On-Demand Detaching Nanosystem for the Spatiotemporal Control of

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Biological and Medical Applications of Materials and Interfaces

On-Demand Detaching Nanosystem for the Spatiotemporal Control of Cancer Theranostics Tianzhi Liu, Qian Wan, Yu Luo, Mengjie Chen, Chao Zou, Ming Ma, Xin Liu, and Hangrong Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b02062 • Publication Date (Web): 15 Apr 2019 Downloaded from http://pubs.acs.org on April 15, 2019

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ACS Applied Materials & Interfaces

On-Demand Detaching Nanosystem for the Spatiotemporal Control of Cancer Theranostics Tianzhi Liu,†,‡ Qian Wan,§ Yu Luo,† Mengjie Chen,║ Chao Zou,§ Ming Ma,† Xin Liu*,§, and Hangrong Chen *,† † 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; § Paul C. Lauterbur Research Center for Biomedical Imaging, Institute of Biomedical and Health Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, People’s Republic of China; ‡ University of Chinese Academy of Sciences, Beijing, 100049, People’s Republic of China; ║Department of Ultrasonography, The Eighth Affiliated Hospital of Sun Yat-Sen University, Shenzhen 518033, People’s Republic of China KEYWORDS: detachable nanotheranostics, hybrid structure, magnetic resonance imaging, magnetic resonance thermometry, high intensity focused ultrasound

ABSTRACT: Engineering multiple theranostic modalities into single nanoscale entity holds great potential to rejuvenate cancer treatments; however, enabling the sophisticated spatiotemporal control of each component for maximizing theranostic improvement and minimizing side effects concurrently remains a challenge. Herein, an intelligent detachable

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“nanorocket” is developed to sequentially manipulate and optimize multi-theranostic processes for magnetic resonance-assisted ultrasound-drug combined therapy (MR-HIFU-Drug). The “nanorocket” is constructed by integrating multi-component (MnCO3, doxorubicin, silica) on the pH-sensitive CaCO3 nanoparticles step by step via cation exchange and controlled heterogeneous nucleation, in which doxorubicin is encapsulated in both carbonates and silica component. The “nanorocket” can initiate sequential detachment in the acidic tumor microenvironment. Specifically, carbonates decompose instantly, releasing Mn2+ as the MR contrast agent and leaving hollow silica nanostructure behind as the HIFU synergistic agent. Consequently, burst release of drug is also triggered, further triggering the degradation of silica, which in turn regulates the slow release of drug from silica matrix. Thus, efficient tumor inhibition is achieved by enhanced HIFU ablation and biphase release of doxorubicin with a step-wise clearance of Mn and Si. This work establishes a system for the systematic spatiotemporal dispatch of diverse theranostic components for the balance of efficacy and safety in cancer theranostics.

INTRODUCTION As nanotechnology flourishes, highly integrated nanosystems, that can execute multiple tasks in a controllable manner, possess great potential to propel interdisciplinary developments, especially for nanomedicine.1-5 By imparting both diagnostic and therapeutic modalities into a single nanoplatform, cancer treatment can be tailored to be more specific for individuals in a certain way, which is referred to as theranostics.6,7 To date, owing to the advance of synthetic methods for nanostructures, various components with diverse theranostic functions have been assembled together (“all in one”) or explored solely (“one for all”) as nanoagents for imaging guided cancer therapies.8-16 However, multifunctional integration within one physical entity


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raises a knotty problem on how to manipulate each theranostic modality separately and logically, because they are required to perform in different spatiotemporal zones in vivo. From a balanced view focusing on efficacy and safety, for imaging guidance, the nanoagent should identify the tumor region immediately, and then rapidly vanish in vivo. For an efficient therapy, the nanoagent should not be “off-line” (disabled/degraded/excreted) until the therapeutic outcome meets expectations. Apparently, here comes a paradox hindering the further translation of nanotheranostic, which has long been ignored in previous research. As the cognition of tumor abnormalities deepens, stimuli-responsive nanosystems (SRNs), which can realize on-demand delivery of specific theranostic effects (e.g., contrast imaging, drug release, photothermal, ultrasound synergism, etc.) under endogenous stimulation (low pH, high redox potential, etc.) in the tumor microenvironment, have elicited broad interests.17-21 Recently, the research of SRNs is stepping forward into an “intelligent” phase, in which SRNs can be programmed with progressively responsive abilities to precisely conduct designated tasks.22-26 However, it is still a daunting challenge to permit elaborate spatiotemporal control of each component within SRNs, thus SRNs could go beyond simply deploy of multi-theranostic effects but optimize each theranostic performance and suppress its side effects concurrently,27 which may solve the previously mentioned issue regarding the independent performance of nanotheranostic as diagnostics and therapeutics. As a state-of-the-art noninvasive tumor ablation technique, magnetic resonance (MR) supported high intensity focused ultrasound (HIFU) surgery is itching for the promotion of MR contrast imaging, HIFU ablation efficacy and the marriage of chemotherapy based on “intelligent” SRNs, to achieve efficient tumor eradication without causing severe collateral damage28,29. Due to the unique glycolytic metabolism30, cancer region is commonly featured with


