br Corresponding author br ef cient
⇑ Corresponding author.
efÞcient source of the antimicrobial [10Ð13], antioxidant , cytotoxicity [14Ð16], bioremediation , and wound healing [18,19]. MgONPs are a bioactive agent with nifty of properties such as bactericidal , analgesic, anti-inßammatory , antioxidant, antidiabetic , and cancer therapy . U.S. food and drug administration (FDA) declared as MgONPs (21CFR184.1431) are biocompatible, biodegradable and low toxicant that can be used in pharmaceutics [24,25].
Existence of the oxygen and superoxide on surface of the MgONPs induce the antibacterial activity through cell wall mem-brane damage, triggering the active oxygen, reactive oxygen spe-cies, lipid peroxidation, electrostatic interaction, alkaline effect, inhibition or alteration in DNA replication, krep cycle, amino acids, nucleotide metabolism and functioning the cellular enzymes [24,26Ð32]. Similarly, silver nanoparticles (AgNPs) also used in medicine, medical devices, disease diagnosis, environmental reme-diation, cosmetics and food industry [33Ð35]. Although there are few reports on the preparation and antibacterial effect of the Ag loaded or doped MgONPs , no reports on the biologic synthesis of Ag-embedded MgONPs and their cytotoxicity in PC-3 cells. Hence the present work aimed to synthesis the Ag-MgONPs using the MCFE of T. aureoviride SKCGW013 and study their characteriza-tion and cytotoxicity in PC-3 cells.
0921-8831/ 2019 The Society of Powder Technology Japan. Published by Elsevier B.V. and The Society of Powder Technology Japan. All rights reserved.
2. Materials and methods
2.1. Chemicals, cell culture, microorganisms, and cell-free extracts preparations
Roswell park memorial institute medium (RPMI 1640; Gibco), penicillin (P), streptomycin (S; Gibco), fetal bovine serum (FBS; Gibco), EZ-CYTOX WST assay kit (EZ-CyTox, Daeil Lab Service, Republic of Korea), Annexin V FITC and PI for Flow Cytometry (Invitrogen, Thermo Þshers scientiÞc, Republic of Korea), 20,70 -Dic hloroßuorescin diacetate (DCFH-DA), 40,6-diamidino-2-phenylin dole (DAPI), acridine orange (AO) and ethidium bromide (EB), tryp-sin, dimethyl sulfoxide (DMSO). Magnesium nitrate hexahydrate (Mg (NO3)2 6H2O), silver nitrate (AgNO3) obtained from Sigma Aldrich, Republic of Korea. All the other chemicals and reagents were purchased from Daejung, Republic of Korea. The human pros-tate cancer cell line (PC-3) was purchased from Korean cell line bank (Seoul, Republic of Korea) and cultured in RPMI 1640 medium with 10% FBS and 1% Streptavidin (P&S). T. aureoviride SKCGW013 (NCBI accession MG940965) were obtained from Plant molecular biotechnology Laboratory, College of Biomedical Sciences, Kang-won National University, Republic of Korea.
2.2. Synthesis and characterization of nanoparticles
Nanoparticles (MgONPs and Ag-MgONPs) were synthesized using the MCFE of strain SKCGW013. For the preparation of MCFE, the strain SKCGW013 was grown in a 250 mL Erlenmeyer ßask containing 100 mL of potato dextrose broth (PDB) in a shaker with agitation of 180 rpm at 28 LC for 4 days. Mycelial biomass was col-lected by Þltration using Whatman No.1 Þlter paper and then washed with sterile distilled water to remove the excess medium residues. A 20 g of wet weight of mycelial biomass was dissolved in 100 mL of Milli-Q deionized water and stirred for 60 min at 180 rpm in 28 LC using a shaker, then Þltered again using the Whatman No.1 Þlter paper. For the synthesis of MgONPs, the 2, 5 and 10 mM of Mg(NO3)2 6H2O was dissolved in 100 mL of MCFE in 250 mL ßask and the solution was kept at 40 LC under darkness for 24 h then the NaOH solution was adding dropwise . For the synthesis of Ag-MgONPs, 2, 5 and 10 mM of AgNO3 and 2, 5 and 10 mM of Mg (NO3)2 6H2O were dissolved in 100 mL of MCFE incu-bated in at 40 LC under darkness for 24 h. The both of synthesized NPs were prepared for the characterization according to the meth-ods described previously  and studied by a high throughput techniques including FTIR (PerkinElmer Paragon 500, USA), XPS (Thermo ScientiÞcTM K-AlphaTM), XRD (XÕpert-pro MPD- PANalytical, Netherland), operated at 40 keV with Cu ja radiation in h-2h, FESEM with EDS (S-4300/HITACHI), TEM with EDS (JEOL-JSM 1200EX, Japan). The particle size was analyzed using PSA (Malvern Mastersizer 2000, Britain).
2.3. Cytotoxicity assay
MgONPs or Ag-MgONPs induced cytotoxicity in the PC-3 cells were investigated using a rapid WST assay kit. In brief, the PC-3 cells were cultured in 5 mL of RPMI 1640 incorporated with 10% of FBS and 1% of antibiotics solutions in a T25 ßask in humidiÞed 5% of CO2 incubator for 24 h. After reaching 80Ð90% conßuence, the cells were harvested by trypsinization, followed by these healthy cells (2 104 cells. mL 1) were dissolved in RPMI 1640 and seeded into 96 well plates (coster) as 1 mL per well. Then the 96 well plates were kept in 5% of CO2 incubator for 24 h to allow the cells to attach in the bottom of the well. After 24 h, the cells were washed with PBS and replaced RPMI1640 medium con-