Preparation, characterization, and antioxidant capacities of selenium nanoparticles stabilized using polysaccharide–protein complexes from Corbicula fluminea

Author’s Accepted Manuscript Preparation, characterization, and antioxidant capacities of selenium nanoparticles stabilized using polysaccharide–protein complexes from Corbicula fluminea Yao-Yao Wang, Wen-Yi Qiu, Ling Sun, Zhi-Chao Ding, Jing-Kun Yan www.elsevier.com/locate/sdj

PII: DOI: Reference:

S2212-4292(18)30346-8 https://doi.org/10.1016/j.fbio.2018.10.014 FBIO354

To appear in: Food Bioscience Received date: 10 April 2018 Revised date: 22 October 2018 Accepted date: 24 October 2018 Cite this article as: Yao-Yao Wang, Wen-Yi Qiu, Ling Sun, Zhi-Chao Ding and Jing-Kun Yan, Preparation, characterization, and antioxidant capacities of selenium nanoparticles stabilized using polysaccharide–protein complexes from Corbicula fluminea, Food Bioscience, https://doi.org/10.1016/j.fbio.2018.10.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Submitted to: Food Bioscience (Original Research MS)

Preparation, characterization, and antioxidant capacities of selenium nanoparticles stabilized using polysaccharide–protein complexes from Corbicula fluminea

Yao-Yao Wang, Wen-Yi Qiu, Ling Sun, Zhi-Chao Ding, Jing-Kun Yan 

School of Food & Biological Engineering, Jiangsu University, Zhenjiang, 212013, Jiangsu, People’s Republic of China

Running Title: Preparation of antioxidative selenium nanoparticles

 Corresponding authors: JK Yan, Tel: +08615952819661, E-mail: [email protected], jkyan27@ ujs.edu.cn 1

Abstract Selenium nanoparticles (SeNP) have recently received increasing attention as potential candidates to replace other forms of Se in diet nutritional supplements. Compared with polysaccharides (PS), polysaccharide–protein complexes (PSP) can be better stabilizers of NP because of their chemical structures. In this study, SeNP were prepared by introducing ascorbic acid to reduce sodium selenite using a PSP isolated from Asian clam (Corbicula fluminea) as a stabilizer and dispersing agent. The particle size, morphology, and structure of the PSP–SeNP were characterized using various spectroscopic and microscopic measurements. Results showed that the PSP–SeNP prepared at different Se/PSP ratios (w/w) showed homogeneous and monodisperse spherical structures of ~40 – 70 nm. Specifically, at the Se/PSP (w/w) ratio of 1:2, the PSP–SeNP with minimum particle size (~43 nm) showed good stability in aqueous medium. Antioxidant activity in vitro showed that the same particles showed significant 1,1-diphenyl-2-picrylhydrazyl radical scavenging ability in a dose-dependent manner and good antioxidant capacity using Trolox equivalent antioxidant capacity and ferric reducing ability of plasma

assays. In addition, these particles showed

low cytotoxic activity against SPCA-1 and HeLa cell lines in vitro. Therefore, the SeNP prepared using PSP might be used as a potential diet nutritional supplement with food and medical applications.

Keywords: Corbicula fluminea; Asian clam; Polysaccharide–protein complexes; Selenium nanoparticles

2

1. Introduction Oxidative stress and overproduction of reactive oxygen species (ROS) have significantly influenced the pathological progression of numerous diseases and several types of cancer (Jayaprakash & Marshall, 2011; Forootanfar et al., 2014). Excessive ROS may cause irreversible oxidative damage to cellular lipids, proteins, and DNA in the human body. ROS, which is most commonly found as superoxide anion radicals (O2·-), hydroxyl radicals (·OH), and hydrogen peroxide (H2O2), are reportedly related to many diseases, such as rheumatoid arthritis, diabetes mellitus, Alzheimer’s disease, coronary heart diseases, nephritis, cancer, arteriosclerosis, and other health disorders associated with aging (Alexandra et al., 2016; Huang et al., 1999). Antioxidants protect the human body from oxidative damage and retard the progress of these diseases to a certain extent by reacting directly with free radicals or improving the activities of antioxidant enzymes (Forootanfar et al., 2014; Jayaprakash & Marshall, 2011). Several synthetic antioxidants, including butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and tert-butylhydroquinone (TBHQ), have been used commercially; however, most of these substances have been reported to be potentially toxic to living organisms (Kong et al., 2014). Therefore, developing novel and efficient alternatives with low toxicity is an ideal strategy to address the current concern for food quality and consumer safety. Clinical and epidemiological evidence has showed that selenium (Se), an essential trace element for human and animal health, protects against oxidative stress and shows excellent antioxidant potential (Jayaprakash & Marshall, 2011; Huang et al., 2007). Se can improve the activities of selenoenzymes, such as Se-dependent glutathione peroxidases, which act as 3