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the weak acidity (pH≈ 6.5), which is a reliable stimulus for the design of SRNs. As a typical biomineral, calcium carbonate has been widely explored as drug nanocarrier and ultrasound contrast nanoagent due to its ultra-high pH sensitivity31,32. Herein, a pH-responsive nanosystem is developed as a detachable “nanorocket” based on the pH-sensitive amorphous calcium carbonate nanoparticles (ACC NPs) for MR-guided/monitored HIFU-drug combined therapy (MR-HIFU-Drug)33,34. MnCO3 and silica component are integrated on ACC NPs via cation exchange and heterogeneous nucleation, during which Doxorubicin (Dox), as the chemotherapy drug, is also encapsulated in both carbonates and silica matrix. Thus, carbonates, Dox and silica are hybridized together as three theranostic stages of the “nanorocket” (Scheme 1a). Similar to a multi-stage rocket that can detach sequentially, such “nanorocket” exhibits a cascade evolution in the acidic extracellular microenvironment (pH= 6.5~6.8) of tumor, rendering the activation and elimination of each component in order. Specifically, the carbonates (MC) decompose instantly, releasing Mn2+ as the MR imaging (MRI) contrast agent for tumor location and leaving hollow silica nanostructure behind as the HIFU synergist to intensify HIFU ablation, which is monitored by MR thermometry (MRT).35,36 Consequently, Dox, located within MC, exhibits a burst release, then activating the “inside-out” degradation of silica, which in turn regulates the slow release of Dox within silica. Such a pHtriggered detachable “nanorocket”, which is comprehensively evaluated in an MRI-MRT-HIFU integrated platform, not only realizes on-demand theranostic improvement for antitumor effects but also enables a step-wise clearance of Mn and Si, representing an intelligent SRN for cancer theranostics with outstanding clinical prospects (Scheme 1b). RESULTS AND DISCUSSION


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Synthesis and Characterizations of P-Dox@MCS. As shown in Figure 1a, the pHresponsive detachable “nanorocket” (P-Dox@MCS) is constructed based on amorphous calcium carbonate (ACC) nanoparticles (NPs) in three steps, i.e., cation exchange with Mn2+ (Supplementary Figure S1, Supplementary Discussion S1), integration of doxorubicin hydrochloride (Dox)-silica composite and physical adsorption of polyvinylpyrrolidone (PVP-30). In the first step, the as-synthesized MC NPs were optimized to have a Mn/Ca molar ratio of 1.12 (Supplementary Figure S2, Supplementary Discussion S1) and maintained nearly the same size and morphology of ACC NPs with a brown color (Figure 1b). Following the assembly of Dox-silica composite onto MC NPs (Supplementary Figure S3, Supplementary Discussion S2), monodispersed Dox@MCS NPs were obtained with a uniform size of less than 200 nm and a rough surface (Figure 1c, Supplementary Figure S4). With the integration of both the imaging (MnCO3) and therapeutic components (Dox-silica), Dox@MCS NPs show an average hydrated diameter size of 178.6 nm (PDI= 0.068) and a negative ζ-potential, distinct from those of ACC and MC (Figure 1d, Supplementary Figure S5). Four major elements (Ca, Mn, Si, and O) all present a uniform distribution in one single Dox@MCS NP according to the scanning transmission electron microscopy (STEM) images and corresponding energy dispersive spectrometry (EDS) results (Figure 1e, Supplementary Figure S6), which could be ascribed to the homogeneous cation exchange of Mn2+ and growth of silica within the ACC matrix. Fourier transform infrared (FTIR) spectrum further confirms the coexistence of carbonate component (CO32-) and silica framework (Si-O-Si, Si-OH, and Si-O) in Dox@MCS (Figure 1f, Supplementary Discussion S3).37,38 Moreover, high-resolution XPS spectra of Mn 2p and Ca 2p confirm that both Mn and Ca in Dox@MCS own the same valence state of +2 in the form of carbonate (MnCO3 and CaCO3) (Supplementary Figure S7, Supplementary Discussion


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S3).39,40 In addition to Mn, Ca, Si, O, and C, the presence of N element is also confirmed in the composition of Dox@MCS as shown in the XPS survey spectrum (Figure 1g), demonstrating the successful incorporation of Dox in the carbonate-silica matrix. Notably, the characteristic absorption band of Dox red-shifts and splits into multiple peaks in the UV−vis absorption spectrum of Dox@MCS, which could be attributed to the chelation of Dox with Ca2+/Mn2+ in MC or the encapsulation of Dox within silica (Figure 1h).41-43 In the final step, the assynthesized Dox@MCS NPs were modified with PVP-30 to finally obtain the biocompatible “nanorocket” (P-Dox@MCS). (Supplementary Figure S8, Supplementary Discussion S3). pH-Responsive Detachment Behavior of P-Dox@MCS. As a transient amorphous mineral, ACC suffers from serious aqueous instability (e.g., dissolution and phase transition), which has impeded the advancement of ACC-based nanomedicine.44-47 Herein, ACC NPs are hybridized in the MnCO3-SiO2 matrix and covered with PVP polymer to form P-Dox@MCS NPs, which show good aqueous stability with negligible morphological and microstructural changes over time in the neutral aqueous condition (pH=7.4) as confirmed by TEM (Supplementary Figure S9a). A slight decrease of average hydrated diameter size and elevated ζ-potential were also observed for P-Dox@MCS after neutral incubation (pH=7.4) for a week, which may be ascribed to the desorption of PVP-30 molecules or the dissolution of surface silica (Supplementary Figure S9b, c). Notably, P-Dox@MCS exhibits ultra-sensitivity to mildly acidic condition (Figure 2a). Specifically, P-Dox@MCS rapidly transformed into a hollow structure in 5 min of acidic incubation (pH=6.6) (Supplementary Figure S10a, b). Then, the shell thickness of these hollow nanostructures decreased when the acidic incubation (pH= 6.6) was extended to 12 h. As the spherical shell of silica became thinner over time, some of the nanoshells deformed or even


