International Journal of Biological Macromolecules 163 (2020) 745–755

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International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

Microwave-assisted rapid synthesis of silver nanoparticles using

fucoidan: Characterization with assessment of biocompatibility and
antimicrobial activity
Sneha S. Rao, K. Saptami, Jayachandran Venkatesan, P.D. Rekha ⁎
Yenepoya Research Centre, Yenepoya (Deemed to be University), Deralakatte, Mangalore, Karnataka 575018, India

a r t i c l e i n f o a b s t r a c t

Article history: Silver nanoparticles (AgNPs) have gained attention due to their exceptional physicochemical properties and
Received 2 April 2020 biological activities, owing to which they are extensively used in biomedical field. We synthesized AgNPs by
Received in revised form 13 June 2020 rapid microwave-assisted method using fucoidan as a reducing agent. The synthesized fucoidan-AgNPs (F-
Accepted 24 June 2020
AgNPs) were characterized for the structural and functional properties. The bactericidal effect and mode of action
Available online 26 June 2020
of F-AgNPs on the pathogenic bacteria and biofilm formation were investigated along with the cytotoxicity
Keywords:
studies. The UV–Visible spectra of the F-AgNPs showed the surface resonance peak at 419 nm. The nanoparticles
Green synthesis were spherical in shape with particle size of 59.5 ± 1.46 nm and polydispersity index (PDI) of 0.3 ± 0.01. Capping
Metal nanoparticles of fucoidan on AgNPs was confirmed by FTIR with characteristic peaks of sulfate group. Silver content of F-AgNPs
P. aeruginosa biofilm inhibition was 87% with 13% contribution from the fucoidan moieties. The F-AgNPs showed antimicrobial activity against
Oxidative stress common pathogenic bacteria and was able to inhibit biofilm formation in Pseudomonas aeruginosa at 20 μg/mL
concentration. The oxidative stress and intracellular protein leakage were observed due to the F-AgNP interaction
with the cell bringing about bactericidal effect. The results highlight the synthesis and bioactivity of AgNPs doped
with organic moieties for applications as antimicrobials.
© 2020 Elsevier B.V. All rights reserved.

1. Introduction cultures as reducing agents to form metallic nanoparticles [17].This

Nanoparticles are extensively studied due to their advantages in
terms of size, stability, strength, activity, large surface area and unique
chemical and biological properties. These properties extend a wide
array of application in nano medicine as antimicrobial agents, wound
dressing, treating diabetic wounds, diagnosis and targeted drug delivery,
and biomedical applications as biosensors [1,2]. The transitional metal
nanoparticles such as silver [3,4], gold [5,6], zinc [7,8], copper [9,10],
iron [11,12] and platinum [13] are gaining attention due to their
versatile properties and wide range of applications as catalytic agents
and antimicrobial agents [14,15]. Among these, silver and gold remain
the most attracted nanoparticles. The synthesis procedure is an
important factor for achieving the size and shape of the nanoparticles
and the latter in turn determines its functional properties.
Nanoparticle synthesis can be achieved by physical, chemical and
mechanical approaches and are generally classified into top-down or
bottom-up methods [16]. Green synthesis is a bottom-up process that
uses natural materials such as plant extractives, bacterial or fungal
⁎ Corresponding author at: Yenepoya Research Centre, Yenepoya (Deemed to be
University), University Road, Deralakatte, Mangalore 575018, Karnataka, India.
E-mail address: rekhapd@yenepoya.edu.in (P.D. Rekha).
results in non-toxic end products making them eco-friendly, cost-
effective, stable and easy to scale up compared to the chemical methods
[18]. The natural sources represent a diverse pool of reducing agents
and include different groups of phytochemicals, pigments, amines,
proteins and carbohydrates that also have stabilizing effect on the
formed nanoparticles, thus eliminating the need to use synthetic and
chemical stabilizers [19]. The phenolic group in the Coffea arabica seed
extracts was used as an effective bio-reductant for synthesizing silver
nanoparticles (AgNPs) of 20–30 nm size at room temperature [20].
Alternatively, natural polymers such as agar and carrageenan from the
seaweeds to form silver nano-composites have been used with
satisfactory results [21]. The synthesis was attributed to the sulfate
and carboxylic groups present in the polymers that readily reduced
the silver to form nanoparticles with size between 37 and 54 nm. The
process duration for the synthesis of AgNPs depend on the reducing
capacity of the reactants used. For example, formation of AgNPs using
the sea weed (Sargassum wightii) extract required 24 h of stirring at
room temperature to form stable NPs of 15–20 nm size [22].
Among the polysaccharides of natural origin, polysaccharides from
seaweeds such as carrageenan, alginate, pullulan and fucoidan are
used for the synthesis due to the presence of functional groups.
Fucoidan is a high molecular weight anionic carbohydrate polymer

