Endogenous Catalytic Generation of O2 Bubbles for In Situ Ultrasound

Subscriber access provided by UNIVERSITY OF ADELAIDE LIBRARIES

Article

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 ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b03772 • Publication Date (Web): 10 Aug 2017 Downloaded from http://pubs.acs.org on August 11, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Nano is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

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*,† Jianlin Shi*,† † State Key Laboratory of High Performance Ceramics and Superfine Microstructures, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai, 200050, P. R. China; ‡ University of Chinese Academy of Sciences, Beijing, 100049, P. R. China; § Second Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, P. R. China KEYWORDS：HIFU ablation, tumor microenvironment, hybrid nanostructure, catalytic nanoreactor, mesoporous organosilica nanoparticles (MONs)

ABSTRACT: High intensity focused ultrasound (HIFU) surgery generally suffers from poor precision and low efficiency in clinical application, especially for cancer therapy. Herein, a multi-scale hybrid catalytic nanoreactor (catalase@MONs, abbreviated as C@M) has been developed as a tumor-sensitive contrast & synergistic agent (C&SA) for ultrasound-guided HIFU cancer surgery, by integrating dendritic-structured mesoporous organosilica nanoparticles (MONs) and catalase immobilized in the large open pore channels of MONs. Such a hybrid

ACS Paragon Plus Environment

1

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 35

nanoreactor exhibited sensitive catalytic activity towards 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 EPR 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 ondemand 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.

Imaging-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-effective 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 the clinical cancer surgeries.3,4 Nevertheless, the current HIFU surgery still suffers from the 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 nanocarrier with phase-transition liquid/solid core as contrast & synergistic agent (C&SA) for HIFU treatment.6-11 Theoretically, upon HIFU


2

Page 3 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

stimulation, gas bubbles will be generated from the temperature-sensitive compound inside the nanocarrier, dramatically changing the in situ acoustic environment of tumor for enhanced ultrasound imaging and HIFU ablation. However, such gas releasing strategy needs the exogenous HIFU pre-stimulation, wherein gas releasing and HIFU irradiation take place simultaneously without pre-imaging guidance. Therefore, in order to achieve higher efficiency and more safety of HIFU surgery, new gas releasing strategies are highly desired to realize precise pre-imaging guidance and 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 Besides, 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 H2O2-enriched microenvironment of malignant tumor could also be exploited to realize the on-demand tumor sensitive gas releasing for HIFU surgery, which could not only break the dependence on HIFU irradiation for gas generation, but also enables the enhanced ultrasound pre-imaging for specific guidance of HIFU ablation. Mesoporous organosilica nanoparticles (MONs) with molecularly 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 traditional mesoporous silica nanoparticles (MSNs) with a pure Si-O-Si frame work.21,22 Moreover, MONs with dendrimer-like 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


3

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 35

However, it is still challenging to synthesize MONs having large pore size (≥ 5 nm) within small particle size (≤ 50 nm), which is considerably favorable for the passive transport of macromolecules into malignant tumor region via EPR (Enhanced Permeability and Retention) effect.26,27 On the basis of our previous work,28 herein, we choose 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 hybrid framework of MONs, catalase@MONs possesses a multi-scale hybrid structure (Scheme 1b). Such an elaborate hybrid nanoreactor is designed to serve as a tumor sensitive C&SA for US-HIFU ablation, since it can realize H2O2triggered sustained O2 bubble release in tumor region without HIFU pre-stimulation, enabling specific ultrasound imaging guidance and enhanced HIFU ablation at much reduced power output (Scheme 1c). RESULTS AND DISCUSSION The thioether-bridged dendritic mesoporous organosilica nanoparticles (MONs) 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 microscopy (TEM) images of the well-defined urchin-like MONs with ultra-large 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. SEM images in Figure S1


4

Page 5 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

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-Emmet-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 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 respectively 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 molecularly 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 hour with a high capacity of 375 mg/g, which is obviously higher than


5

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 35

MSNs with the same particle size (Figure 2b). Since it is reported that the pore size has a significant influence on enzyme immobilization,35 and 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 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 zeta potential 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 the Figure 2d. Owing to the immobilized catalase around the outer surface of MONs, catalase@MONs exhibits further reduced cytotoxicity and hemolysis compared to naked MONs (Figure S5), 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 shows no significant catalase release even under the high ionic strength condition (PBS+0.1 M KCl) and


