Folate-based near-infrared fluorescent theranostic gemcitabine delivery

Article abstract of DOI:10.1021/ja405372k

A series of heptamethine cyanine (1-3) derivatives bearing a carbamate ethyl disulfide group and gemcitabine, an anticancer drug, have been newly synthesized. Their disulfide bonds are readily cleaved by various thiols including glutathione, to result in a subsequent decomposition of the carbamate into amine followed by release of the active gemcitabine, which can be monitored by the fluorescence changes. In the biological experiment, prodrug 1 is preferentially up-taken by folate-positive KB cells over folate-negative A549 cells via receptor-mediated endocytosis to release gemcitabine causing cell death and to emit fluorescence in endoplasmic reticulum. Moreover, it is selectively accumulated in the KB cells which were treated to mice by dorsal subcutaneous injection. This drug delivery system is a new theranostic agent, wherein both therapeutic effect and drug uptake can be easily monitored at the subcellular level, by in vivo and in vitro fluorescence imaging.

Full text of DOI:10.1021/ja405372k

Article
pubs.acs.org/JACS
Folate-Based Near-Infrared Fluorescent Theranostic Gemcitabine
Delivery
Zhigang Yang,† Jae Hong Lee,† Hyun Mi Jeon,‡ Ji Hye Han,‡ Nayoung Park,† Yanxia He,†
Hyunseung Lee,§ Kwan Soo Hong,*,§ Chulhun Kang,*,‡ and Jong Seung Kim*,†
†Department of Chemistry, Korea University, Seoul 136-701, Korea
‡The School of East-West Medical Science, Kyung Hee University, Yongin 446-701, Korea
§Division of MR Research, Korea Basic Science Institute, Cheongwon 363-883, Korea
S
* Supporting Information
ABSTRACT: A series of heptamethine cyanine (1−3) derivatives bearing a
carbamate ethyl disulfide group and gemcitabine, an anticancer drug, have
been newly synthesized. Their disulfide bonds are readily cleaved by various
thiols including glutathione, to result in a subsequent decomposition of the
carbamate into amine followed by release of the active gemcitabine, which
can be monitored by the fluorescence changes. In the biological experiment,
prodrug 1 is preferentially up-taken by folate-positive KB cells over folate-
negative A549 cells via receptor-mediated endocytosis to release
gemcitabine causing cell death and to emit fluorescence in endoplasmic
reticulum. Moreover, it is selectively accumulated in the KB cells which were
treated to mice by dorsal subcutaneous injection. This drug delivery system
is a new theranostic agent, wherein both therapeutic effect and drug uptake
can be easily monitored at the subcellular level, by in vivo and in vitro
fluorescence imaging.
be desirable for in vivo real-time monitoring of drug delivery.
Deep tissue penetration will enhance microscopic imaging, and
weak excitation energy will reduce tissue damage.
INTRODUCTION
■
Over the past several decades, targeted drug delivery systems
(DDSs) have drawn strong attention from the chemistry and
pharmacology fields.1−4 Typically, it is composed of a prodrug
and a targeting unit where they are connected through a linker,
which can be cleaved by intracellular thiols,5−7 enzymes,8−11 or
changes in pH.12,13 The targeting unit can selectively deliver the
system to a specific type of cells, for example, cancer cells via
selective interaction with its receptor on the cell surface
membrane. The crucial advantage of targeted DDS over the
conventional anticancer medications is that it can release the
toxic drug only when it enters the target tumor cells. However,
visualization of these drug delivery processes has been hardly
demonstrated since most of the antitumor drugs (e.g.,
gemcitabine, paclitaxel) are intrinsically nonfluorescent or
weakly fluorescent. Therefore, the DDSs built with a
fluorophore would allow an accurate and quantitative measure-
ment of the drug release process, as recently demonstrated in
our laboratory.14
To date, a number of fluorophores have been employed as
reporters, such as coumarin, pyrene, 1,8-naphthalimide,
xanthenes, squaraine, cyanine, borondipyrromethene difluoride,
and nitrobenzofurazan.15−17 However, several critical draw-
backs were reported for most of them, including poor
penetration of the excitation or emission lights through the
tissues when used in animal or clinical studies.18 In this context,
a reporter with near-infrared (NIR) emission capability would
Heptamethine cyanines (Cy7s) are fluorescent dyes ideal for
use in tumor imaging as they can emit light in the near-infrared
