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Xia Wang, Hangrong Chen,* Yu Chen, Ming Ma, Kun Zhang, Faqi Li, Yuanyi Zheng, Deping Zeng, Qi Wang, and Jianlin Shi*

Perfluorohexane-Encapsulated Mesoporous Silica Nanocapsules as Enhancement Agents for Highly Efficient High Intensity Focused Ultrasound (HIFU)

High intensity focused ultrasound (HIFU) has attracted extensive attention among biological and medical researchers as a repre-sentative non-invasive therapeutic mode because it may be able to be employed as a novel theranostic tool – that is, for diagnostic therapy tailored to individual patients – for simultaneous imaging and site-specific therapy. In contrast to other therapeutic modes (chemotherapy, radiotherapy, etc.), HIFU could provide real-time monitoring before, during, and after non-invasive therapy. How-ever, the current HIFU therapy is still not satisfactory clinically owing to its relatively low therapeutic resolution and efficacy.

To differentiate between normal and abnormal tissues, con-trast agents for HIFU are usually adopted. It is known that microbubbles (MBs), which can transform into acoustic-sensitive gas bubbles during ultrasound scattering, have been extensively researched for ultrasound imaging.[1] Unfortunately, their over-large size (confinement to the vascular space) and instability (short lifespan of microbubbles), which are the result of their generation route and the organic materials themselves, have severely limited their further clinical application.

As far as the therapeutic efficacy is concerned, HIFU is known to be capable of inducing tissue necrosis by converting ultrasound energy to regional hyperthermia when focused. However, the energy will rapidly attenuate with penetration depth; therefore higher ultrasound energy would be needed to achieve a satisfactory therapeutic effect, which will present more potential danger to normal tissues. To destroy malig-nant lesions efficiently and at the same time leave neighboring

© 2012 WILEY-VCH Verlag GAdv. Mater. 2012, 24, 785–791

Dr. X. Wang, H. R. Chen, Y. Chen, M. Ma, K. Zhang, Prof. J. L. ShiState Key Laboratory of High Performance Ceramic and Superfine Microstructures Shanghai Institute of Ceramics Chinese Academy of Science Shanghai 200050, P.R. China E-mail: hrchen@mail.sic.ac.cn; jlshi@sunm.shcnc.ac.cnProf. F. Q. Li, D. P. Zeng, Dr. Q. WangState Key Laboratory of Ultrasound Engineering in Medicine Chongqing Key Laboratory of Ultrasound in Medicine and Engineering College of Biomedical Engineering Chongqing Medical University Chongqing 400016, P.R. ChinaProf. Y. Y. ZhengSecond Affiliated Hospital of Chongqing Medical University Chongqing 400010, P.R. China

DOI: 10.1002/adma.201104033

normal tissues unimpaired, HIFU enhancement agents (EAs) have been strongly recommended to lower the pressure threshold, enlarge the lesion site, and enhance the final thera-peutic efficacy.

It has been reported that organic microbubbles or liposomes exhibit an enhancement effect for HIFU therapy.[2] However, the disadvantages of microbubbles in ultrasound imaging also restrict their applicability in HIFU therapy. The most interesting cavitation-related bio-effect of HIFU therapy is to break down/open cell membranes, which could finally lead to cell death or transient cell closing,[2a] and release of free radicals, causing cell apoptosis.[2b] All of the above processes can be realized success-fully only when bubbles can first be created and then brought into close proximity of target cells.[2c] In addition, when exposed to HIFU, the organic shelled EAs would easily rupture in bolus because of their relatively low strength and low heat-resistance, which is extremely unfavorable for ensuring the necessary cir-culation and enough imaging time for HIFU operation and post-evaluation. Therefore, substitutes for microbubbles as HIFU therapy EAs are now urgently required.

Inorganic materials possess the advantages of high thermal/chemical stability, excellent biocompatibility/degradability, and resistance to erosion under extreme conditions. Especially, inorganic materials with hollow interiors have found significant applications in ultrasound imaging. Elastically shelled micro-capsules with organosilane compositions were initially found to enhance ultrasound resonance.[3] Later, additional boron and perfluoropentane (PFP) vapor were incorporated to strengthen shell robustness and scattering intensity.[4] More recently, a series of cell-level biological effects were also explored by using inorganic microcapsules as the ultrasound imaging agents.[5] In addition to these general inorganic microcapsules, capsules with mesopore channels in the shell could make it possible to encapsulate and sustainedly release guest molecules; these have been extensively investigated for applications in drug delivery. Recently, it was suggested that polyhedral, hollow, silica mes-oporous microcapsules could also provide enhanced contrast of the backscattering signal during ultrasound imaging,[6] how-ever, we could find no reports of the application of mesoporous silica nanocapsules as HIFU EAs by themselves or together with encapsulated temperature-sensitive compounds.

