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Highly Potential Antifungal Activity of Quantum-Sized Silver Nanoparticles

Against Candida albicans

Article in Applied biochemistry and biotechnology · March 2014

DOI: 10.1007/s12010-014-0782-9 · Source: PubMed

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Appl Biochem Biotechnol
DOI 10.1007/s12010-014-0782-9

Highly Potential Antifungal Activity of Quantum-Sized

Silver Nanoparticles Against Candida albicans

Malathi Selvaraj & Prabhu Pandurangan &

Nishanthi Ramasami & Suresh Babu Rajendran &
Sriman Narayanan Sangilimuthu & Palani Perumal

Received: 1 August 2013 / Accepted: 4 February 2014

# Springer Science+Business Media New York 2014

Abstract The antifungal activity of polyvinylpyrrolidone (PVP)-stabilized quantum-sized

silver nanoparticles (SNPs) against the growth of Candida albicans has been demonstrated
in the present study. C. albicans is a known opportunistic human pathogen causing superficial
and systemic infections. Research data carried out on C. albicans so far have shown unequiv-
ocally that it develops resistance against conventional antifungal drugs and that the infections it
causes are difficult to cure with conventional antifungal agents. Hence, it is urgent to find
newer materials for the treatment of infections caused by C. albicans that must be safe for the
host. PVP-capped SNPs were synthesized, and its surface plasmon band was observed at
410 nm. The growth of C. albicans was markedly inhibited when the cells were incubated with
SNP. The minimum inhibitory concentration (MIC) of SNP was determined as 70 ng/ml, and
this value is relatively lower when compared with the conventionally used antifungal drugs
such as amphotericin B (0.5 μg/ml), fluconazole (0.5 μg/ml), and ketoconazole (8 μg/ml). The
viability of SNP-treated cells was checked by measuring the metabolic activity using XTT
assay. Field emission scanning electron microscopic (FE-SEM) and transmission electron
microscopic (TEM) analyses of the cells treated with SNP have lost the structural integrity
to a greater extent.

Keywords Quantum-sized silver nanoparticles . Candida albicans . Antifungal activity .

Minimum inhibitory concentration . Polyvinylpyrrolidone

Introduction

Fungal infections caused by yeasts and molds represent an escalating problem in health care as
advances in modern medicine prolong the lives of severely ill patients. These organisms cause
infection not only in those having HIV, cancer, organ transplant, and surgical operation and
ICU patients but also newborn infants. Fungi, being eukaryotic in nature and more complex
than bacteria, cause infections that are often difficult to diagnose and treat, resulting in

M. Selvaraj : P. Pandurangan : N. Ramasami : S. B. Rajendran : S. N. Sangilimuthu : P. Perumal (*)

University of Madras, Chennai, Tamil Nadu, India
e-mail: palanii7@yahoo.com

P. Perumal
e-mail: palani7@unom.ac.in
 Appl Biochem Biotechnol

unacceptably high mortality rates [1]. In recent years, a rapid increase in microbes that are
resistant to conventionally used antibiotics has been observed [2]. Candida albicans is a
commensal present on the mucosal surfaces of human oral and vaginal cavities and frequently
turns into an opportunistic pathogen causing superficial and systemic infections if the host
immune system is compromised due to immunosuppressive and broad-spectrum antibiotic
therapy, HIV infection, and cancer chemotherapy [3]. Currently, most of the available anti-
fungal agents are based on polyenes (amphotericin B), triazoles (fluconazole, itraconazole,
voriconazole, and posaconazole), and echinocandins (caspofungin, micafungin, and
anidulafungin). However, the administration of these antifungals is often accompanied by
various complications such as amphotericin B toxicity and adverse effects of some azoles
including toxicity and drug interactions [4–7] and yeast resistance to antifungal therapy [8, 9].
A remarkable property observed with C. albicans is that it develops resistance, and the
infections it causes are difficult to treat with conventional antifungal drugs. Therefore, it is
crucial to find newer molecules for the treatment of Candida infections without adverse effect
on the host cells. It has been known, since the ancient times, that silver and its compounds are
effective antimicrobial agents [10, 11]. In particular, due to the recent advances in research on
metal nanoparticles, nano-Ag has received special attention as a possible antimicrobial agent
[12–14]. Therefore, the preparation of uniformly sized silver nanoparticles with specific
requirements in terms of size, shape, physicochemical properties is of great interest in the
formulation of newer pharmaceutical products [15, 16]. Though the biocidal effect and the
mode of action of silver ion are known, the antifungal effects and the mode of action of SNP
against fungi have remained mostly unknown.
Therefore, an attempt has been made in the present investigation to synthesize polyvinyl-
pyrrolidone (PVP)-stabilized quantum-sized silver nanoparticles (SNPs) and to evaluate their
antifungal activity against the growth of C. albicans. The growth of the organism was
markedly inhibited when the cells were incubated with SNP. The minimum inhibitory
concentration (MIC) of SNP has been determined, and the cell viability was checked using
the XTT assay by measuring the mitochondrial dehydrogenase activity of the live cells. FE-
SEM and transmission electron microscopic (TEM) analyses have clearly indicated marked
changes in the integrity of the cells treated with SNP. For the first time, elemental analysis of
the SNP-treated and nontreated cells has been carried out in this study.

