1. health and fitness
2. disease

Myconanosomes: Antibacterial, antifungal and immunomodulatory activities
of silver nanoparticles synthesized from Alternaria brassicae (KF934409)
Faria Fatima1 , Neelam Pathak1 , Sarika Singh2 , Smita Rastogi Ve rma3 and *Preeti Bajpai1
1
2
3
Department of Biosciences, Integral University, Lucknow-226026, India
Toxicology Division, Central Drug Research Institute, Lucknow-226031, India
Department of Biotechnology, Delhi Technological University, Delhi-110042, India
*Corresponding author email: [email protected]
Abstract
Silver nanoparticles (AgNPs) prepared from Alternaria brassicae may exhibit potential
antimicrobial and immunomodulatory activity due to their inimitable character. The purpose of
this study was to evaluate their antibacterial activity, antifungal and immunomodulatory efficacy
on various pathogenic microorganisms and cell lines (J774 and THP1 α). The characterization of
these AgNPs was determined by using differential light scattering, UV-visible spectroscopy and
transmission electron microscopic analyses. Mycologically produced AgNPs were found as
spherical and irregular shaped measuring size range between 55.4 nm to 70.23 nm. The
antimicrobicidal activity of these AgNPs against Staphylococcus aureus, Trichoderma sp. and
Fusarium semitectum was evaluated by agar well diffusion method. Results showed that AgNPs
inhibit the growth of various bacteria and fungi, which may be due to the disruption of cell
membranes, leakage of cytoplasm and DNA degradation. Cytotoxicity analysis of AgNPs on cell
lines revealed its dose dependent effect. Moreover, significant increase of intracellular reactive
oxygen species was characterized in AgNPs treated cells after 4 hr of incubation. Thus, AgNPs
may have a significant advantage over conventional antibiotics as microorganisms are acquiring
resistance against the broad range of available antibiotics. In conclusion, study showed that these
nanoparticles could be used as an antimicrobial agents as well as cost effective and nontoxic
immunomodulatory delivery vehicle.
Keywords: A. brassicae; AgNP; antibacterial; antifungal; cytotoxicity; immunomodulatory
1
1. Introduction
Myco- nanotechnology is an emerging scientific discipline aimed towards studying the
production of nanomaterials or nanostructures with desirable shapes and sizes by fungi. Potential
appliances of myco-nanotechnology have fascinated scientist and researchers to contribute in
providing incremental solutions through green chemistry approaches for targeted drug delivery.
Thus, nano-sized particles have attracted worldwide attention due to their unusual photoelectrochemical, electronic chemical and optical properties.
AgNPs offer unique optical, catalytic and disinfectant properties gaining high scientific and
commercial interest. They are used in broad range of different products ranging from acting as an
antimicrobial agent, coatings of surgical instruments, contraceptive devices, wound dressing and
prostheses [9] to the use in food container systems. Besides that, they are highly attractive for
creation of advanced functional materials. To meet the wide scope of nanostructures, number of
procedure like electrochemical methods, laser ablation method, microwave irradiation method,
thermal decomposition may successfully produce pure well-defined nanoparticles. However,
these procedures are capital- intensive, dangerous, energy use and often create health hazards due
to the usage of toxic chemicals, which lead to production of harmful by-products [12] that makes
their use inappropriate within biological systems.
The safety of nanomaterials marketed during past decades does not appear to be adequately
addressed. There is a persistent requirement to develop new methods which should be safe,
nontoxic, reliable, cost effective, and ecofriendly. The new methods should also be able to assess
the cytotoxicological safety of current and future nanomaterials under different scenarios. Thus,
synthesis of nanoparticles by using bio-organisms like bacteria, fungi, and plant extracts is
compatible with the green chemistry principles [14]. Biosynthesis of metal nanoparticles is a
kind of bottom up approach where the main reaction is the reduction/oxidation reaction. The
enzymes from these microbes possess high redox potential which is usually responsible for
reduction of metal compounds into their respective nanopartic ulates (Figure 1) and synthesized
nanomaterials would be intervened by biological systems. Recently, resistance to commercially
available antimicrobial agents by pathogenic bacteria and fungi has been rising at an alarming
rate and has become a severe problem [8].
