Structure and properties of cholesterol-based hydrogelators with varying hydrophilic terminals: Biocompatibility and development of antibacterial soft nanocomposites

Article abstract of DOI:10.1021/la3038389

The present work demonstrates a rational designing and synthesis of cholesterol-based amino acid containing hydrogelators with the aim to improve the biocompatibility of these amphiphilic molecules. A thorough structure-property correlation of these hydrogelators was carried out by varying the hydrophilic terminal from a neutral amine to a quaternized ammonium chloride. The amphiphiles having a cationic polar head as the hydrophilic domain and cholesterol as the hydrophobic unit showed better water gelation efficiency (minimum gelation concentration (MGC) ～0.9-3.1%, w/v) than the analogous free amines. Presumably, the additional ionic interactions for the quaternized amphiphiles might have played the crucial role in gelation as counterions also got involved in hydrogen bonding with solvent molecules. Hence, the attainment of desired hydrophilic-lipophilic balance (HLB) of hydrophobic cholesterol in combination with the appropriate hydrophilic terminal led to the development of efficient hydrogels. Microscopic investigations revealed the formation of various supramolecular morphologies of hydrogels due to the variation in the molecular structure of the amphiphile. Spectroscopic investigations showed the involvement of hydrogen-bonding, hydrophobic, and π-π interactions in the self-assembled gelation. Importantly, biocompatibility of all the cholesterol-based hydrogelators tested against human hepatic cancer derived HepG2 cells showed increased cell viability than the previously reported alkyl-chain-based amphiphilic hydrogelators. In order to incorporate broad spectrum antibacterial properties, silver nanoparticles (AgNPs) were synthesized in situ within the hydrogels using sunlight. The amphiphile-AgNP soft nanocomposite exhibited notable bactericidal property against both Gram-positive and Gram-negative bacteria.

Full text of DOI:10.1021/la3038389

Article
pubs.acs.org/Langmuir
Structure and Properties of Cholesterol-based Hydrogelators with
Varying Hydrophilic Terminals: Biocompatibility and Development of
Antibacterial Soft Nanocomposites
Sounak Dutta, Tanmoy Kar, Deep Mandal, and Prasanta Kumar Das*
Department of Biological Chemistry, Indian Association for the Cultivation of Science Jadavpur, Kolkata −700 032, India
S
* Supporting Information
ABSTRACT: The present work demonstrates a rational
designing and synthesis of cholesterol-based amino acid
containing hydrogelators with the aim to improve the
biocompatibility of these amphiphilic molecules. A thorough
structure−property correlation of these hydrogelators was
carried out by varying the hydrophilic terminal from a neutral
amine to a quaternized ammonium chloride. The amphiphiles
having a cationic polar head as the hydrophilic domain and
cholesterol as the hydrophobic unit showed better water
gelation efficiency (minimum gelation concentration (MGC)
∼0.9−3.1%, w/v) than the analogous free amines. Presumably,
the additional ionic interactions for the quaternized amphiphiles
might have played the crucial role in gelation as counterions also
got involved in hydrogen bonding with solvent molecules. Hence, the attainment of desired hydrophilic−lipophilic balance
(HLB) of hydrophobic cholesterol in combination with the appropriate hydrophilic terminal led to the development of efficient
hydrogels. Microscopic investigations revealed the formation of various supramolecular morphologies of hydrogels due to the
variation in the molecular structure of the amphiphile. Spectroscopic investigations showed the involvement of hydrogen-
bonding, hydrophobic, and π−π interactions in the self-assembled gelation. Importantly, biocompatibility of all the cholesterol-
based hydrogelators tested against human hepatic cancer derived HepG2 cells showed increased cell viability than the previously
reported alkyl-chain-based amphiphilic hydrogelators. In order to incorporate broad spectrum antibacterial properties, silver
nanoparticles (AgNPs) were synthesized in situ within the hydrogels using sunlight. The amphiphile−AgNP soft nanocomposite
exhibited notable bactericidal property against both Gram-positive and Gram-negative bacteria.
have been reported in the recent past that were used for diverse
applications. However, to utilize them as biomaterials or in
preparing/modifying biomedicinal implants, the prime chal-
lenge faced by chemists is to render them biocompatible.
A simple strategy to improve the biocompatibility of an
amphiphilic gelator could be the inclusion of a biologically
relevant molecule/precursor as one of its segments (either
hydrophobic or hydrophilic). Cholesterol is known for its
abundance and importance in biological systems, and
amphiphiles composed of a cholesterol moiety are expected
to show augmented biocompatibility. The steroidal backbone of
cholesterol is a suitable option as the hydrophobic domain of
the gelator.25−27 Self-assembly of cholesterol-based molecules
has been investigated previously including the host−guest
chemistry of cholesterol and cyclodextrins.32 However, most of
the gelators designed using cholesterol have resulted in gelation
of organic solvents.25−27,33−37 Development of cholesterol-
INTRODUCTION
■
Hydrogels are gaining unremitting importance because of their
ever-expanding applications across scientific disciplines from
advanced materials1−6 to food processing to biomedicine.7,8
Self-assembly of small molecules has been the prime aspect of
supramolecular gelation that leads to the formation of soft
materials such as hydrogels.9−13 Incessant efforts are con-
tinuously on the rise for developing such soft materials with
superior properties and desired applications such as drug
delivery and controlled release,14−16 tissue engineering,17−19
pollutant capture,20 enzyme immobilization matrices,21,22 and
many others.23−31 Hydrogels have earned much interest
particularly for biological applications simply because of their
ability to imbibe water. Although, a large number of small
molecules have been studied for self-assembled gelation in
water, a constant advancement of this inventory is very
important considering its potential applications. Ideally, a
hydrogelator needs an optimum balance between hydro-
phobicity and hydrophilicity that would lead to self-assembled
gelation.31 In this regard, amphiphiles are always preferred as
gelator molecules due to their intrinsic self-assembling ability. A
large number of hydrogelators with varying functional motifs
Received: September 25, 2012
Revised: December 4, 2012
© XXXX American Chemical Society
A
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Article
Chart 1. Structures of Cholesterol-based Amphiphiles
IR (FTIR), uranyl acetate, cholesteryl chloroformate, 2,2′-
(ethylenedioxy)bis(ethylamine), and Amberlite Ira-400 chloride ion
exchange resins were obtained from Aldrich Chemical Co. Reagents
required for preparing the nutrient broth culture medium such as
peptone, yeast extract, and agar powder were purchased from Himedia
Chemical Company, India. The LIVE/DEAD BacLight bacterial
viability kit and LIVE/DEAD viability kit (L-3224) for mammalian
cells were purchased from Molecular Probes, Invitrogen Chemical
Company. High glucose Dulbecco's modified eagle medium (DMEM),
heat inactivated fetal bovine serum (FBS), and trypsin−EDTA
(0.05%) were purchased from Invitrogen. Penicillin−streptomycin
and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide
based functional hydrogels with structure−property correlation
along with its cytocompatibility has not received due attention.
At the same time, the other crucial issue that's needs parallel
attention during the development of self-assembled hydrogels is
their ability to resist microbial infection. Biomedicinal implants
induced infection in patients is a common phenomenon. Apart
from a few examples, supramolecular gels are themselves
inefficient in killing bacteria.38−41 Inclusion of external
bactericidal agents or synthesized in situ like silver nano-
particles (AgNPs) within self-assembled systems would increase
the bactericidal property, and thereby, a superior soft
nanohybrid will be developed.42,43 Therefore, designing a
biocompatible hydrogelator with a possible chance of
accommodating an external antibacterial agent would be of
great significance.