redox centers and prevent free radicals from damaging cells and tissues in vivo (Rayman, 2000). In addition, Se seems to prevent cancer, reduces cancer incidences, and stimulates immune responses (Skalickova et al., 2016). However, Se shows an extremely narrow margin between its lowest acceptable daily intake and its toxicity, thereby limiting its practical applications in food and medicine (Lin & Wang, 2005; Jia et al., 2015). Se nanoparticles (SeNP) have received attention as potential candidates to replace other forms of Se in diet nutritional supplements because of their good bioavailability, biological activity, and low toxicity (Takats et al., 2017; Kong et al., 2014). Several studies showed that SeNP have antioxidant properties and thus can be used as special anti-oxidative drugs (Kong et al., 2014). Furthermore, SeNP in the size range of 5 to 200 nm efficiently scavenge free radicals in vivo and in vitro (Huang et al., 2003; Zhang et al., 2004). SeNP also showed antioxidant activity but less toxicity than Se (Shakibaie et al., 2010). Various methods, such as chemical reduction, hydrothermal synthesis, solvothermal synthesis, sonochemical methods, and biosynthesis, have been developed for the synthesis of SeNP with various sizes and shapes (Chaudhary et al., 2016; Skalickova et al., 2016). However, the SeNP are usually unstable and readily aggregate into large clusters in aqueous solution, leading to decreased bioactivity and bioavailability (Jia et al., 2015; Zhang et al., 2004). Numerous researchers have tried to find a simple and efficient approach for the dispersion and stabilization of SeNP. Polysaccharides (PS) can be effectively used as soft templates for the preparation and stabilization of SeNP due to their eco-friendliness, cost effectiveness, biocompatibility, biodegradability, and easy processability into various hydrogel shapes. Many PS and their derivatives, such as chitosan (CS) (Chen et al., 2015), 4

konjac glucomannan (Zhang et al., 2004), gum arabic (Kong et al., 2014), dextran (Shen et al., 2008), β-glucan (Jia et al., 2015), chondroitin sulfate (Han et al., 2012), carboxymethyl cellulose (Zhang et al., 2004), and carboxymethyl CS (Chen et al., 2015), have been used as stabilizers and dispersants to prepare stable and uniform SeNP in aqueous solutions. For example, Zhang et al. (2010) prepared water-dispersible spherical SeNP using natural hyperbranched polysaccharide extracted from the sclerotia of Pleurotus tuber-regium and Rhizoma Panacis Japonici as the stabilizer and capping agent. Wu et al. (2012) used mushroom polysaccharide–protein complexes (PSP) isolated from the sclerotium of P. tuber-regium as the capping agent for size-controllable and highly stable SeNP in a simple redox system of sodium selenite (Na2SeO3) and ascorbic acid. Wu et al. (2013) also synthesized

functional

SeNP

using

Polyporus

rhinoceros

water-soluble

polysaccharide–protein complexes (PRW) as the capping agent. Compared with PS, these PSP not only have a larger number of terminal hydroxyl groups and high specific surface area, but also have amino groups from the proteins, which are easy to bind to nanomaterials or cell membranes (Zhang et al., 2010; Wu et al., 2013). More recently, a novel PSP from Corbicula fluminea has been prepared using a three-phase partitioning (TPP) technique, and the partially purified PSP showed good radical scavenging and antioxidant activities in vitro (Wang et al., 2017). The present study aimed to prepare water-dispersible SeNP with the PSP from C. fluminea as a stabilizer and dispersing agent using the introduction of ascorbic acid to reduce Na2SeO3. The particle size, morphology, and structure of the SeNP (PSP–SeNP) at different Se/PSP ratios were characterized using UV–Vis absorption spectroscopy, high-resolution 5

transmission electron microscopy (HR-TEM), dynamic light scattering (DLS), X-ray diffraction (XRD), energy-dispersive X-ray spectroscopy (EDX), and Fourier-transform infrared (FT-IR) spectroscopy analyses. The in vitro antioxidant activities and cytotoxicity of the PSP–SeNP were also evaluated.

2. Materials and methods 2.1. Materials and chemicals The Asian clams (C. fluminea), were harvested from Hongze Lake (Suqian, Jiangsu, China), and were obtained from Suqian Chengzihu Food Co. Ltd. (Suqian, Jiangsu, China). The shell of C. fluminea was removed and the whole soft body was kept in ice and was transported to the laboratory (~12 h), stored at -80 °C for 48 h, freeze-dried (FreeZone®, Labconco, Corp., Kansas City, MO, USA), ground into a fine powder using a high speed disintegrator (Model HDV, Dongying Hongjiu Traditional Medicine Machine Co., Dongying, Shandong, China), sieved (60 mesh), and then sealed in airtight plastic bags at 4°C for a maximum of three months. The procedure used for the preparation of PSP from C. fluminea was described in a previous study (Wang et al., 2017). Briefly, the PSP contained an acidic proteoglycan with O-glycosylation and 81.7% carbohydrate mainly composed of glucose, glucosamine and mannose with a molar ratio of 10.8:4.4:1.0. The PSP was composed of a high molecular weight (MW) fraction (2.11 × 106 Da, 27.6%) and a low MW fraction (6.15 × 104 Da, 72.4%)

using high performance gel permeation chromatography (Wang et al., 2017).

