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Phycocyanin-functionalized selenium nanoparticles reverse palmitic acid-induced pancreatic beta cells apoptosis by enhancing cellular uptake and blocking ROS-mediated mitochondria dysfunction Chang Liu, Yuanting Fu, Chang-e Li, Tianfeng Chen, and Xiaoling Li J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 16 May 2017 Downloaded from http://pubs.acs.org on May 16, 2017

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Journal of Agricultural and Food Chemistry

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Phycocyanin-functionalized selenium nanoparticles reverse palmitic

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acid-induced pancreatic beta cells apoptosis by enhancing cellular uptake and

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blocking ROS-mediated mitochondria dysfunction

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Chang Liua#, Yuanting Fua#, Chang-e Lia, Tianfeng Chen a*, Xiaoling Li b*

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a

Department of Chemistry, Jinan University, Guangzhou, China

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b

Institute of Food Safety and Nutrition, Jinan University, Guangzhou, China

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* Corresponding author. Jinan University, Guangzhou 510632, China. Tel.: +86 20

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85227082. E-mail addresses: [email protected] (X LI). [email protected] (T Chen)

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ACS Paragon Plus Environment


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ABSTRACT: Accumulation of palmitic acid (PA) in human bodies could cause

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damage to pancreatic beta cells and lead to chronic diseases by generation of reactive

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oxygen species (ROS). Therefore, it is of great significance to search for nutrition

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available agents with antioxidant activity to protect pancreatic islet cells against

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PA-induced damage. Phycocyanin (PC) and selenium (Se) have been reported to have

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excellent antioxidant activity. In this study, PC-functionalized selenium nanoparticles

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(PC-SeNPs) were synthesized to investigate the in vitro protective effects on INS-1E

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rat insulinoma beta cells against PA-induced cell death. Potent protective effect was

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achieved by regulation of particle size and PC content. Among three PC-SeNPs (165

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nm, 235 nm and 371 nm), PC-SeNPs-235 nm showed the highest cellular uptake and

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the best protective activities. For cell cycle analysis, PC-SeNPs showed better

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protective effect on PA-induced INS-1E cells apoptosis than PC or SeNPs, and

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PC-SeNPs-235 nm exhibited the best effect. Further mechanistic studies demonstrated

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that PA induced overproduction of intracellular reactive oxygen species (ROS),

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mitochondria fragmentation, activation of caspase-3/-8/-9 and cleavage of PARP.

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However, pre-treatment of the cells with of PC-SeNPs effectively blocked these

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intracellular events, which suggest that PC-SeNPs could protect INS-1E cells against

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PA-induced cell apoptosis via attenuating oxidative stress and downstream signalling

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pathways. This finding provides a great promising nutritional approach to protect

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diseases related with islet damage.

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KEYWORDS: PC-functionalized selenium nanoparticles (PC-SeNPs), Oxidative


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stress, Plamitic acid (PA), Antioxidant activities

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INTRODUCTION

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Obesity and hyperglycemia have been major problems exercising the minds of

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modern people around the world and they were reported have relate to the damage of

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islet beta cells (β cells)1,2. Many researchers have reported that the damage of β cells

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is usually accompanied by the increasing production of reactive oxygen species (ROS)

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and impaired antioxidant defenses3,4. Moreover, overproduction of ROS and reactive

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nitrogen species (RNS) result in oxidative stress, which is a deleterious process that

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can be important mediator of damage to cell structures5,6. The levels of antioxidant

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enzymes in islet β cell was low, thus oxidative stress is the core sensitivity of

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diabetes7,8. Therefore the β cells have a long-term chance of survival with the reduced

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ROS level. Palmitic acid (PA), a saturated fatty acid, can cause the dysfunction of

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pancreatic or isolated islets by generation of ROS9.

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Phycocyanin (PC), a natural blue photosynthetic pigment purified from Spirulina, is

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a highly active natural antioxidant, which could significantly activate SOD, GSH-PX

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activity, increase the GSH content and enhance the intracellular antioxidant

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capacity10-12. PC also shows good therapeutic values, such as antioxidant,

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immunomodulation, anti-cancer, anti-inflammatory, blood vessel-relaxing and blood

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lipid-lowering activities and so on11,13-16. Despite the widespread use of PC, there are

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also some limitations in the application of PC for its instability, poor solubility and

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poor penetrability into cells. Beside, PC is sensitive to moisture, light, temperature



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and pH due to the degradation of the protein fraction15,17. Many studies have reported

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that the modification of the protein conformation itself can improve the stability of

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proteins18,19. Our previous studies have indicated that selenium-containing

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allophycocyanin had hepatoprotective effect against the apoptosis induced by

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t-BOOH20.