In this Communication, we report a nanometer-sized inor-ganic enhancement agent (NIEA) for HIFU, which consists of mesoporous silica nanocapsules (MSNCs) as the carriers and an encapsulated temperature-sensitive biocompatible per-fluorohexane (PFH) compound as a bubble generator. Such a

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Scheme 1. Schematic illustration of the fabrication process of MSNC-PFH.

NIEA, denoted as MSNC-PFH, combines the merits of excel-lent stability, modifiable surfaces, sustained release, imaging, and multifunctional integration potentials of MSNCs and the temperature-sensitive function of PFH in one. Interestingly, the MSNCs themselves were found to enhance therapy capabilities. PFH, a highly biocompatible compound with the most favo-rable phase transition temperature (56 °C) of all the perfluoro-carbons (PFCs) known to us, was chosen and encapsulated in the nanocapsules. After exposure to HIFU, PFH bubbles are expected to be released sustainedly via the mesopore channels of MSNCs owing to the HIFU-induced local temperature rise. Besides, HIFU-mediated enhanced permeability and uptake of MSNCs can offer a unique superiority in inducing localized tissue necrosis and drug delivery.[7] To the best of our knowl-edge, this is the first report about NIEAs for HIFU-mediated hyperthermia necrosis of malignant tumors.

The whole synthetic procedure for this special MSNC-PFH is schematically shown in Scheme 1. First, the MSNCs were synthesized according to a selective etching protocol reported in our previous work.[8] Then, the temperature-sensitive PFH was loaded into both the pore network of the shell and the inner cavity by a mild infusion procedure. After being exposed to HIFU under certain conditions, the liquid PFH converted into a large population of small bubbles, which then swelled and merged into bigger ones upon accumulation in targeted tumor tissues. A series of cavitation-related bio-effects, such as direct mechanical oscillation, cell membrane opening, and free-radical release, are expected to be triggered, which enhance the HIFU ablation.

Figures 1a,b are typical transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images of MSNCs. From the images, it can be seen that uniform MSNCs with an average diameter of 300 nm and mesoporous shell thickness of 50 nm have been obtained. The dynamic light scattering (DLS) and zeta potential (versus pH value) data are displayed in Figure S1 in the Supporting Informa-tion. DLS data show that MSNCs have a narrow size distri-bution with an overall hydrodynamic diameter of 346 nm in water. It is demonstrated that the MSNCs are stable in water and do not form aggregates. The N2 adsorption/desorption isotherms of MSNCs (Figure 1c) show a typical MCM-41 structure (type IV) with a Brunauer –Emmett–Teller (BET) surface area of 686 m2 g−1 and pore volume of 0.80 cm3 g−1. A narrow pore size distribution of 3.0 nm was obtained for small mesopores using the Barrett –Joyner–Halenda (BJH) method (Figure 1d). Notably, the relatively large hysteretic loop at higher pressures indicates that large cavities exist in the core, which ensures the high PFH loading capacity in the following experiments.

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The biocompatible PFH molecules, which can significantly improve the enhancement efficacy, were infused into MSNCs to prepare MSNC-PFH. The digital photos of free PFH and MSNC-PFH of the same PFH concen-tration in PBS (Figure 1e) indicate that the prepared MSNC-PFH can be uniformly dis-persed in PBS and PFH molecules remain well-encapsulated within MSNCs in PBS, as indicated by the absence of any free PFH

subnatant. In comparison, apparent phase-separated mixture can be seen in the free PFH in PBS under the same PFH concentration.

To further confirm that PFH can be encapsulated into MSNCs and exhibits temperature-responsive behavior, PFH bubbles were generated and visualized using confocal laser scanning microscopy (CLSM) after heating MSNC-PFH (6 mg mL−1) at 70 °C for 5 s and 10 s (Figure 1f–h). As expected, a large number of microbubbles were observed after the heat treatment of MSNC-PFH, while the blank control (MSNCs alone at the same concentration in PBS) did not generate any microbubbles (Figure S2) in the whole heated area. This indicates that PFH had been successfully encapsulated into the MSNCs and shows the temperature-responsive effect. The swelling and merging of a cluster of neighboring small microbubbles generated larger ones, which could be directly observed by prolonging the heating duration to 10 s. As is known, the capability of ultra-sound imaging and HIFU ablation is predominantly attributed to the cavitation effect of stable bubbles released from the con-trast and EAs.[9] These in vitro results demonstrate the potential of MSNC-PFH for in vivo applications.