(NIR) region, which is capable of deep tissue penetration and
does not get affected by the autofluorescence of tissue.19,20 The
Cy7s in which the meso-position of its conjugation chain is
occupied by amino groups exhibit a large Stokes shift, which
would be advantageous in chemical sensing and biological
imaging, both in vitro and in vivo.21,22 In contrast, the Cy7
derivatives with a simple amide group reveal a rather small
Stokes shift with very weak fluorescence. Therefore, consider-
ing the spectral changes upon drug delivery, designing a Cy7
derivative with an amide group that can be readily transformed
into an amine group by certain intracellular reaction would be a
great idea.23
Herein, we report a new DDS bearing Cy7 dye 1 (Scheme 1)
as an NIR-based reporter. The delivery system is composed of
three critical parts. A folate group is employed as a tumor-
targeting ligand because its receptors are overexpressed on the
surface of most cancer cells.24−27 Second, the Cy7 unit is
adopted as an NIR fluorescent reporter, in which the amide
group can be rapidly decomposed into an amine group upon
Received: May 29, 2013
© XXXX American Chemical Society
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Scheme 1. Chemical Structures of Target Compounds
RESULTS AND DISCUSSION
■
For the achievement of drug delivery with the NIR fluorescent
system, we first designed and synthesized a novel Cy7 dye (5)
with a propargyloxy group at the cyclohexyl position (Schemes
2 and S1). 1,4-Cyclohexanediol was used as the starting
material to react with propargyl bromide to provide 4-
proparyloxycyclohexanol (8). 8 was then oxidized by
pyridinium chlorochromate (PCC) to obtain 4-proparylox-
ycyclohexanone (7) which subsequently generated the
intermediate 6 in the presence of DMF and POCl3
(Vilsmeier−Haack reaction). The intermediate 6 and 1-ethyl-
2,3,3-trimethyl-3H-indolenium iodide salt were heated in acetic
anhydride to obtain a new Cy7 dye (5) in a 65% yield. And
then, the chloro group at the meso-position of 5 was substituted
by ethylamine to give 4 (80%). Compound 4 was subsequently
reacted with phosgene and 2,2′-hydroxyethyl disulfide to
produce 3, which was further reacted with phosgene and
gemcitabine to generate 2. Compound 2 could be conjugated
with 3-azidopropyl-folate (10) via a click reaction to give the
prodrug 1; the 3-azidopropyl-folate (10) was prepared by
coupling of the folic acid and 3-azidopropylamine. All the
detailed procedures, characterization, and HPLC results of the
compounds have been shown in the Supporting Information.
In order to verify the spectral changes of target compounds
after the carbamate decomposition at the central position of the
conjugated chain of the Cy7 derivative, the spectral changes of
the theranostic prodrug 1 upon its reaction with thiol-
containing molecules, including GSH, are investigated and
represented in Figure 1. The disulfide bond is known to be
rapidly cleaved by thiols.7,30 The prodrug 1 herein demon-
strates drastic responses in the colorimetric and fluorescence
disulfide bond cleavage by intracellular thiols. Finally,
gemcitabine is utilized as a model anticancer drug, which
principally functions as a vigorous competitor for a pyrimidine
base in the DNA replication or as an apoptosis inducer through
an interaction with ribonucleotide reductase.28 As summarized
in Scheme 3, upon its reductive cleavage reaction by thiols such
as L-glutathione (GSH),7,29 the disulfide unit generates thiols,
which, in turn, initiate further decomposition of the amide
group and gemcitabine; simultaneously, the intensity of Cy7
fluorescence is enhanced. Thereby, 1 would have the potential
to localize into the tumor cells and to deliver gemcitabine,
which could be fully monitored by in vitro and in vivo real-time
fluorescence imaging.
a
Scheme 2. Synthesis of Targeting Compounds
a
(a) Ethyl iodide, toluene, refluxed for 12 h, 79.6%; (b) NaH, propargyl bromide, DMF, rt, stirred overnight, 70%; (c) PCC, methylene chloride
(MC), rt, stirred for 5 h, 95%; (d) DMF, POCl3, 60°C, 3 h, ca. 75%; (e) anhydrous NaOAc, acetic anhydride, 100 °C, 1 h, 65%; (f) ethylamine in
THF, acetonitrile, 50 °C, 80%; (g) phosgene in toluene, DIPEA, 2-(2-hydroxyethyl)disulfide in DMF, MC, rt, ca. 10%; (h) phosgene in toluene,
DIPEA, DMAP, Gemcitabine in DMF, MC, rt, 63.5%; (i) 3-azidopropylamine, HATU, DMAP, DIPEA, DMF, rt, overnight, 36%; (j) Cu(ClO4)2,
soduim L-ascorbate, DIPEA, 3-azidopropyl-folate, DMF, rt, 22.3%.