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Selenium (Se) is one of the essential trace minerals in human and animals, which

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attracts increasing interest both in pharmaceutical and food industry in recent

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years21-23. Studies have identified that sodium selenite could improve glucose

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homeostasis in type 1 and type 2 diabetic animals24,25. Previous studies also proved

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that Se-PC could inhibit human islet amyloid polypeptide (hIAPP) fibrillation,

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suppress the generation of ROS, and thus show protective effect on hIAPP-mediated

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cell apoptosis and this effect was achieved by attenuating oxidative stress and

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mitochondrial dysfunction26-28. Human erythrocytes could be protected by

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selenium-containing allophycocyania from AAPH-induced oxidative damage through

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inhibition of ROS generation29.

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In the past few decades, emerging studies have indicated the potential application

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of SeNPs and PC in food industry and pharmaceutical industry. However, little

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information about the combination usage of PC and SeNPs was available in protecting

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islet beta cells against diabetes. Therefore, it is of great interested to investigate

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whether there was synergistic action between PC and SeNPs in protecting diseases

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related with islet damage. In this present study, PC-SeNPs with different sizes were

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constructed by selenite/GSH chemical reduction method to improve the antioxidant of


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SeNPs. INS-1E rat insulinoma cell line was selected as the cell model to evaluate the

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in vitro protective effects of PC- SeNPs against PA-induced cell damage (Scheme 1).

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Particle size, PC content, cellular uptake, antioxidant activities, caspase activities,

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ROS generation, mitochondria fragmentation and western blot were evaluated in this

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study. The results showed that PC functionalization could enhance the protective

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effect of SeNPs against PA-induced apoptosis through decreasing oxidative stress.

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This finding demonstrates an effective nutritional approach to protect diseases related

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with islet damage.

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MATERIALS AND METHODS

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Materials. Palmitic acid (PA), Propidium iodide (PI), thiazolyl blue tetrazolium

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bromide

(MTT),

glutathione

(GSH),

4’,6-diamidino-2-phenylindole

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bicinchoninic acid (BCA), sodium selenite and all other chemicals were bought from

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Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO). fetal bovine serum (FBS),

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RPMI-1640 medium and the antibiotic mixture (penicillin-streptomycin) were

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purchased from Invitrogen (Carlsbad, CA). Antibody cleaved caspase-3, Caspase-3,

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Caspase-8, Caspase-9 and cleaved caspase-9 were purchased from Cell Signaling

106

Technology (Beverly, MA). Caspase-3, caspase-8 and caspase-9 substrate were

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obtained from Biomol (Germany).

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Synthesis of PC-SeNPs. PC-SeNPs were prepared by the selenite/GSH chemical

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reduction approach as reported30. Briefly, 0.5 mL of PC with a serious concentration

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(5, 25, 50, 100, 150 mg/L) was mixed with 0.5mL of Na2SeO3 (100 mM), then 2 mL


(DAPI),


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of GSH (100 mM) was drop-wise added into the mixture under stirring. After that,

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deionized water was added to the mixture until the volume was 10 mL. 24 h later, the

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mixture was dialyzed for 72 h in water and lyophilized for using.

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Characterization of PC-SeNPs. The particle size distribution and zeta potential of

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PC-SeNPs in aqueous solution was measured by Malvern Zetasizer Nano ZS

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(Malvern Instruments Limited, Columbia, USA). The sizes and morphologies of SeNPs

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and PC-SeNPs in the dry state were observed by transmission electron (TEM). FT-IR

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(Equinox 55 IR spectrometer, Thermo Fisher, Waltham, USA) and UV-vis (Carry 5000

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spectrophotometer, Palo Alto, USA) were applied to confirm the chemical composition

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of nanoparticles.

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Stability Assay. PC-SeNPs with the PC added concentration of 25, 50 and 100 mg/L

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were selected to evaluate the stability of PC-SeNPs. At 37 ℃, particle size of

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PC-SeNPs in PBS or DMEM were measured by Malvern Zetasizer Nano at

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determined time intervals, respectively.

