Refining hydrogelator design: Soft materials with improved gelation ability, biocompatibility and matrix for in situ synthesis of specific shaped GNP

Article abstract of DOI:10.1039/c1sm05608e

Despite the continuous surge in the development of new supramolecular gels, the prediction of a gelator's structure still remains elusive. It is also imperative to consolidate the existing inventory of gelators and devise ways to make the gels functional.

Full text of DOI:10.1039/c1sm05608e

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PAPER
Refining hydrogelator design: soft materials with improved gelation ability,
biocompatibility and matrix for in situ synthesis of specific shaped GNP†
*
Dibyendu Das, Subhabrata Maiti, Sayanti Brahmachari and Prasanta Kumar Das
Received 6th April 2011, Accepted 23rd May 2011
DOI: 10.1039/c1sm05608e
Despite the continuous surge in the development of new supramolecular gels, the prediction of
a gelator’s structure still remains elusive. It is also imperative to consolidate the existing inventory of
gelators and devise ways to make the gels functional. In the present work, L-phenylalanine based poor
(C-16) or non-gelating (C-12 tail) amphiphiles were converted to excellent gelators with the simple
incorporation of N-terminal protected amino acid/dipeptide at the end of the alkyl tail. More than 6-
fold enhancement in gelation efficiency was observed for amino acid/dipeptides incorporated at the tail
of amphiphile in comparison to the corresponding unmodified alkyl tail. Interestingly, amphiphile with
the tertiary butyloxycarbonyl (Boc) protected amino acid at the tail had better gelation ability than the
amphiphile with the aromatic Fmoc (N-fluorenyl-9-methoxycarbonyl) protecting group. Spectroscopic
investigations (XRD and FTIR) revealed that the modification at the tail compels the amphiphiles to
take a different course of self-assembly than that adopted by their predecessors (alkyl tailed gelator, C-
16). For example, in the case of the amphiphile having a dipeptide at the tail, formation of b-sheet
structure through anti-parallel arrangement between the molecules results in notable improvement in
its gelation ability. Most importantly, these tail modified amphiphiles were capable of in situ synthesis
of gold nanoparticles (GNPs) of specific shape without the help of any external reducing agents in the
newly developed soft materials. The biocompatibility of hydrogels is also crucial for their prolific
biomedicinal functions. MTT assay showed dramatic improvement in the biocompatibility of the tail
modified hydrogelators towards mammalian cells in comparison to the amphiphiles having no amino
acid at the tail.
excellent ability of immobilizing solvents of extreme nature from
Introduction
water, aromatic hydrocarbons to even ionic liquids.7,12,18–28 One
of the intriguing facets of this structure-gelation property ratio-
nale is that when the C-terminal of an aromatic amino acid is
attached with a long hydrophobic tail (C-12 to C-16), quaterni-
zation at the N-terminal yields hydrogelators. However, except
L-tryptophan containing gelators (minimum gelation concen-
tration (MGC) ¼ 0.3%, w/v), the other aromatic amino acids
based amphiphiles exhibited conspicuously poor water gelation
ability (MGC ¼ 5 and 4%, w/v, for L-phenylalanine and
L-tyrosine, respectively).7,19
Supramolecular gels, a fascinating class of soft materials, are
witnessing immense interest in recent times due to their relevance
in diversified fields.1–7 From sensors, actuators in molecular
electronics to tissue engineering and biomedicine, the need for
versatile soft materials has been felt across disciplines.8–17
However, it is often observed that when it comes to predicting
gelator structures, a serendipitous finding has prevailed over
rational designing. In fact foreseeing a gelator design gets even
more tentative when we intend to have task specific applications
of the soft materials. In this context, in recent years we have been
successful in establishing a structural rationale for developing
amino acid/dipeptide based amphiphilic gelators, which has
In the quest of improving gelation ability, continuous efforts
are mostly emphasized on the design and development of new
gelators. At the same time it would be really fascinating to devise
ways for upgrading the existing pool of gelators towards aug-
menting their gelation efficiency with the scope of incorporating
functionality to the developed gels. In this regard, exhaustive
studies at the polar head (from charge to molecular architecture)
of the amphiphilic hydrogelator have been achieved in several
previous reports.7,20,21,24–28 In contrast, variations in the alkyl tail
have been primarily restricted to trifling alterations in their chain
lengths and thereby to its hydrophobicity.11,18,21–23 The same
Department of Biological Chemistry, Indian Association for the Cultivation
of Science, Jadavpur, Kolkata, 700 032, India. E-mail: bcpkd@iacs.res.in;
Fax: +(91)-33-24732805
† Electronic supplementary information (ESI) available: Synthetic
scheme for amphiphiles 1–8; temperature dependent CD spectra; TEM
images of GNPs synthesized by compound 2 and UV-Vis absorbance
1
spectra of GNPs, H NMR spectra and Mass spectra of 1 and 3–8, 13C
NMR spectra of 4–6. See DOI: 10.1039/c1sm05608e
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alkyl tails of these existing amphiphiles can have a more prom-
inent role to play in supramolecular gelation if they are
compelled to participate in additional self assembling interac-
tions through judicious functionalization with a supplementary
hydrogen bonding moiety.
To this end, the present work reports the development of a new
class of amino acid based excellent hydrogelators (Fig. 1) by
a simple structural advancement at the tail of non-gelating or
poor gelating amphiphile. Incorporation of a tertiary butyloxy-
carbonyl (Boc) protected amino acid/dipeptides at the alkyl tail
Fig. 1 Structure of 1–8.
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of L-phenylalanine based cationic amphiphile results in
a remarkable improvement in gelation ability where a non-
gelator (1 having C-12 tail, Fig. 1) gets converted to an efficient
gelator, 5 (MGC ¼ 0.75% w/v, Table 1). In comparison to the C-
16 tail length analogue of L-phenylalanine based amphiphile 2
(MGC ¼ 5.0% w/v), more than a 6-fold enhancement in gelation
ability was observed for 5. The amino acid/dipeptide moiety at
the tail was varied in order to understand the gelation mechanism
and also to establish the structure-gelation property relation-
ships. Insights from XRD and FTIR spectroscopy strongly
indicated that the functionalization at the tail promotes
a different mode of self assembly which consequently enhanced
the gelation ability. The alteration in the supramolecular
arrangement of the self-assembled soft materials was used for in
situ synthesis of gold nanoparticles (GNPs) of different shapes
without the help of external reducing agents. Finally, the cell
viability of the newly developed hydrogelators was tested to
exploit their potentials in diversified biological applications.
