In situ synthesized Ag nanoparticle in self-assemblies of amino acid based amphiphilic hydrogelators: Development of antibacterial soft nanocomposites

Article abstract of DOI:10.1039/c0sm01087a

The present work reports the development of a new class of antibacterial soft-nanocomposites by in situ synthesis of silver nanoparticle (AgNP) within the supramolecular self-assemblies of amino acid (tryptophan/tyrosine) based amphiphilic hydrogelators. Interestingly, the nanoparticle synthesis does not require the use of any external reducing/stabilizing agents. The nanocomposites were characterized by UV-vis spectra, transmission electron microscopy (TEM) images, X-ray diffraction spectroscopy (XRD) and thermo gravimetric analysis (TGA). Encouragingly, these soft nanocomposites showed excellent antibacterial activity against both Gram-positive and Gram-negative bacteria whereas the amphiphiles alone were lethal only toward Gram-positive bacteria. Judicious combination of bactericidal AgNP within the self-assemblies of inherently antibacterial amphiphilic gelators led to the development of soft nanocomposites effective against both type of bacteria. The head group charge and structure of the amphiphiles were altered to investigate their important role on the synthesis and stabilization of AgNP and also in modulating the antibacterial activity of the nanocomposites. The antibacterial activities of soft nanocomposites comprising amphiphiles with cationic head group were found to be more efficient than the anionic soft nanocomposites. Interestingly, these nanocomposites have shown considerable biocompatibility to mammalian cell, NIH3T3. Furthermore, the well-known tissue engineering scaffold, agar-gelatin film infused with these soft nanocomposites allowed normal growth of mammalian cells on its surface while being lethal toward both Gram-positive and Gram-negative bacteria.

Full text of DOI:10.1039/c0sm01087a

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PAPER
In situ synthesized Ag nanoparticle in self-assemblies of amino acid based
amphiphilic hydrogelators: development of antibacterial soft nanocomposites†
*
Anshupriya Shome, Sounak Dutta, Subhabrata Maiti and Prasanta Kumar Das
Received 1st October 2010, Accepted 4th January 2011
DOI: 10.1039/c0sm01087a
The present work reports the development of a new class of antibacterial soft-nanocomposites by in situ
synthesis of silver nanoparticle (AgNP) within the supramolecular self-assemblies of amino acid
(tryptophan/tyrosine) based amphiphilic hydrogelators. Interestingly, the nanoparticle synthesis does
not require the use of any external reducing/stabilizing agents. The nanocomposites were characterized
by UV-vis spectra, transmission electron microscopy (TEM) images, X-ray diffraction spectroscopy
(XRD) and thermo gravimetric analysis (TGA). Encouragingly, these soft nanocomposites showed
excellent antibacterial activity against both Gram-positive and Gram-negative bacteria whereas the
amphiphiles alone were lethal only toward Gram-positive bacteria. Judicious combination of
bactericidal AgNP within the self-assemblies of inherently antibacterial amphiphilic gelators led to the
development of soft nanocomposites effective against both type of bacteria. The head group charge and
structure of the amphiphiles were altered to investigate their important role on the synthesis and
stabilization of AgNP and also in modulating the antibacterial activity of the nanocomposites. The
antibacterial activities of soft nanocomposites comprising amphiphiles with cationic head group were
found to be more efficient than the anionic soft nanocomposites. Interestingly, these nanocomposites
have shown considerable biocompatibility to mammalian cell, NIH3T3. Furthermore, the well-known
tissue engineering scaffold, agar-gelatin film infused with these soft nanocomposites allowed normal
growth of mammalian cells on its surface while being lethal toward both Gram-positive and
Gram-negative bacteria.
antibiotics, to date no bacterial resistance against AgNPs has
Introduction
been reported.4,6,7 However, the AgNPs are known to exhibit
superior antibacterial activity against Gram-negative bacteria
compared to that of Gram-positive bacteria.8,9 Therefore AgNP
based hybrid systems comprising a complementary antibacterial
agent with proven efficacy against Gram-positive bacteria can
lead to the development of versatile antibacterial nano-
composites. Again, it would be indeed beneficial if that anti-
bacterial agent in the composite can simultaneously reduce the
silver ion and stabilize the AgNP instead of using external
reducing agents. Also, the reducing and stabilizing agents should
be preferably biocompatible for the proper utilization of
AgNP-composites in biomedicine including tissue engineering,
drug delivery and so forth.4a,10,11 In this regard, the presence of
natural precursors like amino acid, sugar moiety and so forth in
the structure of reducing/stabilizing agent is expected to intro-
duce biocompatibility within the nanocomposite.4a,7,10–12
Design and development of new antibacterial agents is finding
unremitting significance, particularly in bio-medicinal chemistry,
due to the growing resistance of bacteria against conventional
antibiotics.1–3 To combat this ever increasing threat from path-
ogens, the inventory of existing antibiotics needs to be constantly
upgraded with materials having newer modes of killing. In this
context, silver nanoparticles (AgNPs) are emerging as efficient
antimicrobial agents because of their difference in the mechanism
of killing bacteria.4–7 Importantly, in contrast to the common
Department of Biological Chemistry, Indian Association for the Cultivation
of Science, Jadavpur, Kolkata, 700 032, India. E-mail: bcpkd@iacs.res.in;
Fax: +(91)-33-24732805
† Electronic supplementary information (ESI) available: Zone of
Inhibition for agar-gelatin films containing soft nanocomposites, TEM
images of lyophilized nanocomposites, the fluorescence micrograph of
E. coli and S. aureus before and after treating with AgNP-1 composites
in presence of LIVE/DEADꢀ BacLightꢁ Bacterial Viability Kit,
antibacterial activity of agar-gelatin films against B. Subtilis,
fluorescence images of NIH3T3 cells incubated on agar-gelatin films
containing AgNP-3 and AgNP-4 composites stained with
LIVE/DEADꢀ Viability/Cytotoxicity Kit for mammalian cells. See
DOI: 10.1039/c0sm01087a
To this end, we have recently shown the efficient killing of
Gram-positive bacteria by aromatic amino acid-based amphi-
philic hydrogelators.13,14 The mode of bacteria killing by these
positively charged gelators includes the disruption of cell
membrane, which results in the release of cytoplasmic constitu-
ents leading to cell death.1,13,14 However, these amphiphilic
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gelators with C-16 chain length were unable to kill Gram-nega-
tive bacteria even at high concentrations presumably due to the
existence of an outer layer consisting of lipopolysaccharide and
phospholipids in addition to the peptidoglycan layer as present in
Gram-positive bacteria.13–16 Thus, it is important to develop
systems, which will be equally efficient against both
Gram-positive and Gram-negative bacteria. In this context,
previously13 we have reduced the alkyl chain length to make the
amphiphile (1) effective against both types of bacteria. But
cytotoxicity of amphiphile was found to be increased with
decrease in chain length. This higher toxicity of nano-composites
synthesized from short chain amphiphiles will of course reduce
the scope of their biomedicinal applications. So inclusion of
AgNP within the self-assembled network of these biocompatible
cationic C-16 hydrogelators would be very intriguing with
respect to the killing of Gram-negative bacteria by AgNPs. As
the amphiphiles are intrinsically bactericidal against Gram-
positive bacteria, it is expected that the AgNP based soft nano-
composites will have killing effects against both types of bacteria.
