L-Methionine based phenolic compound mediates unusual assembly of AgNPs and exerts efficient anti-biofilm effect

Article abstract of DOI:10.1039/c6ra06806e

2-(2-Hydroxybenzylamino)-4-(methylthio)butanoic acid (L) together with capping molecules, produced AgNPs with unusual nanostructures that showed efficient inhibition of biofilm growth. Nanoplates of AgNPs self-assemble into micron sized flower-petal like

Full text of DOI:10.1039/c6ra06806e

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ARTICLE
L-Methionine Based Phenolic Compound Mediate Unusual Assembly
of AgNPs and Exert Efficient anti-biofilm effect
V. Vinod Kumar,a Lowrence Rene Christena,b P. Praveen,a Meenakshi Sundaram Muthuraman,b
Received 00th January 20xx,
Accepted 00th January 20xx
Nagarajan Saisubramanianb* and Savarimuthu Philip Anthonya*
2-(2-hydroxybenzylamino)-4-(methylthio)butanoic acid (L) together with capping molecules, produced AgNPs with unusual
nanostructures that showed efficient inhibition of biofilm growth. Nanoplates of AgNPs self-assemble into micron sized
flower-petal like structures in L-PSS-AgNPs whereas mesocrystals with visible voids from smaller nanoplates has formed in
L-SDS-AgNPs. L-PVA- and L-PVP-AgNPs showed polydispersed multi-shaped nanoparticles including nanoprisms, spheres
and plates. The structure-property studies indicate that slow reduction of Ag+ ions and thiophilic interaction of L is
responsible for unusual morphology and organization of AgNPs. Importantly L-capping molecules-AgNPs exhibited strong
biofilm inhibitory effect against four different bacteria, S. aureus, P. aeruginosa, B. subtilis and E. coli of which, the former
two are classical colonizers in burn wounds. BIC (Biofilm inhibitory concentration) and MIC (Minimum inhibitory
concentration) studies further confirmed the true anti-biofilm effect AgNPs. Studies to unravel the mechanism of action
indicated that enhanced anti-biofilm effect could be attributed to altered membrane permeability and integrity caused by
L, which in turn curtails bacterial colonization as revealed by confocal imaging. Thus a simple amino acid modified phenolic
molecule exhibited interesting assembly of AgNPs with remarkable anti-biofilm activity, which holds promise for its
potential use in fabrication of wound healing dressing materials.
DOI: 10.1039/x0xx00000x
www.rsc.org/
as Extracellular Polymeric Substances (EPS) comprised of
polysaccharides, peptides, protein and DNA that provides
increased resistance against antimicrobial agents, disinfectants
and the immune attack. Biofilms on chronic wounds/ medical
devices serve as reservoirs for pathogens which results in
persistent/recurrent infections. Hence the development of new
biomaterials that could curtail biofilm formation on chronic
wounds / on the surface of implantable medical devices is
crucial.
The antimicrobial effect of silver (Ag) is known and has
been used for years in the medical field for antimicrobial
applications.7ꢀ9 Silver nanoparticles (AgNPs) being one of the
predominantly used nanoparticle is well known to display
antimicrobial effect against a wide range of bacteria and fungi
including drug resistant microbial strains due to its increased
surface area.10 AgNPs have been used for decades to treat burn
wounds and to filter microorganisms from water.11 The size,
shape, atomic composition and surface ligands of AgNPs can
modulate its antimicrobial activity.12ꢀ15 More specifically, the
surface ligand structure plays a significant role not only in
controlling the morphology and providing the stability to NPs,
but it also contributes towards augmenting the biological
activity by promoting its adsorption or diffusivity into the
bacterial membrane.16 For example, AgNPs stabilized with
cationic polymers lead to enhanced antibacterial effect due to
the increased permeability.17 Polymer capped AgNPs can be
potentially employed as wound dressing material to prevent
biofilm formation in infected wounds. A recent study has used
AgNPs loaded silk fibroin/carboxy methyl chitosan
Introduction
Exploiting the unique physiochemical properties of metal
nanoparticles for biological applications such as diagnostic and
therapeutic studies have been the focus of material science in
recent years.1ꢀ3 Infectious diseases especially the ones caused by
drug resistant pathogens pose a greater threat to public health
worldwide and hence there is an urgent need to curtail it. Every
year in US alone, more than 2 million people have been
reported to be infected with drug resistant microbes, which
accounts for at least 23000 deaths annually.4 Since microbes
resort to biofilm, instead of planktonic mode of growth, in
many chronic infections and as biofilms, they display 100 to
1000 fold enhanced resistance to antimicrobial agents, of late
intensive research effort has been made to develop effective
antiꢀbiofilm agents. Presence of nutrients and solid substratum
are the prerequisites for biofilm formation by microbes. Most
microbes easily attach to any solid substratum and resort to
biofilm mode of growth.
enmeshed within a complex and heterogeneous matrix known
5ꢀ6
Microbial cells in a biofilms are
a.Department of Chemistry, School of Chemical & Biotechnology, SASTRA
University, Thanjavur-613401, Tamil Nadu, India. E-mail:
philip@biotech.sastra.edu.
b.Department of Biotechnology, School of Chemical & Biotechnology, SASTRA
University, Thanjavur – 613401, Tamil Nadu, INDIA. E-mail: sai@scbt.sastra.edu.
Electronic Supplementary Information (ESI) available: [Absorption spectra, HR-TEM
images of AgNPs with different capping agents, antibiofilm effects of capping agent
alone and confocal fluorescent microscopic images of antibacterial effect of
AgNO3. See DOI: 10.1039/x0xx00000x
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nanocomposite and proved that it exerted superior anti salicylaldehyde (1 equivalent, 10 ml) was added under vigorous
Pseudomonas effect relative to commercially available stirring at room temperature. The solution has turned
AQUACEL®; Ag, which could be attributed to improved immediately bright yellow colour and the stirring was
moisture absorption, retention and water vapor transmission continued for another 30 min. Thenthe reaction mixture was
rate contributed by CMC.18 Similarly PVPꢀ carrageenan cooled in iceꢀbath and NaBH4 (1.5 equivalent) was added in
hydrogel impregnated with 100 ppm of AgNPs retained more portionꢀwise. The bright yellow colour was slowly disappeared
fluid and released ~ 20 % of silver ions / 100 cm2 in 24 h and and neutralization of the reaction mixture produced precipitates
was found to completely mitigate wound colonizers S.aureus, of 1. The precipitate was filtered, washed with cold ethanol and
P.aeruginosa, E.coli and Candida albicans 19
different degree of PVA hydrolysis on the silver ion release 1. Yield = 85 %. H NMR (d6ꢀDMSO)
.
