Potentiation of in Vivo Anticancer Efficacy of Selenium Nanoparticles

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Bioactive Constituents, Metabolites, and Functions

Potentiation of In Vivo Anticancer Efficacy of Selenium Nanoparticles by Mushroom Polysaccharides Surface Decoration Delong Zeng, Jianfu Zhao, Kar-Him Luk, Siu-To Cheung, Ka Hing Wong, and Tianfeng Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00193 • Publication Date (Web): 20 Feb 2019 Downloaded from http://pubs.acs.org on February 21, 2019

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Journal of Agricultural and Food Chemistry

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Potentiation of In Vivo Anticancer Efficacy of Selenium Nanoparticles

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by Mushroom Polysaccharides Surface Decoration

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Delong Zeng a,#, Jianfu Zhao a,#, Kar-Him Luk b, Siu-To Cheung b, Ka-Hing Wong b,* and

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Tianfeng Chen a,*

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a

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China.

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b Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University,

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The First Affiliated Hospital, and Department of Chemistry, Jinan University, Guangzhou 510632,

Hong Kong, China.

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Corresponding Author

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* E-mail addresses: [email protected].

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* E-mail: [email protected] (K. W.)

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Author Contributions

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#

These authors contributed equally to this work.

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1

ACS Paragon Plus Environment


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ABSTRACT: Selenium nanoparticles (SeNPs) are recently emerging as promising anticancer agents

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because of their high bioavailability, low toxicity and remarkable anticancer activities. However, the

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effects of surface physicochemical properties on the biological actions remain elusive. Herein we

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decorated SeNPs with various water-soluble polysaccharides extracted from various mushrooms, to

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compare physical characteristics and anticancer profile of these SeNPs. The results showed that the

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prepared spherical SeNPs displayed particle sizes at 91-102 nm, and kept stable in aqueous solution

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for up to 13 weeks. However, different decoration altered the tumor selectivity of the SeNPs, while

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gastric adenocarcinoma AGS cells showed relative highest sensitivity. Moreover, PTR-SeNPs

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demonstrated potent in vivo antitumor, by inducing caspases- and mitochondria-mediated apoptosis,

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but showed no obvious toxicity to nomal organs. Taken together, this study offers insights into how

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surface decoration can tune the cancer selectivity of SeNPs and provides a basis for engineering

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particles with increased anticancer efficacy.

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Keywords: selenium nanoparticles; mushroom polysaccharide; surface decoration; in vivo anticancer

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efficacy

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INTRODUCTION Cancer has become a leading cause of death worldwide.1-2 Chemotherapy is one of the major

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treatments of cancer. Although effective, it is limited by the side effects and drug-resistance

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developed by cancer cells. Novel high efficacy and low toxicity drugs are still needed for further

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control of cancers. Selenium is a multifunction essential trace element in human and animal

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bodies,3-4 which also displays potent activities in cancer prevention and treatment.5 Large amount of

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selenium-containing compounds have been synthesized and evaluated for their anticancer

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activities.6-14 Selenium nanoparticles (SeNPs) are recently emerging as promising anticancer agents

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because of their high bioavailability, much lower toxicity than selenium compounds and remarkable

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anticancer activities. In our previous studies, we found that SeNPs showed potent anticancer

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efficacy,15 which could be significantly enhanced by conjugation of targeting molecules such as

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RGD peptide,16 folic acid,17 and transferrin. 18 SeNPs may also be effective drug carriers that

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enhance the efficacy of the loaded drugs.19-21 Additionally, SeNPs displayed strong synergism with

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radiotherapy by increasing ROS production.17, 22-23 A multifunction selenium nanosystem we

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recently designed, in which ultra-small SeNPs were combined with bevacizumab and then coated

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with erythrocyte membrane, showed simultaneous cancer radiosensitization and anti-angiogenesis

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activities.24

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However, SeNPs are poor in stability and easy to aggregate and precipitate, which will greatly

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reduce their anti-tumor activities. In addition, unmodified SeNPs cannot selectively target tumor

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cells, causing side effects. In order to increase the stability of nanoparticles in preparation, storage

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and application, stabilizers are often used. Common nanoparticle stabilizers include polyvinyl

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pyrrolidone (PVP),25 sodium methyl cellulose,26 polyvinyl alcohol,27 chitosan,28 and so on. We

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previously found that glucose, sucrose, chitosan, polysaccharide sulfate, degradable 3



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polysaccharides, cyanobacteria protein, amino acids, ATP and other biomolecules could effectively

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regulate the morphology and particle size of SeNPs 29-30 In recent years, increasing studies reported

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the use of biological macromolecules to regulate and stabilize inorganic nanoparticles.31-32

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However, research on the regulation and preparation of SeNPs by biomolecules and its application

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in biomedicine is rare. Mushroom polysaccharides have significant immunomodulatory and anti-

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tumor activities and have been used as natural adjuvants for tumor chemotherapy in Asia for

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decades.33 Mushroom polysaccharide has a large number of hydroxyl groups, this unique chemical

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structure endows it with a strong physical adsorption onto SeNPs, avoiding their accumulation and

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precipitation.31 We had previously constructed several mushroom polysaccharide-decorated SeNPs

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and evaluated their bioactivities. Decoration of mushroom polysaccharides not only increased their