Considering the biocompatibility of these soft nanocomposites it
is preferable to synthesize AgNP in situ within the self-assemblies
of non-toxic hydrogelating amphiphiles.
Results and discussion
Development of new antibacterial materials by the judicious
combination of AgNPs with soft materials like hydrogels would
be of great significance as the resultant soft nanocomposites will
find huge applications in biomedicine.18 In the present work,
AgNP based soft nanocomposites were developed by in situ
synthesis of AgNP within the supramolecular self-assemblies of
aromatic amino acid based amphiphilic hydrogelators (1–4, Chart
1). The head group structure and charge of the amphiphiles were
varied to find their effect on the in situ synthesis of AgNPs and also
on the antibacterial activity of nanocomposites. The polar head of
amphiphiles are comprised of aromatic amino acids, L-trypto-
phan for 1, 3 and 4 and L-tyrosine in case of 2. These two redox
active amino acids are well known for reducing silver and gold
ions to corresponding nanoparticles.19–22 Also the presence of
naturally occurring amino acids in these amphiphiles is advan-
tageous for biocompatibility of the soft composites.13,14 All these
amphiphiles have excellent water gelation ability as large volume
of water gets entrapped within the supramolecular self-assemblies
of these gelators.23,24 Among them, amphiphile 1 forms most
efficient hydrogel with a minimum gelation concentration (MGC)
of 0.3%, w/v (6 mM). Physical gelation is the manifestation of self-
assemblies of amphiphiles at MGC; however the process of self-
assembly starts at much lower concentration of gelator than
MGC.24 With increasing concentration of amphiphile, the
supramolecular self-assembly leads to the formation of hydrogel.
This self-assembled network of the redox active amino acid based
gelators is utilized for in situ synthesis of AgNPs.
In the present work, novel soft nanocomposites have been
developed by the in situ synthesis of AgNP in the supramolecular
self-assemblies of aromatic amino acid (tryptophan and tyrosine)
based amphiphilic hydrogelators with varying head group
structure (1–4, Chart 1). The formation of these soft nano-
composites was characterized by spectroscopic and microscopic
techniques. Encouragingly, the nanocomposites have shown
excellent antibacterial activity against both Gram-positive and
Gram-negative bacteria with minimum inhibitory concentration
(MIC), 10–50 mg mLꢀ1 whereas the amphiphiles alone were either
lethal toward only Gram-positive bacteria or ineffective against
any type of bacteria. Moreover, as confirmed by MTT (3-(4,5-
Synthesis of AgNP within the self-assemblies of amphiphilic
hydrogelators
The critical concentrations at which the amphiphiles, 1–4 begin to
self-assemble in water (critical micellar concentration) are 0.13,
0.08, 0.04 and 0.06 mM, respectively.25 To ensure the synthesis
and stabilization of AgNP within the self-assemblies of the
hydrogelator, the concentration of amphiphiles was always kept
higher than the said critical concentrations. Tollens’ reagent was
used for the synthesis of AgNP within the self-assemblies of all the
amphiphiles. To achieve the optimum reaction condition for
in situ synthesis of AgNP, concentration ratio of hydrogelators to
Tollens’ reagent was varied from 1 : 1 to 10 : 1 and also the
temperature of reaction mixtures changed from 60 to 120 ꢁC. The
said parameters needed for the efficient in situ synthesis of AgNP
were found to be dependent on the structure and self-assembling
behaviour of amphiphiles. In case of 1, Tollens’ reagent was added
to the hot (80 ꢁC) aqueous solution of amphiphile (0.2 mM) with
1 : 1 concentration ratio and the mixture was stirred at 80 ꢁC.
Initially, the AgNP formation was indicated by the change of the
visual appearance of solution from colourless to yellow. The
formation of AgNP was followed by monitoring surface plasmon
resonance (SPR) peak of the nanoparticle at 413 nm in time
dependent UV-visible spectra (Fig. 1A). At the said concentra-
tions of amphiphile 1 and silver ion, it took almost 4 h to complete
the synthesis of AgNP (Fig. 1A). However, in case of other three
hydrogelators (2–4), the required concentration ratio between
amphiphiles and Tollens’ reagent was 10 : 1 (2 mM: 0.2 mM) and
the needed reaction temperatures were 70, 100 and 120 ꢁC,
dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide)
assay, these self-assembled AgNP-composites were found to be
biocompatible to mammalian cells, NIH3T3. Interestingly, the
well-known tissue engineering scaffold,17 agar-gelatin film
infused with these soft nanocomposites allowed normal growth
of mammalian cells on its surface while being lethal toward
bacteria.
Chart 1 Structure of amphiphilic hydrogelators.
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Fig. 1 Time dependent UV-vis spectra of in situ synthesized AgNPs by amphiphile 1 (A) at a: 0; b: 1; c: 2; d: 3; e: 4; f: 5 h, 2 (B) at a: 0; b: 1; c: 2; d: 3; e: 4;
f: 5; g: 6 h: 7 h 3 (C) and 4 (D) a: 0; b: 5; c: 8; d: 12; e: 24; f: 36; g: 48; h: 56 h.
respectively. The characteristic SPR peaks of AgNP were
observed at 410–450 nm depending on the amphiphiles
(Fig. 1B–D). In case of tyrosinate amphiphile (2), in situ reduction
of silver ion was completed within 6 h whereas that took much
longer time (ꢂ2 days) for two negatively charged tryptophan
based dipeptide amphiphiles (3 and 4). Interestingly, the concen-
tration of 1 required (0.2 mM) for the synthesis of AgNPs is
10times lower compared to that which wasused for the other three
amphiphiles (2–4). Both tryptophan and tyrosinate moieties
present at the headgroup of amphiphiles 1 and 2, respectively are
very efficient in synthesizing AgNPs by reducing the silver ion.