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collapsed when the acidic incubation was increased to 24 h (Supplementary Figure S11c). Identified by the corresponding EDS spectra (Supplementary Figure S11a, b, d) and element line scanning (Supplementary Figure S10c), silica turns out to be the only remaining inorganic component of P-Dox@MCS during the acidic incubation (pH= 6.6). Therefore, MC in PDox@MCS is believed to undergo decomposition instantly under H+ stimulus in terms of the protonation of carbonate ions, then initiates the degradation of the remaining silica framework from the inside out as illustrated in Figure 2b. Quantitative element results acquired from ICP (Figure 2c) show that almost 100 % of Ca and Mn (from P-Dox@MCS) were released when the acidic incubation (pH=6.6) lasted for only 5 min, coinciding well with above EDS results. Interestingly, 12.5 % of Si (from P-Dox@MCS) was also released together with Ca and Mn in the first 5 min of acidic incubation (pH=6.6); then, Si was released from P-Dox@MCS, reaching a high released amount of 60.4 % at 48 h post acidic incubation (pH=6.6). In contrast, no significant release of Ca, Mn and Si was found in the neutral condition (pH=7.4) for P-Dox@MCS (Supplementary Figure S12). Moreover, CO2 release was also detected via gas chromatography (GC) and ultrasound imaging once incubated in acidic condition (pH=6.6) for P- Dox@MCS, while negligible CO2 release was observed during neutral incubation (pH=7.4), further validating the rapid breakdown of MC in the acidic condition (pH=6.6) (Figure 2d, Supplementary Figure S13). Furthermore, P-Dox@MCS (6.6 wt% Dox) displays a pH-responsive release behavior of` Dox (Figure 2e). No serious leakage of Dox was found during neutral incubation (pH= 7.4). In contrast, during acidic incubation (pH= 6.6), a burst release of Dox was observed in the first 12 h, after which Dox continued to be released in a relatively slow manner. Such a two-stage release pattern of Dox is well-fitted by a biexponential kinetics equation, representing a typical biphase


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release model with two diverse release rate constants (Supplementary Figure S14, Supplementary Table S1, Supplementary Discussion S4).48,49 Therefore, P-Dox@MCS could render a high concentration of Dox instantly in the tumor region, then largely maintain drug efficacy by sustained release of Dox, which adequately meets the requirement of highly efficient chemotherapy. N, as the characteristic element of Dox, is found to coexist with Si, Mn and Ca in both surface layer and inner layer of Dox@MCS (Supplementary Table S2). Thus, the stepwise control of Dox release is considered to be governed by the pH-triggered compositional and morphological evolution of P-Dox@MCS (MC decomposition and silica degradation). Notably, the valence state of Si decreases from the outer layer to the inner layer of Dox@MCS (Figure 2f), revealing a lower degree of condensation of inner silica than that of outer silica. However, the valence states of Mn and Ca stay the same in the form of MnCO3 and CaCO3 at both the surface layer and the inner layer of Dox@MCS (Supplementary Figure S15). It is verified that Dox can affect the growth of silica nanoparticles due to its opposite charge with initial silica species (monomers or oligomers) formed via hydrolysis of TEOS, thereby obtaining Dox-SiO2 hybrid nanocomposites, which can undergo “inside-out” degradation induced by Dox diffusion/release even in the neutral condition (pH=7.4).50,51 In this study, MC, which has the same charge of Dox (Supplementary Figure S1b), as illustrated in Figure 2g, is believed to interact with initial silica species together with Dox to form the silica framework with a condensation gradient (Supplementary discussion S5). It is worth noting that unlike previously reported Dox-SiO2, PDox@MCS is quite stable in neutral aqueous condition, and shows acid-responsive Dox release and silica degradation, revealing that MC reported in this study, may also act as the anchor of Dox through chelating with its Mn2+ and Ca2+,52 and only when MC decomposes, Dox is free to


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diffuse, thus initiating the “inside-out” degradation of silica simultaneously owing to its condensation gradient. Therefore, due to the homogeneous hybridization, each component (CaCO3, MnCO3, Dox, and silica) within P-Dox@MCS can remain intact without detaching in the normal physiological environments (pH=7.4). However, acidity-triggered MC decomposition in P-Dox@MCS not only facilitates the instant release of Mn2+ and Dox, but also activates a self-regulation mode of the remaining silica-Dox composite subsequently, i.e., silica degradation and slow release of Dox. Such a pH-responsive sequential dispatch of P-Dox@MCS could enable each theranostic component to execute missions in their optimal spatiotemporal zones in the mildly acidic tumor microenvironment for an efficacy-safety balanced cancer treatment. MRI Performance of P-Dox@MCS. As the first detaching component of P-Dox@MCS, MC could decompose to release paramagnetic Mn2+ as the T1 contrast agent for MRI signal enhancement in mildly acidic tumor region. As shown in Figure 3a, P-Dox@MCS exhibits a relatively small longitudinal (r1) and transverse (r2) relaxivity in the neutral condition (pH=7.4), which dramatically rise to 7.17 mM-1 s-1 (r1) and 11.99 mM-1 s-1 (r2) in the acidic condition (pH=6.6). Moreover, a slight increase of r2 was observed for P-Dox@MCS when neutral incubation (pH=7.4) extends to 48 h, indicating a negligible leakage of Mn2+, which accords well with its excellent stability and integrity in neutral aqueous condition (pH=7.4) (Supplementary Figure S16). Owing to the rather low value of r2/r1 (ca. 1.67) in the stimulated tumor microenvironment, P-Dox@MCS could be used for efficient pH-responsive T1 contrast imaging.53,54 As expected, in vitro T1-weighted MR images show that the MR signal is positively correlated with the concentration of Mn in P-Dox@MCS only in the acidic condition (pH=6.6)


