Materials Science & Engineering C 92 (2018) 508–517

Contents lists available at ScienceDirect

Materials Science & Engineering C

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

Preparation of sulfur nanoparticles and their antibacterial activity and T

cytotoxic effect
⁎
Shiv Shankara, Rudra Pangenib, Jin Woo Parkb, Jong-Whan Rhima,
a
Center for Humanities and Sciences, Bionanocomposite Research Center, Department of Food and Nutrition, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu,
Seoul 02447, Republic of Korea
b
Department of Pharmacy, College of Pharmacy and Natural Medicine Research Institute, Mokpo National University, Muan-gun, Jeonnam 58554, Republic of Korea

A R T I C LE I N FO A B S T R A C T

Keywords: Sulfur nanoparticles (SNPs) were prepared using sodium thiosulfate and hydrochloric acid, and the UV–visible
Sulfur nanoparticles spectrum showed the formation of nanoparticulate sulfur. The SNPs were characterized by UV–visible spec-
Chitosan trophotometer, transmission electron microscope (TEM), energy dispersive X-ray spectroscopy (EDX), X-ray
Antibacterial photoelectron spectroscopy (XPS), X-ray diffraction (XRD), and thermogravimetric analysis (TGA). The anti-
Anticancer
bacterial activity and the cytotoxic effects of the SNPs on the human lung carcinoma (A549), mouse colon
Cytotoxicity
carcinoma (CT26), Caco-2, and human fibroblast (CCD-986sk) cells were tested. In addition, the inhibitory effect
of the SNPs on the cancer cell migration was evaluated. The SNPs capped with chitosan (SNP2) exhibited strong
antibacterial activity against Escherichia coli and Staphylococcus aureus. SNP2 also effectively inhibited the
proliferation and migration of cancer cells with minimal toxic effect on normal cells. SNP2 therefore has po-
tential for medical applications, including those used as antibacterial and chemotherapeutic agents.

1. Introduction
In recent centuries, cancer has been a major challenge to public
health. Numerous research works have been focused on the develop-
ment of novel anticancer agents that could selectively induce cell death
or inhibit the growth of cancer cells [1]. Recent breakthroughs in the
nanotechnology have facilitated the development of nanomedicine for
cancer therapy exploiting the unique properties of nanomaterials [2,3].
For instance, nanomaterials such as gold, silver, silica, and carbon na-
noparticles have been intensively explored as drug carriers [4–7].
Sulfur and its plethora of chemically diverse organic and inorganic
compounds are known to exhibit a broad and often diverse spectrum of
biological activities, ranging from antioxidant action to antimicrobial
and even anticancer properties. Sulfur nanoparticles (SNPs) are widely
used as antimicrobial agents, in lithium‑sulfur batteries, and in sulfur-
based photocatalyst [8–13]. However, the application of SNPs for bio-
medical applications such as anticancer agents and drug delivery
system has been limited. Moreover, the large particle size of SNPs, low
reactive activity, and toxicity are the main limiting factors for the use of
a drug delivery agent [14]. To overcome such problems, the surface of
the SNPs should be modified.
Chitosan showed promising features like an auxiliary agent for a
drug delivery system in addition to the applications such as slimming,
wound dressing, and tissue engineering [15]. Chitosan is the only one
natural biopolymer that exhibits a cationic character due to its primary
amino groups, which are responsible for various properties and subse-
quently for its use in drug delivery systems [15]. The surface active
property of chitosan and biological compatibility either in vitro or in
vivo have shed light on its use as a drug delivery molecule that can
improve the stability of nanoparticles (NPs) through conferring steric
stabilization and offering well-dispersed size and homogenous NPs
without aggregation. One of the most valuable properties of chitosan is
its inherent antibacterial action, mainly due to its cationic nature [16].
In addition, chitosan and chitosan derivatives exhibit anticancer ac-
tivity and are used as anticancer drug carriers [17].
Therefore, the present study was focused on the preparation of well-
dispersed SNPs with uniform size and low toxicity using chitosan as
capping and stabilizing agents. The synthesized SNPs were character-
ized using various analytical techniques. The anticancer activity and
cell toxicity of synthesized SNPs were estimated using various cell lines.
2. Materials and methods
2.1. Materials
Reagent grade chemicals such as sodium thiosulfate pentahydrate

⁎
Corresponding author.
E-mail address: jwrhim@khu.ac.kr (J.-W. Rhim).