https://doi.org/10.1016/j.ijbiomac.2020.06.230
0141-8130/© 2020 Elsevier B.V. All rights reserved.
746 S.S. Rao et al. / International Journal of Biological Macromolecules 163 (2020) 745–755
commonly extracted from the sea cucumbers or seaweeds such as
Fucus sp., Sargassum sp., Padina sp., etc., and exhibit properties of
pharmaceutical importance [23]. Its biological activities mainly depend
on the molecular weight, degree of sulfation, occurrence of different
monosaccharide units and their linkages. Fucoidan based AgNPs (F-
AgNPs) have previously been synthesized by overnight stirring and
heating methods [24–27]. The methods so far reported for the
production of F-AgNPs include longer time in extreme conditions. To
overcome the longer time required for the process, microwave based
synthesis are being used for the synthesis of NPs as it offers benefits in
terms of batch to batch consistency in the process with quick formation
of the NPs. Hence, here, we used microwave-assisted method for the
rapid synthesis of AgNPs using the fucoidan by optimizing the reaction
conditions. The synthesized AgNPs were characterized in detail for its
structural and functional properties.
2. Materials and methods
Fucoidan from Fucus vesiculosus (Mol. Wt. 46.4 kDa), silver nitrate
(AgNO3) and dimethyl sulfoxide (DMSO), 2′,7′-Dichlorofluorescin
diacetate (DCFDA) were purchased from Sigma Aldrich (St. Louis, MO,
USA). NIH3T3 (mouse embryonic fibroblast) was obtained from
National Centre for Cell Sciences, Pune, Dulbecco's modified eagle's
medium (DMEM), fetal bovine serum (FBS), antibiotic-antimycotic
solution, trypsin-EDTA, methyl thiazolyltetrazolium (MTT), acridine
orange, and ethidium bromide were purchased from HiMedia, India.
2.1. Preparation of fucoidan-AgNPs
Fucoidan solution (1 mg/mL) and AgNO3 solution (1 mM) were
prepared in distilled water at room temperature. For the synthesis of
the fucoidan mediated AgNPs (F-AgNPs), silver nitrate and fucoidan
solutions were mixed in different ratio (v:v) and subjected to
microwave irradiation (LG MS-2049UW) at 100 °C for different time
duration from 0 to 4 min. Further, based on the optimum ratio and
time, F-AgNPs were bulk synthesized and separated by centrifugation
at 12,000 rpm for 20 min. The supernatant and the F-AgNPs pellet
were collected separately. The collected F-AgNPs were washed with
distilled water, centrifuged, frozen at −80 °C overnight and lyophilized
(FreezeZone benchtop freeze dryers, Labconco Corporation, Missouri,
USA). The lyophilized F-AgNPs were stored at room temperature in
desiccator for further analysis.
2.2. Characterization
UV–visible spectroscopy
The formation of F-AgNPs was followed by UV–visible spectroscopy
(Shimadzu UV – 1800, Kyoto, Japan). Briefly, the different combinations
of AgNO3 and fucoidan solutions subjected to microwave synthesis
were subjected to a spectrophotometric scan in the range of
200–800 nm. Based on the absorption maxima (λ max) the formation
of F-AgNPs was inferred.
Estimation of silver content by ICP-MS
The concentration of silver in the F-AgNPs formed using fucoidan was
determined by the inductively coupled plasma mass spectrometry
analysis (ICPMS). For this both the supernatant and the lyophilized F-
AgNPs were used. The samples were typically digested and reconstituted
in an aqueous matrix containing 2% conc. nitric acid. Diluted and filtered
(Whatman No. 42 filter paper). The silver content in the filtered solutions
was analyzed using ICP-MS (Thermo Scientific iCAP RQ ICP-MS).
Fourier Transform Infrared Spectroscopy (FT-IR)
To determine the chemical interaction between silver and fucoidan,
FT-IR analysis of the F-AgNPs was carried out. Briefly, 10 mg sample was
placed and analyzed directly in a FT-IR instrument (IRSpirit, Shimadzu,
Kyoto, Japan). The IR spectrum was recorded in the scan range of 400 to
4000 cm−1 and interpreted based on the literature.
Dynamic Light Scattering (DLS) analysis
The particle size distribution and the zeta potential of the
synthesized F-AgNPs were obtained by DLS method using Malvern
Zetasizer Nano ZS (Worcestershire, UK). Briefly, the F-AgNPs were
suspended and filtered using Whatman No. 42 filter paper. The filtered
samples were analyzed at 25 °C for a minimum of three cycles and the
particle size was estimated.
X-ray diffraction analysis
The crystalline structure of the F-AgNPs was evaluated using an X-
ray diffractometer (Rigaku miniflex 600, Tokyo, Japan). The lyophilized
F-AgNP samples were scanned at a rate of 4°/min at wavelength of
0.15406 nm and at a 2Ɵ angle pattern. The average crystalline size of
the F-AgNPs was calculated using Debye-Scherrer formula;
L ¼ kλ=βcosθ
where L is the size of the crystalline nanoparticles, k is Scherrer constant
(0.9), λ is the wavelength and β is the angular full-width at half
maximum (FWHM) of the XRD peak at the diffraction angle calculated
using origin software 2017.
Scanning Electron Microscopy (SEM) coupled with Energy Dispersive X-
Ray Analysis (EDX) and Transmission electron microscopy (TEM) analysis
The surface morphology of the synthesized F-AgNPs was studied
using SEM. For this, the F-AgNP sample was coated on the surface of
the double-sided tape, gold-sputtered and analyzed using FE-SEM
(Carl Zeiss, Germany). Scanning electron micrographs collected were
analyzed for surface details. The elemental composition and distribution
were analyzed by Energy-dispersive X-ray spectroscopy (EDX) (Carl
Zeiss, Germany). For TEM analysis, the F-AgNPs were suspended in
water and a drop of the sample was placed on a copper grid and dried.
The morphology of the prepared F-AgNPs was analyzed using TEM-
SAED (Jeol/JEM 2100, Tokyo, Japan).
Antimicrobial activity
The antibacterial activity was tested against both Gram-positive and
Gram-negative bacteria using agar well diffusion method as described
previously [28]. The microorganisms used were; Staphylococcus aureus
(ATCC 25932), Enterococcus faecalis (ATCC 29212), Streptococcus mutants
(ATCC 25932) Pseudomonas aeruginosa (MCC 2080), P. aeruginosa
(PAO1) and Escherichia coli (ATCC 25175). The anti-fungal activity was
tested against Candida albicans (ATCC 90028). Briefly, inoculum was
prepared from overnight cultures of bacteria (0.05-OD600) and
inoculated on to Muller Hinton agar plates using a sterile swab to
prepare a uniform lawn of bacteria. Subsequently, 6 mm diameter
wells were created in the agar plates and 20 μL of F-AgNPs solutions at
different concentrations (2.5, 5, 7.5 and 10 μg/mL) were added. For
C. albicans, Sabouraud's dextrose agar was used and all other procedures