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ABTS·+ Free Radical Scavenging Activity. The antioxidant activities of PC and

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PC-SeNPs were measured by ABTS+ free radical scavenging assay as previously

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described10. Briefly, the tested samples (50 µL) were mixed with ABTS+ reagent (1

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mL) with absorbance of 0.70 ± 0.02 at 734 nm. After mixing for 6 min, the

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absorbance of sample was measured.

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Cellular Uptake Assay. Quantitative analysis of cellular uptake of PC-SeNPs was

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carried out by testing the absorption of Se at determined time points. Briefly, INS-1E

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cells (1.0 × 105 cells /well) were plated in 6-well plates for 24 h, then the culture


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medium was replaced by DMEM medium without phenol red. 2 h later, different

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PC-SeNPs were added to incubate with cells. At 0, 0.5, 1, 2, 3, 4, 6, 8, 10 and 12 h,

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cells were collected and the content of Se in cells was measured by ICP-MS,

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respectively.

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Cell Culture and MTT Assay. INS-1E rat insulinoma cell line was bought from

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American Type Culture Collection (ATCC, Manassas, VA, USA). With fetal bovine

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serum (FBS, 10 %), L-glutamine (2 mM), sodium pyruvate (1 mM), HEPES (10 mM),

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mercaptoethanol (50 µM), penicillin (100 units/mL) and streptomycin (100 µg/ mL),

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the cells were cultured in RPMI-1640 medium at 37 °C in humidified atmosphere (5%

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CO2). Cell viabilities were measured by using MTT assay as described previously31.

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Briefly, INS-1E cells (6 × 104 cells/well) were seeded in 96-well plate at 37 °C for 24

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h. Firstly, the cell cytotoxicity of PC-SeNPs or PA alone was carried out. For

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determine the protective effect of PC, SeNPs and PC-SeNPs on INS-1E cells, the cells

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were pre-incubated with PC, SeNPs and PC-SeNPs for 12 h, and then cells were

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treated with PA, respectively (referred to PC + PA, SeNPs + PA and PC-SeNPs + PA

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in the following description, respectively). After 48 h, the medium was removed and

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MTT reagent was added. At 570 nm, the absorbance was determined by a microplate

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reader (Spectra Max M5, Bio-Tek, Winooski, USA).

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Cell Cycle Analysis. The cell cycle analysis was performed as described previously32.

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The INS-1E cells were treated with different concentration of PC-SeNPs-235 nm for

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12 h and then treated with PA for 2h. At 4 °C, using PBS solution containing 70 %

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cold ethanol, the cells were washed, suspended and fixed for 24 h. Protected from



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light, the cells were then incubated with PI/RNase staining solution (Cell Signaling)

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for 30 min afterwards removing the fixation solution. The staining solution was

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removed by washing 3 times with PBS, and then the cell cycle was analyzed using

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flow cytometry (Beckman Coulter, Miami, FL, 1×106 cells/mL) with cells untreated

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drugs as negative control. Similarly, for determining the protective effect of

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PC-SeNPs, INS-1E cells were pre-incubated with different PC-SeNPs, PC and SeNPs

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for 12 h, and then cells were treated with PA for 2 h.

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Caspase Activity Assay and Western blot analysis. Caspase activity was evaluated

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by meaning of fluorescence intensity by using specific caspase-3, -8 and -9 substrates

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as previous reported33. Briefly, after treatment with PA alone or PC + PA, SeNPs +

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SeNPs, and different size of PC-SeNPs + PA, collecting the cells by centrifugation

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and suspending them with cell lysis buffer. The cell proteins were collecting by

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centrifugation (12000 g, 30 min). Finally, to determine the fluorescence intensity of

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cell lysates, Total cell lysates (100 µg/well) were placed in 96-well plates and then

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specific caspase-3, -8 and -9 substrates were added and incubated at 37℃ for 2 h in

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darkness. Finally, the fluorescence intensity was measured by using a microplate

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reader (Spectra Max M5, Bio-Tek, Winooski, USA) with ex/em wavelengths of

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380/460 nm. The effects of PA, PC, SeNPs and PC-SeNPs on expression levels of the

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protein related to the apoptosis effects were determined by western blot analysis34.