being a natural precursor can possibly augment the biocompat-
ibility of the modified gelators.29,30 Thus, incorporation of an
amino acid as a hydrogen bonding unit at the tail of the
amphiphile would be interesting in view of its influence on
gelation. However, the proposed alteration should not be done at
the cost of losing the critical hydrophobicity of the alkyl tail
needed for supramolecular gelation. In fact, it was observed that
the gelation ability plummets with the truncation or complete
removal of the alkyl tail as the hydrophobic and hydrophilic
balance is severely disturbed.18,23 In order to better understand
the proposed effect, we have used an aromatic amino acid L-
phenylalanine based cationic amphiphile with a hydrophobic tail
of moderate length (twelve carbons, C-12, 1, Fig. 1). Amphiphile
1 has no gelation ability and was soluble in water which is
expected considering the rather poor gelation ability of its longer
alkyl tail (C-16) analogues, 2 with MGC of 5.0% w/v (Table 1).7
The same structural unit as present in the head group (L-
phenylalanine with the quaternized amine) was at first attached
as the hydrogen bonding unit at the tail of the amphiphile 1
through an amide linkage (3, Fig. 1). However, this L-phenyl-
alanine based di-cationic bolaamphiphile (3) did not exhibit any
gelation ability in spite of having an additional H-bonding
moiety. Rather 3 was found to be soluble in water probably due
to the presence of too much hydrophilicity in the amphiphile.
The integration of cationic quaternary group on either side of the
molecule results in the deviation from optimum hydrophilic-
lipophilic balance (HLB) required for hydrogelation. To reduce
the hydrophilicity at the tail, the N-terminal of L-phenylalanine
was protected with the Fmoc (N-fluorenyl-9-methoxycarbonyl)
group (4, Fig. 1) instead of having an additional quaternized
amine. This modification would not only decrease the hydro-
philic content of the amphiphile, but simultaneously will induce
certain degree of hydrophobicity through the planar aromatic
moiety, which is very well known to facilitate self-assembled
gelation due to the strong p–p interaction between the aromatic
rings.21,29,31,32 Indeed, this alteration induced notable water
gelation ability within amphiphile 4 having a MGC of 1.4% w/v
at 25 ꢀC (Table 1). Incorporation of additional H-bonding
moiety along with aromatic rings at the alkyl tail resulted in the
conversion of non-gelator 1 to an efficient hydrogelator 4.
Considering the observed improvement in gelation ability in the
case of 4, it would be interesting to investigate the influence of the
other protecting group of amino acid on the gelation ability of tail
modified amphiphilic hydrogelators. For this purpose, instead of
the Fmoc moiety, the N-terminal of L-phenylalanine was protected
with another hydrophobic but non-aromatic Boc group (5, Fig. 1).
This modification remarkably enhanced the hydrogelation ability
further for amphiphile 5 with a MGC of 0.75% w/v (Table 1). Here
also the non-gelator 1 gets converted to an excellent hydrogelator 5
due to the inclusion of H-bonding moiety at the alkyl tail with
required hydrophobicity. Strikingly, the presence of a non-planar
hydrophobic moiety in 5 resulted in a superior gelation than the
planar Fmoc containing 4. Possibly in the case of 5, the optimum
HLB required for efficient hydrogelation had been achieved which
is to some extent lost in 4 due to the extended aromaticity of Fmoc
moiety. Notably, more than a 6-fold enhancement in gelation
efficiency was observed for 5 in comparison to the L-phenylalanine
based amphiphilic hydrogelator with C-16 alkyl tail, 2 (MGC ¼
5.0% w/v, Table 1). In this context, Smith and co-workers have
Results and discussion
Designing new gelators is a continuous evolution where molec-
ular structure is the key parameter in dictating the self-assembled
supramolecular gelation. In this regard, it is important to
consolidate knowledge on the structure of existing library of
gelator molecules. Judicious analysis on the influence of different
segments of these molecules in gelation can bring in the thought
of including functionality at the needed domain of the weak/non-
gelators to improve its gelation ability. In this context, a library
of amphiphilic hydrogelators based on the conventional surfac-
tant architecture featuring a polar head of either cationic or
anionic charge with a long hydrophobic tail have been reported
in recent years.18,21–23,27,28 In contrast to the most discussed
influence of the polar head, the role of a long alkyl tail in gelation
has always been overlooked except in few works possibly because
of its typical as well as well-established participation mechanism
in self-assembly.11,18,21–23 At this point, we were curious to
investigate whether the influence of this alkyl tail can be made to
be dramatic towards improving the supramolecular gelation
efficiency of an amphiphile and thereby its applications as
functional soft materials.
It is well known from the literature that hydrogen bonding
units like amide bond play an instrumental role in the self-
assembled gelation.22,27–30 In this regard, an amino acid has the
requisites to provide additional hydrogen bonding interactions,
especially when it is attached through an amide bond and also
Table 1 Gelation results of amphiphiles
Amphiphile
Statea
MGC (%, w/v)
1
2
3
4
5
6
7
8
S
TG
S
TG
TG
TG
S
—
5.0
—
1.4
0.75
2.4
—
TG
1.9
a
S: solution (soluble up to 8%, w/v); TG: transparent gel.
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highlighted the important role of protecting groups of the gelators
in modifying the solubility and the saturation point of the system.
This concurrently determines the concentration at which network
formation takes place leading to gelation.33
(at MGC) were investigated by field emission scanning electron
microscopy (FESEM). Fibrous networks were visible in the
microscopic images of all the gels (Fig. 2a–d) and they are
different in their nature and sizes. In the case of 4, the thickness
of the fibers was ꢂ100 to 125 nm (Fig. 2a) and the length varied
up to micrometers. Interestingly, with the simple alteration in
the protection group of the N-terminal end (L-phenylalanine)
from Fmoc (4) to Boc (5), a small but significant change in the
nature of the fiber was observed (Fig. 2b). Presumably, the
molecular structures had an important role in determining
the morphology of the networks. In the case of the dried gel of 5
(Fig. 2b), the networks were formed mainly by fibers of diam-
eters ranging from ꢂ200 nm to even ꢂ1 mm, where the thicker
fibers are entangled with some very thin fibers. It can also be
clearly observed (Fig. 2b) that the intertwined networks were
made from the entanglement of thick fibers with pores and
thereby providing the capillary forces required entrapment of
large number of water molecules (MGC ¼ 0.75% w/v).
