APT 2194 No.

of Pages 9, Model 5G
2 February 2019

Advanced Powder Technology xxx (xxxx) xxx

1

Contents lists available at ScienceDirect

Advanced Powder Technology

journal homepage: www.elsevier.com/locate/apt

2 Original Research Paper

6
4 Biogenic silver embedded magnesium oxide nanoparticles induce the
7
5 cytotoxicity in human prostate cancer cells
8 Kandasamy Saravanakumar, Myeong-Hyeon Wang ⇑
9 Department of Medical Biotechnology, College of Biomedical Sciences, Kangwon National University, Chuncheon, Gangwon 24341, Republic of Korea

11
10
12
a r t i c l e i n f o a b s t r a c t
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2 4
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15 Article history: This work reports the synthesis, characterization, and cytotoxicity of biogenic magnesium oxide nanopar- 28
16 Received 31 August 2018 ticles (MgONPs) and silver embedded magnesium oxide nanoparticles (Ag-MgONPs). The formation of 29
17 Received in revised form 13 December 2018 nanoparticles (NPs) was confirmed by the indications of color changes and precipitations. Field emission 30
18 Accepted 15 January 2019
scanning electron microscopy (FESEM), transmission electron microscopy (TEM) and X-ray diffraction 31
19 Available online xxxx
(XRD) pattern studies showed the agglomeration colloids, porous, spherical, needle-shaped and crystal 32
nature of MgONPs. Hexagonal, and spherical structured nanocrystals of Ag-MgONPs. Energy-dispersive 33
20 Keywords:
X-ray fluorescence spectrometry (EDS) study indicated the existence of Ag, Mg, and O in NPs complex. 34
21 Cancer
22 PC-3
The particle size analysis (PSA) revealed the mean size of 15.09 nm for Ag-MgONPs and 13.68 nm for 35
23 Silver MgONPs. Fourier transform infrared spectroscopy (FTIR) showed the peaks corresponding to amide, car- 36
24 Magnesium oxide boxylic acids, aromatics, alkene and esters from mycelial cell-free extract (MCFE). The absorbed and lat- 37
25 Cytotoxicity tices oxygen of MgO was probably assigned in the formation of Ag-MgONPs as indicated by X-ray 38
26 photoelectron spectroscopy (XPS). Cytotoxicity assay showed the Ag-MgONPs was stronger in inducing 39
the prostate cancer (PC-3) cell death than MgONPs. This work concluded that Ag-MgONPs could be 40
potential therapeutics for cancer therapy. 41
Ó 2019 The Society of Powder Technology Japan. Published by Elsevier B.V. and The Society of Powder 42
Technology Japan. All rights reserved. 43
44

45
46
47 1. Introduction NPs are reported as an efficient source of the antimicrobial [10– 66
13], antioxidant [14], cytotoxicity [14–16], bioremediation [17], 67
48 Globally cancer is one of the recognized hazards to human life. and wound healing [18,19]. MgONPs are a bioactive agent with 68
49 The scientists are eagerly developing the various therapeutic nifty of properties such as bactericidal [20], analgesic, anti- 69
50 agents and precursors to cure the cancer incidents [1]. Among inflammatory [21], antioxidant, antidiabetic [22], and cancer ther- 70
51 the therapeutics, NPs based cancer therapy is more successful apy [23]. U.S. food and drug administration (FDA) declared as 71
52 because of their ability in targeting the cancer cells and low toxic- MgONPs (21CFR184.1431) are biocompatible, biodegradable and 72
53 ity [2]. Human prostate cancer is the development of malignancy low toxicant that can be used in pharmaceutics [24,25]. 73
54 in the prostate, which is a complex heterogeneous disease in Existence of the oxygen and superoxide on surface of the 74
55 man [1,3]. According to American cancer society reports about MgONPs induce the antibacterial activity through cell wall mem- 75
56 180,000 of prostate cancer incidents are detected in the United brane damage, triggering the active oxygen, reactive oxygen spe- 76
57 States during 2016 [1]. Of course, there are several physical or cies, lipid peroxidation, electrostatic interaction, alkaline effect, 77
58 chemotherapeutic approaches are existed to treat cancer, but these inhibition or alteration in DNA replication, krep cycle, amino acids, 78
59 techniques trigger the side effects such as pain, skin erythema, nucleotide metabolism and functioning the cellular enzymes 79
60 atrophy and inducing cytotoxicity to normal cells. Although the [24,26–32]. Similarly, silver nanoparticles (AgNPs) also used in 80
61 NPs/microparticles in inhalation is causing the deposition in medicine, pharmacology, medical devices, disease diagnosis, envi- 81
62 human airways [4–6]. The proper application of the NPs based can- ronmental remediation, cosmetics and food industry [33–35]. 82
63 cer drug or drug delivery system can extensively target the cancer Although there are few reports on the preparation and antibacte- 83
64 cells and induce the ablation via enhanced permissibility and rial effect of the Ag loaded or doped MgONPs [36], no reports on 84
65 retention (EPR) without damaging the normal cells [7–9]. Biogenic biologic synthesis of Ag-embedded MgONPs and their cytotoxicity 85
in PC-3 cells. Hence the present work aimed to synthesis the Ag- 86
⇑ Corresponding author. MgONPs using the MCFE of T. aureoviride SKCGW013 and study 87
E-mail address: mhwang@kangwon.ac.kr (M.-H. Wang). their characterization and cytotoxicity in PC-3 cells. 88

https://doi.org/10.1016/j.apt.2019.01.007
0921-8831/Ó 2019 The Society of Powder Technology Japan. Published by Elsevier B.V. and The Society of Powder Technology Japan. All rights reserved.