remained same. Standard antibiotic discs were used as positive controls
and fucoidan was tested at concentrations between 2.5 and 100 μg/mL.
The plates loaded with samples were incubated upright at 37 °C, and
after 24 h incubation, the zone of growth inhibition was measured.
Effect of F-AgNPs on the Pseudomonas aeruginosa biofilms
Pseudomonas aeruginosa biofilms are always a concern in health care
setting; hence, the biofilm inhibitory activity of F-AgNPs was tested
against this model organism. As the biofilm inhibitory concentration is
always higher than the minimum inhibitory concentration (MIC), at
first the MIC was tested. The MIC of both fucoidan and F-AgNPs was
determined by broth dilution method. In brief, Muller Hinton broth
was inoculated with 105 CFU/mL of P. aeruginosa PAO1 and P. aeruginosa
MCC 2080 cultures. Different concentrations of fucoidan (12.5 to
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200 μg/mL) and F-AgNPs (0.15–100 μg/mL) were added and incubated
at 37 °C for 24 h. The bacterial growth was measured based on the
optical density at 600 nm spectrophotometrically and the MIC was
determined as the lowest concentration at which no visible growth
was observed. For testing the biofilm inhibition by F-AgNPs, the
P. aeruginosa biofilm was developed by growing in Luria-Bertani (LB)
media in 96-well polystyrene plates for 24 h at 37 °C. After incubation,
the planktonic cells were discarded, the biofilm was treated with
different concentrations of F-AgNPs (0.125–20 μg/mL) in LB media
and incubated further for 24 h. After the incubation, the planktonic
cells were removed, the wells containing biofilm were washed with
sterile PBS and fixed using 95% methanol. The fixed cells were strained
with crystal violet dye (0.1% aq.) for 10 min followed by PBS wash to
remove the excess stain from the wells. The stain was solubilized
inacetic acid (33%) and the absorbance of the solution was recorded at
580 nm to quantify the biofilm. The percentage inhibition was
calculated from the untreated control.
Time kill assay
Time kill assay was performed to study the effect of F-AgNPs on the
growth kinetics of the bacteria. Briefly, bacterial cultures were prepared
with an initial OD 600 of 0.05 from overnight culture in Tryptic soya broth
(TSB) and treated with F-AgNPs (10 and 20 μg/mL). The bacterial growth
was measured by recording the OD600 values spectrophotometrically
(FluoSTAR Omega, BMG Labtech, Germany). For comparison, untreated
control was used. The growth curve was plotted using the OD600
readings recorded at different time intervals between 0 and 24 h. Also,
to test the bacterial residual cell viability, the samples were serially
diluted and plated on TSA media. The colony forming units (CFU) were
enumerated using a digital colony counter and represented as CFU/mL.
Estimation of reactive oxygen species (ROS)
To test whether the F-AgNPs treatment results in the oxidative stress
due to the formation of reactive oxygen species (ROS), 2′,7′-
dichlorofluorescein diacetate (DCFDA) method was used as per the
previously described method [29]. For this, the overnight cultures of
P. aeruginosa PAO1 and MCC 2080 strains were treated with F-AgNPs
(10 and 20 μg/mL) in TSB and incubated for 4 h in a shaking incubator
(120 rpm) maintained at 37 °C. For comparison untreated cells were
used as control. After the incubation, the cells were harvested by
centrifugation (5000 rpm, 5 min, 4 °C), re-suspended in sterile PBS
containing 100 μM DCFDA and incubated at 37 °C in dark for 30 min.
The cells collected by centrifugation (5000 rpm, 5 min, 4 °C) were
lysed using alkaline lysis buffer and the lysate was used for measuring
the fluorescent intensity using fluorescence spectrophotometer (F-
2700, Hitachi Tokyo, Japan) at an emission wavelength of 522 nm.
Intracellular protein leakage
Further, to estimate the intracellular protein leakage due to F-AgNP
treatment, overnight cultures of P. aeruginosa PAO1 and MCC 2080
strains were treated with F-AgNPs (10 and 20 μg/mL) in TSB and
incubated for 12 h in the shaking incubator (120 rpm) at 37 °C. After
the treatment the cultures were centrifuged to collect the cell free
supernatant for estimating the intracellular protein leakage by Bradford
method [30]. Respective bacterial cultures without treatment were used
as control. OD595 readings were recorded spectrophotometrically to
estimate the protein content from the standard graph obtained using
bovine serum albumin (BSA).
2.3. Assay for cytotoxicity of F-AgNPs
The cytotoxicity of F-AgNPs was tested at different concentrations
(2.5, 5, 10, 15 20 and 50 μg/mL) in vitro using NIH3T3 cells by MTT
assay as previously described by Mosmann [31]. The NIH3T3 cells
were grown in DMEM containing 10% fetal bovine serum and 1%
antibiotic-antimycotic solution. For MTT assay, 5 × 103 cells/well were
747
seeded in to 96-well plates, incubated for 24 h (37 °C, 5% CO2), treated
with different concentrations of F-AgNPs and further incubated for 24 h.
To the cells, MTT reagent (1 mg/mL) was added treated for 4 h and the
supernatants were removed. The formazan crystals formed were
solubilized using DMSO and the absorbance was recorded at 570 nm
using a multimode micro-plate reader (FluoSTAR Omega, BMG Labtech,
Germany). The percent cell viability was calculated with respect to
untreated control using the formula
OD of sample
Cell viability% ¼  100
OD of control
2.4. Live – dead staining
To visualize the proportion of the live and dead cells after treatment
with F-AgNPs acridine orange - ethidium bromide staining was used.
The NIH3T3 cells were seeded in 24-well plates (1 × 104 cells/well),
allowed to adhere and treated with F-AgNPs. After 24 h of treatment,
the culture supernatants were removed; cells were washed with PBS
and fixed with chilled methanol for 10 min. Acridine orange (2 μg/mL)
and ethidium bromide (2 μg/mL) were added in equal volumes and
incubated at 37 °C for 15 min in the dark. The excess stain was removed,
washed with PBS and images were captured in red and green channels
using a fluorescence imager (ZOE, Bio-Rad, California, USA).
2.5. Statistical analysis
All the experiments were performed at least in triplicates. The data is
presented as mean ± standard deviation. Difference between the
treatments was analyzed using one-way analysis of variance (ANOVA)
and p b 0.05 was considered as significant.
3. Results and discussion
3.1. Green synthesis of F-AgNPs
Among the different combinations of fucoidan and silver nitrate
solutions used for the synthesis, the 9:1 (v/v) fucoidan (1 mg/mL) and
silver nitrate (1 mM) solutions at room temperature subjected to