2
Fig.1 Schematic representation of reduction of metal ions by fungal secretary proteins
In the present study, a phosphate solubilizing fungi was preferred for nanostructure formulation
due to their ability to produce large amount of secretary proteins [16]. Numbers of reports have
showed the biological synthesis of metal nanoparticles but the potential of a phosphate
solubilizing fungi, A. brassicae has not yet been demonstrated. The present investigation is an
effort to synthesize AgNPs from A. brassicae (Accession No. KF934409). Moreover,
antimicrobials of mycological origin have enormous therapeutic potential therefore, it is the need
of time to develop new antimicrobial drugs for the treatment of infectious diseases.
2. Material and methods
2.1 Isolation, screening and characterization of A. brassicae (KF934409)
The fungus was isolated from rhizospheric region of Central Institute of Medicinal And
Aromatic Plants (CIMAP), Lucknow, India and screened by its phosphate solubilizing ability on
Pikovskaya’s medium by using plate assay method [7]. The fungal colonies were subculture on
fresh petriplates containing potato dextrose agar (PDA) media and the plates were incubated in
inverted position for 72 h at 28 ± 3 C and examine for the halo zones around the colony.
Further, the biochemical characterization of the fungus were tested on the basis of morphology
of colony, conidial microscopic analysis, solubilization index (SI), cellulose hydrolysis test and
starch hydrolysis test according to standard protocols [6].
3
The fungus was molecularly characterized by using 18S rRNA sequencing where genomic DNA
was isolated and subjected to high–fidelity PCR using universal primers i.e. forward primer
(5’-GGAAGTAAAAGTCGTAACAAGG-3’)
and
reverse
primer
(5’-TCCTCCGCTTATTGATATGC-3’) and analyzed on 1 % agarose gel. The PCR products
were sequenced bi-directionally using the forward and reverse primers. Homology between this
18S rDNA sequence and the strains available at the public databases (Genbank, EMBL a nd
DDBJ) was determined using BLASTN sequence match routines. The UPGMA (Unweighted
Pair Group Mathematical Average) algorithm was used to perform hierarchical cluster analysis
[13]. The sequences were aligned using CLUSTALW2 program and its phylogenetic a nd
molecular evolutionary analysis were conducted. Sequences analyses were performed by
alignment of the partial 18S rRNA gene sequences to those from the GenBank database, using
the program BLAST (NCBI BLAST® homepage). The nucleotide sequences of 18S rRN A gene
segments determined in this study have been deposited in GenBank database under accession
number KF934409.
2.2 Myco-production of AgNPs and its characterization
The isolated fungus was grown in MGYP (maltose, glucose, yeast potato broth which comprises
of malt extract (0.5 %), glucose (1 %), yeast extract (0.3 %) and peptone (0.5 %). The culture
was incubated at 27° C and harvested after 5 days of growth by sieving followed by extensive
washing with sterile double-distilled water. Initially 20 g of biomass (wet weight) was
transferred to 100 ml deionized water for 48 hr at 27° C in an Erlenmeyer flask and agitated at
150 x rpm for release of secretory proteins. Silver nitrate (1 mM) was added to the Erlenmeyer
flasks and the reaction was allowed to progress under dark conditions for production of AgNPs.
Time-dependent
formation
of
AgNPs
was
observed
by
using
ultraviolet- visible
spectrophotometer (Beckman DU-20 spectrophotometer). The scanning range was 350-650 nm
for AgNPs at a scan speed of 420 nm/min. The data was recorded and analyzed using
“UVWinlab” software.
2.2.1 Differential light scattering (DLS)
The suspensions of AgNPs was prepared in distilled water (dH2 O) by using a bath-sonicator
(ULTRAsonik 57 X, 50/60 Hz, California, USA) prior to size measurements. Dynamic light
4
scattering size measurements were performed with the aid of a Malvern Zeta Sizer Nano ZS
(Malvern Instruments, Worcestershire, UK) operating with version 5.03 of the systems
Dispersion Technology Software (DTS Nano). The sample s for DLS were equilibrated at 25º C
for 3 min before each measurement. The refractive index (RI) of AgNP.dH2 O was 1.330.
2.2.2 Transmission electron microscopy (TEM)
The synthesized AgNPs were characterized by TEM analysis. The samples were prepared by
placing a drop of synthesized nanoparticles over gold-coated negative grid followed by
evaporation of the solvent [22]. TEM analysis was performed on Perkin-Elmer model which was
operated at an accelerating voltage of 1000 kV.