Please cite this article as: K. Saravanakumar and M. H. Wang, Biogenic silver embedded magnesium oxide nanoparticles induce the cytotoxicity in human
prostate cancer cells, Advanced Powder Technology, https://doi.org/10.1016/j.apt.2019.01.007
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2 K. Saravanakumar, M.-H. Wang / Advanced Powder Technology xxx (xxxx) xxx

89 2. Materials and methods further incubated for 24 h in 5% of CO2 incubator. Finally, the cyto- 148
toxicity was measured using the EZ-CYTOX indicator according to 149
90 2.1. Chemicals, cell culture, microorganisms, and cell-free extracts the manufacturer’s instructions. The cells were counted by trypan 150
91 preparations blue dye exclusion method using a hemocytometer under a bright 151
field microscope (Olympus, CKX53 culture microscope, Japan). 152
92 Roswell park memorial institute medium (RPMI 1640; Gibco),
93 penicillin, streptomycin (Gibco), fetal bovine serum (FBS; Gibco), 2.4. AO/EB staining 153
94 EZ-CYTOX WST assay kit (EZ-CyTox, Daeil Lab Service, Republic
95 of Korea), Annexin V FITC and PI for Flow Cytometry (Invitrogen, The nuclear changes and apoptotic body formation of the PC-3 154
96 Thermo fishers scientific, Republic of Korea), 20 ,70 -Dichlorofluores cells were visualized using the fluorescence microscope [38]. The 155
97 cin diacetate (DCFH-DA), 40 ,6-diamidino-2-phenylindole (DAPI), NPs treated or untreated PC-3 cells were harvested and washed 156
98 acridine orange (AO) and ethidium bromide (EB), Trypsin, dimethyl with the PBS. Then, the 25 µL of cell suspension (0.5  106 - 157
99 sulfoxide (DMSO). Magnesium nitrate hexahydrate (Mg (NO3)2- cellsmL1) were incubated with 1 µL of 1:1 of AO and EB for 158
100 6H2O), Silver nitrate (AgNO3) obtained from Sigma Aldrich, South 5 min. The cells were again washed with PBS several times to 159
101 Korea. All the other chemicals and reagents were purchased from remove the residual stain then visualized under the fluorescence 160
102 Daejung, South Korea. The human prostate cancer cells (PC-3) microscope (Olympus, CKX53 culture microscope, Japan). 161
103 was purchased from Korean cell line bank (Seoul, Republic of
104 Korea) and cultured in RPMI 1640 medium with 10% FBS and 1% 2.5. Determination of ROS generation 162
105 antibiotics. T. aureoviride SKCGW013 (NCBI accession MG940965)
106 were obtained from Plant molecular biotechnology Laboratory, The ROS production was measured in PC3 cells by DCFH-DA 163
107 College of Biomedical Sciences, Kangwon National University, the staining method according to the method described earlier [39] 164
108 Republic of Korea. with slight modifications. In brief, the PC3 cells (2  104 - 165
cellsmL1) were seeded in 30 mm confocal glass bottom dish 166
(SPL life sciences, Republic of Korea) containing RPMI 1640 med- 167
109 2.2. Synthesis and characterization of metal nanoparticles
ium and allowed to attach the cells. For the NPs treatment, after 168
the 24 h, the cells were washed with PBS and medium replaced 169
110 Nanoparticles (MgONPs and Ag-MgONPs) were synthesized
with NPs (IC50 concentration) incorporated medium and incubated 170
111 using the MCFE of strain SKCGW013. For the preparation of MCFE,
for 24 h. After the incubation period, the cells were again washed 171
112 the strain SKCGW013 was grown in a 250 mL Erlenmeyer flask
with cold PBS and added the DCFH-DA (50 µm). Then it was incu- 172
113 containing 100 mL of potato dextrose broth (PDB) in a shaker with
bated for 30 min at 37 °C. Finally, the cells washed with the PBS 173
114 agitation of 180 rpm at 28 °C for 4 days. Mycelial biomass was col-
and maintained in 1 mL of PBS. The ROS production assessed using 174
115 lected by filtration using Whatman No.1 filter paper and then
the fluorescence microscope with excitation of 488 nm and emis- 175
116 washed with sterile distilled water to remove the excess medium
sion of 530 nm. 176
117 residues. A 20 g of wet weight of mycelial biomass was dissolved
118 in 100 mL of Milli-Q deionized water and stirred for 60 min at
2.6. Cell nuclear morphology 177
119 180 rpm in 28 °C using a shaker, then filtered again using the
120 Whatman No.1 filter paper. For the synthesis of MgONPs, the 2, 5
DAPI staining was applied to study the cell nucleus morphology 178
121 and 10 mM of Mg(NO3)26H2O was dissolved in 100 mL of MCFE
according to the methods described elsewhere [38] with modifica- 179
122 in 250 mL flask and the solution was kept at 40 °C under darkness
tions. The PC-3 cells were grown in the 30 mm confocal glass bot- 180
123 for 24 h then the NaOH solution was adding dropwise [37]. For the
tom dish containing the RPMI 1640 medium for 24 h and then it 181
124 synthesis of Ag-MgONPs, 2, 5 and 10 mM of AgNO3 and 2, 5 and
was washed with PBS. Then, the cells were treated with an IC50 182
125 10 mM of Mg (NO3)26H2O were dissolved in 100 mL of MCFE incu-
concentration of NPs samples for 24 h. At the end of the treat- 183
126 bated in at 40 °C under darkness for 24 h. The both of synthesized
ments, the cells were again washed with PBS then fixed in 4% 184
127 NPs were prepared for the characterization according to the meth-
formaldehyde for 4 min at ambient temperature. For the DAPI 185
128 ods described previously [17] and studied by a high throughput
staining 300 µL of the 300 nM of DAPI solution was laid on the cells 186
129 techniques including FTIR (PerkinElmer Paragon 500, USA), XPS
for 5 min at room temperature in dark then it was washed again 187
130 (Thermo ScientificTM K-AlphaTM), XRD (X’pert-pro MPD- PANalytical,
with PBS for several times to remove the residual dye and observed 188
131 Netherland), operated at 40 keV with Cu ja radiation in h-2h,
under using the fluorescence microscope with excitation of 189
132 FESEM with EDS (S-4300/HITACHI), TEM with EDS (JEOL-JSM
358 nm and emission of 461 nm. 190
133 1200EX, Japan). The particle size was analyzed using PSA (Malvern
134 Mastersizer 2000, Britain).
2.7. Flow cytometer assay 191

