Counterion-induced modulation in the antimicrobial activity and biocompatibility of amphiphilic hydrogelators: Influence of in-situ-synthesized ag-nanoparticle on the bactericidal property

Article abstract of DOI:10.1021/la104903z

The necessity for the development of new antimicrobial agents due to the ever increasing threat from microbes is causing a rapid surge in research. In the present work, we have shown the efficient antimicrobial activity of a series of amino acid-based hydrogelating amphiphiles through alteration in their counterion. The subtle variation in the counterion from chloride to various organic carboxylates had a significant impact on the antimicrobial properties with notable improvement in biocompatibility toward mammalian cells. Incorporation of a hydrophobic moiety in the counterion augmented the antibacterial property of the amphiphilic hydrogelator as minimum inhibitory concentration (MIC) against the Gram-positive bacterial strain, Bacillus subtilis decreased up to 5-fold (with respect to the chloride) in the case of n-hexanoate. These counterion-varied amphiphilic hydrogelators were also found to be effective against fungal strains (Candida albicans and Saccharomyces cerevisiae) where they exhibited MICs in the range of 1.0-12.5 μg/mL. To widen the spectrum of antibacterial activity, particularly against Gram-negative bacteria, silver nanoparticles (AgNPs) were synthesized in situ within the supramolecular assemblies of the carboxylate hydrogelators. These AgNP-amphiphile soft-nanocomposites showed bactericidal property against both Gram-positive and Gram-negative bacteria. Encouragingly, these carboxylate hydrogelators showed superior biocompatibility toward mammalian cells, HepG2 and NIH3T3, as compared to the chloride analogue at a concentration range of 10-200 μg/mL. Importantly, the AgNP composites also showed sufficient viability to mammalian cells. Because of the intrinsic hydrogelation ability of these counterion-varied amphiphiles, the resulting soft materials and the nanocomposites could find applications in biomedicine and tissue engineering.

Full text of DOI:10.1021/la104903z

ARTICLE
pubs.acs.org/Langmuir
Counterion-Induced Modulation in the Antimicrobial Activity
and Biocompatibility of Amphiphilic Hydrogelators: Influence of
in-Situ-Synthesized AgꢀNanoparticle on the Bactericidal Property
Sounak Dutta, Anshupriya Shome, Tanmoy Kar, and Prasanta Kumar Das*
Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700 032, India
ABSTRACT: The necessity for the development of new
antimicrobial agents due to the ever increasing threat from
microbes is causing a rapid surge in research. In the present
work, we have shown the efficient antimicrobial activity of a
series of amino acid-based hydrogelating amphiphiles through
alteration in their counterion. The subtle variation in the
counterion from chloride to various organic carboxylates had
a significant impact on the antimicrobial properties with notable
improvement in biocompatibility toward mammalian cells.
Incorporation of a hydrophobic moiety in the counterion augmented the antibacterial property of the amphiphilic hydrogelator
as minimum inhibitory concentration (MIC) against the Gram-positive bacterial strain, Bacillus subtilis decreased up to 5-fold (with
respect to the chloride) in the case of n-hexanoate. These counterion-varied amphiphilic hydrogelators were also found to be
effective against fungal strains (Candida albicans and Saccharomyces cerevisiae) where they exhibited MICs in the range of 1.0ꢀ
12.5 μg/mL. To widen the spectrum of antibacterial activity, particularly against Gram-negative bacteria, silver nanoparticles
(AgNPs) were synthesized in situ within the supramolecular assemblies of the carboxylate hydrogelators. These AgNPꢀamphiphile
soft-nanocomposites showed bactericidal property against both Gram-positive and Gram-negative bacteria. Encouragingly, these
carboxylate hydrogelators showed superior biocompatibility toward mammalian cells, HepG2 and NIH3T3, as compared to the
chloride analogue at a concentration range of 10ꢀ200 μg/mL. Importantly, the AgNP composites also showed sufficient viability to
mammalian cells. Because of the intrinsic hydrogelation ability of these counterion-varied amphiphiles, the resulting soft materials
and the nanocomposites could find applications in biomedicine and tissue engineering.
’ INTRODUCTION
chain length and cationic headgroup on the antimicrobial activity and
biocompatibility of different amino acid-based amphiphiles.6a,7a
Although the positive charge and the hydrophobic segment of the
amphiphile have pivotal roles in killing bacteria, the counterion
associated with the headgroup also could play a crucial role in exter-
minating the bacterial membrane by the alteration of its hydropho-
bicity. This change in hydrophobicity and surface charge density
brought by the counterions can simultaneously improve the bio-
compatibility and antimicrobial efficacy of the amphiphiles. In this
regard, the effect of counterion variation on antibacterial properties
of polymeric molecules has recently been studied by Tew et al.10c
However, to the best of our knowledge, such reports involving low
molecular weight amphiphilic gelators have not been investigated.
To investigate the role of the counterion on the antimicrobial
efficacy, in the present work we varied the counterion of trypto-
phan-based amphiphilic hydrogelator from chloride to aromatic-,
aliphatic-, and aromatic-substituted aliphatic carboxylate anions
(Figure 1, 1aꢀg). Variation in the cationic charge density as well
as the hydrophobicity of the counterion enhanced the biocompat-
ibility and antimicrobial activity of the amphiphilic hydrogelators.