The PSP was stored at room temperature (25 °C) in a desiccator for a maximum of 4 wk prior to

further

analyses.

1,1-Diphenyl-2-picrylhydrazyl

(DPPH), 6

6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic

acid

(Trolox),

2,2΄-azinobis

(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and Na2SeO3 were purchased from Sigma Aldrich Chemical Co. (St. Louis, MO, USA). Alamar blue (AB) was purchased from Bio-Rad Laboratories Inc. (Hercules, CA, USA). Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), minimum essential medium (MEM) and antibiotics (penicillin 100 units/mL, streptomycin sulfate 100 μg/mL) were purchased from Gibco Co. (Grand Island, NY, USA). All other chemicals and solvents were of analytical grade from Sinopharm Group Co. Ltd. (Shanghai, China) and used without further purification. Deionized water was obtained using a Millipore-Q pure water system (Milli-Q, Integral, Merck Chemical Technolgy (Shanghai) Co., Ltd., Shanghai, China).

2.2. Preparation of PSP-SeNP PSP-SeNP were prepared based on the method of Jia et al. (2015). PSP (100 mg) was dissolved in 100 mL of deionized water with continuous stirring at 4 °C overnight for complete hydration. Different volumes (2.8–24.3 mL) of Na2SeO3 solutions (0.1 M) were mixed with 100 mL of the PSP aqueous solution (1 mg/mL) and subsequently stirred for 1 min. Afterward, 5 mL of freshly prepared ascorbic acid solution (0.2 M) was added dropwise into the PSP–Na2SeO3 solution, which was stirred for 24 h at 25 °C. The resulting solutions were dialyzed using a dialysis bag (Spectrum Laboratories, Rancho Dominguez, CA, USA) using a nominal cut-off MW of 3.5 kDa against distilled water for 48 h and then freeze-dried to obtain the PSP–SeNP. The PSP–SeNP were prepared at different mass ratios of Se/PSP (1:10, 1:5, 1:2, 1:1, and 4:3, w/w). They were vacuum sealed in airtight plastic bags and 7

stored at 25 °C in a desiccator containing silica gel for up to 30 days. SeNP prepared in the absence of PSP using the same procedure were used as the control. The detailed procedure and possible mechanism for the formation of PSP–SeNP are shown in Fig. 1.

2.3. Characterization of SeNP The UV–Vis absorption spectrum of the sample (1.0 mg/mL) was measured using a Varian Cary 100 spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) in the wavelength range of 190–600 nm at 25 °C. The particle size and morphology of SeNP were observed using TEM (Tecnai 12, FEI Co., Hillsboro, OR, USA) at an accelerating voltage of 120 kV. For TEM analysis, a drop of the SeNP solution was placed on a carbon-coated copper grid (300 mesh) and dried at 25 °C for 30 min. The TEM images were analyzed using the program freeware ImageJ v 1.46r (National Institutes of Health, Bethesda, MD, USA). The Z-average diameter (particle size), polydispersity index (PDI), and zeta potential of the PSP–SeNP solutions (0.1 mg/mL) were determined using DLS analysis with a Zetasizer Nano ZS90 instrument (Malvern Instruments Corp., Malvern, England) using the software that came with the instrument. The elemental composition of the SeNP was determined using energy dispersive X-ray spectroscopy (EDX, Noran, Thermo Fisher Scientific Inc., Madison, WI, USA) attached to the SEM (S4800, Hitachi Ltd., Tokyo, Japan) in back-scattered electron mode after sputtering Pt/Pd onto the specimen. The crystalline structure of the SeNP was determined using a wide-angle XRD 8

(D8-Advance, Bruker, Karlsruhe, Germany). The XRD patterns were measured with Cu Kα radiation (λ= 0.1541 nm) at 40 kV and 30 mA with the 2θ ranging from 5° to 70° with a step speed of 5°/min. The FT-IR spectroscopy of the sample was measured using a Nicolet Nexus 670 FT-IR spectrometer (Thermo Scientific Inc.). The spectrum was collected at ambient conditions in transmittance mode from an accumulation of 64 scans with a 4 cm-1 resolution in the wavenumber ranging from 400 to 4000 cm−1. The sample for testing was mixed with dry potassium bromide (100 mg) and pressed to form a salt disc (10 mm diameter).