135 2.3. Cytotoxicity assay The NPs induced apoptosis was determined using the FITC 192
Annexin V/Dead cell apoptosis kit according to the manufacture 193
136 MgONPs or Ag-MgONPs induced cytotoxicity in the PC-3 cells interactions using the flow cytometer (BD FACS Calibur, BD USA). 194
137 were investigated using a rapid WST assay. In brief, the PC-3 was In brief, the PC-3 cells (1  104 cellsmL1) were grown in the 6 195
138 cultured in 5 mL of RPMI 1640 incorporated with 10% of FBS and well plates (coster) for 24 h in humidified 5% of CO2 incubator. 196
139 1% of antibiotics solutions in a T25 flask in humidified 5% of CO2 After reaching the 80% of confluence the cells were washed with 197
140 incubator for 24 h. After reaching 80–90% confluence, the cells PBS, the medium was replaced with NPs (IC50) incorporated med- 198
141 were harvested by trypsinization, followed by these healthy cells ium and incubated for 24 h then the cells were harvested for the 199
142 (2  104 cells. mL1) were dissolved in RPMI 1640 and seeded into flow cytometer analysis. 200
143 96 well plates (coster) as 1 mL per well. Then the 96 well plates
144 were kept in 5% of CO2 incubator for 24 h to allow the cells to 2.8. Statistical analysis 201
145 attach in the bottom of the well. After 24 h, the cells were washed
146 with PBS and replaced RPMI1640 medium containing different Experiments were performed three times and the descriptive 202
147 concentrations (0–1000 µgmL1) of MgONPs or Ag-MgONPs and statistics, one-way ANOVA (p < 0.05; p < 0.01) and post hoc test 203

Please cite this article as: K. Saravanakumar and M. H. Wang, Biogenic silver embedded magnesium oxide nanoparticles induce the cytotoxicity in human
prostate cancer cells, Advanced Powder Technology, https://doi.org/10.1016/j.apt.2019.01.007
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204 did by use of the statistical software SPSS version 16.0 (SPSS, Chi- reportedly cause the cytotoxicity and antimicrobial effect [17,42] 210
205 cago, IL). The results were shown as mean ± standard error. but there is no work describing accompanied Ag-MgONPs on cyto- 211
toxicity and antimicrobial effects. Although a study reports the sol- 212
gel preparation of Ag-doped MgONPs and their antibacterial activ- 213
206 3. Results and discussion ity, the synergetic cytotoxic effects against cancer cells are 214
unknown [36]. Trichoderma strains are a renowned source for var- 215
207 Biogenic NPs are significantly used in biomedical, food and ious bioactive enzymes and metabolites with therapeutic activities 216
208 chemical industrial applications due to their remarkable properties against various pathogens and diseases [43–45]. Moreover, its cell- 217
209 [40,41]. Green synthesized unaccompanied AgNPs and MgONPs are

Fig. 1. Transmission electron microscopic imaging of MgONPs (a), Electron image for EDS mapping (b), EDS layered image (c), EDS mapping of Mg (d) Energy-dispersive X-ray
spectroscopy analysis (e).

Please cite this article as: K. Saravanakumar and M. H. Wang, Biogenic silver embedded magnesium oxide nanoparticles induce the cytotoxicity in human
prostate cancer cells, Advanced Powder Technology, https://doi.org/10.1016/j.apt.2019.01.007
 APT 2194 No. of Pages 9, Model 5G
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4 K. Saravanakumar, M.-H. Wang / Advanced Powder Technology xxx (xxxx) xxx

Fig. 2. Transmission electron microscopic imaging of Ag-MgONPs (a), Electron image for EDS mapping (b), EDS layered image (c), EDS mapping of Mg (d) Energy-dispersive X-
ray spectroscopy analysis (e).

218 free extracts are known to synthesise the silver and chitosan NPs 3.1. Synthesis and characterization of nanoparticles 223
219 [14,46,47]. Furthermore, this extracts-based fabrication of Ag-
220 MgONPs are not reported. Herein we used the Trichoderma MCFE Several previous studies reported that the microbial or plants 224
221 for the first time to synthesise the Ag-MgONPs in an eco-friendly extracts derived compounds such as carbohydrates, proteins, 225
222 manner for enhanced cancer cell ablation. triterpenoids, steroids, and flavonoids are act as capping or reduc- 226
ing agent for the synthesis of the metallic NPs and Trichoderma 227
strains are able to produce the variety of novel metabolites may 228
involve in synthesis of NPs [14,43,48,49]. The formation of the 229

Please cite this article as: K. Saravanakumar and M. H. Wang, Biogenic silver embedded magnesium oxide nanoparticles induce the cytotoxicity in human
prostate cancer cells, Advanced Powder Technology, https://doi.org/10.1016/j.apt.2019.01.007
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tion, spherical in shape and crystal nature of MgO NPs (Fig. 1a), 235
and it is agreement with earlier reports [42,50,51]. Followed by 236
SEM-EDS studies indicated the presence of the Mg and O forms, 237
it further confirms the effective synthesis of the MgONPs (Supple- 238
mentary Fig. 2a and b). On the other hand, FESEM images showed 239
the colloidal agglomeration of nano-sized, and subsequent forma- 240
tion of the spherical and oval shaped of Ag-MgO NPs (Supplemen- 241
tary Fig. 3a). These FESEM results are in accordance with 242
morphological characteristics of sol-gel based generation of Ag- 243
doped MgONPs [36] with modifications due to the capping of 244
MCFE. The SEM-EDS for Ag-MgONPs indicated the presence of 245
the Ag, Mg and O forms in the synthesized materials (Supplemen- 246
tary Fig. 3b and c). Fig. 1(a–e) shows the TEM images, EDS map- 247
ping, and chromatogram of MgONPs synthesized by MCFE. The 248
results presented the needle-shaped MgONPs crystals, it is in 249
accordance with an earlier report [52]. The EDS results showed 250
the presence of the Mg and O in the specimen of MgONPs, which 251
is agreement with SEM-EDS study. Followed by the TEM-EDS 252
results for Ag-MgONPs showed the hexagonal and spherical struc- 253
tured nanocrystals (Fig. 2a and b). The EDS results confirm the exis- 254
tence of Ag, Mg, and O in mapping as well as in chromatogram 255
were accordance with SEM-EDS (Fig. 2c–e). The PSA results 256
Fig. 3. FTIR analysis of functional group modifications in cell free extract (MCFE), revealed the average size of 15.09 nm for Ag-MgONPs and 257
magnesium oxide nanoparticles (MgONPs), and silver coated magnesium oxide 13.68 nm for MgONPs (Supplementary Fig. 4a,b). The higher size 258
nanoparticles (Ag- MgONPs).
of Ag-MgONPs was attributed due to the capping of Ag on the sur- 259
face of the MgONPs. 260