The high concentration of 2 (2 mM) was needed for the synthesis
of AgNP possibly due to the weaker self-assembled networking of
2 in comparison to that of 1. This difference in the supramolecular
organization led to the formation of weak hydrogel for amphi-
phile 2 with a MGC of ꢂ4%, w/v (82 mM), which is ꢂ14 times
higher than that of amphiphile 1 (0.3%, w/v; 6 mM).23 Even in case
of 3 and 4, despite having the same redox active amino acid
tryptophan in the polar head as in 1, high amphiphile concen-
tration (2 mM) as well as more rigorous conditions (high
temperature and long time) was needed for the synthesis of Ag
nanocomposites. Here also the self-assembling ability is not very
efficient as almost 5 times higher MGC, 30 and 29 mM, respec-
tively were observed for 3 and 4 compared to that of 1.23 Thus, the
self-assembled network of amphiphiles have a crucial influence in
the synthesis of soft nanocomposite particularly in terms of
stabilizing the nanoparticle. The self-assembling properties of
these amphiphiles coupled with their in situ metal ion reducing
ability facilitate the development of AgNP based soft nano-
composites. The synthesized AgNP was centrifuged at 30,000 rpm
and washed thrice with Milli-Q water to remove the excess silver
ions. The pellet was dispersed in 0.2 mM surfactant solution
(5 mL) and lyophilized to obtain nanocomposite powder. This
powder was then weighed and re-dispersed in known volume of
water to prepare desired concentration of nanocomposites for
antibacterial and biocompatibility studies.
Transmission electron microscopy (TEM) images
The AgNP formation within the self-assemblies of amphiphiles
was characterized by acquiring the TEM images of soft nano-
composites before centrifugation (Fig. 2a–d). The AgNPs were
found to be spherical in nature irrespective of the amphiphile
used. The sizes of the nanoparticles were in the range of
10–25 nm (Fig. 2). The formation of AgNPs within the self-
assemblies of amphiphiles was also evident from the observed
supramolecular networks in the respective TEM images of
AgNP-1 to 4. At this point, it was also important to know
whether the shape and the size of AgNPs were retained after
centrifugation/lyophilization since the re-dispersed lyophilized
powders of AgNP-composites in water were used for antibacte-
rial activity and biocompatibility studies. Therefore, the aqueous
solution of lyophilized AgNP-composite was also characterized
by TEM images (Fig. S1, see ESI†). Interestingly, the spherical
nanoparticles with 10–25 nm size were found to be encapsulated
within the supramolecular network of amphiphiles as observed
before centrifugation (Fig. 2). Supramolecular networks of the
amphiphiles in absence of nanoparticles were also evident from
their TEM images (Fig. S2, ESI†).
X-ray diffraction (XRD)
Formation of AgNPs was further confirmed by XRD experi-
ments. XRD spectra of the soft nanocomposites revealed distinct
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Table 1 % of AgNP in the soft nanocomposites
AgNP based nanocomposites
% of AgNP
1
2
3
4
37
28
56
37
varying concentration of amphiphiles. The amount of amphi-
phile present in the nanocomposite was determined from the
calibration curve of amphiphile. Finally, the amount of AgNP in
the nanocomposite was observed to be 28 to 56% (Table 1)
depending on the amphiphiles. AgNP content in the nano-
composites was also determined by TGA.27 In contrast to the
pure amphiphile 1, which left negligible residue after 400 ꢁC
(Fig. 4a), the AgNP-1 composite showed the presence of
considerable amount of residue of AgNP after sharp weight loss
ꢁ
between 200 and 400 C (Fig. 4b). Almost 60% weight was lost
between 200 and 400 ꢁC that correspond to the decomposition of
amphiphile. Thus, the weight percent of AgNP in AgNP-1
composite was 40%, which is in good agreement with the esti-
mated AgNP content from UV-vis absorption spectroscopy
(Table 1).
Fig. 2 TEM images of AgNP synthesized within self-assemblies of
amphiphiles 1 (a), 2 (b),3 (c) and 4 (d).
Antibacterial activity
The antibacterial activity of the amphiphilic hydrogelators as
well as the AgNP based nanocomposites of 1–4 was investigated
using two Gram-positive bacteria Bacillus subtilis (B. subtilis),
Staphylococcus aureus (S. aureus) and three Gram-negative
bacteria Escherichia coli (E. coli), Klebsiella aerogenes (K. aero-
genes) and Klebsiella pneumoniae (K. pneumoniae). Antibacterial
activity was checked in nutrient agar media by spread plate
method.
The amphiphiles (1–2) with quaternary ammonium head
group were highly effective against Gram-positive bacteria
B. subtilis and S. aureus having MIC of 1–10 mg mLꢀ1 (Table 2).
The killing of Gram-positive bacteria by the amphiphile 1 is
consistent with the previous reports.13 On the contrary, the
amphiphiles with negatively charged head group (3–4) were
Fig. 3 XRD patterns of the AgNP based nanocomposites of hydro-
gelator 1 (a), 2 (b), 3 (c) and 4 (d).
four peaks (Fig. 3), which are characteristics for Ag nanocrystal.
The observed peaks at 38.09ꢁ (111), 44.28ꢁ (200), 64.54ꢁ (220),
and 77.42ꢁ (311) were in agreement with the respective planes of
crystalline Ag nanoparticles (Fig. 3).26
Quantification of amphiphiles and AgNP in the nanocomposites
The amount of amphiphile present in the nanocomposites was
measured by UV-vis spectroscopy and thermogravimetric anal-
ysis (TGA). All the hydrogelators (1–4) showed a strong
absorption band at 270–280 nm due to p–p* transition of
aromatic moieties present in the amphiphiles. A standard cali-
bration curve was prepared by measuring the absorbance at
Fig. 4 TGA thermograms of 1 (a) and AgNP-1 composite (b).
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Table 2 MIC of amphiphiles and soft nanocompositesa in mg mLꢀ1
B. subtilis (gm +ve)
S. aureus (gm +ve)
E. coli (gm ꢀve)
K. aerogenes (gm ꢀve)
K. pneumoniae (gm ꢀve)
1
AgNP-1
2
10
20 (7.4)
10
1
—
60 (22.2)
—
—
80 (29.6)
—
—
50 (18.5)
—
10 (3.7)
5
AgNP-2
3
20 (7.8)
200
10 (2.8)
—
75 (21)
—
55 (15.4)
—
60(16.8)
—
AgNP-3
4
AgNP-4
100 (56)
—
200 (74)
150 (84)
—
200 (74)
150 (84)
—
200 (74)
75 (42)
—
100 (37)
75 (42)
—
150 (55.5)
a
Value given in the parentheses describes the AgNP content in the composites.
unable to show antibacterial activity against these two bacteria
except in the case of 3 that killed B. subtilis at a very high MIC,
200 mg mLꢀ1. The outer wall of Gram-positive bacteria is
composed of peptidoglycan layer, which is negatively charged
due to the presence of phosphoryl groups located in the
substituent teichoic and techuronic acid residues as well as
carboxylate groups.28 The cationic charge of the amphiphiles 1
and 2 helped them to interact with the negatively charged cell
membrane of the bacteria. This electrostatic interaction is also
entropically favoured as a large number of counter ions from the
cell membrane are released.29 The lipophilic residues of the
amphiphiles then undergo ‘self-promoted’ transport across the
bacterial membrane followed by the disruption of the cell
membrane and release of cytoplasmic constituents leading to
death of bacteria.30–33 However, this process of bacteria killing
could not take place for anionic amphiphiles (3–4) possibly
because of no favourable interaction with negatively charged cell
membrane of bacteria. In case of Gram-negative bacteria, none
of the hydrogelators (1–4) exhibited any antibacterial activity
even up to the concentration of 200 mg mLꢀ1 (Table 2). In case of
Gram-negative bacteria the peptidoglycan layer is shielded by an
additional outer membrane consisting of lipopolysaccharide and
phospholipids and thus is not exposed to the extracellular envi-
ronment.28 So the cationic amphiphiles (1–2) cannot interact with
the outer cell-membrane as efficiently as in case of Gram-positive
bacteria.