microwave irradiation for 4 min yielded the F-AgNPs. The reaction
mixture was colorless at the initial stage, turned into pale yellow at
1 min, golden yellow at 2 min, light brown at 3 min and dark brown
at four-minutes of microwave radiation (Fig. 1b). The formation of F-
AgNPs was indicated by the occurrence of dark brown color at 4 min,
due to the excitation of AgNPs surface plasma resonance. The UV–
Visible spectra of the suspension containing the F-AgNPs showed the
characteristic peaks due to the presence of AgNPs (Fig. 1a). At the
baseline, there was no observed peak formation, however on
microwave irradiation clear occurrence of peaks at 419 nm was visible
and the peak intensity increased to reach the highest at 4 min of
microwave. Generally, the AgNPs with size ranging between 2 nm to
100 nm show a characteristic peak at 420 ± 5 nm, due to their surface
plasmon. This coincides with the most commonly reported excitation
wavelength of AgNPs [32,33]. Under reducing environment, the
reduction of silver occurs and the remains stable for months. The
microwave induced degradation of fucoidan results in release of
reducing sugars [34] which attributes to the efficient reduction of silver
nitrate to silver nanoparticles. Also, the degradation products of the
reducing agents act as capping agents to give stable nanoparticles [35].
The relative abundance of silver ions present in the pellet and
supernatant collected after the reaction was determined by ICP-MS.
The pellet containing F-AgNPs contained 4.6 ± 0.4 mg (in 100 mL
reaction mixture) of Ag making up to 44.35% of the silver used in the
reaction, while, the supernatant contained 4.4 ± 0.3 mg (in 100 mL
reaction mixture) of Ag making up to 41%. Thus, the 10.37 mg product
748 S.S. Rao et al. / International Journal of Biological Macromolecules 163 (2020) 745–755
Fig. 1. a. UV visible spectra for the biosynthesized F-AgNPs at different time intervals of 0, 1, 2, 3 and 4 mins. The sharp peak at 419 nm at 3rd and 4th min correspond to silver; b. Formation
of F-AgNPs at different times of microwave irradiation with gradual color change from colorless to dark brown at 4th min.
recovered from the 100 mL reaction mixture after microwave
irradiation (4 min) contained 9.0 mg of Ag. This shows that the
recovered product is predominantly made of Ag and small quantity of
the fucoidan moieties. Such data is important to know the percentage
conversion and recovery rates during the synthesis.
3.2. Fourier Transform –Infrared (FT-IR) spectra and functional groups
The interaction between different functional groups involved in the
formation of F-AgNPs was studied using the FT-IR spectroscopy. The
characteristic peaks for fucoidan were observed at 835 cm−1,
1009 cm−1, 1218 cm−1, 1652 cm−1, 1729 cm−1 and 3403 cm−1
(Fig. 2a). The peaks at 835 cm−1, 1009 cm−1, 1218 cm−1 correspond
to the sulfate groups (S_O) [36,37] C\\O\\S stretching of the sulfate
groups in fucoidan [24]. The peaks at 835 cm−1corresponding to the
sulfate groups of fucoidan are evident in the absorption spectrum of F-
AgNPs. The absorption peaks of OH stretching (3403 cm−1) and
1729 cm−1 corresponding to the C_O of the carbohydrate group
observed in the FT-IR spectra of the recovered F-AgNPs strongly indicate
the presence of fucoidan moieties in the product.
3.3. X-ray diffraction (XRD) pattern of F-AgNPs
The XRD patterns of the biosynthesized F-AgNPs shows the
crystalline nature of silver with several Braggs peaks (Fig. 2b). The
peaks at 2Ɵ 38.06, 44.32, 64.58 and 77.3 corresponds to the (111),
(200), (220) and (311) planes of face-centered cubic structure of silver
indicating the signature peaks for the biosynthesized AgNPs (JCPDS 31-
1238). Many studies have reported the presence of silver peaks at these
regions in X-ray diffractogram [38–40] and this is due to the formation
of AgNPs by reduction of Ag+ ions using fucoidan under microwave
energy. The average crystallite size of the F-AgNP calculated from
Debye-Scherer equation is 20.41 ± 11.77 nm and this size reveal the
nano-crystalline nature of synthesized F-AgNPs [41].
3.4. Size and polydispersity index of the F-AgNPs
The hydrodynamic radius and the surface charge of the
biosynthesized F-AgNPs were analyzed using DLS. The average size of
the F-AgNPs was estimated as 59.5 ± 1.46 nm with a polydispersity
index (PDI) of 0.3 ± 0.01 (Fig. 2c). The PDI indicates that the prepared
particles are monodisperse in nature with a narrow range of size
distribution. The zeta potential of the F-AgNPs was – 22.7 ± 2.44 mV,
negative charge confirming the presence of anionic fucoidan. Such
properties are associated with higher particle stability. AgNPs
synthesized using natural extracts exhibit variation in the particle
sizes between 30 and 130 nm [42].The AgNPs synthesized using
carboxymethylated-curdlan or fucoidan by high temperature heating
(100 °C) showed particle size in the range of 40–80 nm [26]. The size
of the nanoparticles mainly depends on the conditions used for the
synthesis such as temperature and pH and it plays an important role
in determining the physical and biological properties [43].
3.5. Surface morphology and atomic distribution
The FESEM of the F-AgNPs showed uniform spherical structures in
the nanometer range representing the nanoparticles (Fig. 2d). Further,
the EDX analysis showed the distribution of silver by an optical
absorption peak at ~3 KeV as well the peaks for carbon and oxygen
from the fucoidan contributing to capping of the AgNPs (Fig. 2e). Similar
observations were reported onearlier on the F-AgNPs and occurrence of
a strong peak at 3 KeV indicating the presence of silver as the key
component [44]. Uniform shape and particle distribution are key
attributes of successful synthesis.
3.6. Transmission Electron Microscopy-selected area electron diffraction
(TEM-SAED)
The TEM observations further confirmed that the biosynthesized F-
AgNPs were highly spherical in shape (Fig. 3a). The coating of AgNPs
is mostly due to the reducing sugars (glucose and xylose) present in
the fucoidan which can act as capping agents [45]. The ring-like
diffraction patterns (Fig. 3b) observed in the SAED analysis corresponds
to the (111), (200), (220), and (311) faced-centered cubic (fcc)
structure of silver in AgNPs [39,46].The particle size measured based
on the TEM shows the size of 36.99 ± 12.39 nm (Fig. 3c). The diffraction
patterns obtained from SAED and XRD analysis confirms the crystalline
nature of the synthesized nanoparticles. The occurrence of these
 S.S. Rao et al. / International Journal of Biological Macromolecules 163 (2020) 745–755 749

