Colloids and Surfaces B: Biointerfaces 134 (2015) 229234

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Colloids and Surfaces B: Biointerfaces

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

A caffeic acid mediated facile synthesis of silver nanoparticles with

powerful anti-cancer activity
Dawei Guo a , Dandan Dou a , Lin Ge a , Zhihai Huang b , Liping Wang a,,1 , Ning Gu b,,1
a
Laboratory of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, Nanjing Agricultural University, 1 Weigang, Nanjing 210095, PR
China
b
State Key Laboratory of Bioelectronics, Jiangsu Key Laboratory for Biomaterials and Devices, School of Biological Science and Medical Engineering,
Southeast University, 2 Sipailou, Nanjing 210096, PR China

a r t i c l e

i n f o

Article history:
Received 24 January 2015
Received in revised form 21 June 2015
Accepted 23 June 2015
Available online 13 July 2015
Keywords:
Silver nanoparticles
Caffeic acid
Anti-cancer activity
Apoptosis

a b s t r a c t
Green synthesis, especially in biological processes, has gained more attention with increasing application
of silver nanoparticles (AgNPs) in biomedical elds. However, the biologically synthesized AgNPs have
been to be anomalous in size and shape in most cases, as well as exhibiting certain difculties when used
in therapy. We used caffeic acid, a naturally plant polyphenol, to prepare the AgNPs in the current study
and also evaluated their anti-cancer activity against the human hepatoma HepG2 cells. Results showed
that the AgNPs could rapidly and simply be synthesized using caffeic acid as both a reducing agent and
stabilizer. The synthesized AgNPs possessed characteristics of having small size, narrow distribution and
high surface negative charge, as well as being stable in aqueous solution. Furthermore, the AgNPs could
enter cells and effectively inhibit viability of tumor cells via induction of apoptosis. In conclusion, a caffeic
acid mediated facile method was successfully developed to prepare the AgNPs as a potential alternative
agent for human hepatoma therapy.
2015 Elsevier B.V. All rights reserved.

1. Introduction
Much time and resources have been devoted to cancer research
in the hope of discovering the most cost-effective and efcacious
strategy for cancer diagnosis, monitoring and treatment. Nanotechnology is envisaged to be the next frontier in the ongoing
development of cancer therapy [1,2], as researchers in the biomedical and material engineering elds are nowadays eagerly working
together to explore the possibility of using nanomaterials as novel
tools for cancer therapy. Silver nanoparticles (AgNPs) have recently
been proved to have great potential in the tumor theranostics
elds, such as their use as nanoprobes for detection and imaging of
tumors, vectors for drug delivery, and inhibitors for suppression of
angiogenesis and tumor growth [35].
Biological synthesis of AgNPs is currently a highly focused
research area [6], considered to be more eco-friendly and costeffective compared to other chemical and physical methods [7,8]
due to reduced use of hazardous reagents and solvents, improved

Corresponding author at: Fax: 86 25 84398669.

Corresponding author at: Fax: 86 25 83272476.
E-mail addresses: wlp71@163.com (L. Wang), guning@seu.edu.cn (N. Gu).
1
Co-senior authors.
http://dx.doi.org/10.1016/j.colsurfb.2015.06.070
0927-7765/ 2015 Elsevier B.V. All rights reserved.

material and energy efciency from the chemical process, and

enhanced design of non-toxic products. Fungi, bacteria, and plants
are commonly used to biologically synthesize nanomaterials [9,10].
The use of plant extracts for synthesis of nanoparticles has been
proved to be advantageous when compared to microbial processes,
because pathogenic bacteria may contaminate the nanoparticles
when used in biomedical elds [11]. Furthermore, the plant-based
materials seem to be best candidates for large-scale production of
nanoparticles [12,13]. The size and shape of nanomaterials provide
efcient control over physical, chemical and biological properties
of nanomaterials, however, the biologically synthesized nanoparticles have been found to be anomalous in size and shape in most
cases, and they also display certain difculties when used for therapy [14].
Polyphenols are key active ingredients found in plant leaves,
roots, latex, seeds and stems, and they are widely used for the
green synthesis of metal nanomaterials due to their inuence on the
nanoparticles reduction process and stability [1517]. It has been
reported that the green synthesis of silver and palladium nanoparticles, with sizes ranging between 20 and 60 nm, was performed
at room temperature using coffee and tea extracts [18], and the
main constituent of the coffee extracts was caffeic acid (Fig. 1).
This is a natural plant polyphenol which is widely distributed in
many agricultural products, such as fruits, vegetables, wine, olive

