intravenous injection via tail vein. As shown in Figure 4a, prior to the injection of C@M, the tumor exhibits a relatively low acoustic signal and then becomes brightened right after the intravenous injection of C@M. In the next 30 min, the echo signal of tumor is continuously enhanced due to the efficient accumulation of C@M in the tumor site and their infiltration and diffusion within tumor tissue via passive EPR effect, wherein many more endogenous H2O2 molecules are gradually decomposed into O2 bubbles under the catalysis of C@M. It is worth noting that this contrast enhancement gradually weakens in about 30 min postinjection and can be hardly detected in 3 h after the intravenous injection (Figure S11), due to the complete consumption of the endogenous H2O2 in the time course. The corresponding quantitative analysis of US signal intensity presents a peak value at 30 min, and the signal enhancement effect could last for almost an hour right after the intravenous injection (Figure S12). For comparison, the intravenous injections of saline and MONs present no significant contrast enhancing effect at different time intervals. Additionally, it is worth noting that the intravenous injection of free catalase induces no echo enhancement in tumor (Figure S13), displaying a totally different phenomenon compared to the ultrasound imaging of tumors receiving

H2O2 and C@M groups reveal the similar enhancement effects of HIFU ablation to a certain extent under two power outputs (70 W/10 s, 100 W/10 s), whereas the C@M+H2O2 group shows the most significant enhancement of gel ablation up to 37 ± 7 and 117 ± 19 mm3 at 70 and 100 W, respectively. Such a remarkable enhancement was approximately the same as that of the free catalase + H2O2 group, which led to ablated volumes of 26 ± 9 and 108 ± 20 mm3 at 70 and 100 W, respectively (Figure S8), indicative of the highly retained catalytic activity of C@M in the tissue-mimicking phantom. As illustrated in Figure 3e, O2 bubbles could be formed within the PAA gel in the presence of C@M and H2O2 without HIFU prestimulation, serving as echo signal amplifier to guide the subsequent HIFU surgery. Meanwhile, the accumulated O2 bubbles could dramatically change the acoustic environment of the PAA gel and intensify the cavitation effect under HIFU irradiations, which finally leads to the great enhancement of HIFU ablation in the tissue-mimicking phantom.44 Therefore, such a H2O2-responsive gas-releasing strategy, facilitated by the hybrid nanoreactor (C@M), makes it feasible for US-HIFU ablation in the in vitro tissue-mimicking model with a greatly reduced power output (70 W/10 s). As is well-known, the cancerous region commonly features constantly produced hydrogen peroxide (H2O2), leading to the accumulation of excessive H2O2 compared to the normal tissue. Therefore, the in vivo ultrasound imaging in three different tumor models (VX2 liver tumors in rabbits, VX2 subcutaneous tumors in rabbits, MB231 xenograft tumors in nude mice) was conducted to further demonstrate the H2O2-responsive gasreleasing strategy by intratumor injection of saline, MONs, and C@M. It is clear that the contrast enhancement of these tumors 9098

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CONCLUSIONS In summary, a multiscale hybrid catalytic nanoreactor, namely, catalase@MONs (C@M), has been developed as both contrast and synergistic agents for ultrasound-guided HIFU cancer surgery, which integrates dendritic-structured mesoporous organosilica nanoparticles and catalase. This hybrid nanoreactor induces H2O2 decomposition in a relatively mild manner, continuously releasing O2 bubbles as the echo amplifier for ultrasound imaging. Furthermore, the accumulative O2 gas leads to greatly enlarged ablated volume under HIFU exposure in the tissue-mimicking phantom due to the enhanced cavitation effect. Such an on-demand H2O2-responsive gasreleasing behavior of C@M further enables the sustained contrast enhancement of ultrasound imaging within the H2O2enriched tumor region and pronounced coagulative necrosis under HIFU exposure of only 80 W. More importantly, this perfluorocarbon-free hybrid nanoreactor realizes the tumorsensitive theranostic function without HIFU prestimulation, which makes HIFU surgery much more precise and efficient for future clinical application.