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(Figure 3b, Supplementary Figure S17), demonstrating its potential to illuminate tumor region for the specific MRI guidance of HIFU ablation. Encouraged by the in vitro results, P-Dox@MCS was further evaluated for its in vivo T1weighted MRI contrast performance in 4T1-tumor-bearing mice via intravenous (i.v.) injection. As shown in Figure 3c, d, the tumor region was brightened instantly at 30 min post-i.v. administration of P-Dox@MCS, then continued to be highlighted in the next 90 min, reaching a maximum MR signal intensity at 2 h post-i.v. administration. Subsequently, the contrast enhancement decayed and could hardly be detected at 24 h post-i.v. administration. Such successive variations in MR signal in tumor is believed to be closely related to the status alteration of Mn2+ in P-Dox@MCS in the tumor region, namely, the rapid dissociation from PDox@MCS and diffusion within tumor tissue, followed by steadily elimination via circulation. In addition, in vivo real-time ultrasound imaging also displays a “first rise then drop” tendency of echo signal in tumor post-i.v. administration, similar to the MR signal changes but within a much shorter period of time (only 2 h) (Supplementary Figure S18), which could be ascribed to the faster generation and dissipation of CO2 gas bubbles along with Mn2+ release during MC decomposition in the mildly acidic tumor microenvironment. Biodistribution and Cytotoxicity of P-Dox@MCS. The biodistribution results of PDox@MCS were obtained in 4T1-tumor-bearing mice by measuring the content of Mn and Si. It is worth noting that Mn and Si have almost the same mass fraction in P-Dox@MCS as confirmed by ICP (Supplementary Table S3). However, a lower content of Mn is verified compared to that of Si not only in the tumor region but also in the major organs post-i.v. administration (Supplementary Figure S19). The quantitative data of the cumulative element content in vivo shows that 20.1 % of Mn in the injected P-Dox@MCS can be detected within the


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major organs and tumor at 2 h post-i.v. administration, then the ratio drops to only 4.2 % at 24 h post-i.v. administration. In contrast, ignoring the low intrinsic content of Si in organs and tumor (Supplementary Figure S20), Si reaches a higher tissue retention of 54.5 % at 2 h post-i.v. administration, which decreases to 29.1 % at 24 h post-i.v. administration (Figure 3e). Moreover, the variations in Mn/Si content in blood over time also differ from each other, displaying diverse blood terminal half-lives of Mn (0.29 h) (Figure 3f) and Si (0.92 h) (Figure 3g). Apparently, Mn and Si in P-Dox@MCS could travel separately in vivo due to its on-demand detachment, which occurs not only in the tumor microenvironment but also in lysosomes (pH= 5) after endocytosis into normal cells. Such in vivo detachment enables the fast release of Mn2+ and initiates the subsequent gradual degradation of silica, resulting in a step-wise clearance of each theranostic component in P-Dox@MCS. Notably, P-MCS, obtained by the integration of MC with only the biocompatible silica component (Supplementary Figure S21), induces negligible cytotoxicity after coincubation with normal cells (BCEC and BRL cells) at low concentrations of Mn (≤ 200 μM), while the cell viability decreases at elevated concentrations of Mn (≥ 400 μM), reflecting the potential toxicity of Mn2+ in cell culture (Supplementary Figure S22a). Therefore, the efficient in vivo elimination of Mn2+ is necessary to avoid its systemic toxicity,55 which can undergo both urinary elimination via kidney and biliary elimination via liver.56,57 In addition, histological examinations reveal no noticeable pathological abnormalities in the major organs 30 days posti.v. injection of P-MCS within a certain concentration range (5~20 mg/kg) in nude mice (Supplementary Figure S23). As expected, P-Dox@MCS demonstrates higher cytotoxicity to 4T1 cancer cells than P-MCS at varied concentrations of Mn due to the release of Dox (Supplementary Figure S22b), which was observed by confocal laser scanning microscopy