The effect of dried in vacuum.
1
δ
7.14ꢀ7.25 (m, 2H),
from PVAꢀAgNPs hydrogels and its antiꢀbiofilm effect against 6.75ꢀ6.84 (m, 2H), 3.85ꢀ3.99 (q, 2H), 3.25ꢀ3.29 (t, 1H), 2.50ꢀ
P.aeruginosa and S.aureus have also been explored.20 These 2.65 (m, 2H), 2.02 (s, 3H), 1.85ꢀ1.95 (m, 2H). 13C NMR
studies clearly suggest that the surface ligands indeed play a (CDCl3)
significant role in increasing or modulating the antibacterial 115.46, 59.60, 46.41, 30.44, 29.75, 14.44. C12H17NO3S
properties of AgNPs. (255.33): calcd. C 56.45, H 6.71, N 5.49; found C 56.70, H
Natural phenolic compounds have displayed versatile 6.48, N 5.62. m/z (LCꢀMS) 256.00 (M+H).
biological properties encompassing antibacterial, antiviral,
δ 171.11, 156.39, 130.39, 129.37, 120.94, 118.78,
S
YNTHESIS OF 1-AGNPS WITH CAPPING AGENTS
antifungal and antiꢀcarcinogenic properties.21,22 One striking Aqueous solution of 1 (0.1 M), AgNO3 (0.1 M) and capping
example is the resistance of stilbenes, grape phenols, to fungal molecules (1 wt % for PSS, PVA and PVP whereas 0.5 M for
colonization.23 Furthermore, the easy ionization properties of SDS) were prepared separately. 1 was dissolved using 1:1
phenols have been successfully exploited for the synthesis of equivalent of NaOH. Then 5 ml of capping molecules and 5 ml
AgNPs.24 The guest interacting properties of phenols have been AgNO3 was mixed together. 5 ml of 1 was added to this
exploited to fabricate surface functionalized AgNPs for mixture with stirring at room temperature. The resulting
selective colorimetric sensing of metal cations and anions in solution was stirred for 30 min and was allowed to stand at
aqueous solution.25ꢀ28 The phenolic molecules can act as room temperature for three days. The colorless solution slowly
reducing, stabilizing as well as surface functionalizing turned to pink to reddish brown depend on the capping
molecules. Herein, we report the unusual nanoꢀassembly of molecules that indicates the formation of AgNPs. The above
AgNPs
using
2ꢀ(2ꢀhydroxybenzylamino)ꢀ4ꢀ(methylthio) concentration for 1, AgNO3 and capping molecules were
butanoic acid (L), a methionine attached phenolic molecule as chosen after series experiments with different combinations.
reducing agent together with capping molecules poly(styerene Increasing the concentration of AgNO3 or 1 or reducing the
sulfonate (PSS), poly(vinyl alcohol) (PVA), poly(vinyl concentration of capping molecules lead to settling down of
pyrrolidone) (PVP) and sodium dodecylsulfate (SDS) that AgNPs slowly. The AgNPs synthesized using PSS, PVA, PVP
exhibited efficient growth inhibition of biofilm. The slow and SDS capping molecules are named as 1ꢀPSSꢀ, 1ꢀPVAꢀ, 1ꢀ
reduction of Ag+ ions and thiophilic interaction of L lead to the PVPꢀ and 1ꢀSDSꢀAgNPs.
formation of interesting nanoꢀassembly of AgNPs such as
flower petals and mesocrystals. Antiꢀbiofilm studies of AgNPs
C
HARACTERIZATION
were evaluated against four different bacteria viz., S. aureus, P. The UVꢀvisible measurements were performed using a Perkin
aeruginosa, B. subtilis and E. coli and efforts to discern Elmer model Lambda 1050, at the resolution of 1 nm. The size
mechanism of antibiofilm effect were undertaken. Based on our and morphology of AgNPs were investigated using High
results, we propose that Lꢀcapping agents – AgNPs could be Resolution Transmission Electron Microscopy (HRꢀTEM,
potentially employed in wound healing applications, to curtail JEOL JEMꢀ2100F). Samples for TEM measurements were
biofilms formed by drug resistant bacteria.
prepared by placing a drop of NPs solution on the graphite grid
and drying it in vacuum.
Experimental Section
ANTIBACTERIAL STUDIES
Methionine, cysteine, ethanol and NaOH were obtained from Minimum inhibitory concentrations (MIC) and Minimum
Ranbaxy fine chemicals. Salicylaldehyde, NaBH4, AgNO3, bactericidal concentration (MBC) for nanocomposites (1ꢀPSSꢀ
poly(vinyl alcohol) (PVA, MWt 40,000), sodium salt of AgNPs, 1ꢀPVAꢀAgNPs, 1ꢀPVPꢀAgNPs and 1ꢀSDSꢀAgNPs)
poly(styerene sulfonate) (PSS, MWt 40,000), sodium and various controls viz., 1, capping molecules (PSS, PVA,
dodecylsulfate (SDS) and poly(vinyl pyrrolidone) (PVP, MWt PVP and SDS) alone as well as 1 with capping molecules (1ꢀ
40,000) were obtained from SigmaꢀAldrich. MillꢀQ water was PSS, 1ꢀPVA, 1ꢀPVP and 1ꢀSDS) and silver nitrate was
used for all the experiments.