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mission electron microscopy (TEM, JEM-2000EX, JEOL, Japan). The

TEM samples were prepared by placing few drops of aqueous dispersions on carbon coated copper grids followed by drying at room
temperature. The nanoparticles mean size was then calculated
from a random eld of TEM images that displayed general morphology of the nanoparticles. The hydrodynamic diameter and zeta
potential of the AgNPs were measured by dynamic light scattering
(DLS), using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK). The nal silver concentration in the aqueous solution
was determined using a graphite furnace atomic absorption spectroscopy (AAS, Z2000, Hitachi, Tokyo).
Fig. 1. Chemical structure of caffeic acid.

oil, and coffee [19]. The caffeic acid has been reported to have a wide
variety of biological properties, including antioxidants, antithrombosis, antihypertensive, antibrosis, and antiviral activities [20].
Several studies have also demonstrated that the caffeic acid exerts
anti carcinogenic effects against various cancer cell lines [2125].
In addition, the combination of plant polyphenols and anti-cancer
molecules is considered to be a novel treatment strategy for cancer
chemotherapy due to their synergistic effect [26,27].
The current work therefore mainly focused on the facile preparation of uniformly sized AgNPs using the caffeic acid as both a
reducing agent and stabilizer under ambient conditions without
additional reagents. The anti-cancer activity of the as-synthesized
AgNPs was subsequently evaluated against human hepatoma
HepG2 cells to explore their potential application in cancer therapy.
2. Materials and methods
2.1. Synthesis of silver nanoparticles
Silver nanoparticles (AgNPs) were synthesized in the current
study using a polyphenol reduction method. The caffeic acid was
utilized as both a reducing agent and stabilizer. A classical synthesis
process is described as follows (Fig. 2); 4 mM (10 L) of caffeic acid
solution was rstly mixed with 1 mL of de-ionized water under
magnetic stirring. The pH value of the solution was adjusted to
10.5 using 0.1 M of sodium hydroxide solution, followed by addition of 8 mM (20 L) silver nitrate solution. The reaction mixture
was subsequently kept under magnetic stirring at room temperature for about 30 min until it turned yellow. The freeze-dried AgNPs
powders were nally obtained after ltration, centrifugation and
lyophilization.

2.3. Cell culture

Human hepatoma (HepG2) cell line was maintained at 37 C in
an incubator with a 5% CO2 humidied atmosphere in RPMI 1640
(Hyclone), supplied with 10% FBS and penicillin/streptomycin mix
(100 U/mL and 100 mg/mL, respectively). The cells were detached
by trypsinization when they reached 80% conuency during subculture.
2.4. Intracellular localization of AgNPs
The HepG2 cells were washed with PBS, detached by trypsinization and centrifuged at 2000 rpm for 5 min after 24 h incubation
with 5 g/mL AgNPs. The medium without nanoparticles was used
as control in the experiment. The supernatants were removed and
cell pellets xed with 2.5% glutaraldehyde in PBS for 24 h, followed
by post-xation in 1% osmium tetroxide (Agar Scientic, Stansted
Essex, England, UK) for 1.5 h. The cell pellets were then dehydrated
through a series of ethanol concentrations (20%, 30%, 40%, 50%,
60%, 70%, and 90%). This was followed by treatment with 2% uranyl
acetate in 95% ethanol (Enblock stain) for 1 h and further dehydration with 100% ethanol for 1 h. The cell pellets were twice treated
with propylene oxide for 15 min each, followed by 1:1 propylene
oxide: araldite resin overnight, and inltration with fresh araldite
resin (3 changes with a gap of 3 to 4 h). This was nally followed
with subsequent embedding in araldite resin at 60 C for 48 h. The
ultra-thin sections were then cut with glass knives in an ultra
microtome (LEICA EM UC6, Netherlands). The sections were then
mounted on copper grids and stained with 1% uranyl acetate and
0.2% aqueous lead citrate. The stained sections were then scanned
with TEM (JEM-2000EX, JEOL) for ultra structural observations at
80 kV.
2.5. Effect of AgNPs on viability of HepG2 cells

2.2. Characterization of AgNPs

The as-synthesized nanoparticles were primarily characterized
by UVvis spectroscopy (Hitachi U-2000, Tokyo, Japan) and trans-

MTT (dimethyl thiazolyltetrazolium bromide) assay was performed to determine the AgNPs cytotoxic effect at various
concentrations against the human hepatoma HepG2 cells. The

Fig. 2. Visual observations of reaction mixtures in preparation process for silver nanoparticles.
(A) deionized water, (B) aqueous solution containing caffeic acid, (C) alkaline solution containing caffeic acid, (D) nanoparticles solution.

