A fluorescence off-on reporter for real time monitoring of gemcitabine delivery to the cancer cells

Article abstract of DOI:10.1039/c3cc42653j

We present the design, synthesis, optical properties and in vitro biological assessments of the theranostic prodrug 6 in which a near IR fluorophore is conjugated with a cancer cell-directing biotin unit; further it is linked with the anti-cancer drug gemcitabine via a self-immolative spacer, a disulfide bond. The prodrug 6 is able to monitor drug delivery and cellular imaging.

Full text of DOI:10.1039/c3cc42653j

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A fluorescence off–on reporter for real time
monitoring of gemcitabine delivery to the
Cite this: DOI: 10.1039/c3cc42653j
cancer cells†
Received 11th April 2013,
Accepted 7th May 2013
Sankarprasad Bhuniya,a Min Hee Lee,a Hyun Mi Jeon,b Ji Hye Han,b Jae Hong Lee,a
Nayoung Park,a Sukhendu Maiti,a Chulhun Kang*b and Jong Seung Kim*a
DOI: 10.1039/c3cc42653j
www.rsc.org/chemcomm
We present the design, synthesis, optical properties and in vitro
biological assessments of the theranostic prodrug 6 in which a near
IR fluorophore is conjugated with a cancer cell-directing biotin
unit; further it is linked with the anti-cancer drug gemcitabine via a
self-immolative spacer, a disulfide bond. The prodrug 6 is able to
monitor drug delivery and cellular imaging.
Theranostics, a special type of drug delivery system (DDS) with
the capability of real time monitoring of drug release, draws
attention from medical or pharmaceutical sciences.1,2 Its unique
feature compared to other DDS systems is that it can generate
notable signals upon drug release to provide information regard-
ing the local drug dosage as well as the distribution of the drug
in tissues. Moreover, an endowed theranostic DDS with a cancer
targeting unit such as biotin or a RGD moiety might be an ideal
candidate for the target-selective drug delivery system, which has
been demonstrated recently.3 However, some of them may not
be compatible with theranostic DDS since the light for the DDS
has to penetrate the tissues which may have strong absorption in
the range of 450 to 530 nm, hence tissue penetration depth of
light of this range is extremely limited.4 This problem may be
circumvented by use of a near infrared-based theranostic system,
which has hardly been reported for theranostic DDS yet. In order
to introduce fluorescing theranostic DDSs compatible with the
biological tissues, we designed a drug delivery system (Scheme 1)
based on a near IR fluorescing BODIPY fluorophore that is
conjugated with a cancer cell-directing biotin unit and linked
Scheme 1 Synthesis of theranostic prodrug 6. Reaction conditions: (i) Et2NH,
MeNO2, EtOH, reflux, 16 h; (ii) NH4OAc reflux, 24 h; (iii) BF3, DIPEA, DCM, 12 h;
(iv) propargyl bromide, K2CO3, acetone, reflux, 12 h; (v) mono-O-DMTr (4,40-dimethoxy-
trityl)-2-hydroxyethyl disulfide, COCl2, DIPEA, DCM, 0 1C–rt, 12 h; (vi) AcOH, DCM,
rt, 24 h; (vii) 4-nitrophenyl chloroformate, DIPEA, DCM, 0 1C–rt, 3 h then
gemcitabine, TEA, DMF, rt, 12 h; (viii) 8, Na-ascorbate, CuSO4, MeOH–H2O, rt,
4 h; (ix) 2-(2-(2-azidoethoxy)ethoxy)ethanamine, EDCI, DMAP, DMF, rt, 6 h.
with the anti-cancer drug gemcitabine (GMC)5 via a self- stable compound; isolation and deprotection of the O-DMTr
immolative spacer, a disulfide bond. Compound 3 was synthe- group by acetic acid in dichloromethane gave 4. Compound 4
sized starting from 40-hydroxychalcone following a previously was further reacted with 4-nitrophenyl chloroformate to give a
reported protocol.6 Themono-O-DMTr-2-hydroxyethyl disulfide reactive intermediate; the intermediate was then reacted with
was formed as an activated intermediate by reacting with gemcitabine to give 5 in moderate yield. Compound 5 was
phosgene which was subsequently reacted with 3 to give a conjugated with biotin derivative 8 to obtain targeted theranostic
prodrug 6 followed by the well known ‘‘click’’ reaction.7
a Department of Chemistry, Korea University, Seoul 136-701, Korea.
To justify the presumed concomitant release of the fluorophore
and GMC, we have carried out UV-Vis and fluorescence study in the
presence of thiols such as DTT. From Fig. 1, it is observed that as
E-mail: jongskim@korea.ac.kr; Fax: +82-2-3290-3121
b The School of East-West Medical Science, Kyung Hee University, Yongin, 446-701,
Korea. E-mail: kangch@khu.ac.kr
the UV absorption at 700 nm gradually increases upon addition
of DTT (Fig. 1a), the fluorescence intensity increases at 720 nm
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c3cc42653j
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Scheme 2 Proposed mechanism for drug release.
Fig. 1 (a) UV-Vis absorption and (b) fluorescence spectra of 6 (10.0 mM) in the
absence and presence of DTT (5.0 mM). Changes in absorbance at 700 nm and
fluorescence intensity at 720 nm as a function of concentration of DTT (0–500 eq.;
0–5.0 mM). All the spectra were acquired 30 min after addition of DTT at 37 1C in
PBS buffer (pH 7.4) containing 4% (v/v) of DMSO and 0.4% (v/v) of Cremophor EL
with excitation lmax = 700 nm.
the similar conditions. Upon the addition of cysteine (Cys) or
homocysteine (Hcy) to 6 changes in the absorption and emission
features similar to those in the case of DTT are seen (Fig. S16, ESI†).
On the other hand, no appreciable spectroscopic changes
were seen upon exposure to non-thiol amino acids or biologically
relevant metal ions. Additionally, we have carried out time-
dependent drug release in the presence of biologically available