Furthermore, it was also observed that the fibers of smaller
diameters (ꢂ100 nm) are coiled together to form thicker fibers
of ꢂ1 mm. The lengths of the fibers ranged up to several
micrometers with various nodes from where the networks
seemed to propagate. Upon incorporation of a diphenylalanine
unit (N-terminal with Boc protection) at the tail of the amphi-
phile, the diameter of the fibrils decreased (Fig. 2c).35 Numerous
thin fibrils (ꢂ50 nm) were visible in the FESEM images, with
some fibrils coiling together to form thicker fibers (ꢂ80–
100 nm). The xerogel of 8 which had a dipeptide of L-alanine
and L-phenylalanine at the tail, also showed intertwined fibrils
(Fig. 2d). Furthermore, the thickness of the fibers was found to
be little larger (ꢂ110 nm) than that observed for 6 with very less
population of thinner fibrils. Intriguingly, in comparison to the
current set of gelators having a N-terminal protected amino
acid/dipeptide residue at the terminal end of the alkyl tail, the
FESEM image of their predecessor gelator with C-16 alkyl tail,
2 (MGC ¼ 5.0% w/v) had a very less intertwined fibrous
morphology.7 Rather very poorly constructed thick fibers (500–
700 nm) were observed for 2. The lower predominance of the
The observation in the preceding paragraph instigated us to
investigate the gelation efficiency of amphiphiles upon intro-
ducing a dipeptide moiety at the tail of 1. To this end, amphiphile 6
was synthesized by attaching a diphenylalanine unit (N-terminal
protected with Boc) at the tail through an amide bond. Once again
the tail modified amphiphile, 6 exhibited improved water gelation
ability with MGC ¼ 2.4% w/v. However, in comparison to the
amphiphilic gelator 5 having a single L-phenylalanine residue at
the tail, the presence of an additional aromatic amino acid
(L-phenylalanine) in the dipeptide unit did not augment the
gelation efficiency presumably due to the loss in the HLB for
amphiphile 6. The loss in the gelation ability due to the added
aromaticity was also observed for amphiphile 7 having a Boc-
protected L-tryptophan-L-phenylalanine dipeptide at the
terminal end of the long chain. This extended aromaticity of
indole moiety in tryptophan even converted a gelator (6) to
a non-gelator (7). This loss of gelation ability could be due to the
variation in the hydropathy indices of the amino acids.12,34
Hydropathy index is the measure of the relative hydrophobic and
hydrophilic properties of the side chain residues present in an
amino acid. According to this series, hydrophobic amino acids
have higher hydropathy indices. Although 6 to 8 have a dipeptide
unit at the tail, the HLB of the amino acid present at the terminal
end have contrasting hydropathy indices (while L-phenylalanine
for 6 and L-alanine for 8 has positive hydropathy number of 2.8
and 1.8, respectively, L-tryptophan for 7 has an index of ꢁ0.9).
Interestingly, this index implies that although both phenylalanine
and tryptophan have aromatic side chain residues, the presence of
L-tryptophan in the tail of 7 incorporates substantial degree of
hydrophilicity to the amphiphile resulting in water solubility. An
extended indole ring containing
a hydrophilic-NH group
presumably imparts this additional hydrophilicity to L-trypto-
phan.33 To avoid this extra aromaticity and hydrophilicity, the
additional amino acid in dipeptide moiety was replaced with
L-alanine (8, the hydropathy index of L-alanine is 1.8). Interest-
ingly, the gelation ability was regained in 8 (MGC ¼ 1.9% w/v,
Table 1) by reducing the aromaticity at the hydrophobic domain
of the amphiphile. Even, the gelation ability increased in 8 with
respect to 6, but it was found to be lower than 5. Considering the
gelation ability of all the designed amphiphiles (4–8), it can be
inferred that the presence of an amino acid or peptide (with the
protected N-terminal) at the tail has a crucial role to induct
gelation ability within these molecules. However, the incorpora-
tion of one aromatic amino acid with an appropriate protecting
group (non-aromatic) at the tail of a non-gelating amphiphile was
found to be the befitting alteration for the development of the
most efficient hydrogelator, 5. All the hydrogels were thermor-
eversible and the gel to sol transition temperature was ꢂ50–60 ꢀC
at their MGC.
Characterization of gels
Scanning electron microscopy (SEM). To investigate the
supramolecular morphologies formed by the amphiphilic
hydrogelators (4–6, 8) in the self assembled state, their dried gels
Fig. 2 (a–d) FESEM images of the dried hydrogels of 4, 5, 6 and 8
respectively at MGC.
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networks with nodes concurrently implies smaller population of
state in D2O (Fig. 4b). Similar shifts in the carbonyl stretching
frequency (at amide-I region) were observed for gelator 4 and 6
(Fig. 4a, c). The intermolecular H-bonding between the carbonyl
groups of one molecule with the amide-NH of another molecule
at the gel state is evident from this shifting.12,21
pores in 2 resulting in weaker gelation.
Atomic force microscopy (AFM). AFM was also utilized to
further investigate the morphology of the gelators 5, 6 and 8.
Expectedly, fibrous morphology of these amphiphiles was found
to be present in the dried gels (Fig. 3). In concurrence with the
observed FESEM images, xerogel of the best gelator, 5 showed
intertwined fibrous network with the fiber diameter ꢂ150–
200 nm (Fig. 3a). However, for 6 the fiber thickness was found to
be ꢂ100 nm (Fig. 3b) and also entangled networks were evident
in the case of gelator 8 (Fig. 3c). Therefore, the microscopic
investigations indicate that the introduction of an amino acid/
dipeptide unit at the terminal end of the alkyl tail significantly
modifies the supramolecular arrangements of the gelators which
concurrently affect the gelation ability.
X-ray diffraction (XRD). In order to further establish that the
presence of a Boc protected amino acid/dipeptide at the tail of an
amphiphilic gelator results in marked alteration in the molecular
packing and the possible orientation of the gelator in the
supramolecular fibrous networks, we have taken the X-ray
diffraction (XRD) spectra of the dried gels of 2, 5 and 6 (Fig. 5).
In case of the weak gelator 2, the small angle XRD peak (Fig. 5a)
corresponds to a d-spacing of 3.5 nm, which is smaller than twice
the fully extended molecular length of the individual molecule
(the length was calculated by the MOPAC AM1 method, CS
Chem. Office). Therefore, in the absence of any hydrogen
bonding unit at the tail of the amphiphile 2, the hydrophobic
interaction helps in the interdigitation of the long alkyl tails.
However, the presence of an amino acid/peptide at the tail of
a new set of gelators could hinder the interdigitation between
alkyl tails. Indeed, the XRD spectra of the gelators 5 and 6 did
not have any small angle peaks which rules out the formation of
any interdigitated bilayer structure through the overlapping of
alkyl tails (Fig. 5b).12,21,39 Instead these gelators showed peaks
within the 8 to 25ꢀ region of 2q. Interestingly, the xerogel
obtained from 5 gave a peak at 2q ¼ 19.35ꢀ which corresponds to
FTIR. At this point we were intrigued to know how this extra
amino acid or dipeptide at the tail of the amphiphile facilitates
the supramolecular self-assembled hydrogelation. As this amino
acid or peptide moieties are linked through amide bonds, the
involvement of hydrogen bonding could be very influential
during the self-assembly of new gelators. To probe this, FTIR
spectra of 4, 5 and 6 were taken both at the self-assembled state
(in D2O) and at non self-assembled state (in chloroform, Fig. 4).