2.3 Antibacterial efficacy
The antibacterial efficacy of synthesized AgNPs was determined by using the agar well diffusion
assay method [4]. Pure cultures of five pathogenic bacteria namely, B. subtilis, B. cereus, S.
aureus, E. coli and E. aeroginosa were procured from National Chemical Laboratory, Pune.
Bacterial stock cultures were maintained at 4° C on nutrient agar media slants and subculture on
nutrient broth media for antibacterial analysis of NPs. 5 mm wells in diameter were prepared and
filled with AgNPs with a range of concentrations (10 µM,
50 µM, 100 µM, 150 µM). Each
experiment was performed in triplicate and the average zone of inhibition, excluding well was
recorded. 1 mM AgNO 3 were used as negative controls. The diameter of such zones of inhibition
was measured for each organism and expressed in centimeter.
2.4 Antifungal efficacy
Disk diffusion method was used to evaluate the antifungal activity of Terconazole against A.
niger, Trichoderma sp. and F. semitectum on PDA. The standard terconazole disks (Fu10; 10
μg/disk) were purchased from Hi-Media (Bangalore, India). To determine the combined effect,
each standard paper disk was further imbued with 25 μl of freshly prepared AgNPs with a
concentrations range of 10 µM, 50 µM, 100 µM, 200 µM. PDA plates were inoculated with a
spore suspension (20 μ l) of the test fungi. Standard antifungal terconazole disks were used as
positive control. The terconazole disks were impregnated with AgNPs, placed onto the PDA
medium and inoculated with tested fungi. The fungal cell filtrate, used for the synthesis of
5
AgNPs was used as negative control. The cultured plates were incubated at 28 ± 4 C for 7 days.
The average inhibition zone, excluding well, for each case was measured.
2.5 Cell lines
Human macrophage cell line THP1 α and mouse macrophage cell line J774 was procured from
the National Center of Cell Sciences, Pune, India and maintained at Animal Tissue Culture
facility of Central Drug Research Institute (CDRI). Cells were cultured in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10 % Foetal bovine serum (FBS) and 1% antibiotic
and antimycotic solution (50,000 units/L of penicillin and 50 mg/L of streptomycin) and 2 mM
glutamine. Cultures were held in 75 cm culture flasks, at 37˚ C and 5 % CO 2 using standard cell
culture methods.
2.5.1 Cytotoxicity assay
Cell viability was assessed by using the MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide] conversion assay [5]. Thus, 1 x 104 cells/ml were seeded on 96well culture plates and incubated with increasing concentrations of nanoparticles (10 μM, 25 μM,
50 μM, 100 μM, 150 μM and 200 μM) for 24 hr at 37° C in CO 2 incubator. The MTT dye was
added to each well and plate was incubated at 37° C for 4 hr. The absorbance of insoluble
formazan salts was assessed at 550
nm using Powerwave XS (BIOTEK,
USA)
spectrophotometer [3]. Data produced were used to plot a dose-reaction curve and the
concentration of these metal nanoparticles required to kill 50 % of cell population (IC 50 ) was
determined.
Cell viability (%) =
Mean OD
× 100
Control OD
2.5.2 Intracellular reactive oxygen species estimation
Intracellular oxidative stress was calculated with the help of 2’, 7’-dichlorofluorescin di-acetate
(DCFH-DA), a well-accepted fluorescent marker for study of intracellular hydroperoxides [18].
The experiment was performed according to the protocol described [15] with slight
modifications. Primarily, healthy confluent cells were harvested and seeded (1000 cells/well)
into black bottomed 96 well plates (Nunc, Denmark) and allowed to adhere for a period of 24 hr
6
prior to exposure. For ROS quantification, both mouse and human macrophage cells were plated
and distributed in triplicates. A working stock of 25 µM DCFH-DA in phosphate buffered saline
(PBS) was prepared and all test concentrations, unexposed negative controls and positive
controls were prepared and exposed to the cells in this working stock. The negative control
consisted of the working stock solely a 25 µM DCFH-DA solution in PBS, the positive control
consisted of 1 µM hydrogen peroxide (H2 O2 ) in 20 µM DCFH-DA/PBS working stock solution
and finally the test concentrations consisted of a continuous range of AgNPs. Lipopolysaccharide
(1 µg/ml) was used as a mitogen for stimulation of macrophages as well as comparison of
phagocytic activity of stimulated and non-stimulated macrophages. The test concentration for
AgNPs was 10µg/ml and the incubation period ranged from 2 hr to 6 hr. The rate of intracellular
oxidative stress was monitored by measuring their fluorescence intensity via fluorometer
(BIOTEK-FLX800’USA) emission at 520 nm (by 485 nm excitation).
