See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/327536686

Biogenic synthesis of silver nanoparticles using extracts of Leptolyngbya JSC-

1 that induce apoptosis in HeLa cell line and exterminate pathogenic bacteria

Article in Artificial Cells · September 2018

DOI: 10.1080/21691401.2018.1499663

CITATIONS READS

6 285

12 authors, including:

Aftab Ahmad Arshad Iqbal

Gift University Beijing Forestry University
65 PUBLICATIONS 803 CITATIONS 31 PUBLICATIONS 143 CITATIONS

SEE PROFILE SEE PROFILE

Sadeeq Ullah Dr Raza

Beijing University of Chemical Technology Shenzhen institute of advanced technology
26 PUBLICATIONS 142 CITATIONS 60 PUBLICATIONS 398 CITATIONS

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Phenolic compounds View project

Senile dementia View project

All content following this page was uploaded by Pengcheng fu on 21 October 2018.

The user has requested enhancement of the downloaded file.

 Artificial Cells, Nanomedicine, and Biotechnology
An International Journal

ISSN: 2169-1401 (Print) 2169-141X (Online) Journal homepage: http://www.tandfonline.com/loi/ianb20

Biogenic synthesis of silver nanoparticles using

extracts of Leptolyngbya JSC-1 that induce
apoptosis in HeLa cell line and exterminate
pathogenic bacteria

Shah Zada, Aftab Ahmad, Sikandar Khan, Xinlu Yu, Kun Chang, Arshad Iqbal,
Adnan Ahmad, Sadeeq Ullah, Muslim Raza, Ajmal Khan, Shahbaz Ahmad &
Pengcheng Fu

To cite this article: Shah Zada, Aftab Ahmad, Sikandar Khan, Xinlu Yu, Kun Chang, Arshad
Iqbal, Adnan Ahmad, Sadeeq Ullah, Muslim Raza, Ajmal Khan, Shahbaz Ahmad & Pengcheng Fu
(2018): Biogenic synthesis of silver nanoparticles using extracts of Leptolyngbya JSC-1 that induce
apoptosis in HeLa cell line and exterminate pathogenic bacteria, Artificial Cells, Nanomedicine, and
Biotechnology, DOI: 10.1080/21691401.2018.1499663

To link to this article: https://doi.org/10.1080/21691401.2018.1499663

Published online: 08 Sep 2018.

Submit your article to this journal

View Crossmark data

Full Terms & Conditions of access and use can be found at

http://www.tandfonline.com/action/journalInformation?journalCode=ianb20
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY
https://doi.org/10.1080/21691401.2018.1499663

Biogenic synthesis of silver nanoparticles using extracts of Leptolyngbya JSC-1

that induce apoptosis in HeLa cell line and exterminate pathogenic bacteria
Shah Zadaa, Aftab Ahmada, Sikandar Khana, Xinlu Yua, Kun Changa, Arshad Iqbalb, Adnan Ahmadc,
Sadeeq Ullaha, Muslim Razaa, Ajmal Khana, Shahbaz Ahmada and Pengcheng Fua,d
a
College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, China; bCollege of Biological Sciences and
Biotechnology, National Engineering Laboratory for Tree Breeding, Beijing Forestry University, Beijing, China; cState Key Laboratory of
Animal breeding, Genetics and Reproduction College of Animal Science and Technology China Agriculture University Beijing, Beijing, China;
d
State Key Laboratory of Marine Resource Utilization in South China Sea, Hainan University, Hainan, China

ABSTRACT ARTICLE HISTORY

Utilizing novel approaches for the green synthesis of metal nanoparticles are of great importance. Received 9 June 2018
Therefore, we reported biogenic synthesis of silver nanoparticles (AgNPs) using extracts of Leptolyngbya Revised 7 July 2018
strain JSC-1, and their significant applications against pathogenic bacteria and cancerous HeLa cell line. Accepted 9 July 2018
The biofabricated AgNPs were characterized by UV–visible spectroscopy, FTIR, SEM, TEM, DLS and zeta-
KEYWORDS
potential. The as prepared AgNPs were assessed for inhibition of bacterial growth and induction of Silver nanoparticles;
apoptosis in HeLa cells by different doses of AgNPs was evaluated. UV–visible spectroscopy and FTIR of nanotechnology;
AgNPs demonstrated the surface plasmon resonance at 413 nm and interaction among extract and Leptolyngbya; antibacterial;
nanoparticles, respectively. Electron microscopy revealed the morphology and DLS demonstrated size anticancer; HeLa
distribution of the particles (10–100 nm). Zeta potential values were between
47 and 0 mV, indicating
stability of the particles. Proliferation of HeLa cells was significantly inhibited and severe cytotoxicity
with higher intracellular uptake were observed after applying high concentration of AgNPs. Efficient
inhibition zones (17 ± 2 and 21 ± 2 mm) were produced at maximum concentration (100 ml from
1 mg ml
1 stock of AgNPs) for Staphylococcus aureus and Escherichia coli, respectively. These findings
reveal that the biofabricated AgNPs possess strong antibacterial activity and ability to induce apoptosis
in cancer cell line (HeLa).

