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Synergistic influence of polyoxometalate surface corona

towards enhancing the antibacterial performance of
Cite this: DOI: 10.1039/c3nr03806h
tyrosine-capped Ag nanoparticles†
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Hemant K. Daima, P. R. Selvakannan, Ahmad E. Kandjani, Ravi Shukla,

Suresh K. Bhargava and Vipul Bansal*

Received 24th July 2013
Accepted 21st September 2013
DOI: 10.1039/c3nr03806h
www.rsc.org/nanoscale
We illustrate a new strategy to improve the antibacterial potential of silver nanoparticles (AgNPs) by their
surface modification with the surface corona of biologically active polyoxometalates (POMs). The stable
POM surface corona was achieved by utilising zwitterionic tyrosine amino acid as a pH-switchable
reducing and capping agent of AgNPs. The general applicability of this approach was demonstrated by
developing surface coronas of phosphotungstic acid (PTA) and phosphomolybdic acid (PMA) around
AgNPs. Our investigations on Gram negative bacterium Escherichia coli demonstrate that in conjugation
with AgNPs, the surface corona of POMs enhances the physical damage to the bacterial cells due to
synergistic antibacterial action of AgNPs and POMs, and the ability of tyrosine-reduced AgNPs (AgNPsY)
to act as an excellent carrier and stabiliser for the POMs. The further extension of this study towards
Gram positive bacterium Staphylococcus albus showed a similar toxicity pattern, whereas these
nanomaterials were found to be biocompatible for PC3 epithelial mammalian cells, suggesting the
potential of these materials towards specific antimicrobial targeting for topical wound healing
applications. The outcomes of this work show that facile tailorability of nanostructured surfaces may
play a considerable role in controlling the biological activities of different nanomaterials.

Introduction issue related to public health.2,3 From this perspective, new

Antibiotic resistance in microorganisms has been recognised as
the single most important issue associated with the ineffective
treatment of infectious diseases. Traditional antibiotics
including ciprooxacin, doxycycline and ceazidime invade
bacterial cells and act upon specic biomolecular targets to
control bacterial growth. In general, these antibiotics either
interact with the genetic material, block cell-division or occa-
sionally trigger autolysins in the targeted bacteria, rather than
causing any physical damage to the bacterial cell wall. There-
fore, as a result of antibiotic action, morphology of the patho-
genic bacteria is typically preserved and bacterial species may
have the possibility to develop resistance against traditional
antibiotics.1 This trend of antibiotic resistance through genetic
mutations preserving physical integrity in bacteria is a critical
NanoBiotechnology Research Lab (NBRL), Centre for Advanced Materials & Industrial
Chemistry (CAMIC), School of Applied Sciences, RMIT University, GPO Box 2476V,
Melbourne, VIC 3000, Australia. E-mail: vipul.bansal@rmit.edu.au; Fax: +61 3 9925
3747; Tel: +61 3 9925 2121
† Electronic supplementary information (ESI) available: XRD analysis (Fig. S1);
cytotoxicity prole (Fig. S2) and optical images of human PC3 cells (Fig. S3)
treated with nanomaterials; FTIR vibrational modes arising from different
materials (Table S1); and relative concentrations of Ag and POMs present in
modied AgNPs used for biological studies (Table S2). See DOI:
10.1039/c3nr03806h
strategies are urgently required to design highly efficient anti-
microbial agents, wherein rapid developments in the eld of
nanotechnology may provide opportunities to control patho-
genic microorganisms via tailored fabrication of
nanomaterials.4
Although antimicrobial properties of ionic silver (Ag+) have
been known since antiquity, the realisation of the antimicrobial
potential of inorganic nanomaterials is a more recent
phenomenon.5 Inorganic nanomaterials are now known to
demonstrate composition, size, shape, chemical functionality
and surface charge-dependent antimicrobial proles.6–8 To this
end, use of inorganic silver nanoparticles (AgNPs) to control
bacterial growth and towards wounds or burns treatment has
been widely investigated.9–12 It is believed that silver binds to the
bacterial membrane,5 which may assist with the disintegration
of bacterial morphology. For instance, Ag+ cations13,14 and
partially oxidised AgNPs have been shown to possess antibac-
terial activity15 by either rupturing the negatively charged
bacterial cell wall or by destabilizing the outer membrane,16
thereby providing them access to the mitochondria and
subsequently interfering with the respiratory chain.17,18 There-
fore, silver possesses high antimicrobial properties and it is also
known to have considerably lower toxicity towards mammalian
cells than towards bacteria.5

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Notably, although the inuence of nanomaterial composi-
tion, size, shape, and surface charge has been reasonably well
studied, the effect of nanoparticle surface corona on antibac-
terial performance is rather not clear. We believe that it is this
aspect, i.e. the tailored modication of the nanoparticle surface,
that may generate new interesting opportunities to develop
efficient antimicrobial agents. To this end, it should also be
noted that the nature has developed special protein-based effi-
cient antibacterial systems such as lysozyme and antimicrobial
peptides to control bacterial growth.19,20 These natural anti-
bacterial systems typically cause bacterial cell lysis and there-
fore microorganisms nd it difficult to gain resistance against
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biology grade Nutrient Agar (NA) medium and Luria–Bertani
(LB) broth (pH 7.2) were purchased from Oxoid and US Bio-
logicals, respectively, and used to grow and maintain the
bacterial cultures at 37
C as per the standard protocol.