killing efficiency against Gram-negative bacteria having MIC
value of 55–75 mg mLꢀ1 (15.4–21 mg mLꢀ1 AgNP). However, the
MIC values for AgNP-3 (75–150 mg mLꢀ1 containing
42–84 mg mLꢀ1 AgNP) and AgNP-4 (100–200 mg mLꢀ1 having
37–74 mg mLꢀ1 AgNP) against Gram-negative bacteria were
comparatively higher than that of AgNP-1/2 composites. The
variation in the antibacterial efficiency of different nano-
composites is presumably due to the effect of amphiphile’s head
group structure, size as well as its charge density. Most impor-
tantly, while the individual amphiphiles were completely inca-
pable of killing Gram-negative bacteria, the AgNP-based soft
nanocomposites of same amphiphiles were lethal to both
Gram-positive and Gram-negative bacteria.
This wide spectrum antibacterial activity of the soft nano-
composites is primarily because of the intrinsic bactericidal
efficiency of AgNP through multimodal mechanism in conju-
gation with the killing efficacy of amphiphiles. Although the
reason behind the antibacterial activity of AgNP is not entirely
understood, the possible mechanisms of action were proposed
in various reports.4,11,34 The AgNP can get attached to the cell
surface of bacteria as well as interact with the sulphur con-
taining protein present in cell membrane, which will hinder the
normal cellular functions leading to the cell death. AgNP can
also penetrate inside the bacterial cell where it can interact with
phosphorus containing DNA or attack the mitochondria
resulting in permanent damage to bacteria. In addition, the
AgNP can release silver ion inside the bacterial cell resulting
oxidative stress responsible for antibacterial activity. As a
whole, the soft composite of AgNP with quaternary ammo-
nium moiety integrated in head group of the amphiphile
(AgNP-1/2) showed better antibacterial efficacy than the soft
nanocomposites of negatively charged amphiphiles, 3/4. It is
clear from the antibacterial activity of AgNP-3/4, AgNP in
these nanocomposites showed its antibacterial activity in
different mechanisms other than only electrostatic interactions.
As mentioned above, the cationic charge around the nano-
particles in AgNP-1/2 can easily localized the bactericidal
AgNP to the negatively charged cell membrane of bacteria
resulting in better antibacterial efficiency compared to AgNP-3/
4. Although 2 contains an extra negative charge in its polar
head, the presence of quaternary ammonium group helped it to
exhibit higher antibacterial efficiency like AgNP-1. Hence, the
multimodal bactericidal efficiency of AgNP in combination
with the inherent bacterial killing ability of amphiphiles made
these soft nanocomposites effective against both Gram-positive
and Gram-negative bacteria.
Importantly, the soft nanocomposites of AgNP showed broad-
spectrum antibacterial activity against both Gram-positive and
Gram-negative bacteria (Table 2). For Gram-positive bacteria,
the MIC of AgNP-1 and AgNP-2 was found to be 10–20 mg mLꢀ1
against B. subtilis and S. aureus. Incorporation of AgNP addi-
tionally could not improve the antibacterial activity of the
composites possibly because of the very efficient inherent killing
ability (MIC-1-10 mg mLꢀ1) of the amphiphiles (1 and 2). The
effect of AgNP was rather observed for AgNP-3 and AgNP-4
composites. These nanocomposites were able to kill Gram-
positive bacteria, although at reasonably high MIC (100–
200 mg mLꢀ1) where the contribution of AgNP was in the range
of 56–84 mg mLꢀ1 (Table 2). Notably, the AgNP induces the
antibacterial activity within the soft nanocomposites of nega-
tively charged amphiphilic hydrogelators, 3 and 4. Encourag-
ingly, in contrast to the individual amphiphiles, more efficient
antibacterial activity of AgNP-composites was observed for
Gram-negative bacteria. The MIC values of AgNP-1 composite
were 50–80 mg mLꢀ1 (Table 2) with 18.5–29.6 mg mLꢀ1 AgNP
content (determined using Table 1). AgNP-2 also showed similar
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properties along with distinct spectral characteristics. SYTO 9
binds to the nucleic acid of both living and dead cells and exhibits
green fluorescence after excitation with BP460–495 nm filter.
Propidium iodide only binds to the nucleic acid of dead cells and
shows red fluorescence. The fluorescence microscopic images of
untreated Gram-negative, E. coli and Gram-positive, S. aureus as
well as that of same bacterial cells treated with 90 and 25 mg mLꢀ1
of AgNP-1 composite, respectively were shown in Fig. S3a and
S3c (ESI†). All the untreated cells exhibited green fluorescence
indicating the living status of bacteria. However, the observed
red fluorescence in the microscopic images (Fig. S3b and S3d,
ESI†) of AgNP-1 treated bacteria further confirms the bacteri-
cidal efficiency of the nanocomposite.
Biocompatibility study
These newly developed antibacterial soft nanocomposites will
find significance in biomedicinal applications only if they exhibit
compatibility with eukaryotic cells. To this end, we investigated
the cytotoxicity of the self-assembled nanocomposites to
mammalian cells, NIH3T3. MTT based cell viability assay was
used to test the cytotoxicity of nanocomposites in the concen-
tration range of 10–200 mg mLꢀ1. AgNP based soft composites
exhibited remarkable biocompatibility, as 85–90% cells remained
alive up to 25 mg mLꢀ1 of nanocomposite (Fig. 6). Even 65–80%
mammalian cells were found to be viable at 75 mg mLꢀ1 of
composite. Interestingly, in this range of nanocomposite
concentration, both Gram-positive and Gram-negative bacteria
were killed by AgNP-1 and AgNP-2. The nanocomposites also
showed 45–60% viability of mammalian cells (Fig. 6) at
75–200 mg mLꢀ1 where AgNP-3 and AgNP-4 are effective in
killing both type of bacterial cell. The nanocomposites with
negatively charged amphiphiles 3 and 4 have shown better cell
viability than positively charged AgNP-1 and AgNP-2. The
quaternary ammonium group in 1 and 2 possibly has preference
over the negatively charged amphiphiles to interact with the cell
membrane of mammalian cells comprised with zwitterionic
lipids. Most importantly, all the soft nanocomposites selectively
attack the bacterial pathogens while they remain biocompatible
Fig. 5 SEM images of bacteria E. coli (a-control, b-treated with
90 mg mLꢀ1 AgNP-1) K. aerogenes (c-control, d-treated with 90 mg mLꢀ1
AgNP-1) B. subtilis (e-control, f-treated with 25 mg mLꢀ1 AgNP-1) and
S. Aureus (g-control, h-treated with 25 mg mLꢀ1 AgNP-1).
Scanning electron microscopy (SEM) images of bacteria
To get an insight into the effect of AgNP-composites on bacteria,
the morphology of Gram-negative (E. coli and K. aerogenes) and
Gram-positive bacteria (B. subtilis and S. aureus) were examined
by taking their SEM images (Fig. 5)35,36 before and after treat-
ment with AgNP-1 composite above MIC (90 mg mLꢀ1 and 25 mg
mLꢀ1 for Gram-negative and Gram-positive bacteria). The
incubation of bacteria with AgNP-1 for 3–4 h showed a signifi-
cant change in their cell morphology. The untreated bacteria
E. coli (Fig. 5a), K. aerogenes (Fig. 5c), B. subtilis (Fig. 5e), and
S. aureus (Fig. 5g) exhibited healthy cell morphology with
preserved integrity of membrane structure. However, after
treatment with the AgNP-1 composite, the SEM images showed
deformation in the structure of bacteria with compromised
membrane integrity of E. coli (Fig. 5b), K. aerogenes (Fig. 5d),
B. subtilis (Fig. 5f), and S. aureus (Fig. 5h).
Fluorescence microscopic imaging of bacteria
We have also studied the antibacterial activity of the AgNP-1
composite by fluorescence microscopy using commercially
available LIVE/DEADꢀ BacLightꢁ bacterial viability kit.13,14,37
The kit contains two nucleic acid binding stains, SYTO 9 and
propidium iodide. These dyes have different cell penetration
Fig. 6 MTT assay based percent cell viability as a function of concen-
tration of AgNP based nanocomposites of amphiphile 1–4.
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to the mammalian cells. Such cell selectivity may have originated
from the difference in the lipid composition of prokaryotic and
eukaryotic cell membrane. The bacterial membranes are rich in
negatively charged lipids for example phosphatidylglycerol and
lipopolysaccharide, while the eukaryotic cell membranes are
mainly composed of zwitterionic lipids such as phosphatidyl-
choline and sphingomyelin.38 As a result, positively charged
nanocomposites are more efficient in discriminating two types of
the cells and preferentially bind to the more negatively charged
bacterial membrane. Also, the thiol (–SH) containing protein
present at the extra cellular membrane of bacteria facilitates its
interaction with AgNP of soft nanocomposites due to the affinity
of silver toward sulphur.11
of inhibition could not be measured in the case of agar-gelatin gel
containing AgNP-4 although the area under AgNP-4 containing
film was free from any bacterial growth (Fig. 7f and S4f, ESI†).
Hence, a tissue engineering scaffold has been made to be
intrinsically antibacterial by simple infusion of soft nano-
composites.
Now it would be very crucial to see whether the incorporation
of AgNP based soft composites (AgNP-1–4) into this agar-
gelatin hybrid has any effect on the biocompatibility of the
bactericidal polymeric film. To this end, we investigated the
attachment and spreading of NIH3T3 mouse fibroblast cells on
agar-gelatin (2 : 1) films with and without AgNP based
composites. The cells were seeded in 24 well plates in presence
and absence of films in DMEM (Dulbecco’s Modified Eagles’
Medium) growth media containing 10% FBS (fetal bovine
serum). The cell viability was followed after 24 h using LIVE/
DEADꢀ viability/cytotoxicity kit for mammalian cells
comprising of calcein-AM (live stain, green fluorescence) and
ethidium homodimer (dead stain, red fluorescence).40 The cells in
the control well without any film and well containing agar-gelatin
film (in absence of nanocomposites) were found alive as the
spindle shaped cells exhibited green fluorescence (Fig. 8a and c)
Development of antibacterial and biocompatible film
The necessity of developing antibacterial materials for bio-
medicinal applications including tissue engineering is on the rise
considering the material induced nosocomial infections in living
systems.39 To this end, utilization of these antibacterial soft
nanocomposites in the development of functional biomaterials
without compromising the biocompatibility would be of great
importance. In this regard, Verma et al. recently reported the
biocompatibility and growth of mice fibroblast cells (NIH3T3)
on the surface of agar-gelatin (2 : 1) hydrogel cross-linked with
glutaraldehyde.17 Although these matrices are safe to eukaryotic
cells but they don’t have any inherent antimicrobial property. In
order to bring in the antibacterial property within this matrix, we
have infused the AgNP based soft nanocomposites into this agar-
gelatin film and tested the antibacterial activity of these hybrid
films. As expected, normal growth of E. coli and B. subtilis were
seen in the agar plate in the absence (Fig. 7a and S4a, ESI†) as
well as in the presence of agar-gelatin film (Fig. 7b and S4b,
ESI†) without soft nanocomposites. Encouragingly, these
nanocomposites (AgNP-1–3) infused polymeric film exhibited
clear zone of inhibition for both Gram-negative, E. coli
(Fig. 7c–e, respectively) and Gram-positive, B. subtilis
(Fig. S4c–e, ESI respectively†) bacteria. The measured zone of
inhibition (Table S1, ESI†) once again indicates the superior
antibacterial activity of AgNP-1 and AgNP-2. However, a zone
Fig. 7 Antibacterial activity of agar-gelatin films against E. coli (a)
control without any film (b) agar-gelatin film without AgNP-composites
and films containing (c) AgNP-1, (d) AgNP-2, (e) AgNP-3 and (f) AgNP-
4 composites.
Fig. 8 Fluorescence microscopic images of NIH3T3 cells incubated in
control well without any film (a,b); on agar-gelatin film (c,d); on agar-
gelatin films containing AgNP-1(e,f) and AgNP-2 (g,h) composites
stained with LIVE/DEADꢀ Viability/Cytotoxicity Kit.
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and no red fluorescence (Fig. 8b and d) was observed. Most
encouragingly, the NIH3T3 cells adhered on these hydrogels
infused with nanocomposites of all the amphiphiles (AgNP-1 to
4) showed green luminescence in microscopic images (Fig. 8e,g
and S5a,c†) indicating their viability to antibacterial agar-gelatin
film. Most of the cells were able to attach and spread on the film
as their spindle shape was retained on the surface of the hydrogel
film. Also, the absence of any red fluorescence eliminates the
possibility of soft nanocomposites induced toxicity to mamma-
lian cells (Fig. 8f,h and S5b,d†). The nanocomposite incorpo-
rated agar-gelatin film showed antibacterial activity because of
its direct interaction with AgNP possibly through the thiol (–SH)
containing protein at the extra cellular membrane of bacteria.
Thus, the synthesized AgNP based soft nanocomposites will find
immense importance as futuristic antibacterial biomaterial.
neutrality. The organic parts were dried over anhydrous sodium
sulphate and concentrated to get the corresponding amines. The
produced amines (1 equivalent) were quaternized with excess
iodomethane using anhydrous potassium carbonate (2.2 equiv-
alent) and catalytic amount of 18-crown-6-ether in dry DMF for
2 h. The reaction mixtures were taken in ethyl acetate and washed
with aqueous sodium thiosulphate and brine solution, respec-
tively. The concentrated ethyl acetate parts were crystallized
from methanol/ether to obtain solid quaternized iodides, which
were then subjected to ion exchange on an Amberlite Ira-400
chloride ion exchange resin column to get the pure desired
amphiphile. Overall yields were in the range of 70–80%. In case
of amphiphile 2, freshly prepared 1 mL of NaOH solution
(22 mM) was added to tyrosine containing quaternary ammo-
nium chloride (10 mg, 22 mmol) to make a clear solution. This
solution was lyophilized to get the corresponding sodium salt of
tyrosine-based amphiphile (2).
Experimental section
The dipeptide-based amphiphiles (3–4, Chart 1) were also
synthesized by the reported protocol. Briefly, methyl ester of an
L-amino acid was coupled with C-16 long chain acid chloride in
dry chloroform and dry pyridine. The ester protected long chain
amide was then purified by column chromatography using
60–120 mesh silica gel and ethyl acetate/hexane as eluent. The
product was hydrolyzed using 1 N NaOH (1.1 equivalent) in
MeOH for 6 h with stirring at room temperature. Solvents were
removed on a rotary evaporator, and the mixture was diluted
with water and then washed with ether, followed by acidification
by 1 N HCl to get the corresponding carboxylic acid. This acid
was then coupled with another methyl ester protected L-amino
acid by using DCC, DMAP and HOBt in dry DCM. The purified
product was obtained by column chromatography using 60–120
mesh silica gel and ethyl acetate/toluene as eluent. The product
was then subjected to hydrolysis by 1 N NaOH (1.1 equivalent) in
MeOH for 6 h with stirring at 45–50 ꢁC. The amphiphilic
dipeptides with free carboxylic acid end were obtained following
the same procedure as described above. Sodium salt of the cor-
responding carboxylic acid was prepared by dissolving the acid in
MeOH and to that 1 equivalent 1 N NaOH (standardized) was
added. After brief stirring, the solvent was evaporated and dried
to get the sodium salt. The formation of sodium salt was
confirmed from FTIR spectroscopy by the disappearance of the
–C]O stretching peak of carboxylic acid ꢂ1720–1728 cmꢀ1 and
also from the improved solubility of the resultant compound in
water.
Materials
Silica gel of 60–120 mesh, L-tryptophan, L-tyrosine, L-valine,
L-isoleucine, n-hexadecylamine, N,N-dicyclohexylcarbodiimide
(DCC), 4-N,N-(dimethylamino) pyridine (DMAP), 1-hydroxy-
benzotriazole (HOBT), iodomethane, solvents and all other
reagents were procured from SRL, India. Water used throughout
the study was Milli-Q water. Thin layer chromatography was
performed on precoated silica gel 60-F254 plates of Merck. CDCl3,
uranyl acetate, Amberlite Ira-400 chloride ion exchange resins
were obtained from Aldrich Chemical Company. Ethylene dia-
minetetraacetic acid (EDTA) and reagents required to prepare the
nutrient broth culture medium like peptone, yeast extract, and
agar powder were purchased from Himedia Chemical Company,
India. The LIVE/DEADꢀ BacLightꢁ bacterial viability kit,
LIVE/DEADꢀ viability/cytotoxicity kit for mammalian cells
were purchased from Molecular Probes, Invitrogen Chemical
Company. All the materials used in the cell culture study such as
gelatin, DMEM, heat inactivated FBS, trypsin from porcine
pancreas and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-
tetrazolium bromide (MTT) were obtained from Sigma Aldrich
Chemical Company. 1H NMR spectra were recorded on
AVANCE 300MHz (BRUKER) spectrometer. UV-vis spectra
were taken in a Cary 50, Varian. Mass Spectrometric (MS) data
were acquired by Electron Spray Ionization (ESI) technique on
a Q-tof-Micro Quadruple mass spectrophotometer, Micromass.
Synthetic procedure
All amphiphiles (1–4, Chart 1) were synthesized and character-
ized following the previously reported protocol.23,24 For the
synthesis of 1 and 2, Boc-protected L-amino acids (10 mmol) were
coupled with n-hexadecylamine (11 mmol) using DCC (11 mmol)
and a catalytic amount of DMAP in the presence of HOBT
(11 mmol). Boc-protected amide was then purified through
column chromatography using 60–120 mesh silica gel and
acetone/hexane as the eluent. Deprotections of Boc-groups were
carried out using trifluoroacetic acid (TFA, 4 equivalent) in dry
DCM (dichloromethane). After 2 h of stirring, solvents were
removed on a rotary evaporator and the mixture was taken in
ethyl acetate. The ethyl acetate part was thoroughly washed with
aqueous 10% sodium carbonate solution followed by brine to
Determination of cmc of the amphiphilic hydrogelators by
surface tension measurement
The critical concentration at which the amphiphiles begin to self-
assemble was measured from the respective critical micellar
concentration (cmc) of the amphiphiles using surface tension
method. The cmc of amphiphiles was measured using a tensi-
€
ometer (Jencon, India) applying the Du Nouy ring method at
25 ꢃ 0.1 ꢁC in water. The cmc values were determined by plotting
surface tension (g) vs. concentration of surfactant with the
accuracy of ꢃ2% in duplicate experiments.
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In situ synthesis of AgNPs
estimated (Table 1). TGA thermograms of representative AgNP-1
composite and 1 were recorded by using a TA SDT Q600 instru-
ment at a heating rate of 20 ꢁC minꢀ1 under a N2 atmosphere.
Amphiphiles 1–4 (Chart 1) were used for in situ synthesis of
AgNPs from Tollens’ reagent, which was prepared by drop wise
addition of 1 N NaOH solution to 5 mM AgNO3 solution
(25 mL) until complete precipitation. The brown precipitate was
dissolved in 25% (w/w) ammonia and the clear Tollens’ reagent
was used for the synthesis of the nanoparticles. To synthesize
AgNPs using amphiphile 1, 5 mg amphiphile was dissolved in
48 mL water in a round bottom flask. The solution was heated to
Microorganisms and culture conditions
The in vitro antimicrobial activity of the amphiphiles and
nanocomposites was investigated against both Gram-positive,
Gram-negative bacteria. The nutrient broth medium containing
peptone (5 g), yeast extract (3 g) in 1 L sterile distilled water at
pH 7.0 was used as a liquid medium and nutrient agar [peptone
(5 g), yeast extract (3 g) and agar (15 g) in 1 L of sterile distilled
water of at pH 7.0] was used as a solid medium in all antibacterial
experiments. All the bacteria were purchased from Institute of
Microbial Technology, Chandigarh, India. The stock solutions
of all the amphiphiles and nanocomposite as well as the required
dilutions were made in autoclaved sterile water. For all the
bacteria, a representative single colony was picked up with a wire
loop and that a loopfull of culture was spread on nutrient agar
slant to give single colonies and incubated at 37 ꢁC for 24 h.