6

Page 7 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

cell culture medium (+fetal bovine serum) (Figure S6).38 Such a strong enzyme-support attachment, enables catalase@MONs a relatively stable hybrid composite in physiological environment without serious catalase leakage problem. Therefore, catalase@MONs was regarded as a whole to measure its catalytic performance for decomposing H2O2 into oxygen bubbles. As expected, substantial gas bubbles were released upon co-incubation of catalase@MONs with 50 µM H2O2 and 10 µM H2O2 (Figure S7). Furthermore, the in vitro ultrasound imaging was carried out to visually observe the O2generating catalytic process in comparison 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 displays completely different oxygen evolving behavior compared to the free catalase. As a specific biocatalyst to decompose H2O2, free catalase could remarkably accelerate the formation of oxygen bubbles in a rapid manner even under 10 µM H2O2. Comparatively, catalase@MONs exhibits 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-depending reaction rate, which dramatically decreases as the catalytic process proceeds.39 While, 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


7

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 35

catalase@MONs could exhibit much higher catalytic stability and O2-generation durability 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. 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 optical transparent tissue-mimicking phantom to visualize the therapeutic outcomes of HIFU in vitro.4143

Meanwhile, ultrasound imaging was applied during the whole theranostic process to record the

change of grey value at specific region. Four-group samples of saline, H2O2 (50 µM), catalase@MONs (1mg/mL in saline, abbreviated as C@M), catalase@MONs+H2O2 (1mg/mL in 50 µM H2O2, abbreviated as C@M+H2O2) were separately injected into the transparent phantom with the same dose, then exposed to the HIFU irradiations at 70W and 100W for 10s, respectively. 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, while the other groups (saline, H2O2, C@M) show no apparent changes of the acoustic environment at the same HIFU irradiation dose (Figure 3a). The corresponding quantitative data in Figure 3b clearly show the significantly increased grey value after injection of C@M+H2O2 upon HIFU exposure. Comparatively, the other groups (saline, H2O2, C@M) exhibit limited increase of the mean grey value even after HIFU irradiation at 100W (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


8

Page 9 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

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: 2

V = π × L × W /6

(1)

(L: the length of the coagulated lesion, W: the width of the coagulated lesion), and the results are summarized in the Figure 3d. Interestingly, compared to the saline group, both H2O2 and C@M groups reveal the similar enhancement effects of HIFU ablation to a certain extent under two power outputs (70 W-10s, 100 W-10s). While, the C@M+H2O2 group shows the most significant enhancement of gel ablation up to 37 ± 7 mm3 and 117 ± 19 mm3 at 70 and 100 W, respectively. Such a remarkable enhancement was approximately the same as that of free catalase+H2O2 group, which led to ablated volumes of 26 ± 9 mm3 and 108 ± 20 mm3 at 70 and 100 W, respectively (Figure S8), indicative of the highly retained catalytic activity of C@M in the tissuemimicking 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 pre-stimulation, 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 tissuemimicking 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 tissuemimicking model with a greatly reduced power output (70W-10 s).


9

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 35

As 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 gas releasing strategy by intratumor injection of saline, MONs, C@M, respectively. It is clear that the contrast enhancement of these tumors can only be detected right after the intratumor injection of C@M (Figure S9). Since 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 minutes, 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 passively enhanced permeability and retention (EPR) effect, wherein, much 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 minutes post-injection, 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


10

Page 11 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

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 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), 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, respectively. As shown in Figure 4b-c, without HIFU pre-stimulation, only C@M group reveals strong echo signal in the tumor region 20 min after intravenous injection and the corresponding mean grey 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. Afterwards, the HIFU irradiation (80W-8s) was applied at tumor sites of the above four groups, and the mean grey value further increased to 161 ± 4.4 for C@M group after HIFU exposure, demonstrating the significant changes of acoustic environment within the tumors. For


11

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 35

comparison, MONs group presents a slight enhancement of the mean grey value after HIFU exposure, while, 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 equation (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 much-enlarged ablated volume (135 ± 30 mm3) after HIFU irradiation at 80 W for 8s. It is worth noting that under such a low power output, negligible tumor damages can be found in either saline or catalase group 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 keep 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 abnormities 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). CONCLUSIONS