Tyrosine (Y)-mediated synthesis of AgNPsY
In a typical experiment, a 300 mL aqueous solution consisting
of 0.1 mM L-tyrosine and 1 mM KOH was allowed to boil.
Under alkaline boiling conditions (pH 10.5), AgNO3 was added
to the above solution, resulting in 0.2 mM equivalent of Ag+
ion concentration. The above solution was further boiled for 5

these ‘nature-made antibiotics’. min, which resulted in a yellow coloured solution consisting of
In the present study, we initially utilise a green and eco- tyrosine-reduced AgNPsY. Additionally, to increase the metal
friendly approach to synthesise tyrosine (Tyr or Y)-capped silver concentration by a factor of three, this AgNPsY solution was
nanoparticles (AgNPsY), wherein tyrosine molecules act as both further boiled to reduce the solution volume to 100 mL. This
reducing and capping agents. The zwitterionic nature of tyro- solution containing AgNPsY was found to be highly stable even
sine and its pH-based charge reversibility allow the tailorability aer concentrating, indicating that AgNPsY were strongly
of a stable surface corona of bioactive ligands, as demonstrated capped by tyrosine amino acid. Further, concentrated AgNPsY
later. Further, we modify the surface of AgNPsY with two solution was dialysed three times against deionised MilliQ
different polyoxometalates (POMs) viz. 12-phosphotungstic acid water at room temperature to remove the excess KOH, unre-
(PTA) and 12-phosphomolybdic acid (PMA). We have chosen duced metal ions and unbound tyrosine molecules, if any.
POMs as bioactive ligands for surface modication due to their
well known biomedical properties including antibacterial, Preparation of AgNPsY@POMs nanocomposites
antiviral and antitumor activities.21–24 Notably, POMs are poly- Concentrated and dialysed AgNPsY were independently surface
anionic clusters of Keggin ions that are formed by self-assembly modied with two different POMs namely PTA and PMA as
of early transition metals with oxygen and phosphorus shown in Scheme 1. To modify AgNPsY with POMs, concen-
atoms,21,25,26 which typically, in their pristine form, loose their trated and dialysed AgNPsY obtained in the previous step were
activity at the physiological pH, therefore limiting their separately mixed with PTA or PMA to obtain 100 mM POM
biomedical applications.21 In the current study, the stability of concentration in these solutions, and incubated for 24 h.
the antimicrobial POMs could be retained by anchoring them Following incubation, these solutions were again subjected to
onto the surface of inherently antimicrobial AgNPsY. This dialysis under ambient conditions (24
C) to remove any
allowed combination of two antimicrobial agents (POMs and
AgNPs) in a single system, leading to AgNPsY@POMs. To
demonstrate the proof-of-concept applicability of such surface
modied materials, their antibacterial performances were
evaluated against the model Gram negative bacterium Escher-
ichia coli and Gram positive bacterium Staphylococcus albus,
whereas biocompatibility towards mammalian system was
evaluated against PC3 human epithelial cells. Since a vast range
of POMs are widely available for different biological action,
including antiviral, antimicrobial and anticancer applications,
we believe that the approach presented here can be equally
extended to other metal nanoparticles and POMs to obtain
desirable biological action.