Interestingly, the n-hexanoate analogue (1d) showed a 5-fold
Development of new antimicrobial agents is a continuous
process by demand due to the growing resistance of microbes
against conventional antibiotics.1ꢀ3 The problem is being further
proliferated by the overuse of designated medicines.4 Hydrogels
with superior biological properties and applications in important
fields like tissue engineering, drug delivery, and design and devel-
opment of biomedicinal scaffolds have attracted a larger section of
chemists in recent times. In this regard, Pochan and co-workers
reported peptide hydrogels and their characteristic properties.5a,b
Amino acid-based cationic hydrogelators are emerging as a class of
antimicrobial agents, which deserves considerable attention for
their effective microbial killing ability.5ꢀ7 These amphiphiles, with
cationic head groups and optimum hydrophobicity, attack the
negatively charged membrane of microbes and facilitate the disin-
tegration of the cell membrane, which cannot get further reformed,
leading to cell death.1,7a,8 The factors that regulate the headgroup
charge density as well as the hydrophobicity of the amphiphile play
an instrumental role in governing the antimicrobial efficacy.9,10
At the same time the effective use of these antimicrobial
amphiphiles in biomedicine is very much dependent on their com-
patibility to mammalian cells. As mentioned, hydrogels with insig-
nificant cytotoxicity along with inherent antimicrobial properties can
be of potential use in the wider domain of biomedicine.5a,b,11
In this context, recently we investigated the effect of hydrophobic
Received: December 9, 2010
Revised:
March 9, 2011
Published: March 29, 2011
r
2011 American Chemical Society
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all other reagents were procured from SRL, India. Water used throughout
the study was Milli-Q water. Thin layer chromatography was performed on
Merck precoated silica gel 60-F254 plates. CDCl3, Amberlite Ira-400
chloride, and Dowex monosphere 550A hydroxide ion exchange resins
were obtained from Sigma-Aldrich Chemical Co. Ethylene diaminetetraa-
cetic acid (EDTA) and reagents required to prepare the Luria Burtani (LB)
and YEPD culture medium like tryptone, peptone, yeast extract, and agar
powder were purchased from Himedia Chemical Co., India. The LIVE/
DEAD BacLight bacterial viability kit was purchased from Molecular
Probes, Invitrogen Chemical Co. All materials used in the cell culture
study, such as Dulbecco’s Modified Eagles’ Medium (DMEM), heat-
inactivated fetal bovine serum (FBS), trypsin from porcine pancreas, and
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide
1
(MTT), were obtained from Sigma Aldrich Chemical Co. H NMR
spectra were recorded on an AVANCE 300 MHz (BRUKER) spectro-
meter. Mass spectrometric (MS) data were acquired by the electron
spray ionization (ESI) technique on a Q-tof-Micro Quadruple mass
spectrophotometer, Micromass.
Synthetic Procedure of Amphiphilic Hydrogelators
(1aꢀg). All cationic amphiphiles (1aꢀg) were synthesized following
the procedures previously reported by us.6a,16,17a,17b Briefly, Boc-
protected L-tryptophan was coupled with n-hexadecylamine using
dicyclohexylcarbodiimide (DCC, 1 equiv) and a catalytic amount of
4-N,N-(dimethyl)aminopyridine (DMAP) in the presence of 1 equiv of
1-hydroxybenztriazole (HOBT) in dry dichloromethane (DCM). Boc-
protected amide was then purified through column chromatography
using 60ꢀ120 mesh silica gel and acetone/hexane as the eluent. The Boc
group in the amide was deprotected by trifluoroacetic acid (TFA, 4
equiv) in dry DCM. After 2 h of stirring, solvents were removed on a
rotary evaporator and the mixture was taken in ethyl acetate. The EtOAc
part was thoroughly washed with aqueous 10% sodium carbonate
solution followed by brine to neutrality. The organic part was dried
over anhydrous sodium sulfate and concentrated to get the correspond-
ing free amine. The primary amine (1 equiv) was then quaternized with
excess methyl iodide using 2.2 equiv of anhydrous potassium carbonate
and a catalytic amount of 18-crown-6-ether in dry DMF for 2 h. The
reaction mixture was taken in ethyl acetate and washed with aqueous
thiosulfate solution and water, respectively. The concentrated ethyl
acetate part was subjected to column chromatography using 60ꢀ120
mesh silica gel and methanol/chloroform as the eluent. Finally, it was
crystallized from methanol/ether to obtain solid quaternized iodide salt,
which was subjected to ion exchange on an Amberlite Ira-400 chloride
ion-exchange resin column to get the pure chloride (1a). Overall yield
was ∼60%. The surfactants 1bꢀg were prepared following the same
experimental procedure as reported earlier. A hydroxide ion-exchange
column made with Dowex monosphere 550A was activated by passing
300 mL of 2 N sodium hydroxide solution and followed by washing with
water (2 L) and methanol (0.5 L). Three times recrystallized 1a (1 g)
dissolved in minimum volume of methanol was then loaded on the
column, and the corresponding hydroxide form was collected as eluent
by passing methanol slowly through this column. This methanolic
solution of the hydroxide form was concentrated in a rotary evaporator
to about 5 mL, and to it 20 mL of distilled water was added. This solution
was neutralized with an equivalent amount of the desired carboxylic acid
and allowed to stir for 1 h. The solution was further concentrated in the
rotary evaporator to ensure that no traces of methanol were present. The
resulting solution was then lyophilized to obtain the surfactant with
desired counterion. The yield of the product was ∼80ꢀ85%.
Figure 1. Amphiphilic hydrogelators with different counterions.
increase in the antibacterial activity against Gram-positive Bacillus
subtilis (B. subtilis) along with enhanced biocompatibility as com-
pared to its chloride analogue (1a). All other organic carboxylates
also showed improved viability toward mammalian cells (HepG2
and NIH3T3). However, counterion variation did not lead to any
activity against Gram-negative bacteria.
It is desirable to broaden the scope of these amphiphilic
hydrogelators to be effective against Gram-negative bacteria by
the inclusion of another antibacterial material without affecting the
inherent property of the soft materials. In this context, Agꢀ
nanoparticles (AgNPs) have emerged as potent antimicrobial
agents with a different killing mechanism.12ꢀ15 Counterion varia-
tion from chloride salt (1a) to the corresponding carboxylates
(1bꢀg) facilitated in-situ synthesis of AgNP using the supramole-
cular assemblies of the tryptophan-based hydrogelators.16 Impor-
tantly, the nanocomposites of these AgNPꢀamphiphiles (1b and
1e) showed efficient bactericidal property against both Gram-
positive and Gram-negative bacteria.