12

Page 13 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

In summary, a multi-scale hybrid catalytic nanoreactor, namely catalase@MONs (C@M), has been developed as both contrast & 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 gas releasing behavior of C@M further enables the sustained contrast enhancement of ultrasound imaging within the H2O2-enriched tumor region, and pronounced coagulative necrosis under HIFU exposure of only 80 W. More importantly, this perfluorocarbon-free hybrid nanoreactor realizes the tumor-sensitive theranostic function without HIFU pre-stimulation, which makes HIFU surgery much more precisive and efficient for future clinical application. 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) was obtained from SigmaAldrich. Synthesis of Thioether-Bridged Dendritic MONs. 2 g cetyltrimethylammonium chloride (CTAC, 25 wt%) and 0.06 g triethanolamine (TEA) were dissolved in 20 mL DI water at 95 ℃ under 300 rpm stirring. After 20 min, the mixture of 1g tetraethyl orthosilicate (TEOS) and 1.3 g bis[3-(triethoxysilyl)propyl]tetrasulfide (BTES) was added dropwise and the resulting solution


13

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 35

was stirred at 95 ℃ for another 4h. The products were collected by centrifugation (20000 r/min for 30 min) and washed for several times with ethanol and water to remove the residual reactants. Then the collected products were extracted twice for 12h with a solution of hydrochloric acid (HCl, 37%) in ethanol (10% v/v) at 78 ℃ by refluxing to remove the template CTAC. Synthesis of 50 nm Mesoporous Silica Nanoparticles (MSNs). 8 g cetyltrimethylammonium chloride (CTAC, 25 wt%) and 0.8 g triethanolamine (TEA) were dissolved in 80 mL DI water at 95 ℃ under 400 rpm stirring. After 1h, 9mL tetraethyl orthosilicate (TEOS) was added dropwise into the above transparent solution, and the resulting mixture was stirred at 95 ℃ for another 1 h. The products were collected by centrifugation and washed for several times with ethanol and water to remove the residual reactants. Then the collected products were extracted twice for 12h with a solution of hydrochloric acid (HCl, 37%) in ethanol (10% v/v) at 78 ℃ by refluxing to remove the template CTAC. Characterization. Transmission electron microscopy (TEM) images were acquired on a JEM2100F electron microscope operated at 200 kV. Scanning electron microscopy (SEM) 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 (DLS) and zeta potential measurement were conducted on a Zetasizer Nanoseries (Nano ZS90). Fourier transform infrared (FTIR) 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).


14

Page 15 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Catalase Immobilization in MONs/MSNs. UV-Vis absorption spectra of catalase solutions with different concentrations (0.125 mg/mL, 0.25 mg/mL, 0.50 mg/mL, 0.75 mg/mL, 1mg/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 ℃ . In order to investigate the saturation loading time and amount of catalase for MONs, three batches of the assynthesized MONs (10 mg) were immersed in 20 mL PBS solution of catalase (pH=7.4, 0.5 mg/mL) and then stirred at 25℃ for 1h, 2h, 4h, respectively. The corresponding supernatant of the above 3 groups were collected for measuring the immobilized amount of catalase by UV-Vis absorption spectra respectively. Moreover, as-synthesized MONs (20 mg) and the same size MSNs (20mg) were dispersed in 20 mL PBS solution of catalase (pH=7.4, 0.5 mg/mL) and then stirred at 25 ℃ for 1h, respectively. The corresponding supernatant were collected for the comparison of immobilized amount of catalase by UV-Vis absorption spectra respectively. The In Vitro Release Profile of Catalase@MONs. Catalase@MONs (1 mg MONs, 348 µg catalase) was immersed in 2 mL 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 h, 4 h, 6 h, 8 h, 10 h, 12 h, 20 h, 24 h, 36 h, 48 h, 60 h, 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 10µM H2O2 solution respectively. Then the mixture of the two groups were spread out on two clean sheet of glass and


15

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 35

covered by a glass slide, excluding all air, respectively. 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 (US) imaging experiment was operated in the degased 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, 10µM) inside, respectively. 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 free catalase and immobilized catalase were immersed in 50 µL H2O2 solution (50 mM), respectively. 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. So that 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 were determined by calculating the amount of decomposed H2O2 in the first 30s of the reaction, respectively. 1 Unit/mg means that 1 mg catalase could decompose 1 µmol H2O2 in 1 min at 25 ℃.