These fresh overnight cultures of all the bacteria were diluted as
required to give a working concentration in the range of 106–109
colony forming units (cfu)/mL before every experiment.
ꢁ
80 C and to it 2 mL of 5 mM Tollens’ reagent was added. The
concentration of both the amphiphile and silver ion (Tollens’
reagent) in the total solution was 0.2 mM. The heating was
continued and AgNP formation was indicated by the generation
of yellow colour in the solution. Time dependent UV-vis spectra
were recorded to determine the completion time of the reaction
by following the SPR peak of the nanoparticle. In the case of
amphiphiles 2, 3, and 4 a similar procedure was followed but the
concentrations of the amphiphiles and Tollens’ reagent were
2 mM and 0.2 mM, respectively. Also, the required temperature
ꢁ
for the synthesis of AgNP was 70, 100 and 120 C, respectively
for 2–4. The solution containing nanoparticles was centrifuged at
30,000 rpm. The pellet was washed thrice by Milli-Q water to
remove excess silver ions. The final pellet was dispersed in 5 mL
of 0.2 mM amphiphile (addition of this excess surfactant was
required for the stabilization of AgNP-composites in nutrient
growth media for bacteria) and lyophilized to get nanocomposite
powder. This powder (1 mg) was re-dispersed in 500 mL water to
get a 2 mg mLꢀ1 stock solution, which was used for antibacterial
and cell-culture study.
Antimicrobial studies
MICs of amphiphiles 1–4 as well as the AgNP based soft
nanocomposites were estimated by spread plate method. MIC
was measured using a series of test tubes containing the amphi-
philes (0.05–200 mg mLꢀ1) in 5 mL liquid medium. Diluted
microbial culture was added to each test tube in identical
concentration to obtain the working concentration of B. subtilis,
7.5 ꢄ 107–1 ꢄ 108 cfu/mL for S. aureus; 5 ꢄ 106–7.5 ꢄ 106 cfu/
mL; for E. coli, 3.75 ꢄ 107–7.5 ꢄ 107 cfu/mL; for K. aerogenes,
1.5 ꢄ 109–6.25 ꢄ 109 cfu/mL; for K. pneumoniae, 1.4 ꢄ 107–7.5 ꢄ
TEM
5 mL of AgNP based composite solution (before centrifugation)
was placed on 300-mesh carbon coated copper grid and dried
under vacuum for 4 h before taking TEM images with a JEOL
JEM 2010 high-resolution microscope. For TEM images of
lyophilized nanocomposites (after centrifugation), the dried
powder was dissolved in Milli-Q water and then placed on copper
grid in similar way as mentioned above. AgNP-1 and AgNP-2
composites were negatively stained with uranyl acetate to
confirm the presence of self-assemblies of amphiphiles.
107 cfu/mL. All the test tubes were then incubated at 37 C for
ꢁ
24 h. Then, 50 mL from each tube was spread onto nutrient agar
plates inside the laminar hood. Finally, plates were incubated for
24 h at 37 ꢁC and the viable cells were counted. The experiments
were performed in triplicate and were repeated twice.
SEM of nanocomposite treated bacteria
E. coli (3.75 ꢄ 107 – 7.5 ꢄ 107 cfu/mL), K. aerogenes (1.5 ꢄ
109 – 6.25 ꢄ 109 cfu/mL) and B. subtilis; (7.5 ꢄ 107 – 1 ꢄ 108 cfu/
mL) S. aureus (5 ꢄ 106 – 7.5 ꢄ 106 cfu/mL) cells (1 mL) were
treated with AgNP-1 composites above MIC at 90 mg mLꢀ1 and 25
mg mLꢀ1 for Gram-negative and Gram-positive bacteria, respec-
tively. The bacterial cells with and without (untreated cells as
control) amphiphile were incubated for 5–6 h. After incubation
the mixtures were centrifuged at 5,000 rpm for 5 min. The media
was removed completely and the cells were redispersed in 0.9 wt%
saline. Finally, 5 mL of the redispersed samples were mounted on
a glass slide and dried under vacuum for 4 h and the SEM images
were taken on JEOL-6700F scanning electron microscope.
XRD
Aqueous solution of AgNP-composites was put on glass slide
and dried under vacuum. XRD measurements were done using
this slide in a Seifert XRD3000P diffractometer where the source
was a Cu-Ka radiation (a ¼ 0.15406 nm) with a voltage and
current of 40 kV and 30 mA, respectively.
Quantification of amount of amphiphiles in AgNP based soft
composites
Amount of amphiphile in the nanocomposites were measured by
UV-vis spectroscopy and TGA. The absorbance value at 280 nm
was measured for different amphiphile concentration (1–4, Chart
1) to obtain a calibration curve. The amount of amphiphile
present in the soft nanocomposite was determined from this
calibration curve and thus % of AgNP in nanocomposites was
Fluorescence microscopic study for bacteria
The LIVE/DEADꢀ BacLightꢁ bacterial kit was used to
examine bacterial cell viability under a fluorescence microscope.
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The kit contains a mixture of two nucleic-acid binding stains,
specifically referred to as SYTO 9 and propidium iodide. The kit
was stored at ꢀ20 ꢁC in dark, which is taken out and thawed at
room temperature just prior to assay. E. coli (3.75 ꢄ 107 – 7.5 ꢄ
107 cfu/mL) and S. aureus (5 ꢄ 106 – 7.5 ꢄ 106 cfu/mL) cells
(1 mL) were treated with AgNP-1 above MIC (90 mg mLꢀ1 for E.
coli and 25 mg mLꢀ1 for S. aureus, respectively) and also
untreated cells were taken in centrifuge tube as control. The
mixtures were centrifuged at 5,000 rpm for 5 min. Then, media
was removed completely and the cells were redispersed in 0.9
wt% saline. Finally, BacLight dye mixture (3 mL) was added and
incubated in dark at room temperature for 15–20 min. After
incubation, 5 mL of the solution mixture was mounted over
microscope slides, which was then air-dried and viewed under the
microscope (BX61, Olympus) using an excitation filter of
BP460-495 nm and a band absorbance filter covering wavelength
below 505 nm.