Experimental section
Chemicals
Silver nitrate (AgNO3), L-tyrosine, phosphotungstic acid (PTA)
and potassium hydroxide (KOH) were purchased from Sigma-
Aldrich and phosphomolybdic acid (PMA) was purchased from
Chem-Supply Pty Ltd. Dialysis tubing cellulose membrane
(12 kDA cut-off) was purchased from Sigma-Aldrich and used
aer processing (boiling twice for 15 min in deionised MilliQ
water). E. coli and S. albus were originally purchased from Scheme 1 Schematic representation of tyrosine-mediated synthesis of silver
Southern Biologicals and maintained in house. Molecular nanoparticles (AgNPsY) and their surface modification with POMs.

Nanoscale This journal is ª The Royal Society of Chemistry 2013


Paper
uncoordinated PTA or PMA molecules, thereby resulting in
POM functionalised AgNPs (AgNPsY@PTA and AgNPsY@PMA). At
each step, thorough dialysis of colloidal solutions was consid-
ered as a crucial step to ensure that the observed antibacterial
effects are indeed due to nanoparticulate systems, and poten-
tially unreduced metal ions, free amino acid molecules and
POMs do not contribute to the antimicrobial prole reported in
this study.
Antibacterial performance of surface-modied nanomaterials
Qualitative and quantitative assessments of the antibacterial
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potential of AgNPsY, AgNPsY@PTA and AgNPsY@PMA were per-
formed against Gram negative bacterium E. coli and Gram
positive bacterium S. albus using a colony counting method.
Briey, 104 bacterial cells were incubated with varying
concentrations (1, 2, 5, and 10 mM equivalent of Ag against
E. coli and 2 mM equivalent of Ag against S. albus) of extensively
dialysed nanoparticles in 1 mL LB broth for 15 min. Following
incubation, different aliquots of bacterial solutions were
plated onto nutrient agar (NA) plates and bacterial colonies
grown overnight at 37
C were counted, which corresponded to
the number of live bacteria in each suspension as a result of
interaction of different nanoparticles with bacteria for 15 min.
The untreated bacterial culture was considered as a control
with 0% cell death, and the antibacterial activity was plotted in
terms of % bacterial cell death as a function of the dose of
different AgNPs.
Cytotoxicity evaluation of surface-modied nanomaterials in
PC3 cells
For the cytotoxicity assessment of AgNPsY, AgNPsY@PTA and
AgNPsY@PMA, MTT assay was performed as detailed previ-
ously.27 Briey, 1  104 human PC3 epithelial cells per mL
were seeded in a at bottomed 96-well tissue culture plate in
the exponential growth phase and incubated for 24 h at 5%
CO2 and 37
C in RPMI medium supplemented with 10% fetal
bovine serum and antibiotics (Life Technologies, USA). Cells
were treated with AgNPsY, AgNPsY@PTA and AgNPsY@PMA to the
nal Ag concentrations of 1, 2, 5 and 10 mM, in addition to
placebo controls. Aer 24 h incubation, the conditioned media
with suspended nanoparticles and dead cells were aspirated
off from the wells and adherent live cells in the wells were
washed once with 200 mL per well of fresh medium. The cells
were further incubated with 100 mL of freshly prepared 0.5 mg
mL1 MTT solution in complete medium at 37
C in the dark
for a period of 6 h and the formazan crystals thus formed were
dissolved in acidied isopropanol. The intensity of the devel-
oped colour was measured using a Perkin Elmer Envision
Microplate reader at 570 nm wavelength. Wells with complete
medium, respective nanoparticles, and MTT, but without cells,
were used as blanks for each tested concentration. All experi-
ments were performed three times in quadruplets, and the
average of all the experiments has been shown as a cell-
viability percentage in comparison with the control experi-
ment, while nanoparticles untreated controls were considered
as 100% viable. For imaging, PC3 epithelial cells grown aer
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24 h of incubation were observed under a Nikon Eclipse T2000
microscope and images were captured using a CCD camera
attached to the microscope.
Characterisation of surface-modied nanomaterials and
treated bacterial cells
AgNPsY and their surface modied variants were thoroughly
characterised at all the stages of synthesis and functionalisation
using spectroscopy tools such as UV-visible, Fourier transform
infrared (FTIR), X-ray photoelectron (XPS), XRD (X-ray diffrac-
tion), atomic absorption (AAS) and inductively coupled plasma
mass (ICPMS) spectroscopy. Additionally, transmission electron
microscopy (TEM) and zeta potential measurements were per-
formed on these nanomaterials. Nano-scanning electron
microscopy (Nano-SEM) was used to record the morphological
changes in bacterial cells aer their treatment with
nanoparticles.