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Measurement of ROS Generation. In order to evaluate ROS accumulation in

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INS-1E cells, the effects of PA and PC-SeNPs + PA on intracellular ROS generation in

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INS-1E cells were detected by DCFH-DA assay and DHE assay34,35. Briefly, INS-1E


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cells were seeded in 96-well plates (1×106 cells/mL). After pre-incubated with 0.8 µM

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PC-SeNPs for 12 h, PA was added into plates and incubated for 2 h. Then, incubating

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the cells with H2DCF-DA (10 µM) or DHE (100 µM) for 30 min at 37 °C. By using a

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microplate reader (Spectra Max M5), the ROS level was determined at 488/525 nm

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for DCF and 300/600 nm for DHE, respectively.

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Fragmentation Analysis. Mitochondrial fragmentation analysis was carried out as

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reported. INS-1E cells were pre-incubated with different PC-SeNPs (0.8 µM) for 12 h,

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then 0.4 mM of PA was added. 2 h later, mitochondria and nucleuses of the cells were

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stained with Mito Tracker Red CMXRos (50 nM, 2 h) and DAPI (1 µg/mL, 20 min),

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respectively. After that, the cells were washed three times with PBS and re-cultured in

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fresh medium. Then the cells were photographed under a monochromatic Cool

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SNAPFX camera (Roper Scientific, New Jersey, USA).

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Western blot analysis. Statistical Analysis. All samples were carried out at least

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three times and data were presented as mean ± SD. Two-tailed Student’s t-test was

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performed for the comparison among the different groups. Statistical significance was

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defined as * p < 0.05.

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RESULTS AND DISCUSSION

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Preparation, Characterization and Stability of PC-SeNPs. Different concentration

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of PC modified SeNPs (PC-SeNPs) were prepared under a simple redox system of

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sodium selenite and glutathione (GSH) in this study. The sizes and morphologies of



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PC-SeNPs were characterized by transmission electron microscope (TEM) and

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Zetasizer Nano ZS particle analyzer. As shown in Figure 1A, SeNPs without PC were

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unstable and accumulated into clumps in aqueous solution. At the concentration of 5

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mg/mL, there also were large gathering of PC-SeNPs. With the increase of

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concentration of PC (25, 50, 100 and 150 mg/L), the nanoparticles had uniform

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spherical shapes and homogeneous particle sizes, which were 165, 235, 371 and 815

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nm, respectively. With the increasing concentration of PC, the size of PC-SeNPs

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became larger. PC-SeNPs with the PC concentration of 25, 50 and 100 mg/mL

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showed better disperse and morphology, so the three PC-SeNPs were chosen to

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evaluate the antioxidant activity. The particle size measured by Malvern Zetasizer

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Nano ZS showed similar tendency with the results of TEM (Figure 1B). The zeta

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potential was increased with the increasing PC content on the surface of SeNPs in a

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certain range (Figure 1C).

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Considering the dimensions of PC

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, ≈10.2 nm × ≈10.2 nm × ≈10.9 nm, the

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thickness of PC layer was used to estimate the number of molecules present in the

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shell of the SeNPs by using the equation as below37:

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Where N is the number of PC molecules per SeNPs; VPC is the PC’s volume

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(3207.2 nm3)38; rSeNPs + PC shell and rSeNPs are the radius of the SeNPs and PC-SeNPs

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respectively.

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As the values obtained from TEM, rSeNPs, rpc-SeNPs-a, rpc-SeNPs-b and rpc-SeNPs-c

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were about 63, 82.5, 117.5 and 185.5 nm, respectively. Thus, the molecules of PC


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layer were 421.9, 1806.5 and 8021.3 for PC-SeNPs with particle size of 165, 235 and

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371 nm, respectively. Furthermore, the content of PC on the surface of PC-SeNPs

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were evaluated by BSA method and the results were shown in Figure 1D. With the

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increasing PC concentration, the PC content on the surface of SeNPs were 3.54, 9.82,

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19.78, 33.45, 38.82 µg/L, respectively.