As a control, the FTIR spectrum of 2 was also taken under
similar conditions. Intriguingly, the carbonyl bonds of the new
gelators showed a splitting in their amide-I stretching frequency.
In the case of 4, the peaks split in two sub peaks where the peak
with large intensity appeared at 1648 cmꢁ1 which was associated
with a weak peak at 1685 cmꢁ1 (Fig. 4a, d). This splitting of the
amide peak was not there in the case of 2 (Fig. 4d). Similarly, in
the case of 5 and 6, the peaks were visibly split in to one strong
and one weak peak (1650 and 1675 cmꢁ1 for 5 and 1639 and
1682 cmꢁ1 for 6, Fig. 4b–d). This splitting of amide peaks is
known to be have been observed in the case of b-sheets in
proteins and peptides where amide bonds are oriented in anti-
parallel mode.36–38 Furthermore, the difference in the nature of
the peaks with respect to the amphiphile having the unmodified
alkyl tail (2), clearly indicates the involvement of the amide
bonds in the formation of antiparallel arrangement during
supramolecular gelation of tail modified amphiphiles. Apart
from this splitting, the carbonyl stretching frequency (amide-I)
which appeared at 1681 cmꢁ1 in chloroform for 5 registered blue
shift (1650 cmꢁ1) when the spectra was taken at the self assembled
ꢀ
a d-spacing of 4.58 A, which matches very well with the distance
between the two peptide backbones present in a conventional
b-sheet. This peak is accompanied by another peak at 2q ¼ 9.63ꢀ
ꢀ
with a d-spacing of 9.4 A (Fig. 5b). These two peaks usually
indicate the presence of cross b-structure between the dipeptide
moieties with an anti-parallel alignment in the peptide
strand.40–42 The distance of 9.4 A is the spacing between two
ꢀ
laminates. Furthermore the peak at 2q ¼ 22.18ꢀ corresponds to
ꢀ
a distance of 4.0 A which indicates the distance between the two
aromatic rings of the amino acid (L-phenylalanine) present at the
tail (Fig. 6a). Similarly, in the case of 6, peaks at 2q ¼ 9.85ꢀ
ꢀ
ꢀ
corresponded to a distance of 9.33 A. The peak at 2q ¼ 19.83
ꢀ
corresponds to a distance of 4.47 A which could be the distance
between the two molecules comprising of peptide backbone.
Here also, a peak at 2q ¼ 21.38ꢀ also indicated the possible
ꢀ
aromatic residue stacking separated by 4.15 A. The spectroscopic
evidence gathered from FTIR and XRD helped us to propose
a schematic representation of the possible self-assembly via an
Fig. 3 AFM images of 5, 6 and 8 at their respective MGC.
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Fig. 4 (a–c) FT-IR spectra of 4–6 in CHCl3 solution (---) and in D2O at gel state (—). (d) The strong and weak amide I transmission band of 2 and 4–6.
antiparallel arrangement of the tail modified gelators (Fig. 6). In
the case of 6, due to the presence of a dipeptide at the tail, the
conformation can be termed as cross b-sheet with alternating
10-membered and 14-membered H-bonding (shown in Fig. 6b).
of the same.43–46 Interestingly, in the case of 6, the presence of one
more L-phenylalanine residue (dipeptide) at the tail resulted in
negative cotton effects in the CD spectra exhibiting a minima at
ꢂ210 nm with a positive peak at 195 nm (Fig. 7b). The spectrum
is characteristic of the b-sheet structure of a protein (Fig. 7b).47–49
However, no such specific CD signature peak was observed for 5.
Despite the amide bonds probably being arranged in an anti-
parallel fashion (in accordance with FTIR and XRD spectra),
the presence of only one amino acid at the tail of the amphiphile
5, rules out the possibility of a conventional b-sheet structure.
The b-sheet hydrogen bonding demands at least the presence of
a peptide bond (two amino acids). Accordingly, a b-sheet CD
profile was observed in the case of the dipeptide incorporated
Circular dichroism (CD). The supramolecular arrangement in
the self-organized aggregates of the tail modified hydrogelators
was also investigated by taking the circular dichroism (CD)
spectra of the aqueous solutions of 5 and 6 (Fig. 7). Amphiphile 5
showed positive cotton effect with two maxima in the CD spectra
(Fig. 7a) at the amide absorption region, 200-225 nm. This could
be attributed to the p–p* transition of the amide bond along
with a shoulder at a longer wavelength from the n–p* transition
Fig. 5 XRD of the dried gels of (a) 2 and (b) 5 and 6.
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Fig. 6 Probable arrangement of 5 and 6 in the self-assembled gel state.
amphiphile 6. Furthermore, with increasing monomer concen-
tration there was a sharp increase in peak intensity in both the
cases (5 and 6). This observation indicates that the formation of
supramolecular network originates from the self-assembled
organization of individual molecules.39 The non-covalent
packing of the molecules was further confirmed from the
temperature dependent CD experiment of 5 and 6 at 0.025%, w/v
(Fig. S1, see ESI)† in water. In both the cases, intensity of the CD
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Accordingly we used the present set of amphiphiles for the
synthesis of GNPs at their respective MGC. Two different ratios
(HAuCl4 : gelator concentrations) were used (1 : 10 and 1 : 20),
keeping the concentration of the gelator fixed at MGC. All the
gelators were capable of synthesizing GNP in situ at room
temperature without the need of any external reducing agents
(see experimental section for details). Formation of GNP was
evident from the corresponding surface plasmon resonance
(SPR) peak in the UV-vis spectra (Fig. S3, ESI†).51,59 The shape
and size of the in situ synthesized GNPs was investigated by
taking their TEM images. In the case of gelator 5, a close view at
the TEM image obtained from a 1 : 10 ratio showed the presence
of some distinctly octahedral shaped GNPs (Fig. 8a, indicated by
arrows and 8b) of 15 to 30 nm dimensions. Also, the corre-
sponding selective area electron diffraction (SAED) pattern
revealed the presence of octahedral gold crystals (Fig. 8c).