OF2-(2-HYDROXYBENZYLAMINO)-4- B.subtilis
BUTANOIC ACID (1)
P.aeruginosa and E.coli) as reported earlier.29
determined for representative gram positive (S.aureus and
S
YNTHESIS
)
and representative gram negative bacteria
(METHYLTHIO
)
(
Reduced Schiff base phenolic chelating ligand (1) was
BIOFILM INHIBITION ASSAY
synthesized by following the reported procedure.25 Typically Inhibition of biofilm formation by 1, capping molecules (SDS,
one equivalent of methionine was dissolved in 20 ml of water PVA, PVP and PSS)separately, 1 with capping molecules (1ꢀ
using 1:1 NaOH. To this solution, ethanol solution of PSS, 1ꢀPVA, 1ꢀPVP and 1ꢀSDS) and nanocomposites (1ꢀPSSꢀ
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AgNPs, 1ꢀPVAꢀAgNPs, 1ꢀPVPꢀAgNPs and 1ꢀSDSꢀAgNPs) computed as reported earlier. Suitable controls were
were determined and quantified by crystal violet staining as maintained.
reported earlier.30 Overnight grown cells of Pseudomonas
MEMBRANE DEPOLARIZATION STUDIES
aeruginosa, E.coli (Gram negative bacterium) and Bacillus The ability of the reducing agent 1 to perturb membrane
subtilis and Staphylococcus aureus (Gram positive bacterium) potential was discerned using the fluorophore DiSc3 [3,3′ꢀ
were diluted (1:100) in sterile Tryptic soy broth and inoculated Dipropylthiadicarbocyanine iodide]
as reported earlier.33
into micro titer plates containing increasing concentrations of DiSc3 partitions to lipid bilayer if the membrane potential is
polymeric nanomaterials as mentioned above in dilute (0.01X) unperturbed. When the membrane potential is perturbed, it
Tryptic Soy Broth, after 18ꢀ24 h the micro titer plates were partitions to the aqueous region wherein, its fluorescence is
washed with PBS to remove unbound cells, dried and stained enhanced. Briefly,midꢀlog phase cells were pelleted,
with 0.1% crystal violet for 15ꢀ20min.The plates were washed resuspended
thoroughly in PBS and dried in an oven at 60°C. Crystal violet hydroxyethyl)piperazinꢀ1ꢀyl]ethanesulfonic
in
5mM
HEPES
buffer
acid]
[2ꢀ[4ꢀ(2ꢀ
and
was extracted using 30% acetic acid for 15ꢀ20min and challenged with 1 at 0.5X MIC. After 30min of incubation, 2µl
absorbance was recorded at 595nm using a microplate reader of DiSc3 [3,3′ꢀDipropylthiadicarbocyanine iodide] was added
(BioRad, USA).
and the readings recorded for 5min (Ex at 622nm and Em at
670nm). Protonophore CCCP was used as a positive control.
CONFOCAL IMAGING
Based on the differential antiꢀbiofilm effect of PVP in ROS ASSAY
combination with 1, we chose PVP and its different derivatives To discern if
1
induced ROS production 2’ꢀ7’
as treatments to visualize antiꢀbiofilm effect by confocal dichlorofluorescin diacetate was used as
imaging. Live dead staining followed by confocal imaging on endogeneous ROS production was
a
probe and
measured
biofilms with and without treatment by PVP and its derivatives spectrofluorimetrically by acquiring fluorescence intensity as
were performed as reported earlier.31 Briefly, biofilms formed reported earlier.34 H2O2 was used as a positive control.
on the surface of cover slips placed inside sterile sixꢀwell plates Fluorescence intensity was normalized with respect growth of
containing 0.1X TSB media. Every 18 h, spent media was each culture.
replaced with fresh sterile media. For sampling, slides were
MOTILITY ASSAYS
removed on Day 2, washed with sterile PBS to remove the Swimming, swarming and twitching motilities for E.coli and
nonꢀadherent cells and stained with a mixture of acridine P.aeruginosa were performed exactly as reported earlier.35
orange (0.2mg/ml) and propidium iodide (0.33mg/ml) and
Results and Discussion
imaged using Olympus FV 1000 confocal microscope with 63X
objective, at a numerical aperture 0.3. For acridine orange,
excitation was done using Multi Argon LASER and for
detecting fluorescence due to propidium iodide, excitation was
performed using Helium Neon LASER. Acridine orange stains
the nucleic acids of live cells which when excited emits green
fluorescence whereas, propidium iodide stains nucleic acids in
membrane compromised or dead cells. Upon excitation,
propidium iodide emits red fluorescence. Thus all viable cells
would appear green and all dead cells would appear red.
SYNTHESIS AND CHARACTERIZATION OF 1 AND AGNPS
L, a methionine amino acid based phenolic molecule, was
synthesized by Schiff base condensation of methionine with
salicylaldehyde followed by imine reduction using NaBH4. We
have been working with phenolic molecules as reducing,
stabilizing and surface functionalizing agents of AgNPs for
selective colorimetric sensing of metal cations and anions.26ꢀ28
The easy ionization potential of phenolic functionality can
reduce silver ions into AgNPs and chelating metal interacting
functionality could stabilize the NPs. Noble metal nanoparticles
such as Ag and AuNPs exhibit strong visible color due to
surface plasmon resonance (SPR) vibration.36 The linear
lipophilic side chain of L along with sulphur group that can
have preferential interactions with noble metal surface might be
useful in controlling AgNPs morphology and enhancing
biological effect. However, L that is expected to act as both
reducing and functionalizing agent did not stabilize AgNPs for
a long time. It produced only black precipitate (Fig. 1a). But
addition of L into AgNO3 in presence of capping molecules
PSS, PVA, PVP and SDS slowly produced stable transparent
colloidal AgNPs solution with different colors (Fig. 1a). It takes
three days to produce AgNPs solution with stable color. L with
other commonly used capping molecules such as polyethylene
glycol (PEG), chitosan and tweenꢀ80 has also been studied but
they also did not produce stable AgNPs. Absorption spectra of
LꢀAgNPs did not show any characteristic peak since it
precipitatedꢀout and confirm that L alone is incapable to
1-N-PHENYLNAPHTHYLAMINE (NPN)-ASSAY
Since 1 preferentially inhibits biofilms formed by gram
negative bacterium, we investigated if the antiꢀbiofilm effect of
1could be due to altered outer membrane permeability. To
assess the extent of outer membrane damage in Pseudomonas
aeruginosa and E.coli (gram negative bacteria) caused by 1, we
performed NPN based membrane permeability assay as
reported earlier.32 NPN exhibits enhanced fluorescence in
phospholipid environment. Typically, Outer Membrane (OM)
of gram negative bacteria prevents the access for hydrophobic
molecules as it contains LPS, increased fluorescence indicates
enhanced OM permeability. Briefly, cells were grown to midꢀ
log phase collected and washed with 5mM HEPES buffer
containing 0.2% glucose at pH 7.5 and resuspended in an equal
volume of the same buffer. NPN was added at a concentration
of 0.5 mM, this was immediately followed by addition of 1 in
increasing concentrations. Fluorescence due to NPN was
measured (Ex350 and Em 420 nm) and NPN uptake factor was
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stabilize AgNPs. The orange pink solution of LꢀPSSꢀAgNPs noted that the formation of flower petal microꢀstructures has
showed absorption at 460 nm whereas LꢀPVAꢀAgNPs (reddish
been observed over the whole sample (Fig. S2). The thin plates
with clear edges form the petal morphologies (Fig. 2b, S2). Lꢀ
PVAꢀAgNPs and LꢀPVPꢀAgNPs showed the formation of
polydispersed AgNPs with diverse morphologies including
triangular prisms, nanorods, spherical NPs and nanoplates (Fig.
2c,d, 3a,b, S3 and S4). TEM images of LꢀSDSꢀAgNPs showed
the formation of nanospheres (200 to 300 nm), which in turn is
comprised of a dense assembly of the primary Ag nanoplates,
with recognizable voids and boundaries between the particles
(Fig. 3c,d and S5). The individual AgNPs are likely to have
length of 20 nm and a diameter of 5ꢀ10 nm. Thus, simple
phenolic chelating ligand, L, in presence of capping agents
reduces Ag+ ions into AgNPs with different morphologies
preferentially nanoplates that further selfꢀassemble into unusual
Fig. 1. (a) Digital images and (b) absorption spectra of AgNPs synthesized using L
without and with different capping molecules (PSS, PVA, PVP and SDS).
brown) showed absorption at 518 nm (Fig. 1b). The pink L-PVP-
AgNPs solution showed broad absorption spectrum with λmax
at 465 nm. L-SDS-AgNPs also showed orange pink color and
exhibited broad spectrum with two absorption peaks at 408
and 566 nm. This looks interesting since other amino acid
(valine, isoleucine, leucine) attached phenolic molecules that
produced stable AgNPs immediately without extra capping
agents showed absorption between 395 to 420 nm.25,26
Further, the presence of capping agents also did not alter the
AgNPs absorption significantly. It is noted that AgNPs prepared
using conventional sodium borohydride (NaBH4) reducing
agents in presence of PSS, PVA, PVP and SDS capping
molecules showed narrow absorption around 410 nm (Fig. S1).
The appearance of different color, broad and longer
wavelength absorption indicates the formation of
polydispersed AgNPs with different morphology that was
confirmed by HR-TEM studies (Fig. 2,3). Typical spherical
AgNPs between 5 to 50 nm commonly show yellow color with
absorption range between 390 - 440 nm.36
and unique nanostructures. This could be attributed to the slow
Fig. 2. HR-TEM images of (a, b) L-PSS-AgNPs and (c, d) L-PVA-AgNPs. The inset in (a)
shows the nanoplates of AgNPs.
evolution of AgNPs by weak reducing effect of (L) and
competing preferential interaction of sulphur group of L with
AgNPs. It is likely that the structure of nanocomposites could
be further aided by the interaction of Lꢀ AgNPs with different
stabilizing agent (PSS, PVA, PVP and SDS). It is noted that
AgNPs synthesized using NaBH4 reducing agent produced
yellow color instantaneously in presence of same four capping
molecules and resulted in the formation of spherical AgNPs
(Fig. S6). L alone without capping molecules failed to produce
stable AgNPs and produced only blackꢀgrey precipitates.
Furthermore, phenolic chelating ligands based on other amino
acids such as alanine and valine has been shown by our group
to produce only spherical AgNPs irrespective of whether
AgNPs were made with or without capping molecules.25 These
result clearly indicate that sulphur atom in the phenolic ligand
together with capping molecules plays significant role in
producing interesting morphologies and unusual assemblies. It
HRꢀTEM studies confirmed the formation of polydispersed
AgNPs with different morphologies that further selfꢀassembled
into unusual nanostructures in presence of capping ligands. Lꢀ
PSSꢀAgNPs revealed the formation of thin plates of AgNPs
with length up to 400 nm and width between 100 – 300 nm
(Fig. 2a,b). Interestingly, the thin plates selfꢀassemble into
micron sized flower petal structures in LꢀPSSꢀAgNPs. It is
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is noted that sulphur atom is known to have strong interaction 160 nM) to exert bactericidal effect against all four bacteria
with noble metal surface such as Ag and Au. Previous studies tested. On the other hand, PVA and PVP capped AgNPs
have shown that thiol based capping molecules can be used for prepared using NaBH4 were more effective in its bacteriostatic
effective controlling of metal NPs size and morphology by and bactericidal effect relative to SDS and PSS capped AgNPs
make use of thiophilic interaction with noble metal surface.37,38
(Table 1). SDS-AgNPs required 2-8 fold higher concentration
relative to its L containing counterpart to show growth
inhibition against the bacteria tested and in fact, PSS-AgNPs
did not show any significant antimicrobial effect up to 160 nM
tested. However, PVP and PVA capped AgNPs exhibited MIC
and MBC at much lower concentration (2-8 folds lower)
compared to L-PVP- and L-PVA-AgNPs.
Orga
S.aureus
B.subtilis
P.aeruginosa
E.coli
nism
MIC( MBC( MIC( MBC( MIC(
MBC(
nM)
MIC( MBC(
nM)
10
nM)
20
nM)
10
nM)
20
nM)
10
nM)
nM)
L-
PSS-
AgNP
s
20
80
20
40
L-
PVA-
AgNP
s
40
40
10
80
160
40
80
80
20
>160
>160
40
40
80
40
80
160
40
>160
>160
80
Fig. 3. HR-TEM images of (a, b) L-PVP-AgNPs and (c, d) L-SDS-AgNPs.
L-
PVP-
AgNP
s
Antibacterial effect
>160
80
We were interested to explore the effect of unusual
morphology and enhanced lipophilic character of L on the
L-
antimicrobial activity
of AgNPs. Minimum Inhibitory
SDS-
AgNP
s
Concentration (MIC) and Minimum Bactericidal Concentration
(MBC) studies were performed to evaluate the antimicrobial
activity of AgNPs (Table 1).
PSS-
AgNP >160
>160
20
>160
10
>160
20
>160
20
>160
40
>160
20
>160
40
L-AgNPs with all four capping molecules exhibited MIC in
the range of 10 nM to 80 nM against three different bacteria
(P. aeruginosa, B. subtilis, S. aureus) and with E. coli, MIC was
higher by 2 fold (20 to 160 nM). When compared with NaBH4
reduced AgNPs, L containing PSS and SDS AgNPs displayed an
enhanced antibacterial effect against all 4 bacteria tested,
probably due to its smaller size (nanoplates) and increased
surface area (Figure 3). Whereas L-containing PVA and PVP
AgNPs exhibited lower antibacterial effect, which could be
attributed to their larger size (Figure 2). Although the reasons
for increased MIC of L containing AgNPs against E. coli relative
to other bacteria was not quite evident, our results discussed
below reveal that L permeabilizes outer membrane of E.coli
more efficiently than it does for P.aeruginosa (Table 3), thus L
capped AgNPs are more likely to enter into E.coli. Despite
increased outer membrane permeability decreased MIC
probably indicates enhanced efflux of L containing AgNPs by E.
coli.
s
PVA-
AgNP
s
10
20
80
PVP-
AgNP
s
40
20
40
20
40
20
40
SDS-
AgNP
s
160
80
160
160
>160
80
160
Table 1. Antibacterial effect of L-capping agent-AgNPs and capping agent-AgNPs
synthesized using NaBH4 without L.
The size, morphology and surface capping molecules of
AgNPs are known to exert strong influence on the
antimicrobial effect.17,39 The capping molecules that provides
higher stability shows weaker antimicrobial activity due to
slow/no release of silver ions. Similarly smaller NPs or
morphologies with higher surface area tend to exhibit
enhanced antimicrobial effects. Hence strongly stabilized
spherical PSS- and SDS-AgNPs without L exhibited lowest
antimicrobial activity. However, L-PSS- and L-SDS-AgNPs, which
Bactericidal studies (MBC) revealed that L-PSS-AgNPs
exerted potent bactericidal effect from 20 to 40 nM. Whereas
L-SDS-AgNPs required a two to four fold higher concentration
to exert bactericidal effect. Both L-PVA- and L-PVP-AgNPs
required much higher (~ 4 fold) concentration of AgNPs (80 to
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showed the formation of nano plates of AgNPs that were which was effective at 80 nM (Fig. 4e-h). It is likely that among
further self-assembled into micron/nano sized flower petals AgNPs without L, PVP-AgNPs release more Ag ions, which
and mesocrystals, displayed enhanced antimicrobial effect. could probably account for enhanced antibiofilm effect. PSS-
Our observations imply that the formation of plate and PVA-AgNPs without L failed to induce discernible anti-
morphologies with higher surface area and self-assembly of biofilm effect in the entire concentration range tested.
nanoplates into micro/bigger nano-structures resulted in Furthermore, we observed that L-PSS-AgNPs were quite
enhanced antimicrobial activity. PVA- and PVP-AgNPs effective as an anti-biofilm agent against all four bacteria
displayed more potent antimicrobial effect relative to L-PVA- tested, whereas PSS-AgNPs without L did not exhibit any anti-
and L-PVP-AgNPs, despite forming poly dispersed biofilm effect. Hence it is plausible that the anti-biofilm effect
nanostructures with altogether different morphology. An of PSS-AgNPs could be mediated by L. The strong anti-biofilm
earlier study showed that NaBH4 reduced AgNPs capped with activity of L-PSS-AgNPs could be of potential value in wound
PVP displayed synergy with antibiotics and mitigated both healing applications. It has been shown in an earlier work that
gram positive and gram negative bacteria effectively relative PSS can favour osseo-integration of artificial ligaments with
to citrate and SDS capped AgNPs.39 In this study, synergy could bone tunnels, which is likely to result in enhanced healing
not be discerned by checkerboard method since L and most of following a surgical reconstruction.42
the capping molecules tested did not display any anti-bacterial
effect. Previous report showed that plant extract reduced
CTAB capped AgNPs displayed enhanced antibacterial effect
against methicillin resistant Staphylococcus aureus (MRSA)
relative to SDS stabilized AgNPs.40 It was also shown that PSS-
Ag nanocomposite exhibited good mobility of AgNPs and
exerted antibacterial effect against S. aureus and S.
epidermidis at higher concentration range (500 µg/ml to 2000
µg/ml) depending upon the bacterial inoculum density.41 Thus
polymers with varying stabilizing effect elicits differential
release of silver ions, which in turn affects the observed MIC
and MBC values. Overall among all AgNPs evaluated, L-PSS-
AgNPs was most effective at a lower concentration against all
four bacteria tested and in fact a strong antibacterial effect
against both S. aureus and P. aeruginosa, which are tough to
eradicate was exhibited by L-PSS-AgNPs at a much lower
concentration of 10-20 nM, implying good enhancement in the
antibacterial effect of AgNPs when L was used as a reducing
and functionalizing agent.
Anti-biofilm studies
The enhanced antibacterial effect of AgNPs when prepared
Fig. 4. Antibiofilm studies of (a) 1-PSS-AgNPs, (b) 1-PVA-AgNPs, (c) 1-PVP-AgNPs, (d) 1-
using L and synergistic effect if any of phenolic structure with
SDS-AgNPs, (e) PSS-AgNPs, (f) PVA-AgNPs, (g) PVP-AgNPs and (h) SDS-AgNPs. SA =
capping molecules prompted us to explore its anti-biofilm
activity. Crystal violet assay is a good indicator of biofilm
biomass. We evaluated the ability of L-PSS-, L-PVA-, L-PVP-
and L-SDS-AgNPs to prevent biofilm formation in two gram
positive bacteria (B. subtilis and S. aureus) and two gram
negative bacteria (P. aeruginosa and E. coli). Various controls
like PSS-, PVA-, PVP- and SDS-AgNPs prepared using NaBH4 as
the reducing agent, capping molecules alone, reducing agent
(L) alone and L with capping molecules were employed.