230 Ag-MgONPs was confirmed by color changes from pale yellow to

231 brown and the MgONPs was confirmed through the observation
3.2. FTIR analysis 261
232 of precipitations (Supplementary Fig. 1). The surface morphology
233 of synthesized MgONPs was visualized using the FESEM and the
In the present study, FTIR analysis was attributed to finding out 262
234 results showed the agglomeration colloids and porous organiza-
the functional molecules form MCFE which responsible for the syn- 263

Fig. 4. XPS analysis of Ag-MgONPs, survey scan (a), high resoulution Ag3d scan (b), Mg1s scan (c) and O1s scan(d).

Please cite this article as: K. Saravanakumar and M. H. Wang, Biogenic silver embedded magnesium oxide nanoparticles induce the cytotoxicity in human
prostate cancer cells, Advanced Powder Technology, https://doi.org/10.1016/j.apt.2019.01.007
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Fig. 5. Effect of MgONPs and Ag-MgONPs treatments on PC3 cells. bright field microscopic observation of morphological changes (a–c), live and dead cells staining by AO/EB
(d–f), ROS generation (g–i), observation of nucleolus damage by DAPI staining (j–l).

Fig. 6. Flow cytometer-based determination of apoptosis stages in PC3 cells untreated (a) treated with MgONPs (b) and Ag-MgONPs (c); upper left-necrosis cells; upper right-
dead cells; Lower left-live cells; Lower right- apoptosis cells.

264 thesis of the NPs (Fig. 3). The absorptions peak of MCFE were 1542 cm1 (C@C, aromatic, weak), 1452 cm1 (CH2, bend, med- 268
265 observed as 3282 cm1 (water, OAH, stretch, strong), 2920 cm1 ium), 1379 cm1 (CH3 bend, s medium), 1291 cm1 and 269
266 and 2851 cm1 (carboxylic acid,OAH stretch, strong), 1739 cm1 1260 cm1 (CAO, aromatic ester, stretch, strong) and 895 cm1 270
267 (C@O aldehyde, strong), 1637 cm1 (C@O, amide, strong), (C@C, alkene bend, strong). Perhaps amide, carboxylic acids aro- 271

Please cite this article as: K. Saravanakumar and M. H. Wang, Biogenic silver embedded magnesium oxide nanoparticles induce the cytotoxicity in human
prostate cancer cells, Advanced Powder Technology, https://doi.org/10.1016/j.apt.2019.01.007
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272 matic, alkene and ester from MCFE were acted as reducing or cap- treatment of Ag-MgONPs induces the more apoptosis necrosis 334
273 ping agents for the synthesis of NPs. The absorptions peak for MgO (Fig. 5a–f), the ROS production (Fig. 5g–i) cellular and nuclear 335
274 NPs were 3693 cm1 (water, OAH, stretch, strong), 3356 cm1 membrane damage than the MgONPs (Fig. 5j–l). Flow cytometer 336
275 (NAH stretch, strong), 1650 cm1 (C@O, amide, strong) results demonstrated that the necrosis cells (4.51%), dead cells 337
276 1437 cm1 (carboxylic acid bend, OAH, medium), 864 cm1 (3.15%), live cells (90.56%), apoptosis cells (1.78%) in untreated 338
277 (CAH, strong), 629 cm1 (CACl, strong). The Ag-MgO NPs showed cells (Fig. 6a). The Ag-MgONPs treated showed the necrosis cells 339
278 absorptions peak in wavelengths including 3315 cm1 (NAH, (48.67%), dead cells (7.3%), live cells (33.16%), apoptosis cells 340
279 stretch, secondary amine), 2920 cm1 (ACAH, stretch, weak), (11.14%) (Fig. 6b). For MgONPs treated showed the necrosis cells 341
280 1785 cm1 (C@O, conjugated acid halide, strong), 1579 cm1 (8.22%), dead cells (1.86%), live cells (88.94.16%); apoptosis cells 342
281 (C@O, amide, strong), 1312 cm1 (NO2, stretch), 1070 cm1 (CAO, (0.98%) (Fig. 6c). These results revealed a higher range of necrosis 343
282 primary alcohol, stretch), 799 cm1 and 720 cm1 (CAH bend, and dead cells in Ag-MgONPs treated one than the MgONPs trea- 344
283 strong). Further the XRD pattern showed the cubic and crystalline ted, which agrees with all the cytotoxicity related assay of this 345
284 nature of MgONPs with the respective diffraction peaks indexed at study. It is known that the MgONPs induce active oxygen, reactive 346
285 (1 1 1), (2 0 0), (2 2 0), (3 1 1), (2 2 2), which is similar to crystallo- oxygen species [16–18] and AgNPs ions penetrate through the can- 347
286 graphic of MgONPs (JCPDS 87-0652) [36], whereas the Ag-MgONPs cer cell membrane and stop the metabolic activity [59]. Therefore 348
287 showed the increased interplanar spacing of Ag-MgONPs at diffrac- the Ag-embedded MgONPs stronger in inducing the cell death 349
288 tion indexed (1 1 1) (Supplementary Fig. 5), which was a strong through the interactions with cancer molecules such as proteins, 350
289 evidence for Ag embedded in MgO [36,53]. DNA and block the replication of cancer cells through ROS produc- 351
tion and activating nucleus damage related apoptosis signaling 352
290 3.3. XPS analysis pathway [59–62]. 353