2.4. Antioxidant properties of PSP-SeNP For the DPPH scavenging assay, the control SeNP, PSP, and PSP-SeNP at different Se/PSP ratios were previously dissolved in distilled water at various concentrations (0.1 - 1.5 mg/mL). Solution (3 mL) was added into 1 mL 0.1 mM DPPH solution in methanol. The reaction mixture was stirred and incubated at 25 oC for 30 min in the dark. The absorbance of the solution was measured at 517 nm using a UV-1600 Spectrophotometer (Beijing Ruili Analytical Instrument Co., Ltd., Beijing, China). The DPPH radical scavenging activity was calculated based on the following equation:  A  A2  Scavenging activity (%) = 1- 1  100% A0  

where A0, A1, and A2 are the respective absorbance values of the blank control (deionized water), the sample with DPPH solution, and the sample with deionized water. Ascorbic acid was used as a positive antioxidant reference. As for the Trolox equivalent antioxidant capacity (TEAC) assay, the TEAC of samples 9

was tested using the ABTS·+ radical, which was generated from the oxidation of ABTS using potassium persulphate at 25 oC in the dark (Re et al., 1999). The sample solution (0.1 mL) was mixed with 3.9 mL diluted ABTS·+ solution at 25 oC for 20 min. The absorbance of the mixture was measured at 734 nm, and the ABTS·+ scavenging activity (in %) was obtained using the following equation:  A Scavenging activity (%) = 1 100%  A0 

where A and A0 represent the absorbance values of ABTS·+ solution with and without the tested samples. The TEAC values were derived from the calibration curve obtained using Trolox (0-30 μM). The ferric reducing ability of plasma (FRAP) assay measured the reducing capacity of a compound to transform ferric tripyridyltriazine complex into ferrous tripyridyltriazine at low pH (Benzie and Strain, 1996). The FRAP reagent was freshly prepared using 3 M sodium acetate buffer (pH 3.6), 0.1 M 2,4,6-tris (2-pyridyl)-s-triazine in 0.4 M HCl, and 0.2 M ferric chloride hexahydrate FeCl3·6 (H2O) at 10:1:1 volume ratio. The FRAP reagent was warmed up to 37 °C, and each sample (905 μL) was added to 95 μL of this reagent. The mixture was incubated at 25 oC for 15 min, and the absorbance was measured at 593 nm and was converted to a FRAP value (μmol Fe2+/g sample) using a calibration curve with ferrous sulfate with concentrations ranging from 0 to 30 μM.

2.5. Cell culture and cytotoxicity assay Human lung adenocarcinoma cell lines (SPCA-1) and human cervical cell lines (HeLa) were used. All cell lines were obtained from the Cell Resource Center of Shanghai Institute of 10

Life Sciences, Chinese Academy of Sciences (Shanghai, China). SPCA-1 cells were cultured in DMEM supplemented with FBS (100 μg/mL), penicillin (100 units/mL), and streptomycin sulfate (100 μg/mL) at 37 °C and 5% CO2 in an incubator with a humidified atmosphere. HeLa cells were cultured in MEM supplemented with FBS (100 μg/mL), sodium pyruvate (0.11 g/L), penicillin (100 units/mL), and streptomycin sulfate (100 μg/mL) at 37 °C and 5% CO2 in an incubator with a humidified atmosphere. SPCA-1 and HeLa cells were harvested in the exponential phase of growth. The AB assay was used to determine the cytotoxicity of the PSP-SeNP (Se/PSP=1:2). In brief, 100 μL of SPCA-1 and HeLa cells were separately seeded in 96-well plates at 4×104 cells/mL and cultured overnight. Then, each cell line was treated using different concentrations of PSP-SeNP (50, 100, and 200 μg/mL) for 72 h. After treatment, the medium was aspirated and 200 μL of fresh phenol-red-free medium containing 5 μg/mL AB was added into the wells. After incubation at 37 °C for 4 – 6 h, when the medium color changed, the absorbance at 570 and 600 nm was measured using a spectrophotometric plate reader (Bio-Tek Instruments, Inc, Winooski, VT, USA). AB assays were done in three replicates for each experiment.

2.6. Statistical analysis All experiments are done in three replicates and the mean ± standard deviation (SD) was used. The statistical analysis was done using analysis of variance (ANOVA) using OriginPro Software Version 8.0 (OriginLab Corp., Northampton, MA, USA). A least significant different test with a significance level of p < 0.05

using triplicate measurements of a given

sample. 11

3. Results and discussion 3.1. Preparation and formation of PSP-SeNP Many reactive amino, hydroxyl, or carboxyl groups in the molecular structure of PS had a role in the formation, stabilization, and growth of SeNP. In addition, the microscopic phase of PS was not a pure solution and significantly affected particle nucleation and growth (Zhang et al., 2004). Water-dispersible and stable SeNP using PSP as a stabilizer and capping agent of SeNP in the simple redox system of selenite and ascorbic acid were prepared. As shown in Fig. 1, a given volume of Na2SeO3 solution (0.1 M) was first added into the PSP aqueous solution (1.0 mg/mL), well mixed, and then a certain amount of ascorbic acid was added to reduce the precursor SeO32− to Se0 atoms in the PSP molecular microenvironment. During synthesis, the increasing Se atoms aggregated into Se nuclei and immediately grew as the redox reaction progressed, further leading to the formation of SeNP. Figs. 2(a) and 2(b) show the digital photographs of the SeNP aqueous solutions in the presence of PSP at 5 different Se/PSP ratios and the control in the absence of PSP for 0 and 7 days. The freshly-prepared SeNP without PSP were orange, which showed the formation of SeNP (Zhang et al., 2004). However, after 7 days, the control SeNP completely aggregated into clusters and precipitated at the bottom of vial, showing a brown yellow color. These aggregations and precipitations could be attributed to the high surface energy of SeNP (Xiao et al., 2017). As shown in Fig. 2(a), in the presence of PSP, the color of the reaction solution slowly turned from light yellow to orange and then turned orange-red with increasing Se/PSP ratios from 1:10 to 1:2 (w/w). The characteristic orange-red color showed the occurrence of 12

