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Renal-Clearable Hollow Bismuth Subcarbonate Nanotubes for Tumor Targeted CT Imaging and Chemoradiotherapy Xi Hu, Jihong Sun, Fangyuan Li, Ruiqing Li, Jiahe Wu, Jie He, Nan Wang, Jianan Liu, Shuaifei Wang, Fei Zhou, Xiaolian Sun, Dokyoon Kim, Taeghwan Hyeon, and Daishun Ling Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.7b04741 • Publication Date (Web): 03 Jan 2018 Downloaded from http://pubs.acs.org on January 3, 2018

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Renal-Clearable Hollow Bismuth Subcarbonate Nanotubes for Tumor Targeted CT Imaging and Chemoradiotherapy Xi Hu,†,‡,⊥ Jihong Sun,§,⊥ Fangyuan Li,†,‡,⊥ Ruiqing Li,†,‡ Jiahe Wu,†,‡ Jie He,§ Nan Wang,†,‡ Jianan Liu,ο,# Shuaifei Wang,†,‡ Fei Zhou,§ Xiaolian Sun,∇Dokyoon Kim,ο Taeghwan Hyeon,ο,# and Daishun Ling*,†,‡,||

†

Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical

Sciences, Zhejiang University, 310058, China ‡

Hangzhou Institute of Innovative Medicine, College of Pharmaceutical Sciences, Zhejiang Uni-

versity, Hangzhou 310058, China §

Department of Radiology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University,

Hangzhou 310020, China ||

Key Laboratory of Biomedical Engineering of the Ministry of Education, College of Biomedical

Engineering & Instrument Science, Zhejiang University, Hangzhou 310027, China ο

Center for Nanoparticle Research, Institute for Basic Science (IBS), Seoul 08826, Republic of

Korea #

School of Chemical and Biological Engineering, and Institute of Chemical Processes, Seoul Na-

tional University, Seoul 08826, Republic of Korea ∇Laboratory

of Molecular Imaging and Nanomedicine (LOMIN), National Institute of Biomedical

Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH), Bethesda, MD 20892, USA

⊥

These authors contributed equally to this work.

1 ACS Paragon Plus Environment

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ABSTRACT: Although metallic nanomaterials with high X-ray attenuation coefficients have been widely used as X-ray computed tomography (CT) contrast agents, their intrinsically poor biodegradability requires them to be cleared from the body to avoid any potential toxicity. On the other hand, extremely small-sized nanomaterials with outstanding renal clearance properties are not much effective for tumor targeting because of their too rapid clearance in vivo. To overcome this dilemma, here we report on the hollow bismuth subcarbonate nanotubes (BNTs) assembled from renal-clearable ultrasmall bismuth subcarbonate nanoclusters for tumor-targeted imaging and chemoradiotherapy. The BNTs could be targeted to tumors with high efficiency and exhibit a high CT contrast effect. Moreover, simultaneous radio- and chemotherapy using drug-loaded BNTs could significantly suppress tumor volumes, highlighting their potential application in CT imaging-guided therapy. Importantly, the elongated nanotubes could be disassembled into isolated small nanoclusters in the acidic tumor microenvironment, accelerating the payload release and kidney excretion. Such body clearable CT contrast agent with high imaging performance and multiple therapeutic functions shall have a substantial potential for biomedical applications.

KEYWORDS: Hollow nanotubes, bismuth subcarbonate, tumor-targeting, CT imaging, chemoradiotherapy, renal clearance