Introduction Bioactive materials in the extracts of photosynthetic

Recently, numerous important technological innovations
occurred in the field of nanotechnology. Nanoscience and
nanotechnology deal with the synthesis of nanoparticles (NPs)
of different chemical compositions [1]. At present, the import-
ance of this field emerges in environmental science because of
wide applications of nanoparticle for removal of toxic chemi-
cals and pollutants from environment [2]. It is a nanorevolution
persisted to unfold the manufacturing of environment friendly
and non-polluting nanomaterials. Chemical synthesis of such
materials might lead to addition of some deadly chemicals on
their surfaces that may have unfavourable effects in medical
applications [3]. Therefore, the researchers are engaged to
think about alternatives and find other ways; hence, green syn-
thesis and assembly of nanoparticles might be a best choice
[4]. The use of eco-friendly materials like whole plant, parts of
plant, algae and cyanobacteria for the synthesis of nanopar-
ticles (NPs) offer several benefits for environment and compati-
bility for biomedical applications [5]. Free metal nanoparticles
due to their unique physical and chemical properties have
potential applications in catalysis, bio-sensing, bio-labelling,
electronics, optical devices and controlled drug delivery [6,7].
cyanobacteria can act as reducing and stabilizing agents and
confer more biocompatibility to biosynthesized NPs [8]. Algae
including cyanobacteria are a group of naturally available
bio-factories for bioactive products [9]. These bioactive com-
pounds might be proteins, carbohydrates, lipids, vitamins,
carotenoids and many other secondary metabolites with a
wide range of biological activities and applications [10].
These bio-nanofactories possess unique ability to synthesize
biochemicals, involved in the synthesis of metallic nanopar-
ticles with high stability [11]. Recently, metal nanoparticles
were synthesized using the extracts of algae such as
Turbinaria conoides, Kappaphycus alvarezii, Laminaria japonica,
Chlorella pyrenoidusa, Stoechospermum marginatum, Sargassum
wightii, Acanthophora spicifera and Sargassum myriocystum [7].
One of the largest group of algae namely cyanobacteria (blue-
green algae) found in natural aquatic environment were also
reported for the same purpose of bio-fabrication of metal
nanoparticles [12].
Because of wide applications of AgNPs in health care, they
might be exploited for cancer treatment, which is one of the
most deadly diseases in the world. Currently, for cancer

Pengcheng Fu fupc@mail.buct.edu.cn College of Life Science and Technology, Beijing University of Chemical Technology, 15 Beisanhuan East Road,
Chaoyang District, Beijing 100029, China; Sikandar Khan uomians@yahoo.com College of Life Science and Technology, Beijing University of Chemical
Technology, 15 Beisanhuan East Road, Chaoyang District, Beijing 100029, China
ß 2018 Informa UK Limited, trading as Taylor & Francis Group
2 S. ZADA ET AL.
treatment different ways are under practice, including surgi-
cal intervention, chemotherapy and radiation, which might
also destroy healthy cells, thus causing other complications
and are harmful for the patients [13]. Hence, there is a dire
need to find new ways of treatment that might have fewer
or no side effects and safer for cancer patients. Nanoparticles
are widely investigated for biological applications and med-
ical purposes due to their unique optical properties, eco-
friendly nature and electrochemical stability. Therefore, in this
study, AgNPs were synthesized using cyanobacterial extracts
and then their apoptotic effects against HeLa cells and some
pathogenic bacteria were investigated.
Materials and methods
Reagents and instruments
The experimental materials including silver salts (analytical
grade) were procured from Sigma Aldrich and the solvent
used throughout the experiments was double distilled water
(Millipore 18.2 MX cm). All other instruments used in the
study were High speed refrigerated centrifuge (JW-3021HR),
UV/Vis spectrophotometer (Shimadzu, 2450), X-ray diffraction
(Powder X-ray-D8 advanced diffractometer, BRUKER), scan-
ning electron microscope (JEOL JEM-3100), EDX (JEOL-JEM
3010), transmission electron microscope (FEI-Tecnai G2 20
TEM), dynamic light scattering and zeta potential (HORIBA
Zetasizer SZ100), FTIR (Nicolet 6700 FT-IR spectrometer), MTT
assay (MAXline microplate reader), ICP-AES (Skyray instru-
ments ICP2060T) and confocal microscope (Leica TCS SP8).
Cyanobacterial cultivation
Leptolyngbya JSC-1 (a siderophilic cyanobacterium) was gifted
by Igor I. Brown (Beijing University of Chemical Technology,
Beijing) [14]. It was grown in modified DH medium (pH 8)
using 4.6 mM 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic
acid (HEPES) as a buffer. The medium was supplemented
with 40 mM iron for its optimum growth. The growth was car-
ried out for 10 days in 500 ml flasks (45 ± 1  C) under 24 h
light condition in a growth chamber [14].
Preparation of extracts
Biomass of Leptolyngbya JSC-1 was acquired by centrifugation
of the culture at 5000g for 12 min. The growth medium
(supernatant) was discarded and the collected pellet (biomass)
was thoroughly washed with distilled water. Finally, the pellet
(biomass) was dried at 60  C and crushed into powder using
an agate mortar. One gram of the powdered biomass was
dispersed in 100 ml of distilled water and maintained at 60  C
for 30 min on mild stirring. The extracted biochemicals from
JSC-1 biomass were separated by centrifugation at 6000 rpm
(10 min) [13].
Synthesis of silver nanoparticles
For the preparation of AgNPs, 10 ml of the JSC-1 extract was
treated with 50 ml of 1 mM silver nitrate (AgNO3) solution
and kept on stirring (500 rpm) at room temperature [15]. The
development of blackish colour in the reaction mixture desig-
nates the synthesis of AgNPs. Optical property such as local-
ized surface plasmon resonance (LSPR) for the AgNPs was
identified by UV–visible spectrometry (Shimadzu, 2450). The
reaction was terminated after SPR bands saturation. The
acquired product in suspension was recovered by centrifuga-
tion at 12,000 rpm for 15 min. The collected AgNPs were
repeatedly washed and freeze-dried [13].
Characterization of nanoparticles
UV–visible spectroscopy
The SPR pattern of AgNPs was investigated by UV–vis spec-
troscopy. The materials were scanned in the range of
200–800 nm [13,15].
Transmission electron microscopy (TEM)
Particles sizes and shapes of the AgNPs were visualized by
transmission electron microscopy. The homogeneous suspen-
sion of biogenic particles of silver were mounted on the car-
bon-coated copper grid, dried in air and then analysed with
FEI-Tecnai G2 20 TEM [15,16].
Scanning electron microscopy and energy-dispersive X-ray
(SEM and EDX)
Surface features and elemental analysis of AgNPs maintained
on graphite grid were studied by SEM and EDX techniques
(JEM-3100 equipped with an EDX unit) [13,15,16].
X-ray diffraction (XRD)
The X-ray diffraction (XRD) profile of the synthesized materi-
als was obtained with X-ray diffractometer (Powder X-ray-D8
advanced diffractometer, Burker) at 2h, 10–100 using Cu Ka
radiation at 40 kV and 30 mA [13,15,16].
Fourier transformed infrared (FTIR)
FTIR spectral bands in the prepared materials (AgNPs) were
determined using a Nicolet 6700 FT-IR spectrometer, @
500–4000 cm
1 in transmittance mode. Samples for FTIR ana-
lysis were prepared using the KBr pellet technique, which
involves mixing thoroughly the as prepared AgNPs with KBr
before forming a pellet at high pressure. IR bands of
Leptolyngbya JSC-1 powder extracts and biosynthesized
AgNPs were compared to evaluate the probable functional
groups involved in AgNPs formation [13,15,16].
DLS and zeta potential
The distribution and size of AgNPs were measured using
dynamic light scattering (DLS) and zeta potential (HORIBA
Zetasizer SZ-100) [17].