Materials and Methods

Chemicals

Antifungal drugs such as amphotericin B and ketoconazole were purchased from HiMedia
(India). Silver nitrate was purchased from Sigma Chemical Co. (USA). An injectable form of
fluconazole was purchased from CIPLA (India). Culture media such as yeast nitrogen base
(YNB) and yeast extract peptone dextrose agar (YEPDA) were purchased from Difco Labo-
ratories (Detroit, MI, USA) and HiMedia (India), respectively. The other solvents used in the
study were of analytical grade procured locally.

Organisms and Growth Conditions

C. albicans used in this study was obtained from the fungal culture collection facility at the
Centre for Advanced Studies in Botany, University of Madras. The organism was cultured on
YNB medium and stored on YEPDA slants at 4 °C until further use.
Appl Biochem Biotechnol

Synthesis and Characterization of Quantum-Sized SNP

The PVP-stabilized quantum-sized SNPs were synthesized as follows. Fifty milligrams

of silver nitrate (AgNO3) was dissolved in 11.25 ml of ethylene glycol to which 1.5 g
of PVP was added. The solution was thoroughly mixed using a magnetic stirrer. The
agitation of the solution was continued for 20 min while increasing the temperature
by 5 °C after every 5 min. The formation of a colloidal, wine-brown-colored solution
confirms the formation of SNP. The SNP prepared as above was diluted to 10−4 and
used for further investigations. The SNP was characterized using UV–Vis spectral
analysis in a spectrophotometer (Lamda-650, Perkin Elmer, USA) and high-resolution
transmission electron microscope (HR-TEM, FEI, Tecnai G2 model T 30 S-twin,
300 kV, Japan).

Determination of MIC of SNP

The determination of MIC of the PVP-stabilized SNP against C. albicans was

performed as per the recommendations of the Clinical Laboratory Standard Institute
(CLSI) [17]. The yeast cells of C. albicans were grown planktonically under aerobic
conditions at 37 °C for 24 h in YNB broth. The cell count was made with hemocy-
tometer, and a standardized cell suspension (1 × 105 cells/ml) was prepared. One
hundred microliters of the cell suspension was dispensed in triplicate microtiter wells
to which 100-μl suspension containing 1.380 to 0.092 μg of nanoparticles in YNB
medium was added and mixed well. The following conventionally used antifungal
agents such as amphotericin B [18], fluconazole, and ketoconazole were used in order
to compare the antifungal potential of SNP. Amphotericin B and ketoconazole were
dissolved in dimethyl sulfoxide (DMSO) and diluted with sterile YNB medium to
obtain drug concentrations ranging from 0.0313 to 1,024 μg/ml. The cells with either
nanoparticles or conventionally used antifungals were incubated at room temperature
for 24 h, and the growth inhibition was measured spectrophotometrically at 600 nm
(Powerserve XS Biotech, USA). Wells with cells without SNP were used as the
control. The MIC was calculated as the lowest amount of SNPs that inhibited 80 %
growth of C. albicans cells under the experimental conditions.

