Supramolecular hydrogelators of N-terminated dipeptides selectively inhibit cancer cells

Article abstract of DOI:10.1039/c1cc15577f

Consisting of N-terminated diphenylalanine, a new type of supramolecular hydrogelators forms hydrogels within a narrow pH window (pH 5.0 to 6.0) and selectively inhibits growth of HeLa cells, which provides important and useful insights for designing molecular nanofibers as potential nanomedicines. The Royal Society of Chemistry 2011.

Full text of DOI:10.1039/c1cc15577f

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COMMUNICATION
Supramolecular hydrogelators of N-terminated dipeptides selectively
inhibit cancer cellsw
Yi Kuang, Yuan Gao and Bing Xu*
Received 9th September 2011, Accepted 20th October 2011
DOI: 10.1039/c1cc15577f
Consisting of N-terminated diphenylalanine, a new type of
supramolecular hydrogelators forms hydrogels within a narrow
pH window (pH 5.0 to 6.0) and selectively inhibits growth of
HeLa cells, which provides important and useful insights for
designing molecular nanofibers as potential nanomedicines.
This communication describes supramolecular hydrogelators
consisting of N-terminated dipeptides that exhibit selective
inhibitory effects against cancer cells. Over the last decade,
peptides, as one class of essential biological molecules, have
emerged as a favourite and useful choice of building blocks for
making nanostructured biomaterials, especially in the form of
nanofibers that serve as the network of hydrogels.1 For
Fig. 1 (A) Molecular structures of the N-terminated dipeptide hydro-
gelators 1 to 3. (B) Illustrated pH response of the hydrogelators.
example, amphiphilic oligopeptides have become a versatile
platform for developing functional, self-assembled nanofibers
ability to serve as hydrogelators and their cytotoxicity. To address
medicine, drug delivery, and anticancer therapeutics.2 A simple
this less explored direction of the development of small peptide
that promise applications in tissue engineering, regenerative
hydrogelators, we conjugate naphthalene to the carboxylate
nanotubes that allow the casting of metal nanowires.3 These
end of a diphenylalanine to generate three N-terminated
dipeptide of phenylalanine also self-assembles to form stiff
dipeptides (1–3, Fig. 1A), which successfully self-assemble into
seminal works have largely stimulated the use of other small
supramolecular nanofibers in water to afford stable hydrogels
peptides as the basic motifs for designing small molecule
with the concentration of less than 0.8 wt% and within a
gelators that not only self-assemble into nanofibers, but also
relatively narrow pH range (pH = 5.0–6.0). The formation of
result in molecular hydrogels, which promise applications
ranging from biomedicine to energy.4 Among the reported
the molecular nanofibers and hydrogels of 1, 2 and 3 likely
arises from both aromatic–aromatic interaction and the proper
molecular hydrogelators containing dipeptide motifs, some
protonation of the N-terminal amine group. Besides that, we find
have already shown useful properties and promising applications,
including separation of protein,5 response to the changes of pH or
that these hydrogelators exhibit significant higher cytotoxicity
temperatures6 or ligand–receptor interactions,7 as matrices for
to HeLa cells than to Ect1/E6E7 cells, a result that represents the
three-dimensional cell-cultures,8 and the formation of vesicles
first example of molecular hydrogelators selectively inhibiting
for the delivery of oligonucleotides.9 These promising results
cancer cells. These results are significant because they may
provide important insights for understanding cell specific
suggest that it is worthwhile to further explore the potentials of
cytotoxicity that are critical for the applications of hydrogelators
molecular hydrogelators containing dipeptide motifs because of
and supramolecular hydrogels.
their versatility, low cost, and well-established chemistry.
Fig. 1A shows the structures of the N-terminated hydro-
Most of the dipeptidic molecular hydrogelators, explored so
far, are C-terminated hydrogelators.10 Though there is one
gelators. 1, 2 and 3 all consist of diphenylalanine and naphthalene
motifs. Their structures differ at the linker between the two motifs
report of a cationic dipeptide that forms vesicles for the
possible delivery of oligonucleotides,9 the properties of the
and the position of substitution on the naphthalene group. 2
has a 2-substituted naphthalene; 1 and 3 both have 1-substituted
N-terminated dipeptides are largely unknown, including their
naphthalene, but the linker of 3 has an extra secondary amine.
These slight structural variations result in difference in the
Department of Chemistry, Brandeis University, 415 South St.,
Waltham, MA 02454, USA. E-mail: bxu@brandeis.edu;
(Table 1). Each of three compounds is able to immobilize a
Fax: +1 781-736-2516; Tel: +1781-736-5201
w Electronic supplementary information (ESI) available: Fig. S1–S5.