UV-vis spectral analysis was performed using a Varian Cary
50 spectrophotometer operated at a resolution of 2 nm, FTIR
spectra were recorded in DRS (diffuse reectance spectros-
copy) mode using a Perkin-Elmer D100 spectrophotometer
with a resolution of 4 cm1, and XPS analysis was performed
using a THERMO K-Alpha XPS instrument at a pressure better
than 1  109 Torr (1 Torr ¼ 1.333  102 Pa). XPS spectra were
background corrected using Shirley algorithm, the core level
binding energies (BEs) were adjusted with reference to the C1s
BE of 285 eV, and core levels were tted and deconvoluted
using a Gaussian–Lorentzian function.28 For XRD, samples
were prepared by drop-casting the nanoparticle solutions on
silicon wafer, and XRD analysis was performed with Cu Ka
radiation using a Bruker AX8 Discover instrument. AAS anal-
ysis of nanoparticles was performed to nd the Ag content
using a Varian AAS spectrophotometer aer dissolution of
samples in aqua regia. ICPMS analysis was carried out using
an Agilent Technologies (7700 series ICP-MS) machine to
analyse the molybdenum (Mo) or tungsten (W) concentration
arising from POMs in the relevant samples. Zeta potential
measurements were performed both in the deionised water
(pH 6.4) and LB broth (pH 7.2) using a Malvern 2000 Zetasizer.
TEM imaging of nanoparticles was carried out aer drop
casting the samples onto a carbon coated copper grid, using a
JEOL 1010 TEM instrument operated at an accelerating voltage
of 100 kV.
Morphological changes in E. coli bacterial cells aer their
treatments with various nanoparticles were visualised using a
FEI Nova Nano-SEM at an electron acceleration voltage of
15 kV. To perform Nano-SEM imaging, bacterial samples
treated with different nanoparticles for 15 min at 1 mM
equivalent of Ag concentration were washed with deionised
water, drop-cast on glass cover slips, which were later mounted
onto an aluminium stub using double-sided carbon tape.
Before imaging, bacterial samples were coated with a 20 Å
thick platinum lm using a precision etching coating system
(Gatan model 682) at 20
rock angle, 40
per sec speed, 25 rpm
rotation and 5 keV beam current to minimise sample charging
during imaging.
Nanoscale

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Results and discussion
Scheme 1 illustrates different steps involved in designing anti-
bacterial nanocomposites used in the present study. Initially,
Ag+ ions are reduced using tyrosine amino acid under alkaline
conditions to form tyrosine-capped AgNPsY. As shown in
Scheme 1A, under alkaline conditions, the phenolic group of
tyrosine acts as a reducing functional group, leading to reduc-
tion of Ag+ ions to form AgNPsY, and during this reduction
process oxidised tyrosine molecules also act as capping agents
to stabilise AgNPsY in the aqueous solution.29,30 The pH of the
dialysed solution containing AgNPsY was found to be 8.6, which
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is well above the isoelectric point of tyrosine (pI  5.66), indi-
cating that the AgNPsY should have an overall negative surface
charge. This was further conrmed by the zeta potential value of
38.8 mV obtained for AgNPsY. In the next step, AgNPsY were
separately surface modied with PTA and PMA molecules to
obtain AgNPsY@PTA and AgNPsY@PMA, respectively as illustrated
in Scheme 1B. Since POMs are highly acidic in nature and
amongst the strongest heteropolyacids, as soon as PTA or PMA
ions were added to AgNPsY, the pH of these solutions instantly
dropped to ca. 2.0–2.5. Since these pH values are signicantly
lower than the pI of tyrosine, it is expected that the surface of
AgNPsY switches to a positively charged state due to the
protonation of tyrosine amine groups at these pH values, which
enables highly negatively charged POM ions to bind electro-
statically to AgNPsY, resulting in AgNPsY@PTA and AgNPsY@PMA.
Notably, aer extensive dialysis of these solutions, the pH of
AgNPsY@PTA and AgNPsY@PMA increased to 3.6 and 4.5, respec-
tively, which is still signicantly below the pI of tyrosine. This
suggests that even aer extensive dialysis, more than 99% of
tyrosine amine groups remain in protonated form, thus
providing sites for strong electrostatic interaction with POMs.