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uptake by cancer cells in cancer therapy,34-35 but also showed strong promotion of bone formation in

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vitro and in vivo.36

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Although the SeNPs decorated with different mushroom polysaccharides showed potent

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activities against cancer cells, whether the physical characteristics, anti-tumor efficacy and tumor

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specificity of SeNPs were affected by the polysaccharides decorated is unknown and their in vivo

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anti-tumor activities have not been explored. In this study, we decorated SeNPs with

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polysaccharides extracted from Pleurotus tuber-regium (PTR), Polyporus rhinoceros (PR),

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Coriolus versicolor (CV) and Ganoderma lucidum (GL), respectively, and evaluated their anti-

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tumor efficacy and tumor specificity against a panel of cancer cell lines. In addition to compare

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their physical characteristics and tumor specific, we established nude mouse model to evaluate their

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in vivo anti-tumor activities and toxicities. Taken together, this study offers insights into how

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surface decoration can tune the cancer selectivity of SeNPs and provides a basis for engineering

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particles with increased anticancer efficacy. 4


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MATERIALS AND METHODS

Materials and Chemicals. Sodium selenite (Na2SeO3), propidium iodide (PI), 4’,6-

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Diamidino-2-phenyindole (DAPI), bicinchoninic acid (BCA) kit, 5,5’,6,6’-

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tetraethylimidacarbocyanine iodide (JC-1) were purchased from Sigma (Shanghai, China). [3-(4,5-

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dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt

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(MTS) was purchased from Promega (Madison, WI). Terminal deoxynucleotidyl transferase dUTP

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nick end labeling (TUNEL) assay kit was purchased from Roche Applied Science (Mannheim,

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Germany). Vitamin C was purchased from Guangzhou chemical reagent factory (Guangzhou,

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China).

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Preparation of Different Mushroom Polysaccharides-decorated SeNPs. The extraction of

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water soluble polysaccharides from Pleurotus tuber-regium, Polyporus rhinoceros, Coriolus

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versicolor and Ganoderma lucidum and the preparation of polysaccharide encapsulated SeNPs were

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conducted as described previously.34-35, 37 Briefly, the polysaccharides stock solution (0.25%) of

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different mushrooms were mixed with sodium selenite solution (25 mM). And then freshly prepared

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ascorbic acid solution (100 mM) was added dropwise into the mixtures under magnetic stirring. The

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mixtures were protected from light and allowed to react at room temperature for 24 h. The solutions

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were then dialyzed against ultra-pure water in the dark with intermittent changes of water until no

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Se could be detected in the outer solution by ICP-AES analysis. Coumarin-6 loaded PTR-SeNPs

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were prepared in the similar procedures with exception that 4 μg/mL coumarin-6 was added to the

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reaction system after the addition of polysaccharides. Characterization of the Different Encapsulated SeNPs. The obtained polysaccharideencapsulated SeNPs were characterized by transmission electron microscopy (TEM), high-

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resolution TEM (HR-TEM), energy dispersive X-ray (EDX), selected area electron diffraction

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(SAED), particle size analysis and Fourier transform infrared spectroscopy (FT-IR). TEM samples

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were prepared by dropping the particle solutions onto holey carbon film on copper grids. The

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images were obtained on an H-7650 Transmission Electron Microscope (Hitachi, Tokyo, Japan) at

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an acceleration voltage of 80 kV. The HR-TEM images and the corresponding SAED patterns were

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taken on a JEOL 2010 high-resolution TEM at a voltage of 200 kV. Elemental composition analysis

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were conducted on an EX-250 system (Horiba, Kyoto, Japan). The size distribution and stability of

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the nanoparticles in aqueous solution were measured on a NanoSight NS300 Instrument (Malvern

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Panalytical, Malvern, UK). FT-IR was recorded on a FT-IR spectrometer (Equinox 55, Bruker,

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Ettlingen, Germany) in the range 4000–500 cm-1.

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Cell Lines and Cell Culture. Human malignant melanoma A375, lung carcinoma A549,

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gastric adenocarcinoma AGS, cervix adenocarcinoma HeLa, hepatocellular carcinoma HepG2,

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foreskin fibroblast Hs68, breast adenocarcinoma MCF-7, osteosarcoma MG-63, pancreatic

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epithelioid carcinoma PANC-1 and prostrate adenocarcinoma PC-3 were gift from Professor Ming-

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Chiu Fung, Division of Life Science of the Chinese University of Hong Kong. A375, A549, Hs68,

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MG63 and PANC-1 were cultivated in DMEM medium. HeLa, HepG2 and MCF-7 were cultivated

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in MEM medium. AGS was cultivated in ATCC-formulated RPMI-1640 medium while PC-3 was

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cultivated in DMEM/F12 medium. All human cancer cell lines and normal cell were supplemented

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with 10% fetal bovine serum and 1% penicillin-streptomycin, except that 10 μg/mL insulin was

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additionally added for MCF-7, in a 37 °C humidified incubator with 5% CO2.