https://doi.org/10.1016/j.msec.2018.07.015
Received 2 November 2017; Received in revised form 22 June 2018; Accepted 5 July 2018
Available online 06 July 2018
0928-4931/ © 2018 Elsevier B.V. All rights reserved.
S. Shankar et al.
and hydrochloric acid were procured from Sigma-Aldrich (St. Louis,
MO, USA). Chitosan (CS-001, viscosity of 110 cp in 1% acetic acid so-
lution at 25 °C and degree of acetylation 90%) was obtained from Fine
Agar Co., Ltd. (Damyang, Jeonnam, Korea), Kanto Chemical Co.
(Tokyo, Japan). Tryptic soy broth (TSB), brain heart infusion broth
(BHI), and agar powder were purchased from Duksan Pure Chemicals
Co., Ltd. (Ansan, Gyeonggi-do, Korea). The cell lines, human lung
carcinoma (A549), mouse colon carcinoma (CT26), human epithelial
colorectal adenocarcinoma (Caco-2), and human fibroblast (CCD-
986sk), were obtained from American Type Culture Collection
(Manassas, VA, USA). The pathogenic test microorganisms such as
Escherichia coli ATCC 25922 and Staphylococcus aureus were obtained
from Korean Collection for Type Cultures (KCTC, Seoul, Korea).
2.2. Synthesis of sulfur nanoparticles (SNPs)
Sodium thiosulphate (STS) solution was prepared by dissolving
2.482 g of solid STS pentahydrate (MW: 248.18) in 900 mL double
distilled water. For the synthesis of sulfur nanoparticles (SNP1), 100 mL
of 0.2 M HCl was added to 900 mL of the STS solution with stirring at
25 °C. For the synthesis of chitosan capped sulfur nanoparticles (SNP2),
2.482 g of STS was first dissolved in 900 mL of distilled water, followed
by the addition of 100 mL of 0.2 M HCl. Then 50 mL of 0.5% chitosan
solution (1% v/v acetic acid solution) was added as a capping agent.
The ratio of molar concentration of STS to HCl was kept 1:2 for all the
tests.
In an acidic solution, sodium thiosulphate undergoes through a
disproportionation reaction to sulfur and sulfonic acid according to
following equation:
Na2S2 O3 + 2HCl → 2NaCl + SO2 + S ↓ + H2 O
SO2 + H2 O → H2 SO3
After mixing the reactants, the reaction was kept for 40 min in a
bath-type sonicator (Ultrasonic Cleaner FS140H, Fisher Scientific,
Pittsburgh, PA, USA) for the completion of the reaction. The SNP1 was
centrifuged and washed with double distilled water for 5–6 times until
the pH of sulfur nanoparticles suspension reached to neutral. In the case
of SNP2, the nanoparticles suspension was centrifuged and washed with
1% (v/v) acetic acid solution to remove unbound chitosan, followed by
washing with double distilled water until the pH of the suspension
reached to neutral.
2.3. Characterization of sulfur nanoparticles
The formation of SNPs was confirmed by the absorption of light
using a UV–visible spectrophotometer (Mecasys Optizen POP Series
UV/Vis, Seoul, Korea). The morphological evaluation of the size and
shape of the SNPs was performed using a transmission electron mi-
croscopy (TEM). For this, 10 μL of sample was dropped on the carbon-
coated copper grid and allowed to dry. The image analysis of SNPs was
performed using JEOL-1010 TEM instrument (Tokyo, Japan) operated
at an accelerating voltage of 200 kV. The particle size distribution of
SNPs was performed using ImageJ software. Elemental analysis of SNPs
was measured by energy dispersive X-ray spectroscopy (EDX) on SEM
instruments (FE-SEM, S-4800, Hitachi Co., Ltd., Matsuda, Japan). X-ray
photoelectron spectroscopy (XPS) measurements were carried out using
a Thermo K-Alpha XPS instrument (Thermo Scientific, NY, USA) at a
pressure of ~1 × 10−9 Torr (1 Torr = 1.333 × 102 Pa). The core-level
spectra from the respective samples were recorded with mono-
chromated aluminum κα radiation (photon energy = 1486.6 eV) at a
pass energy of 20 eV and an electron takeoff angle (angle between the
electron emission direction and surface plane) of 90°. The core level
binding energy was aligned with the C1s (adventitious carbon) binding
energy of 284.6 eV. For the X-ray diffraction (XRD) pattern of the SNPs,
a drop of the sample was cast on a glass slide, air dried and analyzed
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Materials Science & Engineering C 92 (2018) 508–517
Fig. 1. UV–visible spectra of SNPs.
using X-ray diffractometer (PANalytical X'pert pro-MRD diffractometer,
Amsterdam, Netherlands). The spectra were recorded using Cu Kα ra-
diation (wavelength of 0.1541 nm) and a nickel monochromator fil-
tering wave at 40 kV and 30 mA. The diffraction pattern was obtained
at diffraction angles in the range of 2θ = 30–80° with a scanning speed
of 0.4°/min. Fourier transform infrared (FTIR) spectra of chitosan and
SNP2 were recorded by the ATR-FTIR method using an FTIR spectro-
meter (TENSOR 37 spectrophotometer with OPUS 6.0 software,
Billerica, MA, USA) from 4000 to 400 cm−1 with an average resolution
of 32 scans at 4 cm−1. Thermal stability of SNPs was evaluated using a
thermogravimetric analyzer (Hi-Res TGA 2950, TA Instrument, New
Castle, DE, USA). About 10 mg of each sample was taken in a standard
aluminum pan and heated from 30 °C to 600 °C at a heating rate of
10 °C/min under a nitrogen flow of 50 cm3/min. The derivative of TGA
(DTG) was calculated using a central finite difference method, and the
char content of the samples at 600 °C was determined from the TGA
curve. The surface charge of NPs was measured with zeta potential and
particle size using a dynamic light scattering (DLS) method by using
Zeta PALS-zeta potential analyzer (Brookhaven Instruments
Corporation, NY, USA).
2.4. Antibacterial activity
The antimicrobial activity regarding the minimum inhibitory con-
centration (MIC) and minimum bactericidal concentration (MBC) of
NPs against Gram-positive (S. aureus) and Gram-negative (E. coli) pa-
thogenic bacteria was performed using a modified broth microdilution
method recommended by the Clinical Laboratory Standardization
Institute (CLSI) guideline [18,19]. Aliquots (50 μL) of two-fold serially
diluted SNPs and chitosan suspensions were prepared in a 96-well
Microtiter plate to obtain final concentration ranged from 2 to 1024 μg/
mL, followed by the addition of 50 μL of TSB or BHI media. Chitosan
was dissolved in 1% v/v acetic acid solution and serially diluted using
TSB or BHI broths to get the desired concentrations. One hundred mi-
croliters of the bacteria (~106 CFU/mL) was added to each well. After
incubation at 37 °C for 15 h, 10 μL of resazurin (0.4 mg/mL solution)
(Sigma-Aldrich, St. Louis, MO, USA) as a redox indicator was added to
each well and incubated for 3 h. The growth was assessed using a mi-
crotitre plate reader by measuring the optical density (OD) at 595 nm.
The minimum inhibitory concentration (MIC) was defined as the lowest
concentration of the SNPs or chitosan suspension that inhibited the
bacterial growth completely. MBC, the minimum concentration of the
SNPs or chitosan to prevent the bacterial growth completely, was de-
termined with the SNPs that gave MIC by subculturing on fresh TSB and
BHI agar plates. All assays were performed in triplicates.
S. Shankar et al. Materials Science & Engineering C 92 (2018) 508–517