either amorphous or monoclinic SeNP (Zhang et al., 2010). However, when the Se/PSP ratios reached 1:1 and 4:3, the reaction solution had a light color and finally showed a pale yellow. These results suggested that the Se/PSP ratios significantly influenced the formation of SeNP. An excessive amount of PSP in the redox system may be unsuitable for the preparation of SeNP. Thus, the optimum Se/PSP mass ratio was 1:2 (w/w). After storage for 7 days, the PSP-SeNP were well dispersed and showed a homogeneous and stable orange-red color, especially at Se/PSP ratio of 1:2, compared with the control SeNP (Fig. 2b). With Se/PSP ratios lower than 1:5, the PSP-SeNP showed some aggregation or precipitation at the bottom of vial. These results suggested that the presence of PSP in appropriate amounts improved the stability of SeNP. This effect could be attributed to the large number of functional groups, such as hydroxyl and amino groups, in the PSP molecules. Similar results were also found in previous studies (Chen et al., 2015; Xiao et al., 2017; Zhang et al., 2010). UV–Vis spectral analysis was done to investigate the changes in absorption spectra of the PSP-SeNP. During SeNP synthesis, the color of the solution turned from colorless to orange or orange-red, suggesting that the maximum absorption spectrum in the range of 200–400 nm (Liu et al., 2012). Fig. 2(c) shows the UV–Vis absorption spectra of the freshly-prepared PSP–SeNP solutions at 5 different Se/PSP ratios (1:10, 1:5, 1:2, 1:1, and 4:3) as compared with those of the control SeNP. The SeNP showed the maximum absorption peak at about 265 nm, which corresponded to the localized surface plasmon resonance (SPR) of the SeNP (Chen et al., 2015). The maximum absorption peak of the SeNP after stabilization with PSP led to no significant change in the SeNP. In addition, a red shift (~53 nm) occurred for the SeNP relative to that of the reported SeNP (~212 nm) (Shen et al., 2008). 13

This change occurred because the selected preparation method for SeNP significantly influenced the material microstructure and physical properties, and the maximum absorption band of the SeNP in the UV-Vis spectrum was related to its crystallizability (An et al., 2003). The intensity of the absorption peak of the SeNP gradually increased after exposure to the PSP with the increasing Se/PSP ratios from 1:10 to 1:2 (w/w), which showed the increase in SeNP concentration. However, when the Se/PSP ratio exceeded 1:2, the intensity of the maximum absorption peak at ~265 nm decreased with the increase in Se/PSP ratios from 1:1 to 4:3, and the band width also widened. These results suggested that PSP could not sufficiently prevent SeNP from aggregation and even precipitation. Hence, the Se/PSP ratio of 1:2 was considered as the most suitable for the stabilization and dispersion of SeNP in the presence of PSP.

3.2. Morphological and structural characterizations of PSP-SeNP HR-TEM was a reliable technique used to analyze the size and surface morphology of the SeNP (Kong et al., 2014; Zhang et al., 2010). Fig. 3 shows the typical HR-TEM images of the SeNP in the presence of PSP at 5 different Se/PSP ratios as compared with those of the control SeNP. As shown in Fig. 3(a–e), varying the Se/PSP ratios could significantly affect the size and morphology of the PSP–SeNP. The majority of the PSP–SeNP were between 40 and 70 nm as compared with the control SeNP (Fig. 3f), and the size of the PSP–SeNP diminished with the increasing Se/PSP ratios from 1:10 to 1:2. In particular, the PSP–SeNP at the Se/PSP ratio of 1:2 showed a well-monodispersed, homogeneous spherical structure with an average size of ~43 nm, suggesting that the PSP could disperse and stabilize SeNP. 14