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Cell Viability Assay. The anti-proliferation effects of different encapsulated SeNPs on

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different human cancer cells and normal cell were determined by MTS assay as previously

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described.38 Cell at desired cell density was seeded into 96-well plate and incubated at 37 °C in CO2 6


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incubator overnight. Then a serial of concentrations of different SeNPs were added for various time

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points. At the designated time-point, 20 ul of MTS/PMS (2mg/ml MTS, 150 μM PMS) mixture was

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added into each well of 96-well plates and incubated for 4 hours. Absorbance was measured by a

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microplate reader at 490nm. Results were expressed as the percentage of absorbance of treated

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groups relative to the control group. Cytotoxicity Assay. The cytotoxicity of PTR/PR-SeNPs on AGS cancer cells were

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determined by the Cytotoxicity Detection KitPLUS (LDH) (Roche, Mannheim, Germany) and done

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according to the manufacturer’s manual. Briefly, the cells were seeded in 96-well plate and treated

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with different SeNPs as in the cell viability assay. Two control groups, low control and high

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control, were set for each experiment. Low control contained cells and media only and high control

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contained cells and lysis buffer. At the end of treatment, the plates were centrifuged at 250 g for

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10min. Then transfered 100ul/well supernatant carefully into corresponding wells of another 96-

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well plate. Added 100ul reaction mixture to each well and incubated for up to 30min at room

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temperature (protected from light) and stopped the reaction by adding 50uL stop solution. Measured

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the absorbance of the samples at 492nm in a microplate reader. The percentage of cytotoxicity was

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calculated as: Cytotoxicity (%) = (experiment value - low control) / (high control - low control) ×

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100.

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Cell Cycle Pattern Analysis. The effect of PTR/PR-SeNPs on the cell cycle distribution of

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AGS cancer cells were analyzed by the flow cytometry analysis as previously described.39 Cells at

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desired density were seeded into 6-well plate. After incubation at 37 °C CO2 incubator overnight,

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the cells were treated with desired concentrations of different SeNPs for various time. The cells

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were harvested and fixed overnight at -20 °C with 70% ice-cold ethanol. Fixed cells were washed

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twice with PBS (pH7.4) and centrifuge to remove ethanol. Cells were further washed with 1% BSA 7



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and incubated in dark at 4 °C with propidium iodide (PI) staining mixture (1.21mg/ml Tris,

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700U/ml RNase, 50.1ug/ml PI, pH8.0) overnight. Stained cells were analyzed using flow cytometer.

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DNA Damage Analysis. The effect of PTR/PR-SeNPs on the DNA damage of AGS cancer

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cell was assessed by the DNA fragmentation assay.40 Cell at desired cell density was seeded into 6-

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well plate and incubated at 37 °C CO2 incubator overnight. After treatment with desired

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concentrations of different SeNP for various time, the cells were harvested and lysed with DNA

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lysis buffer (200mM Tris-HCl pH8.3, 100mM EDTA & 1%SDS). Proteinase K (10 mg/ml) was

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used to remove proteins. DNA were then precipitated by adding ice-cold ethanol and RNA were

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removed by RNase A (0.2 mg/ml). Finally, equal amount of DNA was loaded to 1.5% TAE agarose

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gel and the gel were ran at 90V for 30 min. The DNA were imaged after ethidium bromide (EB)

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staining.

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Confirmation of Apoptosis. The apoptotic effect of PTR/PR-SeNPs on AGS cancer cell was confirmed by TUNEL-DAPI co-staining assay 18 and Annexin-V-FITC assay.41 For TUNEL assay, cells were seed at desired cell density into 2-cm confocal dishs and treated

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with desired concentration of SeNPs. Then fixed the cell samples with a freshly prepared Fixation

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solution (4% Paraformaldehyde in PBS, pH7.4) for 1h at room temperature. The sample were

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washed twice with PBS and incubated in Permeabilisation solution (0.1% Trition X-100 in 0.1%

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sodium citrate) for 2 min on ice. After wash with PBS again, 50ul TUNEL reaction mixture were

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added to the samples and samples were incubated in a humidified atmosphere for 60 min at 37 °C in

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the dark. Then washed out the buffer and stained with DAPI solution. Images of the samples were

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captured by fluorescence microscopy.

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For Annexin-V-FITC assay, cells were seeded at desired density into 6-well plate and treated

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with desired concentration of SeNPs, then harvested the cells. After wash with ice-cold PBS, the

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cell pellets were resuspended with 1 × binding buffer adjusting the cell density to 2-5 × 105

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cells/mL. The cells were stained with Annexin V-FITC at room temperature for 15 min under

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darkness. Next, washed the cells with 1 × binding buffer then stained with PI (20 ug/mL) and

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analyzed by flow cytometer (BD Accuri C6, San Jose, CA).

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Apoptotic Signaling Pathway Analysis. Cells were seeded in 10-cm dishes. After treatment,

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whole cell protein lysate was extracted with lysis buffer (Cell Signaling Technology, Inc) and the

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cytosolic protein was extracted with the ice-cold buffer A (20 mM Hepes, pH 7.5, 1.5 mM MgCl2,

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10 mM KCl, 1mM EDTA, 1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 10 μg/mL leupeptin,

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aprotinin, pepstatin and 250 mM sucrose). Protein concentration were determined by BCA assay.

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Equal amounts of protein were subjected to Western blot analysis 9 for caspase family and Bcl-2

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family proteins.