However, in the case of a 1 : 20 concentration ratio of HAuCl4 to
gelator (5), no such specific shaped GNP was seen in the corre-
sponding TEM image (Fig. 8d). This shape controlled synthesis
of GNP in the supramolecular network 5 encouraged us to use
the hydrogelators having dipeptides at the alkyl tail (6 and 8) at
MGC under similar experimental conditions. Interestingly, when
chloroaurate ions were reduced by amphiphile 6 (with dipheny-
lalanine at the tail) at a ratio of 1 : 10, the TEM image showed
maximum population (ꢂ80%) of octahedron shaped GNPs
(Fig. 9a–c) with varying sizes of 25 to 45 nm. However, at the
concentration ratio of 1 : 20 (HAuCl4 : 6), GNPs of different
shapes were formed (2D triangular, decahedron, octahedron and
some spherical, Fig. 9d–f). Similarly, in case of 8 at a 1 : 10 ratio,
the predominance of octahedron was also unambiguous (Fig. 9g,
h), while at a 1 : 20 ratio the shape of nanoparticles was found to
Fig. 7 CD spectra of 5 and 6 with varying concentration (%, w/v) in
plain water at 25 ꢀC.
peak gradually decreased with increase in temperature from 20 to
70 ꢀC due to the destruction of the 3D-aggregated structure.
Synthesis of GNP within supramolecular gels
At this point, we were intrigued to exploit these different
supramolecular networks obtained through the refinement in the
gelator’s structure (tail modification) in functional applications
like synthesizing gold nanoparticles (GNPs). In this regard,
development of soft nanocomposites is rapidly coming up in
recent times as advanced functional materials. The low molecular
weight gelators with diversified 3D supramolecular orientation
(fibers, helices, lamellar, etc) have started to gain importance as
templates for the synthesis of metal nanoparticles.50–54 However,
it is always desirable if the synthesis and stabilization of GNPs
(preferably of specific shape) is achieved at room temperature
without the need of any external stabilizing agents.55–58 Amphi-
phile 2 has the ability to reduce HAuCl4 to stable spherical
shaped GNPs (Fig. S2, ESI).† Interestingly, the presence of extra
aromatic amino acid/dipeptide along with amide bond at the tail
of the new set of amphiphilic gelators (Fig. 1) with dissimilar self-
assembled network compared to 2, may influence the nanoscale
environment which will be offered as the breeding ground for the
synthesis of varying shaped GNPs.51,59–64
Fig. 8 TEM images of GNPs (a, b in gel 5) at a 1 : 10 concentration ratio
(HAuCl4 : 5) and (c) the corresponding SAED pattern of the synthesized
GNP. (d) TEM image of GNPs at 1 : 20 concentration ratio
(HAuCl4 : 5). The arrows in (a) indicate octahedral shaped GNPs.
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Fig. 9 (a–c) TEM images of GNPs in gel 6 at a 1 : 10 concentration ratio (HAuCl4 : 6) (c shows a tilted octahedron). (d–f) TEM images of GNPs in gel 6
at a 1 : 20 concentration ratio (HAuCl4 : 6) (g and h) TEM image of GNPs in gel 8 at a 1 : 10 concentration ratio (HAuCl4 : 8). (i) TEM image of GNPs
in gel 8 at a 1 : 20 concentration ratio (HAuCl4 : 8).
be irregular (Fig. 9i). Hence, the supramolecular nanoscale
environment formed due to different mode of self-assembly by
the tail modified amphiphilic gelators led to the synthesis of
distinctly different shaped GNPs.
Cytocompatibility. Notably, hydrogels have received great
attention as potential materials for biological applications; from
antibacterial coatings, tissue engineering scaffolds to drug
delivery systems and so forth.55,65–69 In this regard, structural
refinements of existing hydrogelators with improved biocom-
patibility can further bolster these bio-applications. In previous
works, it has been proposed that the lipophilic chain of amphi-
phile can help in the penetration into the cell membrane resulting
in cytotoxicity.70,71 Thus, it is expected that the presence of an
amino acid/dipeptide unit at the tail of the amphiphile will
presumably diminish the lipophilic content of the alkyl chain and
consequently impede the penetration ability of the amphiphile.
To this end, the biocompatibility of the tail modified gelators (5
and 6) was tested against mammalian cells.72 Indeed, a significant
Fig. 10 MTT assay based percent HepG2 cell viability as a function of
concentration of 1, 2, 5 and 6. Each percent cell viability point represents
enhancement of biocompatibility was observed when Boc pro-
the mean ꢃ SD of six determinations.
tected L-phenylalanine/diphenylalanine was tethered to the tail
of 1. The poor cell viability of less than 35% for 1 and 2 (at
a concentration of 50 mg/mL, incubated with HepG2 cells for 3 h)
Experimental section
enhanced up to ꢂ90% for 5 and 6 (Fig. 10). Even in other
concentrations around a 2.5-fold enhancement in the biocom-
Materials
patibility was observed for the gelators 5 and 6 compared to their
predecessors with no amino acid at the tail (1 and 2). Encour-
agingly, even at higher concentrations of 200 and 400 mg/mL, the
cell viability for both the amphiphiles (5 and 6) was always
greater than 60%. Hence, the incorporation of N-protected
amino acid residues at the alkyl tail results in marked improve-
ment in biocompatibility towards mammalian cells.
All amino acids, di-tert-butyldicarbonate (Boc-anhydride), N,
N0-dicyclohexylcarbodiimide (DCC), 1-hydroxybenzotriazole
(HOBT), Fmoc chloride, solvents and other reagents were
procured from SRL, (India). Trifluoro acetic acid (TFA), iodo-
methane and sodium hydroxide were purchased from Spec-
trochem, India. Amberlite Ira 900 chloride ion exchange resins
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Soft Matter, 2011, 7, 7291–7303 | 7299

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for ion-exchange, 1,12- diaminododecane, HAuCl4 (30 wt.%) in
dilute hydrochloric acid solution, CDCl3 were obtained from
Aldrich chemical co. All the materials used in the cell culture
study such as DMEM, heat inactivated FBS, trypsin from
porcine pancreas and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-
2H-tetrazolium bromide (MTT) were obtained from Sigma
Aldrich Chemical Company. Thin layer chromatography was
performed on Merck precoated silica gel 60-F254 plates. 1H NMR
spectra were recorded in AVANCE 300 MHz (BRUKER)
spectrometer. Mass spectrometric data were acquired by the
electron spray ionization (ESI) technique on a Q-tof-micro
Quadruple mass spectrometer (Micromass).
carbonate and catalytic amount of 18-crown-6-ether in dry DCM
for 2–3 h. The reaction mixture was taken in ethyl acetate and
washed with 5% aqueous sodium thiosulphate solution and
water. The concentrated ethyl acetate part was then purified
through column chromatography using 60–120 mesh silica gel
and methanol/chloroform as the eluent. The column-purified
material was crystallized from methanol/diethylether to obtain
solid quaternized iodide salt. The iodide salt thus obtained was
subjected to ion exchange on Amberlite Ira-400 chloride ion
exchange resin column followed by crystallization from dieth-
ylether to get the pure chlorides (3). Overall yield was 60–70%.