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(CLSM) based on the intrinsic red fluorescence of Dox (Supplementary Figure S24), further confirming the successful delivery of chemotherapy at the cellular level. HIFU Synergism of P-Dox@MCS. After instant detachment, the remaining silica component of P-Dox@MCS in the acidic condition, proceeding with “inside-out” degradation, could act as the HIFU synergist for HIFU ablation, which was first evaluated using a porcine model in the MRI-MRT-HIFU platform (Supplementary Figure S25). Under the MRI guiding modality, the injection site of P-Dox@MCS, which was incubated in acidic condition (pH=6.6) for 5 min, was highlighted in the porcine model due to the released Mn2+ (Figure 4a). Then, MRT shows that the remaining initial hollow silica spheres at the injection site can not only elevate the thermal deposition of HIFU with low powers (< 10 W) (Supplementary Figure S26), but also intensify the hyperthermia effect of HIFU with 20 W (Figure 4b), resulting in enhanced ablation volume of the porcine tissue (P= 0.0239, t= 6.35, df= 2) (Figure 4c). Interestingly, as shown in Figure 4d-f, P-Dox@MCS, after acidic incubation (pH=6.6) for 12 h, still exhibits intensified hyperthermia under 20 W HIFU irradiation, with nearly the same increasing temperature and ablated volume (P= 0.0101, t= 9.87, df= 2) in the porcine model, as those of P-Dox@MCS incubated in acidic condition (pH=6.6) for 5 min, indicating that the degradation of the remaining silica component does not affect its synergic effect for HIFU ablation under such circumstances. Theoretically, HIFU attenuation is the main cause of the temperature increase during HIFU propagation. It is believed that the viscous and thermal transport processes occurring at the interface of nonhomogeneity caused by NPs embedded in the medium, as well as the intrinsic absorption of NPs contribute to the enhancement of HIFU attenuation.58-63 Notably, during acidic incubation, the remaining hollow silica nanospheres not only undergo “inside-out” degradation, but also lose monodispersity and form micron aggregates (Figure 2a,


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Supplementary Figure S27). In addition, in situ injection could benefit the formation of aggregates of NPs in the porcine model. Therefore, we speculate that sufficient amounts of interfaces, comparable to the ultrasonic wavelength, are formed between silica aggregates and porcine tissue, which dominates the HIFU attenuation process, and makes it independent of the degradation degree of silica (Figure 4g). Such a unique HIFU attenuation mechanism of inorganic NPs, makes it superior to the transient gas-based HIFU synergism, and may prolong the enhancement times of HIFU ablation, especially in combating tumors. In Vivo Theranostic Evaluation of P-Dox@MCS. Encouraged by the outstanding performance in contrast MRI and HIFU synergism, P-Dox@MCS was comprehensively evaluated in vivo using 4T1-tumor-bearing mice in the MRI-MRT-HIFU platform to investigate its applicability in an integrated theranostic system and the subsequent anti-tumor effect. After the tumor volume reached 80~100 mm3, 4T1-tumor-bearing mice were divided into 4 groups for different treatments as follows: (1) saline (i.v. injection), (2) saline (i.v. injection) + HIFU, (3) PDox@MCS (i.v. injection) and (4) P-Dox@MCS (i.v. injection) + HIFU. As shown in Figure 5a, the MRI-MRT-HIFU integrated theranostic process was conducted in 4T1-tumor-bearing mice in groups of (2) and (4). Specifically, after i.v. injection of saline or P-Dox@MCS, each mouse was first scanned by MRI for the guidance of HIFU focus at the tumor site, and then HIFU irradiation was applied (10 W for 250 s) under MRT monitoring. As expected, PDox@MCS improves the identification of the locations of tumor sites with enhanced contrast MRI, and then augment the heat deposition on tumors during HIFU irradiation at 2 h post-i.v. injection (Figure 5b). Notably, the corresponding temperature variation curves at tumor sites of each mouse form two thermal gradient surfaces, representing two different tumor heating kinetics for group (2) and group (4) (Figure 5c). Obviously, all mice in group (4) exhibit a faster


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heating rate at the tumor sites compared to that of group (2), resulting in a higher mean maximum temperature at the tumor sites of mice in group (4) after 250 s of HIFU irradiation (P= 0.002, t= 5.85, df= 5) (Figure 5d). Such intensified hyperthermia by HIFU, together with the released Dox, induced significantly higher level of necrosis and apoptosis of cancer cells in group (4) compared to those of other groups one day after different treatments (Supplementary Figure S28). As shown in Figure 5e, tumors in group (1) and (3) grew rapidly and some reached 1000 mm3 on the 9th day post-treatment. Due to ethical concerns about excess tumor volume, mice in group (1) and (3) were all euthanized on the 9th day post-treatment. In contrast, almost all the tumors in group (2) and group (4) were efficiently ablated under HIFU irradiation, leaving only scars at the initial tumor sites on the first day post-treatment (Supplementary Figure S29). Then, the tumor growth was effectively inhibited in group (4) for two weeks post-treatment. However, tumors in group (2) suffered severe recurrence (Supplementary Figure S30). The relative tumor volumes in group (4) present a significant difference over group (2) (P= 0.0097, t= 4.64, df= 4) on the 15th day post-treatment (Figure 5f). Additionally, negligible weight fluctuations are confirmed for all mice during the 15-day observation after different treatments (Supplementary Figure S31). Therefore, the in vivo on-demand detachment feature of PDox@MCS not only achieves a fast and reinforced theranostic modality in the MRI-MRT-HIFU system, but also demonstrates an appreciable long-term antitumor effect without severe side effects, which could be ascribed to the intensified hyperthermia and sustained release of Dox at the tumor site. CONCLUSIONS Distinct from the simplex “all in one” design for nanotheranostics, herein, we demonstrate a multi-stage detachable nanosystem (P-Dox@MCS) for MR-guided/monitored HIFU-drug