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Mitochondrial Membrane Potential (ΔΨm) Measurement. The change of mitochondrial

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membrane potential of AGS cancer cell induced by PTR/PR-SeNPs was measured by JC-1 staining

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as described before.42 Briefly, cells were seeded at desired cell density into 6-well plate and treated

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with desired concentration of SeNPs. Then harvested the cells and washed with ice-cold PBS (pH

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7.4). Resuspended the cells with 1 × JC-1 working solution in the dark at 37 °C for 15 min. After

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wash twice with assay buffer, cells were analyzed by flow cytometer (BD Accuri C6).

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In Vitro Cellular Uptake. The uptake of PTR-SeNPs by AGS cancer cell was quantified by

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the cellular uptake of coumarin-6-loaded PTR-SeNPs as mentioned earlier.34 The efficiency of the

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cellular uptake of PTR-SeNPs was expressed as the percentage of the tested wells over that of the

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positive wells. The intracellular activity of coumarin-6-loaded PTR-SeNPs in AGS cancer cell was

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indicated with LysoTracker® Deep Red and DAPI as mentioned previously.18

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Cellular uptake Mechanism. The cellular uptake mechanism of PTR-SeNPs by AGS cancer

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cell was studied with several endocytosis inhibitors including nystatin, sucrose and 2-deoxy-D-

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glucose.18

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Animal Study. Animal studies were approved by and followed the guidelines of the

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Laboratory Animal Ethics Committee of Jinan University. The in vivo anticancer activities of PTR-

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SeNPs and PR-SeNPs were evaluated in nude mouse model. Three to four-week old Balb/c nude

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mice were inoculated subcutaneously with 2 × 106 MGC-803 gastric cancer cells. The mice were

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divided randomly into 5 groups (n = 10), after the average volume of tumor reached ~ 100 mm3.

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The mice of treated groups were administrated with PTR-SeNPs or PR-SeNPs through intravenous

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injection at dosages of 750 or 2500 μg/kg BW/day, and the mice of control group received vehicle

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(PBS) only. The tumor volume was recorded during the treatment by measuring the tumor length (l)

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and width (w) and calculating by the formula: volume = l × w2/2. The treatment lasted for 27 d. At

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the end of experiment, the mice were euthanized and tumors were dissected and weighed. Major

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organs including heart, liver, spleen, lung, and kidney were isolated and fixed with tumors in

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formalin for hematoxylin and eosin (H&E) staining and immunohistochemical analysis, as

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previously described.12 Hematological analysis were conducted in Blood Test Center of the First

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Affiliated Hospital of Jinan University.

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Statistical Analysis. All results were expressed as mean ± SD from at least three independent

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experiments. Significant differences were tested using Student’s t-test or one-way ANOVA with the

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aid of statistical program Graphpad Prism version 5.0.

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RESULTS

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Synthesis of Mushroom Polysaccharides-decorated SeNPs and Comparison on the

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Physicochemical Characteristics. The preparation of polysaccharide-decorated SeNPs were

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depicted as in Figure 1. Water-soluble polysaccharides were first extracted from four mushrooms,

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Pleurotus tuber-regium, Polyporus rhinoceros, Coriolus versicolor and Ganoderma lucidum,

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respectively. Decoration of SeNPs with polysaccharides were done by dropping ascorbic acid

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solution to the mixture of corresponding polysaccharide and sodium selenite solution. To compare

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the physical and chemical characteristics of the four SeNPs, the morphology, stability and element

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composition were tested. The TEM images showed that the four type of SeNPs were well

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monodispered and nearly spherical particles (Figure 1A-D). PTR-SeNPs had the smallest diameter

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(~12.5 nm), CV-SeNPs were the largest (~20 nm), and PR- and GL-SeNPs were almost equal in

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size (~17 nm). Clear lattice fringes could be observed in all the SeNPs in HR-TEM images. These

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data demonstrated that there was no significant difference of the morphology between the different

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mushroom polysaccharides-decorated SeNPs. The hydrodynamic size of the CV-SeNPs (~102 nm)

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and PR-SeNPs were slightly larger (107 nm, Figure S1 Aa-Da) in aqueous solution. These particle

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sizes were suitable for biological applications.43 We next compared the stability of the four SeNPs

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by measuring their particle sizes in aqueous solution intermittently for up to 13 weeks. As showed

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in Figure S1 Ab-Db, no significant change in particle size of all the SeNPs during the measurement.

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The sizes were comparable to those at the beginning, without aggregation and precipitation. The

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results implied that the four different mushroom polysaccharides could stabilize SeNPs in aqueous

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solution with a similar degree. The SeNPs were further analyzed by SAED (Figure S1Ac-Dc) and

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element composition (Figure S1Ad-Dd). The FT-IR spectra demonstrated the abundance of −OH

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and −NH groups on the polysaccharides (Figure S1Ae-De) which could make them effectively bind 11



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onto the SeNPs. Comparison on Anticancer Activities of Different SeNPs. To compare the anticancer

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profile of the four different SeNPs, a panel of cancer cell lines were treated with various

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concentrations of SeNPs, and the cell viabilities were measured by MTS assay. As shown in Figure

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2A, the four functionalized SeNPs could strongly inhibit the growth of cancer cells. However, the

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inhibition effects of different SeNPs on different cancer cells varied, indicating the tumor specificity

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of the SeNPs were affected by the surface decorating polysaccharides. It was worth noting that

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among the 9 cancer cell lines, gastric cancer cell AGS showed relative high sensitivity to all four

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SeNPs (IC50 = 3.12- 5.58 μM), especially to PR-SeNPs and PTR-SeNPs (Figure S2 and Table S1).

