supporting informationx@bb-zro2 nps. liver vx2 tumor bearing rabbits were utilized as animal model....
TRANSCRIPT
Supporting Information
Ball-in-ball ZrO2 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
ZrO2 NPs.
Three-layer ZrO2 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.
Electronic Supplementary Material (ESI) for Nanoscale.This journal is © The Royal Society of Chemistry 2017
Figure S2. (a-b) FT-IR spectrums of IL, DOX, tetradecanol, BB-ZrO2, keratin and
X@BB-ZrO2. (c) TGA curves of BB-ZrO2, IL@BB-ZrO2 and tetradecanol@BB-
ZrO2. (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
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-ZrO2 system as shown in Figure S2a-b.
TGA was used to investigate the thermal effects of BB-ZrO2, IL@BB-ZrO2 and
tetradecanol@BB-ZrO2 in a range of temperature. Compared with the TGA curve of
BB-ZrO2, 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=41.6194X-0.8088, and R2=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-ZrO2 and
X@BB-ZrO2. (a) Hemolysis test of the IL/tetradecanol/keratin@BB-ZrO2 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-ZrO2 (200, 100, 50, 25,
12.5 and 0 μg mL-1) and (c) X@BB-ZrO2 (50, 25, 12.5, 6.25, 3.13, 1.56 and 0 μg mL-
1).
The as-made X@BB-ZrO2 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-ZrO2 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-ZrO2 system was investigated in HepG-2 cells by MTT assay. As shown
in Figure S3b, the cytotoxicity of IL/tetradecanol/keratin@BB-ZrO2 was more than 80
% even at a high concentration of 200 μg mL-1, indicating the low cytotoxicity of the
IL/tetradecanol/keratin@BB-ZrO2. 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-
ZrO2 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-ZrO2 at different injection dose (400, 200 100, 40, 20
mg kg-1), and different treatment methods at the same injected dose of 40 mg kg-1,
including IL@BB-ZrO2+MW, BB-ZrO2, BB-ZrO2+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,
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).
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 IL@BB-ZrO2+MW, BB-ZrO2, BB-ZrO2+MW, MW, even at a
high injection dose of 400 mg kg-1, the BB-ZrO2 didn’t show significant adverse
effects on the health of mice.
To evaluate the microwave thermotherapy effect of the as-prepared X@BB-ZrO2
NPs on the subcutaneous tumors, ICR mice bearing H-22 tumors were divided into
control, MW, X@BB-ZrO2, IL@BB-ZrO2+MW, X@BB-ZrO2+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-ZrO2, IL@BB-ZrO2 and DOX. In addition to DOX group
(16 mg kg-1), the injected dose was 40 mg kg-1. 10 h post-injection, half of the mice of
X@BB-ZrO2 and PBS; whole of the IL@BB-ZrO2 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
MW groups (IL@BB-ZrO2+MW, MW and X@BB-ZrO2+MW) represented strong
signs of necrosis areas contrast with other groups.
Figure S5. H&E stained images of (a) main organs (liver, heart, spleen, lung and
kidney) and (b) tumors collected from mice in control, IL@BB-ZrO2+MW, X@BB-
ZrO2+MW, MW, DOX and X@BB-ZrO2 groups (all of the scale bars were 100 μm).
Figure S6. The tumor photos taken at 6th 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-ZrO2 NPs. Liver VX2 tumor bearing rabbits were utilized as animal model.
The VX2 tumor bearing rabbits were randomly separated into MW, X@BB-
ZrO2+MW and control group. The as-made X@BB-ZrO2 NPs were injected into the
rabbits of X@BB-ZrO2+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
the tumors were collected. As shown in Figure S6, the representative photos of tumor
in each group were corresponding with the CT imaging results.