See DOI: 10.1039/c1cc15577f
properties of the hydrogelation of these three compounds
large quantity of water and form a stable supramolecular
hydrogel (with more than 99 wt% of water) at a proper pH.
c
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Table 1 Gelation properties and 48 h IC50 of the N-termi-
nated hydrogelators
IC50 (mM/mg/ml)
HeLa Ect1/E6E7
MGCa
Compound (mM/wt%) pH
1
2
3
4.4/0.20 5.0–6.0 142.0 ꢀ 3.6/64.1 ꢀ 1.7 4200/490.2
6.5/0.30 5.0–6.0 4200/493.1 4200/493.1
16.7/0.80 5.0–6.0 150.2 ꢀ 3.1/72.1 ꢀ 1.5 4200/496.1
a
MGC: minimum gelation concentration.
Fig. 3 Rheological properties of hydrogels formed by 1, 2 and 3.
(A) Strain sweep and (B) frequency sweep of gel I ([1] = 0.4 wt%: .),
gel II ([2] = 0.4 wt%: ’) and gel III ([3] = 1.2 wt%: K). Storage
moduli (G0: filled symbols); loss moduli (G0 0: open symbols). Arrow
points to the weak strain overshoot of gel II.
However, under the same pH conditions (Fig. 2A–C), hydro-
gelators 1, 2 and 3, at their optimal gelation concentrations, all
give hydrogels of similar optical appearances despite the different
concentrations. Due to the presence of 1-aminonaphthalene in its
structure,11 the hydrogel of 3 is fluorescent (Fig. 2D).
like 1 and 2, partial protonation derived from the primary
amine of 3 at pH 5.0–6.0 leads to hydrogelation.
Fig. 2E–H demonstrate the phase transition of 3 induced by
the change of pH. At the concentration of 1.2 wt%, 3 forms
the hydrogel at pH 5 to 6, with the pellucidity gradually changing
from transparent to opaque when the pH increases from 5 to 6.
When the pH reaches above 6, 3 becomes insoluble in water
and mainly exists as white precipitates; when the pH is below
5, 3 dissolves well in water and gives a clear solution. The
formation of the hydrogel within a narrow pH range (Fig. 1B)
likely arises from a balance of protonation/deprotonation of
the N-terminal amine. The N-terminal primary amine, at
relatively low pH (e.g., pH o 5.0 in this work), exists as
ammonium groups due to the protonation of the amine group.
The positive charges on the molecules cause the hydrogelators
to repulse each other thus disfavoring the formation of nanofibers
(or the networks of the nanofibers), which results in solutions
of the hydrogelators. At relatively high pH (e.g., pH 4 6.0 in
this work), the N-terminal amines exist as neutral amine
groups due to deprotonation, which significantly increases
the hydrophobicity of the hydrogelators, thus promoting the
aggregation and further precipitation of the hydrogelators.
Only within a narrow pH window of weak acidic conditions,
the N-terminal amine group establishes the subtle balance
of protonation/deprotonation that allows a certain degree of
aggregation (self-assembly and crosslinking of nanofibers) of
the hydrogelators while preserving their strong interactions
with water molecules, thus achieving hydrogelation. Despite
the extra secondary amine in 3, which gives it higher solubility
than the other two hydrogelators, the three hydrogelators
share the same pH window for gelation (Table 1). The higher
pKa (9.7) of secondary amine than primary amine12 makes it to
be protonated within the pH window of hydrogelation, thus,
We use rheology to characterize the viscoelastic properties
of the hydrogels formed by 1, 2 and 3. Fig. 3A shows the
dependence of G0 (storage moduli) and G0 0 (loss moduli) of the
hydrogels over a wide range of strain amplitudes. Unlike gel I
and gel III, whose moduli exhibit a trend of decrease at high
strain amplitude (i.e., strain thinning13), gel II displays a weak
strain overshoot in amplitude response: while G0 decreases
with increasing strain amplitude, G0 0, with the increase of
strain amplitude, rises first before it drops rapidly. This weak
strain overshoot indicates that the molecules of 2 align with
each other and associate into superstructures (in this case,
bundle of fibers) that can resist the deformation up to a certain
strain value (G00 increase) before a large deformation disrupts
the structure (i.e., G0 0 starts to decrease). The strain resistant
structure of gel II explains that G0 of gel II is larger than that
of gel I while the concentration of hydrogelators is the same
(Fig. 3B). In frequency sweep, all three hydrogels have G0 and
G00 essentially independent of frequency, in addition to G0
dominating G0 0 at all frequencies, thus indicating the elastic
nature of the three hydrogels. The mechanical properties of
hydrogels formed by N-terminal hydrogelators classify these
hydrogels as extensively cross-linked networks.14
Transmission electron micrographs (TEM) of uranyl acetate
stained15 samples of the hydrogels reveal the nanostructures of
the matrices of the hydrogels formed by these N-terminal
hydrogelators. As the dominant structures of all the three
hydrogels are nanofibers, each hydrogelator forms the nano-
fibers bearing a slightly different feature. Long and extended
helical nanofibers with the pitch of about 40 nm (Fig. 4A and D)
are the main morphology of the network of gel I; gel II consists
of straight nanofibers that tend to align parallelly (Fig. 4B);
the major morphology of the network of gel III is randomly
tangled nanofibers (Fig. 4C). Unlike the nanofibers in gel I and
gel II, nanofibers in gel III have irregular widths.