(a)

(b) SNP1 SNP2

(c) SNP1 SNP2

Fig. 2. (a) TEM micrographs, (b) particle size distribution histograms, and (c) EDX spectra of SNPs.

Table 1
Particle size, polydispersity index, zeta potential, and antibacterial activity of SNPs.
Sample Particle size by TEM (nm) Particle size by DLS (nm) Polydispersity index Zeta Potential (mV) MIC/MBC (μg/mL)

E. coli S. aureus

SNP1 10–70 230.9 ± 70.8 0.22 ± 0.08 −29.9 ± 7.9 256/ > 512 128/512
SNP2 10–30 129.6 ± 15.2 0.17 ± 0.05 32.6 ± 0.7 16/64 16/32
Chitosan ND ND ND ND > 512/ > 512 > 512/ > 512

ND = not detected.

2.5. In vitro cytotoxicity assay
Cell proliferation assay was performed to evaluate the dose-depen-
dent cytotoxicity of the prepared SNPs. A549, CT26, Caco-2, and CCD-
986sk cells were seeded at a density of 5 × 103 or 1 × 103 cells per well
in 100 μL of Dulbecco's modified Eagle's medium (DMEM; Lonza,
Zurich, Switzerland) containing 10% fetal bovine serum (FBS; Thermo
Fisher Scientific Inc., Waltham, MA) and 1% penicillin/streptomycin
(Thermo Fisher Scientific Inc.), and then incubated at 37 °C for 24 h.
The cells were treated with different concentrations of STS as controls
or SNPs, and incubated for further 48 h. Cell viability was evaluated by
WST-1 assay (Roche Diagnostics, Mannheim, Germany) performed in
accordance with the manufacturer's instructions. Briefly, 10 μL of di-
luted WST-1 solution (5 mg/mL in phosphate-buffered saline [PBS])
was added to each well and incubated for 2 h. The absorbance was then
measured at 450 nm with a microplate reader (multimode plate reader;

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(a) (b)

(c) (d)

(e)

Fig. 3. XPS of SNPs showing (a) S2p of SNP1 (b) S2p of SNP2 (c) C1s of SNP2 (d) O1s of SNP2 and (e) N1s of SNP2.

PerkinElmer, Waltham, MA) and the percentage cell viability was cal-
culated by comparing the values of treated cells with those of untreated
cells.
2.6. In vitro cell migration assay
In vitro cell migration assay was also performed to determine the
optimum concentration of SNPs to inhibit cancer cell migration. For
this purpose, 1 × 104 A549 cells were seeded per well in an Essen
ImageLock 96-well plate (Essen Bioscience, Ann Arbor, MI) and cul-
tured in complete medium at 37 °C for 48 h to form a confluent
monolayer. A cell-free zone (scratch wound) was created in each well
using a wound marker and then washed twice with PBS (pH 7.4). The
cells were further incubated at 37 °C with different concentrations of
STS or SNPs diluted from 10 to 1000 μg/mL with complete medium.
Cell migration was then examined every 12 h by phase contrast imaging
with an IncuCyte Zoom microscope (Essen Bioscience). IncuCyte Zoom
image analysis software was used to automatically detect the cell edges