Fig. 2. Characteristics of the F-AgNPs. Fourier transform infrared spectrum of Fucoidan and F-AgNPs; b. XRD pattern; c. Dynamic light scattering analysis. d. SEM images (scale bar 200 nm)
and; e. EDX spectra showing a peak at 3 KeV for the presence of metallic silver.

patterns is a common feature of AgNPs by green synthesis methods
[47–49].
3.7. Antimicrobial activity
The AgNPs are well reported for their broad range antimicrobial
activity [50]. The antimicrobial efficacy of the biosynthesized F-
AgNPs was tested at different concentrations and showed the
highest zone of growth inhibition at a concentration of 10 μg/mL.
Among the tested microbes, Candida albicans showed the highest
zone of inhibition (21 mm), followed by Pseudomonas aeruginosa
(19 mm), Streptococcus mutants (18 mm) Staphylococcus aureus
(16 mm), Escherichia coli (15 mm) and Enterococcus faecalis
(10 mm) (Table 1, Fig. 4a). Interestingly, at all the tested
concentrations the zone of growth inhibition was obsereved
showing its activity against broad range of microbes.However, the

Fig. 3. a. TEM images of F-AgNPs (scale bar 10 nm); b. SAED patterns support the crystalline F-AgNPs with the occurrence of ring like diffraction patterns; and c. Particle size distribution
pattern.
750 S.S. Rao et al. / International Journal of Biological Macromolecules 163 (2020) 745–755