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As one of the most widely used non-invasive imaging techniques, X-ray computed tomography (CT) plays an important role in the clinical diagnosis of various diseases such as malignant cancers1,2 and pulmonary diseases3, and others4. A large amount of contrast agent is often required for clinical CT imaging due to the relatively low sensitivity. Furthermore, commercial iodinated compound-based agents have shown limited success due to their extremely short half-life in blood4 and adverse secondary effects5. Recently, a wide range of inorganic nanomaterials with high X-ray attenuation effects including bismuth (Bi), gold (Au), platinum (Pt), and tantalum (Ta), have been explored as possible alternatives.6-10 Bi-based nanomaterials have attracted particular interest due to their relatively high X-ray attenuation coefficient (5.74 cm2 kg−1 at 100 keV), low toxicity and cost.11-17 Furthermore, Bi-based nanomaterials were investigated as radio-sensitizers that amplify radiation-induced tumor damage.15,18 However, the U.S. Food and Drug Administration (FDA) asserts that renal clearance of injected metallic nanomaterials is critical in order to avoid the side effects associated with their long-term retention.19 Renal-clearable nanomaterials should have a small size (< 7 nm), which, on the other hand, limits their efficacy for tumor targeting due to their rapid clearance in vivo.20 The development of renal-clearable nanoscale contrast agents that persist in the circulation long enough to allow tumor homing will be able to overcome this dilemma. Controlled assembly of nanoparticles into various shapes and structures for tunable functionality21-25 can promote their targeted delivery26 and allow their controlled elimination from the body27. Herein, we report on the designed assembly of small-sized (BiO)2CO3 nanoclusters (BNCs) into elongated and hollow (BiO)2CO3 nanotubes (BNTs) (Figure 1a). The unique nanostructure of the BNTs promotes their tumor homing and enables the drug loading, which are useful not only for tumor-targeted CT imaging, but also for synergistic radio- and chemotherapy. Interestingly, the BNTs can be disassembled into


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nanoclusters in the acidic tumor microenvironment, leading to accelerated drug release and renal excretion. The BNTs have the following advantages over the previously reported nanoscale contrast agents: i) prolonged persistence in the circulation due to their elongated structure (high aspect ratio) for enhanced tumor homing, ii) hollow interior nanostructure for high drug loading, iii) combination of targeted CT imaging and imaging-guided chemo-/radiotherapy of tumors, and iv) outstanding renal clearance property facilitated by bioresponsive disassembly (Figure 1b). To generate the BNTs, uniform BNCs with an extremely small size of ~1.5 nm were first synthesized by heating a mixture of Bi acetate, oleic acid, hexadecylamine, and 1octadecene (Figure 1c). Among the reactants, oleic acid plays significant roles during the synthesis. Firstly, oleic acid acts as a reagent to form bismuth oleate under heating. Next, oleic acid reacts with organic amine (hexadecylamine, etc.) to produce a trace amount of water, which results in the hydrolysis of bismuth oleate to form bismuth oxide nanoclusters.28 Moreover, similar to bismuth citrate as a source of the carbonate anions,29 bismuth acetate serves not only as a bismuth precursor but also as a possible source of carbonate after the thermal decomposition of acetate anions. The generated carbonate anions subsequently replace one oxide atom of bismuth oxide (Bi2O3), resulting in the formation of (BiO)2CO3. However, the resulting nanoclusters are not suitable for tumor targeting due to their rapid renal clearance and short in vivo circulation time.30,31 Therefore, the nanoclusters were further assembled into elongated nanotubes in the presence of excess oleic acid via a solvothermal reaction. Oleic acid can form a protective shell around the crystal nuclei and confer high dispersibility in non-polar solvents.32,33 However, too much oleic acid can cause nanoparticle clustering.34 After autoclaving the dissolved mixture solution at 200 °C for 4 h, the nanoclusters assembled into open-ended nanotubes with a wall thickness of ~2 nm and an outer diameter of ~8 nm (Figure 1d,e). The solvothermal process


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was turned out to be essential for the tubular assembly, since only thin and soft nanowires were formed when the reaction was performed at 200 °C under atmospheric pressure for 4 h (Figure S1a). However, the ordered assembly can be disturbed by a high chemical potential,35 for instance, by decreasing the amount of oleic acid or increasing the Bi precursor concentration (Figure S1b,c). Similar to oriented attachment and mesoscale self-assembly processes,32 it is possible that oleic acid ligands outside the (BiO)2CO3 nanoclusters are immobilized by adjacent intertwining of fatty acid chains,36 and aligned into mesocrystallike nanotubes, which may be attributed to synergistic steric effects (exerted by the oleic acid ligands), short-range van der Waals force, and dipole-dipole interactions between the nanoclusters.37,38 Moreover, surface oleic acid ligands create a strong steric barrier after cooling, endowing the nanotubes with excellent colloidal dispersibility after the reaction and preventing further interactions between the nanotubes.33