Cell Viability Assay

The metabolic activity of SNP-treated cells were determined by the XTT [2, 3-bis(2-
methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide sodium salt] assay [19].
The XTT tetrazolium salt was dissolved in phosphate-buffered saline (10 mM,
pH 7.4) at a concentration of 0.5 g/l and filter sterilized through a 0.2 μm filter,
and the aliquots were stored at −80 °C. Just before use, an aliquot was thawed, and
10 mM menadione prepared in acetone was added to the XTT solution to a final
concentration of 1 μM. One hundred microliters of culture (1×105 cells/ml) was
dispensed into 96-well plates. Different concentrations of SNP (1.380 to 0.092 μg)
were added to the wells and incubated for 24 h at 37 °C. After 24 h of incubation,
100 μl of the XTT/menadione solution was added to each well and mixed thoroughly.
The plates were incubated in dark at 37 °C for 2–3 h. The reduced formazan-colored
product was measured at 490 nm using a microtiter plate reader (Powerserve XS
Biotech, USA). The concentration of SNP showing 80 % reduction in cell viability
was then calculated.
 Appl Biochem Biotechnol

Field Emission Scanning and Transmission Electron Microscopic Studies

One milliliter of 1×105 cells/ml suspension was incubated with SNPs at their MIC values for
24 h and observed in a FE-SEM (H-7650, Hitachi, Japan) coupled with Energy Dispersive
X-ray Analysis (EDAX) spectrum analysis, and the morphological changes were observed.
The cells grown on YNB medium without SNP served as the control. For TEM, the cells were
fixed with glutaraldehyde (1 %) solution, postfixed with osmium tetroxide for 2 h, and washed
with buffer twice. The cells were dehydrated with increasing concentration of acetone and
embedded in Epon–Araldite resin. Ultrathin sections were made, stained with uranyl acetate
and lead citrate, and observed under TEM (SU 6600 Hitachi, Japan).

Results

Synthesis and Physical Characterization of SNP

The solution containing silver nitrate (AgNO3), ethylene glycol, and PVP turned into a
colloidal, wine-brown-colored solution which indicated the formation of SNP. The SNP
prepared as above was diluted to 10−4 and used for further investigations. The synthesized
SNP showed an absorption peak at 410 nm (Fig. 1a), and this characteristic peak confirmed the
formation of colloidal SNP. The TEM analysis revealed the formation of spherical SNP
measuring about 2 nm (Fig. 1b). The HR-TEM analysis also revealed the formation of a thin
outer layer measuring 0.28 nm (Fig. 1b). This thin layer was made by PVP encompassing SNP
which appeared like core-shell morphology.

Antifungal Activity of SNP

The PVP-stabilized SNP showed antifungal activity against the tested organism. There was a
marked reduction in the growth of the cells when incubated with SNP in a concentration-
dependent manner (Fig. 2a). The MIC of the SNP was determined at 70 ng/ml. The MIC

Fig. 1 a UV–Vis spectra and b HR-TEM image of SNP

Appl Biochem Biotechnol

Fig. 2 MIC values of a SNP and b amphotericin B, fluconazole, and ketoconazole against C. albicans. Each
value represents the mean of three independent experiments

values for the known antifungals such as amphotericin B and fluconazole were observed at
0.5 μg/ml and for ketoconazole at 8 μg/ml (Fig. 2b).

Cell Viability Assay

To verify if the synthesized SNPs have the ability to kill the Candida cells, the cell viability
assay was performed with XTT assay as described in the “Materials and Methods” section. The
metabolic reduction of XTT sodium salt by mitochondrial dehydrogenase forms a colored
water-soluble formazan product, and this was measured spectrophotometrically at 490 nm. The
cells treated with PVP-stabilized SNP showed decreased metabolic activity in a dose-
dependent manner (Fig. 3) and confirmed that the SNPs have the ability to kill or inactivate
the organism. Further, the MIC value of SNP as determined by the XTT assay coincided with
the MIC value obtained through spectrophotometric measurement.