Table 1 3.8. MIC and Biofilm inhibitory activity of F-AgNPs on Pseudomonas

Zone of inhibition (mm) of F-AgNPs against Staphylococcus aureus, Enterococcus faecalis, aeruginosa
Pseudomonas aeruginosa, Escherichia coli, Streptococcus mutants and Candida albicans.

Microbe Zone of inhibition (mm) Pseudomonas aeruginosa is an opportunistic pathogen causing

F-AgNPs nosocomial infections and is difficult to treat due to its ability form
2.5 μg/mL 5 μg/mL 7.5 μg/mL 10 μg/mL
biofilms. The bacteria inside the biofilm environment are resistant to
S. aureus 13 14 15 16
E. faecalis 7 8 9 10 the antibiotics due to the strong matrix developed as result of secretion
E. coli 12 13 14 15 of extracellular polymeric substances [51]. Many antimicrobials fail
P. aeruginosa 16 17 18 19 against the biofilm forming organisms and also, the biofilm inhibitory
S. mutants 15 16 17 18 concentration is several folds higher than the MIC.To establish the activity
C. albicans 16 17 18 21
of F-AgNPs on the P. aeruginosa biofilms, two strains namely, PAO1 and
MCC 2080 were used. The MIC of F-AgNPs was between 5 and 10 μg/mL
against both the strains (Fig. 4b & c). As stated earlier fucoidan alone
fucoidan alone did not display the antimicrobial activity at the tested did not show any activity up to a concentration of 100 μg/mL.The strains
concentrations of 2.5–100 μg/mL. PAO1 and MCC 2080 are strong biofilm formers with biofilm intensity
The AgNPs synthesized using fucoidan from Turbinaria conoides by (OD570) of 0.90 and 0.91 respectively in LB media under control
heating at 75 °C showed growth inhibition against gram negative conditions. The F-AgNPs treatment on the pre-developed biofilm showed
bacteria such as Escherichia coli, Klebsiella pneumoniae, Vibrio cholera, a dose dependent inhibition (Fig. 4b & c). Biofilm inhibition by 50% was
Pseudomonas aeruginosa, Salmonella typhi and Shigella sonnie [25]. achieved with 10 μg/mL concentration of F-AgNP. At the higher

Fig. 4. a. Antimicrobial activity of fucoidan and F-AgNPs at different concentrations (2.5 μg/mL, 5 μg/mL, 7.5 μg/mL and 10 μg/mL) tested on Staphylococcus aureus, Enterococcus faecalis,
Pseudomonas aeruginosa, Escherichia coli, Streptococcus mutants, Candida albicans using agar well diffusion method; b. Biofilm inhibition in P. aeruginosa strains, MCC2080 and PAO1 by
F-AgNPs treatment. The data points are mean ± S.D. c. Biofilm stained by crystal violet showing the reduced intensity of PAO1 and MCC 2080 biofilms treated with different
concentrations of F-AgNPs.
 S.S. Rao et al. / International Journal of Biological Macromolecules 163 (2020) 745–755 751

Fig. 5. a & b. Growth curve for the P. aeruginosa strains PAO1 and MCC 2080 treated with 10 and 20 μg/mL of F-AgNPs at different time points at 37 °C. The untreated group was used as
control. The results are expressed as mean ± S.D. compared to the control; c & d. Bar graphs representing the CFU/mL for the P. aeruginosa strains PAO1 and MCC 2080 strains treated with
10 and 20 μg/mL of F-AgNPs at 12 and 24 h. The results are expressed as mean ± S.D. **p b 0.01, compared to the control.