B
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generate increased electron density on the nitrogen atom.
Then, the amino group undergoes a change in its configuration
to consume some excited energy before liberating the
fluorescence of a much longer wavelength, causing a large
Stokes shift. The carbamate group at the central position of the
conjugating chain of reference 3 is unable to donate electrons
in the ground state, and therefore, it exhibits a similar electronic
distribution at both the ground and first excited states, which
mainly localize at the polymethine chain. This induces a small
Stokes shift. Therefore, when 3 is treated with various thiols in
aqueous solution (Figures S5−S8), the carbamate at the meso-
position of the conjugated chain could be decomposed into the
amine, resulting in a change in the colorimetric absorption at
785 nm (oscillator strength f = 2.32) and 628 nm (f = 2.11),
with an isosbestic point at 668 nm, demonstrating a linear
relationship between A628/A785 vs the concentration of thiols
(0−0.2 mM). In addition, for prodrug 1, the fluorescence at
724 nm (f = 2.45) was 37-fold higher compared to that at 794
nm (f = 2.55, Φf < 0.01), resulting in an increase in the Stokes
shift (96 nm in methanol), and in turn, the fluorescence was
switched on.
Figure 1. Absorption (A) and fluorescence (B) spectral changes of
prodrug 1 (4.0 μM) in the presence of GSH in DMSO-PBS solution
(10 mM, pH = 7.4) (50:50, v/v) at room temperature, λex = 615 nm
and slit width = 5, 10; and the plot of A630/A794 (C) or I735 (D) vs
GSH concentration; all the data were obtained after the incubation of
samples for 2 h under room temperature.
The disulfide bond of Cy7 dye can be selectively cleaved by
thiol-containing molecules to give diverse spectral changes.
When 2 or 3 was incubated with various nonthiol amino acids
and metal ions, their absorption and emission spectra were
hardly changed (Figures 2A, B, S9, and S10). However, upon
spectroscopies to GSH in mixed solvents (DMSO−PBS; v/v
50%, pH = 7.4). Upon reacting with GSH, the absorption of 1
at 794 nm decreased, and a new peak concomitantly appeared
at 630 nm with marked colorimetric changes (Figure 1A). The
gradual increase in the absorption ratio (A630/A794) finally
produced a plateau with GSH concentrations over 0.2 mM
(Figure 1C), and the emission intensity of Cy7 (735 nm)
enhanced 42-fold with good linearity (Figure 1B and 1D).
These results are in harmony with the drug release model
demonstrated in Scheme 2. Upon cleavage of its disulfide bond
with GSH, sulfide anions are generated, which initiate the
intramolecular cyclization of the carbamates at the meso-
position of Cy7 and at the 5′-hydroxyl group of gemcitabine,
inducing fluorescence by spectral changes in Cy731 and the
gemcitabine released. And the drug release upon reaction of
prodrug 1 with GSH was further identified by ESI-MS
spectroscopy where we obviously observed MS peak m/e:
[M1 + H] = 264.05, and [M2 + Na] = 1119.75, [M2 + Na +
H]/2 = 560.40 corresponding to the gemcitabine released and
folate-Cy7 fragment, respectively (Figure S2 and Scheme 3).
The above results lead us to confirm that active Gemcitabine
was released, by concomitantly marked spectral changes of Cy7,
after the disulfide bond cleavage upon GSH treatment.
Figure 2. Changes in the absorption ratio (A) and fluorescence (B) of
2 (2.0 μM) upon addition of various reactants in PBS solution (10
mM, pH = 7.4); 1, blank; 2, Arg; 3, Phe; 4, Asp; 5, Met; 6, Ser; 7, Tau;
8, Ala; 9, His; 10, Pro; 11, Gly; 12, Leu; 13, Asn; 14, Thr; 15, Val; 16,
Lys; 17, Trp; 18, Glu; 19, Ile; 20, Ca2+; 21, Zn2+; 22, Mg2+; 23, Fe2+;
24, GSH; inset, absorption (A) and fluorescence (B) spectral changes
of 2 with addition of GSH (0.25 mM), other amino acids (1.0 mM),
and some metal ions; and C and D, the response of 2 (2.0 μM) to the
variations in pH (4.0−10.0) in the presence and absence of GSH (1.0
mM); (C) the absorbance ratio at two peaks vs pH; (D) fluorescence
change vs varying pH of the solutions (DMSO−water, 10:90, v/v), λex
= 615 nm (incubation for 2 h under room temperature).