Herein, we report cholesterol-based hydrogelators compris-
ing different amino acids linked by an oxyethylene spacer with a
varying hydrophilic terminal (from free amine to quaternized
trimethyl ammonium chlorides, Chart 1). The quaternized salts
(1a−3a) exhibited better hydrogelation efficiency (minimum
gelation concentration (MGC) = 0.9−3.1%, w/v) compared to
the analogous amines (1−3). Spectroscopic and microscopic
characterizations were carried out in detail to understand
different driving forces involved in self-assembled hydro-
gelation. The amphiphilic hydrogelators showed significant
viability toward mammalian cells. Also, hydrogelators 1 and 2
with a free amine at the terminal were used for in situ synthesis
of AgNPs from AgNO3 using sunlight, and the synthesized
nanoparticles were stabilized within the supramolecular hydro-
gels. Although the cholesterol moiety is not directly involved in
synthesis or stabilization of the AgNPs, the amine group
present at the terminal of the gelators assisted in the
stabilization of the nanoparticles. Soft nanocomposites
AgNP−1 and AgNP−2 exhibited excellent bacteria killing
ability against both Gram-positive and Gram-negative bacteria.
1
(MTT) were obtained from Sigma−Aldrich Chemical Co. H NMR
spectra were recorded in an AVANCE 500 MHz (Bruker)
spectrometer. Mass spectrometry (MS) data were acquired by electron
spray ionization (ESI) technique on a Q-tof-micro quadruple mass
spectrometer (Micromass). Elemental analyses were performed on
Perkin−Elmer 2400 CHN analyzer. UV−vis spectra were taken in a
Perkin−Elmer lamda 25 spectrophotometer. Bath sonication was
performed with a Telsonic ultrasonics bath sonicator. A Sorvall RC 6
and Sorvall RC 90 were used for centrifugation and ultracentrifugation,
respectively.
Synthetic Procedure. All amphiphiles (1−3, 1a−3a, Chart 1)
were synthesized and characterized following the general principles of
peptide chemistry described earlier.31 Cholesteryl chloroformate was
coupled with one end of 2,2′-(ethylenedioxy)bis(ethylamine) in dry
dichloromethane (DCM) using a catalytic amount of dry triethylamine
(Scheme S1, Supporting Information). The DCM solution of the
cholesteryl chloroformate was added dropwise to the diamine solution
under cold condition (0−5 °C). After complete addition, the solution
was stirred for 8 h and then washed with water and brine to remove
the excess base and the excess diamine. The DCM part was evaporated
on a rotary evaporator, and solid product was obtained. The product
was purified using column chromatography with chloroform−
methanol as the eluent. The other end of the amine was coupled
with Boc-protected L-amino acids using DCC and a catalytic amount
of DMAP in the presence of HOBT. Boc-protected amide was then
purified through column chromatography using 60−120 mesh silica
gel and acetone−hexane as the eluent. The Boc-group was deprotected
using TFA in dry DCM. After 2 h of stirring, the solvents were
removed on a rotary evaporator, and the mixture was taken in ethyl
acetate. The ethyl acetate part was thoroughly washed with 10%
aqueous sodium carbonate solution followed by brine to neutrality.
The organic portion was dried over anhydrous sodium sulfate and
concentrated to get the corresponding amines (1−3, Chart 1). The
produced amines (1 equiv) were quaternized with excess iodomethane
using anhydrous potassium carbonate (2.2 equiv) and a 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
EXPERIMENTAL SECTION
■
Materials. Silica gel of 60−120 mesh, L-tryptophan, L-alanine, L-
phenylalanine, N, N-dicyclohexylcarbodiimide (DCC), 4-N, N-
(dimethylamino) pyridine (DMAP), 1-hydroxybenzotriazole
(HOBT), Boc anhydride, trifluoroacetic acid (TFA), iodomethane,
solvents, and all other reagents were procured from SRL, India. Water
used throughout the study was Milli-Q water. Thin-layer chromatog-
raphy was performed on precoated silica gel 60-F254 plates of Merck.
CDCl3 and other deuteriated solvents for NMR and Fourier transform
B
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thiosulphate and brine solution, respectively. 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 (1a−3a). Overall yields were in the range 65−80%.
1H NMR of 1 (500 MHz, CDCl3, 25 °C). δ = 1.06−2.33 (m, 43H,
cholestanyl, and 3H, alanine), 3.25−3.59 (m, 12H, oxyethylene), 3.92
(br, 1H, cholestanyl (CH−O(CO)), 4.47 (br, 1H, chiral proton of
amino acid), 5.36 (s, 1H, olefinic cholestanyl), 8.12 (br, 1H, amide).
Elemental analysis calcd (%) for C37H65N3O5: C, 70.32; H, 10.37; N,
6.65; found: C, 70.49; H, 10.53; N, 6.91. MS (ESI): m/z calcd for
C37H65N3O5: 631.4924; found: 632.4997 [M+ + 1].
amphiphile composite solution (before and after centrifugation/
lyophilization).
Circular Dichroism Spectroscopy. Circular dichroism (CD)
spectra of the aqueous solutions of 2, 2a, and 3a at varying
concentrations were recorded by using a quartz cuvette of 1 mm path
length in a Jasco J-815 spectropolarimeter. Temperature-dependent
CD spectra (for 3a) were also recorded at a concentration of 0.05% w/
v from 20 to 90 °C.
FTIR Measurements. FTIR measurements of gelators 2 and 3a in
CHCl3 solution and in the gel state in D2O were carried out in a
Perkin−Elmer spectrum 100 FTIR spectrometer using KBr and CaF2
windows, respectively, with 1 mm Teflon spacers at their MGC.
Fluorescence Spectroscopy. The emission spectra of 8-anilino-
1-naphthalenesulfonic acid (ANS) in varying concentrations of
amphiphiles were recorded on a Varian Cary eclipse luminescence
spectrometer. The probe molecules (ANS) were added to the aqueous
solutions of amphiphiles at varying concentrations at room temper-
ature. An ANS stock solution was prepared in MeOH, and from this
super stock solution, the required amount of ANS solutions was added
to gelators so that the final concentration of ANS solution was 1 ×
10−5 M. The solution was excited at λmax = 360 nm. The emission and
excitation slit widths were 10 nm.
X-ray Diffraction. X-ray diffraction (XRD) measurements were
taken in a Seifert XRD 3000P diffractometer, and the source was Cu
Kα radiation (α = 0.15406 nm) with a voltage and current of 40 kV
and 30 mA, respectively. Gels of 2 and 3a were mounted on the glass
slide and dried under vacuum. The xerogel was scanned from 1° to
40°.
NMR Experiments. Amphiphile 3a was dissolved in D2O (1% w/
v), and temperature-dependent NMR was acquired from room
temperature to 80 °C. In the case of NMR study using varying
solvent proportion, initially, amphiphile 3a was dissolved in DMSO-d6
(1% w/v), and an NMR spectrum was taken. Subsequently, samples
containing 10%, 20%, and 30% water were prepared (keeping the total
solvent volume and amount of gelator constant), and their NMR
spectra were acquired.
1H NMR of 2 (500 MHz, CDCl3, 25 °C). δ = 0.66−2.00 (m, 43H,
cholestanyl), 3.33−3.44 (m, 2H, CH2Ph), 3.47−3.51 (t, 4H,
oxyethylene), 3.52−3.60 (m, 8H, oxyethylene), 3.95 (br, 1H,
cholestanyl (CH−O(CO)), 4.43 (br, 1H, chiral proton of amino
acid), 5.34 (s, 1H, olefinic cholestanyl), 7.20−7.30 (m, 5H, aromatic).