concentration of 20 μg/mL, the biofilm was inhibited by 80%. Disrupting
the biofilm completely is difficult due to the protection of cells in the
thick biofilm matrix. The biofilm architecture contains macromolecules
and can retain or interact with the antimicrobial compounds preventing
its interaction with the bacterial cells [52]. This is one of the reasons for
the failure of antibiotic therapy by the conventional antibiotics against
biofilm forming pathogens.
Some of the AgNPs with additional capping have shown activity
against the bacterial biofilms. For example, silver-nanoparticle-
decorated quercetin showed anti-biofilm effect on a multi-drug
resistant Escherichia coli strain and it was mainly due to the biofilm
inhibitory activity of quercetin [53]. Similarly, the carboxymethyl
tamarind polysaccharide capped AgNPs could inhibit the bacterial cell
division and thereby exerting the lethal effect on the biofilm produced
by drug resistant isolates [54]. The nano-sized silver particles can get
incorporated in the biofilm and migrate to the interior of the biofilms
through the micro-channels present in the architecture [55].The
conventional antibiotics fail to reach the biofilm interior due to their
entrapment and chemical interaction with the charged extra-
polymeric substances. Materials having excellent biofilm inhibitory
potential are suitable for the coating of medical devices intended for
implantation or devices such as catheters.
3.9. Time kill assay
To study the effect of F-AgNPs on the growth of P. aeruginosa strains
PAO1 and MCC 2080 time kill assay was performed. The growth of the
bacteria under normal culture conditions followed a typical sigmoidal
pattern and under the F-AgNP treatment, the growth did not take
place showing a flattened curve (Fig. 5a & b). This shows that F-AgNPs
are able to bring about bactericidal effect as early as 6 h in both the
strains. The CFU calculated from the serially diluted samples were
consistent with the OD600 values showing rapid decrease in the number
colonies after treatment with F-AgNPs (Fig. 5c & d). At 24 h there were a
few colonies of residual bacteria in the treatment, that represent the
cells entrapped in the biofilm. Hence, repeated treatment may be
required to completely eliminate the residual bacteria.
3.10. Mode of action of F-AgNPs on the biofilm forming P. aeruginosa
Due to their small size, AgNPs have the ability to attach to the
bacterial cell wall and penetrate inside to the cytoplasm. During this
process, it can cause structural changes in the cell membrane such as
changes in the permeability of the cell membrane. It is also envisaged
that, AgNPs have the ability of producing high levels of reactive oxygen
species (ROS) and free radical species such as hydrogen peroxide,
superoxide radicle, hydroxyl radical and singlet oxygen that damage
the DNA and the cellular processes causing the bacterial cell killing
[56]. To test whether the F-AgNPs have similar effect on the bacterial
cells, the ROS levels were estimated based on DCFDA method. The
treatment with F-AgNPs induced the significantly higher (p b 0.005)
ROS production at 20 μg/mL concentration compared control and
10 mg/mL F-AgNPs for both the P. aeruginosa strains as measured by
the fluorescent intensity (Fig. 6a, b, c & d). Similar events have been
reported by researchers on C. albicans, S. cerevisiae and other microbes
[57]. Oxidative stress is one of the common mechanisms by which the
antibiotics target the bacterial cells. Increased concentration of the
free radicals interferes with the cell cycle regulation and cellular
metabolic pathways. The ROS can cause intercellular calcium
accumulation resulting in the loss of membrane potential triggering
752 S.S. Rao et al. / International Journal of Biological Macromolecules 163 (2020) 745–755

Fig. 6. a & b. Reactive oxygen species generated in P. aeruginosa strains PAO1 and MCC 2080 treated with 10 and 20 μg/mL of F-AgNPs for 4 h at 37 °C. ROS was detected using DCFDA
method and compared with untreated group control. The results are expressed as mean ± S.D., *p b 0.05 and ***p b 0.005 compared to the control; c & d. Fluorescence spectra for the
treated groups PAO1 and MCC 2080, respectively control and treatment with 10 and 20 μg/mL of F-AgNPs at 522 nm; e & f. Intracellular protein leakage in P. aeruginosa strains PAO1
and MCC 2080 treated with 10 and 20 μg/mL of F-AgNPs for 12 h at 37 °C. The untreated group served as control. The results are expressed as mean ± S.D. **p b 0.01 and ***p b 0.0001.