Biological activity of AgNPs
Growth inhibition study of bacteria
Both strains of bacteria (Staphylococcus aureus and
Escherichia coli) were cultured in LB broth, which were further
inoculated in fresh 10 ml liquid medium. Both the strains
were then treated with different concentrations of biosynthe-
sized AgNPs (0, 25, 50 and 100 mg/ml) under optimum condi-
tions of bacterial growth (37  C and 150 rpm continuous
shaking). Growth of both the strains in LB liquid medium was
measured by optical density (OD) @ k ¼ 600 nm at regular
intervals (1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 h)
using UV–Vis spectrophotometer. The growth curve was
obtained by plotting optical density against time [18].
Well diffusion method
Well diffusion method was used to assess the antibacterial
potential of the biosynthesized AgNPs against two patho-
genic strains (S. aureus and E. coli). Four Petri-dishes were
prepared with Luria Bertani (LB) agar medium having wells of
8 mm diameter at equal distance. After inoculation, the wells
were loaded with different concentrations (0, 25, 50 and
100 ml) from stock solution of AgNPs (1 mg ml
1). The plates
were incubated at 37  C and after 24 h, the inhibition zones
were measured [19].
Cellular damage
Cellular morphologies of S. aureus and E. coli treated with
varying concentrations of AgNPs (0, 25, 50 and 100 ml) were
examined after 24 h of incubation using the standard method
for scanning electron microscopy [16].
Anticancer activity of AgNPs
Anticancer activity of the AgNPs was evaluated by a dose-
dependent manner using HeLa cells of human epithelioid
cervix carcinoma cells. The cells were exposed to 5, 10, 20,
40, 80 and 160 lg ml
1 of AgNPs, in culture media for 24 h
and the viability of cells was determined by MTT assay. The
cells were washed thrice with DMSO and added to each well
having MTT solution (10%) and incubated for 5 h. The
absorbance was measured @ 570 nm and the percent cell
survival was determined as the percent MTT reduction by
cells exposed to different concentrations of AgNPs relative to
control group [20].
AO/EB fluorescent staining for analysis of apoptosis
Cells of human epithelioid cervix carcinoma (HeLa) in the loga-
rithmic growth phase were cultured in 96-well plate contain-
ing Dulbecco's modified Eagle's medium (DMEM) (100 ll/well).
Cells were added to a final concentration of 5  103 ml
1 and
were incubated. HeLa cells were left untreated as control,
treated with only extracts, and different concentrations of
AgNPs, i.e. 5, 10, 20, 40, 80 and 160 mg ml
1. The samples in
96-well plate were divided into eight groups, with 12 samples
in each group corresponding to different treatments. After
being cultured for 24 h, the cell culture suspensions (20 ll
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 3
each) were mounted on glass slides. Dual fluorescent staining
solution (1 ll) containing acridine orange/ethidium bromide
(AO/EB, 100 lg each ml
1) was added. The morphology of
apoptotic cells was examined using a fluorescent micro-
scope [21].
In vitro cellular uptake of AgNPs
In vitro cellular uptake of biogenic AgNPs was determined
after the HeLa cells previously grown in Dulbecco's modified
Eagle's medium (DMEM) in the presence of different concen-
trations of AgNPs, at 37  C for 24 h and adjusted the pH at 7.
These cells were treated with trypsin and collected by centri-
fugation (1600 rpm, for 5 min) and washed the cells with PBS
thrice during centrifugation to remove extracellular AgNPs.
Then resuspend the cells pellet in PBS (2 ml) and according
to Privies protocol the samples were prepared for ICP-AES
analysis [20].
Results and discussion
Synthesis of AgNPs and UV–visible spectroscopy
After mixing the Leptolyngbya JSC-1 extracts with the AgNO3
solution, the colour change occurred from transparent to
dark blackish. The colour change of the mixture was noted
visually (Figure 1(A)). After 20 min of the reaction, both
changes in colour and the absorbance were recorded at
regular intervals, as the colour change was the first evidence
of nanoparticles formation. Generally, the colour change
occurred due to reducing agents released from algal extracts
into solution [21]. This was also supported by UV–visible
spectra, when exposed to light of specific wavelength, the
NPs gave a unique surface phenomenon called surface plas-
mon resonance (SPR); due to this, a specific peak formation
occurred for each kind of NPs by UV–Vis spectroscopy [7].
The synthesis of AgNPs using (Leptolyngbya JSC-1) extract
was monitored by using UV–Vis spectroscopy; the light
absorption pattern of the algal biomass was observed in the
range of 300–700 nm. The silver ions reduction peak of the
SPR occurred at 413 nm (Figure 1(B)). The same peak area for
AgNPs at 413 nm was also reported by other researchers, one
of them was incubation of Agþ with fungal biomass of
Trichoderma koningii [19].
Algal extracts are rich in bioactive molecules and func-
tional groups like –OH, carboxylic and amide groups which
improved the quantitative production of AgNPs and their sta-
ble dispersion. Therefore, algal extracts have the power to be
used for the synthesis of nanoparticles. It is well known that
the extinction coefficient of AgNPs at their SPR band peak is
proportional to the nanoparticle concentration. Thus, the
method publicized powerful potential in producing high-con-
centration of AgNPs with controlled range of particles size
which is the priority for large-scale fabrication and develop-
ment of value-added nano products [22].
4 S. ZADA ET AL.
Figure 1. Confirmation of AgNPs synthesis by visual colour change (A). UV–Vis spectra of the biofabricated AgNPs at different time intervals (B).
TEM analysis of AgNPs
The TEM images confirmed that the newly biofabricated
AgNPs are well dispersed consisting mostly of spherical and
nearly spherical shapes (Figure 2(A)). These micrographs
showed a spherical conformation of majority of the AgNPs
with a size range of 5–50 nm (Figure 2(B)). Selected area elec-
tron diffraction (SAED) of a single spherical particle was
explained and the size measured by TEM analysis was found
to be lower than that observed in DLS analysis. Similar results
were also found by other researchers recently [23–25].
Scanning electron microscopy (SEM) and energy-
dispersive X-ray (EDX) analysis
SEM analysis revealed the morphology and size of the bio-
synthesized AgNPs (Figure 2(C)). It is also evident that the
biomolecules in the extracts of JSC-1 promoted synthesis of
AgNPs. The SEM images of AgNPs reported by other
researchers, from different extracts showed spherical par-
ticles, aggregated spherical particles, irregularly shaped par-
ticles and cubic particles. The moderate particles size
observed were 12, 25, 35 and 50 nm, for AgNPs synthesized
by JSC-1 extracts using water as a solvent [26–29].
EDX of the NPs was performed to investigate the elemen-
tal composition and stoichiometry of the biosynthesized
AgNPs (Figure 2(D)). The EDX spectra revealed the presence
of silver peaks around 3 and 3.1 keV, which were appeared
due to the discharge of different electrons from L and K
shells of silver, respectively. The lower energy peak (3 keV)
was responsible for the outer shell electrons (L) and higher
energy peak (3.1 keV) was responsible for inner shell elec-
trons (K). The carbon and platinum peaks present in the
spectra were mainly due to the carbon adhesive tape and
platinum coating used and other peaks were due to inor-
ganic impurities from the biomolecules in the Leptolyngbya
JSC-1 extract. The EDX data of AgNPs showed that the
weight percentage of Ag was 53.77% [26,27].
XRD and FTIR analysis
The biosynthesized silver nanostructures were further ana-
lysed by XRD that determined the crystalline structure of the
bio-synthesized AgNPs (Figure 3(A)). The XRD pattern of the
AgNPs from 20 to 90 2h using X-ray diffractometer confirmed
that there were five well-defined characteristic diffraction
peaks at 34.5 , 35.1 , 47.1 , 58.4 and 86.7 , these peaks corre-
sponds to the 111, 200, 220, 311 and 422 diffractions, respect-
ively. The XRD features of AgNPs are matched with standard
silver which was reported by JCPDS {file no. 04–0784}. Also
widening of the diffraction peaks was observed which might
be the effect of the size of nanoparticles. As the nanoparticles
are capped by the moieties of JSC-1 biomolecules, therefore,
the background observed was high [26–28].
FTIR was employed to quantify and determine the functional
biomolecules in the JSC-1 extracts which were important for
the reduction of silver ions into relative metal nanoparticles.
The JSC-1 extracts have variety of biomolecules, which might
possibly be involved in the synthesis of these NPs (Figure 3(B)).
The occurrence of many outstanding signatory peaks in the IR
region of electromagnetic spectrum is due to different func-
tional groups. The active and extended band was observed at
3415 cm
1 that confirmed the presence of polyphenolic –OH
group. The second band was observed relative to alkanes group
of C–H stretching vibrations at the absorption band 2859 cm
1,
the third narrow band occurred at 1638 cm
1 indicating the
presence of amide I group, the fourth short band appeared at
1250 cm
1 assigned to C–C stretching of aromatic ring, the fifth
narrow band at 1037 cm
1 corresponds to C–N stretching vibra-
tions of aliphatic amines of protein [28]. The sixth very short
band at 850 cm
1 attached to S–O stretching of sulfonates,
while the last band at 590 cm
1 represented alkyl halides. This
investigation also demonstrated that protein and amino acids
have the capacity to bind metal. It is evident that polysacchar-
ides, sulfonated compounds and amide linkages are strongly
involved in the reduction of silver ions into nanoparticles.
Earlier reports are also in line with this study confirming the
presence of carboxylic, amine, phosphate and hydroxyl func-
tional groups in the algal extract of Sargassum polycystum and
are involved in the reduction of silver ions. The biomolecules
present in algal extract have dual functions of reducing silver
ions and stabilizing the AgNPs [23,30].
Dynamic light scattering (DLS) distribution and
zeta potential
Dynamic light scattering (DLS) analysis is very important and
useful approach to analyse particle size and its distribution in
the nanomaterials. DLS of the biosynthesized AgNPs dis-
played a particle size range of 10–138 nm (Figure 4(A)). The