Development of Silver Nanoparticle (AgNP)-Hydrogela-
tor Soft Nanocomposites. Different carboxylates (1b and 1e) were
used for the in-situ synthesis of silver nanoparticle (AgNP) within the
hydrogel network following a protocol reported by us.16 Briefly, to a
10 mM aqueous solution (1 mL) of amphiphilic hydrogelator 1 mL of
AgNO3 (1 mM) solution was added dropwise. AgNP formation was
’ EXPERIMENTAL SECTION
Materials. Silica gel of 60ꢀ120 mesh, L-tryptophan, n-hexadecylamine,
N,N-dicyclohexylcarbodiimide (DCC), 4-N,N-(dimethylamino)pyridine
(DMAP), 1-hydroxybenzotriazole (HOBT), iodomethane, solvents, and
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indicated by the appearance of a yellow color and further characterized
from its surface plasmon resonance (SPR) peak in UVꢀvis spectra at
420ꢀ460 nm. After the reaction was complete (after 2 days), the
reaction mixture was centrifuged at 30 000 rpm and the pellet was
washed thrice with Milli-Q water. It was done in order to ensure removal
of AgNO3, excess amphiphiles not needed for stabilization of nanopar-
ticles, and also most of the oxidized by products formed in the course of
reduction of metal salts.17c,d The yellowish brown material was lyophi-
lized in order to obtain AgNPꢀamphiphile nanocomposite powder.
This was dispersed in Milli-Q water and used for antibacterial and
biocompatible studies.
Transmission Electron Microscopy (TEM) Images. AgNP
soft composites were characterized by taking their TEM images.
Nanocomposite solutions (5 μL from the reaction mixture) of AgNP-
1b and AgNP-1e were placed on a 300-mesh carbon-coated copper grid,
negatively stained with uranyl acetate after which images were taken
under a JEOL JEM 2010 high-resolution microscope. For TEM images
of lyophilized nanocomposites, first, the dried powder was dissolved in
Milli-Q water. Again, the nanocomposites were negatively stained with
uranyl acetate in order to have a clear perception of supramolecular
association along with the nanoparticles.
test tubes containing the amphiphiles (0.05ꢀ200 μg/mL) in 5 mL of
liquid medium. Diluted microbial culture was added to each test tube in
an identical concentration to obtain the working concentration of B.
subtilis, 7.5 ꢁ 107 ꢀ1 ꢁ 108 cfu/mL, for M. luteus, 1 ꢁ 106ꢀ2.5 ꢁ 106
cfu/mL, for S. aureus, 5 ꢁ 106ꢀ7.5 ꢁ 106 cfu/mL, for E. coli, 3.75 ꢁ
107ꢀ7.5 ꢁ 107 cfu/mL, for K. aerogenes, 1.5 ꢁ 109ꢀ6.25 ꢁ 109 cfu/mL,
for C. albicans, 9.8 ꢁ 104ꢀ1.3 ꢁ 105 cfu/mL, and for S. cerevisiae, 1.5 ꢁ
105ꢀ3 ꢁ 105 cfu/mL. All test tubes were then incubated at 37 °C for 24
or 48 h as required. The optical density of all solutions was measured
before and after incubation at 650 nm. Liquid medium containing
microorganisms was used as a positive control. All experiments were
performed in triplicate and repeated twice. The percentage of error in
each case was less than 5%.
We also tested the antimicrobial activity through the spread plate
method. For these experiments, the same amount of bacterial culture
was added to test tubes containing the desired concentration of
amphiphile solution in liquid medium. The test tubes were then
incubated at 37 °C for 5ꢀ7 h. Then, 50 μL from each tube was spread
onto LB agar plates inside the laminar hood. Finally, plates were
incubated for 24 (for all bacterial strains and for fungi S. cerevisiae)
and 48 h (for fungi C. albicans) at 37 °C, and the viable cells were
counted. The experiments were performed in triplicate and repeated
twice. A similar protocol was followed for the AgNPꢀcomposite
material as the required amount of bacterial culture was added to test
tubes containing desired concentrations of composite solution in liquid
medium.
Thermogravimetric Analysis (TGA). TGA thermograms of
representative AgNP-1b and AgNP-1e composites and amphiphiles
1b and 1e were recorded by using a TA SDT Q600 instrument at a
heating rate of 20 °C minꢀ1 under a N2 atmosphere.
Microorganisms and Culture Conditions. The in vitro anti-
microbial activity of all amphiphilic hydrogelators (1aꢀg) was investi-
gated against pathogenic representatives of Gram-positive bacteria,
Gram-negative bacteria, and fungus. Similarly, the antibacterial activity
of the nanocomposites was tested against a range of Gram-positive and
Gram-negative bacteria. Gram-positive bacteria used in the present
study were B. subtilis, Staphylococcus aureus (S. aureus), and Micrococcus
luteus (M. luteus). Gram-negative strains investigated include Escherichia
coli (E. coli) and Klebsiella aerogenes (K. aerogenes). The nutrient broth
medium containing peptone (5 g) and yeast extract (3 g) in 1 L of 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 7.0] was used as a solid medium in all antibacterial
experiments. The antifungal activity on the two fungi Candida albicans
(C. albicans) and Saccharomyces cerevisiae (S. cerevisiae) was investigated
by YEPD broth dilution and spread plate method. The YEPD medium
(yeast extract (3 g), peptone (10 g), and dextrose (20 g) in 1 L of sterile
distilled water at pH 7.0) was used as a liquid medium, and YEPD agar
(yeast extract (3 g), peptone (10 g), dextrose (20 g), and agar (15 g) in
1 L of sterile distilled water at pH 7.0) was used as a solid medium for the
antifungal studies. All microbial strains were purchased from the
Institute of Microbial Technology, Chandigarh, India. The stock solu-
tions of all amphiphiles and AgNPꢀamphiphile composites as well as
the required dilutions were made in autoclaved sterile water.