D. Guo et al. / Colloids and Surfaces B: Biointerfaces 134 (2015) 229234

231

2.7. Statistical analysis

Data were statistically evaluated by one-way ANOVA test using
SPSS software (v.16.0) (IBM, USA). Means with their standard deviation values were calculated from three replicates. P < 0.05 was
considered to be statistically signicant.
3. Results and discussion
3.1. Characterization of silver nanoparticles

Fig. 3. UVVis adsorption spectrum of AgNP.

The wavelength from maximum absorption peak was 396.5 nm.

assay depended on the reduction of MTT by mitochondrial

dehydrogenase to form a blue formazan product. Mitochondrial
dehydrogenase is an enzyme present in the mitochondria of viable
cells. Briey, a suspension containing approximately 1 104 cells
was added into each well of a 96-well culture plate and incubated
for 24 h at 37 C in a 5% CO2 humidied atmosphere. The cells were
then treated with the AgNPs at various concentrations. The incubation medium was gently removed after 24 h of incubation and
150 L (5 mg/ml) of MTT added into each well, followed by further
incubation for 4 h at 37 C under 5% CO2 humidied atmosphere.
The medium was then carefully removed and 150 L dimethyl sulfoxide (DMSO) added. The formed crystals were dissolved gently by
two to three times pipetting. Absorbance was then measured at two
wavelengths (570 nm for soluble dye and 650 nm for viable cells)
using a microplate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA, USA).

2.6. Apoptosis assay

Apoptosis-mediated tumor cells death was stained using
annexin V-FLUOS/propidium iodide (PI) assay and analyzed with
ow cytometry (CaliburTM , Bectone Dickinson, USA) according to
the manufacturers protocol. The HepG2 cells were incubated at
37 C with 5% CO2 for 24 h in the presence or absence of the AgNPs at
different concentrations. The cells were trypsinized, washed thrice
with PBS, and re-suspended in the incubation buffer consisting of
2 L Annexin V-FLUOS and 2 L PI for 20 min at room temperature
in the dark. The apoptotic/necrotic cells were nally analyzed with
ow cytometry.

The silver nanoparticles (AgNPs) were synthesized via reduction

of silver nitrate using the caffeic acid. This was a one-step silver
nitrate reduction process in which the caffeic acid acted as both
the reducing agent and capping agent. As shown in Fig. 2, the AgNPs
preparation process was visually observed. When the alkaline solution containing the caffeic acid was mixed with AgNO3 solution
under ambient conditions, the reaction mixture turned into light
yellow color within 30 min, which was a clear indication for AgNPs
formation through a wet-chemical process (Fig. 2C and D). In the
current study, the de-protonated hydroxyl and carboxylic groups
from the caffeic acid made it a stronger complexing agent for silver
ions in the presence of an alkaline additive (NaOH). The silver ions
subsequently oxidized the hydroxyl groups into carbonyl groups
in the reduction reaction as the silver ions were simultaneously
reduced into the AgNPs. Several studies have recently reported that
an alkaline medium is a pre-requisite for synthesis of AgNPs using
active reducing components from the bio-resources [2831].
The UVvis spectroscopy plays a signicant role among the analytical techniques used to characterize the nanoparticles. It was
thus used in this study to authenticate the formation and stability of AgNPs in solution [32]. It is known that size inuences the
color of the AgNPs (Fig. 2) due to excitation of nanoparticles surface plasmon resonance (SPR) [33]. The UVvis absorption spectra
from the reaction solution in the present study were monitored in
the 300800 nm range (Fig. 3). The wavelength from the maximum
absorption peak was 396.5 nm, indicating the formation of AgNPs
with small size. The resonance position and peak width depend on
many factors and several physical properties of AgNPs [34]. The
UVvis spectra also demonstrated that the obtained AgNPs were
highly mono-dispersed because the absorbance peaks were sharp
as reported by literature in which a narrower SPR peak indicated a
smaller size distribution [29].
The detailed characterization by transmission electron
microscopy (TEM) analysis in the present study revealed that
the as-synthesized AgNPs had spherical shape and were quite uniform (Fig. 4A). The corresponding size distribution is represented
by histogram from the AgNPs, with 6.67 0.35 nm mean diameter
ranging from 3 nm to 10 nm (Fig. 4B). The TEM images were also in