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In contrast, a much lower cytotoxicity of PR-SeNPs and PTR-SeNPs against the normal cells Hs68

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could be observed with the IC50 values of 225 μM and 113 μM, respectively (Figure 2B, Figure S2

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and Table S1). Additionally, PR-SeNPs and PTR-SeNPs also inhibited the growth of AGS cells in a

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time-depend manner (Figure S3A). According to these results, PR-SeNPs and PTR-SeNPs were

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used for further studies exploring the mechanisms by which SeNPs inhibit the proliferation of AGS

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cells.

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Cellular Uptake of PTR-SeNPs by AGS Cells. Cellular uptake is an important factor

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affecting the anti-tumor activity of nanomedicines.44 To determine the cellular uptake profile of the

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encapsulated SeNPs, AGS cells were incubated with a series of concentrations of couramin-6 (green

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fluorescent)-loaded PTR-SeNPs for different time. The uptake of PTR-SeNPs was monitored by

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fluorescent imaging. As shown in Figure 3A, the fluorescent intensity of couramin-6 in AGS cells

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was increased with time lapse, and after internalization, the PTR-SeNPs were co-localized with

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lysosomes. The uptake of PTR-SeNPs were quantified by determining the fluorescent intensity of

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the cell lysates. Figure 3B showed that the uptake of SeNPs by AGS cells were increased dose- and 12


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time-dependently. We next explored the internalization pathway of PTR-SeNPs. Endocytosis of

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AGS cells were blocked by pre-treatment with different inhibitors before incubation of PTR-SeNPs.

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Quantification of cell lysate fluorescent intensity showed that the uptake of PTR-SeNPs by AGS

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cells was significantly inhibited when pre-treated with high concentration of nystatin, sucrose and

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2-deoxy-D-glucose (Figure 3C), implying that endocytosis plays an important role in the uptake of

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PTR-SeNPs.

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Induction of Apoptosis of AGS Cell by PR-SeNPs and PTR-SeNPs. To understand the

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anticancer mechanism of PR-SeNPs and PTR-SeNPs, we analyzed the cell cycle distribution of

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AGS cells treated with these SeNPs for different time. As shown in Figure 4, treatment with PR-

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SeNPs and PTR-SeNPs resulted in a dose- and time-dependent increase of the sub-G1 population in

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AGS cells, which suggested the induction of apoptosis by the SeNPs. The apoptosis was further

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confirmed by Annexin V/PI double staining and TUNEL assay. Flow cytometry analysis of

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Annexin-V/PI double staining showed that the apoptotic cells, including early (Annexin V+/PI-)

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and late apoptotic (Annexin V+/PI+) subsets, were increased up to more than 53% and 46% when

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AGS cell were treated with high concentration of PTR-SeNPs or PR-SeNPs, respectively (Figure

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5A). In comparison, staurosporine (STS), as a positive apoptosis inducer, resulted in about 10% of

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apoptosis at the concentration of 10 μM. Similar results were observed in TUNEL assay (Figure 5B

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and C) and the DNA fragmentation assay (Figure S4). Taken together, these data demonstrated that

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PR-SeNPs and PTR-SeNPs were effective in inducing apoptotic cell death of AGS cells.

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Apoptotic Signaling Pathways Induced by PR-SeNPs and PTR-SeNPs. Mitochondrion is

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the central regulator of the intrinsic apoptosis pathway.45 Apoptotic signals trigger the loss of

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mitochondrial membrane potential (ΔΨm) and release of pro-apoptotic molecules to activate

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caspase cascade.46 To examine the role of mitochondrion in PR-SeNPs and PTR-SeNPs induced 13



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apoptosis, we used JC-1 to detect the change of mitochondrial membrane potential of AGS cells

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treated with PR-SeNPs or PTR-SeNPs. As shown in Figure 6A and B, PTR-SeNPs and PR-SeNPs

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caused substantial loss of mitochondrial membrane potential in a dose- and time-dependent manner,

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implying the involvement of mitochondrion during the PR-SeNPs and PTR-SeNPs caused

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apoptosis.

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Caspase family proteins are activated sequentially and then play a central roles in the process

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of apoptosis.47 To examine the activation of caspases in AGS cells treated with PR-SeNPs and

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PTR-SeNPs, we measured their cleavage by Western blot. As shown in Figure 7A, the full-length

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caspase-3, -7 and -9 were decreased when AGS cells were treated with the SeNPs, and the active

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(cleaved) forms were increased correspondently. The substrate of caspases PARP was also cleaved,

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which is considered as a marker of apoptosis. Caspase-8, which mediates the death signal from

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outside the cells, were not activated, implying that the extrinsic pathway was not involved in the

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PR-SeNPs and PTR-SeNPs induced apoptosis. These results were in line with the loss of

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mitochondrial membrane potential, which is the upstream signal of the intrinsic apoptosis pathway.