Staphylococcus aureus, BS = Bacillus subtilis , PA = Pseudomonas aeruginosa and EC= E.
Coli.
Comparing BIC (Biofilm inhibitory concentration) with MIC
would reveal whether antibiofilm effect of L-capping agent-
AgNPs arises due to antibacterial effect or they exhibit true
antibiofilm effect. We observed that BIC of L-PSS-, L-PVA-, L-
PVP- and L-SDS-AgNPs was lower than its MIC by 2-16 fold in
S.aureus, with rest of the bacteria, BIC of nanomaterials were
2-64 fold lower than the corresponding MIC, implying that L-
AgNPs with capping molecules exerted a true anti-biofilm
effect, which is independent of its anti-bacterial effect (Table
2). On comparison of BIC of AgNPs with and without L, in case
of S. aureus, P. aeruginosa and B. subtilis, AgNPs with L
displayed 4-32 fold lower BIC relative to AgNPs without L. But
with E. coli, BIC of PVA, PVP and SDS capped AgNPs with and
without L matched with each other. Among capping agents
only PSS capped AgNPs without L displayed a much higher BIC
for all 4 microorganisms tested. Overall our results imply that
AgNPs along with L and capping molecules exert lower BIC and
Our results indicate that L-PSS-, L-PVA-, L-PVP and L-SDS-
AgNPs exhibited significantly stronger anti-biofilm effect
relative to AgNPs prepared using sodium borohydride as
reducing agent (Fig. 4). Untreated biofilms were used as
control. Among AgNPs with L, SDS and PSS capped AgNPs were
most effective at lowest concentration of 10 nM followed by
PVA and PVP capped AgNPs, which exerted their anti-biofilm
effect at 20 nM (Fig. 4a-d). However, among AgNPs using
NaBH4, only PVP capped AgNPs displayed anti-biofilm effect in
the range of 20 to 40 nM, followed by SDS capped AgNPs,
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potent anti-biofilm effect against all 4 bacteria evaluated and formation by gram negative bacteria. Even at higher dilutions,
by virtue of exhibiting true antibiofilm effect, L containing significant biofilm inhibitory effect of L on gram negative
AgNPs can be effectively employed as a dressing material to bacteria was observed; however, L was unable to exert potent
anti-biofilm effect against gram positive bacteria in the entire
Organ
concentration range tested (Fig. 5). Whereas L-PSS-, L-PVA-, L-
S.aureus
B.subtilis
P.aeruginosa
E.coli
ism
PVP- and L-SDS-AgNPs exerted anti-biofilm effect against both
gram positive and gram negative bacteria at a much lower
concentration independent of AgNPs’ antibacterial effect (Fig.
MIC(
BIC(
nM)
MIC(
BIC(
nM)
MIC(
nM)
BIC(
nM)
MIC(
BIC(
nM)
nM)
nM)
nM)
4a-). This differential effect between
L and L-capping
L-PSS-
AgNP
s
molecule -AgNPs prompted us to evaluate a combination of L
with capping molecules but without AgNPs so as to
understand the synergistic effect or modulation of antibiofilm
effect of L by the capping molecule(s). Towards this end, anti-
10
10
10
2.5
2.5
10
40
5
20
5
L-
PVA-
AgNP
s
40
40
10
2.5
80
80
20
2.5
80
160
40
2.5
L-
PVP-
AgNP
s
20
5
2.5
2.5
80
40
2.5
10
2.5
5
L-
SDS-
AgNP
s
PSS-
AgNP
s
>160
10
>160
>160
5
>160
10
2.5
80
10
20
>160
20
>160
80
>160
20
>160
2.5
Fig. 5. Antibiofilm studies of L against gram positive and gram negative bacteria. SA =
PVA-
AgNP
s
Staphylococcus aureus, BS = Bacillus subtilis, PA = Pseudomonas aeruginosa and EC= E.
coli.
PVP-
AgNP
s
biofilm effect of L in combination with capping molecules was
evaluated and capping molecules alone were used as
controls. When polymers were tested alone, SDS exerted anti-
biofilm effect against all four bacteria. PVP inhibited E. coli
biofilms alone, whereas PSS caused a significant but not a
drastic inhibition of biofilms formed by E. coli and P.
aeruginosa. Quite unexpectedly, PVA alone at the highest
concentration tested inhibited biofilms formed by all four
bacteria (Fig. S7). Previous study has shown that SDS can
hinder and even disperse mature biofilms when it was used in
combination with sodium bicarbonate and sodium
metaperiodate.47 SDS loaded nanoporous polymer was also
shown to block bacterial attachment in short term and prevent
biofilm formation in long term.48 SDS due to its anionic and
amphipathic nature could possibly disrupt bacterial
communication through pili49 and nanotubes50 and thereby
prevent attachment and aggregation, which are prerequisites
for biofilm formation.48 PSS also exerted broad spectrum
antimicrobial effect against STD pathogens Chlamydia
20
20
20
20
20
2.5
SDS-
AgNP
s
80
20
80
160
80
80
2.5
curtail biofilm formation on wounds.
Table 2. Antibiofilm effects of L-capping agent-AgNPs and capping agent-AgNPs
synthesized using NaBH4 without L.
Reports on the anti-biofilm effect of silver from AgNPs
against diverse bacteria including clinically relevant P.
aeruginosa and S. aureus are prevalent.43-46 In all these cases
the anti-biofilm effect is predominantly due to the released
silver ions, which acted as a bacteriostatic/ bactericidal agent.
Surprisingly, we observed that with L-PSS-, L-PVA-, L-PVP- and
L- SDS-AgNPs, BIC is much lesser than its corresponding MIC;
implying anti-biofilm effect is not due to released Ag ions but
could be attributed to the capping molecules, methionine
based phenolic chelating ligand (L) or combination of both.