Notably, due to highly acidic/anionic nature of POMs, the
overall charge on POM-modied nanoparticles still remained
negative, with zeta potential values corresponding to 35.0 and
38.8 mV in the case of AgNPsY@PTA and AgNPsY@PMA, respec-
tively, in deionised water. This suggests that POMs form a
strong corona around AgNPsY in deionised water. Since the
presence of salts in bacterial growth medium can potentially
affect the stability of the nanoparticle corona, zeta potential
measurements on AgNPsY, AgNPsY@PTA and AgNPsY@PMA were
Fig. 1 UV-visible absorbance spectra of pristine PTA, pristine PMA and tyrosine-
reduced silver nanoparticles (AgNPsY) before and after their surface modification
with POMs.
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also performed by transferring these particles to the LB
medium, which revealed the values of 40.4, 36.1 and
40.7 mV. A subtle increase in the zeta potential values aer
transferring particles from deionised water (pH 6.4) to LB broth
(pH 7.2) suggests that the surface corona of these nanomaterials
remains stable under physiological conditions.
Illustrated in Fig. 1 are the UV-visible absorbance spectra of
AgNPsY and their POM composites. Pristine AgNPsY showed an
SPR band with maxima at 420 nm corresponding to metallic Ag,
along with a shoulder feature at 280 nm due to molecular
transitions within Ag-bound tyrosine molecules. Surface modi-
cation of AgNPsY with POMs did not result in any signicant
spectral shis in the Ag SPR feature, however additional
features due to surface-bound POM molecules were observed.
Additionally, post-POM modication, the POM features in
AgNPs showed blue shis due to the dielectric environment of
the Ag surface, such that a 255* nm feature in pristine PTA was
shied to 250 nm in AgNPsY@PTA, and a 215* nm feature in
pristine PMA shied to 205 nm in AgNPsY@PMA. Occurrence of
POM absorbance bands along with their blue shis conrms
the formation of a strong anionic POM corona around AgNPsY.
TEM images and particle size histograms corresponding to
modied AgNPs are presented in Fig. 2. AgNPsY obtained using
tyrosine as a reducing and capping agent are spherical in shape
with an average diameter of 18.2 nm with 1.9 nm standard
deviation. Aer surface modication of these AgNPsY with PTA
and PMA, the average diameter of resulted AgNPsY@PTA and
AgNPsY@PMA nanoparticles increased to 32.5 and 30.4 nm,
respectively, with a SD of 5.5 nm in both cases. This further
conrms the formation of a stable POM surface corona in
AgNPsY@POMs composites.
Since POMs are crystalline solids, the presence of POMs on
the surface of AgNPsY was evaluated using XRD analysis, which
Fig. 2 TEM images, mean particle diameters along with standard deviation and
particle size distribution histograms corresponding to (A) AgNPsY, (B) AgNPsY@PTA
and (C) AgNPsY@PMA. In TEM images, scale bars correspond to 50 nm.
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revealed the presence of either PTA or PMA molecules on the
surface of AgNPsY (Fig. S1†). Additionally, FTIR spectroscopy
was employed to provide evidence that the tyrosine and POMs
used for surface modication of AgNPs are stable and present
on the surface of nanoparticles (Fig. 3). Carbonyl stretching
vibration from the carboxylate ion in tyrosine shis from 1607
cm1 in the case of pristine tyrosine to 1656 cm1 in AgNPsY
(Fig. 3A). As shown in Scheme 1, this shi may be attributed to
formation of a quinone type structure on the surface of AgNPsY
due to oxidation of the phenolic group in tyrosine while tyrosine
molecules act as a reducing agent for Ag+ ions.29,31 Binding
modes of POMs to AgNPsY were further analysed by comparing
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the FTIR spectra originating from pristine POM molecules and
AgNPsY (Fig. 3B and C). Keggin structures of POMs
(PTA–H3PW12O40 or PMA–H3PMo12O40) consist of a cage of
either tungsten (W) or molybdenum (Mo) atoms linked by
oxygen atoms with the phosphorus atom at the center of the
tetrahedra32,33 and within this Keggin structure, oxygen atoms
form distinct bonds in both cases of PTA (P–O, W–O–W, and
W]O) and PMA (P–O, Mo–O–Mo, and Mo]O), which have
distinguishable infrared signatures. P–O (1075 cm1 and
1058 cm1 for PTA and PMA, respectively) corresponds to an
asymmetric stretching vibrational mode between phosphorus
and oxygen atoms at the center of the Keggin structure; W–O–W
or Mo–O–Mo (874 cm1 and 864 cm1 for PTA and PMA,
respectively) corresponds to bending vibrational modes of
oxygen atoms that form a bridge between the two tungsten or
molybdenum atoms within the Keggin structure; and W]O or
Mo]O (972 cm1 and 950 cm1 for PTA and PMA, respectively)
corresponds to the asymmetric stretching of terminal oxygen
atoms. The strong binding of POM molecules to the surface of
AgNPsY is clearly evident from the shis observed in the
vibrational modes of POM molecules in AgNPsY@PTA and
AgNPsY@PMA, which are summarised in the ESI (Table S1†).