Fig. 3 Effect of PVP-stabilized silver nanoparticles on the cell viability. Each value represents the mean of three
independent experiments
 Appl Biochem Biotechnol

Field Emission Scanning Electron Microscopic Studies

The cells treated with SNP exhibited noticeable structural changes (Fig. 4c, d) when compared
with the control cells (Fig. 4a, b). The SNP-treated cells that appeared deformed exhibited
surface shrinkages when compared with the control. The SNP-treated cells aggregated into
clumps, remained attached to the adjacent cells (Fig. 4c, d), forming EPS (extracellular
polysaccharide)-rich biofilms. The SNP adhered onto the biofilm and cellular surfaces, besides
being concentrated at the cellular interjections in the biofilm. The EDAX spectrum of SNP-
treated cells showed the presence of Ag peak (Fig. 5b) along with other inorganic elements
present in the medium, but in the case of untreated cells, the Ag peak was absent (Fig. 5a). The
elemental analysis clearly indicated adherence of the SNP on the cell surface leading to cell
damage and arrest of growth.

Transmission Electron Microscopic Studies

The cells treated with SNP were highly deformed (Fig. 6b, c, e, f), and the cells had shrunken to a
greater extent (Fig. 6b, c, e, f). Alteration in the cell wall and cell membrane was also observed.
Pronounced increase in the number and enlargement of vacuole was evident with reduction in the
cytoplasm, and the cells started to disorient from its original shape. A significant portion of cells
showed accumulation of granules in the cytoplasm and vacuoles. C. albicans cells treated with
SNP also showed signs of pseudohypha and hypha morphogenesis.

Fig. 4 a, b Structural changes due to interaction of SNP with C. albicans cells: control. c, d SNP-treated
Candida cells
Appl Biochem Biotechnol

Fig. 5 EDAX analysis of C. albicans cells treated a in the absence and b in the presence of SNP

Fig. 6 TEM analysis of C. albicans cells treated a, b in the absence and b, c, e, f in the presence of SNP
 Appl Biochem Biotechnol

Discussion

Nanosized inorganic particles have increasingly become an important material in the devel-
opment of novel nanodevices that are being used in numerous physical, biological, biomedical,
and pharmaceutical applications [20, 21]. Among them, nano-Ag has proved to be highly toxic
to a variety of microorganisms and potentially inhibited the growth of both bacteria and fungi
[22, 23] excepting few strains that showed resistance [24]. An attempt has been made in the
present investigation to synthesize quantum-sized SNPs which are stabilized with PVP
and to evaluate their antifungal activity against C. albicans. The PVP had reduced the
silver nitrate leading to the formation of colloidal SNP, and the formation was
manifested as a change in color from colorless to wine brown. As is evident from
Fig.1a, the SNP exhibited characteristic absorption peak at 410 nm and had confirmed
the formation of SNPs. The reducing and stabilizing capabilities of PVP in the
synthesis of SNP have been well documented. The coordination of Ag+ ion and O
atom of the carbonyl group of PVP facilitates the reduction of Ag+ ion with the lone
pair of electrons of N atom from pyrrolidone ring that resulted in the formation of
PVP-stabilized SNP [25–27]. The nanoparticles appeared spherical, and as it is highly
evident from Fig. 1b, there was a conspicuous presence of an ultrathin cap formed by
PVP. The monodispersed SNP had a mean primary grain size of 2–3 nm and endured higher
stability due to the ultrathin (0.28 nm) stabilizing cap of PVP that precluded aggregates.
The SNPs were evaluated for its antifungal activity against the cells of C. albicans. There
was a marked reduction in the growth of the cells when incubated with SNP and the reduction
in growth occurred in a concentration-dependent manner (Fig. 2a). In the present investigation,
the MIC value of the SNP was observed at 70 ng/ml which is comparatively lower than the
MIC values obtained with conventional antifungal agents such as amphotericin B (0.5 μg/ml),
fluconazole, and ketoconazole (8 μg/ml; Fig. 2b). The antifungal activity of SNP observed in
the present study agrees with an earlier report wherein comparatively higher antimicrobial
activity has been observed when the SNPs were stabilized with stabilizing agents. The MIC
value of SNP observed in the present study has been much lower when compared with the
MIC values of the study reported previously [28]. The results have clearly indicated that
SNP has a potential as an antifungal agent in treating fungal infectious diseases. It has
been reported that the clinical applications of several antimicrobial agents have been
restricted as they bring about cytolysis of human erythrocytes. Nano-Ag showed low
hemolytic activity, while amphotericin B shows a slightly higher hemolytic activity
that could be fatal in patients who are treated with this agent for fungal infection [29].
As is evident in Fig. 2, the growth of the C. albicans was inhibited effectively by
PVP-capped SNP even at low concentration. The efficacy of the SNP was better when
compared with the conventionally used antifungal drugs.
The C. albicans cells treated with SNP have lost their structural integrity and have
presumably induced the production of extracellular polymeric substances in which the
cells were interconnected thus giving a biofilm-like appearance. Biofilms are formed
in response to a wide array of environmental clues that include mechanical perturba-
tions, nutritional constraints, and exposure to harmful metabolites (antibiotics). In the
present investigation, the SNPs have strikingly inhibited the test strain at very low
concentration (70 ng/ml), and SEM images showed the accumulation of SNP at the
cellular interjections in the biofilm and fungal cell wall. The SNPs have been reported
to detrimentally interact with cell membrane resulting in breakdown of the membrane
permeability barrier in prokaryotic and eukaryotic systems [29, 30]. It has been
reported about the oxidative damage of the cell membranes due to the release of
Appl Biochem Biotechnol