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The mitochondrial outer membrane integrity is regulated by the Bcl-2 family proteins.48 We

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therefore measured the expression levels of a panel of Bcl-2 family proteins. As showed in Figure

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7B, the expression levels of anti-apoptotic members, Bcl-2 and Bcl-XL, were significantly decreased

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while Mcl-1 and phosphorylated Bcl-2 were nearly unchanged after treatment of PR-SeNPs or

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PTR-SeNPs. The activation of apoptotic signal was also promoted by the upregulation of the pro-

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apoptotic proteins, including Bad, Bax, Bim, Bid, Puma and Bak (Figure 7B). Taken together, these

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data demonstrated that PR-SeNPs and PTR-SeNPs could induce apoptosis of AGS cells by

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regulating the Bcl-2 family proteins to decrease the integrity of mitochondrial outer membrane and

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activation of caspases. The possible apoptosis signaling pathway induced by PR- and PTR-SeNPs 14


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were depicted in Figure7C. In vivo Anticancer Activities of PR-SeNPs and PTR-SeNPs. The in vivo anticancer

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activities of mushroom polysaccharides-decorated SeNPs haven’t been evaluated before. Therefore,

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we tested the in vivo efficacy of PR-SeNPs and PTR-SeNPs by using nude mouse xenograft model.

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Because AGS cells failed to graft on nude mice in our pre-experiments, another gastric cancer cells

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MGC-803 were used. The cells were inoculated subcutaneously on nude mice which then received

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treatment with PTR-SeNPs, PR-SeNPs or vehicle. Tumor growth curves were shown in Figure 8A

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which demonstrated potent tumor inhibition effects of PTR-SeNPs and PR-SeNPs. Images of

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tumors dissected from mice and representative images of mice in different treatment groups were

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shown in Figure 8B and C respectively. Tumor sections were further analyzed by

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immunohistochemical staining (Figure 8D). Ki67 is a proliferation marker, which significantly

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decreased in the SeNPs treated groups, especially in the high dose groups. In the contrast, the tumor

318

suppressor p53 were strongly induced by the treatment of SeNPs. VEGFR2, which plays a critical

319

role in angiogenesis, displayed similar inhibition tendency to Ki67. Moreover, results of TUNEL

320

assay showed that high dose of SeNPs treatment induced remarkable apoptosis in tumor tissues. In

321

H&E staining, larger areas of necrosis were found in the groups of high dose of SeNPs treatment. In

322

summary, the results above clearly showed that PR-SeNPs and PTR-SeNPs were effective in

323

inhibiting the growth of tumors in vivo.

324

Furthermore, the toxicity of PR-SeNPs and PTR-SeNPs to the mice was evaluated by

325

examination of the H&E-staining sections of major organs and blood biochemical indexes. As

326

shown in Figure 9A, no obvious damage could be observed in the organ sections, including lung,

327

liver, spleen, kidney and heart. Blood biochemical indexes, LDLC and GLB, reflecting the level

328

blood fat and liver function, were increased in the model group, which were alleviated by the 15



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329

treatment of SeNPs (Figure 9B). These data together demonstrated potent anti-tumor activity of PR-

330

SeNPs and PTR-SeNPs in vivo and a relative low toxicity to the mice.

331

DISCUSSION

332

To increase the stability and efficacy of nanoparticles in medical use, surface decorators were

333

commonly used. We had previously found that decoration of polysaccharides from certain

334

mushrooms significantly increase the stability, cellular uptake and in vitro anti-tumor potency of

335

SeNPs.34-35 However, whether there is difference of physical characteristics and cancer type

336

selectivity between different mushroom polysaccharides-decorated SeNPs were unclear. Thus, we

337

synthesized and compared the physical characteristics and anticancer activities of four mushroom

338

polysaccharides-decorated SeNPs in the present study. And the in vivo efficacy of mushroom

339

polysaccharide-decorated SeNPs were evaluated for the first time.

340

Stability is an important characteristic affecting the application of nanoparticles. Naked

341

SeNPs are very unstable and will precipitate in the solution soon after synthesized. 34-35 Due to the

342

abundant hydroxyl groups of the mushroom polysaccharides, they had strong physical adsorption to

343

SeNPs, which could effectively avoid their accumulation and regulate their particle size. The

344

modified SeNPs in our study were stable in aqueous solution for up to 13 weeks. Another

345

interesting result found in the present study is that SeNPs decorated with different mushroom

346

polysaccharides inhibited the growth of different cancer cells in varied degrees. As the four

347

mushroom polysaccharide-modified SeNPs were similar in physical characteristics, we assumed

348

that the difference of anti-cancer profiles of these SeNPs were derived from the chemical

349

differences of the polysaccharides. The chemical component and molecular structure of the

350

polysaccharides covering the SeNPs may affect the interaction of the nanoparticles and cell

351

membranes and therefore affect their cellular uptake behavior and the signalling inside the cells. It 16


Page 17 of 33


352

would be interested for us to further explorer the chemical structure of the mushroom

353

polysaccharides and their relation with anti-cancer efficacy in the future.

354

The in vivo anti-tumor activity of polysaccharide-modified SeNPs had not been evaluated

355

before. Here, we used xenograft model to demonstrate the tumor inhibition efficacy in nude mice.

356

The tumor growth of the PT/PTR-SeNPs treated group was significantly slower than the control

357

group, confirming the in vivo activity of polysaccharide-modified SeNPs for the first time. In

358

additionally, the SeNPs showed no evident toxicity to the mice, implied by the results of blood

359

biochemical indexes and histochemical staining of organ tissues. These data demonstrated an

360

effective way to change cancer selectivity of SeNPs by decoration of mushroom polysaccharides

361

and suggested their potential of further usage in clinical.