2.6 Statistical analysis
All the experiments were conducted in triplicates and results were expressed as mean ± SD. Oneway analysis of variance (ANOVA) with a Dunnett’s test was performed for the multiple
comparisons for normally distributed samples with homogenous variance. Statistically
significant differences were set at p < 0.05.
3 Results
3.1 Screening and fungal characterization
The rhizospheric sample was collected, serially diluted and plated on Pikovskaya’s media to
check their phosphate solubilization efficiency. After 7 d of incubation, a fungal colony was
observed, identified, as dark ash colour, looking like cotton having irregular edges and 1.2 cm
in diameter. On Pikovskaya’s medium, the transparency of the media is a primary indicator of
phosphate solubilization to be visualized as halo zone formation around the colony. Thus
among all, the fungal isolate FCK 20 was found as an efficient fungus on the basis of SI, pH
and phosphatase enzyme production. This fungus was further characterized morphologically,
biochemically and at molecular level. The SI of selected fungus was recorded with size of 2.9
cm in comparison with others strains. Further, colony morphology was analyzed on the
Sabouraud agar medium and filamentous structural pattern was analyzed microscopically after
7
lactophenol staining. Moreover, this fungus gave a negative test for starch hydrolysis and
cellulose degrading ability.
For molecular characterization, the genomic DNA was amplified using 18S rRNA specific
primers (ITS 4 and ITS 5). An amplicon of 500 bp was observed on 1 % agarose gel. The DNA
sequencing of the amplified product and its BLAST analysis confirmed the fungus as A.
brassicae (KF934409). The phylogenetic analysis (Figure 2) revealed the fungus to belong to
Pleosporacae family and the genus was Alternaria.
Fig. 2 Phylogenetic analysis of A. brassicae (KF934409)
3.2 Synthesis and size estimation of AgNPs
AgNPs were mycological produced from A. brassicae (KF934409). 1 mM of silver nitrate were
added to the fungal filtrate under magnetic stirring which was used as reducing and stabilizing
agent for 1 mM of silver nitrate. Appearance of a brownish color in solution (Figure 3) reflected
the formation of AgNPs in the reaction mixture which is due to the surface plasmon resonance,
8
exhibited by the AgNPs. The control sets did not demonstrate any color change under similar
experimental conditions.
Fig.3 Nanoparticles formation on the basis of colour change due to surface plasmon resonance
3.2.1 Ultraviolet-visible spectrophotometric analysis
Ultraviolet- visible spectrophotometer showed no evidence of absorption in the range of 380-750
nm for the fungal extract. Whereas the AgNO 3 exposed fungal extract showed a distinct
absorption at around 350 nm and 650 nm, with a peak at 370 nm for AgNPs [Figure 4 (a)].
Fig.4 (a) UV-VIS spectrophotometry of AgNP nanoparticles and 4 (b) DLS spectrum of Ag
nanoparticles
3.2.2 Dynamic light analysis (DLS)
This technique enables the particle size determination by measuring the random changes in the
intensity of light scattered from a suspension or solution. DLS spectra showed an intensity of
9
92.7 nm for AgNPs [Figure 4 (b)]. This size variation is due to oxidation of metal salts into their
respective nanoparticles in the presence of enzymes.
3.2.3 Transmission electron microscopic (TEM) analyses
The morphology of AgNPs is spherical and the TEM micrographs suggest that particle diameters
ranged from 48.4 to 65.23 nm (Figure 5). The dimensions of AgNPs were small enough and
imaged as poly dispersed small and large spherical nanoparticles with variable diameter. This
was further confirmed by the representative images recorded from the uniformly dispersed dropcoated film of the AgNPs on grid.