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S. Shankar et al.
(a)
(b)
Fig. 4. (a) XRD pattern of SNPs and (b) FTIR of chitosan and SNP2.
and to generate an overlay mask, which was used to calculate the re-
lative cell migration rate.
2.7. Statistical analysis
For cytotoxicity and cell migration assays, one-way analysis of
variance (ANOVA) was performed followed by Tukey's multiple-com-
parison test using the SPSS software (SPSS Inc., Chicago, IL, USA).
3. Results and discussion
3.1. Synthesis and characterization of sulfur nanoparticles
During the synthesis of SNPs, color change in the reaction mixture
indicated the formation of SNPs. Initially the colorless solution of STS
became turbid with yellowish color after the formation of SNPs. The
progress in the formation of SNPs was also monitored using the
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Materials Science & Engineering C 92 (2018) 508–517
UV–visible spectrophotometer as shown in Fig. 1. The UV–visible
spectra of SNPs recorded in the range of 250–500 nm showed a clear
peak around 280 nm indicating the formation of SNPs [20]. Sur-
yavanshi et al. also found the maximum absorption peak of SNPs
around 290 nm [20].
According to the stoichiometry of the reaction, 1 mol of sodium
thiosulphate reacts with 2 mol of mono-basic acid to precipitate sulfur
[21]. The concentration of acid, capping agents, and ultrasonication
played a major role in determining the size of the SNPs. TEM was used
to determine the morphology (size and shape) of the nanoparticles, and
the resulting TEM images of the SNPs are shown in Fig. 2a. The size of
the sulfur nanoparticles without a capping agent was not uniform, the
average diameter was 45.05 ± 15.55 nm, and the size ranged from
10 nm to 70 nm. When chitosan was used as a capping agent, the SNPs
were uniform in size, and the size was in the range of 10–30 nm with a
mean diameter of 15.80 ± 7.82 nm (Fig. 2b). Choudhari and Paria
found the large particle size of SNPs (~1 μm) when STS was treated
S. Shankar et al.
(a)
(b)
Fig. 5. (a) TGA and (b) DTG thermograms of SNPs.
with HCl [21]. However, in the present study, the particle size of SNPs
was smaller than 100 nm, which might be due to the completion of the
reaction in the bath type sonicator. The ultrasonication treatment
prevented the particles from aggregation. However, the average par-
ticle size of the SNPs determined by the dynamic light scattering (DLS)
method was bigger than 100 nm (Table 1). The average particle size of
these nanoparticles estimated by the TEM analysis was smaller than
those determined by the DLS method, which was because the DLS
provides information on the hydrodynamic radius of particle in a so-
lution [18]. The zeta potential values of SNPs in milliQ water are also
presented in Table 1. SNP1 showed the zeta potential value of
−29.9 ± 7.9, however, SNP2 (chitosan capped SNPs) showed the zeta
potential value of 32.6 ± 0.7 which was attributed to the cationic
capping. The high value of zeta potential indicates the high stability of
SNPs in aqueous solution. The elemental analysis of SNP was performed
using EDX and the results are presented in Fig. 2c. The EDX spectrum of
the SNP shows a single peak around 2.4 keV, confirming the purity of
the SNP. For SNP2, low intensity carbon and oxygen peaks were found,
indicating the capping of chitosan on the sulfur nanoparticles.
Fig. 3 represents the XPS spectra of the S2p level of SNP1 and S2p,
C1s, O1s, and N1s levels of SNP2. The binding energy of S2p level
observed in the SNP1 was 164.01 and 165.18 eV. However, in the case
of SNP2, the binding energies of S2p level were 162.23, 163.53, 164.66,
168.1, and 169.22 eV. The change in binding energy of S2p in SNP2
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Materials Science & Engineering C 92 (2018) 508–517