362

Overall, comparing with previous study on SeNPs, this study makes improvement on the

363

following issues. Firstly, we have compared the difference in physical characteristics and anticancer

364

efficacy of SeNPs decorated with polysaccharides from different mushrooms, while the previous

365

papers were intended to investigate whether the decoration of mushroom polysaccharides could

366

stabilize SeNP and increase its uptake by cancer cells. The results of present study demonstrated the

367

different species of polysaccharide determined the cancer selectivity of SeNPs they decorated. It

368

would be a pioneer work that guide the future design of tumor selective SeNPs for cancer therapy

369

based on surface decoration. Secondly, the present study is a pioneer study on the in vivo anticancer

370

activity and action mechanisms of polysaccharide-decorated SeNPs. However, previous studies just

371

focused on in vitro anticancer activity of PTR-SeNPs. Herein we have examined the changes of

372

Bcl-2 family proteins which are central regulators in the process of endogenous apoptosis pathway.

373

In addition, the in vivo anti-cancer efficacy of polysaccharide-decorated SeNPs had been evaluated

374

in the present study, which haven’t been reported before. Indeed, not all the agents that display 17



375

good anticancer efficacy in vitro show similar potency in vivo. So, our present study could provide

376

solid data demonstrating the in vivo tumor inhibition effect and the underlying molecular

377

mechanisms of the mushroom polysaccharide-decorated SeNPs. Taken together, this study offers

378

insights into how surface decoration can tune the cancer selectivity of SeNPs and provides a basis

379

for engineering particles with increased anticancer efficacy.

Page 18 of 33

380 381

AUTHOR INFORMATION

382

Corresponding Authors

383

* E-mail addresses: [email protected]. [email protected].

384

Author Contributions

385

#

These authors contributed equally to this work.

386 387

Funding

388

This work was supported by Natural Science Foundation of China (21877049), National Program for

389

Support of Top-notch Young Professionals (W02070191), YangFan Innovative & Entepreneurial

390

Research Team Project (201312H05), Fundamental Research Funds for the Central Universities,

391

Shenzhen's strategic emerging industries development fund (JCYJ20130401152508660).

392

Notes

393

The authors declare no competing financial interest.

394

Supplementary Information

395

The Supporting Information is available free of charge on the ACS Publications website at DOI:

396

10.1021/acs.jafc.XXXXX.

18


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397

Characterization of the four mushroom polysaccharide-decorated SeNPs; the dose-response

398

curves of the PTR- and PR-SeNPs against cancer and normal cells; time-dependent inhibition of

399

growth of AGS cells by PRT- and PR-SeNPs; the IC50 values of different polysaccharides-

400

decorated SeNPs against different cell lines; agarose gel electrophoresis of DNA extracted from

401

PR/PTR-SeNPs treated AGS cells(PDF).

402 403

References

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Figure Captions Figure 1. Preparation of Pleurotus tuber-regium (A), Polyporus rhinoceros (B), Coriolus versicolor (C) and Ganoderma lucidum (D) polysaccharide-decorated SeNPs and characterization of their morphology by TEM and HR-TEM. Figure 2. Tumor growth inhibition effects of mushroom polysaccharide-decorated SeNPs. (A) The IC50 of PTR-, PR-, CV- and GL-SeNPs against nine different cancer cells measured by MTS assay. (B) The IC50 of PTR- and PR-SeNPs against normal fibroblasts cells Hs68 measured by MTS assay. Figure 3. The uptake mechanism and intracellular localization of coumarin-6-loaded PTR-SeNPs. (A) Localization of PTR-SeNPs in AGS cells were visualized by staining the cells with DAPI, LysoTracker® Deep Red after treatment with coumarin-6-loaded-PTR-SeNPs. (B) The uptake of coumarin-6-loaded PTR-SeNPs by AGS cells were quantified by measuring the fluorescent intensity of lysates from cells treated with a serial of concentrations of PTR-SeNPs for the indicated time. (C) The uptake of coumarin-6-loaded PTR-SeNPs in AGS cells pre-treated with the indicated endocytosis inhibitors. Figure 4. Flow cytometry analysis of AGS cells treated with PTR- and PR-SeNPs. (A) Representative PI staining flow cytometry histograms of the AGS cells treated with IC50 (3 μM) or 10 × IC50 (30 μM) of PTR- or PR-SeNPs for different time. (B) Statistic bar charts of three replicated flow cytometry analyses showing the distribution of cells in different cell cycle phase. Sub-G1 population was considered as the apoptotic subset. Figure 5. Validation of apoptosis in AGS cells treated with PTR- or PR-SeNPs. (A and B) Apoptosis of AGS cells treated with PTR- or PR-SeNPs was detected by Annexin-V/PI double staining (A) and TUNEL assay (B). (C) Statistics bar chart of the percent of TUNEL-positive cells in (B). Staurosporine (STS) was used as a positive control of apoptosis inducer. 22