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combined cancer therapy based on a multi-component hybrid nanostructure, which is constructed using pH-sensitive ACC NPs via cation exchange with Mn2+, subsequent integration of silicaDox composite and surface modification with PVP. Such a nanosystem can remain stable and intact in normal physiological environment (pH= 7.4), while responding instantly to mild acidity (pH=6.6) in the tumor microenvironment, in which carbonates (MnCO3 and CaCO3) decompose completely in 5 min, releasing Mn2+ as the MRI contrast agent and leaving hollow silica spheres behind as the HIFU synergistic agent. Meanwhile, a stage-wise release (biphasic release) pattern of Dox is also activated which further induces the “inside-out” degradation of remaining silica component. Consequently, P-Dox@MCS presents a fast MRI enhancement of tumor within 2 h and efficient in vivo clearance of Mn in 24 h, which is faster than that of Si. Furthermore, MRT reveals that the remaining silica nanostructure can promote the thermal deposition of HIFU under MRI guidance, not only in ex vivo porcine model but also at the tumor site in vivo, which together with sustained release of Dox, inhibits tumor growth effectively. In brief, P-Dox@MCS realizes on-demand sequential separation of its imaging and therapeutic components in vivo, which are sequentially actuated and eliminated during the theranostic process, thus achieving a balance between high theranostic efficacy and safety. Such a smart material perfectly meets the clinical demand and may inspire the design of intelligent nanoagents for other theranostic modalities. EXPERIMENTAL SECTION Chemicals and Reagents. Tetraethyl orthosilicate (TEOS), ammonium solution (25−28%), ammonium bicarbonate (NH4HCO3), polyvinylpyrrolidone (PVP-30, average relative molecular mass of 30000) and ethanol were purchased from Sinopharm Chemical Reagent Company. Calcium chloride dihydrate (CaCl2·2H2O), manganese chloride tetrahydrate (MnCl2·4H2O) were


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purchased from Sigma-Aldrich. Doxorubicin hydrochloride (Dox) was obtained from Beijing Hua Feng United Technology Co., Ltd.. Synthesis of Dox@MnCO3&CaCO3-SiO2 (Dox@MCS) NPs. 5 mg of Dox, 3.4 mL of ammonium solution were added to 75 mL of MC NPs (Ca/Mn molar ratio: 1.12) ethanol dispersion and stirred for 1 hour at 25 ℃. Then, 40 μL of TEOS was added to the above mixture and stirred for another 24 hours at 25 ℃. The final product (Dox@MCS) was collected by centrifugation and washed with ethanol for several times. Surface Modification of Dox@MCS. 100 mg of PVP-30 were dissolved in 100 mL of assynthesized Dox@MCS ethanol dispersion (1 mg/mL), then stirred for 4 h at 25 ℃. The PVPDox@MCS NPs (P-Dox@MCS) were finally collected by centrifugation and washed with ethanol and water for several times. In Vitro Dox Release from P-Dox@MCS. 10 mg of P-Dox@MCS were encapsulated into a dialysis bag and put into 25 mL of saline with different pH (pH= 7.4 and 6.6). Then, the releasing process was performed in a shaking table at a shaking speed of 80 rpm at 37 ℃, the released Dox was monitored by UV-vis spectra at a time-course manner. All the experiments were carried out in triplicate and independently. Data were taken as mean ± standard error. Relaxivity Characterization. Relaxation time (T1, T2) measurement was operated in the 0.5 T NMI20 Analyzing and Imaging System (Shanghai NIUMAG Corporation, Shanghai, China) for P-Dox@MCS in saline of different pH (pH= 7.4 and 6.6) with a given Mn concentration of 0.5, 1, 2, 4 mM. Test parameters were as follows, T2: TR =7500 ms, TE =0.49996 ms, RFD =0.08 ms; T1: TR =6000 ms, TE =1 ms, RFD =0.2 ms. The corresponding relaxation rate (r1, r2) were obtained through linear fitting the inverse T2/T1 (1/T2 or 1/T1) as a function of Mn concentration.


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In Vitro T1-weighted MR Imaging. In vitro T1-weighted MR Imaging (MRI) was conducted at 25 °C using a 7.0 T Bruker Biospec small animal MRI system (Bruker, Inc., Billerica, MA). PDox@MCS at different Mn concentrations (0.25, 0.5, 0.75, 1, 2, 4 mM) were dispersed in saline solution at different pH (pH= 7.4 and 6.6), then placed in centrifuge tubes for MRI scanning. The T1-weighted MR scanning parameters were as follows, TR= 800 ms, TE= 7.0 ms, slice thickness= 1 mm, field of view (FOV)= 30 × 30 mm2. The intensity of MR images was quantified using RadiAnt DICOM Viewer. Animal Model. Four-week old male BALB/c nude mice were purchased from Guangdong Medical Experimental Animal Center. 4T1-tumor-bearing BALB/c nude mice were supplied by Shenzhen Institutes of Advanced Technology (SIAT). All experiments involving animals were ethically and scientifically approved by animal usage and care regulations of SIAT. In Vivo Ultrasound imaging. P-Dox@MCS in saline (200 μL, [Mn]= 200 μg, [Si]= 215 μg) was intravenously injected into 4T1-tumor-bearing nude mice (n= 3). Meanwhile, the tumor sites were monitored by B-mode ultrasound imaging (Vevo 2100-VisualSonics FujiFilm, MicroScan Transducers MS250 (40 MHz), mechanical index (MI)= 0.5) during the whole process. Hyper echoes were detected, and post-gray scale values were recorded using software GrayVal 1.0 (Chongqing Haifu Technology, Chongqing, China). In Vivo T1-weighted MR Imaging. In vivo T1-weighted MR Imaging was operated on a 3.0 T clinical MR scanner (TIM TRIO, Siemens, Germany) pre and post-intravenously (0.5 h, 1 h, 2 h, 6 h, 24 h) injected of P-Dox@MCS in saline solution (200 μL, [Mn]= 200 μg, [Si]= 215 μg) into 4T1-tumor-bearing mice (n= 3). The MRI scanning parameters were as follows, TR= 546 ms,