Synthesis of amphiphiles 4, 5
Synthesis of amphiphiles
Fmoc Chloride (0.8 equivalent) and Boc anhydride (0.8 equiva-
lent) respectively were added drop wise into a solution of diamine
III (1 equivalent) in DCM (Scheme S1, ESI).† The solutions were
stirred overnight and the mixture was washed with brine to
remove the unreacted diamines. The mono protected amines
were then isolated and purified through column chromatography
using 60–120 mesh silica gel and methanol/chloroform as the
eluent. The amine (1 equivalent) thus obtained was quaternized
with excess iodomethane using anhydrous potassium carbonate
and catalytic amount of 18-crown-6-ether in dry DCM for 2–3 h.
The reaction mixture was taken in ethyl acetate and washed with
5% aqueous sodium thiosulphate solution and water. The
concentrated ethyl acetate part was then purified through
column chromatography using 60–120 mesh silica gel and
methanol/chloroform as the eluent. The column-purified mate-
rial was crystallized from methanol/diethylether to obtain solid
quaternized iodide salt. The iodide salt thus obtained was sub-
jected to ion exchange on Amberlite Ira-400 chloride ion
exchange resin column followed by crystallization from dieth-
ylether to get the pure chlorides (4, 5). Overall yield was 40–50%.
A generalized synthetic scheme for all the amphiphiles is given in
Scheme S1 (ESI).†
Synthesis of amphiphile 1 and 2
The amphiphiles were synthesized following the reported
protocols.7,19 Briefly, Boc-protected L-phenylalanine was
coupled with n-dodecylamine (for 1) and n-hexadecylamine (for
2) using DCC (1 equivalent) and catalytic amount of DMAP in
presence of 1 equivalent of HOBT in dry DCM. The product was
then purified through column chromatography using 60–120
mesh silica gel and acetone/hexane as the eluent. The obtained
amide was then deprotected by trifluoroacetic acid in dry DCM
by stirring the reaction for about 2 h. After removing the solvents
on a rotary evaporator, the mixture was then taken in ethyl
acetate and thoroughly washed with 10% aqueous sodium
carbonate solution followed by brine to neutrality. The organic
part was then collected over anhydrous Na2SO4 and evaporated
to dryness to get the primary amine. The primary amine
(1 equivalent) thus obtained was quaternized with excess iodo-
methane using anhydrous potassium carbonate and catalytic
amount of 18-crown-6-ether in dry DCM for 2–3 h. The reaction
mixture was taken in ethyl acetate and washed with 5% aqueous
sodium thiosulphate solution and water. The concentrated ethyl
acetate part was then purified through column chromatography
using 60–120 mesh silica gel and methanol/chloroform as the
eluent. The column-purified material was crystallized from
methanol/diethylether to obtain solid quaternized iodide salt.
The iodide salt thus obtained was subjected to ion exchange on
Amberlite Ira-400 chloride ion exchange resin column followed
by crystallization from diethylether to get the pure chlorides
(1, 2). Overall yield was 60–70%.
Synthesis of amphiphiles 6–8
The Boc-protected amino acids (L-phenylalanine/L-tryptophan/
L-alanine) were selectively coupled with the diamine compound
(III) in a similar way (Scheme S1, ESI)† as described above in
case of 3. The Boc-protected amino acid (0.8 equivalent) in dry
DCM was added drop wise into a dry DCM solution of the
compound III (1 equivalent), HOBt (1.1 equivalent) DCC (1.1
equivalent) and DMAP (1.1 equivalent) under ice cold condition.
The reaction was stirred for 12 h at room temperature and usual
base work-up was done to remove the unreacted materials. The
mono Boc-protected amine was then isolated through column
chromatography using 60–120 mesh silica gel and methanol/
chloroform as the eluent. Finally the amine was quaternized with
excess iodomethane using anhydrous potassium carbonate and
catalytic amount of 18-crown-6-ether in dry DCM for 2–3 h. The
reaction mixture was taken in ethyl acetate and washed with 5%
aqueous sodium thiosulphate solution and water. The concen-
trated ethyl acetate part was then again purified through column
chromatography using 60–120 mesh silica gel and methanol/
chloroform as the eluent. The iodide salt thus obtained was
subjected to ion exchange on Amberlite Ira-400 chloride ion
exchange resin column followed by crystallization from dieth-
ylether to get the pure chlorides (6–8). Overall yield was 30–35%.
Synthesis of amphiphile 3
In brief, Boc-protected L-phenylalanine was coupled with 1,12-
dodecylamine using DCC (2 equivalents) and catalytic amount
of DMAP in presence of 2 equivalent of HOBT in dry DCM. The
mixture was stirred overnight and the solution was washed with
HCl followed by sodium carbonate to isolate the coupled
compound. The compound was then column purified using 60–
120 mesh silica gel and acetone/hexane as the eluent. The
obtained compound was then deprotected using TFA as
described above. The diamine III (Scheme S1, ESI)† was then
reacted with excess iodomethane using anhydrous potassium
7300 | Soft Matter, 2011, 7, 7291–7303
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Characterization data for all the amphiphiles are given below
except for 2 which was reported earlier.7 1H NMR, mass spectra
of all other amphiphiles and 13C NMR of most of the gelators are
provided in the ESI (Fig. S4–S10).†
15H, aromatic protons), 8.68 (s,1H, CH2NHCOCHNMe3); 13C
NMR (75 MHz, CDCl3): d 170.94, 170.17, 164.90, 162.33, 136.21,
129.64, 129.34, 129.25, 128.98, 128.75, 128.58, 127.79, 127.14,
126.96, 118.16, 80.54, 76.63, 73.52, 52.80, 39.65, 37.84, 32.88, 29.41,
29.34, 29.24, 29.16, 28.15, 26.77. ESI-MS: m/z: calcd for
C47H70N5O5: (the 4ꢀ ammonium ion, 100%): 784.5371, found
784.5347 (M+).
Data for 1. Anal calcd (%) for C24H43N2OCl: C, 70.12; H,
10.54; N, 6.81. Found: C, 70.02; H, 10.41; N, 6.76; 1H NMR (300
MHz, CDCl3, 25 ꢀC, TMS) d ¼ 0.81 (t, 3H, -(CH2)11–CH3),
1.08–1.23 (br, 20H, –CH2(CH2)10CH3), 2.81–3.11 (m, 2H,
CHCH2Ph), 3.16–3.19 (d, 2H, NHCH2(CH2)10), 3.39 (s, 9H, N
(CH3)3), 5.71–5.81 (q, 1H, COCHNMe3), 7.16– 7.29 (m, 5H,
aromatic protons), 8.74 (s, 1H, CONHCH2); ESI-MS: m/z: calcd
for C24H43N2O: (the 4ꢀ ammonium ion, 100%): 375.3370, found
375.3374 (M+).