31

The X-ray photoelectron

spectroscopy (XPS) analysis of the BNTs reveals two major asymmetrical peaks centered at 158.6 and 163.8 eV (Figure S2a,b), corresponding to the Bi 4f7/2 and Bi 4f5/2 peaks of Bi3+ ions, respectively.11,39,40 The Bi:O ratio of approximately 2:5 determined by XPS and energy dispersive spectroscopy (Figure S2c) matches well with stoichiometric ratio of (BiO)2CO3. All reflection peaks in the powder X-ray diffraction pattern (Figure S2d) could be indexed to the (BiO)2CO3 structure (PDF No. 41-1488). To assess the utility of the elongated BNTs for CT imaging and therapeutic application (Figure 2a), hydrophilic ligands were used to modify the surface of the BNTs, obtaining a stable aqueous dispersion. Mixed surfactants (lecithin PL-100M and polyvinylpolypyrrolidone (PVP)) were applied to increase the colloidal stability of the BNTs in water by providing a combination of electrostatic and steric stabilization.41,42 The BNTs are well dispersed in aqueous solution, with a mean hydrodynamic diameter of ~95 nm (Figure 2b), which remain stable over 3 days in 10% fetal bovine serum solution (Figure S3a). The


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small-angle X ray diffraction results, thermogravimetric analysis curve data, and infrared spectra reveal the formation of an outer stabilizer layer on the BNTs after surface modification (Figure S3b-d). An analysis of the in vitro CT contrast efficiency shows that the Hounsfield unit value of either BNTs or BNCs is much higher than that of iohexol at equivalent molar concentrations owing to the larger X-ray attenuation coefficient of Bi (Figure 2c and Figure S4). After intravenous (i.v.) injection of the BNTs, normal rats were scanned by CT to evaluate (Figure S5) their effectiveness for in vivo imaging as compared to BNCs, and the biodistribution of the BNTs was studied by inductively coupled plasma mass spectrometry (ICP-MS) analysis (Figure S6). We further investigated the pharmacokinetics of the BNTs and BNCs. Both BNTs and BNCs followed two-compartment pharmacokinetics with distribution half-lives (t1/2α) of 0.46 ± 0.25 h and 0.09 ± 0.04 h, respectively (Figure 2d). The blood-elimination half-life (t1/2β) for the BNTs was 26.99 ± 0.41 h, which is about 7 times longer than that of the BNCs (3.77 ± 1.53 h). These results suggest that elongated BNTs offer feasibility of tumor accumulation in contrast to BNCs because the long t1/2β is critical for the enhanced permeation and retention (EPR) of nanomaterials.43 To demonstrate the tumor targeting of BNTs in vivo, same doses of BNTs and BNCs were administered to subcutaneous tumor-transplanted mice by i.v. injection, and their tumor-targeting capacity and biodistribution were assessed by CT imaging. The CT value in tumor tissue was increased at 1 h post-BNT injection and reached a maximum at 9 h, which is consistent with their relatively long t1/2β and indicated efficient accumulations in tumors. In contrast, BNCs showed a limited signal enhancement in tumor tissues after i.v. administration of the same dose (Figure 2e,f). The Bi level of BNTs in the tumor was ~8.09% ID g−1 tissue at 1 h post-injection and the value increased to ~18.5% ID g−1 tissue at 9 h, as determined by ICP-MS analysis. For the case of BNCs, a smaller number of BNCs (5.88% ID g−1 tissue) remained in the tumor tissue 1 h after injection, and the


