A supramolecular hydrogelator of curcumin

Article abstract of DOI:10.1039/c4cc03139c

Here we report on the first supramolecular hydrogelator of curcumin and the evaluation of its inhibition capacity towards cancer cells and tumor growth.

Full text of DOI:10.1039/c4cc03139c

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A supramolecular hydrogelator of curcumin†
Chengbiao Yang,a Zhongyan Wang,b Caiwen Ou,c Minsheng Chen,c Ling Wangb
Cite this: Chem. Commun., 2014,
50, 9413
and Zhimou Yang*ab
Received 28th April 2014,
Accepted 29th May 2014
DOI: 10.1039/c4cc03139c
www.rsc.org/chemcomm
Here we report on the first supramolecular hydrogelator of curcumin
and the evaluation of its inhibition capacity towards cancer cells and
tumor growth.
Supramolecular hydrogels1 of therapeutic agents have recently
attracted extensive research interest as drug delivery carriers
because of their several advantages,2 such as the high and con-
trollable drug loading, sustained and responsive drug release
properties, and good biocompatibility.3 Supramolecular hydrogels
are formed by hydrogelators via non-covalent interactions.4 Up to
now, several kinds of therapeutic agents including anti-cancer,5–7
anti-bacteria,8 and anti-inflammatory drugs6,9 have been converted
into hydrogelators through conjugation with small molecules,
Scheme 1 The chemical structures of pro-gelator
possible gelator catalyzed by glutathione (GSH).
and
especially peptides.10 The resulting hydrogels can be used directly to improve its solubility in aqueous solutions and its bioavail-
as injectable hydrogels for the topical treatment11 or after dilution ability, it has been formulated into micelles and nanospheres, or
as nanofiber dispersions12 by intravenous injection, and they have incorporated into liposomes or polymeric hydrogels.19 However,
shown constant release of therapeutic agents and excellent inhibi- there is no supramolecular hydrogelator of Cur. We planned to
tion capacity towards cancer cells13,14 and bacteria.15 For their develop a hydrogelator of it and generate a hydrogel for its
application in cancer therapy, anti-cancer drugs taxol7,12,14,16 and delivery. We therefore designed the molecule Cur-FFE-ss-ERGD,
camptothecin5,6 have been converted into hydrogelators by several shown in Scheme 1, as a pro-gelator because the dipeptide
groups. In order to expand the scope of hydrogelators of anti-cancer diphenylalanine (FF) has been widely used to construct supra-
drugs and develop more of these hydrogels for combinatory molecular hydrogelators. We also demonstrated that disulfide
therapy, there is a need to develop hydrogelators of other anti- bond reduction was a biocompatible method for hydrogelation.7
cancer drugs. In this study, we report on the first supramolecular Therefore we believed that, upon the addition of glutathione
hydrogelator of the anti-cancer drug curcumin (Cur).
(GSH), the pro-gelator would be converted to a possible hydro-
Cur has been widely used as a food additive (e.g. curry) and gelator, thus resulting in hydrogelation.
has shown anti-bacterial17 and anti-cancer18 properties. In order
Following our previously published procedure,7 we first pre-
pared Fmoc-CS which contains a disulfide bond. The compound
was then directly used for standard Fmoc solid phase peptide
synthesis to produce the peptide FFE-ss-ERGD. We then prepared
the Cur derivative with a carboxylic acid by reacting Cur with
glutaric anhydride, which coupled with the peptide to achieve the
title compound. The pure compound was obtained by reverse
phase high performance liquid chromatography (HPLC) and it
a State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Bioactive
Materials, Ministry of Education, and College of Life Sciences, Nankai University,
and Collaborative Innovation Center of Chemical Science and Engineering,
Tianjin 300071, P. R. China. E-mail: yangzm@nankai.edu.cn
b College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research,
Nankai University, Tianjin 300071, P. R. China
c Department of Cardiology, Zhujiang Hospital of Southern Medical University,
showed a very good water solubility of up to 5 wt% (50 mg mLꢀ1
in phosphate buffer solution (PBS, pH = 7.4).