indicates the interaction of sulfur with capped chitosan. In addition, the
presence of carbon, nitrogen, and oxygen indicates the capping of
chitosan on SNP2.
The powder X-ray diffraction (XRD) patterns of all the SNPs are
presented in Fig. 4a. All the SNPs were polycrystalline and exhibited the
characteristic diffraction peaks corresponding to the SNPs. The posi-
tions and intensities of the diffraction peaks are in good agreement with
the literature values for sulfur with S8 structure (74-1465 from JCPDS
PDF Number) [22,23].
Fig. 4b shows the FTIR spectra of chitosan and SNP2, showing the
structural changes in chitosan before and after capping of SNP2 with
chitosan. The absorption band from 3450 to 3200 cm−1 with a peak at
3290 cm−1 is assigned to eOH and eNH stretching vibrations in chit-
osan [24]. The band at 2923 and 2877 cm−1 attributed to the eCH
stretching. The band at 1640 cm−1 is due to C]O stretching band of
the acetyl group (amide I). The band at 1549 cm−1 assigned to eNH
bending and stretching (amide II) [25]. A peak at 1408 cm−1 associated
with OeH bending vibration and the peak at 1064 cm−1 assigned to
CeO stretching. A weaker peak at 1258 cm−1 assigned to the asym-
metric stretch of the CeOeC bridging. The absorption band at
1152 cm−1 and 899 cm−1 ascribed to the saccharide structure of chit-
osan [26]. There was a change in the intensity and location of some
peaks in SNP2, indicating the interaction of chitosan with SNP2.
3.2. Thermal stability of SNPs
Fig. 5 shows the TGA and DTG thermograms of the SNPs. All SNPs
exhibited two-step pattern of thermal degradations. The initial weight
change observed at 80-100 °C was due to the evaporation of moisture in
the SNPs. The main thermal degradation was observed between 300
and 400 °C which was presumably due to the partial degradation of
sulfur. The char content after 600 °C was between 45 and 50% of the
initial weight which indicated that the SNPs are thermostable not de-
graded completely at the tested temperature range.
3.3. Antibacterial activity of SNPs
Though antimicrobial activity of sulfur nanoparticles has been
suggested, not many works have been done on the evaluation of the
antibacterial potential of SNPs. Recently, Choudhury et al. reported that
nano-sized sulfur (SNP) exhibited antibacterial activity against tested
Gram-positive and Gram-negative bacteria, while the elemental sulfur
failed to show any bacterial growth inhibition [9]. However, Suleiman
et al. found that sulfur nanoparticles showed significant antibacterial
activity against the Gram-positive bacterium, i.e., S. aureus, but showed
no activity against E. coli [27]. Therefore, in the present study, E. coli
and S. aureus were chosen for the test and found all the SNPs were
active against both organisms (Table 1). However, the degree of anti-
bacterial activity depended on the type of SNPs. The SNP1 showed
MIC/MBC values of 256/ > 512 and 128/512 μg/mL against E. coli and
S. aureus, respectively. SNP2 showed MIC/MBC values of 16/64 and
16/32 μg/mL against E. coli and S. aureus, respectively. However,
chitosan exhibited MIC/MBC values of > 512/ > 512 and > 512/
> 512 μg/mL against E. coli and S. aureus, respectively. The high an-
tibacterial activity of SNP2 as compared to SNP1 might be due to the
cationic surface charge of SNP2 because of chitosan. The positive
charge of SNP2 might help for the binding of the SNP2 to the negatively
charged bacterial cell membrane. Though the exact mechanism of an-
tibacterial action of the SNPs is not known, it is believed that the SNPs
bind with bacterial cell wall and cause membrane rupture leading to
lysis of cell membrane and cell death [28].
3.4. Cytotoxicity assay
3.4.1. In vitro cytotoxic effect of SNPs on cancer cells
Cell viability assay was performed to evaluate the in vitro
S. Shankar et al. Materials Science & Engineering C 92 (2018) 508–517

Sodium thiosulphate
(a) SNP1
SNP2
110

100

90 **
***
**
Cell viability (%) 80
###
70 ***

60

50
$$$ ### ###
###
*** ***
40 *** $$$
###
***
30

20

10

0
0 10 50 100 200 500 1000

Concentration ( g/mL)

Sodium thiosulphate
(b) SNP1
SNP2
110

100
* *
* **
90
*** ###
80 *** ## ***
*** $
Cell viability (%)

###
70 ***
###
60
***
50 ***
### ###
$$$
40 ### *** *** *** ***
***
30

20

10

0
0 10 50 100 200 500 1000

Concentration ( g/mL)

Fig. 6. In vitro cytotoxic effect of sodium thiosulphate (STS) and sulfur nanoparticles (SNPs) on (a) A549 and (b) CT26 cells. Statistical analysis: one-way ANOVA
followed by Tukey's multiple-comparison test. Each value represents the mean ± standard deviation (n = 5 for each group). ⁎ p < 0.05, ⁎⁎ p < 0.01, ⁎⁎⁎ p < 0.001
compared to control. ## p < 0.01, ### p < 0.001 compared to STS. $ p < 0.05, $$$ p < 0.001 between SNP1 and SNP2.

cytotoxicity of the SNPs with different concentrations of A549 cells
(Fig. 6a). Cell viability was over 80% at 100 μg/mL of each SNP.
However, cell viability for SNP1 and SNP2 was significantly reduced at
concentrations above 200 μg/mL compared to STS. Cells treated with
500 μg/mL of SNP1 and SNP2 showed cell viability of 28.2% ± 2.7%
and 59.5% ± 8.1%, respectively. The cell viability was decreased by
60.3% and 51.7%, respectively, compared with STS (80.1% ± 3.7%)
at 1000 μg/mL of SNP1 and SNP2. However, STS did not severely in-
hibit A549 cell viability at the concentration ranging from 10 to
1000 μg/mL.
SNP1 and SNP2 also showed dose-dependent cytotoxic effects on
CT26 cells at the concentration above 50 μg/mL (Fig. 6b). SNP2 was the
most cytotoxic compared to STS, but SNPs did not significantly affect
cell viability at 10 μg/mL. However, dose-dependent inhibition of cell
viability was observed in CT26 cells treated with STS, SNP1, or SNP2.
Cells treated with 100 μg/mL STS, SNP1, and SNP2 showed decrease in
the cell viability of 86.3% ± 3.7%, 70.6% ± 3.8%, and
58.2% ± 9.5%, respectively. In addition, treatment with SNP1 and
SNP2 at 500 μg/mL showed cytotoxic effects of 1.65 and 1.63 times,
respectively, on CT26 cells compared to STS.
Previous studies have shown that copper is an essential cofactor for
the proliferation and survival of cancer cells by activation of mitogen-
activated protein kinase (MAPK)/extracellular activation kinase (ERK)/
kinase (MEK) [29]. Thus, the depletion of copper in the cells by che-
lating agents or copper traps has been suggested as a promising strategy
to improve cancer therapy, and several copper chelators have been