apoptosis [58]. The compromised cellular pathways trigger the cellular
transcription towards apoptosis failing to survive the stress.
Intracellular protein leakage is also another important mechanism of
action by AgNPs. The F-AgNPs treatment at concentrations of 10 and
20 μg/mL released significantly higher protein to the culture
supernatant of the bacteria compared to the untreated control
(p b 0.01). The protein contents in the supernatants of PAO1 and MCC
2080 cultures respectively were 54.82 ± 1.42 μg/mL and 103.26 ±
0.29 μg/mL with 20 μg/mL F-AgNPs (Fig. 6e & f). It has been shown
that the silver ions of the AgNPs damage of the bacterial membrane by
binding and blocking the thiol and amino groups [29,59].The membrane
integrity is the key for the bacterial cell-functioning and transport of
nutrients and metabolites as well preventing the entry of xenobiotics
and toxic substances. The cells with destabilized membranes undergo
apoptosis and reflected as cell killing by the treatment with F-AgNPs.
The anti-bacterial activity of AgNPs in Gram-negative bacteria is
stronger than in Gram-positive bacteria due to the cell wall thickness,
which is made up of peptidoglycan [59]. The adhesion of AgNPs on
bacterial cell wall is attributed to the positive charge in the AgNPs
which assures electrostatic attraction between negatively charged
bacterial cell membrane [60]. The anti-fungal activity exerted by
AgNPs in Candida is achieved by cell membrane permeabilization by
the NPs, manifesting disruption of the cell leading to inhibition of the
budding [61].
3.11. Biocompatibility of F-AgNPs
It is well documented that AgNPs are relatively cytotoxic to
mammalian cells also. Here, the biocompatibility of the F-AgNPs was
tested in vitro using NIH3T3 cells at a wide range of concentrations
coveringthe doses of antibacterial and anti-biofilm effects (2.5 to
50 μg/mL). The MTT assay results showed that at concentrations of
2.5, 5 and 7.5 μg/mL, the cell viability remained at 97.3%, 91.4% and
87.5% respectively indicating the cytocompatibility of the synthesized
 S.S. Rao et al. / International Journal of Biological Macromolecules 163 (2020) 745–755
Fig. 7. a. Effect ofF-AgNPs on the viability of NIH3T3 cells treated for 24 h. Results are based on the MTT assay and cell viability compared to the untreated control. The results are expressed
as mean ± S.D., *p b 0.05 and **p b 0.01, compared to control group; b. Live dead staining using acridine orange and ethidium bromide of NIH3T3 cells after 24 h treatment with 2.5 μg/mL
and 50 μg/mL F-AgNPs. Green represents living cells and red the dead cells. Scale bar 100 μm.
F-AgNPs (Fig. 7a). Earlier studies on AgNPs prepared using different
methods on diverse cell lines showed toxicity at higher doses. The
AgNPs prepared using the bark extracts of Toxicodendron vernicifluum
containing alcoholic, aromatic and phenolic groups showed no toxicity
on NIH3T3 cells up to 320 μg/mL [62]. It is assumed that the coating
with biopolymers reduces the toxicity of AgNPs that can be suitable
the tissue engineering purposes. AgNPs with a coating of low
molecular weight chitosan reduced its toxicity on HS27 human
fibroblast cells compared to uncoated AgNPs [63]. AgNPs synthesized
using natural extracts containing phenolics and flavanoids from
plants such as Arnebia hispidissima and Terminalia bellirica showed
no toxicity against normal cells [64,65].The cytotoxicity of the
AgNPs also depends on the concentration of the silver present in
the decorated/capped preparations. Here, the Ag content in the F-
AgNP was 44% in the prepared material. Hence, it is also important
to know the silver/metal content relative to the organics present in
the total material.
The live dead staining of the NIH3T3 cells treated with F-AgNPs is
represented in Fig.7b. The cells showed presented viability with intact
morphology as indicated by the intense green fluorescence due to the
acridine orange. The results indicate that the F-AgNPs at lower
concentration were least toxic to the normal NIH3T3 cells in conformity
with the MTT results. Considering the higher activity of the AgNPs in
lower concentrations, it could be considered at lower concentrations
at which it is compatible to normal cells for various biomedical
applications.
4. Conclusion
The microwave mediated biosynthesis of AgNPs using fucoidan was
rapid and the F-AgNPs showed characteristics and properties that can
be useful for antimicrobial applications including against biofilms.
Detailed characterization of the F-AgNPs confirmed size shape,
crystallinity and presence of functional groups due to fucoidan. The
synthesized F-AgNPs contained 87% Ag and remaining as organics
contributed from the fucoidan. The F-AgNPs exhibited an effective broad
range antimicrobial activity against Staphylococcus aureus, Enterococcus
faecalis, Pseudomonas aeruginosa, Escherichia coli, Streptococcus mutants
and Candida albicans. The study outlined a rapid and green synthesis
method for AgNP production using fucoidan as a reducing and stabilizing
agent. Its excellent antimicrobial activity, biofilm inhibition potency and
biocompatibility can make it ideal for the use as coating material in
medical devices.
753
Acknowledgements
Authors acknowledge the funding support from DST-SERB, Govt. of
India (EMR/2016/003113). Authors also acknowledge the help
provided by Dr. Ashwini Prabhu in the cell culture experiments.
Author contribution
Sneha S Rao: Performed experiments, analyzed data and co-wrote
the manuscript
K Saptami: Helped in conducting antimicrobial studies and data
analysis
Jayachandran Venkatesan: Conceptualised the study, involved in the
research and helped in data analysis
PD Rekha: Conceptualised the study, analyzed data and co-wrote
and approved the manuscript
All authors read and approved the manuscript.
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