These vibrational shis are in agreement with previously
reported salt-like complexation of amino acid–POM struc-
tures.34,35 Hence, FTIR spectroscopy provides strong conrma-
tion that the tyrosine amino acid and POMs used for surface
modication of AgNPs provide a stable corona around
nanoparticles.
The presence of colloidal silver (Ag0) in all the samples, as
well as the presence of PTA or PMA (W or Mo, respectively), was
further conrmed by XPS analysis. The core level binding
Fig. 3 FTIR spectra of [A] tyrosine (curve 1) and AgNPsY (curve 2); [B] PTA (curve
1) and AgNPsY@PTA (curve 2); and [C] PMA (curve 1) and AgNPsY@PMA (curve 2).
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energies (BEs) of C1s, N1s, O1s, Ag3d, W4f and Mo4f obtained
from XPS in different samples correlate well with the literature
values, conrming that AgNPs were coated with POMs in the
respective samples.29,35 For instance, the core level XPS spec-
trum of Ag3d originated from AgNPsY could be decomposed
into two chemically distinctive spin–orbit pairs (Fig. 4A). These
two distinct BEs appeared at 368 and 371.5 eV, respectively,
wherein, the lower BE component is attributed to the metallic
Ag nanocore, while the low-intensity higher BE component
arises from the unreduced metal ions, indicating that a very
small fraction of Ag+ ions remain bound to the surface of
AgNPsY. As illustrated in Fig. 4B, the C1s core level spectra
recorded from the surface of AgNPsY could be deconvoluted into
three chemically distinct components at 285, 286.6 and
288.4 eV. The higher BE component observed at 288.4 eV can be
assigned to the carboxylate carbon and the 286.6 eV BE peak is
attributed to the C–N and C–O species present in tyrosine amino
acid bound to the nanoparticle surface. Moreover, W4f (Fig. 4C
for AgNPsY@PTA) and Mo4f (Fig. 4D for AgNPsY@PMA) signals
obtained from PTA and PMA modied nanocomposites
conrmed the presence of POMs in respective samples. Thus,
the XPS analysis conrmed that the employed surface modi-
cation strategy produced effective and stable surface coronas
around AgNPsY to produce composite nanomaterials.
In depth physicochemical characterisation of POM surface
modied AgNPs established clearly that our synthesis strategy
resulted in controlled systems with a stable POM surface
corona. Since AgNPs as well as POM molecules employed here
are independently considered antibacterial, the coexistence of
these systems may have the potential to show enhanced anti-
bacterial performance. Therefore, antibacterial abilities of these
composite nanoparticulate systems were explored in a dose-
dependent manner against Gram negative bacterium E. coli.
The amount of Ag present in extensively dialysed samples was
determined by AAS, whereas the amount of W (from PTA) and
Mo (from PMA) present in these samples was assessed by
ICPMS. To perform antibacterial tests, the amount of Ag was
Fig. 4 Core level XPS spectra of (A) Ag 3d and (B) C1s from AgNPsY, and those of
(C) W 4f in AgNPsY@PTA and (D) Mo 4f in AgNPsY@PMA.
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than those present in AgNPsY@PMA and AgNPsY@PTA, respec-

tively. Such a signicant increase in the antibacterial activity of
POM-functionalised Ag nanocomposites can be attributed to
the synergistic effect due to the presence of two antibacterial
materials in a single system, while AgNPsY also act as a POM
stabiliser and carrier inside the bacterial cells. Notably, it was
recently illustrated that silver has the potential to disrupt
numerous cellular processes in Gram-negative bacteria
including disulphide bond formation, metabolism and iron
homeostasis, which ultimately leads to higher amounts of
reactive oxygen species production and enhanced membrane
permeability.36,37 These promising features of Ag-based ‘antibi-
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Fig. 5 Antibacterial activity of (A) surface-functionalised nanomaterials and (B) otics’ are likely to potentiate the action of POMs aer AgN-
pristine PMA and PTA molecules. PsY@POM uptake by the bacterial cell, resulting in high
synergistic biological action demonstrated by AgNPsY@POM
nanocomposites.