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Sodium thiosulphate
(a) SNP1
SNP2
110
##
###
100
*
** **
90 $$$
$$$ ***
***
80 *** ***
Cell viability (%) *** ***
#
70 *** ***
$$
##
60 ***

50
***
40 ***
$$$
###
$
30 *** #
***
20

10

0
0 10 50 100 200 500 1000

Concentration ( g/mL)

Sodium thiosulphate
SNP1
(b) SNP2
220
###
###
200 ***
***

180

160 ### ###

$
*** ***
Cell viability (%)

###
140 $
*
### $$$
$$$ ###
120 #
###

100 $$$
$$
* *
*** **
80

60

40 ***
*** ***

20

0
0 10 50 100 200 500 1000

Concentration ( g/mL)
Fig. 7. In vitro cytotoxic effects of STS and SNPs on (a) Caco-2 and (b) CCD-986sk cells. Statistical analysis: one-way ANOVA followed by Tukey's multiple-
comparison test. Each value represents the mean ± standard deviation (n = 5 for each group). ⁎ p < 0.05, ⁎⁎ p < 0.01, ⁎⁎⁎ p < 0.001 compared to control. #
p < 0.05, ## p < 0.01, ### p < 0.001 compared to STS. $ p < 0.05, $$ p < 0.01, $$$ p < 0.001 between SNP1 and SNP2.

shown to be effective in inhibiting tumor growth [30,31]. Liu et al. also
prepared polyethylene glycol-coated sulfur nanoparticles (Nano-S) and
showed that Nano-S could effectively detain copper, thus inhibiting the
proliferation of melanoma and breast cancer cells via inactivation of the
MEK/ERK pathway followed by selective deceleration of mitosis in
cancer cells [1]. Thus, the anti-proliferative activity of SNPs against
A549 and CT26 cells may also be due to the chelation of intracellular
copper and decrease in the expression of proteins responsible for mi-
tosis.
3.4.2. In vitro cytotoxic effects of SNPs on Caco-2 and CCD-986sk cells
Fig. 7a shows the in vitro cytotoxicity of different concentrations of
SNPs against Caco-2 cells. Cells treated with < 100 μg/mL of SNPs
exhibited > 80% cell viability. However, when SNP1 or SNP2 was
treated at a concentration of 200 μg/mL or more, cell viability was
significantly reduced. Cells treated with 200 μg/mL of SNP1 or SNP2
showed cell viability of 53.9% ± 4.4% and 69.6% ± 9.7%, which
decreased to 18.7% ± 4.0% and 32.4% ± 3.8% when treated with
1000 μg/mL, respectively. On the other hand, cell viability for STS re-
mained above 70% even when the concentration increased to 500 μg/
mL.
Fig. 7b shows the in vitro cytotoxicity of SNPs against CCD-986sk
cells. Here, the fibroblasts showed significant proliferation in the pre-
sence of SNP1 at concentrations up to 200 μg/mL. The cell viability at
200 μg/mL SNP1 was 185% ± 16.4%, which was 2.37 and 2.03 times
higher than that at the same concentration of STS and SNP2,

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S. Shankar et al. Materials Science & Engineering C 92 (2018) 508–517

Sodium thiosulphate Control

(a) SNP1 Sodium thiosulphate (200 µg/mL)
SNP2
(b) SNP1 (200 µg/mL)
110 100 SNP2 (200 µg/mL)
$$$
### ### ### ### ###

100 *** *** *** *** *** 90

Inhibition of cell migration (%)

90 *** 80

Relative cell migration (%)

### ***
80 *** 70

70 60

60 *** 50
***
50 40
40 30 ###
$ ***
30 ** 20
20
10
10 $$$
0 ###
***
0
0 10 50 100 200 500 1000 0 10 20 30 40 50

µg/mL)
Concentration (µ Time (h)

(c)

Fig. 8. In vitro inhibitory effects of STS and SNPs on A549 cell migration. (a) Inhibition of cell migration at 48 h after treatment as a percentage of unrecovered area
(set at 100%). (b) Time course of inhibitory effects of STS and SNPs on in vitro A549 cell migration. (c) Representative micrographs showing migration of A549 cells
treated with vehicle (complete medium), STS (200 μg/mL), SNP1 (200 μg/mL), and SNP2 (200 μg/mL). Statistical analysis: one-way ANOVA followed by Tukey's
multiple-comparison test. Each value represents the mean ± standard deviation (n = 5 for each group). ⁎⁎ p < 0.01, ⁎⁎⁎ p < 0.001 compared to control. ###
p < 0.001 compared to STS. $$$ p < 0.001 between SNP1 and SNP2. Scale bar in (c), 300 μm.