Elemental analysis calcd (%) for C43H69N3O5: C, 72.94; H, 9.82; N,
5.93; found: C, 72.51; H, 9.59; N, 5.74. MS (ESI): m/z calcd for
C43H69N3O5: 707.5237; found: 708.5300 [M+ + 1].
1H NMR of 3 (500 MHz, CDCl3, 25 °C). δ = 0.65−2.29 (m, 43H,
cholestanyl), 2.68−2.77 (d, 2H, CH2−indole), 3.05−3.48 (m, 12H,
oxyethylene), 4.44 (s, 1H, cholestanyl (CH−O(CO)), 4.83 (br, 1H,
chiral proton of amino acid), 5.32 (s, 1H, olefinic cholestanyl), 7.01−
7.48 (m, 5H, aromatic), 8.57 (s, 1H, amide). Elemental analysis calcd
(%) for C45H70N4O5: C, 72.35; H, 9.44; N, 7.50; found: C, 72.49; H,
9.62; N, 7.12. MS (ESI): m/z calcd for C45H70N4O5: 746.5346; found:
747.5391 [M+ + 1].
1H NMR of 1a (500 MHz, CDCl3, 25 °C). δ = 0.67−2.33 (m, 43H,
cholestanyl, and 3H, alanine), 3.39 (s, 9H, N(CH3)3), 3.55 (t, 4H,
oxyethylene), 3.57−3.68 (m, 8H, oxyethylene), 4.34 (br, 1H,
cholestanyl (CH−O(CO)), 5.05 (br, 1H, chiral proton of amino
acid), 5.35 (br, 1H, olefinic cholestanyl), 8.83 (s, 1H, amide).
Elemental analysis calcd (%) for C40H72N3O5Cl: C, 71.17; H, 10.75;
N, 6.23; found: C, 71.24; H, 10.53; N, 5.98. MS (ESI): m/z calcd for
+
C40H72N3O5 : 674.5466; found: 674.5472 [M+].
In Situ Synthesis of AgNPs. Amphiphiles 1 and 2 (Chart 1) were
used for in situ synthesis of AgNPs. Hydrogels (400 μL) of the
corresponding amines were prepared in accordance with the MGC
(Table 1). To the hydrogel, 50 μL of AgNO3 solution was added to
1H NMR of 2a (500 MHz, CDCl3). δ = 0.64−2.29 (m, 43H,
cholestanyl), 3.06−3.20 (m, 2H, CH2Ph), 3.28−3.46 (m, 12H,
oxyethylene), 3.39 (s, 9H, N(CH3)3), 4.43 (m, 1H, cholestanyl
(CH−O(CO)), 5.25 (br, 1H, chiral proton of amino acid), 5.32 (br,
1H, olefinic cholestanyl), 7.21−7.31 (m, 5H, aromatic), 8.89 (s, 1H,
amide). Elemental analysis calcd (%) for C46H76N3O5Cl: C, 73.56; H,
10.20; N, 5.59; found: C, 73.12; H, 10.65; N, 5.71. MS (ESI): m/z
Table 1. Minimum Gelation Concentration (MGC) of
Amphiphiles
+
calcd for C46H76N3O5 : 750.5779; found: 750.5784 [M+].
amphiphile
MGC (% w/v)
1H NMR of 3a (500 MHz, CDCl3, 25 °C). δ = 0.66−2.49 (m, 43H,
cholestanyl), 2.94−2.98 (m, 2H, CH2−indole), 3.18−3.36 (m, 12 H,
oxyethylene), 3.48 (s, 9H, N(CH3)3), 4.33 (br, 1H, cholestanyl (CH−
O(CO)), 5.30 (br, 1H, chiral proton of amino acid), 5.35 (br, 1H,
olefinic cholestanyl), 7.09−7.22 (m, 2H, aromatic), 7.36−7.39 (d, 1H,
aromatic), 7.54−7.60 (m, 2H, aromatic), 8.88 (br, 1H, amide).
Elemental analysis calcd (%) for C48H77N4O5Cl: C, 72.96; H, 9.82; N,
7.09; found: C, 72.67; H, 9.48; N, 7.35; MS (ESI): m/z calcd for
1
5.3
4.0
2
a
3
ins
1a
2a
3a
3.1
1.5
0.9
a
ins: insoluble.
+
+
C48H77N4O5 : 789.5888; found: 789.5891 [M ].
Preparation of Hydrogel. The required amounts of the
compounds were added in 400 μL of water in a screw-capped vial
with an internal diameter (i.d.) of 10 mm and slowly heated until the
solid was completely dissolved. The solutions were then cooled to
room temperature without any disturbance. After 1 h, the formation of
gel was confirmed by stable to inversion of the glass vial.
Microscopy Studies. Field emission scanning electron microscopy
(FESEM) was performed on a JEOL-6700F microscope. A piece of
hydrogel was mounted on a glass slide and dried for a few hours under
vacuum. A Au coating was employed for 90 s before imaging.
Transmission electron microscopy (TEM) images were acquired on a
JEOL JEM 2010 high-resolution microscope. For TEM images, 5 μL
of solution (10 times more dilute than MGC) was placed on a 300
mesh carbon coated copper grid. The sample was negatively stained
with 2% uranyl acetate solution (2 μL) and dried under vacuum for 4
h before imaging. A similar procedure was followed for the AgNP−
maintain a gelator/AgNO3 concentration of 10:1. In the case of 1, the
final gelator concentration was 75 mM, and the AgNO3 concentrtion
was ∼7.5 mM. In the case of 2, the gelator concentration was 50.6
mM, and that of AgNO3 was ∼5 mM. The mixture was kept in bright
sunlight, and after 5 min, a change in color from colorless to yellow
was noted. UV−vis spectra confirmed the synthesis of AgNPs. The
solution containing nanoparticles was centrifuged at 30 000 rpm. The
pellet was washed twice with Milli-Q water to remove excess silver
ions and amphiphiles. The final pellet was lyophilized to get the
nanocomposite powder. This powder was dispersed in water and used
for antibacterial study.
Microorganisms and Culture Conditions. The in vitro
antimicrobial activity of the nanocomposites was investigated against
both Gram-positive and Gram-negative bacteria. The nutrient broth
medium containing peptone (5 g) and yeast extract (3 g) in 1 L of
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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 at pH 7.0) was used as a solid medium in all
antibacterial experiments. All the bacteria were procured from the
Institute of Microbial Technology, Chandigarh, India. The stock
solutions of the nanocomposites as well as the required dilutions were
made using autoclaved sterile water. For all the bacteria, a
representative single colony was picked up with a wire loop, and
that loopful of culture was spread on a 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 106−109 colony forming units (cfu)/mL
before each experiment.
concentrations (5−100 μg/mL) in the microtiter plate. The cells were
incubated for 6 h at 37 °C under 5% CO2. Then, 15 μL of the MTT
stock solution (5 mg/mL) in phosphate-buffered saline (PBS) was
added to the above mixture, and the cells were further incubated for 3
h. The precipitated formazan was dissolved thoroughly in DMSO, and
the absorbance at 570 nm was measured using a BioTek ELISA reader.
The number of surviving cells was expressed as percent viability =
(A570(treated cells) − background/A570(untreated cells) − back-
ground) × 100.