Fig.5 Transmission electron microscopic analysis of AgNP nanoparticles
3.3 Antibacterial activity of AgNPs
The bactericidal activity of AgNPs were studied using the pathogenic strains of bacteria as S.
aureus, E. coli, B. subtilis, B. cereus and E. aerogenes using agar well diffusion method. After
the incubation time, zone of inhibition (clear zones) were observed against all the tested
microbes. The results recorded in centimetres for AgNPs are shown in Figure 6 (a) respectively.
The effectiveness of AgNPs could be attributed to the fact that their larger surface area enabled
them to have a better contact with the microbial cell wall. This is further supported by the
observation that size dependent interaction of AgNPs with bacteria leads to its antibacterial
activity [21]. The exact mechanism by which the reaction occurs is still largely unknown.
10
However, some literature proposes that Ag interact with the thiol groups of proteins, which is
necessary for microbial respiration ability [10]. Ag might also interact with phosphorus
containing compounds like DNA disturbing the replication process or preferably by their attack
on the respiratory chain [10]. The direct interaction of Ag with cell membrane of bacteria has
also been shown which consequently breaks the membrane. Earlier experimental evidences have
also advocated the failure of replication ability by the DNA after treatment with silver ions [17].
The comparative histogram demonstrated that the best antibacterial efficacy of AgNPs was
against B. cereus followed by S. aureus. The marked increase in antibacterial activity was
demonstrated with increasing concentration of AgNPs [Figure 6 (a)]. In addition, the efficacy of
AgNPs was also found to be enhanced in combination with the antibiotic rather than alone.
Zone of inhibition (cm)
3.5
E. aerogenes
3
B. subtilis
E.coli
S. aureus
B.cereus
2.5
2
*
1.5
1
0.5
0
10 µM
50 µM
100 µM
150 µM
Ab
Ab+AgNP
Concentration (µM)
(a)
Zone of inhibition (cm)
1.6
A.flavus
Trichoderma
Fusarium semitectum
1.4
1.2
1
0.8
0.6
0.4
0.2
0
10 µM
50 µM
100 µM
200 µM
Concentration (µM)
(b)
Fig. 6 Comparative analysis of (a) antibacterial activity and (b) antifungal activity of AgNP for
different pathogenic bacteria and fungi. Results are presented in relative units compared with
controls(Ab) which is not shown in the graph. Different signs (* and **) letters indicate
significant differences (p < 0.05).
11
3.4 Antifungal activity of AgNPs
The colloidal AgNPs inhibited the growth of the fungus (A. niger, Trichoderma sp. and F.
semitectum) which was seeded in the Muller Hinton agar plate and formed a zone of inhibition
around the central cavity. The zone of inhibition with diameter of 1 cm was recorded in case of
A. niger, 1.2 cm in Trichoderma sp. and 1.1 cm in case of F. semitectum [Figure 6 (b)]. The
antifungal activity is due to the formation of insoluble compounds by inactivation of sulfhydryl
groups in the fungal cell wall and disruption of membrane bound enzymes and lipids, which
causes cell lysis [1]. We have observed that Trichoderma sp. and F. semitectum are susceptible
to the lethal effects of the prepared silver due to the smaller size of AgNPs.
3.5 Cell lines
To determine the biomaterial capability of nanoparticles with no toxic effects, the cell viability
and cytotoxicity assays were performed. Cell viability was determined by a standard MTT
conversion assay.
3.5.1 In vitro cytotoxicity assay
Treatment of AgNPs to J774 and THP1 α cell lines exhibited mild cytotoxicity in a dose
dependant manner. At low doses (10 µg) no cytotoxic effects were observed whereas at high
doses of 100-150 µg of AgNPs mild cytotoxicity was observed. Long-time exposure resulted in
additional toxicity to the cells and reached to maximum dead cells after 24 hr of incubation,
which might be due to over-accumulation of metal nanoparticles within the cell (Figure 7).
Fig.7 Dose-dependent effect of AgNP over cell viability using MTT assay on J774 and THP1 α
cells. Results are presented in relative units compared with controls. . Different signs (* and **)
letters indicate significant differences (p < 0.05).