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amount showed a gradual decrease thereafter (Figure 2g). The elongated BNTs can penetrate leaky tumor blood vessel walls more efficiently than spherical nanoparticles44,45 to reach the interstitium of tumor tissues46. On the other hand, small-sized BNCs can be cleared more rapidly by the kidney and consequently have a much short circulation time (short blood-elimination half-life, t1/2β), leading to lower tumor accumulation.28,29 In addition to CT imaging, BNTs exhibit the drug loading and radiotherapy sensitization capabilities, which can facilitate the CT imaging-guided cancer therapy (Figure 3a). Doxorubicin (DOX), a representative chemotherapeutic agent, was loaded into the hollow interior of the BNTs (BNTs/DOX) for delivery into malignant tumor tissues. The ultraviolet absorption (Figure 3b) and fluorescence (Figure S7a) spectra reveal strong characteristic peaks corresponding to the BNTs/DOX after removal of excess free drug molecules. The loading efficiency ([loaded drug]/[total drug], w/w) of the BNTs/DOX is ~53% and the loading ratio ([loaded drug]/[BNTs], w/w) is ~12%. The release of the loaded DOX from the BNTs/DOX is pH-dependent (Figure 3c); the enhanced release at pH 6 can be attributable to the decrease in hydrophobic interactions between DOX and BNTs, and can provide a therapeutic effect based on the targeted accumulation of BNTs in tumor tissues, where the environment is slightly acidic. Moreover, DOX fluorescence can be detected in the cytoplasm of Huh-7 cells by CLSM after the incubation with the BNTs/DOX for 4 h, demonstrating their efficient cellular uptake (Figure S7b,c). There is greater uptake of the BNTs by human Huh-7 liver tumor cells than by human L02 normal liver cells as determined by flow cytometry analysis (Figure S7c), implying that the elongated BNTs have a higher affinity for tumor cells.47 Furthermore, the BNTs show a dose-dependent radiosensitizing capacity upon exposure to a low dose of X-rays (2 Gy) (Figure 3d), since Bi can increase the production of cellular reactive oxygen species (ROS) and generate shortrange secondary electrons upon X-ray irradiation.15,18 We further evaluated the chemo-


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/radiotherapeutic effects via clonogenic assay. There was ~13.1 % colony formation of Huh-7 cells after BNTs + X-ray treatment, which is much lower than those of BNT (~97.4 %) or X-ray (~32.1 %) treatment. Moreover, BNTs/DOX + X-ray treatment shows a nearly complete suppression of the colony formation, demonstrating the synergistic chemo-/radiotherapeutic effects to inhibit cell proliferation (Figure S7d). DOX is known to stimulate cellular ROS production and interfere with the activity of DNA topoisomerase II.48 Using a fluorescent probe dichlorodihydrofluorescein diacetate to detect ROS in Huh7 tumor cells (Figure S7e), we found that the various combinations of BNTs, X-ray irradiation, and DOX increased the cytoplasmic and nuclear ROS levels, where BNTs/DOX combined with X-ray irradiation resulted in the most significantly enhanced fluorescence intensity, suggesting the potential for synergistic tumor therapy. DNA damage and cytotoxicity caused by the radio-/chemotherapy were evaluated by immunofluorescence labeling of gamma-histone γ-H2AX (Figure 3e,f, and Figure S7f) and 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium (MTT) cell viability assay (Figure 3g), respectively. A few H2AX foci were observed in the X-ray-irradiated and DOX-treated groups, whereas none was observed in phosphate-buffered saline (PBS-) or BNTs-treated groups. Although the density of γ-H2AX foci was higher in the DOX + X-ray group, it was higher in the BNTs/DOX + X-ray group, reflecting more extensive double-stranded DNA damage resulting from the synergistic effects of radio-/chemotherapy. A quantitative analysis reveals that the density of γ-H2AX foci is 2.54 and 4.41 times higher in the BNTs/DOX + X-ray group than those in the BNTs/DOX and X-ray only groups, respectively. As expected, X-ray irradiation aggravates the cytotoxicity of BNTs/DOX and decreases the cell survival rate to ~27.6% even at relatively low concentrations of Bi and DOX. Therefore, we conclude that the combination of the chemotherapy with the DOXloaded BNTs and the Bi-mediated radiosensitization again show the synergistic effects on