thiols such as Cys, Hcy, GSH, Trx as shown in Fig. S18 (ESI†). We
observed that the reactivity order was Cys > Hcy > GSH > Trx. To
confirm the drug release and fluorescence change at the cellular
level, we have carried out in vitro cellular imaging in A549 cells
with treatment of 6. Fig. 3b displays a strong fluorescence
response when excited at 740 nm using a two photon laser. In
contrast to that observed in Fig. 3c little fluorescence intensity
was observed as the cells were pretreated with NEM, a strong
modifier of the reactive biological thiols.8 This experimental
result implied that only biologically available thiols can trigger
the drug release as suggested by in vitro solution tests.
In order to demonstrate the preference of the prodrug 6 to
the cancer cells, we investigated the fluorescence enhancement of
6 with A549 cells9 and WI38 cells,10 the biotin receptor positive
and negative cell lines, respectively. As shown in Fig. 4a, the
fluorescence enhancement was dramatically affected in the A549
cells and a strong fluorescence signal concomitantly appeared.
In contrast, the negative cells, WI38 cells (Fig. 4b), remain dark
even under similar conditions. The overexpression of the biotin
receptors is strongly anticipated for many cancer cells, which
Fig. 2 (a) Time-dependent fluorescence spectral changes observed when 6
(10.0 mM) was treated with DTT (5.0 mM). (b) Changes in fluorescence intensity
at 720 nm recorded as a function of time with (K) and without (n) DTT. All the
spectra were acquired 30 min after addition of DTT at 37 1C in PBS buffer (pH 7.4)
containing 4.0% (v/v) of DMSO and 0.4% (v/v) of Cremophor EL with excitation
lmax = 700 nm.
and reaches the saturation point in the presence of 100 equiv.
of DTT (Fig. 1c and d). Furthermore, we have evaluated the
theranostic efficacy of 6 at a fixed concentration of DTT
(5.0 mmol) with respect to time.
From Fig. 2a and b, it is observed that the absorbance and
the fluorescence of probe 6 are fully consumed and reach the
saturation point within 10 min. This observed result implies
that by the action of DTT, the vulnerable S–S bond is cleaved
and subsequently active GMC and the fluorophore (7) (Scheme 2)
are generated as observed previously by us in our another
study.8 Compound 6 was treated with 5.0 mmol GSH at 37 1C
for 2 h and then the aliquot was subjected to MS analysis; we
obtained mass for gemcitabine (Mw = 262) and 7 (Mw = 966.3) as
seen in Fig. S17 (ESI†). These data strongly support the drug
release upon GSH treatment.
Fig. 3 (a) Phase contrast and (b) confocal microscopy images of A549 cells
treated with 5.0 mM of 6 in PBS buffer, total incubation period of 30 min;
(c) cells were pretreated with NEM (1.0 mM) in PBS buffer for 30 min, then
treated with 5.0 mM of 6 in PBS buffer, incubation period of 25 min. Cell images
were obtained using two photon excitation at 740 nm and a long-path (650–720 nm)
emission filter.
To evaluate the possibility of interferences from biologically
relevant analytes, the reactions of 6 with various thiols,
nonthiol amino acids, and metal ions were investigated under
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Additionally, the cytotoxicity of 6 was evaluated by determining
the viability of cells after incubation with 6 using MTT assay as
shown in Fig. S19 and S20 (ESI†). The cell viability was
decreased in a dose-dependent manner. This observation
implied that prodrug 6 is able to release active drugs and it is
applicable, as a useful, activatable theranostic prodrug, in non-
invasive imaging and delivery of a potent drug.
In conclusion, we have reported an activatable theranostic
agent, 6 and its synthesis, characterization, spectroscopic proper-
ties, anti-cancer effects, as well as biological applications. The
disulfide bond cleavage by intracellular thiol species leads
to release of the active GMC and fluorescence enhancement.
The co-localization experiments using a commercially available
lysosome-selective dye as well as an endoplasmic reticulum-
selective dye demonstrated that compound 6 localized to the
endoplasmic reticulum presumably through the receptor
mediated endocytosis. According to confocal microscopic experi-
mental studies, our final prodrug only goes to biotin receptor-
positive A549 tumor cells, compared to biotin receptor-negative
WI38 cells. Therefore, our drug delivery system (DDS) could
provide a powerful new strategy for the specific tumor targeting
drug delivery and non-invasive cellular imaging.
Fig. 4 (a) A549 cells were treated with 5.0 mM of 6 in PBS buffer, total
incubation period of 25 min; (b) WI38 cells were treated with 5.0 mM of 6 in
PBS buffer, incubation period of 25 min. Cell images were obtained using two
photon excitation at 740 nm and a long-path (650–720 nm) emission filter. The
left side panels show the confocal microscopy images of A549 and WI38 cells; the
right side panels show nonconfocal phase contrast images.
This work was supported by the CRI project (20120000243) (JSK)
and by Basic Science Research Program (2012R1A1A2006259) (CK)
through the National Research Foundation of Korea funded by
Ministry of Education, Science and Technology.
Notes and references
Fig. 5 Confocal microscopic images of colocalized experiments in A549 cells.
(a) and (d) Fluorescence images of A549 cells incubated with 6 (10.0 mM) for 25 min.
(b) Fluorescence image of A549 cells incubated with 6 (10.0 mmol) as well as Lyso
Tracker Blue DND-167 (0.05 mM) for 25 min. (c) Overlay of the merged images of
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c
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