12
3.5.2 Intracellular reactive oxygen species estimation
The ROS estimation has shown that AgNP exposure to J774 and THP1 α cells caused time
dependant increased production of ROS at respective concentration indicating macrophage
stimulation. The maximum amount of ROS was produced uptill 4 hr of incubation, which
declined at 6 hr (Figure 8). Here, the comparable ROS generation by AgNPs, from nonstimulated
cells
to
mitogen-stimulated
cells
was
observed
which
indicates
the
immunomodulatory potential of the synthesized AgNPs. This may be due to the small size of
AgNPs as these could easily penetrate through the cell wall in an appreciable number [15].
The maximum free radical activity was obtained by the J774 cells [Figure 8 (a)] and THP1 α
cells [Figure 8 (b)] after 4 h of incubation with AgNPs. Free radical generation and analogous
encapsulation efficacy capability of AgNPs revealed the potential role of AgNPs as drug /
vaccine delivery vehicle for macrophages as well as also
indicate towards their
immunomodulatory activity.
Fig. 8 ROS estimation in (a) J774 cell lines and (b) THP1 α cell lines after incubation with
AgNPs at various time points (2 h, 4 h, 6 h). Results are presented in relative units compared
with controls. . Different signs (* and **) letters indicate significant differences (p < 0.05).
4. Discussion
In the present study, a phosphate solubilizing fungal isolate, FCK 20 was isolated and subjected
to its characterization. The sequence of 18S rRNA of the fungal strain FCK 20 was submitted to
GenBank with an accession number of (KF934409). The homology search using BLAST
13
indicated a close genetic relatedness of the strain FCK 20 with the rRNA sequence of A.
brassicae (18S: 100 % similarity with the reference sequence Ba nkIt1680606 Seq5) in NCBI
database. Such a higher identical value confirmed the strain FCK 20 to be A. brassicae.
Myconanotechnology has become an extensive field of study involving chemistry, physics,
engineering, computing, electronics, energy, agriculture and biomedicine. In the realm of
biomedicine, nanotechnology is widely touted as one of the next promising and important
approaches to diagnose and treat various ailments [11]. Since physical and chemical methods of
metal nanoparticle production are expensive and involve incorporation of toxic chemicals
therefore their biological synthesis (bacterial, fungal and plant extract) would be preferred. This
owes to their ease of availability, nontoxic nature and quicker synthesis, which prompted us to
use this phosphate solubilizing fungus A. brassicae for the synthesis of AgNPs. In best of our
knowledge this is the pioneer study showed the use of fungus for the production of nanoparticles.
The confirmation of synthesis of AgNPs was based on surface plasmon r esonance involving
color alteration. Further, in the UV/VIS absorption, a strong peak located at 370 nm was
observed. Average particle size calculated from DLS data was found to be 65.7 nm for AgNPs.
Furthermore, the TEM image of the synthesized AgNPs has validated the formation of spherical,
nanoparticles, which was 50 nm in diameter. This indicated the reduction of Ag+ to elemental
silver (Ag). The UV/VIS spectra of AgNPs was observed at 380 nm and showed their spherical
shape. Additionally, the synthesized AgNPs were stable in solution over a time of three months
at room temperature. Nano-silver is an effective and a fast-acting microbicide against a broad
spectrum of pathogenic bacteria and fungi thus can be utilized in various processes in the
medical field [9]. Our findings are in agreement to the previous reports and synthesized AgNPs
have showed the considerable antibacterial activities along with the standard antibiotic
(tetracycline) against B. cereus. The antibacterial activity of AgNPs is due to the permeability of
the cell membrane [23] or formation of free radicals [20] or interaction of AgNPs with the thiol
groups of many enzymes thus inactivating them. Beside this, the moderate antifungal activity of
AgNPs against Trichoderma sp. and F. semitectum was due to formation of insoluble compounds
by inactivation of sulfhydryl groups in the fungal cell wall and disruption of membrane bound
enzymes and lipids, causing cell lysis [19].
The MTT assay determines the mitochondrial activity of the cells which, reduce the soluble,
yellow MTT into an insoluble, purple formazon. The reduction of MTT to formazon indicates
14
the decrease in mitochondrial metabolism of the cells. Therefore, the absorption of formazon
formed, directly co relates to the number of cells whose mitochondrial metabolism is intact even
after the exposure of AgNPs. Dose response of AgNPs in J774 cells and THP1 α cells showed
the decrease in the reduction of MTT to formazon with the increasing concentration and time of
exposure. However, AgNPs were found to be significantly more toxic to THP1 α cells as
compared to J774 cells, which could be attributed to the intrinsic anticancer property of AgNPs.