16

Page 17 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

In Vitro Evaluation of H2O2-Triggered Synergistic Effect of C@M for HIFU Ablation. The in vitro assessment of 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 catalase@MONs dispersion and 0.5mL H2O2 solution were injected to the same site inside the transparent phantom (n=3) simultaneously, where the concentration of catalase@MONs and H2O2 were 1mg/mL and 50 µM, respectively. Then high intensity ultrasound was focused at the injection site at different operating parameters (70W for 10s and 100 W for 10s). 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 post-grey 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 (L: the length of the coagulated lesion, W: 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 1mL 1mg/mL catalase@MONs, 1 mL catalase PBS solution, 1mL 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 96-well culture plates at a density of 8000 cells per well and left to adherence for 12 h, respectively. Then the two plates of cancer cells were cultured with the MONs and catalase@MONs dispersions with different concentrations (400 ppm, 200 ppm,


17

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 35

100ppm, 50 ppm, 25 ppm) for 24 h respectively. 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 2mL abovementioned human blood and washed with PBS solution for six times, then diluted to 20 mL with PBS solution for further use. Then, 0.3 mL diluted RBCs solution was added into a) 1.2 mL PBS solution as a negative control; b) 1.2 mL deionized water as a positive control; c) 1.2 mL PBS suspensions of MONs or catalase@MONs at concentrations ranging from 15.63-1000 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, respectively. 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. The New Zealand white rabbits with VX2 liver tumors and VX2 subcutaneous tumors, 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 B-mode ultrasonic monitoring system (Chongqing Haifu Technology, China) was employed for the characterization. In the


18

Page 19 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

experiment described below, the therapeutic and diagnostic frequency was set at 3.5 and 1.1 MHz, respectively. Ultrasound Imaging of 3 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), C@M (MONs: 1mg/mL, catalase: 372 µg/mL), respectively. 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 post grey scale values were recorded using software GrayVal 1.0 (Chongqing Haifu Technology, Chongqing, China). The experimental process above was repeated for 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), C@M (MONs: 1mg/mL, catalase: 372 µg/mL) via tail veins at the same dose of 200 µL, respectively. 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 grey scale values were recorded using software GrayVal 1.0 (Chongqing Haifu Technology, Chongqing, China). The 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 nude mice (n=3), the blood (10 µL) was taken out at different time


19

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 35

intervals (2 min, 5 min, 10 min, 30 min, 1 h, 2 h, 4 h, 6 h, 12 h, 24 h) and melt by chloroazotic acid. The Si content in blood was determined by the ICP-OES. Evaluation of the Contrast & 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), C@M (MONs: 1mg/mL, catalase: 372 µg/mL) via tail veins at the same dose of 200 µL, respectively. 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 8s. The corresponding pre-, in- and post- grey 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 2,3,5-Triphenyltetrazolium chloride (TTC) to clearly observe the ablated regions, and further stained with hematoxylin and eosin (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 hematoxylin and eosin (H&E) staining after 1 day and 7 days feeding, respectively. In addition, The mice without any treatment were used as the control group. Statistical Analysis. Data were expressed as mean ± s.e.m. Differences between groups were assessed using the paired, two-sided Student t-test. ***P < 0.001 was considered highly significant.


20

Page 21 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

ASSOCIATED CONTENT Supporting Information Available: Supplementary figures from Figure S1 to Figure S16 as supporting information are included, and this material is available free of charge on the ACS Publications website. AUTHOR INFORMATION Corresponding Author * Address correspondence to [email protected]. 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), 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.