respectively. However, the cell viability was reduced to 37.1% ± 3.0% 21.8, and 30.0 times greater inhibition of cell migration than the con-
at 1000 μg/mL SNP1. The cell viability was > 75% up to 200 μg/mL trols (3.30% ± 1.8%), respectively. In addition, the migration of A549
STS, but significantly decreased down to 33.2% ± 0.2% at 1000 μg/ cells was completely inhibited by treatment with SNP1 or SNP2 at a
mL. SNP2 did not show cytotoxicity at a concentration range of concentration of 500 μg/mL or more. However, cells treated with
10–1000 μg/mL, but the cell viability at 1000 μg/mL SNP2 was 500 μg/mL STS showed ~20% cell migration. Fig. 8b and c illustrate
109% ± 13.4%. the process of wound closure driven by the migration of A549 cells at
These results indicate that SNP2 has a greater cytotoxic effect on different time points. The scratched portion of the control was com-
tumor cells while minimizing toxicity to normal cells compared to STS pletely covered after 48 h, but a detectable gap of the monolayer was
and other SNPs. This may result in the inhibition of MEK/ERK signaling still observed after treatment with 200 μg/mL SNP1 or SNP2. The re-
by copper deficiency because SNP2 selectivity between cancer cells and covery rates at 48 h after treatment with SNP1 and SNP2 were
normal cells is higher due to differences in the MEK/ERK pathway. 28.0% ± 7.8% and 0.0% ± 0.0%, respectively. The significant in-
hibitory effect of SNP2 on cell migration may be due to the activity of
3.4.3. Cell migration assay the sulfur present in the nanoparticles incorporating chitosan as a
As shown in Fig. 8a, cell migration was significantly inhibited by capping agent. Previous studies have shown that sulfur treatment in-
STS, SNP1, and SNP2 at the concentration above 200 μg/mL. After 48 h, hibits the expression and activation of epithelial growth factor receptor
cells treated with 200 μg/mL of STS, SNP1, and SNP2 showed 16.3, and increases the expression of Bax in cancer cell lines, thereby

516
S. Shankar et al.
inhibiting the proliferation of cancer cells [32]. In addition, chitosan
showed growth inhibitory activity in cancer cells through nuclear
fragmentation and chromatin condensation, which was confirmed by
detection of intercytosomal DNA cleavage [33]. Wimardhani et al. also
demonstrated the anticancer activity of chitosan in oral squamous cell
carcinoma by inducing apoptosis and cell cycle arrest [34]. In addition,
inhibition of cancer cell migration can also be attributed to the higher
affinity of positively charged SNP2 with the negatively charged cancer
cell membrane, and also to the enhanced cellular uptake of chitosan
nanoparticles by endosomes overcoming the permeability barriers of
the epithelia [35,36]. Thus, SNP2 can be applied as an effective an-
ticancer therapy due to its dose-dependent cytotoxic activity and in-
hibitory effect on the cancer cell migration. However, more in vivo
studies are needed to determine the optimal dose for anticancer activity
of SNP2 in various cancer cell-bearing animal models.
4. Conclusions
The SNPs were synthesized using HCl and sodium thiosulfate. The
size of SNPs was affected by the capping agents. The TEM results
showed that the SNP1 had an irregular size, while SNP2 has uniform
particle size. The SNPs showed surface plasmonic resonance near
280 nm. XRD peaks indicated that the SNPs have the S8 structure. SNP2
showed greater antibacterial activity against E. coli and S. aureus
compared to SNP1. In addition, SNP2 effectively inhibited the pro-
liferation and migration of cancer cells with minimal toxic effect on
normal cells. SNP2 therefore has potential for medical applications,
including those used as antibacterial and chemotherapeutic agents.
Acknowledgments
This research was supported by the Agriculture Research Center
(ARC 710003) program of the Ministry of Agriculture, Food and Rural
Affairs, Korea, and Korea Research Fellowship Program through the
National Research Foundation of Korea (NRF) funded by the Ministry of
Science, ICT and Future Planning (2016H1D3A1903910).
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