The freeze-dried ampules of all microbial strains were opened, and a
loopful of culture was spread to give single colonies on the respective
solid agar (LB agar for bacterial strains and YEPD agar for fungal strains)
media and incubated for 24 h (for all the bacterial strains and for fungi S.
cerevisiae) and 48 h (for fungi C. albicans) at 37 °C. For all microbial
strains, a representative single colony was picked up with a wire loop and
spread on an agar slant to give single colonies. The slants were incubated
at 37 °C for the respective time. These incubated cultures of all the
microorganisms were diluted as required to give a working con-
centration in the range of 106ꢀ109 and 104ꢀ105 colony forming units
(cfu)/mL for bacteria and fungus, respectively, before every experiment.
Antimicrobial Studies. Minimum inhibitory concentrations
(MICs) of 1aꢀg were estimated by both broth dilution and spread
plate method, and those for the AgNPꢀcomposites were estimated
through the spread plate method. MIC was measured using a series of
FESEM of Hydrogelator and Nanocomposites-Treated
Bacteria. B. subtilis (7.5 ꢁ 107ꢀ1 ꢁ 108 cfu/mL) and S. aureus (5 ꢁ
106ꢀ7.5 ꢁ 106 cfu/mL) cells (1 mL) were treated with compound 1e
above MIC at 8.0 and 2.0 μg/mL, respectively. Similarly, E. coli (3.75 ꢁ
107ꢀ7.5 ꢁ 107 cfu/mL) and K. aerogenes (1.5 ꢁ 109ꢀ6.25 ꢁ 109
cfu/mL) cells were treated with AgNP-1e above MIC at 150.0 and
50.0 μg/mL, respectively. The bacterial cells with and without (untreated
cells as control) amphiphile/nanocomposites were incubated for 30ꢀ
40 min. After incubation the mixtures were centrifuged at 10 000 rpm for
10 min. The medium was removed completely, and the cells were
redispersed in 0.9 wt % saline. Finally, 5 μL of the redispersed samples
was mountedona glass slideand dried undervacuumfor4 h, andthe SEM
images were taken on a JEOL-6700F scanning electron microscope.
Fluorescence Microscopic Study. The LIVE/DEAD BacLight
bacterial kit was used to examine bacterial cell viability under a fluorescence
microscope. 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 ꢁ 107ꢀ7.5 ꢁ 107 cfu/mL) and S. aureus (5 ꢁ
106ꢀ7.5 ꢁ 106 cfu/mL) cells (1 mL) were treated with AgNP-1e and
amphiphile 1e above MIC (150.0 μg/mL for E. coli and 5.0 μg/mL for S.
aureus, respectively), and also untreated cells were taken in a centrifuge tube
as control. The mixtures were centrifuged at 5000 rpm for 5 min. Then,
medium was removed completely, and the cells were redispersed in 0.9 wt
% saline. Finally, BacLight dye mixture (3 μL) was added and incubated in
the dark at room temperature for 15ꢀ20 min. After incubation, 5 μL 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 BP460ꢀ495 nm and a band absorbance filter covering
wavelengths below 505 nm.
Cell Cultures. Human hepatic cancer-derived HepG2 cells and
Mouse fibroblast NIH3T3 cells were obtained from the National Center
for Cell Science (NCCS), Pune (India), and maintained in DMEM
medium supplemented with 10% FBS, 100 mg/L streptomycin, and 100
IU/mL penicillin. Cells were grown in a 25 mL cell culture flask and
incubated at 37 °C in a humidified atmosphere of 5% CO2 to
approximately 70ꢀ80% confluence. A subculture was performed every
2ꢀ3 days. After 48ꢀ72 h, medium was removed to eliminate the dead
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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 viability of the cationic amphiphilic molecules
(1aꢀg) and nanocomposites AgNP-1b and AgNP-1e.
Table 1. MIC Value (μg/mL) of Amphiphilic Hydrogelators
against Gram-Positive Bacteria and Fungi
Gram-positive bacteria
fungi
Cytotoxicity Assay. Cytotoxicities of the cationic amphiphilic
molecules (1aꢀg) and nanocomposites AgNP-1b and AgNP-1e were
assessed by the microculture MTT reduction assay as used in the
literature.6a,7a This assay is based on the reduction of a soluble tetra-
zolium salt by mitochondrial dehydrogenase of the viable cells to an
insoluble colored product, formazan. The amount of formazan formed
can be measured spectrophotometrically after dissolution 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 attributed to the killing of the cells or inhibition
of cell proliferation by the amphiphiles. Cells were seeded at a density of
15 000 cells per well in a 96-well microtiter plate for 18ꢀ24 h before the
assay. A stock solution of all amphiphiles and AgNPꢀcomposites were
prepared in autoclaved water. Sequential dilutions of these stock
solutions were done during the experiment to vary the concentrations
of amphiphiles (10ꢀ200 μg/mL) in the microtiter plate. The cells were
incubated with amphiphiles/nanocomposites for 3 h at 37 °C under 5%
CO2. Then, 15 μL of MTT stock solution (5 mg/mL) in phosphate
buffer saline was added to the above mixture, and the cells were further
incubated for another 4 h. The precipitated formazan was dissolved
thoroughly in DMSO, and absorbance at 570 nm was measured using
BioTek Elisa Reader. The number of surviving cells was expressed as
percent viability = (A570(treated cells) ꢀ background/A570(untreated
cells) ꢀ background) ꢁ 100.
amphiphile B. subtilis M. luteus S. aureus C. albicans S. cerevisiae
1a
1b
1c
1d
1e
1f
10.0
8.0
5.0
2.0
5.0
4.0
3.0
2.5
3.5
2.5
2.0
2.2
3.0
2.2
1.0
2.0
1.0
0.5
1.0
0.9
0.5
1.0
2.0
1.0
1.0
3.0
1.5
2.5
10.0
12.5
10.0
8.0
7.5
15.0
12.5
1g
biocompatible soft materials. To investigate the role of the
counterion, the chloride counterion in 1a was replaced with
aromatic carboxylates like benzoates (1b) and salicylates (1c).