Hence, we explored the anti-biofilm roles of individual
components that make up the nanocomposite viz., L, capping
molecules and L with capping molecules, against the four
representative bacteria reported in this study.
trachomatis and Neisseria gonorrhoeae.51
A biopolymer
isolated from marine sponge has shown to exert anti-adhesive
effect against V. alginolyticus, V. harvei and V.
parahaemolyticus. Similarly, chitosan was found to hinder
adherence, biofilm formation and interfere with mature
biofilms in S. mutans compared to commercially available
mouth washes.52-54 Based on these studies, it is evident that
polymers do exhibit anti-biofilm effect. The anti-biofilm effect
of PVA against all four bacteria is a hitherto unreported
interesting observation and the detailed mechanism bio-film
As we observed a differential anti-biofilm effect between
PSS-, PVA-, PVP- and SDS-AgNPs with and without L, the role of
L in exerting anti-biofilm effect was explored. Interestingly at a
concentration of 160 nM, L completely mitigated biofilm
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growth inhibition would be explored in future studies combinations, only L-PVP displayed anti-biofilm effect against
separately.
all four bacteria tested. In addition, PVP-AgNPs without L also
displayed anti-biofilm effect against all bacteria albeit at higher
concentrations. Hence, the confocal live dead imaging of
biofilms formed by P. aeruginosa and S. aureus (classical
colonizers in wounds), were performed subsequent to the
following treatments viz., PVP alone, L-PVP combination, L-
PVP-AgNP. PVP-AgNPs with NaBH4 and AgNO3 solutions were
used as controls. For treatment with L alone, live dead staining
was performed only with gram negative bacteria since L was
effective only against gram negative bacteria hence, despite
being effective, its utility as a potential wound dressing
material is limited, because wounds are typically colonized by
polymicrobial communities comprised of both gram positive
and gram negative bacteria, which inevitably forms biofilms
and thus we evaluated the antibiofilm effect of L along with
capping molecule and AgNPs.
P. aeruginosa and S. aureus are classical colonizers of
chronic/burn wounds. With P. aeruginosa, treatment by L
alone, L-PVP and L-PVP-AgNPs resulted in impaired
colonization (Fig. 7). In contrast, treatment with controls, PVP-
AgNPs and AgNO3, caused increased proportion of dead cells,
which contributed to its anti-biofilm effect (Fig. S8, S9).
Similarly, L, L-PVP and L-PVP-AgNPs caused impaired
colonization with S. aureus, whereas PVP-AgNPs and AgNO3
exhibited enhanced killing, probably due to the released silver
ions (Fig. 8). Hence, the results from confocal imaging broadly
shows that with P. aeruginosa and S. aureus, the anti-biofilm
effect of individual components that make up the NPs and the
Fig. 6. Antibiofilm studies of (a) L-PSS, (b) L-PVP, (c) L-PVA and (d) L-SDS compositions.
SA = Staphylococcus aureus, BS = Bacillus subtilis , PA = Pseudomonas aeruginosa and
EC= E. coli. X axis label Conc (nM +%) signifies concentration of ligand (L) in nM in
combination with the concentration of the polymer in %.
Among the combination of L with PSS, PVA, PVP and SDS
capping molecules, only L-PVP exerted anti-biofilm effect
against all the four bacteria tested (Fig. 6). PVP alone did not
display significant anti-biofilm effect (Fig. S7) and L was
effective only against gram negative bacteria. The phenolic
ligand L in combination with PVP was able to effectively inhibit
biofilms formed by all four bacteria (Fig 6). So it is evident that
L in combination with PVP modulates anti-biofilm activity of
PVP. Whereas when used in combination with other three
capping molecules (PVA, PSS and SDS), L could not retain its
anti-biofilm effect against P. aeruginosa. In fact L-PSS inhibited
only E.coli and L-SDS and L-PVA lost their biofilm inhibitory
effect against both P.aeruginosa and E.coli, but was effective
against B.subtilis. Thus, our results imply that among capping
agents, SDS and PVA alone at higher concentration and among
combination of L with capping agents L-PVP at slightly higher
concentrations and from L- -capping agent – AgNPs, L-PSS- and
L-SDS-AgNPs at lower concentrations were effective in
preventing biofilm formation by all four bacteria tested.
Although polymer alone and polymer in combination with L
itself displayed effective antibiofilm effect albeit at higher
concentration, L containing polymer capped AgNPs were made
because, AgNPs have been shown in many earlier studies to
heal chronic wounds and polymer capped AgNPs that release
Ag in a sustained manner have been proven as a good wound
dressing material.55 This is supported by the earlier report
which showed that biological dressings usually lead to wound
deepening and chronic wound formation in burn wounds,
which can be abrogated by impregnating dressing materials
with AgNPs.21
Fig. 7. Live-dead staining and confocal imaging of P.aeruginosa treated with (a) L, (b)
PVP, (c) L-PVP and (d) L-PVP-AgNPs. P. aeruginosa was grown for 3 days on the surface
of a glass slide in 0.1 X nutrient broth and tryptic soy broth respectively, stained with
acridine orange and propidium iodide and imaged using confocal microscopy.
L-PVP-AgNPs can be predominantly attributed to impaired
colonization, which correlates well with the observation of
BIC< MIC and also with the fact that the anti-biofilm effect of
L-PSS-, L-PVA-, L-PVP- and L-SDS-AgNPs is true anti-biofilm
effect, which is independent of its antibacterial effect due to
released silver ions.
Mechanism of action of L
In order to gain insight on the mechanism of anti-biofilm effect
exerted by L, different studies such as membrane permeability,
membrane potential, membrane integrity, ROS generation and
motility assays were performed.