Figure 2. TEM micrographs of biosynthesized AgNPs (A and B). SEM images of the silver nanoparticle (C) and EDX spectrum of AgNPs confirming its composition (D).
Figure 3. XRD spectrum of AgNPs displaying characteristic diffraction peaks (A) and FTIR spectrum of the biosynthesized AgNPs shows different functional groups
involved in capping (B).
average expected size of AgNPs was identified to be 71.5 nm.
The variation in particles size as determined by two techni-
ques (DLS and TEM) may be ascribed to the fact that these
techniques are based on different detection methods and
principles. Besides, DLS measures the hydrodynamic diameter
of the particles which might be augmented by water mole-
cules, thus, the resulting particles size is generally broader
than that obtained from TEM analysis [31–33].
Zeta potential analysis of the biosynthesized AgNPs was found
as a single sharp peak between
47 and 0 mV while having a
maximum intensity at
24.4 mV (Figure 4(B)). It indicates that the
negatively charged moieties are present on the surface of the
AgNPs that expanded in the medium. The repulsion among the
particles might be due to the negative values that proved that
the particles are very stable. Low zeta potential values of particles
suggest no flocculation and no tendency to assemble together
due to repulsion forces among particles [33,34].
Inhibition of bacterial growth and antibacterial activity
Antibacterial activities of the biosynthesized AgNPs were per-
formed for E. coli and S. aureus and the growth rates were
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 5
measured by optical densities at regular intervals after treat-
ing with different doses of AgNPs (0–100 ml from stock solu-
tion 1 mg ml
1) (Figure 5(A,B)). It was found that the growth
of both bacteria was decreased with increasing the concen-
tration of AgNPs and vice versa. The minimum inhibitory con-
centration (MIC) of AgNPs was 25 ml while the growth rates
of both strains of bacteria were quite low at 50 and 100 ml.
The antibacterial potency of the biosynthesized AgNPs
was inspected against the pathogenic bacteria (E. coli and S.
aureus). The diameter of inhibition zones were measured as a
score, indicating that the diameter of inhibition zones
increases with increasing the concentration of AgNPs. No
inhibition zone was noted in the control (having no AgNPs).
The average diameter of inhibition zones were 9 ± 2 mm,
15 ± 2 mm and 21 ± 2 mm at 25, 50 and 100 ml, respectively
for E. coli (Figure 5(C)). Inhibition zones for S. aureus were
measured as 7 ± 2 mm, 12 ± 2 mm and 17 ± 2 mm at 25, 50
and 100 ml concentrations of AgNPs, respectively (Figure
5(D)). The maximum inhibition zone was observed at 100 ml
concentration of AgNPs for E. coli (21 ± 2 mm), suggested the
efficacy of the biosynthesized NPs against the pathogenic
bacterial strains. Comparatively higher activity against E. coli
6 S. ZADA ET AL.