Ag+ from SNPs and their detrimental action on bacterial membrane-bound proteins
resulting in loss of cellular integrity and osmoticum culminating in acute toxicity to
the cells [29]. The similar conclusive results are lacking in fungal systems; however,
the current understanding of membrane depolarization, dynamics, and permeability
corroborates with the mode of action reported in bacteria. Quite recently,
transcriptomic analysis of Saccharomyces cerevisiae exposed to SNP confirmed the
potential damage to the cell wall and transmembrane proteins and upregulation of
cell-wall-strengthening genes in surviving cells [31]. Such changes result in the
release of glucose and trehalose [29] which, according to our hypothesis, could
eventually be used up in biofilm formation, as enhanced biofilm formation had been
reported in glucose-rich conditions [3]; nevertheless, the membrane damages caused
by the SNPs were severe to mend and hence result in cell death.
Investigations on the mode of SNP entry into the fungal cells are scarce, and it persists as an
intriguing query like the organization of the fungal cell wall itself, to be probed in detail. The
highly heterogeneous C. albicans cell wall has a central core composed of branched β-1,3-
glucan cross-linked with chitin which, in turn, covalently bound to β-1,6-glucan and α and β-
mannans [32, 33] interwoven into a fine matrix with ultrafine porosity, permeable to small
macromolecules of Mr 620 and 0.81-nm hydrodynamic radius (RH) [34, 35]. In the present
study, we hypothesize that the endosomal trafficking systems involved in the uptake and
release of macromolecules and nutrients may be involved in the uptake of SNP. Of the various
types of endocytosis, fluid phase endocytosis has been documented to be involved in assim-
ilation of Lucifer yellow, an impermeable dye by C. albicans and found to be accumulated in
the vacuole. Assimilation of polystyrene beads (40 nm) and CdSe/ZnS quantum dots (20 nm)
by sycamore plants’ protoplasts and cells in cell cultures further substantiates the involvement
of fluid phase endocytosis in transport of macromolecules and synthetic nanomaterials across
the cell wall [36]. Moreover, in the present study, the TEM image (Fig. 6b) showed a cluster of
endocytic vesicles with SNP accumulated at the margins of the enlarged central vacuole. We
extrapolate these ideas and resolve to the involvement of fluid phase endocytosis involvement
in assimilation of quantum-sized SNP (2–3 nm).
Our TEM results support the previous reports claiming significant changes in the mem-
brane structures along with the dramatic enlargement and increase in the number of vacuoles
on SNP treatment. Intense vacuolation has occurred in response to environmental cues and
cellular stress in C. albicans; however, it has demonstrated the vitality of vacuoles in
filamentous growth which may aid in survival and host tissue invasion through mutational
studies [37]. In the present study, SNP treatment induced severe environmental stress that had
resulted in vivid morphological changes like enlargement of existing vacuoles and formation
of numerous vacuoles in the cytoplasm (Fig. 6b, c, e, f). The emergence of polarized growing
points in SNP-treated cells, which is considered as one of the survival strategies under stress
conditions, is evidence of the antifungal activity of SNP. A delay or arrest of cell cycle
progression in C. albicans often results in a terminal phenotype, different from
pseudohyphae and hyphae in the ability to divide, and hence eventually dies [38].
We observed very few highly elongated pseudohypha-like cells (Fig. 6e) and highly
distorted hyphae with multiple vacuoles (Fig. 6f). In addition, the vast majority of the
examined cells showed emergence of small evagination structures but failed to
progress further. It could be inferred that the SNP treatments have impacted the cell
cycle, and these observations also corroborate with the reported fungistatic, fungicidal,
and cell cycle impedance activity of SNPs against C. albicans [29, 39].
In this study, we have evaluated the viability of the cells treated with SNP by
measuring the mitochondrial dehydrogenase activity with XTT sodium salt. The
 Appl Biochem Biotechnol