In order to understand whether the observed anti-biofilm
effect was due to reduced viability or impaired colonization,
we performed confocal live/dead imaging of a representative
L-AgNPs with appropriate controls. L-PVP-AgNPs was chosen
for confocal imaging because among L and capping molecules
NPN was used to assess membrane permeability. NPN
shows enhanced fluorescence in lipid environment compared
to aqueous environment. If the test compound affects
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membrane permeability, NPN will partition to lipid quantified by spectrofluorimetry. Our observations showed
environment resulting in enhanced fluorescence. Since L that only in case of P. aeruginosa, L caused 50% increase in
caused anti-biofilm effect exclusively against gram negative endogenous ROS relative to untreated cells, which was
bacteria, the effect of L on the outer membrane (OM) incidentally 50 % lower than ROS formed due to treatment
permeability in gram negative bacteria was explored. The with hydrogen peroxide. Interestingly the fluorescence
results showed that treatment with L induced a significant obtained in P. aeruginosa with hydrogen peroxide treatment
increase in NPN uptake factor of 7.8 for P. aeruginosa and 8.77 was much lower compared to other cells, even though the
for E.coli (Table S1). A high NPN uptake factor implies that the reasons are unclear, fluorescence quenching/protection
OM of gram negative bacteria is permeablized by L. Thus the against oxidative stress by bacterial pigments might be
observed decreased colonization of bacteria especially with P. responsible for this observation. An earlier study has shown
aeruginosa in confocal imaging by L could be due to the that Staphyloxanthin protects S.aureus against oxidative
increased permeability that might interfere with intracellular stress.57 In all other cases L did not cause any difference in ROS
accumulation of quorum sensing (QS) molecule, thereby production relative to untreated cells and in fact it appeared
hindering biofilm formation. A recent study has shown that that L quenched ROS production in E. coli, B. subtilis and S.
myco-fabricated AgNPs inhibited quorum sensing, secretion of aureus. Membrane potential remained unperturbed due to L
virulence factors and biofilm formation in P. aeruginosa.56
treatment and motility also remained unaffected due to L
treatment (data not shown) revealing that altered membrane
permeability and membrane integrity is likely to account for
anti-biofilm effect of L.
Conclusion
Bacterial biofilms serve as a source of recurrent/ persistent
infections particularly with respect to infections on chronic
wounds and biofilms formed on implantable medical devices.
Due to multifarious factors like EPS, heterogeneity,
degradation, sequestration, slow growth phenotype etc.,
biofilms display antimicrobial resistance and are highly
recalcitrant. By their mere presence and secretion of virulence
factors, biofilms trigger a persistent inflammatory response,
which significantly impairs wound healing. As biofilms are
tough to eradicate, constant demand for non- toxic
biomaterials as wound dressing materials with good anti-
biofilm properties continues unabated. Our study showed that
methionine phenolic chelating ligand (L) produced nanoplates
of AgNPs with unusual assembly of nanostructures that
resulted in the strong enhancement of anti-biofilm activity of
polymer capped AgNPs. The effective inhibition of biofilms at
lower concentration (BIC, Biofilm inhibitory concentration)
compared to antimicrobial activity (MIC, Minimum inhibitory
concentration) suggested the true anti-biofilm effect displayed
by L-capping agent-AgNPs. Furthermore, L-PVP-AgNPs also
exhibited stronger biofilm inhibitory effect against all four
bacteria relative to the individual components as well as
AgNPs without L. Studies to unravel the mechanism of action
indicate that the increased anti-biofilm activity of L-AgNPs-
capping agents could be due to altered membrane
permeability and integrity caused by L. Thus a simple amino
acid based phenolic chelating ligand led to usual assembly
AgNPs and resulted in enhanced anti-biofilm activity which
could be potentially exploited as a wound dressing material for
wound healing applications.
Fig.8. Live-dead staining and confocal imaging of S. aureus treated with (a) L, (b) PVP,
(c) L-PVP and (d) L-PVP-AgNPs. S. aureus was grown for 3 days on the surface of a glass
slide in 0.1 X nutrient broth and tryptic soy broth respectively, stained with acridine
orange and propidium iodide and imaged using confocal microscopy.
Membrane integrity was evaluated by quantifying release of
protein (A280) and release of nucleic acids (A260) due to
treatment with L and Triton X 100 (positive control), untreated
bacterial cells were used as control. Relative to untreated E.
coli cells, significant protein leakage (A280 of 0.368 Vs 0.819)
was observed in L treated cells, which was equivalent to ~ 1/2
of protein released due to treatment with Triton X 100 (A280
of 1.815). P. aeruginosa also showed a similar trend with L
causing increased protein leakage relative to untreated cells
(A280 of 0.304 vs 0.709), whereas Triton X 100 treatment
caused ~ 3 fold increase in protein release (A280 of 1.994).
Even with nucleic acids, significant leakage was observed due
to L treatment in both E. coli (A260 of untreated 0.34; 1
treated 0.54; Triton X treated 1.94) and P. aeruginosa (A260 of
untreated 0.348; 1 treated 0.509 and Triton X 100 treated
1.203). Thus it is likely that L exerts its anti-biofilm effect in
gram negative bacteria by altering membrane permeability
which concomitantly results in leakage of protein and nucleic
acid from the bacterial cells.
The ability of L to induce endogenous ROS production in all
four bacteria were explored by 2′,7′ dichlorofluorescin
diacetate (DCFH- DA) method as reported earlier.34 2′,7′
dichlorofluorescin (DCFH) is considered as a good indicator of
overall oxidative status of the cell.57 Hydrophobic non
fluorescent DCFH-DA penetrates into the cell where it is
cleaved by esterases and oxidized by cellular ROS to
fluorescent 2,7-dichlorofluorescein (DCF) that can be
Acknowledgement
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Financial support from the Department of Science and
Technology, New Delhi, India (DST Fast Track scheme no.
SR/FT/CS-03/2011(G) and DST–FIST Grant no: SR/FST/ETI-
331/2013) is acknowledged with gratitude. We thank CRF,
SASTRA University for providing infrastructural support in the
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L-Methionine Based Phenolic Compound Mediate unusual Assembly of
AgNPs and Exert Efficient anti-biofilm effect
Methionine based phenolic chelating ligand together with PSS, PVA, PVP and SDS capping
agents produced unusual self-assembly of AgNPs and strong anti-biofilm effect against four
different bacteria including S.aureus and P.aeruginosa, which are classical colonizers in burn
wounds, in presence of PSS, PVA, PVP and SDS capping agents.