21

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 35

(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.; Lee, F. T.; Livraghi, T.; McGahan, J.; Phillips, D. A.; Rhim, H.; Silverman, S. G.; Soc Interventional Radiology, Techn. Image-Guided Tumor Ablation: Standardization of Terminology and Reporting Criteria. Radiology 2005, 235 (3), 728739. (3) Feng, Y. J.; Hu, L.; Chen, W. Z.; Zhang, R.; Wang, X.; Chen, J. Y. Safety of UltrasoundGuided High-Intensity Focused Ultrasound Ablation for Diffuse Adenomyosis: A Retrospective Cohort Study. Ultrason. Sonochem. 2017, 36, 139-145. (4) Zhang, L.; Zhang, W. Y.; Orsi, F.; Chen, W. Z.; Wang, Z. B. Ultrasound-Guided High Intensity Focused Ultrasound for the Treatment of Gynaecological Diseases: A Review of Safety and Efficacy. Int. J. Hyperthermia 2015, 31, 280-284. (5) Zhou, Y.; Wang, Z. G.; Chen, Y.; Shen, H. X.; Luo, Z. C.; Li, A.; Wang, Q.; Ran, H. T.; Li, P.; Song, W. X.; Yang, Z.; Chen, H. R.; Wang, Z. B.; Lu, G. M.; Zheng, Y. Y. Microbubbles from Gas-Generating Perfluorohexane Nanoemulsions for Targeted Temperature-Sensitive Ultrasonography and Synergistic HIFU Ablation of Tumors. Adv. Mater. 2013, 25, 4123-4130. (6) Chen, Y.; Chen, H. R.; Sun, Y.; Zheng, Y. Y.; Zeng, D. P.; Li, F. Q.; Zhang, S. J.; Wang, X.; Zhang, K.; Ma, M.; He, Q. J.; Zhang, L. L.; Shi, J. L. Multifunctional Mesoporous Composite Nanocapsules for Highly Efficient MRI-Guided High-Intensity Focused Ultrasound Cancer Surgery. Angew. Chem. Int. Ed. 2011, 50, 12505-12509. (7) Niu, D. C.; Wang, X.; Li, Y. S.; Zheng, Y. Y.; Li, F. Q.; Chen, H. R.; Gu, J. L.; Zhao, W. R.; Shi, J. L. Facile Synthesis of Magnetite/Perfluorocarbon Co-Loaded Organic/Inorganic Hybrid Vesicles for Dual-Modality Ultrasound/Magnetic Resonance Imaging and ImagingGuided High-Intensity Focused Ultrasound Ablation. Adv. Mater. 2013, 25, 2686-2692.


22

Page 23 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

(8) Wang, X.; Chen, H. R.; Zheng, Y. Y.; Ma, M.; Chen, Y.; Zhang, K.; Zeng, D. P.; Shi, J. L. Au-Nanoparticle Coated Mesoporous Silica Nanocapsule-Based Multifunctional Platform for Ultrasound Mediated Imaging, Cytoclasis and Tumor Ablation. Biomaterials 2013, 34, 20572068. (9) Zhang, K.; Chen, H. R.; Li, F. Q.; Wang, Q.; Zheng, S. G.; Xu, H. X.; Ma, M.; Jia, X. Q.; Chen, Y.; Mou, J.; Wang, X.; Shi, J. L. A Continuous Tri-Phase Transition Effect for HIFUMediated Intravenous Drug Delivery. Biomaterials 2014, 35, 5875-5885. (10) Wang, X.; Chen, H. R.; Zhang, K.; Ma, M.; Li, F. Q.; Zeng, D. P.; Zheng, S. G.; Chen, Y.; Jiang, L. X.; Xu, H. X.; Shi, J. L. An Intelligent Nanotheranostic Agent for Targeting, Redox-Responsive Ultrasound Imaging, and Imaging-Guided High-Intensity Focused Ultrasound Synergistic Therapy. Small 2014, 10, 1403-1411. (11) Ma, M.; Xu, H. X.; Chen, H. R.; Jia, X. Q.; Zhang, K.; Wang, Q.; Zheng, S. G.; Wu, R.; Yao, M. H.; Cai, X. J.; Li, F. Q.; Shi, J. L. A Drug-Perfluorocarbon Nanoemulsion with an Ultrathin Silica Coating for the Synergistic Effect of Chemotherapy and Ablation by HighIntensity Focused Ultrasound. Adv. Mater. 2014, 26, 7378-7385. (12) Szatrowski, T. P.; Nathan, C. F. Production of Large Amounts of Hydrogen Peroxide by Human Tumor Cells. Cancer Res. 1991, 51, 794-798. (13) Chen, H. C.; Tian, J. W.; He, W. J.; Guo, Z. J. H2O2-Activatable and O2-Evolving Nanoparticles for Highly Efficient and Selective Photodynamic Therapy Against Hypoxic Tumor Cells. J. Am. Chem. Soc. 2015, 137, 1539-1547. (14) Fan, W. P.; Bu, W. B.; Shen, B.; He, Q. J.; Cui, Z.; Liu, Y. Y.; Zheng, X. P.; Zhao, K. L.; Shi, J. L. Intelligent MnO2 Nanosheets Anchored with Upconversion Nanoprobes for Concurrent