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the cytotoxicity against the tumor cells. To investigate the in vivo antitumor activity of the BNTs/DOX, tumor volume (Figure 3h) and body weight (Figure 3i) of tumor-bearing mice were monitored over the course of treatment. Compared to the DOX-treated mice, those in the BNTs/DOX group show enhanced tumor suppression, likely due to the tumor-homing efficacy of BNTs as a drug vehicle. Tumor growth was further inhibited by X-ray exposure. Tumor growth rate in the BNTs + X-ray group is much slower than that in the X-ray group, implying a greater radio-sensitization effect of the BNTs. Interestingly, the tumor inhibition rate of the BNTs/DOX + X-ray group is nearly 83.5%, which is significantly higher than that of the BNTs/DOX (32.2%) and BNTs + X-ray (42.4%) groups (Figure 3j and Figure S8). Moreover, the marked decrease in body weight observed in the free DOX + X-ray group is alleviated by BNTs/DOX + X-ray treatment, demonstrating that the systemic toxicity of free DOX is minimized as a result of more efficient DOX delivery to tumor tissues. Moreover, hematoxylin and eosin (H&E) staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and Ki-67 immunolabeling (Figure 3k) reveal extensive tumor cell nuclear breakdown, apoptosis, and inhibition of proliferation following the BNTs/DOX + X-ray treatment. Importantly, we found that the oriented assembly of the BNTs could be compromised by exposure to slightly acidic environment of tumors, which induces the disassembly of the BNTs into BNCs that are smaller than the size threshold for efficient renal filtration (~8 nm) (Figure 4a).49,50 This disassembly process is expected to avoid potential long-term toxicity.51 It is declared by FDA that all injected imaging agents must be cleared to avoid long-term accumulation.19 Since large-sized metallic nanoparticular contrast agents (> 6-8 nm) generally can be trapped by reticuloendothelial system (RES) organs for long time or are slowly excreted through the hepatic route,52 the long-term safety evaluation that re-


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quired for FDA approval may continue to discourage the efforts for clinical translation.20 Nevertheless, the disassembly-induced renal clearance of BNTs as reported here, provides an alternative strategy to realize the renal clearance of relatively large nanoparticles. The BNTs are stable in PBS (pH 7.4; Figure S9a) and 10% fetal bovine serum/PBS solutions (pH 7.4; Figure S3a and Figure S9b) in vitro. However, they gradually disassemble when incubated in PBS (pH 5.5) at 37 °C and most of them are reverted to the original nanocluster form after 48 h (Figure 4b). Transmission electron microscopy (TEM) analysis reveals that incubation of Huh-7 tumor cells with BNTs results in the shortening of the nanotubes within the cells (Figure S9c), indicating the disintegration of the tubular assembly. Examination of urine (Figure 4c) by TEM and clearance routes (Figure 4d) by ICP-MS confirms endogenic disassembly and kidney excretion in tumor-bearing mice. We speculate that the slightly acidic microenvironment of tumors perturbs the balance of steric effects exerted by the oleic acid ligands, van der Waals forces, or dipole-dipole interactions in the nanotube system. Factors other than the pH may contribute to the disassembly of the BNTs in vivo, which requires further investigation. Moreover, BNTs not only are cytocompatible with both Huh-7 tumor cells and L02 normal cells (Figure 4e), but also show no obvious toxicity in serum biochemical analyses (Figure 4f), hematology tests (Figure S10), and histological examinations (Figure 4g). In summary, we demonstrate the controlled assembly of BNCs into hollow BNTs for use as a high-resolution CT contrast agent and for CT imaging-guided radio-/chemotherapy of tumors. The elongated BNTs with high aspect ratios homed to and are taken up and retained by tumors. The BNTs enhance X-ray-induced DNA damage in vivo because of their strong radiosensitivity and exerted chemotherapeutic efficacy via drugs loaded into their hollow structure; combined radio-/chemotherapy using the BNTs/DOX shows an excellent therapeutic efficacy. The elongated nanotubes are subsequently disassembled into their


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original nanocluster form in the acidic microenvironment of tumors for renal clearance. Thus, the controlled assembly and disassembly strategy described here provides a basis for the development of next-generation CT contrast agents that are renal clearable, multifunctional, and effective for tumor treatment.


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Methods. BNCs synthesis and assembly into BNTs. Bismuth acetate (40 mg), hexadecylamine (0.20 g), and oleic acid (2.0 ml) were added to 1-octodecene (8 ml), and the mixture was heated to 100 °C for 15 min with stirring to obtain small-sized BNCs. The mixture was transferred into a 15 ml autoclave with a Teflon liner and heated to 200 °C for 4 h to induce assembly. After allowing the solution to cool naturally to room temperature, the precipitate was collected by centrifugation, washed three times with ethanol, and dispersed in chloroform. Surface modification of BNTs and drug loading. Mixed surfactants were used to enhance the water-dispersibility of the BNTs via surface modification. Chloroform (3 ml) containing lecithin (PL-100M; 100 mg) and PVP (100 mg) was added dropwise to 2 ml of the organic solvent-dispersible BNTs (20 mg Bi) in chloroform under magnetic stirring. The dispersion was dried by rotary evaporation under vacuum for 1 h before adding 3 ml of distilled water. Uniformly dispersed and stable BNTs were obtained after sonication for complete hydration. Excess surfactants were removed by centrifugation. DOX-loaded BNTs were obtained by mixing DOX and BNTs in chloroform for 24 h, followed by removal of free DOX by centrifugation and surface modification. In vitro CT imaging. To assess CT contrast efficiency, BNTs were dispersed in water with Bi concentrations ranging from 0 to 0.32 M. In vitro CT imaging was performed at 120 kVp using a third-generation dual-source multi-detector CT scanner (SOMATOM Force; Siemens Healthcare AG, Erlangen, Germany). Imaging parameters were as follows: thickness, 0.1 mm; pitch, 0.5; 120 kVp, 200 mA; field of view, 108 × 108; matrix, 512 × 512 pixels; gantry rotation time, 0.25 s; and table speed, 73.7 cm/s. Thin-section axial images were reformed into coronal images by multiplanar reconstruction. The threedimensional (3D) reconstructed images were obtained using Syngo 3D Workstation (CT


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2007S VB30, v.3.8.1; Siemens). In vitro cellular uptake. Cellular uptake of the BNTs was evaluated by incubating Huh7 tumor cells with different concentrations of BNTs ([Bi] = 0, 2.5, 5 mM) for 24 h, followed by two washes with PBS. Cell pellets obtained by centrifugation were covered with 1% agarose solution, and in vitro CT imaging was performed at 120 kVp. In vivo CT imaging. BNTs solution (Bi = 0.12 mM kg−1) was administered to healthy rats or tumor-bearing mice by i.v. injection. CT images were acquired prior to and at several time points after injection. Clonogenic assay. Huh-7 cells (~2000) were seeded in 6-well plates and incubated at 37 °C for 24 h. The cells were treated with Dulbecco’s Modified Eagle’s Medium (DMEM), BNTs (20 µg ml−1 Bi) or BNTs/DOX (20 µg ml−1 Bi and 5 µg ml−1 DOX) for 24 h (n = 3). Next, the 6-well plates were subjected to X-ray irradiation (sham or 6 Gy). Then, the cells were washed and subsequently incubated at 37 °C for 7 to 10 d. Finally, the cells were fixed by 4% paraformaldehyde and stained with Giemsa dye. The survival fractions of the colonies were counted to evaluate the effects of different treatments. Immunofluorescence detection of γ-H2AX. To evaluate DNA damage after BNTs treatment, Huh-7 cells were seeded in glass-bottomed culture dishes at a density of 5 × 104 dish-1 for 24 h and divided into six groups (control, BNTs, BNTs/DOX, X-ray, BNTs + Xray, and BNTs/DOX + X-ray). After treatment with DMEM, BNTs (20 µg ml−1 Bi) or BNTs/DOX (20 µg ml−1 Bi and 5 µg ml−1 DOX) for 24 h, cells were subjected to X-ray irradiation (6 Gy) or left without irradiation. After 2 h, cells were fixed with 4% paraformaldehyde for 15 min, washed with PBS, blocked with 1% bovine serum albumin in PBS for 1 h at room temperature, and incubated with mouse monoclonal anti-γ-H2AX antibody (dilution 1:1000) for 1 h at room temperature. After three washes with PBS, cells were incubated with Alexa Fluor555-conjugated goat anti-mouse IgG (H+L) (1:2000) and