Fig. 4. The morphology and size distribution of AgNPs synthesized by caffeic acid reduction method.
(A) The morphology of AgNPs depicted by transmission electron microscopy (TEM). Scale bar is 50 nm. (B) Size distribution histogram obtained by size analysis of several
TEM images of particles. The mean diameter was 6.67 0.35 nm.

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Fig. 5. Hydrodynamic diameter and zeta potential measurement of AgNPs in an aqueous solution.
Hydrodynamic diameter and surface charge of AgNPs were measured by dynamic light scattering (DLS), using a Malvern Zetasizer Nano (8.72 0.89 nm and 37.6 2.24 mV,
respectively).

Fig. 6. Uptake and localization of AgNPs in HepG2 cells.

(A) The representative image of control cells (B) The representative images of AgNPs-treated cells for 24 h. Nanoparticle aggregates visible in endosomes as black and electron
dense spots indicated by arrows. (C) Magnied view of the selected region inside cells showed that the cluster was composed of individual nanoparticles. Scale bars represent
500 nm in (A) and (B), and 200 nm in (C).

concordance with results obtained from the UVvis spectroscopy.

These results indicated that the caffeic acid-mediated AgNPs had
narrow particle size distribution when compared to the AgNPs
synthesized using the plant extracts [32,34,35].
The hydrodynamic diameter and zeta potential of the AgNPs
were measured by dynamic light scattering (DLS) using a Malvern
Zetasizer Nano. The average size of the AgNPs in colloidal solution was 8.72 0.89 nm (Fig. 5A). A negative (37.6 2.24 mV) zeta
potential was observed in the present study, posing an ideal surface
charge (Fig. 5B). High zeta potential absolute value designates high
electrical charge on the surface of the nanoparticles, causing strong
repulsive force among the particles for withholding agglomeration
[36]. The caffeic acid-mediated AgNPs therefore possessed high stability in colloidal solution as a result of their high zeta potential
value.

intake of the particles was successful but also retention of their

shapes and sizes without further agglomeration in the process. Further experiments are thus needed to completely elucidate their
uptake mechanism.
3.3. Effect of AgNPs on viability of HepG2 cells
The unique physico-chemical properties of the nanomaterials
could result in unexpected and novel biological responses compared to their pristine counterparts [38]. Several investigators

3.2. Intracellular localization of AgNPs in HepG2 cells

Uptake and localization of nanomaterials by cells is an important factor for evaluating nanotoxicity [37]. The uptake of the AgNPs
by the HepG2 cells was thus investigated using the TEM analysis, to conrm that the AgNPs successfully entered the cells. The
non-treated cells that were free of the AgNPs were used as control (Fig. 6A). The results showed that the HepG2 cells that were
treated with 5 g/mL AgNPs evidently internalized the nanoparticles aggregates and were mainly localized in the endosomes
(Fig. 6B), revealing that the internalization of the AgNPs in the
HepG2 cells was through endocytosis. A better view of the image at
higher magnication (Fig. 6C) clearly demonstrated clusters composed of individual nanoparticles as indicated by arrows inside the
cells. The average dimension of the particles matched with that
of the as-synthesized nanoparticles, conrming that not only the

Fig. 7. Effect of AgNPs on viability of hepatoma HepG2 cells.

The cell viability was determined using a MTT assay after a 24-h exposure of the ultra
ltrate (UF) or AgNPs at multiple concentrations. The data are summary of three
independent experiments and expressed as the means SD. *Denotes a signicant
reduction in cell viability (P < 0.05) compared to the control.

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233

Fig. 8. Apoptosis of HepG2 cells induced by AgNPs.

