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1 Electronic Supplementary Material (ESI) for Nanoscale. This journal is The Royal Society of Chemistry 2017 Supporting Information Ball-in-ball ZrO 2 Nanostructure for Simultaneous CT Imaging and Highly Efficient Synergic Microwave Ablation and Tri-stimuli Responsive Chemotherapy of Tumor Dan Long, Meng Niu, Longfei Tan, Changhui Fu, Xiangling Ren, Ke Xu, Hongshan Zhong, Jingzhuo Wang, LaifengLi, Xianwei Meng Figure S1. (a) TEM image and (b) diameters histograms distribution of tree-layer ZrO 2 NPs. Three-layer ZrO 2 NPs have been synthesized through the improved template method and the particles size was 325±3 nm as shown in Figure S1 (a-b). The unique physical and chemical properties multi-layer nanomaterials need to be further explored and found. They are expected to be used for further biological applications.

2 Figure S2. (a-b) FT-IR spectrums of IL, DOX, tetradecanol, BB-ZrO 2, keratin and 2. (c) TGA curves of BB-ZrO 2, 2 and ZrO 2. (d) Standard curve of DOX at 483 nm under different concentrations (0, 1, 5, 10, 20 and 30 μg ml -1 ). To further verify the existence of IL, DOX and tetradecanol, FT-IR was used to determine functional groups in the as-prepared system (Figure S2a). The characteristic peaks of IL appeared at 840 cm -1 (the absorption peak of P-F), 1573 and 1469 cm -1 (the imidazole skeleton vibration) and 1167 cm -1 (imidazole ring stretching vibration). The characteristic peaks of 1621, 1580, 1438 and 1498 cm -1 were the characteristic peaks of the aromatic ring in DOX. The peaks of tetradecanol appeared at 3631, 3296 cm -1 (respectively from the free stretching vibration and intermolecular

3 hydrogen bond of O-H), 1065 cm -1 (C-O) and 683 cm -1 (plane bending peak of O-H), respectively. The characteristic peaks of keratin appeared at 539 cm -1 (-S-S-), 760 cm - 1 (the C=O stretching vibration in the COOH) and 3550 cm -1 (the O-H stretching vibration of -COOH). All of the characteristic peaks of IL, DOX, keratin and tetradecanol could be found in the X@BB-ZrO 2 system as shown in Figure S2a-b. TGA was used to investigate the thermal effects of BB-ZrO 2, IL@BB-ZrO 2 and tetradecanol@bb-zro 2 in a range of temperature. Compared with the TGA curve of BB-ZrO 2, the loading capacity of IL and tetradecanol was 5.1% and 10.0%, respectively (Figure S2c). The loading and encapsulation efficiency of DOX were calculated by the standard curve established at 483 nm. The linear equation as shown in Figure S2d was Y= X , and R 2 =0.99, where Y represents the absorbance, and X represents the concentration of DOX. Figure S3. In vitro cytotoxicity test of IL/tetradecanol/keratin@BB-ZrO 2 and X@BB-ZrO 2. (a) Hemolysis test of the IL/tetradecanol/keratin@BB-ZrO 2 NPs under different concentrations (1000, 500, 250, 125 and 62.5 ug ml -1 ). HepG-2 cells viability by MTT assay of (b) IL/tetradecanol/keratin@BB-ZrO 2 (200, 100, 50, 25, 12.5 and 0 μg ml -1 ) and (c) X@BB-ZrO 2 (50, 25, 12.5, 6.25, 3.13, 1.56 and 0 μg ml - 1 ).