)
Guangzhou 510280, P. R. China
† Electronic supplementary information (ESI) available: Synthesis and character-
ization, chemical structures, confocal images, and TEM images. See DOI: 10.1039/
c4cc03139c
We then tested its gelation ability by disulfide bond
reduction. As shown in Fig. 1A, the addition of 4 equiv. of
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We also obtained the IC50 value of the pro-gelator, gelator,
peptide, and Cur against HepG2, HeLa, and MCF-7 cells. After
incubating the cells with different compounds at different
concentrations for 48 h, an MTT assay was performed. As
shown in Fig. 2B, the pro-gelator exhibited an IC50 value of
8.1, 8.4, and 9.5 mM against HepG2, MCF-7 and HeLa cells,
respectively, which is very similar that of Cur. The peptide
without Cur showed no obvious toxicity to the cells at a
concentration of 5 mM. The gel showed a decreased inhibition
capacity to three cells and its IC50 value was 27.8, 52.9, and
53.2 mM against HepG2, HeLa, and MCF-7 cells, respectively.
These observations suggest a superior inhibition capacity of the
pro-gelator compared to the gelator in the nanofiber form.
In order to understand the superior inhibition capacity of
the pro-gelator than the gelator, we obtained confocal fluores-
cence microscopy images of MCF-7 cells treated with a solution
of the pro-gelator and the gelator in the form of nanofibers.
Fig. 3 shows overlay images of the MCF-7 cells at a 4 h time
point (excitation wavelength = 488 nm). The pro-gelator was
distributed evenly in the cytoplasm of the cells, as indicated by
the bright green fluorescence in the entire cytoplasm of the
cells (Fig. 3A and Fig. S7, ESI†). However, we observed much
weaker fluorescence in cells treated with the nanofibers and
the fluorescence signal was not evenly distributed in the
cells (Fig. 3B and Fig. S7, ESI†). We then used liquid
chromatography-mass spectrometry (LC-MS) to determine the
concentration of the compounds in the cells when pro-gelator
and gelator solutions were used to treat the cells. We found that
the concentration of the gelator was 6.8, 5.3, and 4.9 times
higher than that of the pro-gelator in MCF-7, HeLa, and HepG2
cells, respectively (Fig. S8, ESI†), indicating a much higher
cellular uptake of the nanofibers than of the pro-gelator. It
has been demonstrated that self-assembled molecules have
higher cellular uptake than single molecules.20 The weaker
fluorescence in cells treated with the nanofibers was due to
the well-known phenomenon of aggregation induced quenching.
These observations also suggest that the nanofibers changed the
distribution of Cur, thus resulting in its less reduced inhibition
capacity to cancer cells.
Fig. 1 (A) Rheology with the dynamic time sweep mode for a PBS solution
containing 0.5 wt% of the pro-gelator with 4 equiv. GSH (inset: optical
image of the formed gel) and (B) a TEM image of the formed gel.
GSH to a PBS solution of the pro-gelator (0.5 wt%, 5 mg mLꢀ1
)
resulted in the formation of a clear yellowish hydrogel (Fig. 1A,
inset) after 1.5 h at 37 1C. We then characterized the mecha-
nical properties of the hydrogel by rheology. As shown in
Fig. 1A, when GSH was added to the pro-gelator solution, the
value of the storage modulus (elasticity or G0) became more
dominant than the loss modulus (viscosity or G00) after about
1 h. Both G0 and G00 exhibited a weak frequency dependency
from 0.1 to 100 rad sꢀ1 (Fig. S4, ESI†), suggesting an elastic
network in the gel. We then characterized the nanostructures
in the hydrogel by transmission electron microscopy (TEM,
Fig. 1B). We observed filamentous structures in the gel and the
diameter of the fibrils was about 25–35 nm. These uniform and
flexible fibrils were longer than 2 mm and entangled with each
other to form a network for the gel formation.
