Carbohydrate Polymers 199 (2018) 641–648

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

One-pot, large-scale green synthesis of silver nanoparticles-chitosan with T

enhanced antibacterial activity and low cytotoxicity
Jitrada Wongpreechaa, Duangporn Polpanichb, Teeraporn Suteewongc, Chariya Kaewsanehad,
⁎
Pramuan Tangboriboonrata,
a
Department of Chemistry, Faculty of Science, Mahidol University, Rama 6 Road, Phyathai, Bangkok 10400, Thailand
b
NANOTEC, National Science and Technology Development Agency (NSTDA), 130 Thailand Science Park, Phahonyothin Road, Khlong Luang, Pathum Thani 12120,
Thailand
c
Department of Chemical Engineering, Faculty of Engineering, King Mongkut’s Institute of Technology Ladkrabang, Ladkrabang, Bangkok 10520, Thailand
d
School of Bio-Chemical Engineering and Technology, Sirindhorn International Institute of Technology (SIIT), Thammasat University, Pathum Thani, 12121, Thailand

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

Keywords: Silver nanoparticles stabilized with chitosan (AgNPs-CS) were synthesized based on the one-pot green process in
Silver nanoparticle an autoclave, in which CS acts as reducing agent as well as stabilizer. Effects related to temperature and pressure
Chitosan input on particle formation were systematically investigated. Mechanism taking place during particle nucleation
One-pot green process and growth was proposed. The data from UV–vis absorption, X-ray diffraction pattern and morphology con-
Antibacterial
firmed the formation of AgNPs-CS with face-centered cubic (fcc) structure. The synthesized AgNPs-CS showed
Cytotoxicity
the effective antibacterial activity against both E. coli and S. aureus. The minimum bactericidal concentration
values of 39.1 and 312.5 μg/ml for E. coli and S. aureus, respectively, did not show cytotoxicity to L-929 fi-
broblast. Moreover, the covering of CS on the surface of AgNPs-CS was proven to reduce the cytotoxicity when
compared with commercial citrate-stabilized AgNPs. Considering simple and mild process, this synthesis ap-
proach would be helpful for development of benign AgNPs-based antibacterial agent.

1. Introduction of properties of NPs and their functions.

Silver nanoparticles (AgNPs) are one of metal NPs globally used in
large amount in many fields, e.g., catalysis and hygiene products (Ray,
Yu, & Fu, 2009). AgNPs and their oxidized species (Ag+) possess the
biocidal effect to microbes including E. coli, S. aureus, V. cholera, C.
albicans and P. aeruginosa (Biao et al., 2017; Jung et al., 2008; Tran &
Le, 2013) due to the electrostatic attraction with negatively charged
bacterial cell and change membrane permeability leading to cell death
(Dakal, Kumar, Majumdar, & Yadav, 2016). In addition, Ag+ can in-
teract with bacterial DNA or proteins through an electron-release me-
chanism which resulted in losing DNA replication ability and protein
denaturation (Sharma, Yngard, & Lin, 2009). The higher stability
against oxidation of AgNPs minimizes excessive exposure of Ag+, en-
ables them a reservoir for Ag+ and ensures the long-term use (López-
Carballo, Higueras, Gavara, & Hernández-Muñoz, 2013). Owing to the
oligodynamic action, AgNPs display broad killing spectrum and low
possibility for development of microbial resistance. Since the particle
size, shape and surface properties affect their antimicrobial activities
(Pal, Tak, & Song, 2007), intensive studies are carried on optimization
Among several techniques used for the synthesis of AgNPs, i.e.,
physical, chemical and biological methods, the chemical reduction of
Ag+ has been mostly employed (Iravani, Korbekandi, Mirmohammadi,
& Zolfaghari, 2014). Besides its convenience, this method provides
AgNPs with better control size and morphology via the reduction both
in organic and in aqueous media. However, the frequently used redu-
cing agents, e.g., hydrazine, sodium borohydride, dimethyl formamide
and elemental agents, are potentially hazardous (Sarkar, Banerjee,
Kundu, Madras, & Chatterjee, 2015). With focusing on the green
chemistry, natural compounds, e.g., glucose, soluble starch and chit-
osan, have attracted considerable research interest as safer alternatives,
reducing and stabilizing agents (Murugadoss & Chattopadhyay, 2007;
Raveendran, Fu, & Wallen, 2003; Venkatesham, Ayodhya,
Madhusudhan, Babu, & Veerabhadram, 2014; Vigneshwaran, Nachane,
Balasubramanya, & Varadarajan, 2006). Moreover, the green process is
cost-effective and easily scaled up.
As a natural cationic polymer, obtained from deacetylation of chitin,
chitosan (CS) is biocompatible, non-toxic, biodegradable, and has an-
tibacterial properties. Due to its significant content of primary amines

⁎
Corresponding author.
E-mail address: pramuan.tan@mahidol.ac.th (P. Tangboriboonrat).

https://doi.org/10.1016/j.carbpol.2018.07.039
Received 5 April 2018; Received in revised form 7 July 2018; Accepted 12 July 2018
Available online 19 July 2018
0144-8617/ © 2018 Elsevier Ltd. All rights reserved.
J. Wongpreecha et al.
and hydroxyl groups, this polymer has high affinity towards metal ions
either by chelation or by an ion exchange route which supports the
nucleation as well as stabilization of the synthesized AgNPs
(Murugadoss & Chattopadhyay, 2007). Several research groups re-
ported the use of CS as mild reducing agent and stabilizing agent for
fabrication of AgNPs and showed their antibacterial and catalytic ac-
tivities (Biao et al., 2017; Huang, Yuan, & Yang, 2004; López-Carballo
et al., 2013; Murugadoss & Chattopadhyay, 2007; Venkatesham et al.,
2014). López-Carballo et al. developed the controlled release anti-
bacterial CS films consisting of AgNPs synthesized in situ during the
neutralization of the CS acetate film with NaOH (López-Carballo et al.,
2013). Hydroxyl ions accelerate the reduction reaction of Ag+ and the
formation of AgNPs by increasing the reducing power of CS. In addi-
tion, CS acts as a carrier of AgNPs, allows the slow and extends the
release of Ag+ in a liquid medium while maintains the antimicrobial
activity. The presence of NaOH causes the coexistence of elementary
silver and silver oxide. The specific interactions between the primary
amine and amide groups of CS and the surface of AgNPs have been
recently proved by using the synthesized CS-functionalized AgNPs in
aqueous solution via the one-step hydrothermal method using Teflon-
lined autoclave at 140 °C for 4 h (Biao et al., 2017). Although, the
AgNPs displayed monodispersity, good stability and antibacterial ac-
tivities against both gram-positive and gram-negative bacteria and
fungus, the reaction was carried out at high temperature for long re-
action time. By applying pressure at 15 psi, Venkatesham et al. pre-
pared AgNPs using CS as reducing agent and stabilizer in an autoclave
at 120 °C for 50 min without any toxic chemicals (Venkatesham et al.,
2014). These NPs showed significant catalytic activity toward reduction
of 4-nitrophenol to 4-aminophenol and antibacterial activity on E. coli
and M. luteus. However, a deeper understanding of the underlying na-
noparticle formation is still missing inspite of the fact that the shape,
morphology and degree of crystallinity of AgNPs depend significantly
on the synthetic methods. The CS-functionalized AgNPs (AgNPs-CS)
with high crystallinity and good stability in large-scale fabrication are
still demanded and, hence, its formation mechanism needs to be elu-
cidated.
In the embodied of work, the insight mechanism of AgNPs-CS for-
mation is unfolded by investigating the synthesis parameters, i.e.,
temperature and pressure. The AgNPs-CS were synthesized via a one-
step green process in an autoclave, in which CS acts as reducing agent
as well as stabilizer. The synthesized particles were fully characterized
in terms of UV–vis absorption, particle morphology, X-ray diffraction
pattern and zeta potential value. Effectiveness of this biocomposite as
active antibacterial component is tested against E. coli and S. aureus. Its
cytotoxicity to mouse fibroblast in culture is also examined and com-
pared with the commercial citrate-stabilized AgNPs.
2. Experimental
2.1. Materials
AgNO3 (Aldrich, Reagent Plus), ethanol (Carlo Erba, Analytical) and
fluorescein isothiocyanate (FITC) (Aldrich) were used as received. CS
stock solution (1%w/v) was prepared by dissolving CS (Ta Ming
−
Enterprise, %deacetylation of 94.85, Mw = 1.7 × 105 g. mol−1) in
acetic acid (1%v/v, J.T. Baker, Glacial), stirred overnight before fil-
tering. Commercial AgNPs stabilized by citrate (AgNPs-citrate;
Nanocomposix Inc.) for comparison of cytotoxicity was used as re-
ceived. The antibacterial test was carried out using E. coli (ATCC
25922) and S. aureus (ATCC 6538). The L-929 cells were used to ex-
amine the in vitro cytotoxicity. Deionized (DI) water was applied
throughtout the work.
2.2. Preparation and characterizations of AgNPs-CS
0.3%w/v CS solution (10 ml) was mixed with 0.4%w/v AgNO3
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Carbohydrate Polymers 199 (2018) 641–648
solution (10 ml) while stirring at 700 rpm for 5 min. The mixture was
kept under pressure of 15 psi in an autoclave (Hirayama, HVA-85) at
105, 110, 115 and 120 °C for 50 min. In parallel, the experiment
without applying pressure were carried out at room temperature, 60, 90
and 120 °C for 8 h.
The formation of AgNPs-CS was traced using UV–vis spectro-
photometer (Agilent, Cary 60) at scan rate of 600 nm.min−1. The di-
lution factor of samples was kept constant for each set of studies. The
crystallographic structure of AgNPs was determined by X-ray dif-
fractometer (XRD; Bruker, D8 Discover) using Cu Kα radiation (wave-
length of ca. 0.154 nm). From XRD peak profile, full width at half
maximum (FWHM), standing for the most intense plane, was analyzed
and then used for calculating crystalline size by the Scherrer Equation.
0.94λ
D=
βcosθ
where D is the average crystallite domain size perpendicular to the
reflecting planes (nm), λ is the X-ray wavelength (nm), β is the FWHM,
θ is the diffraction angle (rad). Particle morphology was investigated
under transmission electron microscopy (TEM; FEI, TECHNAI G2). The
particle size was measured with ImageJ program, considering > 150
particles from multiple TEM micrographs. The hydrodynamic diameter
(Dh) of AgNPs-CS at 25 °C was measured by using Zeta sizer (Zeta Sizer,
Malvern, Nano ZS). The sample was diluted by DI water and measured
thrice. The presence of CS on the surface of AgNPs was confirmed using
zeta potential measurement (Zeta Sizer, Malvern, Nano ZS) at pHs 2–12
and FITC fluorescent labeling of particles for confocal laser scanning
microscopy (CLSM) (OLYMPUS, FV10i-DOC) (Wei, Wang, Zou, Liu, &
Tong, 2012). The weight composition of AgNPs-CS was determined by
Inductively Coupled Plasma (ICP) spectrometry (PerkinElmer Optima
8000, emission mode). AgNO3 solution was used as a calibration
standard. The AgNPs-CS was performed with 5%w/v HNO3.
2.3. Antibacterial study
The antibacterial activity of AgNPs-CS against gram-negative (E.
coli, ATCC 25922) and gram-positive (S. aureus, ATCC 6538) bacteria
was examined using the agar disk diffusion method adopted by the
National Committee for Clinical Laboratory Standards (Ortez, 2005).
The disk (6 mm in diameter) containing AgNPs-CS suspension (40 μl)
was placed on a surface of Mueller-Hinton Agar (MHA) previously
seeded with inoculums test microorganism (20 μl, ∼108 CFU. ml−1).
The plate was incubated at 37 °C for 24 h in an incubator (Memmert,
UNB400). The diameter of inhibition zone surrounding the film disk
was then measured. The statistical significance was determined by one-
way analysis of variance (ANOVA) followed by Turkey HSD Post-Hoc
test, using SPSS PASW Statistics 18 (SPSS Inc., Chicago, USA). Statis-
tical significance was defined as p < 0.05.
To determine the lowest concentration of AgNPs-CS required to kill
of a particular microorganism or minimum bactericidal concentration
(MBC), the broth dilution method was applied (Ortez, 2005). The two-
fold dilutions of AgNPs-CS (0.4%wt) in tryptic soy broth (TSB) were
dispensed in 48-well plates. Each sample well was inoculated with E.
coli and S. aureus (∼108 CFU/ml) at 37 °C. After overnight incubation,
the sample in well that did not possess turbidity caused by the presence
of bacteria was streaked onto MHA plate. After being incubated at 37 °C
for 24 h, the MBC was then determined from the dilution showing no
bacterial growth.
2.4. In vitro Cytotoxicity study
As suggested by ISO 10993-5, mouse fibroblast cells, NCTC clone
929 [L-929, derivative of Strain L] (ATCC® CCL1™) was used for cyto-
toxicity test by the clonogenic assay (Iso, 1999). The L-929 cells were
trypsinized and seeded at density of 400 cells/well in 6-well plates and
then incubated at 37 °C under 5% CO2 atmosphere for 24 h. After that,
J. Wongpreecha et al.
the cells were treated with CS, AgNPs-CS or commercial AgNPs-citrate
(10 μl), which were serially diluted in the completed medium (1990 μl),
i.e., DMEM supplemented with 10% fetal bovine serum (FBS) and 1%
Penicillin Streptomycin. Plates were incubated for 14 days and the
colonies were then fixed with methanol before being stained with
methylene blue. The number of living cell colonies on each well were
counted under optical microscopy (OM; Olympus, CKX41) and survival
fraction was determined (Qian et al., 2014). Each assay was made in
triplicate and only colonies containing at least 50 cells were counted.
3. Results and discussion
3.1. Analysis of AgNPs-CS
The formation of AgNPs-CS was analyzed using UV–vis spectro-
photometry. The absorption spectra of AgNPs-CS synthesized at 105,
110, 115 and 120 °C under pressure of 15 psi for 50 min and those of
AgNPs-CS synthesized at room temperature, 60, 90 and 120 °C without
applying pressure for 8 h are shown in Fig. 1(a). Fig. 1(b) reveals TEM
images of AgNPs-CS prepared at different autoclaving temperatures for
50 min under applied pressure.
Since an absorption peak occurs due to the surface plasmon re-
sonance (SPR) of nano-sized Ag, the absence of SPR peak after mixing
AgNO3 and CS in unpressurized condition at room temperature or 60 °C
for 8 h indicates that the AgNPs were not formed. Once the reaction
temperature was raised to 90 °C or 120 °C, the broad SPR peak with low
intensity at ca. 420 nm appeared due to the formation of AgNPs. The
SPR peak locating between 410 and 450 nm might refer to spherical
morphology of AgNPs (Zaheer & Rafiuddin, 2012). Increasing the re-
action temperature enhanced the absorption intensity implying the
increase in the formation of AgNPs (Jiang, Chen, Chen, Xiong, & Yu,
2011). Previously, Murugadoss et al. synthesized AgNPs-CS composite
as a heterogeneous catalyst by reduction of AgNO3 onto CS powder in
the presence of NaOH (Murugadoss & Chattopadhyay, 2007). They
found that CS powder in neutral pH condition at 95 °C did not react
with AgNO3. On the other hand, heating the mixture at > 95 °C using
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Carbohydrate Polymers 199 (2018) 641–648
Fig. 1. (a) UV–vis absorption spectra of
AgNPs-CS synthesized at 105, 110, 115
and 120ºC under pressure of 15 psi for
50 min (–) and at room temperature,
60, 90 and 120ºC without applying
pressure for 8 h (—) and (b) TEM
images of AgNPs-CS prepared at 105,
110, 115 and 120ºC for 50 min in au-
toclave under applied pressure.
NaOH as catalyst was essential in the reduction of Ag+ to AgNPs. The
large full width at half maximum (FWHM) of the absorption peaks in
Fig. 1(a) implies the broad size distribution of AgNPs-CS synthesized at
90 °C or 120 °C without applying pressure (Agnihotri, Mukherji, &
Mukherji, 2014). Under applied pressure, it was clearly seen that the
increase in autoclaving temperature from 105 to 120 °C caused the blue
shift of SPR peaks and decreased the FWHM value corresponding to the
decrease of size and dispersity of the prepared particles. At 120 °C, the
symmetrical SPR peak with high intensity implies the formation of a
larger amount of monodispersed AgNPs particles (Schatz & Van Duyne,
2002). This correlated well with the TEM images shown in Fig. 1(b) and
indicated that the applied pressure reaction produced larger con-
centration of AgNPs in a much shorter reaction time (50 min). Although
Biao et al. synthesized CS-functionalized AgNPs by hydrothermal
method using Teflon-lined autoclave, the reaction was carried out at
higher temperature (140 °C) and longer reaction time (4 h) (Biao et al.,
2017). Upon operation in the autoclave, the pressure gives the higher
temperature than the boiling point of medium (water), causing the
higher energy of water molecule. Increasing the energy of a system
gives rise to the higher average kinetic energy. In this case, the particles
move faster which yields the larger collision energy. To form a stable
nucleus, the collision between one atom and existing nucleus is a
dominant process (Chen & Wu, 2000). This is possibly the reason why,
in our pressure-assisted system, the growth process would be superior
to the nucleation. Without applying pressure, the reduction rate of Ag+
at lower energy was slow and only few nuclei were formed at the early
stage of the reaction. Then, Ag+, generated at the latter period, mainly
collide with the existing nuclei in the system and therefore led to the
formation of larger particles.
The XRD pattern of powder AgNPs-CS synthesized under applied
pressure at 120 °C for 50 min is displayed in Fig. 2.
In Fig. 2, the XRD pattern of powder AgNPs-CS exhibits diffraction
peaks at 2θ of 38.12°, 44.45°, 64.56° and 77.87°, corresponding to the
reflection plane indices (hkl) of (111), (200), (220) and (311), respec-
tively, as per available literature of standard silver values (JCPDS PDF
card 04-0783). These diffraction patterns are in good agreement with
J. Wongpreecha et al. Carbohydrate Polymers 199 (2018) 641–648

Fig. 2. XRD pattern of powder AgNPs-CS synthesized under applied pressure at 120 °C for 50 min.

the characteristic of Ag with face-centered cubic (fcc) (Venkatesham

et al., 2014). The crystallite size of the synthesized AgNPs-CS, calcu-
lated using the Scherrer equation with (111) as the most intense plane,
was 12.7 nm.
The morphology and diameter of AgNPs-CS were investigated under
TEM and the data are shown in Fig. 3.
Fig. 3(a) shows mostly spherical and well dispersed AgNPs-CS. The
diameter of particle obtained from TEM images in Fig. 3(b) was larger
than the crystallite size calculated from XRD. This might be due to the
fact that XRD measures the size of coherent diffraction domains in
AgNPs, which is equal to the diameter of a single-crystal particle and
smaller than the actual diameter of twinned particles that have more
than one diffraction domain (Agnihotri et al., 2014). The assumption is
consistent with high magnification TEM image as seen in the inset of
Fig. 3(a). It was clearly observed that the AgNPs of > 20 nm possess
grain boundary and twinning, suggesting polycrystallinity. The me-
chanism of the formation of AgNPs-CS in one-pot is proposed as shown
in Fig. 4.
When mixing AgNO3 with CS solution under mild conditions, i.e.,
room temperature and short reaction time, the coordination between
Ag+ and lone pair electrons of nitrogen and/or oxygen on CS backbone,
depending on pH, can be occurred (Chudobova et al., 2013). As pre-
viously reported, the nitrogen atom in the amines might lose one
electron to form its oxidized form, and Ag+ acquired the electron to
become Ag° (Murugadoss & Chattopadhyay, 2007). In the present
study, the reaction was carried out at pH 4 which is significantly lower
than the pKa of CS and amine moieties are in protonated form (-NH3+).
Thus, electrostatic attractive interaction dominates only the complex
between Ag+ and oxygen atom of CS and is a major driving force of the
reaction. The high temperature and applied pressure possibly act as the
catalyst for the reduction of Ag+ to Ag° by lone pair electron of oxygen
in CS, resulting in the formation of Ag nuclei (single crystal). Con-
centration gradient of Ag+ and pressure might induce the diffusion of
Ag+ from bulk solution to compensate the reduced ion as well as the
coalescence of nearby Ag nuclei to Ag clusters (polycrystallinity) upon
the change in CS conformation. The Ag grows by merging neighboring Fig. 3. (a) TEM images at low and high magnification (inset) and (b) histogram
Ag clusters on surrounding CS and further coalesces until the particles of diameter of AgNPs-CS.
have certain stability. CS chains then provide steric and electrostatic
stabilization to the resulting composites. presents hydrodynamic diameter (Dh) distribution curve of AgNPs-CS at
To confirm the presence of CS on the surface of AgNPs, the zeta pH 4 whereas Fig. 5(c) and (d) show confocal images of unlabeled and
potentials at various pHs (2–12) are plotted in Fig. 5(a). Fig. 5(b) FITC-labeled AgNPs-CS, respectively.

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J. Wongpreecha et al. Carbohydrate Polymers 199 (2018) 641–648

Fig. 4. Proposed mechanism for the formation of AgNPs-CS in one-pot.

Fig. 5(a) reveals the high positive zeta potential of AgNPs-CS of >
+ 30 mV at pH < 10 due to the protonation of amino groups of CS
(Kaewsaneha, Jangpatarapongsa, Tangchaikeeree, Polpanich, &
Tangboriboonrat, 2014) and suggests high stability of particles. The
zero and negative zeta potentials are possibly due to the fact that AgNPs
precipitate and are oxidized as previously reported (Long, Wu, & Chen,
2007). Since glucosamine residues of CS are almost fully protonated
under acidic condition providing the mutual electrostatic repulsion, the
narrow size distribution curve of AgNPs-CS at pH 4 (average
Dh = 74.7 nm) with polydispersity index (PDI) of 0.294 is observed in
Fig. 5(b). The composition of Ag in AgNPs-CS, determined by ICP, was
29.7 wt% as shown in Supplementary data, Fig. S1. Therefore, the ex-
istence of ca. 70 wt% CS on the surface of AgNPs was further clarified
by labeling with FITC fluorescent dye. The reaction between amine
group in CS and isocyanate group in FITC yields thiourea bond con-
ducting in confocal laser scanning microscopy studies (Wei et al.,
2012). The FITC-labeled AgNPs-CS were observed in Fig. 5(d) as bright
green dots whereas the unlabeled AgNPs-CS in Fig. 5(c) did not show
any fluorescent signal. To verify the stability of the AgNPs-CS, the ab-
sorption measurement of newly-prepared and stored particles (after 1, 3
and 6 months) was performed and the data are shown in Supplementary
data, Fig. S2. The absorption peaks of newly-prepared and stored na-
nocomposites were nicely overlayed. The results demonstrated that the
synthesized AgNPs-CS exhibited good long-term stability without any
aggregation, indicating the role of CS as steric and electrostatic stabi-
lizer as aforementioned. In addition, these data also implied that the

Fig. 5. (a) Zeta potentials of AgNPs-CS as a function of pH, (b) hydrodynamic diameter (Dh) distribution of AgNPs-CS at pH 4; and confocal images of (c) unlabeled
and (d) FITC-labeled AgNPs-CS.

645
J. Wongpreecha et al.
Fig. 6. Inhibition zone of E. coli and S. aureus in the presence of AgNPs-CS with 0.1–0.5%w/v AgNO3 compared with the negative control (DI water) from the disk
diffusion test *a-h on bar graph denote a statistically significant difference within and between groups for each response (p < 0.05).
oxidation of Ag° is prohibited or significant slowed down by the pre-
sence of macromolecular shell of CS.
3.2. Antibacterial activity of AgNPs-CS
The antibacterial activity against E. coli and S. aureus of AgNPs-CS
synthesized at 0.1-0.5%w/v AgNO3 was evaluated using the disk dif-
fusion method. The clear zone around the disk for AgNPs-CS samples at
all concentrations relating to the zone of inhibition where bacteria are
unable to grow, comparing with negative control (DI water), is ob-
served and displayed in Supplementary data, Fig. S3. The diameter of
inhibition zone of CS and AgNPs-CS samples was measured and then
plotted versus AgNO3 concentration as presented in Fig. 6.
It can be seen that CS (at the same concentration used for synthe-
sizing AgNPs-CS) can prevent only the growth of S. aureus while AgNPs-
CS can inhibit the growth of both bacteria. The inhibition zone in-
creases with increasing AgNO3 concentration. This indicated that
AgNPs strongly enhance the antibacterial activity of AgNPs-CS. The
antibacterial activity of CS might come from the contact-inhibitory
mechanism between negatively charged teichoic acid in peptidoglycan
layer on the bacteria surface and positively charged of protonated
amine moieties on CS backbone (Vallapa et al., 2011). On the contrary,
AgNPs perform antibacterial activities by disrupting cell wall of bac-
teria via several pathways. For example, they adhere to the cell mem-
brane through electrostatic interaction, leading to the alteration of its
permeability and respiration chain (Dakal et al., 2016). They can also
penetrate into bacteria cell and then release Ag+, which interacts with
thiol groups and/or phosphates moieties in bacterial DNA or protein
(Dakal et al., 2016; López-Carballo et al., 2013). Either by direct cell-
AgNPs contact or by releasing Ag+ could be synergistic mechanism to
produce a strong antibacterial activity of both types of bacteria. In-
creasing concentration of AgNO3 affects both size and the number of
particles (Huang et al., 2004). Enlargement of particle size lowers their
specific surface area as well as contact surface with bacteria, which
consequently reduces antibacterial activity (Agnihotri et al., 2014). On
the other hand, generating more particles will add up the inhibitory
opportunity. This was the reason why no statistically significant dif-
ference in antibacterial efficacy was observed when concentration of
AgNO3 is > 0.4%w/v. This concentration was, therefore, chosen to
prepare AgNPs for further study, presumably that it balances the impact
of size/number of particles and antibacterial properties. The MBC va-
lues or the lowest concentration of AgNPs-CS that destroys bacteria of
the chosen AgNPs-CS (synthesized using 0.4%w/v AgNO3) against both
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Carbohydrate Polymers 199 (2018) 641–648
types of bacteria were determined, as shown in Supplementary data,
Fig. S4. The MBC values of AgNPs-CS were found to be 39.1 μg/ml and
312.5 μ g/ml for E. coli and S. aureus, respectively.
3.3. In vitro Cytotoxicity study
Since AgNPs show strong antibacterial activity, it might induce
cytotoxicity, genotoxicity and immunological responses (Zhang, Wang,
Chen, & Chen, 2014), caused by the strong oxidative nature of AgNPs
and the release of Ag+ to biological environments (He, Zhou, Wamer,
Boudreau, & Yin, 2012). The previous toxicity study of Ag+ released
from AgNPs in a commercially available dressing to keratinocytes and
fibroblasts showed that fibroblasts were more sensitive to the particles
(Poon & Burd, 2004). Therefore, L-929 fibroblast cells were used as a
model for the cytotoxicity test of AgNPs-CS and CS using the clonogenic
assay in our work. The result revealed that AgNPs-CS at their MBC
value did not show any significant cytotoxicity against this cell line.
Survival fraction of L-929 cells at MBC values for both types of bacteria
was > 0.99. The elongated spindle-shaped morphology of L-929 cells
incubated with AgNPs-CS at 39.1 and 312.5 μg/ml, in Fig. 7(c) and (d)
at day 6 is similar to that of negative control (DI water) in Fig. 7(a) and
different from the cells of positive control (0.4%w/v AgNO3 solution) in
Fig. 7(b) which detached from the plate. Therefore, it could be assumed
that the use of AgNPs-CS at their MBC value can effectively inhibit the
growth of bacteria but not toxic to normal cell.
The toxicity of commercial AgNPs-citrate (60.0 ± 4.0 nm) on cell
growth inhibition was also examined and compared with that of the
synthesized AgNPs-CS (37.2 ± 16.4 nm). The number of cell colonies
incubated with 2.5–20 ppm of NPs at day 14 and survival fraction are
presented in Fig. 8.
In the case of AgNPs-citrate, the survival fraction of cells varying
from 0.64 to 0.93 was inversely proportional to the mass concentration
of Ag from 20 to 2.5 ppm of Ag. The high survival fraction of the syn-
thesized AgNPs-CS of > 0.90 at all Ag mass concentrations was closed
to that of CS (0.99) and the negative control (1.00) which confirm their
effective reduction of cytotoxicity. This might be contributed by the
presence of macromolecule layer of biocompatible CS that controls the
release rate of Ag+ and/or reduces contact killing between surface of
AgNPs and L-929 fibroblast cells. Recently, Gliga et al. have showed
that the amount of released Ag+ from 10 nm AgNPs-citrate that present
in cell medium was higher than poly(vinylpyrrolidone) (PVP) coated
particles (Gliga, Skoglund, Wallinder, Fadeel, & Karlsson, 2014). Their
explanation is based on high displacement rate of the electrostatically
J. Wongpreecha et al.
Fig. 7. Morphologies of L-929 cells incubated with (a) negative control (DI water), (b) positive control (0.4%w/v AgNO3 solution), (c) 39.1 μg/ml AgNPs-CS and (d)
312.5 μg/ml AgNPs-CS (at day 6).
Fig. 8. Survival fraction of L-929 fibroblasts at various mass concentrations of
Ag in AgNPs-citrate and AgNPs-CS compared with CS and negative control. *a-b
on bar graph denote a statistically significant difference within and between
groups for each response (p < 0.05).
and loosely bound citrate with medium components (e.g., amino acids),
high ionic strength medium, when compared to the polymeric stabi-
lizer, PVP. Rapid breakdown of the stabilizing layer evidently affects
the particle stability and promotes the release of Ag+.
4. Conclusions
The AgNPs-CS was synthesized using AgNO3 as metal precursor and
CS as reducing agent and stabilizer in one-step batch process autoclave
without producing toxic waste. Increasing the reaction temperature
played a key role in the formation of AgNPs. In addition, the applied
pressure enhanced the formation of AgNPs, confirmed by UV–vis
spectrophotometry, XRD and TEM, in shorter reaction time. The zeta
potential values of AgNPs-CS of > + 30 mV at pHs 2–10 and the in-
significant shift in UV–vis absorption peak after storage for 1, 3 and 6
647
Carbohydrate Polymers 199 (2018) 641–648
months indicated the long-term particle stability. The synthesized
particles showed high antibacterial efficiency with increasing AgNO3
concentration but no statistically significant difference at concentra-
tion > 0.4%w/v. Their MBC values against E. coli and S. aureus were 39
μ g/ml and 312.5 μ g/ml, respectively, without notable cytotoxicity to
L-929 fibroblast cells. The cytotoxicity of AgNPs-CS was lower com-
pared to that of AgNPs-citrate. CS having a negligible effect on fibro-
blast cells but distinctly affected the bacterial cell is the suitable sta-
bilizer of AgNPs for bacterial growth inhibition with relatively low
cytotoxicity.
Acknowledgments
This research project is supported by Mahidol University. The
scholarships from the Thailand Graduate Institute of Science and
Technology, NSTDA to J.W. and W.R. are greatly acknowledged. The
authors thank the Center of Nanoimaging, Faculty of Science and the
Center for Research and Innovation, Faculty of Medical Technology,
Mahidol University, for facility support.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.carbpol.2018.07.039.
References
Agnihotri, S., Mukherji, S., & Mukherji, S. (2014). Size-controlled silver nanoparticles
synthesized over the range 5–100 nm using the same protocol and their antibacterial
efficacy. RSC Advances, 4(8), 3974–3983.
Biao, L., Tan, S., Wang, Y., Guo, X., Fu, Y., Xu, F., et al. (2017). Synthesis, characterization
and antibacterial study on the chitosan-functionalized Ag nanoparticles. Materials
Science and Engineering: C, 76, 73–80.
Chen, D.-H., & Wu, S.-H. (2000). Synthesis of nickel nanoparticles in water-in-oil mi-
croemulsions. Chemistry of Materials, 12(5), 1354–1360.
Chudobova, D., Nejdl, L., Gumulec, J., Krystofova, O., Rodrigo, M. A. M., Kynicky, J.,
et al. (2013). Complexes of silver (I) ions and silver phosphate nanoparticles with
hyaluronic acid and/or chitosan as promising antimicrobial agents for vascular grafts.
J. Wongpreecha et al.
International Journal of Molecular Sciences, 14(7), 13592–13614.
Dakal, T. C., Kumar, A., Majumdar, R. S., & Yadav, V. (2016). Mechanistic basis of an-
timicrobial actions of silver nanoparticles. Frontiers in Microbiology, 7, 1831. https://
doi.org/10.3389/fmicb.2016.01831.
Gliga, A. R., Skoglund, S., Wallinder, I. O., Fadeel, B., & Karlsson, H. L. (2014). Size-
dependent cytotoxicity of silver nanoparticles in human lung cells: The role of cel-
lular uptake, agglomeration and Ag release. Particle and Fibre Toxicology, 11(1), 1–17.
He, W., Zhou, Y.-T., Wamer, W. G., Boudreau, M. D., & Yin, J.-J. (2012). Mechanisms of
the pH dependent generation of hydroxyl radicals and oxygen induced by Ag nano-
particles. Biomaterials, 33(30), 7547–7555.