Figure 4. Dynamic light scattering (A) and Zeta potential (B) of the biosynthesized silver nanoparticles, illustrating the particle size distribution and surface charge
value, respectively.

Figure 5. Growth variation of E. coli and S. aureus (A and B, respectively) at different concentrations (0, 25, 50 and 100 ml) of AgNPs after incubation for 24 h. Error
bars indicate the triplicates in experiment where p values was .05. Inhibition zones in culture plates at varying concentrations of AgNPs like 0, 25, 50 and 100 ml
indicated as a, b, c and d for E. coli while A, B, C and D for S. aureus, respectively.

may be due to the structural variation and cell wall compos-
ition in both species. The thin peptidoglycan layer in E. coli
would produce less barriers for particles to enter the cells. The
probable mechanism of action of the noble metal nanopar-
ticles to eradicate bacteria might be the membrane damage,
creation of reactive oxygen species (ROS), inhibition of some
essential enzymes and DNA damage, etc. [7]. Among these
mechanisms, the most effective was found to be the ROS pro-
duction and cell membrane disruption leading to leakage of
nucleic acids and protein from bacterial cells [16,19].
Cell membrane disruption
SEM micrographs displayed a sever membrane disruption of
bacterial cells exposed to four different concentrations of
biogenic AgNPs (0, 25, 50 and 100 ml from stock solution
1 mg ml
1). The extracellular morphology of treated cells indi-
cated pores formation in both strains at 25 ml AgNPs (Figure
6(B,b)). Subsequent leakage of the cytoplasmic materials and
cell membrane disruption was observed at 50 and 100 ml of
AgNPs (Figure 6(C,c,D,d)), respectively. In comparison to con-
trol group (Figure 6(A,a)), the cellular deformation was found
to be more severe in case of E. coli than S. aureus [34,35].
Anticancer activity using HeLa cell line
The anticancer efficiency of biofabricated AgNPs were eval-
uated using HeLa cell line as the target and both percent via-
bility (Figure 7(A)) and cancer cell mortality rate (Figure 7(B))
were examined. HeLa cells treated with several concentrations
 ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 7

Figure 6. SEM micrographs showing cellular damage of both S. aureus (A0 B0 C and D) and E. coli (a0 b0 c and d) treated with varying concentrations of AgNPs (0,
25, 50 and 100 ml, respectively).