intratumor injection of free catalase (Figure S14), which could be ascribed to the decomposition or deactivation of free catalase during blood circulation via intravenous injection.45,46 Furthermore, the blood half-time of C@M was calculated to be 0.61 h (Figure S13), according to the variation of Si element concentration in the blood of nude mice after intravenous injection, demonstrating that C@M could indeed accumulate into the tumor site by blood circulation and retain its high catalytic activity during blood circulation owing to the dendritic pore structure of MONs for the efficient storage and protection of catalase. Considering the excellent responsive imaging performance of C@M, in vivo US-HIFU surgery was further operated to verify the capability of C@M in serving as both contrast and synergistic agents on nude mice bearing MB231 xenograft tumors. Specifically, saline, catalase (400 μg/mL), MONs (1 mg/mL), and C@M (MONs: 1 mg/mL, immobilized catalase: 372 μg/mL) were intravenously injected at the same dose to four groups of mice with tumors. As shown in Figure 4b,c, without HIFU prestimulation, only the C@M group reveals a strong echo signal in the tumor region 20 min after intravenous injection and the corresponding mean gray value increases from 66 ± 2.2 to 119 ± 5.1; such an on-demand ultrasound imaging enhancement is highly beneficial for the precise tumor location before the subsequent HIFU treatment. Afterward, the HIFU irradiation (80 W/8 s) was applied at tumor sites of the above four groups, and the mean gray value further increased to 161 ± 4.4 for the C@M group after HIFU exposure, demonstrating the significant changes of acoustic environment within the tumors. For comparison, the MONs group presents a slight enhancement of the mean gray value after HIFU exposure, whereas the other control groups (saline, catalase) show no notable changes of the acoustic environment within the tumors at the same dose of HIFU exposure. As shown in Figure 4d, the TTC (2,3,5-triphenyltetrazolium chloride) staining results indicate different levels of ischemic damages of tumor tissues induced by HIFU ablation, and the corresponding ablated volumes of each group can be calculated by using eq 1. In accordance with the results of in vitro assessments, C@M facilitates the generation of O2 gas within the H2O2-enriched tumor region, which finally leads to the greatly enlarged ablated volume (135 ± 30 mm3) after HIFU irradiation at 80 W for 8 s. It is worth noting that under such a low power output, negligible tumor damage can be found in either saline or catalase groups after HIFU exposure. Nevertheless, the MONs group exhibits a certain degree of synergistic effect for HIFU ablation. The H&E (hematoxylin-eosin) staining results of tumor tissues after HIFU ablation shown in Figure 4e reveal that almost all the tumor cell structures remain intact in the saline and catalase groups, and only a few denatured cells can be found in the MONs-treated group. However, the massive karyopyknosis and karyorrhexis are clearly observed from the ablated tumor site treated with C@M under the same power output, demonstrating the remarkable synergistic effect of C@M for HIFU ablation. As for the normal major organs (heart, liver, spleen, lung, kidney), no significant pathological abnormalities could be found after intravenous injection of C@M compared to control nude mice, indicating the high biocompatibility of C@M for in vivo US-HIFU surgery (Figure S16).

EXPERIMENTAL METHODS Chemicals. Tetraethyl orthosilicate (TEOS), triethanolamine (TEA), ethanol, and hydrochloric acid (HCl, 37%) were purchased from Sinopharm Chemical Reagent Company. Bis[3-(triethoxysilyl)propyl]tetrasulfide (BTES), cetyltrimethylammonium chloride (CTAC, 25 wt %), and catalase (powder, from bovine liver, 2000− 5000 units/mg protein) were obtained from Sigma-Aldrich. Synthesis of Thioether-Bridged Dendritic MONs. Two grams of CTAC (25 wt %) and 0.06 g of TEA were dissolved in 20 mL of DI water at 95 °C under 300 rpm stirring. After 20 min, the mixture of 1 g of TEOS and 1.3 g of BTES were added dropwise, and the resulting solution was stirred at 95 °C for another 4 h. The products were collected by centrifugation (20000 rpm for 30 min) and washed several times with ethanol and water to remove the residual reactants. Then the collected products were extracted twice for 12 h with a solution of HCl (37%) in ethanol (10% v/v) at 78 °C by refluxing to remove the template CTAC. Synthesis of 50 nm Mesoporous Silica Nanoparticles. Eight grams of CTAC (25 wt %) and 0.8 g of TEA were dissolved in 80 mL of DI water at 95 °C under 400 rpm stirring. After 1 h, 9 mL of TEOS was added dropwise into the above transparent solution, and the resulting mixture was stirred at 95 °C for another 1 h. The products were collected by centrifugation and washed several times with ethanol and water to remove the residual reactants. Then the collected products were extracted twice for 12 h with a solution of HCl (37%) in ethanol (10% v/v) at 78 °C by refluxing to remove the template CTAC. Characterization. Transmission electron microscopy images were acquired on a JEM-2100F electron microscope operated at 200 kV. Scanning electron microscopy images were obtained on a field emission Magellan 400 microscope (FEI Company). Nitrogen adsorption−desorption isotherms and pore size distribution were tested on a Micrometitics Tristar 3000 system. Dynamic light scattering and ζ-potential measurement were conducted on a Zetasizer Nanoseries (Nano ZS90). Fourier transform infrared spectrum was obtained on a Thermo Scientific spectrometer (NICOLET iS10). Raman spectrum was measured using a Thermo Scientific spectrometer (DXR microscope with a 532 nm laser). Catalase Immobilization in MONs/MSNs. UV−vis absorption spectra of catalase solutions with different concentrations (0.125, 0.25, 0.50, 0.75, 1 mg/mL) were taken to draw the corresponding calibration curve obtained from the absorbance at 405 nm of different concentrations of catalase based on Beer−Lambert Law at 25 °C. In order to investigate the saturation loading time and amount of catalase for MONs, three batches of the as-synthesized MONs (10 mg) were immersed in 20 mL of PBS solution of catalase (pH 7.4, 0.5 mg/mL) 9099