According to previous reports, the size of PSP–SeNP (~43 nm) was almost similar to those of SeNP prepared from chitosan (~50 nm) (Chen et al., 2015), dextran (~36 nm) (Shen et al., 2008), expolysaccharides from Cordyceps sinensis (~50 nm) (Xiao et al., 2017), and PSP from Polyporus rhinoceros (~50 nm) (Wu et al., 2013). The shape always remained sphere-like in all reports. When the Se/PSP ratio exceeded 1:2, the amount of PSP was less than the requirement for the dispersion and stabilization of SeNP, and its function as a surface decorator was weak. Hence, a small amount of nonhomogeneous spherical PSP–SeNP was obtained, and partial aggregation was observed (Figs. 3d and 3e). Fig. 3f shows that the control SeNP aggregated to a larger cluster in the absence of PSP, further suggesting that PSP has an important role in dispersing and stabilizing SeNP. The monodisperse, homogeneous spherical PSP–SeNP (~43 nm) were obtained using addition of PSP to the redox system of sodium selenite and ascorbic acid at the Se/PSP ratio of 1:2. HR-TEM results were in good agreement with that of UV-Vis spectral analysis. Fig. 4(a) shows the intensity distribution profiles of particle sizes for the PSP–SeNP at different Se/PSP ratios. The SeNP prepared in the presence of PSP showed one modal peak and relatively narrow size distributions. The PSP–SeNP with Se/PSP ratio of 1:2 had the minimum particle size (~104 nm), which was consistent with the results of HR-TEM observation. Compared with the TEM results shown in Fig. 3, the particle size measured using DLS was larger than that using TEM. This difference occurred because the size measured using DLS was a hydrodynamic diameter, whereas the particle size determined using TEM was an actual diameter after drying (Zhang et al., 2010). In addition, the PDI values for the PSP–SeNP at different Se/PSP ratios of 1:10, 1:5, 1:2, 1:1, and 4:3 were 0.212, 15

0.191, 0.216, 0.210, and 0.228, respectively, suggesting that these PSP–SeNP were satisfactorily stabilized in aqueous solutions. Zeta potential was used to evaluate the actual status of NP and their stability in the solution (Molina et al., 2011). Fig. 4(b) shows the zeta potential of the PSP–SeNP at different Se/PSP ratios. All PSP–SeNP showed negative zeta potential (p < 0.05). In particular, the PSP–SeNP with Se/PSP (w/w) ratio of 1:2 showed the largest negative zeta potential (–17.7 mV) among other PSP–SeNP, suggesting that the greater stability of PSP–SeNP (Se/PSP = 1:2) in an aqueous medium. According to a previous report (Patil et al., 2007), negative zeta potential was beneficial for the NP uptake in the A549 cells. Collectively, these results suggested that the negatively charged PSP decorated on the surface of SeNP, and the PSP–SeNP were electrostatically stabilized using the negatively charged PSP shells, thereby providing good stability in the aqueous solution. XRD analysis was done to determine and confirm the crystalline nature of PSP–SeNP. Fig. 4(c) shows a typical XRD pattern of the control SeNP and the PSP–SeNP at the Se/PSP ratio of 1:2. For the control SeNP, the two characteristic diffraction peaks at 2θ corresponding to ~24 and 30°respectively, confirming the presence of crystalline Se. These results were in agreement with the database of the Joint Committee on Powder Diffraction Standard (JCPDS file No. 06-362, http://jcpds.crystalstar.org/). However, the two typical peaks disappeared after exposure to PSP as compared with those of the control SeNP, suggesting that the PSP–SeNP was amorphous. This change occurred because as a soft template, the PSP macromolecules were decorated on the surface of the SeNP and destroyed the crystallinity of Se, thus leading to the occurrence of amorphous SeNP. Similar results were observed in previous reports (Jia et al., 2015; Xiao et al., 2017). EDX was used to investigate the elemental composition and distribution of the PSP–SeNP at the Se/PSP ratio of 1:2, and the 16

results are shown in Fig. 4(d). The strong signal at approximately 1.5 keV in the EDX spectrum was assigned to Se Kα, and the atomic percentage value for Se was 7.05%, thereby confirming the presence of SeNP. Several other peaks of C, O, and Na were found in the EDX spectrum and may be attributed to the PSP molecules that could be involved in stabilizing and capping of SeNP. XRD and EDX analyses also confirmed the composition and crystallinity of the PSP–SeNP at the Se/PSP ratio of 1:2.

3.3. Interaction mechanism of SeNP and PSP FT-IR analysis was done to characterize the interaction between PSP and SeNP. Fig. 5 shows the FT-IR spectra of PSP and the PSP–SeNP at the Se/PSP ratio of 1:2. In the FT-IR spectrum of PSP, the absorption peaks were at 3380, 2910, 1643 (amide I), and 1541 (amide II) cm−1, which corresponded to the characteristic stretching vibrations of O-H (or N-H) and C-H groups of the polysaccharides and the secondary –CO-NH- group of the proteins in PSP molecules (Wang et al., 2017). The PSP–SeNP showed an FT-IR spectrum similar to that of PSP. The absorption peaks shifted from 3380 and 1543 cm−1 to 3328 and 1530 cm−1, respectively, suggesting an interaction between O-H and -CO-NH- groups of PSP and Se atoms (Chen et al., 2015). Basing on these findings, it could be concluded that the main interaction between PSP and SeNP occurred in the Se-O and Se-N bonds, further leading to the stable spherical structure of PSP-decorated SeNP. In a previous study (Wang et al., 2017), the PSP isolated from C. fluminea using the TPP technique combined with dialysis was an acidic proteoglycan, which was attributed to the presence of acidic amino acids, such as aspartic acid and glutamic acid. The large number of hydroxyl and amino groups in the molecular structure of PSP might be responsible for 17