We use the MTT cell viability assay to examine the cytotoxicity
of the N-terminal hydrogelators against HeLa cells and its
counterpart cell line, Ect1/E6E7 cells, after treatment for
48 hours. HeLa, an epithelial cell line from cervical tumor, is
the most widely used cell line in biology; Ect1/E6E7 is an
immortalized normal epithelial cell line from cervix. These two
cell lines serve as each other’s reference, making a cancer–
normal cell pair that is largely used as model cell pair in cancer
studies.16 Though hydrogelators 1, 2 and 3 exhibit similar
Fig. 2 Optical images of (A) gel I ([1] = 0.4 wt%), (B) gel II ([2] =
0.4 wt%), (C) gel III ([3] = 1.2 wt%). (D) A fluorescent image of gel
III (lex = 345 nm). Compound 3 (1.2 wt%) exists (E) as a clear
solution at pH o 5.0, (F) a transparent gel at pH = 5.0, (G) an
opaque gel at 5.0 o pH o 6.0, and (H) precipitates at pH 4 6.0.
c
12626 Chem. Commun., 2011, 47, 12625–12627
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is serum-free, the growth of HeLa cells requires serum, so the
N-terminated hydrogelators may interact with some essential
proteins through ionic bonds and consequently inhibit growth
of HeLa cells. This ability to distinguish cancer and normal cells
has not been discovered on any of the C-terminal hydrogelators
(though it is possible for the precursors of hydrogelators18), which
makes it a unique property of the N-terminated hydrogelators
that warrants further investigation.
In conclusion, we design and synthesize a series of dipeptides
based hydrogelators with exposed N-terminal amine that, unlike
their C-terminal analogues, forms hydrogels only within a
narrow pH range. These N-terminated hydrogelators, as a new
group in the family of dipeptide based hydrogelators, not only
provide an alternative option for designing functional hydro-
gelators that allow bioactive groups to be conjugated through
their C-terminals, but also have a unique property of selective
inhibition of the growth of HeLa cells, which is of significance to
the understanding of cellular response to hydrogels and hydro-
gelators and may eventually lead to a route for the applications of
supramolecular hydrogels and hydrogelators in cancer therapies.
This work was partially supported by NIH (R01CA142746).
We thank the EM facility at Brandeis University for assistance.
Fig. 4 The TEM images of negative stained (A) gel I, (B) gel II, (C)
gel III, and (D) the magnified image of the helical nanofibers in gel I.
Notes and references
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cytotoxicity towards same cell lines, it is clear that 1, 2 and 3 all
have lower cytotoxicity against Ect1/E6E7 cells than against
HeLa cells (Fig. 5). Same cytotoxicity tests were performed on
another cancer cell line T98G to verify if the cytotoxicity of the
N-terminated hydrogelators only occurs on HeLa cells or is a
general behavior on cancer cells. IC50 values of N-terminated
hydrogelators on T98G (Fig. S1, ESIw) are in the same order of
magnitude with HeLa cells and 2 is also being slightly less toxic
than 1 and 3, which demonstrate that N-terminated hydrogelators
are generally toxic towards cancer cells. 2 differs from the other
two compounds as it has 2-substituted naphthyl instead of the
1-substituted naphthyl group. The cytotoxicity of 2 on HeLa cells
is also lower than that of 1 or 3, thus implying a possible
correlation between substitution patterns with the cytotoxicity.
The difference of the cytotoxicity against the two cell lines
increases with the concentration, and the difference is as large
as 40% (in the case of 3) at the highest concentration tested.
The cause of difference in cytotoxicity of the N-terminated
hydrogelators toward cancer and normal cell lines, we speculate,
unlikely originates from the positive charges provided by the
hydrogelators since the excess of positive charge should also
lead to the death of Ect1/E6E7 cells, but might lie in one or the
combination of the following reasons: first, normal cells might
have a poor permeability to the N-terminated hydrogelators;
second, as acidification is a significant feature of cancer cells,17
normal cells have a pH of 7.4–7.6, so the N-terminated
hydrogelators are less likely to self-assemble in normal cells
than in cancer cells; third, while the growth of Ect1/E6E7 cells
c
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Chem. Commun., 2011, 47, 12625–12627 12627