23

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 35

pH-/H2O2-Responsive UCL Imaging and Oxygen-Elevated Synergetic Therapy. Adv. Mater. 2015, 27, 4155-4161. (15) Zhu, W.; Dong, Z.; Fu, T.; Liu, J.; Chen, Q.; Li, Y.; Zhu, R.; Xu, L.; Liu, Z. Modulation of Hypoxia in Solid Tumor Microenvironment with MnO2 Nanoparticles to Enhance Photodynamic Therapy. Adv. Funct. Mater. 2016, 26, 5490-5498. (16) Song, G.; Chen, Y.; Liang, C.; Yi, X.; Liu, J.; Sun, X.; Shen, S.; Yang, K.; Liu, Z. Catalase-Loaded TaOx Nanoshells as Bio-Nanoreactors Combining High-Z Element and Enzyme Delivery for Enhancing Radiotherapy. Adv. Mater. 2016, 28, 7143-7148. (17) Yang, F.; Hu, S.; Zhang, Y.; Cai, X.; Huang, Y.; Wang, F.; Wen, S.; Teng, G.; Gu, N. A Hydrogen Peroxide-Responsive O2 Nanogenerator for Ultrasound and Magnetic-Resonance Dual Modality Imaging. Adv. Mater. 2012, 24, 5205-5211. (18) Olson, E. S.; Orozco, J.; Wu, Z.; Malone, C. D.; Yi, B.; Gao, W.; Eghtedari, M.; Wang, J.; Mattrey, R. F. Toward In Vivo Detection of Hydrogen Peroxide with Ultrasound Molecular Imaging. Biomaterials 2013, 34, 8918-8924. (19) Hou, R.; Wang, Y.; Xu, Y.; Zheng, Y.; Ma, M.; Hu, B. Theranostic Hollow/Mesoporous Organosilica Nanospheres Enhance the Therapeutic Efficacy of Anticancer Drugs in Metastatic Hormone-Resistant Prostate Cancer. RSC Adv. 2016, 6, 94058-94067. (20) Ma, M.; Yan, F.; Yao, M. H.; Wei, Z. J.; Zhou, D. L.; Yao, H. L.; Zheng, H. R.; Chen, H. R.; Shi, J. L. Template-Free Synthesis of Hollow/Porous Organosilica-Fe3O4 Hybrid Nanocapsules toward Magnetic Resonance Imaging-Guided High-Intensity Focused Ultrasound Therapy. ACS Appl. Mater. Interfaces 2016, 8, 29986-29996.


24

Page 25 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

(21) Chen, Y.; Shi, J. L. Chemistry of Mesoporous Organosilica in Nanotechnology: Molecularly Organic-Inorganic Hybridization into Frameworks. Adv. Mater. 2016, 28, 32353272. (22) Huang, P.; Qian, X. Q.; Chen, Y.; Yu, L. D.; Lin, H.; Wang, L.; Zhu, Y. F.; Shi, J. L. Metalloporphyrin-Encapsulated Biodegradable Nanosystems for Highly Efficient Magnetic Resonance Imaging-Guided Sonodynamic Cancer Therapy. J. Am. Chem. Soc. 2017, 139, 12751284. (23) Yang, Y.; Bernardi, S.; Song, H.; Zhang, J.; Yu, M.; Reid, J. C.; Strounina, E.; Searles, D. J.; Yu, C. Anion Assisted Synthesis of Large Pore Hollow Dendritic Mesoporous Organosilica Nanoparticles: Understanding the Composition Gradient. Chem. Mater. 2016, 28, 704-707. (24) Yang, Y. N.; Wan, J. J.; Niu, Y. T.; Gu, Z. Y.; Zhang, J.; Yu, M. H.; Yu, C. Z. StructureDependent and Glutathione-Responsive Biodegradable Dendritic Mesoporous Organosilica Nanoparticles for Safe Protein Delivery. Chem. Mater. 2016, 28, 9008-9016. (25) Ni, Q. Q.; Teng, Z. G.; Dang, M.; Tian, Y.; Zhang, Y. L.; Huang, P.; Su, X. D.; Lu, N.; Yang, Z. L.; Tian, W.; Wang, S. J.; Liu, W. F.; Tang, Y. X.; Lu, G. M.; Zhang, L. J. Gold Nanorod Embedded Large-Pore Mesoporous Organosilica Nanospheres for Gene and Photothermal Cooperative Therapy of Triple Negative Breast Cancer. Nanoscale 2017, 9, 14661474. (26) Ma, M.; Zheng, S.; Chen, H. R.; Yao, M. H.; Zhang, K.; Jia, X. Q.; Mou, J.; Xu, H. X.; Wu, R.; Shi, J. L. A Combined “RAFT” and “Graft From” Polymerization Strategy for Surface Modification of Mesoporous Silica Nanoparticles: towards Enhanced Tumor Accumulation and Cancer Therapy Efficacy. J. Mater. Chem. B 2014, 2, 5828-5836.