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TE= 13.0 ms, slice thickness= 1.1 mm, field of view (FOV)= 36 × 52 mm2. The intensity of MR images was quantified using RadiAnt DICOM Viewer. In Vitro (Porcine Model) Theranostic Evaluation in MRI-MRT-HIFU Platform. In vitro theranostic assessment of P-Dox@MCS was carried out using porcine model in the MRI-MRTHIFU platform. 2 mg of P-Dox@MCS was dispersed in 0.5 mL saline (pH= 6.6) and incubated for 5 min, then injected into porcine tissue (n= 3). 0.5 mL saline (pH= 6.6) was also injected into the same porcine tissue next to the injection site of P-Dox@MCS. Then the porcine tissue was pre-scanned by T1-weighted (TR= 600 ms, TE= 7.8 ms) and T2-weighted (TR= 6230 ms, TE= 82 ms) MR imaging for the location of injection sites. The focus of the ultrasound transducer was moved to the injection sites and sonicated at different operating parameters (5.8 W for 250 s, 7.6 W for 250 s, 9.5 W for 250 s, 20 W for 160 s). During sonication, the temperature changes at the injection sites were monitored by MR thermometry. After HIFU irradiation of 20 W for 160 s, the volume of the coagulated lesion with white color was calculated by the following equation: 𝑉 = п × 𝐿 × 𝑊2/6#(1) (where L is the length of the coagulated lesion, W is the width of the coagulated lesion). The experimental process above was repeated for P-Dox@MCS after acidic incubation (pH= 6.6) for 12 h. In Vivo Theranostic Evaluation in MRI-MRT-HIFU platform. In vivo theranostic assessment of P-Dox@MCS was carried out on 4T1-tumor-bearing nude mice in the MRI-MRTHIFU platform via intravenous (i.v.) injection. BALB/c nude mice bearing 4T1 xenograft tumors (n= 6) were divided into 4 groups after the tumor volume reached 80~100 mm3 for different treatments: (1) saline (i.v. injection, 200 μL), (2) saline (i.v. injection, 200 μL) + HIFU, (3) P-


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Dox@MCS (i.v. injection, in 200 μL saline, [Mn]= 200 μg), (4) P-Dox@MCS (i.v. injection, in 200 μL saline, [Mn]= 200 μg) + HIFU. For group (2) and (4), each 4T1 tumor-bearing nude mice were pre-scanned by T1-weighted (TR= 600 ms, TE= 7.8 ms) and T2-weighted (TR= 2740 ms, TE= 84 ms) MR imaging for the location of tumor sites at 2 h post-i.v. injection. Then the focus of the ultrasound transducer was moved to the tumor sites and sonicated at 10 W for 250 s. Meanwhile, the temperature changes at the tumor sites were monitored by MR thermometry. For each group, one nude mouse was sacrificed, tumors were harvested for H&E and TUNEL staining at the first day post each treatment. Then, the tumor size (length and width) and body weight were measured every 2 days. The tumor volume was calculated using the following formula: 𝑉 = 𝐿 × 𝑊2/2#(2) (where L is the length of tumor, W is the width of tumor), which was then divided by the pretreatment tumor volume to obtain the relative tumor volume. 4T1 tumor-bearing nude mice in group (1) and (3) were euthanized when tumor volume reached 1000 mm3 on the 9th day posttreatment due to ethical concerns about excess tumor volume. 4T1 tumor-bearing nude mice in group (2) and (4) were euthanized on the 15th day post-treatments. ASSOCIATED CONTENT Supporting Information Available: Supplementary methods, figures (Figure S1-Figure S31), tables (Table S1-Table S3) and discussions (Discussion S1-Discussion S5) are included as supporting information. This material is available free of charge via the Internet on the ACS Publications website (http://pubs.acs.org).


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AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] * E-mail: [email protected] ORCID Hangrong Chen: 0000-0003-0827-1270 Notes The authors declare no competing ﬁnancial interest ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Grant No. 51772316, 81327801, and 81527901), the National Key Research and Development Program of China (Grant No. 2017YFB0702602), the Key Project of International Cooperation and Exchange of NSFC (No. 81720108023) and the Natural Science Foundation of Shanghai (Grant No. 18ZR1444800).

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SCHEMES AND FIGURES

Scheme 1. (a) Multi-component integration within one nanosystem. (b) Spatiotemporal control of cancer theranostics via pH-triggered multi-stage detachment of the hybrid nanosystem (“nanorocket”) in the MRI-MRT-HIFU platform.