We therefore monitored the release profile of Cur from
the gel in vitro under physiological temperature conditions
(at 37 1C). We added 0.25 mL PBS buffer solution on top of
0.25 mL of the gel formed from 0.5 wt% Cur-FFE-ss-ERGD at a
24 h time point. The upper solution was completely removed at
desired time intervals to measure the accumulated amount of
Cur released from the gel and a fresh PBS buffer solution
(0.25 mL) was then added. We only observed the release of the
original Cur and no Cur derivatives were released from the gel,
suggesting hydrolysis of the ester bond. As shown in Fig. 2A,
the gel exhibited a constant release profile at a rate of about
0.8 mg mLꢀ1 per hour during the 24 h experimental period. About
1.9 mg mLꢀ1 Cur was released during the 24 h. These observations
suggest its big potential for the sustained and long term delivery
of Cur.
We opted to compare the in vivo anti-cancer capacity of the
pro-gelator solution and the solution of nanofibers in a mouse
tumor model (4T1-luciferase breast tumors in the mammary fat
Fig. 2 (A) The accumulative amount of curcumin released from the gel
formed from a PBS buffer containing 0.5% wt pro-gelator and (B) cyto-
toxicity of the pro-gelator, the gel, and curcumin against HepG2, HeLa,
and MCF-7 cells.
Fig. 3 Confocal fluorescence microscopy images of MCF-7 cells treated
with (A) the pro-gelator and (B) the formed nanofibers containing 25 mM
curcumin (excitation wavelength = 488 nm) at a 4 h time point.
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pad of female mice). When the volume of breast tumors reached
about 13 mm3, we injected the same dosage (2.5 mg kgꢀ1 of
Cur ꢁ 4 every other day, Cur was dissolved in an excipient
mixture of polyethylene glycol 400, propylene glycol, and
polysorbate 80 (40 : 58 : 2)) of different formulations of Cur into
mice through the caudal vein. As shown in Fig. 4, the solution
of nanofibers exhibited a similar anti-tumor growth efficacy to Cur.
The pro-gelator showed an enhanced anti-tumor growth capacity
over the nanofibers and Cur. The final volume of the tumors was
about 4675, 4518 (*P = 0.0446), 4207 (*P = 0.0233), and 2992%
(***P o 0.0001) higher than the original volume of the tumors
(13 mm3) for the PBS control group, Cur, the nanofibers, and the
pro-gelator, respectively. There was no obvious body weight loss in
the groups of mice administrated with different forms of Cur
(Fig. S9, ESI†), compared to the control group of mice treated
with PBS. These results, in combination with the results giving the
in vitro inhibition capacity to cells, suggest that the pro-gelator is a
more promising candidate than the gelator in nanofibers for
cancer therapy.
In summary, a new hydrogelator based on Cur was reported
in this study. The resulting hydrogel formed by disulfide bond
reduction and could sustainably release the original Cur through
ester bond hydrolysis. Though the cellular uptake of the nano-
fibers of the Cur–peptide conjugate was much higher than that
of solutions of the pro-gelator, the nanofibers possessed a lower
potency to inhibit cancer cells in vitro and in vivo than the
pro-gelator solution. Therefore, the hydrogel might only be
applied for the topical treatment of cancers. The results also
indicate that, in order to achieve better inhibition capacity of Cur
nanofibers on cancer cells, the nanofibers are required to be
responsive to a pH change after endocytosis and must dissociate
into single molecules. Our study provides useful information to
design nano-materials to deliver the anti-cancer drug, curcumin.
This work is supported by NSFC (31271053, 51373079, and
81301311).
20 Y. Cai, Y. Shi, H. Wang, J. Wang, D. Ding, L. Wang and Z. Yang, Anal.
Chem., 2014, 86, 2193.
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Chem. Commun., 2014, 50, 9413--9415 | 9415