Fluorescence Microscopy Study to Check Cell Viability. The
LIVE/DEAD viability/cytotoxicity kit for mammalian cells was used to
examine cell viability under a fluorescence microscope. The kit
contains a mixture of two nucleic acid binding stains, specifically
referred to as Calcein AM (component A) and ethidium homodimer-1
(EthD-1, component B). The acetomethoxy derivative of Calcein
(Calcein AM) has the ability to pass through the cell membrane. After
its transportation into the cell, the esterase enzyme present in live cells
removes the acetoxy group. This form of the compound then
intercalates with the DNA and results in an enhancement of
fluorescence, and a bright green color can be observed. This binding
occurs in the presence of the esterase enzyme, which is present only in
live cells. Ethidium homodimer can only pass through damaged cell
membranes; thus, it gets incorporated only into dead cells. It does not
require any enzyme and shows red fluorescence upon binding with
DNA in the dead cells. The kit was stored at −20 °C in the dark,
which is taken out and thawed at room temperature just prior to assay.
A portion (4 μL) of the supplied 2 mM EthD-1 stock solution
(component B) was added to 2 mL of sterile, tissue culture-grade PBS,
and the mixture was vortexed to ensure thorough mixing. This gave an
approximately 4 μM EthD-1 solution. A portion (1 μL) of the supplied
4 mM calcein AM stock solution (component A) was then added to
the 2 mL of EthD-1 solution and vortexed. The resulting
approximately 2 μM calcein AM and 4 μM EthD-1 working solution
was then added directly (500 μL) to HepG2 cells treated with the
amphiphile having a concentration of 50 μg/mL and incubated for 24
h. After incubation with the fluorescent dye, the cells were observed
under the Olympus IX51 inverted microscope using an excitation filter
of BP460−495 nm and a band absorbance filter covering wavelength
below 505 nm. The bright green color resulting from the enhanced
fluorescence of DNA-intercalated calcein indicated the presence of
viable cells. When the images were taken using the excitation filter
BP530−550 and a band absorbance filter covering wavelength below
570 nm, negligible red fluorescence was observed, which confirmed
the absence of dead cells rather ensured the abundance of live cells.
Antibacterial Studies. Minimum inhibitory concentrations
(MICs) of the AgNP-based soft nanocomposites were estimated by
the liquid broth method and the spread plate method.42 The MIC was
measured using a series of test tubes containing the composites (5−
150 μg/mL) in 5 mL of liquid medium. The diluted microbial culture
was added to each test tube in identical concentrations to obtain the
working concentration for Escherichia coli, 3.75 × 107 to 7.5 × 107 cfu/
mL; for Bacillus subtilis, 7.5 × 107 to 1 × 108 cfu/mL; for
Staphylococcus aureus, 5 × 106 to 7.5 × 106 cfu/mL; and for
Pseudomonas aeruginosa, 9 × 107 to 1.2 × 108 cfu/mL. All the test
tubes were then incubated at 37 °C for 24 h. The optical density of all
the solutions was measured before and after incubation at 650 nm.
Liquid medium containing microorganisms was used as a positive
control. For the spread plate method, 50 μL 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.
Fluorescence Microscopy Study. The LIVE/DEAD BacLight
bacterial viability 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 the dark; it was taken out and
thawed at room temperature just prior to the assay. E. coli (3.75 × 107
to 7.5 × 107 cfu/mL) was treated with AgNP−2 above MIC and also
untreated cells were taken in a centrifuge tube. The mixtures were
centrifuged at 5000 rpm for 5 min at 4 °C. Then, the media was
removed completely, and the cells were redispersed in 0.9 wt % saline.
Finally, 3 μL of the BacLight dye mixture was added and incubated in
the dark at room temperature for 15−20 min. After incubation, 5 μL of
the solution mixture was mounted over microscope slides, which were
then air-dried and viewed under the light microscope (BX61,
Olympus) using an excitation filter of BP460−495 nm and a band
absorbance filter covering wavelength below 505 nm.
Cell Cultures. Human hepatic cancer derived HepG2 cells were
obtained from the National Center for Cell Science (NCCS), Pune
(India), and maintained in DMEM supplemented with 10% FBS, 100
mg/L streptomycin, and 100 IU/mL penicillin. Cells were grown in 25
mL cell culture flasks 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, the 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 amphiphiles.
Cytotoxicity Assay. Cell viability of amphiphiles was assessed by
the microculture MTT reduction assay.42 This assay is based on the
reduction of a soluble tetrazolium salt by mitochondrial dehydrogen-
ase of the viable cells to a water-insoluble colored product, formazan.
The amount of formazan formed can be measured spectrophoto-
metrically 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 composites. Cells were seeded at a density 15 000 cells per well in
a 96-well microtiter plate for 18−24 h before the assay. Stock solutions
of all the amphiphiles were prepared in water. Sequential dilutions of
these stock solutions were done during the experiment to vary the
RESULTS AND DISCUSSION
■
Designing small molecules that can entrap solvents resulting in
the formation of supramolecular gels has fascinated chemists for
long. In recent past, efforts have been made to include “task-
specific functionalities” in the molecular structures of hydro-
gelators to widen their prospect in different disciplines
including biomedicinal applications.1−18 In order to improve
the biocompatibility and biodegradability, gelator structure is
often tethered with a biomolecular precursor.44,45 In previous
work, we have developed several amino acid based amphiphilic
hydrogelators that comprised long alkyl chain as the hydro-
phobic unit.31,42 However, most of these gelators failed to show
significant viability toward mammalian cells particularly at a
longer time scale. It is believed that the lipophilic chain
penetrates the cell membrane resulting in the death of cells.46,47
In this context, we have made efforts to improve the
biocompatibility of amphiphilic hydrogelators by varying the
counterions at the hydrophilic end and also by introducing an
amino acid moiety at the hydrophobic tail to reduce the
penetration ability of the alkyl chain.42,48 Instead of diminishing
the lipophilic character of the alkyl tail, we thought of replacing
the hydrophobic unit with a biologically relevant precursor such
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Figure 1. FESEM images of dried gel of (a) 2, (b) 2a, and (c) 3a ; TEM images of dried gels of (d) 2 (arrows indicate the presence of thin fibers),
(e) 2a, and (f) 3a.
as cholesterol. In the present work, we have exploited this
steroidal unit as the hydrophobic moiety to develop
amphiphilic hydrogelators with the objective of improving the
cytocompatibility of the molecule.
amine 3 was found to be insoluble in water. The amphiphile in
combination of cholesterol and tryptophan linked by an
oxyethylene diamine spacer might have lost the critical
hydrophilic−lipophilic balance (HLB) required for even its
solubilization in water.
Structure Correlation of the Amphiphilic Molecules
with Their Gelation Properties. Cholesterol and the amino
acid were linked together using an oxyethylene diamine spacer.
The C terminal of the amino acid was connected to the spacer
through an amide linkage, and the steroid moiety was tethered
to the other end of oxyethylene diamine (Chart 1).
Amphiphilic compounds were synthesized using L-alanine, L-
phenylalanine, and L-tryptophan having a free primary amine at
the N terminal (1−3, Chart 1). The synthetic procedures
(Scheme S1, Supporting Information) are illustrated in the
Experimental Section. We have initially used an aliphatic amino
acid, L-alanine, with an amine at the polar head (1, Chart 1).
This cholesterol-based amphiphilic compound showed weak
hydrogelation ability with an MGC of 5.3% w/v (Table 1). It
was apparent that the crucial balance between hydrophobicity
and hydrophilicity for 1 was not optimum, which is essential for
superior gelation. However, the presence of hydrogen-bonding
units such as an amide, an oxyethylene spacer, and a free amine
group might have facilitated the self-assembled gelation of 1 in
water, although its MGC was high. Cholesterol possibly
contributed toward the gelation through its hydrophobic
interactions. The presence of an aromatic moiety is known to
facilitate self-assembly of molecules in water through π−π
interaction.31 With this comprehension, we used aromatic
amino acids instead of L-alanine. In the case of the L-
phenylalanine-based amine (2), gelation efficiency moderately
improved as the MGC slightly decreased to 4.0% w/v (Table
1). This decrease in the MGC was presumably due to the
additional π−π interaction between the aromatic rings in 2.
Hence, an extended aromaticity possibly could result in more
efficient hydrogelation for cholesterol based amphiphilic amine.
We introduced the L-tryptophan amino acid (3) comprising an
extended aromatic system (indole moiety). Surprisingly, free
Previously, we have observed that, in the case of long chain
amphiphilic amino acid based compounds, modification of a
primary amine to a quaternized ammonium chloride resulted in
the formation of efficient hydrogelators.48,49 Thus, to improve
the hydrogelating efficiency of the present cholesterol-based
amphiphiles, we transformed the primary amines to the
corresponding quaternized trimethylammonium chloride salts
(1a−3a, Chart 1) keeping all other functional moieties
unaltered. In the case of the alanine-based quaternized
amphiphile (1a, Chart 1), enhancement in the gelation
efficiency was noted (MGC = 3.1% w/v, Table 1) compared
to that was observed for the corresponding free amine, 1
(MGC = 5.3% w/v). In addition to the alteration of HLB, the
ionic interactions also played a crucial role in gelation as the
counterion got involved in hydrogen bonding with solvent
molecules. The hydrogelation ability of this cholesterol-based
alanine-containing quaternized amphiphile is noteworthy
because the corresponding hydrophobic long alkyl chain
(instead of cholesterol) quaternized amphiphile of alanine
failed to exhibit any hydrogelation ability (as reported
earlier).50 This certainly points to the fact that the hydrophobic
cholesterol moiety is responsible for self-aggregating inter-
actions that facilitated the supramolecular hydrogelation. The
quaternized ammonium chloride of phenylalanine (2a) showed
even more efficient hydrogelation (MGC, 1.5% w/v, Table 1).
Almost 2.7-fold improvement in the gelation efficiency in 2a
was observed with respect to the precursor free amine (2, MGC
= 4.0% w/v). The additional effect of aromatic π−π interaction
along with the ionic interaction of the polar head further
bolstered the self-assembled gelation. Strikingly, the quaterniza-
tion of the tryptophan-containing cholesterol-based amphiphile
(3) led to the development of the superior hydrogelators (3a)
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Figure 2. Concentration-dependent CD spectra of (a) 2, (b) 2a, and (c) temperature-dependent heating and cooling cycle for 3a (0.05% w/v).
with a MGC of only 0.9% w/v (Table 1). Although free amine
3 was insoluble in water, quaternization of the same resulted in
the formation of the most efficient hydrogelator, 3a. The
transformation of the primary amine to the quaternary
ammonium headgroup resulted in the development of efficient
gelators possibly due to the attainment of optimum HLB in the
amphiphiles required for self-assembled gelation. Thus, the
complementary effect of the cholesterol-based hydrophobic
segment and the tailor-made hydrophilic end led to the
development of efficient amphiphilic hydrogelators. In previous
instances, the presence of an aromatic moiety was found to be a
prerequisite for the hydrogelation of amphiphilic molecules
having a long alkyl chain as the hydrophobic unit.31,49,50
However, in the present case, the hydrophobic cholesterol
could induce hydrogelation in presence of both aliphatic (1 and
1a) as well as aromatic amino acids (2, 2a, and 3a).
Gel-to-Sol Transition Temperature (Tgel). The gel-to-sol
transition temperatures (Tgel) of all the hydrogels were
measured at varying concentrations of gelators. Hydrogels
were thermoreversible in nature, turning to sol when heated
and again forming semisolid gels upon cooling down to room
temperature. Tgel values of the hydrogels were between 45 and
60 °C at corresponding MGCs, which is quite similar to those
reported for long alkyl chain based hydrogels (Figure S1, see
the Supporting Information). Also, in concurrence with
previous studies, Tgel values were found to increase with
increasing concentration of gelators.31
Microscopy Studies. In order to find out the structural
morphologies of the hydrogels at the self-assembled state, they
were visualized through FESEM and TEM. Among the free
amines, 2 was chosen as a representative for its better gelation
ability. The FESEM image of 2 showed supramolecular
networks comprising nanometer-sized fibrils. Individual fibers
of ∼40 nm in thickness were entangled to form thicker fibers
(Figure 1a). We also carried out microscopic investigation of
hydrogels of quaternized salts 2a and 3a (once again chosen for
their superior water gelation ability) to find out whether
modification at the molecular level brought any changes in
supramolecular self-assembly. The amphiphilic gelator 2a
featuring L-phenylalanine at the polar head exhibited supra-
molecular meshlike structures comprising fibers having a
diameter of ∼1 μm and several micrometers in length. The
supramolecular fibrils were interconnected with each other
thereby forming a greater network (Figure 1b). However, in the
case of 3a, (L-tryptophan at the polar head), porous structures
(or spherical voids) were observed in the self-assembled state in
the corresponding FESEM image (Figure 1c) with pore
diameters varying from 0.5 to 2 μm.44 The zoomed image in
TEM also shows similar voids within the supramolecular mesh
of the self-assembled amphiphiles (Figure S2, Supporting
Information). In a few reports, it was observed that
modification of a particular polymeric molecule with cholesterol
resulted in the formation of porous networks. Such a difference
in supramolecular morphologies is quite intriguing and might
be due to the change in the molecular architecture of the
amphiphiles that modulated the self-assembling behavior of the
cholesterol-based hydrogels. A similar difference in supra-
molecular architecture was also noted in TEM images of the
corresponding hydrogels. The TEM image of 2 exhibited
supramolecular structures that are interconnected with thin
fibers (Figure 1d). The fibers are visible at the terminal of the
supramolecular networks. Hydrogels of 2a showed fibrillar
meshlike morphology, while 3a revealed spherical voids in the
self-assembled state (Figure 1e,f). Self-association of the
amphiphiles generally results in the formation of fibrillar or
other supramolecular structures that entraps solvent mole-
cules.51 However, the combination of a cholesterol moiety as
the hydrophobic unit and a quaternized ammonium group as
the hydrophilic domain resulted in a supramolecular hydrogel
of different self-assembling morphologies.
CD Spectroscopy. Gelation is a phenomenon that is visibly
identified from the “stable to inversion” nature of the
amphiphilic solution at MGC. However, self-association of
these amphiphiles initiates at a much lower concentration,
which transforms into higher order aggregates with increasing
concentration of amphiphile resulting in the formation of gel at
MGC. The nature of supramolecular aggregation that depends
on the molecular structure of the amphiphile was investigated
using CD spectroscopy. Hydrogelator 2 showed a negative
cotton effect with double minima peaks at 208 and 228 nm
(Figure 2a). These peaks became more prominent with the
increase in the concentration of amphiphile from 0.0025% to
0.1% w/v. The observed nature of the CD spectrum with
double minima is a characteristic of an α-helical structure of
proteins.52 Therefore, this α-helical type of arrangement of 2 in
the self-assembled state led to the formation of fibril network
structures that was observed in the corresponding microscopic
images (Figure 1). Nevertheless, there was a positive peak with
weak intensity at a longer wavelength (335 nm) in the CD
spectra of 2. This might be due to the n−π* transition involved
with the amide bond of the amphiphile.49 Interestingly, with
modification of the amine to 2a, the nature of supramolecular
association was found to be different. The amphiphile showed
positive cotton effects with two peaks at 203 and 213 nm
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Figure 3. FTIR spectra of (a) 2 and (b) 3a; (black line: gel state in D2O; red line: sol state in CHCl3).