Data for 7. Anal calcd (%) for C49H71N6O5Cl: C, 68.47;
1
H, 8.33; N, 9.78. Found: C, 68.49; H, 8.21; N, 9.83; H NMR
(300 MHz, CDCl3, 25 ꢀC, TMS): d ¼ 0.90–1.22 (m, 16H,
CH2(CH2)8CH2), 1.30 (s, 9H, OC(CH3)3), 1.38–1.58 (m,
4H,
CH2(CH2)8CH2),
3.03–3.24
(m,
8H,
CH2Ph,
NHCH2(CH2)10CH2), 3.40 (s, 9H, N(CH3)3), 3.43–3.44 (m, 2H,
CH2-indole), 4.29–4.30 (m, 1H, NHCHCH2Ph), 4.58–4.59
(m, 1H, OCONHCH-Trp), 5.07 (t, 1H, COCHNMe3), 6.40 (s,
1H, NHCHCONHCHCO), 6.85 (s, 1H, OCONHCH), 7.05 (s,
2H, CONHCH2, CH2NHCOCHNMe3), 7.11–7.34 (m, 15H,
aromatic protons), 8.74 (s, 1H, CHNHC); 13C NMR (75 MHz,
CDCl3): d 171.63, 170.41, 164.77, 142.44, 136.40, 133.12, 129.46,
129.27, 128.59, 128.32, 128.23, 127.34, 126.54, 123.60, 121.47,
118.87, 118.40, 111.54, 79.78, 73.57, 69.72, 52.24, 39.86, 39.58,
39.30, 39.19, 39.02, 32.57, 32.48, 29.25, 29.09, 29.00, 28.85, 28.36,
26.61, 26.40. ESI-MS: m/z: calcd for C49H71N6O5: (the 4ꢀ
ammonium ion, 100%): 823.5480, found 823.5471 (M+).
Data for 3. Anal calcd (%) for C36H60N4O2Cl2: C, 66.34; H,
1
9.28; N, 8.60. Found: C, 66.41; H, 9.31; N, 8.53; H NMR (300
MHz, CDCl3, 25 ꢀC, TMS):
d
¼
0.88–1.25 (m, 20H,
CH2(CH2)10CH2), 2.98–3.08 (m, 4H, CHCH2Ph), 3.23–3.25 (d,
4H, NHCH2(CH2)10CH2), 3.68 (s, 18H, N(CH3)3), 5.59 (t, 2H,
COCHNMe3), 7.26–7.32 (m, 10H, aromatic protons), 8.71 (s, 2H,
CONHCH2); ESI-MS: m/z: calcd for C36H60N4O2: (the 4ꢀ
ammonium ion, 100%): 580.4705, found 290.2354 (M+)/2.
Data for 4. Anal calcd (%) for C48H63N4O4Cl: C, 72.47; H, 7.98;
N, 7.04. Found: C, 72.39; H, 8.01; N, 7.13; 1H NMR (300 MHz,
CDCl3, 25 ꢀC, TMS): d ¼ 0.83–1.26 (m, 20H, CH2(CH2)10CH2),
2.88–3.20 (m, 8H, CH2Ph, NHCH2(CH2)10CH2), 3.37 (s, 9H, N
(CH3)3), 4.15(t, 1H, COCHNH), 4.28–4.38 (m, 3H, OCH2CH),
5.57 (s, 1H, CONHCH2), 5.84 (t, 1H, COCHNMe3), 6.14 (s, 1H,
OCONHCH), 7.20–7.74 (m, 18H, aromatic protons), 8.77 (s, 1H,
CH2NHCOCHNMe3); 13C NMR (75 MHz, CDCl3): d 170.84,
164.97, 143.79, 141.32, 136.83, 129.72, 129.44, 128.98, 127.79,
126.99, 73.77, 67.05, 52.82, 47.15, 39.65, 39.57, 39.11, 32.85, 29.42,
29.32, 28.60, 26.66; ESI-MS: m/z: calcd for C48H63N4O4: (the 4ꢀ
ammonium ion, 100%): 759.4844, found 759.4849 (M+).
Data for 8. Anal calcd (%)for C41H66N5O5Cl: C, 66.15; H, 8.94;
N, 9.41. Found: C, 66.10; H, 8.91; N, 9.33; 1H NMR (300 MHz,
CDCl3, 25 ꢀC, TMS): d ¼ 0.98–1.32 (m, 20H, CH2(CH2)10CH2),
1.37 (s, 9H, OC(CH3)3), 1.51–1.62 (m, 3H, CHCH3), 3.03–3.23
(m, 8H, CH2Ph, NHCH2(CH2)10CH2), 3.41 (s, 9H, N(CH3)3),
4.03–4.04 (d, 1H, NHCHCH2Ph), 4.68 (t, 1H, OCONHCH-Ala),
5.61 (t, 1H, COCHNMe3), 7.04 (s, 2H, CONHCH2,
NHCHCONHCHCO), 7.19–7.32 (m, 10H, aromatic protons),
8.43 (s, 2H, OCONHCH, CH2NHCOCHNMe3); ESI-MS: m/z:
calcd for C41H66N5O5: (the 4ꢀ ammonium ion, 100%): 708.5058,
found 708.5048 (M+).
Data for 5. Anal calcd (%) for C38H61N4O4Cl: C, 67.78; H,
1
9.13; N, 8.32. Found: C, 67.69; H, 9.01; N, 8.23; H NMR (300
MHz, CDCl3, 25 ꢀC, TMS):
d
¼
1.16–1.25 (m, 20H,
Preparation of hydrogels
CH2(CH2)10CH2), 1.39 (s, 9H, OC(CH3)3), 2.93–3.24 (m, 8H,
CH2Ph, NHCH2(CH2)10CH2), 3.49 (s, 9H, N(CH3)3), 4.29 (t,
1H, COCHNH), 5.13 (t, 1H, COCHNMe3), 5.97 (s, 1H,
CONHCH2), 6.03 (s, 1H, OCONHCH), 7.20–7.37 (m, 10H,
aromatic protons), 8.90 (s, 1H, CH2NHCOCHNMe3); 13C NMR
(75 MHz, CDCl3): d 164.87, 155.34, 136.93, 129.55, 129.26,
128.74, 79.74, 73.71, 55.84, 52.49, 39.39, 32.65, 29.39, 29.34,
29.12, 28.99, 28.48, 28.19, 26.72, 26.52; ESI-MS: m/z: calcd for
C38H61N4O4: (the 4ꢀ ammonium ion, 100%): 637.4687, found
637.4602 (M+).