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FT-IR and UV-vis were employed to characterize the structure and formation of

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PC-SeNPs. As shown in Figure 2A, the present of two special peaks around 1656 and

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1204 cm-1 in the spectrum of PC and PC-SeNPs were assigned to the characteristic

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absorption of amide group. The peaks around 1412 cm-1 in the spectrum were

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confirmed the stretching vibration of carboxyl group. These indicated the success bind

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of PC on the surface of SeNPs. Meanwhile, the UV absorption of PC-SeNPs at

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wavelength of 268 and 623 nm in the spectrum of PC and PC-SeNPs also suggested

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the success bind of PC on the surface of SeNPs (Figure 2B).

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Stability studies of PC-SeNPs were also carried out under different conditions.

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As shown in Figure 2C, there was little change in the size of PC-SeNPs (235 nm) in

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the first 15 days and it became slightly larger on the 30th day in PBS. However, the

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sizes of PC-SeNPs (165 nm) and PC-SeNPs (371 nm) became large on the 3 th or 5 th

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day, respectively. These demonstrated that SeNPs modified with 50 mg/L of PC were

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much stable than other PC-SeNPs. The possible reason was that the zeta potential of

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PC-SeNPs (165 nm, -17.5 mV) was lower than PC-SeNPs (235 nm, -33.3 mV), so the

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repulsion force was smaller and thus they were much easier to in aqueous solution.

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Meanwhile, the massive of proteins adsorbed onto the surface of SeNPs were easy to



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connect with each other, which caused the unstability of PC-SeNPs (371 nm). Besides,

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under the physiological condition, with the present of 10 % fetal bovine serum (FBS)

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in DMEM medium, the sizes of these three PC-SeNPs maintained constant during 48

246

h incubation (Figure 2D). The possible reason was that the negative-charged proteins

247

in FBS could be adsorbed to the surface of nanoparticles, which increased the stability

248

of the nanoparticles. In this study, GSH instead of ascorbic acid (Vc) was selected as

249

the reducing agent for preparing SeNPs, because GSH is weak alkaline in water

250

solution which is beneficial for the stability of PC 17.

251 252

ABTS+ Free Radical Scavenging Activities. The antioxidant activity of SeNPs and

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PC-SeNPs with different sizes were evaluated by ABTS·+ assay. The antioxidant

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activities of tested specimens were corresponding to inhibition percentage. As Figure

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3A shown, the three different PC-SeNPs showed higher inhibition ability of ABTS

256

oxidation than SeNPs, which suggesting that PC-SeNPs had higher antioxidant

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activity. With the highest PC content, PC-SeNPs with the size of 371 nm exhibited the

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highest antioxidant among the three nanoparticles and the possible reason was that PC

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also had strong antioxidant activity. To validate influence of PC content on the

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antioxidant, different concentration PC-SeNPs (235 nm) were given to the cells and

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the results showed that the higher concentration of PC-SeNPs, the stronger

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antioxidant activity (Figure 3B).

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Cellular Uptake of PC-SeNPs. Cellular uptake experiments were carried out on


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INS-1E cells. In the first two hours, as shown in Figure 4, the cellular uptake of

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PC-SeNPs-165 nm showed a rapid rate and then slowed down in the following time,

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while the PC-SeNPs-235 nm and PC-SeNPs-371 nm showed similar cellular uptake

268

rate. The reason of this phenomenon might be particle size was the limiting factor for

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cellular uptake in the first. As time went on, PC-SeNPs-235 nm showed the highest

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cellular among the three nanoparticles. Nevertheless, the cellular uptake of

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PC-SeNPs-371 nm had been persistently slowing. The possible reasons were as

272

follows: Firstly, the particle size is one of the important factors which influence the

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cellular. Particles with small size have relatively large surface area and curvature,

274

which benefit to the cellular uptake39. Moreover, the zeta potential of particle is

275

another important influence factor40,41. Negative charged particles could enter the cell

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easily and the stronger the negative, the more particles enter into cells which the

277

reason was that negative charged were hard to combined with BSA in circulation. In

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summary, PC-SeNPs with 235 nm and charged -33.3 mV showed the highest cellular

279

uptake by INS-1E cells.

