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pre-incubation with GSH–OEt for 18 h to investigate DOX release
triggered by elevated GSH. Uptake of the SST–DOX conjugate into
MCF-7 cells is clearly observed by CLSM (Fig. S16 in ESI†) and the
conjugate already induces cytotoxicity (Fig. 4, IC50 = 70 mM,16,17
Fig. S15 in ESI†) but less than free DOX (IC50 = 55 mM). By increasing
the intracellular GSH concentrations, the cytotoxicity of SST–DOX
(IC50 = 44 mM) increases significantly according to the one-way
ANOVA test, indicating GSH-stimulated cleavage and cytotoxicity.
Bis-sulfide conjugation represents a versatile approach to build
structurally precise bioconjugates that are stable under physiological
conditions but efficiently cleaved at high GSH concentrations
(10 mM) as present in many tumor cells. Compared to the more
frequently used disulfide connections, bis-sulfide linkers offer some
distinct advantages such as high structural integrity under physio-
logical conditions since no disulfide scrambling occurs and
improved stability at elevated GSH concentrations as present in
the cell plasma of healthy cells. Therefore, bis-sulfide linkers propose
great potential for tumor targeting by offering drug release in tumor
cells and reducing non-specific cleavage in healthy cells.
Fig. 3 Confocal microscopy images of 4 incubated with MCF-7 for 24 h.
(A) GSH–OEt preincubation for 18 h. (B) No GSH–OEt preincubation (green
shows the emission of FITC, whereas red indicates SR emission). (C) Quanti-
fication of GSH mediated cleavage of FITC–SR–SST (4) based on CLSM images
A and B, calculated by the ratio of the total emission intensities of FITC and SR.
(D) Calcium flux induced by SST–DOX (EC50 = 13 nM) in SSTR2 expressing
Chem-1 cells (red) and wild type Chem-1 cells (black).
This concept has been verified by connecting the tumor targeting
moiety SST and the anticancer drug DOX by the bis-sulfide linker. SST
in SST–DOX still activates SSTR2 nearly as potent as native SST which
is important for cell-type selective uptake. DOX delivery into MCF-7
cells that are of interest for therapeutic applications and controlled
release at high GSH levels has been demonstrated. In addition, the
bioorthogonal azide group has been introduced apart from DOX that
the peak for 1c decreases in intensity accompanied by the appearance offers the attachment of various other drug molecules in a single
of 2c and 3. The kinetics of the cleavage of 1c has been determined synthetic step. The multifaceted bioconjugates reported herein com-
(ESI†) and the identity of the signals has been confirmed using bine the additive features of their subunits, i.e. bioactivity, cell-specific
MALDI-MS (Fig. S14 in ESI†). As shown in Fig. 4B, 88% of a uptake and cell-type dependent intracellular release and pave the way
DOX–glutathione conjugate is released from SST after 22 h, to smart biotherapeutics for targeted therapies.
indicating significant drug release.
Notes and references
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Fig. 4 (A) HPLC profile of the cleavage of SST–DOX (1c, 50 mM) with 10 mM
GSH. Arrows indicate increase or decrease of the peak area with time. (B) The
percentage of released DOX over time calculated from the percentage of the
integrated peak areas of 2c + 3 in (A). (C) Cytotoxicity of 1c on MCF-7 cells with
or without pre-treatment of 10 mM GSH–OEt. (**) p o 0.01 and (***) p o 0.001.
1118 | Chem. Commun., 2014, 50, 1116--1118
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