(Figure 2b). In all the cases, higher ordered structures were
formed from the association of individual amphiphiles that
showed increase in the peak intensity with increasing gelator
concentration.
clearly indicates the presence of intermolecular hydrogen
bonding within the amide moieties of the neighboring
molecules. We also observed that the νN−H (amide A)
stretching frequency appeared in the range 3440−3445 cm−1 in
the nonself-assembled state in CHCl3. However, at the gel state
in D2O, the corresponding peak appeared in the range 3405−
3415 cm−1. This decrease is also characteristic of hydrogen-
bonded N−H stretching. Thus, hydrogen bonding is obviously
playing the crucial role in the process of self-assembled
hydrogelation.
The noncovalent supramolecular association of the self-
assembled hydrogel was studied using temperature-dependent
CD spectroscopy of 3a (chosen for its superior hydrogelation
ability). Initially, CD spectra of 3a in water were acquired at 20
°C (with an amphiphile concentration of 0.05% w/v) (Figure
2c). A positive cotton effect in the amide absorption region
(210 nm) was accompanied with a negative peak at 230 nm and
a shoulder at longer wavelength (270 nm). This particular
pattern of CD spectra is analogous to the β-sheet arrangement
of the peptide backbone,52 which has mostly dominated the
self-assembly of gelator 3a. Thus, a significant change in
supramolecular packing of the molecules in the self-assembled
state was observed with variation in the molecular structure of
the amphiphiles. The CD spectrum of 3a was further recorded
at 90 °C at the same concentration. The nature of the peaks
was similar to the spectra taken at 20 °C; however, the peak
intensity notably decreased. Hence, at higher temperature,
there was significant loss of the higher ordered aggregates that
formed through the supramolecular association of the
amphiphile. With lowering of temperature further to 20 °C,
the self-assembly of the gelator molecule was restored as the
observed CD spectrum almost matched with that of the initial
spectra taken at 20 °C (Figure 2c). Temperature-dependent
CD spectra clearly revealed the thermoreversible nature of the
self-assembled hydrogels because of the noncovalent supra-
molecular assembly, the nature of which depends on the
structure of amphiphile.
FTIR Spectra. FTIR spectroscopy is a powerful tool to
elucidate noncovalent interactions, especially hydrogen bond-
ing present within self-associated systems.31 To find out the
involvement of amide moieties toward hydrogen-bonding
interactions in self-assembled gelation, FTIR spectra of 2 and
3a (taken as representatives from both free amine and
quaternized category as efficient gelators) were recorded in
CHCl3 (for sol) and D2O (for gel state). It was observed that,
in the sol state, the amide-I (carbonyl) stretching frequencies
were at 1682 and 1699 cm−1 for 2 and 3a, respectively (Figure
3a, b). However the same peaks were shifted to lower
frequencies at 1670 and 1673 cm−1 for 2 and 3a, respectively
in D2O (Figure 3a,b). Such a decrease in the amide-I stretching
Luminescence Studies. Hydrophobic interaction among
the molecules is an important factor in the supramolecular
association in particular for hydrogelation.31 Involvement of
hydrophobic interaction in the case of cholesterol-based amino
acid containing gelators was investigated using an external
hydrophobic fluorophore, ANS. We have doped ANS (1 × 10−5
M) in the aqueous solution of amphiphilic hydrogelators with
varying concentration and noted the change in the nature of the
fluorescence peak. We have taken amphiphiles 2 and 3a as
representative examples. In the case of both gelators, the
emission peak of ANS increased initially with a blueshift (475
nm) with respect to native ANS (510 nm in water). In the case
of 2, the intensity of the ANS peak increased up to a
concentration of 1% w/v, which is 4 times below the MGC
(Figure S3a, Supporting Information). The increase in the
fluorescence intensity of ANS can be attributed to the
enhanced hydrophobicity in its microenvironment. Also, the
increase in the emission intensity was accompanied with a
further blueshift of the peak (∼3 nm). With further increase in
the concentration of 2, the intensity of the ANS peak was
decreased along with generation of an extra peak at a lower
wavelength (395 nm). Similarly, in the case of 3a, initially at
very low concentration, the ANS peak appeared at 474 nm, the
intensity of which increased with a blueshift (465 nm, Figure
S3b, Supporting Information) up to an amphiphile concen-
tration of 0.1% w/v (which is 9-fold lower than the MGC).
Such characteristic behavior of ANS clearly indicates the
participation of hydrophobic interaction during the self-
assembly. The emission intensity notably decreased with
increase in the concentration of 3a with another shoulder
peak appearing at lower wavelength. Thus, the gelation process
of the amphiphiles seemed to undergo via an intermediate state
of self-association at which ANS experienced maximum
hydrophobicity. Molecules within the self-assembled hydrogel
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1
Figure 4. (A) H NMR spectra of 3a (1% w/v) in D2O with varying temperature and (B) in DMSO-d6 with increasing H2O content.
Figure 5. Pictorial representation of the plausible packing of cholesterol-based amphiphilic hydrogelator 3a in the self-assembled state (counterions
were not shown).
might have experienced a less hydrophobic environment
compared to that of the intermediate state of gelation possibly
due to the well-ordered network at the gel state. At higher
concentrations of gelators, the splitting of the fluorescence
peaks may be a result of anisotropic heterogeneity or formation
of amphiphile bound ANS.31,53
At 40 °C, broad peaks started to appear at δ 7.33 ppm that got
shifted and separated to sharper peaks at 7.51 and 7.82 ppm at
80 °C (Figure 4A). This clearly indicates the involvement of
the aromatic rings in supramolecular gelation. At the gel state
(in D2O), the aromatic rings are involved in a strong π−π
interaction. As a result, the protons are not in their
characteristic spinning motion and could not exhibit sharp
individual peaks in the NMR spectra being in the aggregated
forms.49 With an increase in temperature, the intermolecular
noncovalent interactions get destroyed, and the amphiphile
loses its gelation efficiency leading to the transformation from
gel-to-sol where the protons showed their characteristic signals.
Similarly, participation of hydrogen-bonded interactions
NMR Spectroscopy. 1H NMR experiments were per-
formed to understand further the intermolecular interactions
involved in the self-assembled gelation. In a temperature-
1
dependent NMR study, H NMR of 3a (1% w/v) in D2O
hardly exhibited any peaks in the aromatic region at room
temperature. However, with an increase in the temperature, the
initially broad peak of aromatic protons appeared, which
gradually transformed to a sharper peak at a higher δ value at 80
°C (which is much above the Tgel of the gelator) (Figure 4A).
1
during gelation was studied through H NMR experiments
using 1% w/v 3a in DMSO-d6 with an increasing amount of
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water. Initially, the NMR of 3a (1% w/v) was recorded in
DMSO-d6 where the amphiphile was in the nonself-assembled
state. The indole N−H proton appeared at δ 11.03 ppm. Also
in DMSO-d6, amide protons showed characteristic sharp signals
at 8.33 and 8.49 ppm, since there is no self-assembling and H
bonding (Figure 4B). Water content in that system was then
gradually increased up to 30%. In general, the signals of the
NMR protons were broadened accompanied by a shift at lower
δ value. At 30% water content, the indole N−H proton was
observed at 10.73 ppm, and the amide protons were shifted to
7.99 and 8.32 ppm, respectively (Figure 4B). This upfield shift
clearly suggests the involvement of indole N−H and amide
protons in H bonding and also the contribution of the π−π
stacking interaction between the indole moiety to the self-
aggregation of the amphiphiles in the gel state.54 With an
increase in water content, self-assembly initiated, which
instigated the intermolecular packing of the molecules and
other noncovalent interactions.