25

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 35

(27) Hobbs, S. K.; Monsky, W. L.; Yuan, F.; Roberts, W. G.; Griffith, L.; Torchilin, V. P.; Jain, R. K. Regulation of Transport Pathways in Tumor Vessels: Role of Tumor Type and Microenvironment. P. Natl. Acad. Sci. USA 1998, 95, 4607-4612. (28) Wu, M. Y.; Meng, Q.; Chen, Y.; Du, Y. Y.; Zhang, L.; Li, Y.; Zhang, L.; Shi, J. L. LargePore Ultrasmall Mesoporous Organosilica Nanoparticles: Micelle/Precursor Co-Templating Assembly and Nuclear-Targeted Gene Delivery. Adv. Mater. 2015, 27, 215-222. (29) Alptekin, O.; Tukel, S. S.; Yildirim, D.; Alagoz, D. Characterization and Properties of Catalase Immobilized onto Controlled Pore Glass and Its Application in Batch and Plug-Flow Type Reactors. J. Mol. Catal. B: Enzym. 2009, 58, 124-131. (30) Shi, J.; Jiang, Y.; Wang, X.; Wu, H.; Yang, D.; Pan, F.; Su, Y.; Jiang, Z. Design and Synthesis of Organic-Inorganic Hybrid Capsules for Biotechnological Applications. Chem. Soc. Rev. 2014, 43, 5192-5210. (31) Guo, L.; Li, J.; Zhang, L.; Li, J.; Li, Y.; Yu, C.; Shi, J. L.; Ruan, M.; Feng, J. A Facile Route to Synthesize Magnetic Particles within Hollow Mesoporous Spheres and Their Performance as Separable Hg2+ Adsorbents. J. Mater. Chem. 2008, 18, 2733-2738. (32) Liu, J.; Yang, Q.; Zhang, L.; Jiang, D.; Shi, X.; Yang, J.; Zhong, H.; Li, C. ThioetherBridged Mesoporous Organosilicas: Mesophase Transformations Induced by the Bridged Organosilane Precursor. Adv. Funct. Mater. 2007, 17, 569-576. (33) Hanefeld, U.; Gardossi, L.; Magner, E. Understanding Enzyme Immobilization. Chem. Soc. Rev. 2009, 38, 453-468. (34) Kim, J.; Grate, J. W.; Wang, P. Nanostructures for Enzyme Stabilization. Chem. Eng. Sci. 2006, 61, 1017-1026.


26

Page 27 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

(35) Santos, J. C. S. d.; Barbosa, O.; Ortiz, C.; Berenguer-Murcia, A.; Rodrigues, R. C.; Fernandez-Lafuente, R. Importance of the Support Properties for Immobilization or Purification of Enzymes. ChemCatChem 2015, 7, 2413-2432. (36) Itoh, T.; Ishii, R.; Matsuura, S.; Hamakawa, S.; Hanaoka, T.; Tsunoda, T.; Mizuguchi, J.; Mizukami, F. Catalase Encapsulated in Mesoporous Silica and Its Performance. Biochem. Eng. J. 2009, 44, 167-173. (37) Wang, Y. J.; Caruso, F. Mesoporous Silica Spheres as Supports for Enzyme Immobilization and Encapsulation. Chem. Mater. 2005, 17, 953-961. (38) Gao, Z.; Zharov, I. Large Pore Mesoporous Silica Nanoparticles by Templating with a Nonsurfactant Molecule, Tannic Acid. Chem. Mater. 2014, 26, 2030-2037. (39) Aebi, H., Catalase In Vitro. In Methods in Enzymology, Elsevier, 1984; Vol. 105, pp 121126. (40) Kondo, A.; Murakami, F.; Kawagoe, M.; Higashitani, K. Kinetic and Circular Dichroism Studies of Enzymes Adsorbed on Ultrafine Silica Particles. Appl. Microbiol. Biotechnol. 1993, 39, 726-731. (41) Choi, M. J.; Guntur, S. R.; Lee, K. I.; Paeng, D. G.; Coleman, A. A Tissue Mimicking Polyacrylamide Hydrogel Phantom for Visualizing Thermal Lesions Generated by High Intensity Focused Ultrasound. Ultrasound Med. Biol. 2013, 39, 439-448. (42) Takegami, K.; Kaneko, Y.; Watanabe, T.; Maruyama, T.; Matsumoto, Y.; Nagawa, H. Polyacrylamide Gel Containing Egg White as New Model for Irradiation Experiments Using Focused Ultrasound. Ultrasound Med. Biol. 2004, 30, 1419-1422.