This might be due to small particle size of AgNPs with enormous specific surface area, which
facilitated further expression and dissolution of ions, leading to increased toxicity. AgNPs are
highly reactive, exhibit oxidative potential, contains the ability to bind with biomolecules like
proteins and DNA and consequently caused the disturbance in the functioning of bimolecules
[2].
In both cell lines J774 and THP1 α a significant increase in the levels of ROS was observed in
comparison to the unexposed controls. The maximum amount of ROS was produced after 4 hr of
incubation with AgNPs, which declined at 6 h. ROS plays an important role in triggering cellular
pathways that can lead to cellular death by either causing nuclear damage or by contributing in
cell membrane rupture mechanisms. Exposure of AgNPs to cells caused increased production of
ROS, suggested their potential use as an anti- microbicide, occupying its application in
agriculture. Moreover, these nanoparticles can also act as immunomodulatory agent alone or in
combination with established therapeutic immunomodulatory agents. As these NPs are simply
engulfed by the macrophages, they also pose themselve s as targeted drug / vaccine delivery
vehicle to macrophages thereby a boom for development of a potent chemotherapeutic vehicle
for diseases involving macrophages viz., leishmaniasis, tuberculosis etc. Hence, care has to be
taken to utilize this marvel well and in a good, efficient and effective ways. Researchers should
also understand its limitations and taking excessive care for its effect on environment and human
health.
In future, these bio-synthesized nanomaterials (encapsulation) may lead to enhancement of
agricultural productivity like slow release of phosphorus from fertilizers and its effective uptake
by plants. Fungus A. brassicae contains phosphatase enzyme whose main function is to
solubilize the insoluble form of phosphorus in to soluble one. Thus, the bio-nanoparticles
prepared from this fungus may display slow release of encapsulated enzyme and hence may
improve phosphate solubilization. These synthesized myco-nanoparticles may be mixed with
15
fertilizers to increase the uptake of phosphorus nutrient. However, in future, this strategy needs
to be validated.
5. Conclusion
The present study showed the mycogenic production of AgNPs with particular emphasis on
procedure of synthesis and its potential applications in human welfare as well as in other allied
sectors. AgNPs were synthesized from fungus A. brassicae which has phosphate solubilizing
ability. Findings showed that synthesized nanoparticles were spherical in shape and has a
promising antimicrobial agent against both gram-positive / gram- negative bacteria and
pathogenic fungi also. However, higher doses of AgNPs exhibited the cytotoxicity and ROS
production in J774 and THP1α cells in time dependant manner. . Observations have also showed
the bactericidal activity of synthesized nanoparticles thus implementing their role as
antimicrobicidal agent. In conclusion findings showed that biosynthesized silver nanoparticles
could be use in agriculture and also have antimicrobicidal activity which could be utilize for
human welfare. However, further studies are required to fully characterize the mechanistic and
toxicity aspects of synthesized nanoparticles.
Conflict of interest
The authors have no conflict of interest.
Acknowledge ments
The authors are highly thankful to Vice Chancellor, Integral University for his support and
encouragement. We sincerely thank Mr. Sharma ITRC, Lucknow for carrying out TEM analysis
of nanoparticles. The grant of UGC-Maulana Azad National Fellowship is gratefully
acknowledged.
References
1. B. Dorau, R. Arango, and F. Green, “Forest Products Society pp. Proceedings of the 2nd
Wood-Frame Housing Durabili and Disaster Issues Conference,” 133, Las Vegas, NV.
2004.
16
2. B. Reidy, A. Haase, A. Luch, and K. Dawson, “Mechanisms of AgNP release,
transformation and toxicity: A critical review of current knowledge and recommendations
for future studies and applications,” Lynch, Materials, vol. 6, pp. 2295-2350, 2013.
3. C. Lam, J. James, and R. Mccluskey, “Pulmonary toxicity of single-wall carbon
nanotubes in mice 7 and 90 days after intra-tracheal instillation,” Tox Sci, vol. 77, pp.
126–34, 2004.
4. C. Perez, M. Pauli, and P. Bazerque, “An antibiotic assay by agar-well diffusion
method,” Acta Biologiae et Medecine Experimentaalis, vol. 15, pp.113-115, 1990.