4 The as-made 2 NPs were proved to have a good microwave heating effect and could significantly increase the ablation area via experiments in vitro, which was expected to be used in further experiments in vivo. Therefore, the biocompatibility should be taken into consideration. The hemolysis test result (Figure S3a) of the as-prepared IL/tetradecanol/keratin@BB-ZrO 2 NPs under different concentrations shows no obvious hemolysis, the hemolysis rates were lower than 5% even at the highest concentration of 1000 μg ml -1. The cytotoxicity of the as-made and X@BB-ZrO 2 system was investigated in HepG-2 cells by MTT assay. As shown in Figure S3b, the cytotoxicity of IL/tetradecanol/keratin@BB-ZrO 2 was more than 80 % even at a high concentration of 200 μg ml -1, indicating the low cytotoxicity of the IL/tetradecanol/keratin@BB-ZrO 2. However, the viability of the cells decreased rapidly after DOX was loaded (Figure S3c), the viability was lower than 80% when the concentration was higher than 12.5 μg ml -1, indicating that the as-made X@BB- ZrO 2 have favorable lethality to tumor cells. To further validate the toxicity of the as-prepared materials, the systematic toxicity in vivo experiment was utilized. Healthy ICR mice were randomly assigned into 10 groups (n=5): IL@BB-ZrO 2 at different injection dose (400, , 40, 20 mg kg -1 ), and different treatment methods at the same injected dose of 40 mg kg -1, including IL@BB-ZrO 2 +MW, BB-ZrO 2, BB-ZrO 2 +MW, MW and control group. 10 h post-injection, the mice of microwave irradiation groups were irradiated by microwave for 5 min. After 14 days, the mice were terminated and main organs (liver,

5 Figure S4. H&E stained images of main organs (liver, heart, spleen, lung and kidney) collected from each group (all of the scale bars were 100 μm).

6 heart, spleen, lung and kidney) were collected with 4% formalin solution for histochemistry analysis. Compared with control group, the H&E stained images (Figure S4) of main organs (liver, heart, spleen, lung and kidney) collected from mice in each group indicated no obvious abnormalities. The results demonstrated the good biocompatibility of 2 +MW, BB-ZrO 2, BB-ZrO 2 +MW, MW, even at a high injection dose of 400 mg kg -1, the BB-ZrO 2 didn t show significant adverse effects on the health of mice. To evaluate the microwave thermotherapy effect of the as-prepared X@BB-ZrO 2 NPs on the subcutaneous tumors, ICR mice bearing H-22 tumors were divided into control, MW, X@BB-ZrO 2, IL@BB-ZrO 2 +MW, X@BB-ZrO 2 +MW and DOX groups. ICR mice bearing H-22 tumors (tumor size in any direction not exceeding 10 mm) were divided into 6 groups (n=5 per group). The mice were tail-intravenously injected with PBS, X@BB-ZrO 2, IL@BB-ZrO 2 and DOX. In addition to DOX group (16 mg kg -1 ), the injected dose was 40 mg kg h post-injection, half of the mice of X@BB-ZrO 2 and PBS; whole of the IL@BB-ZrO 2 groups were irradiated by a microwave ablation antenna at a power of 2 W for 5 min. When the tumor size of mice was more than 20 mm in any direction, the mice were sacrificed and the main organs and tumors were collected for further histochemistry analysis. Compared with control group the H&E stained images of major organs (liver, heart, spleen, lung and kidney) collected from mice in each group showed no significantly pathologies (Figure S5a). The results demonstrated the good biocompatibility of the different treatments. As shown in Figure S5b, the tumors of

7 MW groups 2 +MW, MW and 2 +MW) represented strong signs of necrosis areas contrast with other groups.

8 Figure S5. H&E stained images of (a) main organs (liver, heart, spleen, lung and kidney) and (b) tumors collected from mice in control, 2 +MW, ZrO 2 +MW, MW, DOX and X@BB-ZrO 2 groups (all of the scale bars were 100 μm). Figure S6. The tumor photos taken at 6 th day postoperative, the white tissue in red circle is tumor and the surrounding of the red circle is normal liver tissue (all of the scale bar were 10 mm). To further investigate the therapeutic effect in the deep tumor of the as-made X@BB-ZrO 2 NPs. Liver VX2 tumor bearing rabbits were utilized as animal model. The VX2 tumor bearing rabbits were randomly separated into MW, X@BB- ZrO 2 +MW and control group. The as-made X@BB-ZrO 2 NPs were injected into the rabbits of X@BB-ZrO 2 +MW group via auricular vein at the dose of 3.2 mg kg -1, and the MW and control group were injected with saline. After 8 h, the tumor site of each group rabbits was irradiated by 10 W MW for 2 min. Then the therapeutic effect was monitored by CT in real time. 6 days after treatments, the rabbits were sacrifice and

9 the tumors were collected. As shown in Figure S6, the representative photos of tumor in each group were corresponding with the CT imaging results.

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