Not only dual spectral changes of prodrug 1 upon incubating
with various thiols were observed, the reference compound 2
without a folate group also exhibited marked colorimetric
absorption and dramatic fluorescence switched-on.
For further characterization of the spectral properties of 1,
the references 3 and 4 were prepared and subjected to a
calculation through the Gaussian 09 software suite32 (Figures
S3−S4). The absorption and emission bands at 628 and 724
nm for 4, and at 785 and 794 nm for 3 in methanol, indicate
that 4 produces a much larger Stokes shift (by 96 nm) than 3
(by only 9 nm). This marked Stokes shift is ascribed to the
ethylamine group at the meso-position of the polymethine
chain. In the ground state, the electron density is mainly
distributed on the Cy7 backbone, excluding the centrally
positioned amine. However, upon excitation, the charge is
transferred from the polymethine chain to ethylamine to
the addition of GSH (1.0 mM) at pH > 6, both absorption
ratios (A615/A780) and fluorescence intensities of 2 and 3
increased (Figures 2C, D and S11). A slight decrease in the
fluorescence intensity at pH > 8.5 may be attributed to the
instability of Cy7 under such alkaline conditions. Besides, 4 is
stable enough to maintain >95% of its fluorescence intensity for
60 min at 37 °C (pH = 7.4, PBS buffer; Figure S12). To
investigate the DDS dynamics, a time-course experiment was
C
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conducted, and the results are shown in Figures S13 and S14.
The prodrug 1 incubated with various concentrations of GSH
in DMSO−PBS (v/v, 50%) at 37 °C completed the disulfide
cleavage reaction in 15 min to give rise to enhanced
fluorescence. Therefore, it is noticeable that the compounds
are stable under physiological pH (7.4) and that their
fluorogenic reactions are selective to the thiol species (GSH)
in the cells, which proves them to be adequate for further use in
biological systems in vitro and in vivo.
reconfirming that the fluorescence enhancement of 1 is mainly
triggered by intracellular thiols, as depicted in Scheme 3.
To identify the intracellular location of gemcitabine release
by the theranostic prodrug 1, colocalization experiments were
carried out using confocal laser microscopy, and the results are
shown in Figure 4. Both Lyso-sensor Blue DND-167 (A) and
To gain insight into the drug release action of 1 in the cells,
we investigated whether the folate moiety can guide the probe
preferentially to folate receptor-positive tumor cells or folate
receptor-negative tumor cells.33 For this, prodrug 1 was
incubated with folate receptor-positive KB cells and folate
receptor-negative A549 cells. As shown in Figures 3 and S15,
Figure 4. Confocal laser fluorescence microscopy images of KB cells
with prodrug 1 and Lyso-Sensor Blue DND-167 (1.0 μM) or ER-
tracker Red (0.5 μM). (A) is the image of Lyso-Sensor Blue DND-
167, collected by a 370−550 nm filter upon excitation at 740 nm (two-
photon excitation); (D) is the image from ER-tracker Red (0.5 μM),
collected by a 530−570 nm band path filter with excitation at 514 nm;
the images of the cells treated with prodrug 1 (1.0 μM) excited at 633
nm acquired by a 650−750 nm filter are shown in (B) and (E), and
the merged images are shown in (C) and (F) (scale bar = 20 μm).
Figure 3. Confocal microscopy images of KB and A549 cells treated
with prodrug 1 (1.0 μM) at various times (0, 5, 10, and 20 min); the
fluorescent images were obtained via a Leica Confocal Laser
Microscope (Leica TCS SP2); excited at 633 nm and detected at
650−750 nm (scale bars = 20 μm).
ER-tracker Red (D) can be visualized in green, and the probe
(B and E), in red. The yellow area in the merged images (C)
and (F) implicates the location where the probe and organelle
selective trackers coexist. From the merged images, it is clearly
noticeable that the yellow region of (F) is predominant
compared to that of (C). In addition, in a parallel experiment
with Mito-tracker Green FM, the merged image failed to show
an intensely yellow image as in the case with ER-tracker Red
(Figure S18). Therefore, it is noteworthy that thiol-induced
disulfide cleavage of 1 occurred in the endoplasmic reticulum
(ER), resulting in the release of gemcitabine, which presumably
diffuses into the cell nucleus where it replaces one of the
building blocks of nucleic acids, cytidine, during DNA
replication. This process leads to forming a faulty nucleoside,
leading to apoptosis.28
the KB cells exhibit strong fluorescence intensity within 20 min
of incubation with prodrug 1 (1.0 μM), whereas the A549 cells
showed little fluorescence signal under similar experimental
condition whereas the difference in the fluorescence intensity
decreased gradually with a longer incubation (Figure S16). As a
result, it can be concluded that prodrug 1 can enter the cells
aided by the interaction of its folate moiety with a folate
receptor on the tumor cells, possibly via receptor-mediated
endocytosis.