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ACS Nano and then stirred at 25 °C for 1, 2, and 4 h. The corresponding supernatant of the above three groups were collected for measuring the immobilized amount of catalase by UV−vis absorption spectra. Moreover, as-synthesized MONs (20 mg) and the same size MSNs (20 mg) were dispersed in 20 mL of PBS solution of catalase (pH 7.4, 0.5 mg/mL) and then stirred at 25 °C for 1 h. The corresponding supernatant was collected for the comparison of the immobilized amount of catalase by UV−vis absorption spectra. In Vitro Release Profile of Catalase@MONs. Catalase@MONs (1 mg MONs, 348 μg catalase) were immersed in 2 mL of PBS solution (pH 7.4). The releasing process was performed in a shaking table at a shaking speed of 80 rpm at 37 °C, and the released catalase in the supernate was monitored by UV−vis spectra after centrifugation at a given time (2, 4, 6, 8, 10, 12, 20, 24, 36, 48, 60, 72 h). The experimental procedure above was repeated for PBS solution (+0.1 M KCl) and cell culture medium (+fetal bovine serum (FBS)) with the same concentration of catalase@MONs. H2O2-Triggered Bubble Generation of Catalase@MONs in Vitro. Two batches of catalase@MONs solution (1 mg/mL) were mixed with 50 μM and 10 μM H2O2 solution. Then the mixture of the two groups was spread out on two clean sheets of glass and covered by a glass slide, excluding all air. The generation of the H2O2-triggered gas bubbles from the mixture was observed by optical microscopy. H2O2-Triggered Ultrasound Imaging in Vitro. In vitro ultrasound imaging experiment was operated in degassed water with a cellulose tube inside, which is for sample loading. Three batches of 2 mL catalase@MONs solution were injected into the cellulose tubes with H2O2 solutions of different concentrations (0.1 M, 1 mM, 10 μM) inside. The corresponding US enhancing images were acquired under the same B-mode US imaging with a 3.5 MHz center frequency probe (Philips Medical Systems, Bothell, WA). The experimental procedure above was repeated for free catalase solution with the same concentration of catalase (250 ppm). Kinetic Studies of Catalase before/after Immobilization. The enzyme kinetic studies of free catalase and immobilized catalase were measured with catalase assay kit (S0051 from Beyotime Biotechnology, China) in an undirect way. Specifically, 0.5 mg of free catalase and immobilized catalase was immersed in 50 μL of H2O2 solution (50 mM). During the H2O2 decomposition reaction, the remaining H2O2 at different proceeding time was reduced by the chromogenic substrate, which was catalyzed by peroxidase, producing the red complex (N-(4-antipyryl)-3-chloro-5-sulfonate-p-benzoquinonemonoimine), which has a characteristic absorption peak at 520 nm. The amount of decomposed H2O2 at a certain period could be obtained by measuring the corresponding absorbance at 520 nm with the microplate spectrophotometer. Furthermore, the enzymatic activity of free catalase and immobilized catalase was determined by calculating the amount of decomposed H2O2 in the first 30 s of the reaction. One unit/mg means that 1 mg catalase could decompose 1 μmol of H2O2 in 1 min at 25 °C. In Vitro Evaluation of H2O2-Triggered Synergistic Effect of C@M for HIFU Ablation. The in vitro assessment of the synergistic effect of catalase@MONs for HIFU ablation was carried out with the tissue-mimicking phantom made by polyacrylamide gel containing egg white. A JC HIFU tumor treatment system equipped with B-mode ultrasonic monitoring system (Chongqing Haifu Technology, China) was employed for the characterization. In the experiment described below, the therapeutic and diagnostic frequency was set at 3.5 and 1.1 MHz, respectively. Specifically, 0.5 mL of catalase@MONs dispersion and 0.5 mL of H2O2 solution were injected to the same site inside the transparent phantom (n = 3) simultaneously, where the concentrations of catalase@MONs and H2O2 were 1 mg/mL and 50 μM, respectively. Then high intensity ultrasound was focused at the injection site at different operating parameters (70 W for 10 s and 100 W for 10 s). Meanwhile, the injection site was monitored by the B-mode ultrasound imaging apparatus of the HIFU system during the whole process. Hyper echoes were detected, and the pre-, in-, and postgray scale values were recorded using software GrayVal 1.0 (Chongqing Haifu Technology, Chongqing, China). After the HIFU irradiation, the volume of the coagulated lesion with white color was calculated by the