kept constant in all the samples so that the effect of POM The mode of interaction of these nanocomposites with E. coli
surface corona could be compared. Relative concentrations of cells was further conrmed by monitoring the morphological
Ag and W/Mo present on the surface of modied AgNPs used for changes and cellular disruption using Nano-SEM (Fig. 6). The
antibacterial studies are shown in the ESI (Table S2†). Fig. 5A SEM images of bacteria without treatment show an intact cell
compares the antimicrobial performance of AgNPsY, architecture with rod-like appearance and a size of ca. 3 mm 
AgNPsY@PTA and AgNPsY@PMA composites. As expected, the 1 mm (Fig. 6A). However treatment of bacteria for 15 min with
toxicity of pristine AgNPsY was found to increase in a dose- 10 mM equivalent of Ag present in AgNPsY, AgNPsY@PTA and
dependent manner with 1 mM equivalent of Ag causing ca. 36% AgNPsY@PMA (Fig. 6B–D, respectively) revealed distinct
bacterial cell death that increased to over ca. 94% at 10 mM Ag morphological changes indicative of signicant damage to the
concentration. AgNPsY@POM showed signicantly better anti- cellular integrity. The higher level of physical damage caused to
microbial performance over AgNPsY, wherein PTA-functional- the bacterial cells by POM-functionalised materials (Fig. 6C–D)
ised materials showed higher activity over PMA-functionalised in comparison to those treated only with AgNPsY (Fig. 6B) is also
materials. Since high concentrations of pristine AgNPsY (5 and evident from SEM micrographs. For instance, pristine AgNPsY
10 mM) are themselves highly toxic, the degree of increase in caused signicant roughening of the bacterial cell, along with
antimicrobial performance of AgNPsY@POMs over AgNPsY some disruption of bacterial cell wall and membrane (Fig. 6B).
nanoparticles is more comparable from the lower Ag dosages Conversely, in comparison to AgNPsY, AgNPsY@PTA (Fig. 6C) and
(1 and 2 mM). For instance, at a xed Ag dose of 1 mM, AgNPsY AgNPsY@PMA (Fig. 6D) treatment resulted in high levels of
cause ca. 36% bacterial cell death, which increases up to 66% physical damage to bacterial cells, wherein complete
and 85%, respectively, in the case of AgNPsY@PMA and
AgNPsY@PTA, respectively. It is however noteworthy that loading
of PMA molecules is less in AgNPsY@PMA than that of PTA in
AgNPsY@PTA (Table S2†), which may be attributed to the lower
antibacterial performance of the former. To further validate
whether the presence of Ag and POMs within a single system
acts synergistically to enhance the antibacterial performance of
these nanocomposites, bacterial cells were independently
exposed to pristine POM molecules in a concentration-depen-
dent manner (Fig. 5B). These control experiments revealed that
pristine POM molecules had signicantly lower antibacterial
potential than AgNPsY@POM even at much higher concentra-
tions than those present in AgNPsY@POM. For instance, while
Table S2† shows that the amount of POM corona present in
1 mM AgNPsY is 0.198 mM PTA and 0.042 mM PMA, Fig. 5B
depicts that 0.5 mM PTA and PMA cause only 8% and 12%
bacterial cell death. Conversely, the comparison of the anti-
bacterial proles of AgNPsY, AgNPsY@PMA and AgNPsY@PTA at
1 mM Ag concentration in Fig. 5A indicates that the presence of
0.198 mM PTA and 0.042 mM PMA in 1 mM AgNPsY coronas leads
Fig. 6 SEM micrographs of E. coli cells (A) before and (B–D) after treatment with
to more than 50% and 30% enhanced antibacterial perfor- (B) AgNPsY, (C) AgNPsY@PTA and (D) AgNPsY@PMA. Insets show the higher
mance, respectively, over AgNPsY. Notably, the above compared magnification images of the respective panels. Scale bars in the main figures and
0.5 mM pristine POM concentration is 12 and 2.5 times higher insets correspond to 5 mm and 500 nm, respectively.

Nanoscale This journal is ª The Royal Society of Chemistry 2013


Paper
disintegration of the bacterial cells is seen to such an extent that
the E. coli cells exhibited big holes in their morphology.