5. F. Mosmann, “Methods: Rapid calorimetric assay for cellular growth and survival:.
Application to proliferation and cytotoxicity assay,” J. Immunol,
vol. 65, pp.55–63,
1983.
6. G. Garrity, “Springer-Verlagpp. Bergey's Manual of Systematic Bacteriology,” In
Bergey's Manual of Systematic Bacteriology pp. 742 New York, 2001.
7. I. Pikovskaya, “Mobilization of Phosphate in Soil in Connection With Their Vital
Activities of Some Microbial Species,” Microbiologiya, vol. 17, pp. 362-370, 1948.
8. J. Wright, K. Lam, D. Hansen, and R. Burrell, “Efficacy of top ical silver against fungal
burn wound pathogens,” Am. J. Infest. Control, vol. 27, pp. 344-350, 1999.
9. J.W. Alexander, “History of the medical use of silver. Surgical Infections,” vol. 10, pp.
289-292, 2009.
10. K, Yliniemi, and M.Vahvaselka, “Antimicrobial activity of colloidal AgNPs prepared by
sol-gel method,” Chem, vol. 18, pp.199, 2008.
11. K. Hartman, L. Wilson, and M. Rosenblum, “Detecting treating cancer with
nanotechnology,” Mol Diagn Ther, vol. 12, pp. 1–14, 2008.
12. K. N. Thakkar, S. S. Mhatre, R. Y. Parikh, “Biological synthesis of metallic
nanoparticles,” Nanomedicine: Nanotechnology, Biology, and Medicine, pp. 1016, 2009.
13. L.A.S Dias, and A.C. Alfenas (Ed.), “Eletroforese de Isoenzimas e Proteínas Afinspp.
Fundamentos e Aplicações em Plantas e Microrganismos, UFV, Viçosa Análises
multidimensionais, pp. 405–473, 1998.
14. Li. Huang, J. Q. Sun, D. Lu, and Y. Su, “Biosynthesis of silver and gold nanoparticles by
novel sundried Cinnamomum camphora leaf,” Nanotechnology, vol. 18, pp. 11-15, 2007.
17
15. N. Swarnakar, K. Thanki, and S. Jain, “Effect of co-administration of CoQ10-loaded
nanoparticles on the efficacy and cardiotoxicity of doxorubicin- loaded nanoparticles,”
RSC Advances, vol 3, pp. 14671, 2013.
16. P. Mohanpuria, N. K. Rana and S.K. Yadav, “Biosynthesis of nanoparticles
: Technological concepts and future applications,” J. Nanopart, vol. 10, pp. 507–517,
2008.
17. Q. Feng, G. Chen, F. Cui, T. Kim, and J. Kim, “A mechanistic study of the antibacterial
effect of silver ions on Escherichia coli and Staphylococcus aureus,” J Biomed Mater
Res, vol. 52, pp. 662–668, 2000.
18. R. Cathcart, E, Schwiers, and B. N. Ames, “Detection of picomole levels of
hydroperoxides using a fluorescent dichlorofluorescin assay,” Anal. Biochem, vol. 134,
pp. 111–116, 1983.
19. R. Liao, and J. Talbot, “Assessment of the effect of amphotericin B on the vitality of
Candida albicans,” Antimicrobial agents and chemotherapy, vol 43, pp. 1034-1041
1999.
20. S. Hussain, K. Hess, J. Gearhart, and K. Geiss, “In Vitro: Toxicity of Nanoparticles in
BRL 3A Rat Liver Cells,” J. Toxicol, vol. 19, pp. 975–983, 2005.
21. S. Pal, Y. Tak, and J. Song, “Does the antibacterial activity of AgNPs depend on the
shape of the Nanoparticle? A study of the gram- negative bacterium E. coli,” Appl.
Environ. Microbiol, vol. 73, pp. 1712-1720, 2007.
22. V. Germain, J. Li, D. Z. and M.P. Pileni, “Stacking faults in formation of silver
nanodisks,” J. Phys Chem B, vol. 107, pp. 8717–8720, 2003.
23. Y. Gavrieli, Y. Sherman, and S. Bensasson, “Identification of programmed cell death in
situ via specific labeling of nuclear DNA fragmentation,” J. cell Biol vol. 119, pp. 493–
501, 1992.
18