In vitro cell viability tests were performed on MCF-7, HeLa, and HepG2 cell lines. MSNC-PFH with different concentra-tions (25, 50, 100, 400, and 600 μg mL−1) were added to and incubated with the cells for 24 h. Cell viabilities higher than 80% at all of the studied concentrations were found (Figure S3). Further internalization by MCF-7, HeLa, and HepG2 cell lines of MSNC-PFH at a concentration of 200 μg mL−1 (Figure S4) revealed a high uptake of MSNC-PFH EA by these cells.

The coagulative necrosis in vitro was investigated on degassed bovine livers, which could be determined in real-time B-mode ultrasound imaging based on the change of gray scale at the focus (Figure 2) and the related quantitative necrosis volume (Figure 3), as shown in the 3D schematic illustration in Figure 3c.

0.3 mL MSNCs, MSNC-PFH (both 6 mg mL−1 in PBS), and PBS control were separately injected into bovine livers and sub-sequently exposed to HIFU irradiation at a low power output of 70 W for 10 s. It was found that MSNCs themselves lead to a significant gray scale difference before and after exposure (middle panels in Figure 2a), and therefore enhance coagulative ablation. Nevertheless, there have been no reports about abla-tion effects by HIFU hyperthermia using inorganic hollow nan-oparticles in previous studies.

No pronounced difference existed between MSNCs and MSNC-PFH at this low power output, which can be attributed to the limited local temperature rise at this ultrasound energy. The transient temperature at HIFU focus at 70 W for 10 s was determined to be around 40 °C; that is to say, at such a low

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Figure 1. Typical TEM (a) and SEM (b) images, N2 sorption isotherms (c), and corresponding pore size distribution curve (d) of MSNCs. e) Digital photos of free PFH and MSNC-PFH at the same PFH concentration in phosphate buffered saline (PBS; arrow indicates the phase-separation of PFH from PBS). f–h) Confocal laser scanning microscopy (CLSM) images of MSNC-PFH at room temperature (f), heated at 70 °C for 5 s (g), and at 70 °C for 10 s (h).

power the enhanced ablation effect results mostly from MSNCs. Similar results were found in bovine livers in vitro with a lower dosage (0.2 mL) of MSNC-PFH (Figure S5). In contrast, no necrosis formed in tissues that were injected with only PBS under this condition.

Noticeably, there are significant gray scale changes (bottom panels in Figure 2b) and quantitative necrotic differences (Figure 3b) after HIFU exposure at 120 W for 5 s between MSNCs and MSNC-PFH, because a much higher temperature of 60 °C could be reached at 120 W for 5 s, which is already above the phase-transition temperature of PFH (56 °C). As shown in the schematic 3D representation (Figure 3c), the

© 2012 WILEY-VCH Verlag GmAdv. Mater. 2012, 24, 785–791

maximal dimension (length) of lesions was determined according to the z-axis (L), while the width was measured along a perpendicular x-axis (W). Successive lesions were generated in the same plane (x,y) by moving the HIFU generator. The ablated volumes (V [mm3]) were calculated using[10]

V = π × L × W2/6 (1)

In particular, the ablated volume with MSNC-PFH here is calculated to be twice that with only MSNCs or PBS control (Figure 3d). The higher necrosis efficiency by MSNC-PFH at 120 W, therefore, could be ascribed to the enhanced ultrasonic absorption in tissues and intensified cavitation caused by highly

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Figure 2. Typical in vitro B-mode ultrasound images before (left column) and after (right column) HIFU exposure on degassed bovine livers at 70 W for 10 s (a) and 120 W for 5 s (b).

echogenic phase-transitioned PFH bubbles. A significant differ-ence (P value) of necrosis volume exists between using PBS, MSNCs, and MSNC-PFH. It can be concluded that the HIFU ablated volume using MSNC-PFH at 120 W for 5 s is signifi-cantly higher than using MSNCs or PBS control. The enhanced effect of MSNC-PFH in HIFU ablation is defined according to its enhanced ablation effect found by comparison with MSNCs and the PBS control, respectively.

