Construction of highly stable selenium nanoparticles

Nano Res.

Electronic Supplementary Material

Construction of highly stable selenium nanoparticlesembedded in hollow nanofibers of polysaccharide andtheir antitumor activities

Zhaohua Ping1, Ting Liu2, Hui Xu1, Yan Meng1, Wenhua Li2, Xiaojuan Xu1 (), and Lina Zhang1 ()

1 College of Chemistry & Molecular Sciences, Wuhan University, Wuhan 430072, China 2 Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan 430072, China

Supporting information to DOI 10.1007/s12274-017-1590-7

S1 Materials and methods

S1.1 Synthesis of FITC conjugated AF1

The synthetic method of AF1 labeling to the fluorescein isothiocyanate isomer I (FITC, Sigma, USA) which

covalently reacts with hydroxyl groups is according to the reported procedure [S1]. Briefly, AF1 (200 mg), FITC

(30 mg), pyridine (100 μL, Sinopharm, China) and dibutyltin dilaurate (20 μL, Sinopharm, China) were first

dissolved in DMSO (20 mL, Sigma, USA). The reaction mixture was heated for 4 h at 100 °C and precipitated

with 4 volumes of ethanol by centrifugation (6,000 rpm, 10 min). The precipitations were repeated four times in

total to remove the unbound FITC. The final sample was finally obtained after drying at 60 °C, which coded as

FITC-AF1.

S1.2 Fluorescence spectroscopy and fluorescence microscopy

BALB/c mice (7–8 weeks old, female, weighting 20 ± 2 g) and Sprague-Dawley (SD) male rats (6 week old, female,

150–200 g) were purchased from Center for Animal Experiment ABSL-Ⅲ Laboratory of Wuhan University

(Hubei, China). All experiments were conducted under approved procedures. FITC-AF1 was dispersed in water

(10 mg·mL–1) and intraperitoneally administrated to SD rats (10 mg·kg–1 body weight) after starving for 24 h.

The blood samples were collected from orbit in heparinized tubes before or after administration at the time

points of 0 h, 0.25 h, 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 4 h, 5 h, 6 h, 9 h, 12 h, 24 h, 36 h, and 48 h, and centrifuged at

5,000 r·min–1 for 15 min to get the serum sample. The blood samples were kept in dark before analysis. Finally,

the amount of fluorescence of the plasma samples was determined under the condition with emission at 488 nm

and absorption at 525 nm by microplate reader (TECAN, SPARK10M, Austria).

Address correspondence to Xiaojuan Xu, [email protected]; Lina Zhang, [email protected]

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The BALB/c mice were killed after intraperitoneally administration of FITC-AF1 for desired time, and the major

organs including heart, liver, spleen, kidney and lung were collected , which were then kept at −86 °C and made

into frozen section for observation by fluorescence microscope (NIKON ECLIPSE TI-SR, Japan).

S2 Synergy analysis

The synergistic effect between AF1 and SeNPs was analyzed by CompuSyn software based upon the Chou-

Talalay method [S2]. Briefly, the Chou-Talalay method uses the following equation: (D)1/(Dx)1 + (D)2/(Dx)2 where

(D)1 and (D)2 are the doses of drug 1 and drug 2 that have x% effect when used in combination, and (Dx)1 and

(Dx)2 are the doses of drug 1 and drug 2 that have the same x% effect when used alone. When the combination

index (CI) is equal to 1.0, this equation represents the conservation isobologram and indicates additive effects.

Synergy is present when the CI is less than 1.0. The combination is additive when CI equals to 1.0, and antagonistic

when it is more than 1.0.

S3 Histology and TUNEL assay

The major organs including heart, liver, spleen, kidney and lung were collected and immediately fixed in 4%

formalin for further histopathological examination. The tissues of organ samples embedded in paraffin blocks

and HE staining were carried out by the standard protocol. Digital images were obtained on an Olympus CX31

light microscope (magnification, 100×). The identity and analysis of the pathology slides were blind to the

pathologist. TUNEL assay was employed to determine in situ apoptotic DNA breaks by using a commercial

apoptosis detection kit following manufacturer’s instructions. Formalin-fixed, paraffin-embedded 4 μm thick

tumor sections were subjected to the assay. TUNEL data were analyzed by a researcher blind to the nature using

fluorescence microscopy (magnification, 200×).