dispersing and stabilizing SeNP in an aqueous medium. Therefore, basing on the above findings, a possible mechanism underlying the formation of SeNP in the presence of PSP was proposed (Fig. 1). In the preparation of PSP–SeNP, the precursor SeO32− was first dispersed in the aqueous PSP microenvironment. SeO32− was reduced to the element Se0 using ascorbic acid in situ, and the number of Se0 continually increased with the redox reaction and then further grew into SeNP. PSP was a negatively charged macromolecule having a number of hydroxyl and amino groups in the solution, leading to a microenvironment with good suspension, emulsification, and stabilization. Then, the SeNP were adsorbed and capped with the PSP molecules in situ using the interaction of Se-O and Se-N bonds between PSP and SeNP (Fig. 5). This change prevented NP from coalescing and agglomerating with each other, slowed down and controlled the growth of the NP, leading to a good water-dispersible and stable SeNP. As a result, the negatively charged PSP on the surface of Se gave SeNP capping and stability. A similar mechanism for the construction of PS-stabilized SeNP was also predicted in previous studies (Han et al., 2012; Jia et al., 2015; Xiao et al., 2017; Zhang et al., 2010).

3.4. Antioxidant activities of PSP-SeNP in vitro According to recent reports, SeNP showed excellent free radical scavenging capacities and antioxidant activities in vitro and in vivo (Huang et al., 2003; Torres et al., 2012; Xiao et al., 2017; Zhang et al., 2004). The size of the SeNP had an important role in their antioxidant property, that is, small SeNP had stronger antioxidant activity (Torres et al., 2012). For example, Peng et al. (2007) found that the size of the SeNP significantly affected their biological activity; as expected, 5 – 200 nm SeNP could effectively scavenge free radicals in 18

vitro. In the current study, therefore, the PSP-SeNP (~40 – 70 nm, from HR-TEM observation) increased the antioxidant activity of SeNP. DPPH radical was conventionally used as a stable free radical to assess the free radical scavenging ability of antioxidants and could be effectively scavenged with antioxidants using the donation of hydrogen to form a stable DPPH molecule (Matthaus, 2002). Fig. 6(a) shows the scavenging ability of PSP and PSP–SeNP at different ratios on DPPH radical compared with the control SeNP and ascorbic acid as a positive reference. All PSP–SeNP showed strong DPPH radical scavenging ability in a dose-dependent manner at 0.1 – 1.5 mg/mL. At the concentration of 1.5 mg/mL, the DPPH radical scavenging ability of PSP–SeNP at 5 different ratios were 70, 77, 83, 79, and 53%, respectively. Among the complexes, the PSP–SeNP at the Se/PSP ratio of 1:2 showed the strongest DPPH radical scavenging ability, which was markedly stronger than that of PSP (~35%, 1.5 mg/mL) and control SeNP (~11%, 1.5 mg/mL) but weaker than that of ascorbic acid (~96%, 1.0 mg/mL). These results may be due to the smallest size of the PSP–SeNP at the Se/PSP ratio of 1:2 as compared with other SeNP. Kong et al. (2014) found that the DPPH radical scavenging activity of gum arabic-decorated SeNP was ~85% at 4.0 mg/mL, which was lower than that of the PSP–SeNP at the Se/PSP ratio of 1:2. Chen et al. (2015) reported that the scavenging ability of CS–SeNP on DPPH radical reached ~93% at a concentration of 0.6 mmol/L. In the TEAC and FRAP assays, the PSP, PSP–SeNP at different Se/PSP ratios, and the control SeNP total antioxidant capacity was measured [Figs. 6b and 6c]. With the increasing Se/PSP ratio from 1:10 to 4:3, both TEAC and FRAP values of the PSP–SeNP initially increased and then decreased, and the PSP–SeNP at the Se/PSP ratio of 1:2 showed the highest antioxidant capacity with the TEAC value of

226 μmol Trolox/g sample and the

FRAP value of 150 μmol Fe2+/g sample. In particular, the PSP–SeNP at the Se/PSP ratio of 1:2 with the smallest particle size showed higher TEAC and FRAP values than that of PSP 19

(~104 μmol Trolox/g sample, and ~45 μmol Fe2+/g sample, respectively) and the control SeNP (~17 μmol Trolox/g sample, and ~25 μmol Fe2+/g sample, respectively), showing a similar result as that of DPPH radical scavenging. Thus, from the above results, the SeNP in the presence of PSP, especially at the Se/PSP ratio of 1:2, could increase the antioxidant properties of the SeNP, which was in good agreement with the previous reports (Chen et al., 2015; Torres et al., 2012; Xiao et al., 2017).