amount of formazan product was directly correlated with the number of live cells. As
the SNP concentration increased, there was a decrease in the reduced formazan
product implying that the SNPs have prevented the enzymatic respiration. It has been
reported that SNP disrupts the mitochondrial integrity and induces cytochrome c
release and promotes apoptosis through phosphatidylserine exposure and the activation
of metacaspases. The mechanism of SNP in mitochondria-dependent apoptosis in
C. albicans has not been fully elucidated; however, it has been envisaged that the
SNP induces programed cell death through ROS accumulation especially OH·[39].
The outstanding activity of PVP-coated SNP has also been proved against bacteria
and viruses. It has been reported that the PVP-coated SNP ranging from 1 to 10 nm
is inhibiting the HIV-1 virus from attaching to the host cells preferably via binding to
gp120 glycoprotein knobs [40]. In a similar study, PVP-coated SNP was shown to
reduce the respiratory syncytial virus infection by 44 % [41]. The mechanism of SNP
uptake and accumulation has been reported for the bacteria such as Vibrio cholera,
Pseudomonas aeruginosa, and Salmonella typhi. It has been observed that the attach-
ment of the 10-nm-sized SNP to the bacterial cell membranes and inside the cells
occurs [42]. The inactivation of lactate dehydrogenase (LDH) activity and increased
protein leakage was observed with SNP-treated Staphylococcus aureus and
Escherichia coli [43]. Irregular pit generation on E. coli cell surfaces was observed
in cells treated with differentially shaped SNP [44]. As far as the cytotoxic effects of
the PVP-stabilized SNP are concerned, there is no conclusive recorded evidence of
cytotoxicity on human fibroblast and Paramecium caudatum at recommended MIC
values against pathogenic microbes [45, 46]. Ruparelia et al. [47] also reported the
strain-specific antimicrobial activity of 3-nm-sized SNP against E. coli and S. aureus.
The inhibitory potential was observed 40–50 % greater than the copper nanoparticles.
Reports on reduced efficacy of PVP-stabilized SNPs over nonstabilized or surfactant-
stabilized SNPs stated that PVP coating results in slower or reduced release of Ag+ [44],
while other reports claimed that the bonding of Ag to PVP is accountable for the reduced
antimicrobial activity over free Ag encapped within foamy carbon [40]. Interestingly, our
results contradict the above claims and exhibited a superior antifungal activity because of the
quantum size and high stability of the PVP-stabilized SNP. Assimilation of nanoparticles is
inversely proportional to its size, quantum-sized particles finding greater entry than
microdimensional particles, and directly correlates to the level of toxicity [42, 48].
Also, PVP encapsulation provides sustained release of Ag from SNPs that could
ensure prolonged antifungal activity.

Conclusion

The present study offers an unequivocal proof that PVP-stabilized quantum-sized

SNPs work as a potent antifungal agent at very low concentration. The MIC values
obtained were comparatively lower when compared with the MIC values of known
antifungals. Furthermore, the viability of the yeast cell treated with PVP-stabilized
SNP has been evaluated, and the results very well coincide with the MIC values.
While SEM images confirmed the interaction of SNP onto the cell wall and biofilm,
TEM analysis gives a picture of the loss of cellular integrity, vacuolation, and cell
cycle arrest resulting in deformation when incubated with the PVP-stabilized SNP. It
could turn out to be an effective and safe alternative to conventional oral and topical
antifungal agents with due investigations for clinical applications.
Appl Biochem Biotechnol

Acknowledgments The authors thank the National Centre for Nanoscience and Nanotechnology, MHRD and
DST-INSPIRE for financial support in the form of a research grant and junior research fellowships. Thanks is
also due to Mr S. Prathap Augustine, technician, NCNSNT, for assisting us with FE-SEM and TEM imaging.

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