XRD. In order to get detailed insight toward the molecular
packing of the amphiphiles in the self-assembled gel state, we
have performed wide-angle XRD of dried gels of 2 and 3a. The
thin film of dried hydrogel 2 gave a peak at 2θ = 10.5° that
corresponds to a d spacing of 8.42 Å. This is accompanied with
another broad peak at 2θ = 21.2° that corresponds to a d
spacing of 4.12 Å (Figure S4, Supporting Information). The
peak at higher 2θ (21.2°) designates the intermolecular
distance while the smaller 2θ (10.5°) was due to the spacing
between two consecutive layers. Similar peaks were also
observed in the xerogel of 3a, where the distance between
the two stacked layers was 9.5 Å (2θ = 9.39°) and that between
the two amphiphiles was 3.8 Å (2θ = 23.33°) (Figure S4,
Supporting Information and Figure 5).
An ordered arrangement of the cholesterol-based amino acid
containing amphiphilic gelators at the self-assembled state can
be suggested involving H-bonding, π−π stacking, and hydro-
phobic interactions on the basis of the above-discussed
spectroscopic and microscopic experiments (representative
example is given for 3a in Figure 5). The hydrophobic units
were stacked on one another with the hydrophilic terminals
stretched away on either side. H bonding between the amide
groups of the alternate molecules and the indole N−H with
H2O facilitated the self-association of the amphiphiles.
Additionally, π−π stacking between the aromatic groups (in
the case of 2, 2a, and 3a) contributed toward the gelation
phenomena (Figure 5). Individual molecules were oriented in a
layer-by-layer fashion that developed the higher ordered
packing in the three-dimensional network of the hydrogel.52
Cell Viability. The prime objective of choosing cholesterol
as the hydrophobic moiety in designing amphiphilic hydro-
gelators was to ensure that these newly developed gelators have
substantial biocompatibility. To this end, we have investigated
the cell viability of these amphiphilic hydrogelators toward
human hepatic cancer derived HepG2 cell line by MTT-based
assay (details given in the Experimental Section). Encourag-
ingly, all the hydrogelating amphiphiles showed significant
biocompatibility after a 6 h incubation period. At a
concentration of 5 μg mL−1, more than 95% of the cells were
found alive (Figure 6). With an increase in the concentration of
the amphiphile, a gradual decrease in the biocompatibility was
observed. However, even at a concentration of 100 μg mL−1,
55−70% cell viability was noted (Figure 6). Amphiphiles at
further higher concentration showed precipitation effects in the
cell culture media and as a result could not be used for MTT
Figure 6. MTT-based % cell viability of HepG2 cells with varying
concentration of amphiphilic hydrogelators (incubation period was 6
h).
experiments. The cationic gelators (1a−3a) exhibited slightly
enhanced cytotoxicity compared to that of the neutral amines
(1, 2).
The positively charged amphiphiles obviously had an
electrostatic interaction with the eukaryotic cell membrane
comprising zwitterionic lipids such as phosphatidylcholine and
sphingomyelin. However, at comparable concentrations,
cholesterol-based amphiphilic gelators exhibited notably higher
biocompatibility than the corresponding alkyl chain based
amino acid containing hydrogelators previously studied (Figure
S5, Supporting Information).The viability of cells in the
presence of amphiphile 2 was also checked using the Live/
Dead viability kit for mammalian cells. The bright green color
resulting from the enhanced fluorescence of DNA-intercalated
calcein indicated that most of the cells were alive. However,
negligible red fluorescence confirmed the absence of dead cells.
(Figure S6, Supporting Information). The hydrophobic long
chain is presumed to play a crucial role in penetrating the cell
membranes after favorable electrostatic interaction between the
cell surface and the polar headgroup. However, by substituting
the alkyl chain with cholesterol, the present hydrophobic unit is
possibly less effective in disrupting the eukaryotic cell
membrane. Moreover, the cholesterol-rich eukaryotic cell
membranes presumably could impede the inclusion of an
additional steroidal moiety at the cell surface. So, we have been
successful in bringing down the cytotoxicity of hydrogelators by
the inclusion of a cholesterol moiety in the amphiphile’s
structure. Thus, the rationally designed hydrogelators in the
present study having significant cytocompatibility might be
considered for the development of biomaterials/biomedicinal
implants in future.
Synthesis of AgNPs within Hydrogels and Antibacte-
rial Properties. Nosocomial infections are most common
obstacles associated with the development of implants for
biomedicinal use. In this context, many previously reported
hydrogelators were not found to be intrinsically antimicrobial.
However, a serious effort and attention is necessary to make
them capable of killing microbes.55−57 In this regard, recently, a
few cationic amphiphilic hydrogelators have shown effective
bactericidal property against Gram-positive bacteria; however,
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most of them were incapable to kill Gram-negative strains. On
the contrary, those amphiphilic hydrogelators also did not
exhibit substantial mammalian cell viability. In order to develop
effective antibacterial as well as biocompatible soft materials, we
used these cholesterol-based amphiphilic hydrogels for syn-
thesis and stabilization of AgNPs (well-known antibacterial
agents). Utilizing self-assembled systems for synthesis of metal
nanoparticles (MNPs) has been an interesting subject of
research in the recent past.42,43,55,58−60 In fact, efforts have been
concerted in developing gelator molecules with the ability to
synthesize and/or stabilize MNPs within the hydrogel matrix so
that the composite soft material can exhibit superior mechanical
or biological properties.42,43,55,58−60 Herein, we have synthe-
sized AgNPs in situ within cholesterol-based hydrogels by using
sunlight irradiation at physiological pH using AgNO3 as the
precursor. Sunlight-mediated AgNP synthesis is considered to
be a green technique for preparation of AgNPs that does not
involve the use of toxic chemicals or harmful UV irradiation.60
Amphiphiles 1 and 2 were used for this purpose with an
assumption that the terminal amine will provide additional
stability to the synthesized AgNPs along with the supra-
molecular networks of the hydrogel.59 The quaternized chloride
salts (1a−3a) obviously could not be used for the synthesis of
AgNPs from AgNO3 due to the precipitation of AgCl.
Hydrogels 1 and 2 were prepared at their MGCs (Table 1),
and AgNO3 was added to maintain a gelator/AgNO3
concentration at 10:1. The hydrogels (after addition of
AgNO3) were kept in sunlight, and after 5 min, transformation
of the colorless gel to yellow was observed. We monitored the
AgNP formation through UV−vis absorbance of diluted
aqueous solutions of the hydrogels. In the case of 1, a broad
peak around 420 nm was observed, while a relatively sharp peak
at 418 nm for 2 indicated the formation of AgNPs (Figure S7,
Supporting Information). Formation of AgNPs was also
followed by TEM that revealed the presence of stabilized
nanoparticles within the supramolecular network of hydrogel 2
(Figure 7a). Magnification of the same image showed
monodisperse nanoparticles of an average diameter of 8 nm
(Figure 7b).