(A) Representative images. (B) Summary of distributions of cell status. The data are summary of three independent experiments and expressed as the means SD. *Denotes
a signicant reduction of apoptosis (P < 0.05) compared to the control.

have reported that the AgNPs can induce toxicity to cancer cells
and therefore be utilized as potent therapeutic agents for cancers
[3,3941]. Moreover, a recent investigation has showed that treatment with AgNPs can delay repair of X-ray induced DNA damage in
the HepG2 cells, suggesting the potential application of the AgNPs
in combination with radiotherapy for cancer treatment [42]. The
viability of the HepG2 cells in the present study, after 24-h exposure to AgNPs, was determined using the MTT assay. The results
showed that the cell viability was 76.1% at a low concentration
(1.25 g/mL) and further decreased with increasing nanoparticle
concentrations (Fig. 7). It is reported that oxidative stress is one
of the critical mechanisms of cytotoxicity induced by AgNPs [43].
The shape, size, and zeta potential are several important aspects
that inuence the toxicity of metal and metal oxide nanoparticles
through elevating reactive oxygen species (ROS) [44]. Due to the
physico-chemical differences, some AgNPs are broken down in the
lysosomes, and then release silver ions that bring about oxidative
stress [45]. To further clarify whether other factors affect the toxicity of the AgNPs to HepG2 cells, the AgNPs solution was centrifuged
using Millipore tube and the toxicity of the collected ultra ltrate
was investigated against the HepG2 cells. The results showed that
the cell viability was not signicantly inuenced by the ultra ltrate (Fig. 7). The caffeic acid had no signicant inuence on the
viability of the HepG2 cells at up to 20 g/mL concentration (Data
not shown) as reported previously [23,25]. These results clearly
demonstrated that the AgNPs cytotoxicity against the HepG2 cells
originated from the as-synthesized nanoparticles themselves.
3.4. Measurement of apoptosis in HepG2 cells treated with AgNPs
Previous reports have demonstrated that the AgNPs are
cytotoxic through their interaction with the mitochondria and
induction of the apoptosis pathway via production of ROS
[39,40,45,46]. In addition, oxidative stress can lead to genotoxic
stress and even upregulation of p53 gene [47,48], which is a
signicant advantage for nanomaterial application as anti-cancer
nanomedicine since upregulation of p53 gene triggers apoptosis [49]. Indeed, starch-capped AgNPs could be considered as the
potential anti-cancer agents to treat colon cancer cells that act in
a p53-dependent manner [50]. In the present study, the annexin
V-FLOUS/PI assay was carried out to evaluate the cytotoxic effects
of the AgNPs in terms of apoptosis (Fig. 8). The results showed that
the early apoptotic cells and late apoptotic cells were drastically

increased after 24 h treatment with the AgNPs at various concentrations (1.2520 g/mL). However, there were no signicant
changes in the percentage of necrotic cells. These results suggested
that the apoptosis was mediated by direct anti-tumor effect from
the as-prepared AgNPs. These ndings generally provide in vitro
evidence for the anti-cancer effect of the caffeic acid-mediated
AgNPs via the induction of apoptosis, indicating that the AgNPs
exhibit their potential application for hepatoma therapy. Moreover, a few studies suggests that exposure of endothelial cells to
nanoparticles indirectly results in endothelial cell leakiness by activating apoptotic events [5152]. This effect is also caused by the
physical interaction between negatively charged nanoparticles and
endothelial cells adherens junction, which is called nanoparticleinduced endothelial leakiness (NanoEL) [53]. NanoEL may be useful
in increasing leakiness from blood vessel to the tumor especially
when there is minimal enhanced permeability and retention (EPR)
effect, like in immature solid tumor [54].
4. Conclusion
In summary, we have herein reported a facile and rapid
approach for green synthesis of AgNPs using caffeic acid as both
a reducing agent and stabilizer at ambient conditions. Compared
to the classical biological synthesis procedures with irregular
morphologies and widely sized nanoparticles, the caffeic acidmediated AgNPs have comparatively spherical shape and uniform
size, with 6.67 0.35 nm mean diameter, and zeta potential of
37.6 2.24 mV, suggesting that they are also stable in aqueous
solution. Moreover, our data demonstrated that the AgNPs can
enter cells via endocytosis and effectively inhibit growth of the
HepG2 cells via apoptosis induction. Taken together, this study
suggests that the AgNPs, synthesized via the one-step caffeic acid
mediated reduction method, may be a potential therapeutic agent
for cancer.
Acknowledgments
This work was supported by the grants from the Natural Science
Foundation of Jiangsu Province in China (BK20140716), the China
Postdoctoral Science Foundation Funded Project (2015M571771),
the National Key Basic Research Program of China (2011CB933503),
and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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