S4 Results and discussion

Figure S1 SAED image of SeNPs.

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Figure S2 SEM image of selenium nanoparticles in the AF1 aqueous solution (1 mg·mL−1).

Figure S3 Photographs of SeNPs in water in the presence (a) and absence (b) of AF1 in the redox reaction after storage for 1 day.

Figure S4 FT-IR spectra of AF1 and AF1-Se.


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Figure S5 In vitro cellular uptake of FITC-AF1. MCF-7 cells were stained by 100 nM of lyso-tracker DND-99 (red fluorescence) for 30 min and 10 μg·mL−1 of Hoechst 33342 (blue fluorescence) for 20 min, and then treated with 50 μg·mL−1 FITC-AF1 (green fluorescence) for different time and visualized under a fluorescence microscope.

Figure S6 Growth inhibition of AF1, SeNPs and AF1-Se on selected cancer and normal cells. (a)–(c) Cell viability of cancer cells (MCF-7, MDA-MB-231, MDA-MB-468, Hep3B, HepG2, HCCLM9, BEL7402, Huh-7 and HepG2) treated with different concentrations of AF1, SeNPs and AF1-Se for 72 h. (d) Cell viability of normal cells (HBL-100, L02 and 293T) treated with different concentrations of AF1-Se for 72 h. Cell viability was determined by a colorimetric MTT assay. The data represent the average of at least three independent experiments ± SD.


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Figure S7 Induction of synergetic anti-cancer effect by AF1-Se. (a) Fraction affected (Fa) of AF1-Se, AF1 and SeNPs against MCF-7 cells. Cells were treated with various concentrations of AF1-Se, AF1 and SeNPs for 72 h, and the cell viability was determined by a colorimetric MTT assay, and shown as an isobologram graph. (b) The cell viability was converted to combination index (CI) according to the Chou-Talalay equation. Synergy is present when the CI is less than 1.0. The combination is additive when CI equals 1.0, and antagonistic when it is more than 1.0.

Figure S8 AF1-Se induces cell apoptosis and cell cycle arrest in cancer cells (a) Statistical analysis of apoptotic cells of MCF-7 cells treated with AF1-Se. (b) Statistical analysis of cell cycle distribution of MCF-7 cells treated with AF1-Se. *p < 0.05, **p < 0.001 when compared with the control. The data represent the average of at least three independent experiments ± SD.

Figure S9 The images stained with HE of main organs including heart, liver, spleen, lung and kidney from the control mice and treated mice for 21 days (magnification 100×).


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Figure S10 AF1 and AF1-Se induce apoptosis in MCF-7 tumor cells. (a) TUNEL assay for detect apoptotic cells in MCF-7 tumor tissues from female BABL/c athymic nude mice treated with PBS, 5 mg·kg−1 AF1, 5 mg·kg−1 AF1-Se and 10 mg·kg−1 AF1-Se (magnification 200×). (b) The tissue proteins were extracted, and PARP1, PdCD4, Bcl-2, cleaved-casepase9 and pro-casepase3 were detected by western blotting analysis using their specific antibodies with β-actin as the loading control. (c) The digital results were determined by quantitative densitometry. *p < 0.05, **p < 0.001 when compared with the control. The data represent the average of at least three independent experiments ± SD.

Figure S11 Mean serum concentration-time curve of FITC-AF1 after intraperitoneal injection of 10 mg·kg−1 FITC-AF1.


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Figure S12 Fluorescence microscopy images of the main organs including heart, liver, spleen, lung and kidney from the control mice and treated mice (10 mg·kg−1 FITC-AF1) for different time (magnification 200×).

References

[S1] Tromp, R. H.; van de Velde, F.; van Riel, J.; Paques, M. Confocal scanning light microscopy (CSLM) on mixtures of gelatine and

polysaccharides. Food Res. Int. 2001, 34, 931–938.

[S2] Chou, T. C.; Talalay, P. Quantitative analysis of dose-effect relationships: The combined effects of multiple drugs or enzyme

inhibitors. Adv. Enzyme Regul. 1984, 22, 27–55.

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Construction of highly stable selenium nanoparticles