Figure 7. Percent viability of cancer cells after treating with AgNPs (0, 5, 10, 20, 40, 80 and 160 ml) for 24 h (A), cancer cells mortality rate (B) and cytotoxicity to
HeLa cells studied by MTT assay (C). Errors bars indicate that the experiments were carried out in triplicates and where p value was .05 as calculated using
student’s t test. The further division of (C) such as i, ii, iii, iv, v, vi, vii and viii correspond to different treatments, e.g. extracts only, 0, 5, 10, 20, 40, 80 and 160 ml,
respectively.

of biogenic AgNPs (0, 5, 10, 20, 40, 80 and 160 ml from
1 mg ml
1 stock) for a period of 24 h, and then the morph-
ology of these cells were studied by using MTT assay (Figure
7(Ci–viii)). Significant difference in terms of decline in number
of viable HeLa cells were observed after treating them with
biogenic AgNPs as compared to extracts only, where p value
was .05 as calculated using student’s t test.
Cellular viability and mortality were determined after
applying AgNPs by MTT assay and it was observed that the
number of viable cells was decreased with increasing concen-
tration of biogenic AgNPs. Finally, 55% viability was detected
at the highest concentration (160 ml). The “extract only” did
not show any significant cytotoxicity towards HeLa cells
[36,37]. This indicated that the AgNPs become more potent
after reduction of silver ions by the extracts utilized, showing
high mortality rate (45%) when applied to HeLa cells (Figure
7(B,Cviii)). Our findings are in line with the previous studies,
suggesting potential use of metallic NPs against cancer
cells [38–40].
Analysis of apoptosis
The apoptotic morphological changes in HeLa cells caused
by AgNPs were studied by using dual staining AO/EB. It was
found that HeLa cells showed apoptotic features after treat-
ment with AgNPs, such as condensed nuclei, membrane
8 S. ZADA ET AL.

Figure 8. Control/untreated HeLa cells (A), HeLa cells treated with only extract of JSC-1 (B) and HeLa cells treated with different concentrations of AgNPs, i.e. 5, 10,
20, 40, 80 and 160 mg ml
1 (C, D, E, F, G and H, respectively).

Figure 9. Normal cells viability after treatment with different concentrations of AgNPs as determined by MTT assay (A). Intracellular accumulation of AgNPs by
HeLa cells using ICP-AES (B). Errors bars indicate that the experiment were carried out in triplicates and where p  .05.

blebbing, apoptotic bodies, etc. The fluorescence from green
to red shows the level of damage and death of the cell. The
stained cells (green, control) were characterized as viable with
no damage occurred, while the apoptotic effect started from
early apoptotic (light red), mid apoptotic with condensed
chromatin (bright red), late apoptotic and nonviable cells (dark
red) (Figure 8(A–H)). With increasing concentration of AgNPs,
the change in fluorescence switched from greenish to dark
red, evidently described that the cells are going to die. In case
of plant extracts only, no such features were observed even at
the higher concentration tested (Figure 8(B)). Thus, these were
our preliminary findings that the AgNPs capped with bioactive
components of JSC-1 extract possess potential to cure the
deadly cancer. An in-depth study is required to further evalu-
ate the molecular changes occurred in these cells and also
mechanism of action of the said AgNPs against HeLa cell line
and other cancerous cells [41,42].
Analysis of specificity and cellular uptake of AgNPs by
HeLa cell line
Specificity of the as prepared AgNPs for HeLa cells was deter-
mined after treating the normal cells with different concentra-
tions of AgNPs. The bio-synthesized AgNPs were well tolerated
by normal human cells and did not caused any significant tox-
icity up to 80 mg ml
1. However, about 30% mortality was
observed at the highest dose (160 mg ml
1) of AgNPs (Figure
9(A)). This enhanced anticancer activity and specificity of bio-
genic AgNPs for cancerous cells while low cytotoxicity to nor-
mal cells could be of significant importance in the treatment
of cancer cells. This specificity could be attributed to the less
compact cellular architecture of cancerous cells that allow
penetration of AgNPs into cytoplasm of the cells. Similar find-
ings have been reported by other researchers [20,43,44].
On the other hand, ICP-AES studies were carried out to con-
firm the cellular uptake of AgNPs by HeLa cells. It was observed
that increasing the dose of AgNPs the amount of intracellular
AgNPs accumulation were increased (Figure 9(B)). ICP-AES data
suggested that diffusion might be the probable mechanism of
AgNPs’ entry into the cells; thus, it might lead the cancer cell to
display apoptotic features and ultimately dies. A previous study
indicated that AgNPs (under cell culture condition) displayed
lesser endocytosis and clathrin pits formation [20].
Dissolution of silver ions from AgNPs
The cytotoxicity of AgNPs is basically associated with the dis-
solution and release of toxic silver ions (Agþ) in biological