Huang, H., Yuan, Q., & Yang, X. (2004). Preparation and characterization of metal–chi-
tosan nanocomposites. Colloids and Surfaces B: Biointerfaces, 39(1-2), 31–37.
Iravani, S., Korbekandi, H., Mirmohammadi, S. V., & Zolfaghari, B. (2014). Synthesis of
silver nanoparticles: Chemical, physical and biological methods. Research in
Pharmaceutical Sciences, 9(6), 385–406.
Iso, E. (1999). Biological evaluation of medical devices-part 5: Tests for cytotoxicity: in vitro
methods. German version EN ISO1–8.
Jiang, X. C., Chen, W. M., Chen, C. Y., Xiong, S. X., & Yu, A. B. (2011). Role of tem-
perature in the growth of silver nanoparticles through a synergetic reduction ap-
proach. Nanoscale Research Letters, 6(1), 1–9.
Jung, W. K., Koo, H. C., Kim, K. W., Shin, S., Kim, S. H., & Park, Y. H. (2008).
Antibacterial activity and mechanism of action of the silver ion in Staphylococcus
aureus and Escherichia coli. Applied and Environmental Microbiology, 74(7), 2171–2178.
Kaewsaneha, C., Jangpatarapongsa, K., Tangchaikeeree, T., Polpanich, D., &
Tangboriboonrat, P. (2014). Fluorescent chitosan functionalized magnetic polymeric
nanoparticles: Cytotoxicity and in vitro evaluation of cellular uptake. Journal of
Biomaterials Applications, 29(5), 761–768.
Long, D., Wu, G., & Chen, S. (2007). Preparation of oligochitosan stabilized silver na-
noparticles by gamma irradiation. Radiation Physics and Chemistry, 76, 1126–1131.
López-Carballo, G., Higueras, L., Gavara, R., & Hernández-Muñoz, P. (2013). Silver ions
release from antibacterial chitosan films containing in situ generated silver nano-
particles. Journal of Agricultural and Food Chemistry, 61(1), 260–267.
Murugadoss, A., & Chattopadhyay, A. (2007). A ‘green’ chitosan–silver nanoparticle
composite as a heterogeneous as well as micro-heterogeneous catalyst.
Nanotechnology, 19(1), 1–9.
Ortez, J. H. (2005). Disk diffusion testing. In M. B. Coyle (Ed.). Manual of antimicrobial
susceptibility testing (pp. 39–52). Washington DC: American society for microbiology
Inc.
Pal, S., Tak, Y. K., & Song, J. M. (2007). Does the antibacterial activity of silver nano-
particles depend on the shape of the nanoparticle? A study of the gram-negative
648
Carbohydrate Polymers 199 (2018) 641–648
bacterium Escherichia coli. Applied and Environmental Microbiology, 73(6), 1712–1720.
Poon, V. K. M., & Burd, A. (2004). In vitro cytotoxicity of silver: Implication for clinical
wound care. Burns, 30(2), 140–147.
Qian, Y., Qiu, M., Wu, Q., Tian, Y., Zhang, Y., Gu, N., et al. (2014). Enhanced cytotoxic
activity of cetuximab in EGFR-positive lung cancer by conjugating with gold nano-
particles. Scientific Reports, 4, 1–8.
Raveendran, P., Fu, J., & Wallen, S. L. (2003). Completely “green” synthesis and stabi-
lization of metal nanoparticles. Journal of the American Chemical Society, 125(46),
13940–13941.
Ray, P. C., Yu, H., & Fu, P. P. (2009). Toxicity and environmental risks of nanomaterials:
Challenges and future needs. Journal of Environmental Science and Health. Part C,
Environmental Carcinogenesis & Ecotoxicology Reviews, 27(1), 1–35.
Sarkar, K., Banerjee, S. L., Kundu, P. P., Madras, G., & Chatterjee, K. (2015).
Biofunctionalized surface-modified silver nanoparticles for gene delivery. Journal of
Materials Chemistry B, 3(26), 5266–5276.
Schatz, G. C., & Van Duyne, R. P. (2002). In J. M. Chalmers, & P. R. Griffiths (Eds.).
Handbook of vibrational spectroscopy. New York: Wiley.
Sharma, V. K., Yngard, R. A., & Lin, Y. (2009). Silver nanoparticles: Green synthesis and
their antimicrobial activities. Advances in Colloid and Interface Science, 145(1–2),
83–96.
Tran, Q. H., & Le, A.-T. (2013). Silver nanoparticles: Synthesis, properties, toxicology,
applications and perspectives. Advances in Natural Sciences: Nanoscience and
Nanotechnology, 4(3), 1–20.
Vallapa, N., Wiarachai, O., Thongchul, N., Pan, J., Tangpasuthadol, V., Kiatkamjornwong,
S., et al. (2011). Enhancing antibacterial activity of chitosan surface by hetero-
geneous quaternization. Carbohydrate Polymers, 83(2), 868–875.
Venkatesham, M., Ayodhya, D., Madhusudhan, A., Babu, N. V., & Veerabhadram, G.
(2014). A novel green one-step synthesis of silver nanoparticles using chitosan:
Catalytic activity and antimicrobial studies. Applied Nanoscience, 4(1), 113–119.
Vigneshwaran, N., Nachane, R. P., Balasubramanya, R. H., & Varadarajan, P. V. (2006). A
novel one-pot ‘green’ synthesis of stable silver nanoparticles using soluble starch.
Carbohydrate Research, 341(12), 2012–2018.
Wei, Z., Wang, C., Zou, S., Liu, H., & Tong, Z. (2012). Chitosan nanoparticles as particular
emulsifier for preparation of novel pH-responsive Pickering emulsions and PLGA
microcapsules. Polymer, 53(6), 1229–1235.
Zaheer, Z., & Rafiuddin (2012). Silver nanoparticles to self-assembled films: Green
synthesis and characterization. Colloids and Surfaces B: Biointerfaces, 90, 48–52.
Zhang, T., Wang, L., Chen, Q., & Chen, C. (2014). Cytotoxic potential of silver nano-
particles. Yonsei Medical Journal, 55(2), 283–291.