Therefore, from the antimicrobial experiments and SEM
imaging of E. coli cells, it is clear that modication of AgNPs
with a POM corona causes irreversible E. coli cell damage and
ultimately cell death by disruption of the integrity of these
bacterial cells. Since the POM corona on the surface of AgNPs
employs physical modes of action against E. coli by causing pore
formation, cell wall cleavage and cell lysis, it is likely that such
tailored nanomaterials may offer signicant opportunities to
control pathogenic bacteria by preventing them to develop
resistance.
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Furthermore, since the cell wall compositions of Gram
negative and Gram positive bacterial strains are signicantly
different, the antibacterial activity of these nanomaterials was
also evaluated against Gram positive bacterium S. albus. This
experiment was performed at a 2 mM Ag concentration, as this
nanoparticle concentration showed the highest inuence of
surface corona in the case of E. coli (Fig. 5A). The trend of the
antibacterial activity of AgNPsY, AgNPsY@PMA and AgNPsY@PTA
against S. albus was found to be similar to that in the case of E.
coli, however these nanomaterials showed less antibacterial
activity against the Gram positive S. albus, wherein the observed
cell death corresponded to 31%, 49% and 57%, respectively.
Additionally, the cytotoxicity of these nanomaterials was
evaluated against human PC3 epithelial cells in a concentra-
tion-dependent manner. Interestingly, all the nanoparticles
including AgNPsY, AgNPsY@PMA and AgNPsY@PTA did not show
either any signicant toxicity (Fig. S2†) or change in PC3 cell
morphology and cell density up to the highest tested Ag
concentration of 10 mM (Fig. S3†). Although the reasons for the
selective toxicity of these materials towards the tested bacterial
strains are not entirely clear at this stage, it has been demon-
strated that the toxicity of Ag to human cells is considerably
lower than to bacteria.5 Nevertheless, the outcomes of the
current study show that AgNPs with PTA and PMA surface
coronas have the potential to offer bacterial targetability char-
acteristics under in vivo settings.
Conclusions
This study demonstrates the important role that organic surface
coronas surrounding nanoparticles may play towards control-
ling nanomaterial properties for biological applications. In a
vast majority of existing studies, the effect of nanoparticle
surface coronas on biological modes of action is oen neglected
or overlooked, which is now receiving considerable attention.
Due to this reason, nanomaterials of similar composition, size
and shapes, however those prepared using different synthesis
routes or in different laboratories, show different biological
proles. In the current study, we have employed a facile
approach to obtain a stable surface corona, wherein initially a
green approach involving zwitterionic amino acid tyrosine
allows synthesis of charge-switchable AgNPsY. These charge-
switchable tyrosine moieties further allow facile generation of a
stable POM surface corona, as shown for PTA and PMA in this
study. The signicant variability in the iso-electric points of
This journal is ª The Royal Society of Chemistry 2013
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Nanoscale
20 natural amino acids and their characteristic charge-switch-
ability feature is attractive for their employment as natural
building blocks for surface functionalisation. Therefore, the
proposed strategy offers signicant opportunities to tailor-
design a variety of organic coronas for a range of biological
applications. Moreover, the antibacterial activity test of POM-
functionalised AgNPsY reveals that the POM surface corona
plays a signicant role in causing a high degree of physical
damage to the bacterial cells, which is likely to offer opportu-
nities to control pathogenic bacteria by preventing them from
developing resistance. The ability of these surface-functional-
ised materials to cause specic toxicity against bacteria without
causing damage to the tested human epithelial PC3 cells may
offer opportunities to employ them for topical wound healing
applications, wherein the control of bacterial infections to allow
the growth of new epithelial cells is considered critically
important. The choice of an appropriate inorganic material and
POM molecules may further extend the application window of
these materials from antibacterial in the current case to anti-
viral, anticancer, etc. in the future.
Acknowledgements
HKD gratefully acknowledges Government of India for a
National Overseas Scholarship. VB acknowledges the Australian
Research Council (ARC) for the award of an APD Fellowship and
research support through the ARC Discovery (DP0988099,
DP110105125) and Linkage (LP100200859) grant schemes. VB
also acknowledges the generous support of the Ian Potter
Foundation for establishing a multimode spectroscopy facility
used in this study. Authors acknowledge the support of RMIT
Microscopy and Microanalysis Facility for technical assistance
and providing access to characterisation facilities. The assis-
tance of Mr. Paul Morrison for acquiring ICPMS data and Ms.
Zahra Homan for acquiring AAS data is duly acknowledged.
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This journal is ª The Royal Society of Chemistry 2013