To provide further evidence for thiol-induced disulfide bond
cleavage and concomitant fluorescence-On, fluorescence
emission from probe 1 was studied in the presence of a strong
thiol reacting agent, N-ethylmalemide (NEM); the results are
shown in Figure S17. The fluorescence intensity of 1 in the cells
decreased as the concentration of NEM increased (0−1.0 mM),
A cell viability experiment was performed to understand the
cytotoxicity profile of the prodrug 1. As demonstrated in Figure
Scheme 3. Drug (Gemcitabine) Release from the DDS by Treatment of GSH and Fluorescence Switching-on of Cy7
D
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5, 1 and 2 are independently applied to KB and A549 cells. The
cytotoxicity of 1 prefers KB cells to A549 cells whereas a
successfully deliver the gemcitabine into KB tumor cells in
living mice.
CONCLUSIONS
■
In conclusion, a novel NIR DDS composed of three distinctive
parts has been developed: a folate moiety for targeting tumor
cells with folate receptors, a nonfluorescent model drug
gemcitabine, and a Cy7 dye conjugated with a cleavable
disulfide unit employed as a signaling reporter. The carbamate
group of the prodrug 1 is decomposed into an amine upon
reacting with various thiols to give marked absorption changes
and enhanced fluorescence intensity. This prodrug exhibits
selective cytotoxicity toward folate-positive KB cells over folate-
negative A549 cells. We also found that the prodrug 1 is mainly
located in the ER of the cells and that the drug was delivered
upon reaction with cellular thiols. Furthermore, in vivo
fluorescence imaging experimentation upon intravenous
injection of the prodrug into the mice tail confirms that the
prodrug 1 is selectively taken up by KB over A549 tumor tissue
and its active gemcitabine is safely released. Therefore, our NIR
DDS could provide a powerful new strategy for tumor targeted
drug delivery, which can be easily monitored by cellular and
tissue fluorescence imaging.
Figure 5. Cell viability assay of 1 and 2 in KB and A459 cell lines,
respectively; all compounds were incubated with the cells for 72 h, and
the cell viability was observed via MTT assay.
parallel experiment using probe 2 shows high toxicity but no
preference to the cell type (Figure S19). The difference in the
drug sensitivity of these cells under the prodrug 1 treatment is
obviously due to the presence of the folate receptors on the KB
tumor cells. However, compared to 1, the KB cells are
somehow susceptible to 2. It seems to be delivered via simple
diffusion which is more efficient than 1′s delivery without any
cell preference. Nonetheless, together with the results described
in Figure 3, it is confirmed that the folate moiety is able to
guide the prodrug 1 to the folate receptor positive KB tumor
cells, followed by receptor-mediated endocytosis, resulting in
apoptosis by gemcitabine drug delivery.
The fluorescent switching-on of prodrug 1 has been
examined after intravenous injection of the prodrug into the
xenograft tumor animal model. Tumor-bearing mice were
prepared by subcutaneous injection of KB and A549 (3 × 106
each) cells into the right and left back of the mice (n = 3 each).
After 10 days of tumor inoculation, 0.1 mM/kg (DMSO:PBS =
50:50) of prodrug 1 was administered via the tail vein and
tumors were dissected from the mice after 24 h. Fluorescence
images of cryo-sectioned tumor tissues of 7-μm thickness were
obtained with excitation of Cy7 at 710/775 nm (Figure 6).
Fluorescence signals in the tumor site were detected only in
tissues bearing the KB tumor cells (about 1 cell per several 104
μm2 tissue sections), implying that the prodrug 1 shows the
tumor-targeting potential in vivo, which enables 1 to
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental details; synthesis and characteristic data (1H
NMR, 13C NMR, mass spectra); fluorescence spectra; CLSM
and HPLC data. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
jongskim@korea.ac.kr (J.S.K.); kangch@khu.ac.kr (C.K.);
kshong@kbsi.re.kr (K.S.H.)
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the CRI project (J.S.K., 2009-
0081566) of NRF, a grant from Korea Basic Science Institute
(K.S.H., D33400), and the “Cooperative Research Program for
Agriculture Science & Technology Development (Project No.
PJ008959)” Rural Development Administration, Republic of
Korea.
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