Encouragingly, the amphiphilic hydrogelators 1b and 1c showed
improvement in their bactericidal property as the MIC value
decreased against Gram-positive B. subtilis (Table 1). The
salicylate counterion (1c, MIC 5.0 μg/mL) was twice more
effective than the chloride analogue (1a, MIC 10.0 μg/mL) in
killing bacteria presumably due to the increased hydrophobicity
of the counterion. This inspired us to incorporate more hydro-
phobicity in the counterion in the form of an aliphatic carboxylate
(n-hexanoate, 1d). Interestingly, 1d was more efficient than the
chloride (1a) and aromatic carboxylates (1b and 1c), with a MIC
value of 2.0 μg/mL (Table 1), which is 5-fold better than 1a. To
confirm the role of the hydrophobic counterion on the bacter-
icidal property, we used acetate (1e) as the counterion instead of
n-hexanoate (1d). The antibacterial activity slightly decreased
with reduced hydrophobicity of counterion in the case of 1e
(MIC = 5.0 μg/mL) compared to 1d, but 1e is still a more
effective bactericide than 1a. Thus, the hydrophobicity of the
counterion appears to have a considerable influence in regulating
the antibacterial activity of the amphiphiles.
On the basis of the preceding results, we aimed to enhance the
hydrophobic character of the counterion using aromatic-substi-
tuted aliphatic counterions such as phenyl acetate (1f) and
naphthyl acetate (1g). Interestingly, both of these amphiphilic
hydrogelators showed substantially improved antibacterial activ-
ity against Gram-positive bacteria B. subtilis (MIC 4.0 and
3.0 μg/mL for 1f and 1g, respectively) compared to that of
chloride analogue (1a). However, among all the counterions,
n-hexanoate (1d) exhibited the best bacteria killing ability pre-
sumably due to the presence of hydrophobicity at the headgroup
region induced from the lipophilic character of counterion.
The influence of the counterion on the antibacterial activity of
these amphiphilic hydrogelators (1aꢀg) was also tested against
two other Gram-positive bacteria M. luteus and S. aureus. In all
cases, very efficient and comparable bactericidal property was
observed with MIC values in the range of 0.5ꢀ3.5 μg/mL. The
chloride analogue (1a) itself is an efficient antibacterial amphi-
phile having very low MIC (1.0 and 2.5 μg/mL for M. luteus and
S. aureus, Table 1), which did not improve further upon variation
of counterion. However, the observed similar antibacterial
efficacies of all the organic carboxylates would become significant
if they are simultaneously more biocompatible than the chloride
analogue (1a).
’ RESULTS AND DISCUSSION
Synthesis and development of antimicrobial agents effective
against a large spectrum of microbes along with considerable
biocompatibility have obvious significance in medicinal chemis-
try.1ꢀ3 In particular, soft materials like hydrogels, if developed
from such antimicrobial compound, would have great potential
in biomedicine.5a,b,6,11,18 In this context, efforts have been made
to tune the antimicrobial properties by varying different segments
of amphiphilic molecules.9,10c,19 However, to the best of our
knowledge, there has been no report on the role of the counter-
ion in modulating the antibacterial property or biocompatibility
of low molecular weight amphiphilic hydrogelators. Despite
being an intrinsic component of ionic amphiphiles, the influence
of the counterion has always been overlooked from physico-
chemical investigation to biochemical applications. In order to
investigate the counterion-induced variation in the antimicrobial
activity and biocompatibility of amphiphilic hydrogelators, we
altered the counterion of L-tryptophan-based amphiphilic hydro-
gelator (1a) from chloride to various organic carboxylates
(1bꢀg, Figure 1). All amphiphiles had an inherent property of
hydrogelation having a minimum gelation concentration (MGC)
from 0.3% to 3.8%, w/v.16,17a,17b
The antibacterial activity of these amphiphilic hydrogelators
was investigated against three Gram-positive bacteria B. subtilis,
S. aureus, and M. luteus, two Gram-negative bacteria E. coli and
K. aerogenes, and two fungi C. albicans and S. cerevisiae. The
minimum inhibitory concentration (MIC) values were deter-
mined by broth dilution and the spread plate method.
The hydrogelator (1a, Figure 1) had earlier shown potent
antibacterial activity against Gram-positive bacteria having MIC
values in the range of 1.0ꢀ10.0 μg/mL.6 Also, the compatibility
of 1a to mammalian cells needs to be improved particularly at
higher concentration for its effective use in developing
These amphiphilic hydrogelators will definitely gain substan-
tial importance if they also have antifungal activity along with
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their antibacterial property. The antifungal activity was tested
against yeast and fungus, C. albicans and S. cerevisiae, respectively.
The chloride salt (1a) was 10 times more effective against the
yeast, C. albicans with MIC of 1.0 μg/mL compared to that
against S. cerevisiae (MIC = 10.0 μg/mL, Table 1). Here also the
alteration of counterion to organic carboxylates did not yield
further enhancement in the antifungal activity for C. albicans
having MIC = 1.0ꢀ3.0 μg/mL for 1bꢀg. However, against the
fungal strain S. cerevisiae the aliphatic carboxylates were once
again found to be most effective with MIC values of 8.0 and 7.5
μg/mL for 1d and 1e, respectively (Table 1). As a whole the
amphiphilic gelators are lethal toward both Gram-positive bac-
teria and fungi and thus could be used as efficient antimicrobial
agents. However, counterion variation of the tryptophan-based
amphiphilic hydrogelator from chloride (1a) to organic carbox-
ylates (1bꢀg) failed to bring in any killing ability (up to 200
μg/mL) against the Gram-negative bacteria.