27

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 35

(43) Maxwell, A. D.; Wang, T. Y.; Yuan, L.; Duryea, A. P.; Xu, Z.; Cain, C. A. A Tissue Phantom for Visualization and Measurement of Ultrasound-Induced Cavitation Damage. Ultrasound Med. Biol. 2010, 36, 2132-2143. (44) Khokhlova, V. A.; Bailey, M. R.; Reed, J. A.; Cunitz, B. W.; Kaczkowski, P. J.; Crum, L. A. Effects of Nonlinear Propagation, Cavitation, and Boiling in Lesion Formation by High Intensity Focused Ultrasound in a Gel Phantom. J. Acoust. Soc. Am. 2006, 119, 1834-1848. (45) Shieh, F. K.; Wang, S. C.; Yen, C. I.; Wu, C. C.; Dutta, S.; Chou, L. Y.; Morabito, J. V.; Hu, P.; Hsu, M. H.; Wu, K. C.; Tsung, C. K. Imparting Functionality to Biocatalysts via Embedding Enzymes into Nanoporous Materials by a de Novo Approach: Size-Selective Sheltering of Catalase in Metal-Organic Framework Microcrystals. J. Am. Chem. Soc. 2015, 137, 4276-4279. (46) Wang, X.; Niu, D. C.; Li, P.; Wu, Q.; Bo, X.; Liu, B.; Bao, S.; Su, T.; Xu, H. X.; Wang, Q. Dual-Enzyme-Loaded Multifunctional Hybrid Nanogel System for Pathological Responsive Ultrasound Imaging and T2-Weighted Magnetic Resonance Imaging. ACS Nano 2015, 9, 56465656.


28

Page 29 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

SCHEMES AND FIGURES

Scheme 1. (a) Schematic diagram of the synthetic procedures for thioether-bridged MONs and the following catalase immobilization in MONs. (b) Schematic illustration of the multi-scale hybrid nanostructure of catalase@MONs. (c) Schematic representation of the ultrasound-guided HIFU therapy mediated by catalase@MONs through endogenous in situ catalytic reaction in the H2O2-enriched cancer region.


29

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 35

Figure 1. a-c) TEM images of MONs at different magnifications (inset: the 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).


30

Page 31 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

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 (DLS) and Zeta 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, respectively. f) The catalytic reaction kinetics of free catalase and catalase@MONs towards H2O2 decomposition, respectively.


31

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 35

Figure 3. a) In vitro ultrasound images of the transparent phantom before and after the injections of saline, H2O2, C@M, C@M+H2O2, followed by HIFU exposure at different irradiation powers. b) The corresponding mean grey values of the ultrasound images of different treatment groups. c) Digital photos of the transparent phantom after the injections of saline, H2O2, C@M, C@M+H2O2, followed by HIFU exposures at different irradiation powers. d) The 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


32

Page 33 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

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 tissuemimicking phantom.


33

ACS Nano

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 35

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, C@M, followed by HIFU exposure at 80 W for 8 s, respectively. c) The corresponding mean grey 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, C@M, followed by HIFU exposure at 80 W for 8s. *** 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, C@M, followed by HIFU exposure at 80 W for 8 s, respectively.


34

Page 35 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

For Table of Contents Only A multi-scale hybrid nanoreactor is constructed based on catalase-immobilized mesoporous organosilica nanoparticles (catalase@MONs, abbreviated as C@M). C@M facilitates ondemand tumor-sensitive O2 gas generation in a continuous and mild manner without HIFU prestimulation, enabling real-time ultrasound contrast imaging and subsequent enhanced HIFU ablation at a relatively low power output, which is highly desired in clinical ultrasound-guided HIFU surgery.


35

Endogenous Catalytic Generation of O2 Bubbles for In Situ Ultrasound

Recommend Documents