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Figure 1. Preparation and characterizations of the “nanorocket”. (a) A schematic illustration of the synthetic process of P-Dox@MCS (“nanorocket”). (b) TEM images of ACC and MC. Insets: the corresponding digital photographs of the sample dispersed in ethanol. (c) TEM and SEM images of Dox@MCS NPs. (d) Dynamic light scattering (DLS) results of ACC, MC and Dox@MCS. (e) Bright-field STEM image and corresponding EDS element mapping of Dox@MCS. (f) Fourier transform infrared (FTIR) spectrum of Dox@MCS. (g) Survey XPS spectrum of Dox@MCS. Inset: the high resolution XPS spectra of N 1s. (h) UV-vis absorption spectra of Dox and Dox@MCS in deionized (DI) water.


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Figure 2. The pH-activated detachment behavior of P-Dox@MCS in vitro. (a) TEM images of PDox@MCS incubated in acidic condition (pH=6.6) at different time points. (b) A schematic illustration of the pH triggered compositional and morphological evolution process of PDox@MCS. (c) Element release profile of P-Dox@MCS incubated in acidic conditions (pH=6.6) at different time points (n=3). (d) Carbon dioxide release amounts and (e) drug release profiles (n=3) of P-Dox@MCS incubated in neutral (pH=7.4) and acidic (pH=6.6) conditions. (f) High resolution XPS spectra of Si 2p of Dox@MCS pre- and post-etching at different levels. (g)


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A schematic illustration of the formation mechanism of Dox-silica composite on MC with a condensation gradient of silica.

Figure 3. MRI performance and pharmacokinetics/biodistribution of P-Dox@MCS. (a) Plots of 1/T1 and 1/T2 versus Mn2+ concentration of P-Dox@MCS in neutral (pH=7.4) and acidic (pH=6.6) conditions. (b) In vitro T1-weighted MR images of P-Dox@MCS at different Mn2+ concentrations in neutral (pH=7.4) and acidic (pH=6.6) conditions. (c) In vivo T1-weighted MR images and (d) the corresponding MR signal intensity of the tumor area of subcutaneous 4T1tumor-bearing mice (n= 3) pre- and post-i.v. injection of P-Dox@MCS at different time points. (e) Summary of Mn/Si biodistribution percentage in major organs and tumor at 2 h and 24 h post-i.v. injection of P-Dox@MCS in mice (n= 3). (f, g) Plots of the Mn/Si concentration in


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blood versus time post-i.v. injection of P-Dox@MCS in mice (n= 3) to determine the blood halftime of Mn/Si.

Figure 4. Ex vivo porcine model evaluations of P-Dox@MCS in the MRI-MRT-HIFU theranostic platform. In vitro MRI guiding/MRT monitoring HIFU irradiation of porcine tissue injected with saline and P-Dox@MCS dispersion (incubated in pH=6.6 saline for 5 min): (a) T1weighted and T2-weighted MR images of the porcine tissue before HIFU irradiation (20 W); (b) MR thermography images and corresponding temperature rising curves of the porcine tissue during HIFU irradiation (20 W); (c) Digital photograph of the porcine tissue, and corresponding ablated volumes after HIFU irradiation (20 W) (n=3), * represents statistically significant differences in the ablated volume at P< 0.05. In vitro MRI guiding/MRT monitoring HIFU


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irradiation of porcine tissue injected with saline and P-Dox@MCS dispersion (incubated in pH=6.6 saline for 12 h): (d) T1-weighted and T2-weighted MR images of the porcine tissue before HIFU irradiation (20 W); (e) MR thermography images and corresponding temperature rising curves of the porcine tissue during HIFU irradiation (20 W); (f) Digital photograph of the porcine tissue, and corresponding ablated volumes after HIFU irradiation (20 W) (n=3), * represents statistically significant differences in the ablated volume at P< 0.05. (g) A schematic illustration of the HIFU attenuation process of hollow silica nanoparticles embedded in the porcine model.


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Figure 5. In vivo evaluations of P-Dox@MCS in the MRI-MRT-HIFU theranostic platform. (a) A schematic illustration of the in vivo MRI guiding/MRT monitoring HIFU surgery on 4T1tumor-bearing mice treated with P-Dox@MCS. (b) T1-weighted and T2-weighted MR images, and MR thermography images of mice treated with HIFU and HIFU+P-Dox@MCS before and during HIFU irradiation. (c) The curved surfaces of temperature increases in groups of saline (i.v. injection) + HIFU and P-Dox@MCS (i.v. injection) + HIFU (formed with all of the temperature increase curves at the tumor sites of 4T1-tumor-bearing mice (n=6) in above two groups during HIFU irradiation at 2 h post-i.v. injection) (d) Mean maximum temperature increases (ΔT) at tumor sites in groups of saline + HIFU and P-Dox@MCS + HIFU (n=6) at 2 h post-i.v. injection, ** represents statistically significant differences in the maximum ΔT at P< 0.01. (e) Time-dependent relative tumor volume curves after different treatments. (f) Relative tumor volume at 15 days post-treatment of groups (saline+HIFU and P-Dox@MCS+HIFU, n= 5), ** represents statistically significant differences of the relative tumor volume at P< 0.01.


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For Table of Contents Only A pH-responsive detachable “nanorocket” (P-Dox@MCS) is developed based on a hybrid nanostructure composed of carbonate, drug and silica, which can enable logical spatiotemporal control of each theranostic component for magnetic resonance-guided/monitored ultrasound-drug combined therapy (MR-HIFU-Drug) via a sequential evolution of composition and morphology (multi-stage detaching) in acidic tumor region.


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On-Demand Detaching Nanosystem for the Spatiotemporal Control of

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