The bacterial killing process possibly takes place in two steps.
First, the positive charge of amphiphile infuses into the negatively
charged bacterial membrane primarily driven by electrostatic
interaction. This interaction is immediately followed by the ‘self-
promoted’ transport of the hydrophobic alkyl tail of the amphiphile
through the peptidoglycan layer, resulting in disruption of the cell
membrane and release of cytoplasmic constituents leading to death
of the bacterium.20ꢀ25 An optimum hydrophobicity facilitates
incorporation of amphiphiles within the lipid bilayer membrane
of bacteria.7b,8 Obviously, the hydrophobicity of the counterions
also has an important role in assisting the amphiphiles to penetrate
the bacterial membrane. To this end, the hydrophobic character of
aliphatic counterions akin to the lipophilicity of alkyl tail seems to
be ideal for bactericidal property. Thus, the observed improvement
in the antibacterial activity with increasing hydrophobicity of the
counterion is in accordance with this mechanism. However, in the
caseoffungus, thenegativechargedensityoftheoutermembraneis
not as high as in the case of bacteria. Therefore, not all positively
charged antibacterial compounds may have antifungal activity.8c
Interestingly, the counterion-varied amphiphiles showed superior
killing efficiency against the Gram-positive bacteria and fungi
(Table 1) compared to magainin 2 (MIC = 50ꢀ80 μg/mL), the
well-known standard antimicrobial peptide (AMP) consisting of 23
amino acids.
Although the amphiphiles with varying carboxylates (1bꢀg)
displayed excellent antimicrobial activities against Gram-positive
bacteria and fungi, they were incapable of showing any bacter-
icidal property against Gram-negative bacteria. The cell mem-
brane of Gram-negative bacteria contains an extra layer
comprising of lipopolysaccharide (LPS) and phospholipids in
addition to the peptidoglycan layer.26,27 This added protection
due to the presence of LPS layer in the case of Gram-negative
bacteria is possibly the main reason of the ineffectiveness of the
amphiphilic hydrogelators (1aꢀg). In order to expand their
antimicrobial domain, we utilized the supramolecular assemblies
of the tryptophan-based hydrogelators for in-situ synthesis of
silver nanoparticles (AgNPs). AgNPs are well known for their
killing efficacy against multidrug-resistant microbes including
Gram-negative bacteria.12ꢀ15 Thus, integration of AgNPs within
the supramolecular self-assemblies of these amphiphilic hydro-
gelators may lead to the development of soft nanocomposites,
which are expected to be effective against Gram-negative bacteria
in addition to Gram-positive bacteria and fungi.
Figure 2. TEM images of AgNP (negatively stained with uranyl
acetate) synthesized using amphiphile 1b (a) and 1e (c) as prepared;
nanocomposites after centrifugation followed by lyophilization of 1b (b)
and 1e (d).
amphiphiles and the stabilizing network of the supramolecular
hydrogel. Two carboxylates (1b and 1e, taken as representative
hydrogelators) were used for the in-situ synthesis of AgNP within
the hydrogel network from silver nitrate under mild conditions in
the absence of any external reducing agent. Interestingly, coun-
terion variation from chloride (1a) to organic carboxylates also
facilitated the in-situ synthesis of AgNPs from AgNO3 as the
resulting Agꢀcarboxylates salts have greater solubility in water
compared to AgCl.16 The reaction mixture was centrifuged and
washed with Milli-Q water in order to remove excess silver
nitrate. The final pellet was then lyophilized to get the nano-
composite powder. This was then dissolved in Milli-Q water to
form a stock solution and diluted as required for the antimicro-
bial and cytotoxicity studies.
Development of soft nanocomposites and the presence of
AgNPs within the supramolecular self-assemblies of amphiphilic
hydrogelators were investigated by transmission electron micro-
scopy (TEM). The samples (taken from the reaction mixture of
1b and 1e as well as that after lyophilization) were negatively
stained with uranyl acetate before taking the TEM images
(Figure 2). In all cases, the presence of monodispersed AgNPs
was observed along the self-assembled fibers of the amphiphilic
hydrogelators. The AgNP content within the soft composites was
determined by thermogravimetric analysis (TGA). In contrast to
the pure amphiphiles (1b and 1e), which left negligible residue
after 400 °C (Figure 3), the corresponding nanocomposites
showed the presence of a considerable amount of AgNP residue
after sharp weight loss between 200 and 400 °C. In the case of
AgNP-1b almost 43% weight loss was observed due to decom-
position of amphiphile between 200 and 400 °C, indicating the
presence of ∼57% of AgNP within the nanocomposite
(Figure 3A). Similarly, in the case of 1e, the amount of AgNP
present in the soft composite was found to be ∼39% (Figure 3B).
The antibacterial activity of the AgNPꢀnanocomposites of 1b
and 1e was investigated by a spread plate method against
two Gram-positive bacteria B. subtilis and S. aureus and two
Synthesis of AgNPꢀamphiphile nanocomposites was
achieved by utilizing the reducing ability of tryptophan-based
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Figure 3. TGA thermograms of 1b and nanocomposite AgNP-1b (A) and 1e and nanocomposite AgNP-1e (B).