media. The release of Agþ depend upon the pH value and
O2 concentration in the medium and is also affected by the
availability of reducing sugars, chlorides, sulphides and sul-
phur containing components. The mechanism of dissolution
of AgNPs in biological media, either cell culture media or in
cells has hardly been investigated. However, preliminary stud-
ies on the dissolution of AgNPs in NaCl solution (10 mM) viz
far below the isotonic concentration (154 mM) indicate that
even under such mild conditions quick degradation of the
particles occur. In general, the dissolution of NPs can be
ascribed to their surface functionalization. It is well known
that AgNPs can be oxidized in aqueous solutions exposed to
air (Equation (1)) resulting in the release of Agþ under acidic
conditions (Equation (2)) [45,46].
4Ag0 þ O2 ! 2Ag2 O (1)
2Ag2 O þ 4Hþ ! 4Agþ þ 2H2 O (2)
Conclusion
The bioactive components in the extract of cyanobacteria
Leptolyngbya JSC-1 have great potential to be used as reducing
and capping agents for biosynthesis of stable AgNPs. As men-
tioned in our previous study of gold nanoparticles synthesis with
the same extracts, it was observed that the JSC-1 extract has a
strong redox potential which can be exploited for the synthesis of
a variety of metal NPs. Currently, the as prepared AgNPs have
strong antibacterial activities against both E. coli and S. aureus.
The most important aspect of this study was the application of
the biogenic AgNPs against the deadly cancer (HeLa cell line). Our
preliminary findings unveil that the AgNPs capped with JSC-1
extracts depicted excellent anticancer activity against HeLa cells.
This might be an area of interest for other researchers to further
investigate and disclose more important findings.
Disclosure statement
All the authors declare that there is no potential conflict of interests.
Funding
This study was supported by CSC (China Scholarship Council) via a schol-
arship to S.Z (No. 2015GXZU57), and by Fundamental Research Funds for
the Central Universities in China (No. YS0417).
References
[1] Saleh T, Ahmed E, Yu L, et al. Silver nanoparticles improve struc-
tural stability and biocompatibility of decellularized porcine liver.
Artif Cell Nanomed B. 2018. Available from: https://doi.org/10.
1080/21691401.2018.1457037
[2] Shankar SS, Rai A, Ahmad A, et al. Rapid synthesis of Au, Ag, and
bimetallic Au core–Ag shell nanoparticles using Neem (Azadirachta
indica) leaf broth. J Colloid Interf Sci. 2004;275:496–502.
[3] Nadagouda MN, Varma RS. Green synthesis of silver and palla-
dium nanoparticles at room temperature using coffee and tea
extract. Green Chem. 2008;10:859–862.
[4] Barabadi H, Ovais M, Shinwari ZK, et al. Anti-cancer green biona-
nomaterials: present status and future prospects. Green Chem
Lett Rev. 2017;10:285–314.
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 9
[5] Singh M, Kalaivani R, Manikandan S, et al. Facile green synthesis
of variable metallic gold nanoparticle using Padina gymnospora,
a brown marine macroalga. Appl Nanosci. 2013;3:145–151.
[6] Lewis LN. Chemical catalysis by colloids and clusters. Chem Rev.
1993;93:2693–2730.
[7] Zada S, Ahmad A, Khan S, et al. Biofabrication of gold nanopar-
ticles by Lyptolyngbya JSC-1 extract as super reducing and stabi-
lizing agents: Synthesis, characterization and antibacterial activity.
Microb Pathogenesis. 2018;114:116–123.
[8] Dumur F, Guerlin A, Dumas E, et al. Controlled spontaneous gen-
eration of gold nanoparticles assisted by dual reducing and cap-
ping agents. Gold Bull. 2011;44:119–137.
[9] Inbakandan D, Venkatesan R, Khan SA. Biosynthesis of gold nano-
particles utilizing marine sponge Acanthella elongata (Dendy,
1905). Colloid Surface B. 2010;81:634–639.
[10] Kim S-K. Handbook of marine macroalgae: biotechnology and
applied phycology. New Jersey: John Wiley & Sons; 2011.
[11] Song JY, Kim BS. Rapid biological synthesis of silver nanoparticles
using plant leaf extracts. Bioprocess Biosyst Eng. 2009;32:79.
[12] Gadd GM. Heavy metal accumulation by bacteria and other
microorganisms. Experientia 1990;46:834–840.
[13] Rajeshkumar S, Malarkodi C, Paulkumar K, et al. Algae mediated
green fabrication of silver nanoparticles and examination of its anti-
fungal activity against clinical pathogens. Int J Met. 2014;2014:1.
[14] Brown II, Bryant DA, Casamatta D, et al. Polyphasic characteriza-
tion of a thermotolerant siderophilic filamentous cyanobacterium
that produces intracellular iron deposits. Appl Environ Microb.
2010;76:6664–6672.
[15] Ahmad A, Syed F, Shah A, et al. Silver and gold nanoparticles
from Sargentodoxa cuneata: synthesis, characterization and anti-
leishmanial activity. RSC Adv. 2015;5:73793–73806.
[16] Ahmad A, Wei Y, Syed F, et al. Isatis tinctoria mediated synthesis
of amphotericin B-bound silver nanoparticles with enhanced pho-
toinduced antileishmanial activity: a novel green approach. J
Photoch Photobio B. 2016;161:17–24.
[17] Dinesh D, Murugan K, Madhiyazhagan P, et al. Mosquitocidal and
antibacterial activity of green-synthesized silver nanoparticles from
Aloe vera extracts: towards an effective tool against the malaria
vector Anopheles stephensi?. Parasitol Res. 2015;114:1519–1529.
[18] Baghbani-Arani F, Movagharnia R, Sharifian A, et al. Photo-cata-
lytic, anti-bacterial, and anti-cancer properties of phyto-mediated
synthesis of silver nanoparticles from Artemisia tournefortiana
Rchb extract. J Photoch Photobio B. 2017;173:640–649.
[19] Tripathi R, Gupta RK, Shrivastav A, et al. Trichoderma koningii assisted