following equation: V = π × L × W2/6 (where L is the length of the coagulated lesion, W is the width of the coagulated lesion). The experimental process above was repeated for the control groups (injections of 1 mL saline, 1 mL 50 μM H2O2, and 1 mL 1 mg/mL catalase@MONs, 1 mL catalase PBS solution, 1 mL catalase + H2O2 (catalase: 271 μg/mL, H2O2: 50 μM)). In Vitro Cytotoxicity Test of MONs and Catalase@MONs. Human breast cancer cells (MDA-MB-231) were seeded in two 96well culture plates at a density of 8000 cells per well and left to adherence for 12 h. Then the two plates of cancer cells were cultured with the MONs and catalase@MONs dispersions with different concentrations (400, 200, 100, 50, 25 ppm) for 24 h. The corresponding cell viability was determined by the CCK-8 reduction assay. Hemolysis Assay of MONs and Catalase@MONs. Human blood anticoagulated with citric acid−dextrose anticoagulant was kindly provided by Shanghai Blood Center. Typically, the red blood cells (RBCs) were obtained by centrifugation (2000 rpm, 10 min) from 2 mL of the above-mentioned human blood and washed with PBS solution six times, then diluted to 20 mL with PBS solution for further use. Then, 0.3 mL of diluted RBC solution was added into (a) 1.2 mL of PBS solution as a negative control, (b) 1.2 mL of deionized water as a positive control, and (c) 1.2 mL of PBS suspensions of MONs or catalase@MONs at concentrations ranging from 15.63 to 1000 ppm. The mixture of all the groups were gently vortexed and incubated at room temperature for 2 h and then centrifuged (4000 r/ min, 5 min) to obtain the supernatants. The absorbance of the supernatant at 541 nm was measured by UV−vis adsorption characterization. The hemolysis percentages of MONs and catalase@ MONs were calculated by the following equation: hemolysis% = (Asample − A(−)control)/(A(+)control − A(−)control). A is the absorbance intensity of UV−vis adsorption spectra. In Vivo Assessment of Tumor-Sensitive Theranostic Functions of C@M. All experiments involving animals were ethically and scientifically approved by the university and complied with Practice for Laboratory Animals in China. New Zealand white rabbits with VX2 liver tumors and VX2 subcutaneous tumors and nude mice bearing MB231 xenograft tumors were supplied by the Laboratory Animals Center of Second Affiliated Hospital of Chongqing Medical University. A JC HIFU tumor treatment system equipped with Bmode ultrasonic monitoring system (Chongqing Haifu Technology, China) was employed for the characterization. In the experiment described below, the therapeutic and diagnostic frequency was set at 3.5 and 1.1 MHz, respectively. Ultrasound Imaging of Three Different Tumor Models by Intratumor Injection. Three groups of New Zealand white rabbits with VX2 liver tumors (n = 3 per group) were intratumor injected with saline, MONs (1 mg/mL), and C@M (MONs: 1 mg/mL, catalase: 372 μg/mL). Meanwhile, the injection tumor sites were monitered by B-mode ultrasound imaging (Philips Medical Systems, Bothell, WA) during the whole process. Hyper echoes were detected, and postgray scale values were recorded using software GrayVal 1.0 (Chongqing Haifu Technology, Chongqing, China). The experimental process above was repeated for the other two tumor models with different dose (VX2 subcutaneous tumors on rabbits, MB231 xenograft tumors on nude mice). Ultrasound Imaging on Nude Mice Bearing MB231 Xenograft Tumors by Intravenous Injection. Four groups of nude mice bearing MB231 xenograft tumors (n = 3 per group) were injected with saline, catalase (400 μg/mL), MONs (1 mg/mL), and C@M (MONs: 1 mg/mL, catalase: 372 μg/mL) via tail veins at the same dose of 200 μL. Meanwhile, the tumor sites were monitered by B-mode ultrasound imaging (Philips Medical Systems, Bothell, WA) during the whole process. The corresponding ultrasonic images were captured at different time intervals (0 min, 10 min, 30 min, 1 h, 3 h), and the gray scale values were recorded using software GrayVal 1.0 (Chongqing Haifu Technology, Chongqing, China). Evaluation of Blood Half-Time of Catalase@MONs in Blood of Nude Mice. Catalase@MONs (200 μL, MONs: 1 mg/mL, catalase: 372 μg/mL) in PBS solution was intravenously injected into 9100