291 XPS analysis attributed to examine the chemical states of metal

4. Conclusion 354
292 NPs such as Ag-MgONPs and MgONPs. The survey scan spectrum
293 exhibited the signals of magnesium (Mg), silver (Ag), oxygen (O)
This work demonstrated the characterization and cytotoxicity 355
294 and residual carbon (C) in biogenic synthesized Ag-MgONPs
activity of the biogenic MgONPs and Ag-MgONPs. The high 356
295 (Fig. 4a). The characteristics of metallic silver (Ag°) were indicated
throughput biomaterials characterizations revealed the successful 357
296 by XPS based bimodal peak of Ag 3d were observed at 374.84 (AgI)
fabrication of the biogenic NPs. Particularly characterization anal- 358
297 and 368.82 (AgIII) could be were indexed as Ag 3d 5/2, and Ag 3d 3/2.
ysis of XPS and EDS results strongly evidenced the formation of 359
298 Another two peaks were located in 373.94 eV (AgII) and (367.95 eV
Ag-MgONPs by bonding of Ag-O-Mg. The cytotoxicity assays 360
299 AgIV) as a characteristics for Ag+ (AgAO) (Fig. 4b) [36,54]. The Mg
revealed Ag-MgONPs induced cell death in PC-3 through the acti- 361
300 1 s scan showed the bimodal peaks at 1303.23 eV, 1303.99 and
vation of ROS production followed by cellular and nucleus damage. 362
301 1304.83 which correspondence to Mg2+ (Fig. 4c). This results
This work is worthy for the further study on molecular mecha- 363
302 clearly revealed that the Ag and Mg existed in Ag-MgONPs as a
nisms of Ag-MgONPs induced cancer cell death. 364
303 chemical state of Ag° and Mg2+. Also, the XPS analysis of Ag-
304 MgONPs was showed the MgO related oxygen was evidenced with
305 the peak values of 531.46 eV, 532.38 eV, and 533.46 eV (Fig. 4d) Conflict of interest 365

306 which in accordance with earlier work [36]. In case of the survey
307 scan spectrum of MgONPs were showed the presence of the mag- The authors declare no competing financial or other conflicts of 366

308 nesium (Mg), sodium (Na), Oxygen (O), residual carbon (C) and alu- interests. 367

309 minum (Al) (Supplementary Fig. 6a). The high-resolution survey

310 scan displayed the peaks located at 1302.49 eV, 1303.32 eV, and Acknowledgment 368
311 1304.41 indexed the Mg2+ which existed in MgONPs (Supplemen-
312 tary Fig. 6b), whereas the O1s scan showed the peaks at 530.47 eV, This work was supported by Korea Research Fellowship Pro- 369
313 531.31 eV, 532.27 eV and 535.35 eV was corresponding to lattice gram through the National Research Foundation of Korea (NRF) 370
314 oxygen Mg (Supplementary Fig. 6c) and which is similar to previ- funded by the Ministry of Science, ICT and Future Planning 371
315 ous reports [36]. Overall the XPS results indicated that the (2017H1D3A1A01052610). 372
316 absorbed and lattices oxygen of MgO probably assigned the forma-
317 tion of the Ag-MgO NPs as Mg-O----Ag; AgAO bond [36,55,56].
Appendix A. Supplementary material 373

318 3.4. Cytotoxicity assay

Supplementary data to this article can be found online at 374
https://doi.org/10.1016/j.apt.2019.01.007. 375
319 Cytotoxicity of these two metallic NPs was tested against PC-3
320 using WST assay. The cell viability was significantly decreased with
References 376
321 the decrease of the NPs concentration (Supplementary Fig. 7). The
322 calculated inhibitory concentration (IC50) was 125 µgmL1 for Ag- [1] A. Afkham, L. Aghebati-Maleki, H. Siahmansouri, S. Sadreddini, M. Ahmadi, S. 377
323 MgNPs and 218.75 µgmL1 for MgONPs. This results demonstrated Dolati, N.M. Afkham, P. Akbarzadeh, F. Jadidi-Niaragh, V. Younesi, M. Yousefi, 378
324 the Ag-MgNPs induced the more cell death than the MgONPs due Chitosan (CMD)-mediated co-delivery of SN38 and Snail-specific siRNA as a 379
useful anticancer approach against prostate cancer, Pharmacol. Rep. 70 (2018) 380
325 to the presence of the silver ions which reported as a cytotoxic 418–425. 381
326 agent against various cancer cells lines such as HT 29 cells [57], [2] H. Padinjarathil, M.M. Joseph, B.S. Unnikrishnan, G.U. Preethi, R. Shiji, M.G. 382
327 A549, Hep2, HeLa, COLO 205 and SH-SY5Y [58]. Moreover few ear- Archana, S. Maya, H.P. Syama, T.T. Sreelekha, Galactomannan endowed 383
biogenic silver nanoparticles exposed enhanced cancer cytotoxicity with 384
328 lier studies are indicated that the silver-doped NPs easily penetrate 385
excellent biocompatibility, Int. J. Biol. Macromol. 118 (2018) 1174–1182.
329 through the cell membrane and induce the free radicals, ROS pro- [3] D. Datta, M. Aftabuddin, D.K. Gupta, S. Raha, P. Sen, Human prostate cancer 386
330 duction and trigger the nucleus damage [58]. Hence in the present hallmarks map, Sci. Rep. 6 (2016) 30691. 387
[4] M. Rahimi-Gorji, T.B. Gorji, M. Gorji-Bandpy, Details of regional particle 388
331 study, we further studied the effect of IC50 concentrations of these 389
deposition and airflow structures in a realistic model of human
332 two NPs on cell death by AO/EB staining, ROS generation followed tracheobronchial airways: two-phase flow simulation, Comput. Biol. Med. 74 390
333 by nucleus damage by DAPI staining. The results exhibited that (2016) 1–17. 391