Page 23 of 33


Figure 6. Mitochondrial member potential is decreased in PR-SeNPs and PTR-SeNPs-caused apoptosis. Loss of mitochondrial member potential in AGS cells treated with PTR- or PR-SeNPs was examined by JC-1 staining. Shown are representative flow cytometry dot plots (A) and statistic bar charts (B). Figure 7. Apoptotic pathways triggered by PTR- or PR-SeNPs. (A) Activation of caspases in AGS cells treated with PTR- or PR-SeNPs was examined by Western blot. (B) The changes of expression of Bcl-2 family proteins in AGS cells after treatment of PTR- or PR-SeNPs were examined by Western blot. (C) The hypothetic signaling pathway by which the polysaccharide-decorated SeNPs induced apoptosis of AGS cells. Figure 8. In vivo anticancer efficacy of PTR- and PR-SeNPs tested in nude mice xenograft model. (A) The growth curves of xenograft tumor inoculated on nude mice treated with 750 μg/kg (L) or 2500 μg/kg (H) of PTR-/PR-SeNPs or vehicle (PBS). (B and C) The images of tumors and representative mice in each group taken at the end of treatment. (D) Tumor inhibition effects of PTRand PR-SeNPs were examined by immunohistochemical and H&E analysis. Figure 9. Toxicities of PR- and PTR-SeNPs to mice were evaluated by H&E staining of tissue sections (A) and hematological analysis (B).

23



Page 24 of 33

Figure 1. Preparation of Pleurotus tuber-regium (A), Polyporus rhinoceros (B), Coriolus versicolor (C) and Ganoderma lucidum (D) polysaccharide-decorated SeNPs and characterization of their morphology by TEM and HR-TEM.

24


Page 25 of 33


Figure 2. Tumor growth inhibition effects of mushroom polysaccharide-decorated SeNPs. (A) The IC50 of PTR-, PR-, CV- and GL-SeNPs against nine different cancer cells measured by MTS assay. (B) The IC50 of PTR- and PR-SeNPs against normal fibroblasts cells Hs68 measured by MTS assay.

25



Page 26 of 33

Figure 3. The uptake mechanism and intracellular localization of coumarin-6-loaded PTR-SeNPs. (A) Localization of PTR-SeNPs in AGS cells were visualized by staining the cells with DAPI, LysoTracker® Deep Red after treatment with coumarin-6-loaded-PTR-SeNPs. (B) The uptake of coumarin-6-loaded PTR-SeNPs by AGS cells were quantified by measuring the fluorescent intensity of lysates from cells treated with a serial of concentrations of PTR-SeNPs for the indicated time. (C) The uptake of coumarin-6-loaded PTR-SeNPs in AGS cells pre-treated with the indicated endocytosis inhibitors.

26


Page 27 of 33


Figure 4. Flow cytometry analysis of AGS cells treated with PTR- and PR-SeNPs. (A) Representative PI staining flow cytometry histograms of the AGS cells treated with IC50 (3 μM) or 10 × IC50 (30 μM) of PTR- or PR-SeNPs for different time. (B) Statistic bar charts of three replicated flow cytometry analyses showing the distribution of cells in different cell cycle phase. Sub-G1 population was considered as the apoptotic subset.

27



Page 28 of 33

Figure 5. Validation of apoptosis in AGS cells treated with PTR- or PR-SeNPs. (A and B) Apoptosis of AGS cells treated with PTR- or PR-SeNPs was detected by Annexin-V/PI double staining (A) and TUNEL assay (B). (C) Statistics bar chart of the percent of TUNEL-positive cells in (B). Staurosporine (STS) was used as a positive control of apoptosis inducer.

28


Page 29 of 33


Figure 6. Mitochondrial member potential is decreased in PR-SeNPs and PTR-SeNPs-caused apoptosis. Loss of mitochondrial member potential in AGS cells treated with PTR- or PR-SeNPs was examined by JC-1 staining. Shown are representative flow cytometry dot plots (A) and statistic bar charts (B).

29



Page 30 of 33

Figure 7. Apoptotic pathways triggered by PTR- or PR-SeNPs. (A) Activation of caspases in AGS cells treated with PTR- or PR-SeNPs was examined by Western blot. (B) The changes of expression of Bcl-2 family proteins in AGS cells after treatment of PTR- or PR-SeNPs were examined by Western blot. (C) The hypothetic signaling pathway by which the polysaccharide-decorated SeNPs induced apoptosis of AGS cells.

30


Page 31 of 33


Figure 8. In vivo anticancer efficacy of PTR- and PR-SeNPs tested in nude mice xenograft model. (A) The growth curves of xenograft tumor inoculated on nude mice treated with 750 μg/kg (L) or 2500 μg/kg (H) of PTR-/PR-SeNPs or vehicle (PBS). (B and C) The images of tumors and representative mice in each group taken at the end of treatment. (D) Tumor inhibition effects of PTRand PR-SeNPs were examined by immunohistochemical and H&E analysis.

31



Page 32 of 33

Figure 9. Toxicities of PR- and PTR-SeNPs to mice were evaluated by H&E staining of tissue sections (A) and hematological analysis (B).

32


Page 33 of 33


The table of contents

Herein we decorate selenium nanoparticles (SeNPs) with water soluble polysaccharides extracted from mushrooms to compare their physical characteristics and evaluate their effects on in vivo anticancer efficacy and the underlying action mechanisms. Taken together, this study offers insights into how surface decoration can tune the cancer selectivity of SeNPs and provides a basis for engineering particles with increased anticancer efficacy.

33


Potentiation of in Vivo Anticancer Efficacy of Selenium Nanoparticles

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