280 281

PC-SeNPs Inhibits PA-induced Cytotoxicity. As reported, islet β cells could be

282

damage by exposure to high concentration of free fatty acids (FFA) and palmitic acid

283

(PA) is one of the most representative one in FFA42. Consequently, the effect of

284

nanoparticles on PA-induced INS-1E cells damage was examined. As illustrated in

285

Figure 5 A, PC-SeNPs-235 nm slightly inhibited INS-1E cells growth even when the

286

concentration raised up to 4 µM, as indicated by the the cell viability of INS-1E cells



287

was 74.5% under this concentration treatments. PA significantly inhibited INS-1E

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cells proliferation in a concentration dependent manner with its IC50 value less than

289

1µM. And 0.4 mM of PA induced INS-1E cells death was about 20%, which was

290

suitable for the next experiments (Figure 5B). PC and SeNPs could not reversed PA

291

induced INS-1E cells death when the concentration varied from 0.25 µM to 1µM

292

(Figure 5C). Interestingly, we found that, pre-incubating the cells with PC-SeNPs for

293

12 h dramatically restrained PA inhibition in the cells proliferation as can be seen in

294

the cell viability of PC-SeNPs-165 nm, PC-SeNPs-235 nm, PC-SeNPs-371 nm were

295

about 91.2%, 98.4% and 82.4%, respectively, which demonstrated that PC modified

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SeNPs could decrease cell damage caused by PA. Moreover, among the three

297

PC-SeNPs, PC-SeNPs-235 nm displayed the best protective effect and the possible

298

reason might be PC-SeNPs-235 nm show the highest cellular uptake, the relatively

299

small particle size and the relatively higher PC content (Figure 5 D). In all, these

300

results indicated that PC-SeNPs could protect INS-1E cells from PA-induced

301

cytotoxicity in vitro.

302 303

Effects of PC-SeNPs on the cell cycle distribution induced by PA. Cell cycle arrest

304

and apoptosis are two major action modes which cause cell death43,44. In order to

305

investigate the mechanisms of PA induced cells death the protective effects of

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PC-SeNPs, we carried out flow cytometry assay to examine the cell cycle distribution

307

and apoptosis of PA, PC, SeNPs and PC-SeNPs. The premier cell cycle pattern of

308

INS-1E cells without PA-induced treated with PI was set as control group. As shown


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in Figure 6, PA treatment increased the population in sub-G1 from 3.7% to 30.6%,

310

which indicated that PA induced INS-1E cells death mainly through inducing

311

apoptosis. PC and SeNPs alone slightly decreased the cell apoptosis from 30.6% to

312

23.1% and

313

PC-SeNPs-235 nm and PC-SeNPs-371 nm) pretreatment significantly protect PA

314

induced INS-1E cells apoptosis without other phases obviously changes and

315

PC-SeNPs-235 nm appears to exhibited the most potent protective effects, as reflected

316

by the decreased population of sub-G1 from 30.6 % to 22.6 %, 19.4 % and 22.3 %

317

respectively. To further determine the effect of PC-SeNPs-235 nm, the dose-course

318

effects of PC-SeNPs-235 nm were evaluated. The results demonstrated that

319

PC-SeNPs-235 nm pretreatment notably protected INS-1E cells apoptosis induced by

320

PA. Taken together, these results demonstrated that PC-SeNPs effectively blocked the

321

apoptosis effects induced by PA.

24.3% respectively.

However,

PC-SeNPs

(PC-SeNPs-165

nm,

322 323

PC-SeNPs Inhibits PA-induced Caspase Activation. Evidences have implicated that

324

caspases play an essential role in apoptosis regulation46,47, therefore, we next

325

conducted fluorimetric assay to determine whether caspases activation were

326

involved in PA-induced apoptosis. As indicated in Figure 7A, PA significantly

327

induced caspase-8/9 and caspase-3 activation in INS-1E cells, which indicated the

328

activation of both extrinsic and intrinsic mediated apoptosis signaling pathway.

329

However, these effects were significantly inhibited by the pretreatment of PC and

330

SeNPs. Moreover, pre-incubation of PC-SeNPs dramatically reversed PA-induced



331

caspase-8/-9 and caspase-3 activation, as indicated by the decreased fluorescence

332

intensity in PC-SeNPs pre-treatments. Among them, PC-SeNPs-235 nm appeared to

333

display the most protective effects against PA induced caspases activation. These

334

results were further certified Western blotting results. As illustrated in Figure 7B,

335

exposure of INS-1E cells to PA resulted in significant cleavage of Caspase-3/-8/-9 and

336

PARP. PC and SeNPs displayed slightly protective effects against PA induced INS-1E

337

cells apoptosis as can be seen in the activation of caspase-3/8/9 and PARP. However,

338

PC-SeNPs pretreatment strongly restrained the caspases activation and PARP

339

cleavage induced by PA which indicated the protective effects of PC-SeNPs against

340

the apoptosis inducing capacity of PA towards INS-1E cells. Together, these results

341

clearly demonstrated that PC-SeNPs exhibited strongly protective effects against

342

PA-induced death receptor and mitochondria mediated apoptosis towards INS-1 cells.