biogenic synthesis of silver nanoparticles and evaluation of their anti-
bacterial activity. Adv Nat Sci: Nanosci Nanotechnol. 2013;4:035005.
[20] Ullah S, Ahmad A, Wang A, et al. Bio-fabrication of catalytic plat-
inum nanoparticles and their in vitro efficacy against lungs cancer
cells line (A549). J Photoch Photobio B. 2017;173:368–375.
[21] Subbaiya R, Saravanan M, Priya AR, et al. Biomimetic synthesis of
silver nanoparticles from Streptomyces atrovirens and their poten-
tial anticancer activity against human breast cancer cells. IET
Nanobiotechnol. 2017;11:965–972.
[22] Kim D-Y, Saratale RG, Shinde S, et al. Green synthesis of silver nano-
particles using Laminaria japonica extract: Characterization and seed-
ling growth assessment. J Clean Prod. 2018;172:2910–2918.
[23] Anandalakshmi K, Venugobal J, Ramasamy V. Characterization of
silver nanoparticles by green synthesis method using Pedalium
murex leaf extract and their antibacterial activity. Appl Nanosci.
2016;6:399–408.
[24] Kasithevar M, Saravanan M, Prakash P, et al. Green synthesis of sil-
ver nanoparticles using Alysicarpus monilifer leaf extract and its
antibacterial activity against MRSA and CoNS isolates in HIV
patients. J Interdi Nanomed. 2017;2:131–141.
[25] Lee S-W, Chang S-H, Lai Y-S, et al. Effect of temperature on the
growth of silver nanoparticles using plasmon-mediated method
under the irradiation of green LEDs. Materials 2014;7:7781–7798.
[26] Omar HH, Bahabri FS, El-Gendy AM. Biopotential application of
synthesis nanoparticles as antimicrobial agents by using
Laurencia papillosa. Int J Pharmacol. 2017;13:303–312.
10 S. ZADA ET AL.
[27] Vivek M, Kumar PS, Steffi S, et al. Biogenic silver nanoparticles by
Gelidiella acerosa extract and their antifungal effects. Avicenna J
Med Biotechnol 2011;3:143.
[28] Gnanajobitha G, Paulkumar K, Vanaja M, et al. Fruit-mediated syn-
thesis of silver nanoparticles using Vitis vinifera and evaluation of
their antimicrobial efficacy. J Nanostruct Chem. 2013;3:67.
[29] Roy S, Anantharaman P. Biosynthesis of Silver Nanoparticles by
Chaetomorpha antennina (Bory de Saint-Vincent) Kutzing with Its
Antibacterial Activity and Ecological Implication. J Nanomed
Nanotechnol 2017;8:2.
[30] Rajeshkumar S, Malarkodi C, Gnanajobitha G, et al. Seaweed-
mediated synthesis of gold nanoparticles using Turbinaria con-
oides and its characterization. J Nanostruct Chem. 2013;3:44.
[31] Roy S, Mukherjee T, Chakraborty S, et al. Biosynthesis, character-
isation & antifungal activity of Silver nanoparticles synthesized by
the fungus aspergillus Foetidus mtcc8876. Dig J Nanomater Bios
2013;8:197–205.
[32] Gurunathan S, Jeong J-K, Han JW, et al. Multidimensional effects
of biologically synthesized silver nanoparticles in Helicobacter
pylori, Helicobacter felis, and human lung (L132) and lung carcin-
oma A549 cells. Nanoscale Res Lett. 2015;10:35.
[33] Roda E, Barni S, Milzani A, et al. Single Silver Nanoparticle
Instillation Induced Early and Persisting Moderate Cortical
Damage in Rat Kidneys. Ijms. 2017;18:2115.
[34] Carlson C, Hussain SM, Schrand AM, et al. Unique cellular inter-
action of silver nanoparticles: size-dependent generation of react-
ive oxygen species. J Phys Chem B. 2008;112:13608–13619.
[35] Park H-J, Kim JY, Kim J, et al. Silver-ion-mediated reactive oxygen
species generation affecting bactericidal activity. Water Res.
2009;43:1027–1032.
[36] Guilger M, Pasquoto-Stigliani T, Bilesky-Jose N, et al. Biogenic sil-
ver nanoparticles based on trichoderma harzianum: synthesis,
characterization, toxicity evaluation and biological activity. Sci
Rep. 2017;7:44421.
View publication stats
[37] Panzarini E, Mariano S, Dini L, editors. Investigations of the toxic
effects of glycans-based silver nanoparticles on different types of
human cells. AIP Conf Proc. New York, NY: AIP Publishing; 2017.
[38] Datkhile KD, Durgawale PP, Patil MN. Biogenic silver nanoparticles
are equally Cytotoxic as Chemically Synthesized silver nanopar-
ticles. Biomed Pharmacol J. 2017;10:337–344.
[39] Sadat Shandiz SA, Shafiee Ardestani M, Shahbazzadeh D, et al.
Novel imatinib-loaded silver nanoparticles for enhanced apoptosis
of human breast cancer MCF-7 cells. Artif Cell Nanomed B.
2017;45:1082–1091.
[40] Yakop F, Abd Ghafar SA, Yong YK, et al. Silver nanoparticles
Clinacanthus nutans leaves extract induced apoptosis towards oral
squamous cell carcinoma cell lines. Artif Cell Nanomed B.
2018;1:1–9.
[41] Dini L, Panzarini E, Serra A, et al. Synthesis and in vitro cytotoxicity
of glycans-capped silver nanoparticles. Nanomater Nanotechno.
2011;1:10.
[42] Ovais M, Khalil AT, Raza A, et al. Green synthesis of silver nano-
particles via plant extracts: beginning a new era in cancer thera-
nostics. Nanomedicine 2016;11: 3157–3177.
[43] Moulton MC, Braydich-Stolle LK, Nadagouda MN, et al. Synthesis,
characterization and biocompatibility of “green” synthesized silver
nanoparticles using tea polyphenols. Nanoscale 2010;2:763–770.
[44] Amooaghaie R, Saeri MR, Azizi M. Synthesis, characterization and
biocompatibility of silver nanoparticles synthesized from Nigella
sativa leaf extract in comparison with chemical silver nanopar-
ticles. Ecotox Environ Safe. 2015;120:400–408.
[45] Graf C, Nordmeyer D, Sengstock C, et al. Shape-Dependent
Dissolution and Cellular Uptake of Silver Nanoparticles. Langmuir
2018;34:1506–1519.
[46] Xiu Z-m, Zhang Q-b, Puppala HL, et al. Negligible particle-spe-
cific antibacterial activity of silver nanoparticles. Nano Lett.
2012;12:4271–4275.