The required amount of the amphiphiles was added in 1 mL
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 cooled to room temperature without any
disturbance. The formation of gel was confirmed by stable
to inversion of the glass vial. Gelation study was checked up to
8% w/v.
Microscopic studies
Data for 6. Anal calcd (%) for C47H70N5O5Cl: C, 68.80; H, 8.60;
N, 8.54. Found: C, 68.69; H, 8.65; N, 8.53; 1H NMR (300 MHz,
CDCl3, 25 ꢀC, TMS): d ¼ 1.01–1.25 (m, 20H, CH2(CH2)10CH2),
1.31 (s, 9H, OC(CH3)3), 2.87–3.24 (m, 10H, CH2Ph,
NHCH2(CH2)10CH2), 3.46(s, 9H, N(CH3)3), 4.24 (t, 1H,
NHCHCONHCH2), 4.60 (t, 1H, OCONHCHCO), 4.82 (t,
1H, COCHNMe3), 5.76 (s, 1H, CONHCH2), 6.20 (s, 1H,
NHCHCONHCHCO), 6.48 (s,1H, OCONHCH), 7.05–7.33 (m,
FESEM was performed on a JEOL-6700F microscope. A piece
of hydrogel was mounted on glass slide and dried for few hours
under vacuum before imaging. The morphology of the dried gels
was studied using atomic force microscopy (Veeco, mod-
elAP0100) in non contact mode. A piece of gel was mounted on
a silicon wafer and dried for a few hours under vacuum before
imaging. TEM measurements were performed on JEOL JEM
2010 microscope. 4 mL of diluted particle suspension in water was
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placed on 300-mesh Cu coated TEM grid and dried under
vacuum for 4h before taking the TEM images.
Cytotoxicity MTT assay
The biocompatibility of the amphiphiles was done by microcul-
ture MTT reduction assay as reported.72 This assay is the result
from the reduction of a soluble tetrazolium salt by mitochondrial
dehydrogenase of the viable tumor cells to an insoluble colored
formazan product. The amount of formazan product produced
can be spectrophotometrically calculated after dissolution of the
dye in DMSO. The enzyme activity and the amount of the for-
mazan produced is proportional to the number of alive cells. The
decrease in absorbance value can be attributed to the killing of
the cells or inhibition of the cell proliferation by the amphiphiles.
HepG2 cells were seeded at a density of 20 000 cells per well in
a 96-well microtiter plate for 18–24 h before the evaluation.
Concentrated stock solutions (5 mg/mL) of the amphiphiles were
sequentially diluted during the experiment to vary the concen-
trations of the amphiphiles (50–400 mg/mL) in the microtiter
plate. The HepG2 cells were incubated with amphiphile 1, 2, 5
FTIR measurements
FTIR measurements of the gelators 2, 4–6 in CHCl3 solution and
in gel state in D2O were carried out in a Perkin Elmer Spectrum
100 FT-IR spectrometer using KBr and CaF2 windows, respec-
tively with 1 mm teflon spacers at their MGC.
X-ray diffraction (XRD)
XRD measurements were taken in Seifert XRD 3000P diffrac-
tometer and the source was Cu-Ka radiation (a ¼ 0.15406 nm)
with a voltage and current of 40 kV and 30 mA, respectively. Gel
of 2, 5, 6 was mounted on the glass slide and dried under vacuum.
The xerogels were scanned from 1–40ꢀ.
ꢀ
and 6 for 3 h at 37 C under 5% CO2. Then, 10 mL MTT stock
Circular dichroism (CD)
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 BioTekꢀ
Elisa Reader. The number of surviving cells were expressed as
CD spectra of the aqueous solutions of compound 5, 6 at varying
concentration were recorded by using a quartz cuvette of 1 mm
path length in a Jasco J-815 spectropolarimeter. Varying
temperature CD spectra were also recorded for those compounds
ꢀ
at concentration 0.025%, w/v from 20 to 70 C.
percent viability
¼
(A570(treated cells)
ꢁ
background/
A
570(untreated cells) ꢁ background) ꢄ 100.
Preparation of GNPs within the hydrogel
Conclusion
The required amount of gelators (50, 7.5, 24 and 19 mg of
compound 2, 5, 6 and 8 respectively) were taken in 1 mL water and
then slowly heated to make a homogeneous solution. Then this
solution was cooled at room temperature to form the gel. To this
gel, HAuCl4 solution was added very slowly for 5–10 min at room
temperature under slow magnetic stirring. The molar concentra-
tion ratio of gelator and HAuCl4 was maintained at both 10 : 1
and 20 : 1. Molar concentration of HAuCl4 in gel 2, 5, 6, 8 was at
10.5, 1.1, 2.9, 2.6 mM respectively when gelator : HAuCl4 was at
10 : 1. In the case of a 20 : 1 ratio of gelator to HAuCl4, the
concentration of HAuCl4 was accordingly decreased maintaining
constant the gelator concentration. The pH of the final reaction
mixture was adjusted to 9–10 with a standard 1 N NaOH solution.
Gradually the yellow colored HAuCl4 solution became colorless
(Au+3 to Au+1) and turned to ruby red (Au+1 to Au0) indicating the
formation of GNPs. In all cases, 48–72 h was needed for complete
conversion.
In summary, the present work finds a rational synthetic approach
to significantly augment the gelation ability of existing gelators
where a hydrogen bonding moiety like amino acid/dipeptide was
judiciously tethered to the tail of an amphiphile. The structure-
property relationship was sketched out by investigating a library
of tail modified gelators. Spectroscopic and microscopic inves-
tigations indicated the formation of anti-parallel hydrogen
bonding which eventually forms b-sheet in case of the amphiphile
having dipeptide at the tail. The developed gelators had
improved functions which can find potential applications in the
widely different fields, from material science to biomedicine.
Furthermore, the altered self assembly route adopted by the
structurally refined amphiphiles results in the conversion of
a non-gelator to an excellent gelator which in principle takes us
closer to the goal of rational designing of efficient gelators with
improved applications.
Cell cultures
Acknowledgements
Human hepatic cancer derivedHepG2 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 70–80%
confluence. A subculture was performed every 2–3 days. After
48–72 h, the medium 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 viability of the cationic
amphiphilic amphiphiles (1, 2, 5, 6).
P.K.D. is thankful to the Department of Science and Tech-
nology, India for financial assistance. D.D., S.M. and S.B.
acknowledge the Council of Scientific and Industrial Research,
India for their Research Fellowships. We are thankful to Dr Sisir
Debnath for valuable help.
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This journal is ª The Royal Society of Chemistry 2011
Soft Matter, 2011, 7, 7291–7303 | 7303