Preparation of agar-gelatin gel film
The mixture (1 wt%) of agar and gelatin at ratio of 2 : 1 taken in
PBS (pH 7.4) was dissolved by heating the solution. In case of
AgNP containing film preparation, nanocomposite solution was
added to this homogeneous mixture of agar and gelatin so that 50
mg mLꢀ1 of AgNP-1/2 and 200 mg mLꢀ1 of AgNP-3/4 could be
attained. Then, glutaraldehyde (0.15 wt%) was added to the hot
solution for cross linking of the components in the solution. Each
solution (1 mL) was poured into 24-wells tissue culture flask and
was allowed to form gel at room temperature for overnight. Then
plates containing gels were dried in an incubator for 12 h at 50 ꢁC
to form thin films. These prepared films were treated with
0.1 mM glycine for 1 h to block the remaining aldehyde group.
The films were then washed several times with Milli-Q water to
remove excess glycine and then with PBS to neutralize the
surface. These films were then again dried and UV sterilized
overnight and kept in sterile vacuum desiccators for further
experiment.
Cell cultures
Antibacterial activity of agar-gelatin films
Mouse fibroblast NIH3T3 cells was obtained from National
Center for Cell Science (NCCS), Pune (India), and maintained in
DMEM medium supplemented with 10% FBS, 100 mg Lꢀ1
streptomycin and 100 IU/mL penicillin. Ceꢁlls were grown in
25 mL cell culture flask and incubated at 37 C in a humidified
atmosphere of 5% CO2 to approximately 70–80% confluence.
Media was changed after every 2–3 days and subculture was
performed every 7 days. After 7 days media was removed to
eliminate the dead cells. Next, the adherent cells were detached
from the surface of the culture flask by trypsinization. Cells were
now in the exponential phase of growth for checking the toxicity
of the soft nanocomposites.
The antibacterial activity of agar-gelatin films against E. coli and
B. Subtilis was followed in nutrient agar plates. For this, each
bacterium was cultured on nutrient agar slant at 37 ꢁC for 24 h.
These overnight cultures of bacteria were diluted as required to
get a concentration ꢂ105 cfu/mL. This bacteria containing
solution (1 mL) was added to nutrient agar plate. After the agar
plate became dry in 30 min, the agar-gelatin films with and
without AgNP was placed on the middle of the agar plates. The
plates were then incubated for 24 h at 37 ꢁC. The bacteria killing
ability of the films was followed by measuring the zone of
inhibition in the agar plate.
Cytotoxicity assay
Biocompatibility of agar-gelatin films
Cytotoxicities of nanocomposites of AgNP-amphiphiles (1–4)
were assessed by the microculture MTT reduction assay. This
assay is based on the reduction of a soluble tetrazolium salt by
mitochondrial dehydrogenase of the viable cells to water insol-
uble coloured product, formazan. The amount of formazan
formed can be measured spectrophotometrically after dissolu-
tion of the dye in DMSO. The activity of the enzyme and the
amount of the formazan produced is proportional to the number
of cells alive. Reduction of the absorbance value can be attrib-
uted to the killing of the cells or inhibition of cell proliferation by
the composites. Cells were seeded at a density of 15,000 cells per
well in a 96-well microtiter plate for 18–24 h before the assay.
Stock solutions of all the composites were prepared in water.
Sequential dilutions of these stock solutions were done during
the experiment to vary the concentrations (10 to 200 mg mLꢀ1) in
the microtiter plate. The cells were incubated for 4 h at 37 ꢁC
under 5% CO2. Then, 15 mL MTT stock solution (5 mg mLꢀ1) in
phosphate buffer saline was added to the above mixture and the
cells were further incubated for 4 h. The precipitated formazan
was dissolved thoroughly in DMSO and absorbance at 570 nm
was measured using BioTekꢀ Elisa Reader. The number of
surviving cells were expressed as percent viability ¼ (A570(treated
cells) ꢀ background/A570(untreated cells) ꢀ background) ꢄ 100.
The NIH3T3 cell attachment studies were done on control well
(24 well plate) without any film and on agar-gelatin films with
and without AgNP based composites. Films were incubated with
DMEM media (1 mL) containing 10% FBS. After 3 h the media
was removed and 5 ꢄ 105 cells were seeded in each well with 1 mL
media. The cells were incubated for 24 h in a 5% CO2 atmo-
sphere. After 24 h the adherent cells were washed with PBS and
cell viability was examined under a fluorescence microscope
using the LIVE/DEADꢀ viability/cytotoxicity kit for mamma-
lian cells. The kit contains a mixture of two nucleic acid binding
stains, specifically referred to as Calcein AM and Ethidium
ꢁ
homodimer-1 (EthD-1). The kit was stored at ꢀ20 C in dark,
and was taken out and thawed at room temperature just prior to
assay. The supplied 2 mM EthD-1 stock solution (4 mL) was
added to 2 mL of sterile, tissue culture-grade PBS and the
mixture was vortexed to ensure thorough mixing. The supplied
4 mM calcein AM stock solution (1 mL) was then added to the
2 mL EthD-1 solution and vortexed. The final stock solution
(500 mL) of calcein AM (2 mM) and EthD-1 (4 mM) was then
added directly to each well containing NIH3T3 cells. After
30 min incubation with the fluorescent dye, the cells were viewed
under Olympus IX51 inverted microscope (ex/em ꢂ495 nm/
515 nm for calcein AM and ex/em ꢂ 495 nm/635 nm) for EthD-1.
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13 S. Roy and P. K. Das, Biotechnol. Bioeng., 2008, 100, 756.
14 R. N. Mitra, A. Shome, P. Paul and P. K. Das, Org. Biomol. Chem.,
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15 J. W. Costerton and K. J. Cheng, J. Antimicrob. Chemother., 1975, 1,
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In summary, the newly developed AgNP based soft nano-
composites by in situ synthesis of AgNPs within the self-assem-
blies of amphiphilic hydrogelators have potent antibacterial
activity against both Gram-positive and Gram-negative bacteria.
Importantly, the intrinsic Gram-positive bacteria killing efficacy
of the cationic amphiphiles was complemented in the presence of
AgNPs, as the nanocomposite were lethal towards both types of
bacteria. Furthermore, the structure of the amphiphile was also
altered in order to understand the specific roles of the charge and
nature of the head group on the synthesis and stabilization of
AgNPs as well as in modulating the bactericidal efficacy.
Encouragingly, when these AgNP-composites were incorporated
into well know tissue engineering scaffold agar-gelatin films, they
were able to kill both Gram-positive and Gram-negative bacteria
despite being non-toxic toward mammalian cells. Therefore, the
designed soft nanocomposite promises to have immense impli-
cations in biomedicine including tissue engineering.
Acknowledgements
P.K.D. is thankful to Department of Science and Technology,
India for financial assistance. A.S., S.D. and S.M. acknowledge
Council of Scientific and Industrial Research, India for Research
Fellowships.
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