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Hoechst 33342 (1:6000) for 1 h at room temperature in the dark. After three washes with PBS, cells were imaged by CLSM (A1R; Nikon, Tokyo, Japan). The density of γ-H2AX foci was determined using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Cytotoxicity study. Huh-7 hepatocellular carcinoma cells or L02 normal liver cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C in an atmosphere of 5% CO2 and 95% air. A total of 104 cells well-1 were seeded in 96-well plates with 200 µl culture medium. After 24 h, cells were treated with various concentrations of BNTs (0, 0.08, 0.16, 0.31, 0.63, 1.25, 2.50, and 5.00 mM) for 24 h. The medium was replaced with 200 µl of fresh medium containing 10% MTT reagent, yielding purple formazan crystals. After incubation for 4 h, the medium was replaced with 200 µl dimethylsulfoxide. The absorbance of each well at 570 nm was measured with a microplate reader (Bio-Rad, Hercules, CA, USA). In addition, cells grown in 96-well plates (104 well-1) were incubated with various concentrations of BNTs or BNTs/DOX with or without X-ray irradiation. After incubation for 24 h, the standard MTT assay was carried out to measure assess cell viability and evaluate the in vitro efficacy of BNTs for radio-/chemotherapy. In vivo antitumor efficacy test. BALB/c nude mice (Shanghai SLAC Laboratory Animal Co., Shanghai, China) subcutaneously inoculated with Huh-7 cells were randomly divided into the following eight groups (n = 5 each) when tumor size was ~100 mm3: saline; BNTs (25 mg kg−1 Bi); DOX solution (6.25 mg kg−1 DOX); BNTs/DOX (25 mg kg−1 Bi and 6.25 mg kg−1 DOX); X-ray; BNTs + X-ray (25 mg kg−1 Bi); DOX solution + X-ray (6.25 mg kg−1, DOX); BNTs/DOX + X-ray (25 mg kg−1 Bi and 6.25 mg kg−1 DOX). Each group was injected via the tail vein once a week and X-ray groups were irradiated (6 Gy) once a week. Tumor volume (tumor volume = width2 × length/2) and body weight were measured


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every 2 days. At the end of the experiment, tumors were harvested, weighed, and prepared for H&E staining, TUNEL, and Ki-67 immunodetection. Animal experiments were carried out in accordance with institutional guidelines and were approved by Zhejiang University Laboratory Animal Center. In vivo metabolism studies. Tumor-bearing mice were reared in metabolic cages after i.v. injection of BNTs (0.12 mM kg−1). Urine and feces were collected from each mouse at various time points and solubilized in 70% nitric acid. After incubation at 60 °C for 12 h, samples were centrifuged at 10000 rpm for 5 min; the supernatant was diluted 100 fold with distilled water and analyzed by ICP-MS. Serum biochemistry test and blood panel examination. Blood samples (1–1.5 ml, n = 5) were collected from the orbit before and after BNTs injection, and 500 µl of serum obtained from each animal was centrifuged at 8000 rpm for 15 min. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and creatinine (CREA) were measured. In addition, complete blood panel measurements were performed with the blood samples obtained from the mice at 6 h, 1d, and 7d postinjection. Histopathological examination. Healthy nude mice were administered normal saline (control group) or BNTs by i.v. injection. After 1, 7, and 15 days, mice were sacrificed and the heart, liver, spleen, lung, and kidney were removed and processed for H&E staining.


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ASSOCIATED CONTENT Supporting Information Experimental details, TEM images of (BiO)2CO3 assemblies under different reaction conditions, STEM and Elemental analysis of BNTs, XPS spectrum and XRD pattern of BNTs, SAXS patterns of BNTs, TEM image and CT value of BNCs, serial CT coronal images of rat after i.v. injection of BNTs, in vivo biodistribution of BNTs, drug loading and in vitro therapeutic effect of BNTs, tumor weights measurement, TEM images of BNTs in PBS and FBS/PBS solutions, TEM images of tumor cells incubated with BNTs, hematology data of mice after BNT administration. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Correspoding Author *E-mail: [email protected] Author Contributions X. H., J. S., and F. L. contributed equally. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We gratefully acknowledge the financial support by the National Key Research and Development Program of China (2016YFA0203600), the National Natural Science Foundation of China (51503180, 51703195, 51611540345, 81430040, 81571738), “Thousand Talents Program” for Distinguished Young Scholars (588020*G81501/048), the Research Center Program for the Institute of Basic Science (IBS-R006-D1) and Fundamental Research Funds for the Central Universities (520002*172210161).