Table 2. MIC Value (μg/mL) of AgNP-1b and AgNP-1e
Composites against Gram-Positive and Gram-Negative
Bacteria
Gram-positive bacteria
Gram-negative bacteria
nanocomposites
B. subtilis
S. aureus
E. coli
K. aerogenes
AgNP-1b
AgNP-1e
5.0
3.0
2.0
2.0
50.0
25.0
20.0
100.0
Gram-negative bacteria E. coli and K. aerogenes. As the amphi-
philic hydrogelators themselves were antibacterial against
Gram-positive bacteria, it was expected that the corresponding
nanocomposites would also be equally active against the same
Gram-positive bacteria. Encouragingly, the nanocomposites of
1b and 1e exhibited approximately 40% improvement (Table 2)
in antibacterial activity against B. subtilis compared to the amphi-
philes alone (Table 1). A notable decrease in the MIC was
observed for AgNP-1b (5.0 μg/mL) and AgNP-1e (3.0 μg/mL)
in comparison to that of corresponding hydrogelator (8.0 and
5.0 μg/mL for 1b and 1e, respectively, Table 1). However, the
nanocomposites showed almost similar antibacterial activity
against S. aureus as observed in the case of the amphiphile
alone (Table 1 and 2). Most interestingly as to our objective,
the nanocomposites exhibited antibacterial activity against Gram-
negative bacteria as well. The observed MIC values for AgNP-1b
and AgNP-1e against the Gram-negative E. coli were 50 and
100 μg/mL, respectively. Importantly, these nanocomposites
exhibited substantially improved bactericidal property against K.
aerogenes with MICs of 20ꢀ25 μg/mL (Table 2). The hydro-
gelators, which were inactive up to a concentration of 200 μg/mL,
displayed efficient antibacterial activity in the form of AgNP soft
composites against Gram-negative bacteria, presumably due to the
intrinsic bactericidal property of AgNP.
Thus, a wide spectrum of antibacterial activity is achieved by
these soft nanocomposites using the inherent multimodal killing
mechanism of AgNPs along with the killing ability of cationic
amphiphiles. Bactericidal killing of AgNPs has also been inves-
tigated earlier, though the exact killing mechanism is not yet
understood.12ꢀ15,28 AgNPs may act by attacking the phos-
phorus-containing DNA or interacting with the mitochondria
causing permanent damage and cell death.12b,15b They may also
interact with the sulfur-containing proteins, diminishing the
normal functioning of the cell. In addition, the AgNPs can release
Figure 4. SEM images of B. subtilis ((a) control and (b) treated with
8.0 μg/mL of 1e) and S. aureus ((c) control and (d) treated with
2.0 μg/mL 1e).
silver ions inside the bacterial cell, resulting in oxidative stress
leading to damage of cells.12d,15b
In order to obtain visual insight of bacteria killing, FESEM images
of bacteria before and after treatment with the antimicrobial agents
(amphiphilic hydrogelator as well as its AgNP-nanocomposites)
were acquired.7a,29,30 The effect upon treating Gram-positive
bacteria with amphiphilic hydrogelator 1e (taken as repre-
sentative) is shown in Figure 4. The concentration of 1e used for
this experiment was higher than its respective MIC (8.0 μg/mL for
B. subtilis and 2.0 μg/mL for S. aureus). Both untreated bacteria
showed the presence of normal cells (Figure 4a and 4c) with
appropriately preserved cell membranes. However, irregularly
shaped dead bacteria were observed upon treatment with 1e
(Figure 4b and 4d). The disrupted membrane resulted in the loss
of cytoplasmic constituents leading to cell death. Similar killing of
Gram-negative bacteria was also visualized through the microscopic
image (Figure 5) upon treating E. coli and K. aerogenes with soft
nanocomposites AgNP-1e (150.0 and 50.0 μg/mL, respectively).
The lethal effect of the nanocomposites was clearly seen from the
FESEM images of the treated bacteria (Figure 5b and 5d) in
comparison to the control (untreated) bacteria (Figure 5a and 5c).
Even in this case, the treated cells revealed compromised mem-
brane with deformation in the shape of bacteria.
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Figure 6. Fluorescence micrograph of E. coli and S. aureus before and
after treating with nanocomposite and amphiphile in the presence
of LIVE/DEAD BacLight bacterial viability kit: (a) control E. coli,
(b) E. coli treated with 150 μg/mL of AgNP-1e, (c) control S. aureus,
and (d) S. aureus treated with 2.0 μg/mL of 1e.
Figure 5. SEM images of E. coli ((a) control and (b) treated with
150 μg/mL AgNP-1e) and K. aerogenes ((c) control and (d) treated with
50 μg/mL AgNP-1e).
We also studied the antibacterial activity of hydrogelator 1e
and nanocomposite AgNP-1e by fluorescence microscopy using
the commercially available LIVE/DEAD BacLight bacterial
viability kit.6a,7a,31 The kit is composed of two nucleic acid
binding stains, known as SYTO 9 and propidium iodide. These
dyes have different cell penetration properties as well as different
spectral characteristics. SYTO 9 binds to the nucleic acid of both
living and dead cells, while propidium iodide can only bind to the
nucleic acid of dead cells. Consequently, all live cells show green
fluorescence, and dead cells with compromised cell membrane
appear red under the fluorescence microscope. Figures 6a and 6c
show the control image of E. coli and S. aureus, while Figures 6b
and 6d show the fluorescence images of the E. coli and S. aureus
cells treated with AgNP-1e (150 μg/mL) and 1e (2 μg/mL),
respectively, above their MIC values. All untreated bacterial cells
exhibited green fluorescence (Figure 6a and 6c), indicating that
they were alive. However, the antimicrobial activity of both
hydrogelator and soft nanocomposite was once again confirmed
from the observed red fluorescence for bacterial cells treated with
nanocomposite AgNP-1e (for E. coli, Figure 6b) and 1e (for
S. aureus, Figure 6d).
Biomedicinal use of such antimicrobial hydrogelators as well
as their soft nanocomposites will find great importance only
if they are biocompatible. Ideally, an antimicrobial agent should
be lethal to microbes and pathogens but at the same time must
be safe toward mammalian cells. In fact, it is very important
to find molecular architectures which will widen this diff-
erence (antimicrobial activity versus mammalian cell viability)
even more.