In addition, we also found that free PFH molecules, not encapsulated in MSNCs carriers, presented an obvious phase separation from PBS owing to their complete insolubility. Intra-venously injected PFH molecules in the form of PFH-PBS mixture might diffuse and become quickly dispersed in blood and thus no significant enhancement would be detected, which demonstrates the vital importance of encapsulation of PFH in MSNCs.

Furthermore, therapeutic effects of MSNC-PFH were evalu-ated on the ablation of rabbit liver tumors in vivo (Figure 4a) 10 min after injection of either MSNC-PFH or PBS control. When only PBS control was injected, no ultrasonic echoes could be detected in the B-mode ultrasonic imaging system even at 400 W for 2 s. This can be attributed to the ineffectiveness of

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PBS alone and also the short exposure time (2 s), which was not long enough to cause thermal accumulation to harm tumor tissues in the absence of EAs. In contrast, when the EA MSNC-PFH was injected, the tumor could be monitored and ablated in 2 s at as low as 120 W (bottom panels in Figure 4a) during this ultrasound-guided HIFU treatment. Such a significant enhancement in gray scale (143 dB) using MSNC-PFH, as com-pared to 0 dB when using only PBS, clearly suggests that the ability to destroy malignant lesions with EAs while leaving the surrounding normal tissues unimpaired, as we expected, is very nearly within reach.

The related macroscopic appearance of tumor tissues ablated with MSNC-PFH (Figure S6) shows a sharply demarcated hem-orrhagic volume on gross inspection. This suggests that MSNC-PFH has the potential for occluding blood vessels in cancerous tissues, thus diminishing the blood supply to the tumor, when employed as a NIEA in HIFU therapy.

To further reveal the accumulation behavior of MSNC-PFH in tumors and other organs, the in vivo bio-distributions of Si (nanogram of Si per milligram of tissues) in the tumors and in heart, liver, spleen, lung, and kidney (Figure S7) at different time intervals after injection of NIEA were investigated and

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Figure 3. Digital photos of ablated bovine livers at a) 70 W for 10 s and b) 120 W for 5 s. The scale bar indicates 1 cm. c) Schematic 3D illustration of ablation process on bovine livers in vitro. d) The corresponding necrotic volume after injection of 0.3 mL PBS control, MSNCs, and MSNC-PFH (each column is the average of three data). ** and *** represent significant differences in necrotic volumes found by comparing MSNC-PFH with MSNCs or the PBS control at P ≤ 0.01 and P ≤ 0.001, respectively.

measured by inductively coupled plasma optical emission spec-trometry (ICP-OES) and CLSM.

Results of ICP-OES analysis (Figure S7a) in the related organs indicated that Si accumulations in spleen, liver, and lung increased significantly initially and then decreased. In more detail, the Si content in lung, spleen, and liver reached the maximum values of 156, 57.8, and 128 ng mg−1 at 2, 8, and 8 h, respectively, and decreased accordingly to 47.2, 33.6, and 50.4 ng mg−1 24 h after injection. Such a time-dependent bio-distribution of Si is thought most probably to derive from the capture from the circulation by phagocytic cells in these reticu-loendothelial system (RES) organs, and the subsequent biodeg-radation and excretion of MSNC-PFH. Importantly, the concen-trations of Si in tumors were measured to be 20.5 ng mg−1 in the first 10 min, which then increased gradually to 33.0 ng mg−1 24 h after injection, which demonstrated a gradual but signifi-cant nanoparticle accumulation during the time after injection. The possible reason for this could be the enhanced permeability and retention (EPR) effect due to the leaky vasculature and poor lymphatic drainage in tumor,[11] leading to the continuous accu-mulation of the nanoparticles from the circulation system.

The CLSM images of the targeted tissues (Figure S7b) 10 min after injection of MSNC-PFH clearly showed strong fluorescence in lung, and also in liver and spleen, and in the meantime, fluorescence could also be clearly observed in the tumors, which confirmed the significant uptake of MSNC-PFH

© 2012 WILEY-VCH Verlag GAdv. Mater. 2012, 24, 785–791

in these organs and the tumors as well. The continuous accu-mulation of the MSNC-FPH in the tumors (from 1 to 24 h after injection of MSNC-PFH) demonstrates that these nanoparticles can be used as an efficient carrier to deliver PFH to tumors, and the MSNC-PFH may be a highly promising EA for HIFU therapy of cancers.