3.5. Cytotoxicity assay in vitro The PSP–SeNP with the Se/PSP ratio of 1:2 and the smallest particle size was selected to evaluate in vitro cytotoxicity using the AB assay against SPCA-1 and HeLa cell lines. As shown in Fig. 7, the effect of PSP–SeNP at the Se/PSP ratio of 1:2 on cell viability of SPCA-1 and HeLa cells showed a dose-dependent decrease with the concentration of PSP–SeNP ranging from 50 – 200 μg/mL. Moreover, cell viability remained ~75% at or above the test concentration. These results suggested that the PSP–SeNP showed relatively low cytotoxic activity in vitro against the two cancer cell lines, which was in line with a previous study (Luesakul et al., 2016). Similarly, Zhai et al. (2017) found that SeNP, stabilized using CS with different MW showed relatively lower cytotoxicity on BABLC-3T3 and Caco-2 cells. Moreover, Forootanfar et al. (2014) confirmed the lower cytotoxicity of the biogenic SeNP on the MCF-7 cell line as compared with selenium dioxide.

4. Conclusions The PSP isolated from C. fluminea can be used as a stabilizer and capping agent for the preparation of SeNP in aqueous solutions. These results showed that the Se/PSP ratio had significant effects on the size, morphology, structure, and stability, as well as antioxidant properties of the SeNP in the presence of PSP, and the PSP had an important role in 20

improving the stability and antioxidant activity of SeNP. In particular, at the Se/PSP ratio of 1:2 (w/w), the PSP–SeNP showed homogeneous and monodisperse spherical structures with an average particle size of ~43 nm. Moreover, the PSP–SeNP showed strong antioxidant activities and low cytotoxicity against SPCA-1 and HeLa cells in vitro. Therefore, it could be concluded that the PSP–SeNP having stronger antioxidant capacities with low cytotoxicity can be explored as potential diet nutritional supplement for applications in the food and medical fields. Further studies on antioxidant activity in vivo and its mechanism of action are underway.

Conflict of Interest The authors confirm that they have no conflicts of interest with respect to the work described in this manuscript.

Acknowledgements This study was supported financially by the National Natural Science Foundation of China (31671812), the Natural Science Foundations of Jiangsu Province (BK20140542, BK20150501), the Jiangsu Overseas Research and Training Program for University Prominent Young and the Middle-aged Teachers and Presidents and the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions.

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Figure Legends:

Fig. 1. Detailed experimental procedure and possible mechanism of PSP-SeNP. Fig. 2. Photographs of the SeNP aqueous solutions in the presence of PSP at different Se/PSP ratios (1:10, 1:5, 1:2, 1:1, and 4:3) and the control SeNP in the absence of PSP for 0 (a) and 7 days (b); (c) UV–Vis absorption spectra of the PSP–SeNP solutions at different Se/PSP ratios (1:10, 1:5, 1:2, 1:1, and 4:3). Fig. 3. TEM images of PSP–SeNP at different Se/PSP ratios (a) 1:10, (b) 1:5, (c) 1:2, (d) 1:1, (e) 4:3, and (f) the control SeNP. Fig. 4. (a) The intensity distributions of particle sizes for the PSP–SeNP at different Se/PSP ratios; (b) zeta potentials of PSP–SeNP at different Se/PSP ratios; (c) typical XRD patterns of the control SeNP and PSP–SeNP at the Se/PSP ratio of 1:2; (d) Typical EDX spectra of PSP-SeNP at the Se/PSP ratio of 1:2 from SEM. Bars with different letters are statistically different (p < 0.05). Fig. 5. FT-IR spectra of (a) PSP and (b) PSP–SeNP at the Se/PSP ratio of 1:2. Fig. 6. Antioxidant activities of the control SeNP, PSP and PSP–SeNP at different Se/PSP ratios by scavenging ability on (a) DPPH, (b) TEAC assay, and (c) FRAP assay. Each value is expressed as a mean ± SD (n=3). Bars with different letters are statistically different (p < 0.05). Fig. 7. Effect of PSP–SeNP at the Se/PSP ratio of 1:2 on cell viability of SPCA-1 and HeLa cells determined by AB assay. Each value is expressed as a mean ± SD (n=3). Bars with different letters are statistically different (p < 0.05). 28

Fig. 1 Yan et al.

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Fig. 5 Yan et al.

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Fig. 7 Yan et al.

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Graphical Abstract A given volume of Na2SeO3 solution was first added into an aqueous polysaccharide–protein complex (PSP) solution, well mixed, and then various amounts of acscorbic acid was added to reduce the precursor SeO32− to Se0 atoms. During synthesis, the Se atoms increased and aggregated into Se nuclei and immediately grew as the redox reaction progressed, further leading to the formation of SeNP.

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Highlights ◆ PSP from C. fluminea was used as a stabilizer and disperser for the formation of SeNP. ◆ Se/PSP ratio significantly affects physicochemical and antioxidant properties of SeNP. ◆ PSP-SeNP (Se/PSP=1:2) with minimum particle size (~43 nm) showed good stability. ◆ PSP–SeNP (Se/PSP=1:2) showed strong antioxidant activities and low cytotoxicity.

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