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392 [5] M. Rahimi-Gorji, O. Pourmehran, M. Gorji-Bandpy, T.B. Gorji, CFD simulation of [30] Z.-X. Tang, B.-F. Lv, MgO nanoparticles as antibacterial agent: preparation and 478
393 airflow behavior and particle transport and deposition in different breathing activity, Braz. J. Chem. Eng. 31 (2014) 591–601. 479
394 conditions through the realistic model of human airways, J. Mol. Liq. 209 [31] S.M. Dizaj, F. Lotfipour, M. Barzegar-Jalali, M.H. Zarrintan, K. Adibkia, 480
395 (2015) 121–133. Antimicrobial activity of the metals and metal oxide nanoparticles, Mater. 481
396 [6] M. Prasad, U.P. Lambe, B. Brar, I. Shah, J. Manimegalai, K. Ranjan, R. Rao, S. Sci. Eng., C 44 (2014) 278–284. 482
397 Kumar, S. Mahant, S.K. Khurana, H.M.N. Iqbal, K. Dhama, J. Misri, G. Prasad, [32] J.K. Salem, I.M. El-Nahhal, T.M. Hammad, S. Kuhn, S.A. Sharekh, M. El-Askalani, 483
398 Nanotherapeutics: an insight into healthcare and multi-dimensional R. Hempelmann, Optical and fluorescence properties of MgO nanoparticles in 484
399 applications in medical sector of the modern world, Biomed. Pharmacother. micellar solution of hydroxyethyl laurdimonium chloride, Chem. Phys. Lett. 485
400 97 (2018) 1521–1537. 636 (2015) 26–30. 486
401 [7] Z. Tang, Y. Zhou, H. Sun, D. Li, S. Zhou, Biodegradable magnetic calcium [33] M. Akter, M.T. Sikder, M.M. Rahman, A.K.M.A. Ullah, K.F.B. Hossain, S. Banik, T. 487
402 phosphate nanoformulation for cancer therapy, Eur. J. Pharm. Biopharm. 87 Hosokawa, T. Saito, M. Kurasaki, A systematic review on silver nanoparticles- 488
403 (2014) 90–100. induced cytotoxicity: physicochemical properties and perspectives, J. Adv. Res. 489
404 [8] J. Kim, Y. Piao, T. Hyeon, Multifunctional nanostructured materials for 9 (2018) 1–16. 490
405 multimodal imaging, and simultaneous imaging and therapy, Chem. Soc. [34] S.-J. Yu, Y.-G. Yin, J.-F. Liu, Silver nanoparticles in the environment, Environ. 491
406 Rev. 38 (2009) 372–390. Sci. Processes Impacts 15 (2013) 78–92. 492
407 [9] A. Raza, U. Hayat, T. Rasheed, M. Bilal, H.M.N. Iqbal, ‘‘Smart” materials-based [35] V. Edwards‐Jones, The benefits of silver in hygiene, personal care and 493
408 near-infrared light-responsive drug delivery systems for cancer treatment: a healthcare, Lett. Appl. Microbiol. 49 (2009) 147–152. 494
409 review, J. Mater. Res. Technol. (2018), https://doi.org/10.1016/j. [36] Y. Cai, D. Wu, X. Zhu, W. Wang, F. Tan, J. Chen, X. Qiao, X. Qiu, Sol-gel 495
410 jmrt.2018.03.007. preparation of Ag-doped MgO nanoparticles with high efficiency for bacterial 496
411 [10] K. Kathiresan, N.M. Alikunhi, S. Pathmanaban, A. Nabikhan, S. Kandasamy, inactivation, Ceram. Int. 43 (2017) 1066–1072. 497
412 Analysis of antimicrobial silver nanoparticles synthesized by coastal strains of [37] T. Jin, Y. He, Antibacterial activities of magnesium oxide (MgO) nanoparticles 498
413 Escherichia coli and Aspergillus niger, Can. J. Microbiol. 56 (2010) 1050–1059. against foodborne pathogens, J. Nanopart. Res. 13 (2011) 6877–6885. 499
414 [11] A. Arévalo-Gallegos, J.S. Garcia-Perez, D. Carrillo-Nieves, R.A. Ramirez- [38] S. Pajaniradje, K. Mohankumar, R. Pamidimukkala, S. Subramanian, R. 500
415 Mendoza, H.M. Iqbal, R. Parra-Saldívar, Botryococcus braunii as a bioreactor Rajagopalan, Antiproliferative and apoptotic effects of sesbania grandiflora 501
416 for the production of nanoparticles with antimicrobial potentialities, Int. J. leaves in human cancer cells, BioMed. Res. Int. 2014 (2014) 474953. 502
417 Nanomedicine 13 (2018) 5591–5604. [39] G.-Z. Jiang, J.-C. Li, Protective effects of ginsenoside Rg1 against colistin sulfate- 503
418 [12] M. Bilal, T. Rasheed, H.M.N. Iqbal, C. Li, H. Hu, X. Zhang, Development of silver induced neurotoxicity in PC12 cells, Cell. Mol. Neurobiol. 34 (2014) 167–172. 504
419 nanoparticles loaded chitosan-alginate constructs with biomedical [40] R. Mohanraj, Chapter 4 - antimicrobial activities of metallic and metal oxide 505
420 potentialities, Int. J. Biol. Macromol. 105 (2017) 393–400. nanoparticles from plant extracts, in: A.M. Grumezescu (Ed.), Antimicrobial 506
421 [13] M. Bilal, T. Rasheed, H.M.N. Iqbal, H. Hu, W. Wang, X. Zhang, Macromolecular Nanoarchitectonics, Elsevier, 2017, pp. 83–100. 507
422 agents with antimicrobial potentialities: a drive to combat antimicrobial [41] W.J. Stark, P.R. Stoessel, W. Wohlleben, A. Hafner, Industrial applications of 508
423 resistance, Int. J. Biol. Macromol. 103 (2017) 554–574. nanoparticles, Chem. Soc. Rev. 44 (2015) 5793–5805. 509
424 [14] K. Saravanakumar, R. Chelliah, D. MubarakAli, E. Jeevithan, D.-H. Oh, K. [42] J. Suresh, G. Pradheesh, V. Alexramani, M. Sundrarajan, S.I. Hong, Green, 510
425 Kathiresan, M.-H. Wang, Fungal enzyme-mediated synthesis of chitosan synthesis and characterization of hexagonal shaped MgO nanoparticles using 511
426 nanoparticles and its biocompatibility, antioxidant and bactericidal insulin plant (Costus pictus D. Don) leave extract and its antimicrobial as well 512
427 properties, Int. J. Biol. Macromol. 118 (2018) 1542–1549. as anticancer activity, Adv. Powder Technol. 29 (2018) 1685–1694. 513
428 [15] R. Tahir, B. Muhammad, L. Chuanlong, M.N.I. Hafiz, Biomedical potentialities of [43] K. Saravanakumar, C. Yu, K. Dou, M. Wang, Y. Li, J. Chen, Synergistic effect of 514
429 taraxacum officinale-based nanoparticles biosynthesized using methanolic Trichoderma-derived antifungal metabolites and cell wall degrading enzymes 515
430 leaf extract, Curr. Pharm. Biotechnol. 18 (2017) 1116–1123. on enhanced biocontrol of Fusarium oxysporum f. sp. Cucumerinum, Biol. 516
431 [16] T. Rasheed, M. Bilal, H.M.N. Iqbal, C. Li, Green biosynthesis of silver Control 94 (2016) 37–46. 517
432 nanoparticles using leaves extract of artemisia vulgaris and their potential [44] K. Saravanakumar, R. Vivek, N. Sithranga Boopathy, L. Yaqian, K. Kathiresan, J. 518
433 biomedical applications, Colloids Surf. B: Biointerfaces 158 (2017) 408–415. Chen, Anticancer potential of bioactive 16-methylheptadecanoic acid methyl 519
434 [17] K. Saravanakumar, R. Chelliah, S. Shanmugam, N.B. Varukattu, D.-H. Oh, K. ester derived from marine Trichoderma, J. Appl. Biomed. 13 (2015) 199–212. 520