343 344

Protective effect of PC-SeNPs Against ROS Overproduction. ROS and RNS

345

generation have been found to play important role in the oxidative damage of islet

346

cells. The excess amount of ROS could attack many components of DNA, thus causes

347

DNA damage48. To evaluate the effect of PC-SeNPs on ROS scavenging, INS-1E

348

cells were pre-incubated with 0.4 mM PA to induce the overproduction ROS. As

349

shown in Figure 8A, ROS in PA-induced INS-1E cells increased to 153% as the

350

control group was 100%. However, when INS-1E cells pre-incubated with PC-SeNPs,

351

the generation of ROS in PA-induced INS-1E cells significantly reduced. It was

352

103.2% when treated with PC-SeNPs-235 nm, which had the best protective effect.


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Similarly, they were 110.3% and 142.3% for PC-SeNPs-165 nm and PC-SeNPs-371

354

nm, respectively. Furthermore, the representative DHE fluorescence images also

355

showed the similar results as shown in Figure 8B. In general, PC-SeNPs could

356

protect INS-1E from the damage by PA by eliminating ROS and PC-SeNPs-235 nm

357

showed the best protective effect.

358 359

PC-SeNPs Inhibits PA-induced Mitochondrial Fragmentation. Mitochondria are

360

remarkably dynamic organelles and the importance of energy generated by

361

mitochondria has long been appreciated. Mitochondrial dynamics are important

362

indications for the quality control of mitochondria49,50. Oxidative stress could cause

363

mitochondrial malfunction and enhance metabolism, which promoted the release of

364

apoptogenic factors from mitochondria inner membrane space and initiates apoptotic

365

cascades51. The cells treated with PA for 2 h and washed twice by PBS, then stained

366

by mito-Red for 2 h, mitochondrial fragmentation were observed under fluorescence

367

microscopy. As shown in Figure 9, the mitochondria were in good condition without

368

any for the control group, while 0.4 mM PA incubation induced obvious disruption of

369

mitochondria structure. Significantly reduction in these changes were observed when

370

pre-treated different PC-SeNPs and PC-SeNPs-161 nm showed the best effect. The

371

morphological improvements indicated that PC-SeNPs could restrain the PA-induced

372

damage to mitochondria in INS-1E cells.

373 374

Funding



375

This work was supported by National High-level personnel of special support

376

program, National High Technology Research and Development Program of China

377

(SS2014AA020538), Science Foundation for Distinguished Young Scholars of

378

Guangdong Province (S2013050014667), YangFan Innovative & Entepreneurial

379

Research Team Project (201312H05), Guangdong Special Support Program and

380

Guangdong Frontier Key Technological Innovation Special Funds (2014B050505012)

381

and Fundamental Research Funds for the Central Universities.

382 383

Notes

384

The authors declared no competing financial interest.

385

# Chang Liu and Yuanting Fu contributes equally to this manuscript.

386 387

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534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550



551

Scheme 1

552 553

Scheme 1. Rational design of PC-SeNPs to protect INS-1E cells from oxidative

554

damage and the underlining mechanisms. (A) The core-shell structure of

555

PC-SeNPs. (B) Proposed structure of PC-SeNPs. (C) and (D) The action mechanism

556

of PC-SeNPs-caused protective effect on PA-induced INS-1E cells.

557 558 559 560 561 562 563


Page 26 of 36

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564

Figure 1

565 566

Figure 1. The morphology and particle size of PC-SeNPs. (A) TEM images of

567

PC-SeNPs with different PC concentration, and the concentrations from a to f were 0,

568

5, 25, 50, 100, 150 mg/L, respectively. (B) The particle size distribution of PC-SeNPs

569

with different PC concentration. (C) Zeta potential of PC-SeNPs with different PC

570

concentration. (D) Effects of added PC concentration in the synthesis reaction on the

571

conjugated surface PC contents in PC-SeNPs.