The VX2 liver tumor model in rabbits was used to investi-gate the passive targeting effect because of the easier and more significant nanoparticle accumulation in liver than in other organs. Besides, the transient opening of cell membranes induced by the ultrasound would lead to an enhancement of the EPR effect, known as “ sonoporation”, which may contribute, to some extent, to the accumulation of Si in the tumor.[12]

It is believed that the present results are very promising owing to the low dosage (2 mL of 6 mg mL−1) of the EAs and mild ther-apeutic power (120 W for 2 s) employed, which can readily meet the demands for remarkably enhanced therapeutic efficiency and indispensable bio-safety in clinical applications in future.

Pathological examinations of related tumor tissues after HIFU ablation revealed that the overlying normal tissues were not injured during the HIFU therapy. Without MSNC-PFH, the highly compact and aggregated tumor nucleus/tissues stayed intact and no denatured cells could be found even at the power of 400 W for 2 s. In comparison, with MSNC-PFH, no spared viable cells or tissues within the exposure area existed in a patho-logical slice after ablation at 120 W for 2 s (Figure 4c). Destroyed

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Figure 4. a) Typical in vivo B-mode ultrasonic images before (left) and after (right) HIFU expo-sure on rabbit liver tumors. b,c) Pathological examinations of related tumor tissues after HIFU ablation at 400 W for 2 s using PBS control (b) and at 120 W for 2 s using MSNC-PFH (c).

cells, large vacuoles, and irregular widening of the liver tumor could be found in ultra-structural examinations, too. Besides, sharply demarcated un-ablated and ablated regions in the tumor could be observed both by hematoxylin and eosin (H&E) staining microscopy and CLSM images (Figure S8), which verifies the accuracy of HIFU therapy. The significant pathological differ-ence under exposure to HIFU with and without MSNC-PFH further confirms the applicability and efficacy of this NIEA.

Very interestingly, when low dosages (2 mL of 6 mg mL−1) and ablating power (200 W for 5 s) are used, a stable imaging effect and long circulation period can be found in gray scale value changes of ablated rabbit liver tissues in vivo (Figure S9). The gray scale value was 154 dB 100 s after injection of MSNC-PFH, which was well maintained at 151 dB at 7 min and at 125 dB at 23 min after injection. The pathological regression of permanent ischemia and fibrosis appeared in rabbit liver tissues on the 22nd day after HIFU ablation with MSNC-PFH (Figure S10), which further evidenced the effectiveness of MSNC-PFH as EA for HIFU.

Combined with its strong imaging signals (Figure S11) in phase inversion (PI) mode at a mechanical index (MI) of 1.1, the present nanometer-sized MSNC-PFH can well satisfy and balance the size requirements for easy uptake by tumor tissues for PFH (or other guest molecules) delivery and its functioning as an EA for highly effective HIFU imaging and therapy.

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In conclusion, we have demonstrated a nanometer-sized inorganic HIFU EA (denoted as MSNC-PFH) for the first time, which integrates a stable, functional meso-porous silica nanocapsule with encapsulated temperature-sensitive perfluorohexane in its cavity. This newly developed EA had a pronounced coagulative necrosis effect on bovine liver tissues at the low power of 70 W when only mesoporous silica nanocapsules were employed, and the ablated volume was much enlarged at 120 W when MSNC-PFH was used as the EA. In vivo experiments indicated that the tumor could be ablated in just 2 s at as low as 120 W when MSNC-PFH was injected as compared to no detectable necrosis in cancerous tissues even at 400 W for 2 s when only PBS control was injected. The present results demonstrate that this HIFU EA can be developed as a highly prom-ising theranostic agent for effective HIFU imaging and therapy owing to its high sta-bility, efficient PFH loading and release, enhanced tumor ablation capability, and easy uptake by target tissues.

Experimental SectionExperimental materials and methods as well as additional results can be found in the Supporting Information.

Supporting InformationSupporting Information is available from the Wiley Online Library or from the author.

AcknowledgementsWe gratefully acknowledge financial support from the National Basic Research Program of China (973 Program, Grant No. 2011CB707905), National Natural Science Foundation of China (Grant Nos. 51072212, 51132009), and the Science and Technology Commission of Shanghai (Grant Nos. 10QH1402800, 11nm0506500).

Received: October 20, 2011Published online: January 5, 2012

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