We initially investigated the cytotoxicity of amphiphile 1a
against human hepatic cancer-derived HepG2 cells and mouse
fibroblast NIH3T3 cells. Encouragingly, the amphiphile showed
sufficient selectivity as more than 90% of cells (HepG2, Figure 7)
were viable up to a concentration of 20 μg/mL, which is higher
than the concentration required to kill the microbes. However,
with further increase in concentration up to 200 μg/mL, the
viability gradually decreased to 45%. The reason for this selective
detrimental effect toward microbes could be due to the difference
in composition of the bacterial cell membranes with mammalian
cells. The bacterial membranes are rich in negatively charged
lipids such as phosphatidylglycerol and lipopolysaccharide,
while the eukaryotic cell membrane is primarily composed of
zwitterionic lipids such as phosphatidylcholine and sphingo-
myelin.32 Due to this difference in charge, the cationic amphi-
philes preferentially get adsorbed onto the bacterial mem-
branes.33,34 Again, the presence of cholesterol in the eukaryotic
membrane also gives additional protection to the mammalian
cells.35 Since the charge and composition of the membranes
determines their fate in the presence of the cationic amphiphiles,
we expected that the amphiphiles with different counterions
(1bꢀg) having varying degrees of hydrophobicities could have
an impact on determining the viability of the cells. Encouragingly,
upon changing the counterions from chloride to carboxylates, the
biocompatibility toward mammalian cells significantly improved.
In the case of most of the carboxylates, 60% of the HepG2 cells
were viable even up to a high concentration of 200 μg/mL
(Figure 7a). For instance, in the case of 1c ∼75% of cells
(HepG2) were viable at concentrations of 200 μg/mL. Again,
at a similar concentration of 1d 69% cells were viable. For all
amphiphiles, ∼90% cells (HepG2) were viable at 20 μg/mL
(Figure 7a). It is noteworthy here that the MIC values of these
amphiphiles toward Gram-positive bacteria and fungi are much
less than 20 μg/mL (Table 1).
The lower cytotoxicity of the carboxylates (1bꢀg, Figure 1)
can be explained on the basis of the larger size of the counterions
used. The hydrophobic counterions probably helped in diffusion
of the charge density over a larger surface area, thus decreasing
the probability of eukaryotic cell penetration. On the other hand,
the same augmented hydrophobicity of the counterions was
responsible for the improved efficiency against the microbes
(most pronounced effect in case of B. subtilis).
To generalize the biocompatibility on different cell lines, we
investigated the cytotoxicity of the amphiphilic hydrogelators on
NIH3T3 mammalian cells (Figure 7b). Here also, all organic
carboxylates showed lesser cytotoxicity throughout the concen-
tration range of 10ꢀ200 μg/mL (Figure 7b). An encouraging
increase in eukaryotic cell viability is clearly noted for the
amphiphilic hydrogelators when the chloride salt (1a) was
transformed to the corresponding organic carboxylates
(1bꢀg). Thus, we were successful in tuning the molecular
properties of the amphiphilic hydrogelator through counterion
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Figure 7. MTT-based % viability of (a) HepG2 and (b) NIH3T3 cells using 1aꢀg.
Figure 8. MTT-based % viability of (a) HepG2 and (b) NIH3T3 cells using AgNP-1b and AgNP-1e composites.
variation for developing antimicrobial soft materials with
improved viability toward mammalian cells.
work, which could be a useful strategy in the development of
potent antimicrobial agents. The bactericidal effects of these
amphiphiles against Gram-positive bacteria and fungi were
widened by in-situ synthesis of AgNPs within the supramolecular
assemblies of the hydrogelators in the absence of any external
reducing agent. The nanocomposites thus developed exhibited a
broad spectrum activity against both types of bacteria. Impor-
tantly, the AgNPꢀcomposites also showed considerable cyto-
compatibility to mammalian cells.
As we have seen previously that incorporation of nanoparticles
has in fact expanded the effectiveness of the amphiphiles to
microbes, it was very important to know whether they are
nontoxic toward eukaryotic cells as well. To this end, we investi-
gated the biocompatibility of the AgNPꢀcomposites against
mammalian cell lines HepG2 and NIH3T3. At a concentration of
20 μg/mL, both nanocomposites (AgNP-1b and AgNP-1e)
showed more than 80% viability (Figure 8). Encouragingly
enough, the cells were more than 60% viable up to a concentra-
tion of 200 μg/mL for both cell lines (Figure 8). Thus, the
counterion varied amphiphilic hydrogelators, and their nano-
composites offer considerable cell viability and wide spectrum
antimicrobial activity at very low concentrations, making them
potential candidates for diversified biomedicinal applications.
’ AUTHOR INFORMATION
Corresponding Author
*Fax: þ(91)-33-24732805. E-mail: bcpkd@iacs.res.in.
’ ACKNOWLEDGMENT
P.K.D. is thankful to the Department of Science and Technol-
ogy, India, for financial assistance. S.D., A.S., and T.K. acknowl-
edge the Council of Scientific and Industrial Research, India, for
Research Fellowships.
’ CONCLUSION
This work demonstrates the effect of systematic counterion
variation of an amphiphilic hydrogelator molecule on antimicro-
bial activity as well as biocompatibility. Inclusion of hydrophobic
counterions resulted in enhancement of the antibacterial activity.
Encouragingly, the biocompatibility of the carboyxlate hydro-
gelators toward eukaryotic cells was also found to be improved.
Attainment of this selectivity was an important aspect of this
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dx.doi.org/10.1021/la104903z |Langmuir 2011, 27, 5000–5008