572 573 574 575



576

Figure 2

577 578

Figure 2. The spectroscopy and stability of PC-SeNPs. (A) The FT-IR spectrum of

579

PC, SeNPs and PC-SeNPs. (B) The UV-vis spectrum of PC, SeNPs, and PC-SeNPs

580

with different size. (C) The stability of PC-SeNPs in PBS. (D) The stability of

581

PC-SeNPs in DMEM.

582 583 584 585 586 587 588


Page 28 of 36

Page 29 of 36


589

Figure 3

590 591

Figure 3. The antioxidant activities of PC-SeNPs determined by ABTS·+ assay. (A)

592

The comparison antioxidant activities of SeNPs and PC-SeNPs with different particle

593

size. (B) The antioxidant activities of different concentration PC-SeNPs (235 nm).

594 595 596



597

Figure 4

598 599

Figure 4. Cellular uptake of nanoparticles by INS-1E cells. INS-1E cells were

600

treated with 0.8 µM of PC-SeNPs, and cells were collected and the content of Se were

601

measured at determined time point with ICP-MS.

602 603 604 605 606 607 608 609 610


Page 30 of 36

Page 31 of 36


611

Figure 5

612 613

Figure 5. Cell viability assay of INS-1E cells under different condition by MTT

614

assay. (A) Cell viability of INS-1E cells treated with different concentration of

615

PC-SeNPs-235 nm. (B) Cell viability of INS-1E cells treated with different

616

concentration of PA. (C)

617

concentration of PC or SeNPs for 2 h before treated PA, respectively. (D) Cell

618

viability of cells pre-incubated with different PC-SeNPs for 12 h before treated with

619

PA for 2 h. n=3, p < 0.05.

Cell viability of the cells pre-incubated with different

620 621 622



623

Figure 6

624 625

Figure 6.Different effects of PC-SeNPs on the cell cycle induced by PA as

626

examined by propidium iodide (PI)-flow cytometric analysis. (A) Cells (2 × 104

627

cells/mL) were treated with PC, SeNPs, different form or concentreation of PC-SeNPs

628

and PA for indicated times. (B) and (C) Quantitative cell cycle distribution data for

629

INS-1E in every group. n=3.

630 631 632 633 634


Page 32 of 36

Page 33 of 36


635

Figure 7

636 637

Figure 7. PC-SeNPs Inhibits PA-induced Caspase Activation. (A) INS-1E cells

638

pre-incubated with PC, SeNPs and different PC-SeNPs (0.8 µM) for 12 h before

639

treated with 0.4 mM of PA for 2 h and the influence was determined by synthetic

640

fluorogenic substrate (n =3, P < 0.05). (B) Inhibition of PARP and caspase cleaved in

641

the apoptosis pathway and equal protein loading was confirmed by Western analysis

642

of β-actin in the protein extracts.

643 644 645 646 647 648 649 650 651 652



653

Figure 8

654 655

Figure 8. ROS generation of PA-induced INS-1E cells prevented by PC-SeNPs.

656

(A) Changes in intracellular ROS generation in INS-1E cells pre-incubated with 0.8

657

µM of PC-SeNPs for 12h and then exposed to 0.4 mM of PA for 2 h. Then cells were

658

stain with DHE-DA for 30min. (B) Representative DHE fluorescence images of

659

INS-1E cells exposed to PA after treated with different PC-SeNPs. n = 3.

660 661


Page 34 of 36

Page 35 of 36


662

Figure 9

663 664

Figure 9. PC-SeNPs Inhibits PA-induced Mitochondrial Fragmentation. The

665

representative images of mitochondrial fragmentation in INS-1E cells after treatment

666

with 0.8 µM of PC-SeNPs for 12 h and 0.4 mM PA for 2 h, after that cells were

667

stained by Red Mito-Tracker for 2 h and stained by DAPI for 20 min. Mitochondria

668

fragmentation was measured by fluorescence microscope.

669 670 671 672 673 674 675 676



677

TOC graphic

678 679

TOC: Rational design of PC-SeNPs to protect INS-1E cells from oxidative

680

damage and the underlining mechanisms. (A) The core-shell structure of

681

PC-SeNPs. (B) Proposed structure of PC-SeNPs. (C) and (D) The action mechanism

682

of PC-SeNPs-caused protective effect on PA-induced INS-1E cells.

683


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