DOI: 10.1021/acsnano.7b03772 ACS Nano 2017, 11, 9093−9102

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ACS Nano nude mice (n = 3), and the blood (10 μL) was taken out at different time intervals (2 min, 5 min, 10 min, 30 min, 1 h, 2 h, 4 h, 6 h, 12 h, 24 h) and melted by chloroazotic acid. The Si content in blood was determined by the ICP-OES. Evaluation of the Contrast and Synergistic Effect of C@M for Ultrasound-Guided HIFU Ablation by Intravenous Injection. Four groups of nude mice bearing MB231 xenograft tumors (n = 6 per group) were injected with saline, catalase (400 μg/mL), MONs (1 mg/mL), and C@M (MONs: 1 mg/mL, catalase: 372 μg/mL) via tail veins at the same dose of 200 μL. Meanwhile, the tumor sites were monitered by B-mode ultrasound imaging apparatus of the HIFU system. After the intravenous injection for 20 min, high intensity ultrasound was focused at the echo enhanced area of the tumor sites at 80 W for 8 s. The corresponding pre-, in- and postgray scale values were recorded using software Gray Val 1.0 (Chongqing Haifu Technology, Chongqing, China). After that, the as-obtained tumor sections were isolated and stained with TTC to clearly observe the ablated regions and further stained with H&E for histopathological analysis by optical microscope. In Vivo Biocompatibility Assay. Catalase@MONs (200 μL, MONs: 1 mg/mL, catalase: 372 μg/mL) in PBS solution was intravenously injected into two groups of nude mice (n = 6). They were sacrificed, and their major organs (heart, liver, spleen, lung, kidney) were harvested for H&E staining after 1 and 7 day feeding. In addition, the mice without any treatment were used as the control group. Statistical Analysis. Data were expressed as mean ± standard error. Differences between groups were assessed using the paired, twosided Student t test. ***P < 0.001 was considered highly significant.

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ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b03772. Figures S1−S16 (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Yu Chen: 0000-0002-8206-3325 Hangrong Chen: 0000-0003-0827-1270 Jianlin Shi: 0000-0001-8790-195X Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was financially supported by China National Funds for Distinguished Young Scientists (Grant No. 51225202), Shanghai International Cooperation Project (16520710200), National Natural Science Foundation of China (Grant No. 51402329), and Science Foundation for Youth Scholar of State Key Laboratory of High Performance Ceramics and Superfine Microstructures (Grant No. SKL201404). REFERENCES (1) Goldberg, S. N.; Charboneau, J. W.; Dodd, G. D.; Dupuy, D. E.; Gervais, D. A.; Gillams, A. R.; Kane, R. A.; Lee, F. T.; Livraghi, T.; McGahan, J. P.; Rhim, H.; Silverman, S. G.; Solbiati, L.; Vogl, T. J.; Wood, B. J. Int Working Grp Image-guided, Tumor. Image-Guided Tumor Ablation: Proposal for Standardization of Terms and Reporting Criteria. Radiology 2003, 228 (2), 335−345. (2) Goldberg, S. N.; Grassi, C. J.; Cardella, J. F.; Charboneau, J. W.; Dodd, G. D.; Dupuy, D. E.; Gervais, D.; Gillams, A. R.; Kane, R. A.; 9101

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DOI: 10.1021/acsnano.7b03772 ACS Nano 2017, 11, 9093−9102