435 Kathiresan, M.-H. Wang, Green synthesis and characterization of biologically [45] K. Saravanakumar, S. Mandava, R. Chellia, E. Jeevithan, R.S. Babu Yelamanchi, 521
436 active nanosilver from seed extract of Gardenia jasminoides Ellis, J. D. Mandava, W. Wen-Hui, J. Lee, D.-H. Oh, K. Kathiresan, M.-H. Wang, Novel 522
437 Photochem. Photobiol., B 185 (2018) 126–135. metabolites from Trichoderma atroviride against human prostate cancer cells 523
438 [18] A. Nabikhan, S. Kandasamy, K. Kandasamy, Fabrication of antimicrobial cotton and their inhibitory effect on Helicobacter pylori and Shigella toxin producing 524
439 fabrics by treating with biogenic metal nanoparticles and their effect on Escherichia coli, Microb. Pathog. 126 (2019) 19–26. 525
440 clinical pathogens, Pharm. Nanotechnol. 3 (2015) 19–25. [46] P.K. Seetharaman, R. Chandrasekaran, S. Gnanasekar, G. Chandrakasan, M. 526
441 [19] M. Bilal, T. Rasheed, H. Iqbal, H. Hu, X. Zhang, Silver nanoparticles: Gupta, D.B. Manikandan, S. Sivaperumal, Antimicrobial and larvicidal activity 527
442 biosynthesis and antimicrobial potentialities, Int. J. Pharmacol. 13 (2017) of eco-friendly silver nanoparticles synthesized from endophytic fungi 528
443 832–845. Phomopsis liquidambaris, Biocatal. Agric. Biotechnol. 16 (2018) 22–30. 529
444 [20] M.K. Patel, M. Zafaryab, M.M.A. Rizvi, V.V. Agrawal, Z.A. Ansari, B.D. Malhotra, [47] K. Saravanakumar, M.-H. Wang, Trichoderma based synthesis of anti- 530
445 S.G. Ansari, Antibacterial and cytotoxic effect of magnesium oxide pathogenic silver nanoparticles and their characterization, antioxidant and 531
446 nanoparticles on bacterial and human cells, J. Nanoengineering cytotoxicity properties, Microb. Pathog. 114 (2018) 269–273. 532
447 Nanomanufacturing 3 (2013) 162–166. [48] K. Saravanakumar, D. MubarakAli, K. Kathiresan, N. Thajuddin, N.S. Alharbi, J. 533
448 [21] L. Jahangiri, M. Kesmati, H. Najafzadeh, Evaluation of analgesic and anti- Chen, Biogenic metallic nanoparticles as catalyst for bioelectricity production: 534
449 inflammatory effect of nanoparticles of magnesium oxide in mice with and a novel approach in microbial fuel cells, Mater. Sci. Eng., B 203 (2016) 27–34. 535
450 without ketamine, Eur. Rev. Med. Pharmacol. Sci. 17 (2013) 2706–2710. [49] K. Saravanakumar, S. Shanmugam, N.B. Varukattu, D. MubarakAli, K. 536
451 [22] S. Moeini-Nodeh, M. Rahimifard, M. Baeeri, M. Abdollahi, Functional Kathiresan, M.-H. Wang, Biosynthesis and characterization of copper oxide 537
452 improvement in rats’ pancreatic islets using magnesium oxide nanoparticles nanoparticles from indigenous fungi and its effect of photothermolysis on 538
453 through antiapoptotic and antioxidant pathways, Biol. Trace Elem. Res. 175 human lung carcinoma, J. Photochem. Photobiol., B 190 (2019) 103–109. 539
454 (2017) 146–155. [50] V. Srivastava, Y.C. Sharma, M. Sillanpää, Green synthesis of magnesium oxide 540
455 [23] K. Krishnamoorthy, J.Y. Moon, H.B. Hyun, S.K. Cho, S.-J. Kim, Mechanistic nanoflower and its application for the removal of divalent metallic species 541
456 investigation on the toxicity of MgO nanoparticles toward cancer cells, J. from synthetic wastewater, Ceram. Int. 41 (2015) 6702–6709. 542
457 Mater. Chem. 22 (2012) 24610–24617. [51] G. Sharma, R. Soni, N.D. Jasuja, Phytoassisted synthesis of magnesium oxide 543
458 [24] H. Elmotasem, H.K. Farag, A.A.A. Salama, In vitro and in vivo evaluation of an nanoparticles with Swertia chirayaita, J. Taibah Univ. Sci. 11 (2017) 471–477. 544
459 oral sustained release hepatoprotective caffeine loaded w/o Pickering [52] L. Jianping, Q. Longzhen, Q. Baojun, Controlled synthesis of magnesium 545
460 emulsion formula – containing wheat germ oil and stabilized by magnesium hydroxide nanoparticles with different morphological structures and related 546
461 oxide nanoparticles, Int. J. Pharm. 547 (2018) 83–96. properties in flame retardant ethylene–vinyl acetate blends, Nanotechnology 547
462 [25] D.-R. Di, Z.-Z. He, Z.-Q. Sun, J. Liu, A new nano-cryosurgical modality for tumor 15 (2004) 1576. 548
463 treatment using biodegradable MgO nanoparticles, Nanomedicine: [53] Y.Ö. Altıntasß, U.H. Emrah, D. Caner, Highly efficient room temperature 549
464 Nanotechnol. Biol. Med. 8 (2012) 1233–1241. synthesis of silver-doped zinc oxide (ZnO:Ag) nanoparticles: structural, 550
465 [26] C.J. Hewitt, S.R. Bellara, A. Andreani, G. Nebe-von-Caron, C.M. McFarlane, An optical, and photocatalytic properties, J. Am. Ceram. Soc. 96 (2013) 766–773. 551
466 evaluation of the anti-bacterial action of ceramic powder slurries using multi- [54] R. Janardhanan, M. Karuppaiah, N. Hebalkar, T.N. Rao, Synthesis and surface 552
467 parameter flow cytometry, Biotechnol. Lett. 23 (2001) 667–675. chemistry of nano silver particles, Polyhedron 28 (2009) 2522–2530. 553
468 [27] Y.H. Leung, A.M. Ng, X. Xu, Z. Shen, L.A. Gethings, M.T. Wong, C.M. Chan, M.Y. [55] J.T. Newberg, D.E. Starr, S. Yamamoto, S. Kaya, T. Kendelewicz, E.R. Mysak, S. 554
469 Guo, Y.H. Ng, A.B. Djurišić, P.K. Lee, Mechanisms of antibacterial activity of Porsgaard, M.B. Salmeron, G.E. Brown, A. Nilsson, H. Bluhm, Formation of 555
470 MgO: non-ROS mediated toxicity of MgO nanoparticles towards escherichia hydroxyl and water layers on MgO films studied with ambient pressure XPS, 556
471 coli, Small 10 (2014) 1171–1183. Surf. Sci. 605 (2011) 89–94. 557
472 [28] J. Sawai, H. Kojima, H. Igarashi, A. Hashimoto, S. Shoji, T. Sawaki, A. Hakoda, E. [56] Y. Rao, W. Wang, F. Tan, Y. Cai, J. Lu, X. Qiao, Sol–gel preparation and 558
473 Kawada, T. Kokugan, M. Shimizu, Antibacterial characteristics of magnesium antibacterial properties of Li-doped MgO nanoplates, Ceram. Int. 40 (2014) 559
474 oxide powder, World J. Microbiol. Biotechnol. 16 (2000) 187–194. 14397–14403. 560
475 [29] K. Krishnamoorthy, G. Manivannan, S.J. Kim, K. Jeyasubramanian, M. [57] R. Sriranjani, B. Srinithya, V. Vellingiri, P. Brindha, S.P. Anthony, A. 561
476 Premanathan, Antibacterial activity of MgO nanoparticles based on lipid Sivasubramanian, M.S. Muthuraman, Silver nanoparticle synthesis using 562
477 peroxidation by oxygen vacancy, J. Nanoparticle Res. 14 (2012) 1063.