We tested the antibacterial activity of these nanocomposites
through the broth dilution method against both Gram-positive
and Gram-negative bacteria to determine its potentials in
biomedicinal applications. The MIC (the lowest amphiphile
concentration at which no viable bacterial cell is present) values
for AgNP−1 were 70 and 50 μg mL−1 against Gram-positive
bacteria B. subtilis and S. aureus, respectively (Table 2). The
Table 2. MIC of Soft Nanocomposites in μg/mL
Gram-positive bacteria
Gram-negative bacteria
nanocomposite
B. subtilis
S. aureus
E. coli
K. aerogenes
AgNP−1
AgNP−2
70
30
50
20
100
60
75
50
MIC values in the case of Gram-negative bacteria were a little
higher (for E. coli, 100 μg mL−1, and for P. aeruginosa, 75 μg
mL−1, Table 2). Interestingly, the soft nanocomposite AgNP−2
exhibited better bactericidal property compared to that of
AgNP−1 having MIC values of 30 and 20 μg mL−1 for Gram-
positive B. subtilis and S. aureus, respectively, and MIC values of
60 and 50 μg mL−1 for Gram-negative E. coli and P. aeruginosa,
respectively (Table 2). It is well known that antibacterial
activity of AgNPs is dependent on the shapes and size of the
nanoparticles.61 Here, also, the AgNPs were synthesized in a
different supramolecular environment formed by hydrogelators
1 and 2. Also, the UV absorbance spectrum of AgNP−2
showed a sharp peak while that of AgNP−1 showed a broader
peak. This indicated a broad size distribution of nanoparticles in
the case of AgNP−1, which possibly reduces its antibacterial
efficacy. The killing ability of these soft composites against the
Gram-negative bacteria is relatively weaker than that against the
Gram-positive bacteria due to the presence of an extra layer of
protection by lipopolysaccharide in addition to the peptidogly-
can membrane for the former.39,42,55
Antibacterial activity was also investigated through the spread
plate method. For this purpose, the same amount of bacterial
cultures (E. coli) were added to test tubes containing AgNP−2
(80 μg mL−1, higher than the MIC value) and also in the
absence of the nanocomposite (control). After 5−7 h
incubation, 50 μL from each test tube was spread on agar
plates and kept at 37 °C for 24 h. We observed that, in absence
of the soft nanocomposite (the control plate), there was normal
growth of bacteria. However, no viable bacterial cell was seen in
the agar plate treated with AgNP−2 (Figure S9, Supporting
Information). Hence, the present AgNP−amphiphile compo-
sites exhibited broad spectrum antibacterial activity due to the
bactericidal property of AgNPs.
We also studied the antibacterial activity of AgNP
composites by fluorescence microscopy using the commercially
available LIVE/DEAD BacLight bacterial viability kit. 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 character-
istics. 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 membranes
appear red under the fluorescence microscope. Figure S10a (see
the Supporting Information) showed the control image of E.
coli, while Figure S10b showed the fluorescence images of the
E. coli cells treated with AgNP−2 (80 μg/mL) above their MIC
values. All untreated bacterial cells exhibited green fluorescence
Figure 7. TEM images of (a) AgNPs synthesized within self-
assemblies of hydrogelator 2; (b) magnified image of AgNPs.
This AgNP hydrogel composite was centrifuged, and the
pellet was washed twice with water to remove unreacted silver
ions. It was then lyophilized and dispersed in water. The
negatively stained TEM image of this dispersed nano-
composites showed similar sized nanoparticles within supra-
molecular fibrillar network (Figure S8, Supporting Informa-
tion).
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(Figure S10a, Supporting Information), indicating that they
were alive. However, the antimicrobial activity of the soft
nanocomposite was further confirmed from the observed red
fluorescence for bacterial cells treated with AgNP−2 (Figure
S10b, Supporting Information).
CONCLUSION
■
Cholesterol-based cationic and neutral hydrogelators compris-
ing different amino acids (aliphatic and aromatic) were
designed and synthesized. The amino acid at the polar head
bearing cationic charge exhibited better gelation ability
compared to the corresponding free amine containing
amphiphiles. Different spectroscopic and microscopic techni-
ques were employed to determine the involvement of different
noncovalent driving forces such as intermolecular H-bonding,
π−π stacking, and hydrophobic interactions of cholesterol in
the self-assembled gelation. Designing of amphiphilic hydro-
gelators using a cholesterol moiety as the hydrophobic segment
was successfully achieved. A judicious combination of hydro-
philic terminal with the cholesterol led to the development of
efficient hydrogels. Importantly, cholesterol-based amphiphiles
showed improved biocompatibility as compared to those having
a long alkyl chain as the hydrophobic part. Thus, introducing a
steroidal moiety can be an effective strategy for decreasing the
cytotoxicity of the system. Also, these biocompatible hydrogels
were transformed to antibacterial matrix by in situ synthesis of
AgNPs. The soft nanocomposite showed broad spectrum
antibacterial properties. Development of such soft materials
with significant cell viability complemented by antibacterial
property can be an effective step for designing smart
biomaterials in future.
ASSOCIATED CONTENT
* Supporting Information
■
S
(13) Pal, A.; Basit, H.; Sen, S.; Aswal, V. K.; Bhattacharya, S.
Structure and Properties of Two Component Hydrogels Comprising
Lithocholic Acid and Organic Amines. J. Mater. Chem. 2009, 19,
4325−4334.
(14) Barnard, A.; Posocco, P.; Pricl, S.; Calderon, M.; Haag, R.;
Hwang, M. E.; Shum, V. W. T.; Pack, D. W.; Smith, D. K. Degradable
Self-Assembling Dendrons for Gene Delivery: Experimental and
Theoretical Insights into the Barriers to Cellular Uptake. J. Am. Chem.
Soc. 2011, 133, 20288−20300.
Synthetic procedure of amphiphiles, Tgel of the amphiphilic
gelators, TEM image of 3a, luminescence spectra of ANS
within self-assemblies of 2 and 3a, XRD spectra of gels, cell
viability of alkyl chain based hydrogelators, fluorescence live/
dead images of HepG2, UV−vis spectra of AgNP, TEM image
of AgNP−2, growth of E. coli on agar plates in the absence and
presence of AgNP−2, and fluorescence images of control and
AgNP−2 treated bacteria. This material is available free of
charge via the Internet at http://pubs.acs.org.
(15) Boekhoven, J.; Koot, M.; Wezendonk, T. A.; Eelkema, R.; van
Esch, J. H. A Self-Assembled Delivery Platform with Post-production
Tunable Release Rate. J. Am. Chem. Soc. 2012, 134, 12908−12911.
(16) Jadhav, S. R.; Chiou, B. S.; Wood, D. F.; Hoffman, G. D.; Glenn,
G. M.; John, G. Molecular Gels-based Controlled Release Devices for
Pheromones. Soft Matter 2011, 7, 864−867.
AUTHOR INFORMATION
Corresponding Author
*Fax: +(91)-33-24732805; e-mail: bcpkd@iacs.res.in.
■
(17) Lee, K. Y.; Mooney, D. J. Hydrogels for Tissue Engineering.
Chem. Rev. 2001, 101, 1869−1880.
Notes
The authors declare no competing financial interest.
(18) Jayawarna, V.; Ali, M.; Jowitt, T. A.; Miller, A. F.; Saiani, A.;
Gough, J. E.; Ulijn, R. V. Nanostructured Hydrogels for Three-
Dimensional Cell Culture Through Self-Assembly of Fluorenylme-
thoxycarbonyl−Dipeptides. Adv. Mater. 2006, 18, 611−614.
(19) Bieser, A. M.; Tiller, J. C. Supramolecular Self-Organization of
Potential Hydrogelators on Attracting Surfaces. Supramol. Chem. 2008,
20, 363−367.
ACKNOWLEDGMENTS
■
P.K.D. is thankful to Department of Science and Technology,
India, (SR/S1/OC-25/2011) for financial assistance. S.D., T.K.,
and D.M. acknowledge Council of Scientific and Industrial
Research, India, for research fellowships.
(20) Jadhav, R. S.; Vemula, P. K.; Kumar, R.; Raghavan, S. R.; John,
G. Sugar-Derived Phase-Selective Molecular Gelators as Model
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Co-networks Derived from Aqueous Solutions for Biocatalysis in
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