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Figure 1. Design and mechanism of hollow bismuth subcarbonate nanotubes for tumortargeting CT imaging, chemoradiotherapy and disassembly-induced renal clearance. (a) Schematic illustration of the formation and shaping of BNTs. (b) Schematic illustration of tumor-homing capacity of elongated BNTs, CT imaging-guided radio-/chemotherapy mediated by the BNTs/drug, and subsequent BNTs disassembly and renal clearance. (c) TEM of nanoclusters. (d,e) TEM image (d) and scanning electron micrograph (SEM) image (e) of the BNTs after complete assembly under solvothermal conditions (200 °C) for 4 h.

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Figure 2. Enhanced tumor uptake and tumor imaging of elongated BNTs. (a) Tumor targeting and accumulation of the BNTs. (b) Particle size distribution of the BNTs and BNCs in aqueous phase solution. (c) In vitro CT value (in Hounsfield unit, HU) as a function of BNTs and iohexol concentration, which demonstrates that Hounsfield unit value of the BNTs is much higher than that of iohexol at equivalent molar concentrations. Insert above: CT images obtained at different BNTs concentrations. Insert below: CT images of Huh-7 cells incubated with increasing concentrations of the BNTs (0, 2.5, and 5 mM Bi for 1, 2, and 3, respectively) for 24 h. (d) Time profile of Bi concentration in blood after a single i.v. injection of the BNTs and BNCs (n = 6). (e,f) Reconstructed 3D CT images (e, BNTs [top] and BNCs [bottom]; yellow dashed circle: tumor) and CT values (f) of a subcutaneous tumor-transplanted mouse after i.v. administration of the BNTs or BNCs. (g) Bi content in tumor tissues 1, 9, and 24 h after i.v. administration of the BNTs or BNCs.


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Figure 3. Synergistic radio-/chemotherapy of tumors using the BNTs. (a) Schematic illustration of the imaging and therapeutic applications of the BNTs. (b) Ultraviolet spectra of the BNTs, free DOX, and BNTs/DOX in water. (c) Cumulative release profiles of DOX from BNTs/DOX at pH 7.4 and 6.0. (d) Sensitizer enhancement ratio in Huh-7 cells treated with the BNTs or BNTs irradiated with X rays at 0, 2, 4, 6, and 8 Gy. (e) Representative confocal laser scanning micrographs of DNA damage after indicated treatments (with or without X-ray irradiation at 6 Gy) showing Hoechst 33342 staining and γ-H2AX immunolabeling to visualize cell nucleus and DNA fragmentation, respectively. Scale bar = 20 µm. (f) Quantitative analysis of the density of γ-H2AX foci. (g) Viability of Huh-7 cells after indicated treatments (with or without X-ray irradiation at 6 Gy). (h-j) Tumor growth curves (h), body weight (i), and tumor gross appearance (j) following the treatments. *P < 0.05, **P < 0.01, ***P < 0.001. (k) Micrographs showing H&E staining, TUNEL staining, and Ki-67 immunoreactivity in tumor sections from mice following the treatments.


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Figure 4. Disassembly-induced renal clearance and safety profiles of the BNTs. (a) Schematic illustration of the pH-associated disassembly of the BNTs and their renal clearance in vivo. (b) Representative TEM images of BNTs incubated in PBS (pH 5.5) for 6, 12, and 48 h. (c) TEM images of urine collected from a tumor-bearing mouse 3 h after injection of BNTs. (d) Bi in urine and feces of tumor-bearing mice at indicated time points after i.v. injection of BNTs (n = 2). (e) Viability of Huh-7 tumor cells incubated with the BNTs for 24 h. (f) Results of serum biochemical analysis, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and creatinine (CREA) levels in mice (n = 5) administered the BNTs. (g) Histological examination of heart, liver, spleen, lung, and kidney tissues by H&E staining (40×). Tissue samples were obtained from nude mice sacrificed 1, 7, and 15 days after saline (200 µl) or BNTs (200 µl) administration. Scale bar = 200 µm.


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