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563 Clerodendrum phlomidis leaf extract and preliminary investigation of its [60] K. Satyavani, S. Gurudeeban, T. Ramanathan, T. Balasubramanian, Toxicity 571
564 antioxidant and anticancer activities, J. Mol. Liq. 220 (2016) 926–930. study of silver nanoparticles synthesized from suaeda monoica on Hep-2 cell 572
565 [58] J.R. Nakkala, R. Mata, S.R. Sadras, Green synthesized nano silver: synthesis, line, Avicenna J. Med. Biotechnol. 4 (2012) 35–39. 573
566 physicochemical profiling, antibacterial, anticancer activities and biological [61] M. Somayyeh, A. Hammed, S. Delavar, A. Motallebi, A. Ali Anvar, R.-N. Jafar, M. 574
567 in vivo toxicity, J. Colloid Interface Sci. 499 (2017) 33–45. Reza, Shokrgozar, toxicity study of nanosilver (NanocidÒ) on osteoblast, Cancer 575
568 [59] S. Rajeshkumar, C. Malarkodi, M. Vanaja, G. Annadurai, Anticancer and Cell Line (2011). 576
569 enhanced antimicrobial activity of biosynthesizd silver nanoparticles against [62] M.M. El-Sheekh, H.Y. El-Kassas, Algal production of nano-silver and gold: their 577
570 clinical pathogens, J. Mol. Struct. 1116 (2016) 165–173. antimicrobial and cytotoxic activities: a review, J. Genet. Eng. Biotechnol. 14 578
(2016) 299–310. 579
580

Please cite this article as: K. Saravanakumar and M. H. Wang, Biogenic silver embedded magnesium oxide nanoparticles induce the cytotoxicity in human
prostate cancer cells, Advanced Powder Technology, https://doi.org/10.1016/j.apt.2019.01.007