Angewandte
Chemie
lysates and culture medium for liquid chromatography–mass
spectrometry (LC-MS) analysis and determined the intra-
cellular concentrations of the precursors, the hydrogelators,
and the relevant proteolyzed products. After incubation with
SKOV3 or A2780cis cells for 4 h, more than 85% of the
precursors (l-1 or d-1) turned into the corresponding hydro-
gelators (l-2 or d-2; Table 1). Moreover, the intracellular
Table 1: The intracellular concentrations of the precursors and hydro-
gelators in SKOV3 and A2780cis cells.
Compd.
Precursor (1) [mm]
Hydrogelator (2) [mm]
Ratio[a]
Figure 2. Cell viability of ovarian cancer cells incubated with the
l-1[b]
d-1[b]
d-1[c]
62
16
69
431
108
582
6.95
6.75
8.43
precursors with and without cisplatin (CDDP). A) The cell viability of
SKOV3 cells incubated with the precursors d-1 or l-1 alone, or in
combination with CDDP for 72 h. B) The cell viability of A2780 cells
and A2780cis cells incubated with the precursors d-1 alone, or in
combination with CDDP for 72 h (***=pꢀ0.001, ****=pꢀ0.0001).
[a] ratio of hydrogelator to precursor after 4 h. [b] The cell lysates of
SKOV3 cells were collected after 4 h incubation with 20 mm (15 mgmLÀ1
)
of d-1 or with 50 mm (37 mgmLÀ1) of l-1 at 378C. [c] The cell lysates of
A2780cis cells were collected after 4 h incubation with 100 mm
(73 mgmLÀ1) of d-1 at 378C.
the inhibition of SKOV3 is about 80% or 86%, respectively.
The higher efficacy exhibited by l-1 agrees with the higher
uptake and incubation concentration of l-1.
concentrations of the hydrogelators were all above 100 mm,
which indicates the intracellular self-assembly of the hydro-
gelators. The cumulative intracellular concentration of l-
1 and l-2 was also about 10-fold higher than the incubation
concentration of l-1, and the cumulative intracellular con-
centration of d-1 and d-2 was about 5-fold higher than the
incubation concentration of d-1. These results not only
indicate that the cellular uptake of l-1 is more efficient than
that of d-1, but also confirm that the selective retention of
hydrogelators inside the cells originates from ester bond
cleavage catalyzed by CES. A fluorescent esterase substrate,
6-CFDA,[18] also confirmed high esterase activity in SKOV3
cells (Supporting Information, Figure S7). We also analyzed
the culture medium containing l-1 (or d-1), which was
incubated with SKOV3 cells or A2780cis cells. After 4 h
incubation with SKOV3 cells, about 19% of l-1 in the
medium turned into l-2 (Supporting Information, Table S2),
and the concentration of l-1 in the medium decreased from
50 mm to 39 mm; about 15% of d-1 converted d-2, and the
concentration of d-1 in the medium decreased from 20 mm to
16 mm. A similar trend was also observed in A2780cis cells.
These results further validate the hypothesis that intracellular
enzymatic conversion of the precursors catalyzed by CES
results in the intracellular self-assembly of the hydrogelators.
To evaluate the effect of intracellular self-assembly of l-2
or d-2 for cisplatin-based combination therapy, we test the
cell viability of three ovarian cancer cell lines by incubating
them with the mixture of precursors and cisplatin (CDDP).
After 72 h, the mixture of CDDP (6 mgmLÀ1) with d-
1 (15 mgmLÀ1) or l-1 (37 mgmLÀ1) inhibits about 74% or
87%, respectively, of SKOV3 cells (Figure 2A). In contrast,
d-1 (15 mgmLÀ1) or l-1 (37 mgmLÀ1) alone is almost innoc-
uous to the cells, and CDDP (6 mgmLÀ1) alone inhibits only
48% SKOV3 cells. We also used another method to treat the
SKOV3 cells, in which the d-1 (or l-1) were added 12 h after
the addition of CDDP to SKOV3 cells. As shown in Fig-
ure 2A, 72 h after the addition of d-1 (15 mgmLÀ1) or l-
1 (37 mgmLÀ1) following the addition of CDDP (6 mgmLÀ1),
We also tested the combination of CDDP and d-1 for
treating A2780cis (cisplatin-resistant) and A2780 (cisplatin-
sensitive) cells. d-1 (15 mgmLÀ1) alone hardly exhibited any
cytotoxicity to A2780cis cells (Figure 2B). The combination
of d-1 and CDDP inhibited 70% of A2780cis cells, which is
double the activity of CDDP. The combination of d-1 and
CDDP significantly inhibits A2780 cell viability and decreases
the viability of A2780 from about 38% (without adding d-1)
to only 9%. Since SKOV3 and A2780cis are two drug-
resistant ovarian cell lines, CDDP shows lower inhibition
ability against these two cell lines compared with A2780 cells.
These results confirm that the addition of the precursors of
self-assembling small molecules in combination with cisplatin
drastically boosts the activity of cisplatin against drug-
resistant ovarian cancer cells. The IC50 values of l-1 and d-
1 against the ovarian cancer cells are 62–94 mm and 48–69 mm,
respectively (Supporting Information, Table S2), but their
concentrations for the combination therapy can be lower than
IC50 values because EISA accumulates the hydrogelators
intracellularly. Furthermore, the intracellularly formed nano-
fibers (of d-1) are about seven times more effective against
HeLa cells than the nanofibers of the dipeptides reported
previously (Nap-FF[8]; Supporting Information, Table S3).
To verify the critical role of enzyme-instructed self-
assembly, we synthesize a control compound (3), which
replaces the ester bond in d-1 by an amide bond (Supporting
À
À
À
À
Information, Scheme S2). This change ( COO to CONH
) renders 3 resistant to CES. Control compound 3 (500 mm)
alone hardly inhibited SKOV3 cells after 72 h incubation
(Supporting Information, Figures S8A,S9A). After 72 h
incubation with SKOV3 cells, while CDDP (6 mgmLÀ1
)
alone causes about 40% cell death, the mixture of 3
(15 mgmLÀ1) and CDDP (6 mgmLÀ1) inhibited only about
32% of SKOV3 cells. The innocuous effects of 3 also exclude
the possibility that l-1 or d-1 act as a surfactant to inhibit cell
survival. A similar trend is observed in A2780cis cells
(Supporting Information, Figures S8B, S9B). These results
further confirm that enzyme-instructed self-assembly inside
Angew. Chem. Int. Ed. 2015, 54, 13307 –13311
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim