A Glutathione (GSH)-Responsive Near-Infrared (NIR) Theranostic Prodrug for Cancer Therapy and Imaging

Article abstract of DOI:10.1021/acs.analchem.6b01135

To reduce the side effects of chemotherapy, nontoxic prodrugs activated by the tumor microenvironment are urgently required for use in cancer treatment. In this work, we developed prodrug 4 for tumor-targeting treatment and imaging of the anticancer drug release in vivo. Taking advantage of the high glutathione (GSH) concentration in cancer cells, the disulfide bond in prodrug 4 was cleaved, resulting in the release of an active anticancer drug and a near-infrared (NIR) fluorescence dye turn-on. Furthermore, contrast to the free anticancer drug, the prodrug exhibited higher cytotoxicity to hepatoma cells than that to normal HL-7702 cells. Thus, prodrug 4 is a promising platform for specific tumor-activatable drug delivery system, because of its favorable features of in situ and in vivo monitoring of the drug release and therapeutic efficacy.

Full text of DOI:10.1021/acs.analchem.6b01135

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Article
A GSH-responsive NIR Theranostic Prodrug for Cancer Therapy and Imaging
Fanpeng Kong, Ziye Liang, Dongrui Luan, Xiaojun Liu, Kehua Xu, and Bo Tang
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01135 • Publication Date (Web): 24 May 2016
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Analytical Chemistry
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A GSH-responsive NIR Theranostic Prodrug for Cancer Therapy
and Imaging
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Fanpeng Kong, Ziye Liang, Dongrui Luan, Xiaojun Liu, Kehua Xu* and Bo Tang*
College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized
Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Eduꢀ
cation, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University, Jinan
250014, P. R. China.
ABSTRACT: To reduce the side effects of chemotherapy, nontoxic prodrugs activated by the tumor microenvironment are urgentꢀ
ly required for use in cancer treatment. In this work, we developed prodrug 4 for tumorꢀtargeting treatment and imaging of the antiꢀ
cancer drug release in vivo. Taking advantage of the high GSH concentration in cancer cells, the disulfide bond in prodrug 4 was
cleaved, resulting in the release of an active anticancer drug and an NIR fluorescence dye turnꢀon. Furthermore, contrast to the free
antiꢀcancer drug, the prodrug exhibited higher cytotoxicity to hepatoma cells than that to normal HLꢀ7702 cells. Thus, prodrug 4 is
a promising platform for specific tumorꢀactivatable drug delivery system due to its favorable features of in situ and in vivo monitorꢀ
ing of the drug release and therapeutic efficacy.
Cancer is one of the most lifeꢀthreatening diseases in the world.
Although various approaches for cancer therapy have been
developed, such as gene therapy,1ꢀ2 photodynamic therapy,3
photothermal ablation4 and radiation therapy,5ꢀ6 chemotherapy
remains a front line therapy for cancer in combination with
surgery. However, the main challenge of cancer chemotherapy
is the selective elimination of tumor cells without affecting
normal tissues. In an ideal case, drugs would be applied in
vivo at exactly the minimally needed therapeutic concentration
and would precisely target cancer cells. To achieve this goal,
the use of theranostic systems,7ꢀ12 which contain an inactive
anticancer drug and an imaging reagent, has received attention
as a promising cancer chemotherapeutic approach due to its
imaging capability and selective elimination of cancer cells. In
these systems, masked anticancer drugs and imaging reagents
are conjugated by tumorꢀsensitive linkers. In the tumor microꢀ
environments, such as those with low pH,13 high ROS level,14
high expressed enzymes15ꢀ16 and high GSH concentration,17 the
breaking of linkers is followed by the release of the antiꢀ
cancer drugs and the activation of the imaging reagents.
CPT, for monitoring cancer treatment in vivo.27 However, the
capability of tracking drug releases in vivo in these studies is
limited by poor photostability, low quantum yield, or water
solubility. Thus, there is an urgent need for monitoring drug
releases in vivo; NIR fluorophores, which has excellent propꢀ
erties, is one way to monitor drug releases in vivo.
Compared with traditional cyanine dyes, merocyanine dyes28ꢀ31
showed unique properties of photostability and high fluoresꢀ
cence quantum yield that are favorable for improving the qualꢀ
ity of the image in vivo. Although merocyanine dyes have
been employed as signal reporters of various probes,32ꢀ39 they
have rarely been used in theranostics prodrug systems to date.
Herein, we describe a novel NIR theranostic prodrug 4 for
cancer treatment obtained by conjugating a merocyanine dye
and an anticancer drug camptothecin (CPT)
40ꢀ41
via disulfide
linker. The disulfide bond in the prodrug 4 can be cleaved by a
high concentration of glutathione (GSH) in cancer cells,42ꢀ45
resulting in the anticancer drug release and NIR fluorescence
turnꢀon. Surprisingly, compared with free CPT, 4 showed efꢀ
fective pharmacodynamic effects in cancer cells and low cytoꢀ
toxicity to normal cells. This cancer cellꢀtargeting ability make
4 a promising prodrug platform with high efficacy and reꢀ
duced sideꢀeffects.
In theranostics, fluorophores are usually used as imaging reaꢀ
gents for monitoring the release of the drugs. However, fluorꢀ
ophores used for theranostic prodrugs, such as coumarin,18
BODIPY,19 1, 8ꢀnaphthalimide20 and xanthene,21ꢀ22 suffer from
short wavelength emission that is not suitable for monitoring
the drug release in vivo. Nearꢀinfrared (NIR) fluorophores
with characteristics such as deep tissue penetration, minimal
damage to the biological samples and low background interꢀ
ference23ꢀ25 are ideal candidate fluorophores for theranostics
prodrug systems. To date, very few theranostics prodrug sysꢀ
tems using NIR fluorophores have been reported. Kim et al.
developed a theranostic prodrug bearing the Cy7 dye as a NIR
fluorescence reporter.26 Zhu et al. described dicyanomethꢀ
yleneꢀ4Hꢀpyran (DCM)ꢀbased theranostic prodrug, DCMꢀSꢀ
EXPERIMENTAL SECTION
Materials and Instruments
Cy7.Cl and compound 1 was synthesized in our laboratory46ꢀ48
Bis (2ꢀhydroxyethyl) disulfide was purchased from Alfa Aesar
Chemical Ltd. (Tianjin, China). Nꢀ(4ꢀacerylphenyl)malemide
(NEM), glutathione (GSH), Lꢀcysteine (Lꢀcys), Lꢀ
Homocysteine (Hcy), ascorbic acid (Vc) , NꢀacetylꢀLꢀcysteine
(NAC), 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium
.
bromide
(MTT)
and
4ꢀ(2ꢀhydroxyethyl)ꢀ1ꢀ
piperazineethanesulfonic acid (HEPES) were all purchased
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Page 2 of 6
from SigmaꢀAldrich Co. Ltd. Triphosgene, 3ꢀNitrophenol, 4ꢀ
nitrophenylꢀchloroformate, (S)ꢀ(+)ꢀCamptothecin(CPT), and
DLꢀDithiothreitol were obtained from Aladdin Chemical
Company (Shanghai, China). N,NꢀDimethylformamide (DMF),
Triethylamine, Dichloromethane, Methanol, SnCl2ꢀH2O, Hyꢀ
drochloric Acid, Sodium hydroxide, Sodium sulfate, pyridine,
tetrahydrofuran, 4ꢀdimethylaminopyridine, Ether, Aluminum
oxide were purchased from Sinopharm Chemical Reagent. Co.
Ltd. (Shanghai, China). MitoꢀTracker Green was purchased
from Beyotime Institute of Biotechnology (Shanghai, China).
The silica gel for the flash chromatography was purchased
from Qingdao Haiyang Chemical Co. (China). Sartorius ulꢀ
trapure water (18.2 Mꢁ cm) was purified with Sartorius Ariꢀ
um 611 VF system (Sartorius AG, Germany). The solvents
were used after appropriate distillation or purification.
MeOH (100:0 then 20:1) to afford 11.6 mg (33 %) of the
compound 3. H NMR (400 MHz, CDCl3 ): δ 1.54 (s, 3H),
1
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1.63 (s, 6H), 1.90 (s, 2H), 2.65 (s, 2H), 2.73 (s, 2H), 3.03 (s,
4H), 3.93 (s, 2H), 4.39 (s, 4H), 5.09 (s, 1H), 6.26 (s, 1H),
7.25ꢀ7.37 (m, 7H), 7.92 (s, 1H), 8.07 (s, 1H), 8.63 (s, 2H),
10.75 (s, 1H). 13C NMR (100 MHz, CDCl3 ): δ12.6, 20.3, 28.2,
38.1, 41.0, 50.6, 60.8, 62.45, 76.7, 77.0, 77.4, 105.1, 11.7,
116.6, 122.8, 129.0, 135.2, 141.0, 153.8, 162.6, 176.3. HR MS
[MꢀI] +: m/z calcd 577.2189, found 577.2188.
Synthesis of compound 4
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The solution of camptothecin (83.6 mg, 0.24 mmol) in CH2Cl2
(10 mL) were added DMAP (61.1 mg, 0.5 mmol) and triphosꢀ
gene (23.7 mg, 0.08 mmol). The reaction mixture was stirred
at room temperature for 15 min. Then a solution of the comꢀ
pound 3 (70.4 mg, 0.1 mmol) in CH2Cl2 (20 mL) was slowly
added into above mixture. The mixture was stirred at room
temperature over night, poured into diethyl ether. The crude
product produced was filtered and purified via chromatogꢀ
raphy (neutral alumina) eluting with CH2Cl2: MeOH (100:0
Highꢀresolution mass spectral analyses were carried out on
Bruker maXis ultraꢀhigh resolutionꢀTOF MS system. 1H NMR
and 13C NMR spectra were taken on Bruker Advance 300
MHz and 400 MHz spectrometer (Bruker, Germany). The
fluorescence spectra measurements were performed with FLSꢀ
920 Edinburgh fluorescence spectrometer. Absorption spectra
were recorded on UVꢀ1700 UV−vis spectrophotometer (Shiꢀ
madzu, Japan). All pH measurements were performed with a
pHꢀ3c digital pH meter (Shanghai Lei Ci Device Works,
Shanghai, China) with a combined glasscalomel electrode.
The MTT assay was measured with a microplate reader (RT
6000, Rayto, United States). The fluorescence imaging studies
were performed with a Leica DMI6000 fluorescence microꢀ
scope (Leica Co., Ltd., Germany). The in vivo fluorescence
imaging was carried out using an IVIS LuminaⅢ in vivo imꢀ
aging system.
1
then 20:1) to afford 73.8 mg (68%) of the compound 4. H
NMR (400 MHz, CDCl3 ): δ 1.54 (s, 3H), 1.63 (s, 6H), 1.90 (s,
2H), 2.65 (s, 2H), 2.73 (s, 2H), 3.03 (s, 4H), 3.93 (s, 2H), 4.39
(s, 4H), 5.09 (s, 1H), 6.26 (s, 1H), 7.25ꢀ7.37 (m, 7H), 7.92 (s,
1H), 8.07 (s, 1H), 8.63 (s, 2H), 10.75 (s, 1H). 13C NMR (100
MHz, CDCl3 ): δ12.6, 20.3, 28.2, 38.1, 41.0, 50.6, 60.8, 62.45,
76.7, 77.0, 77.4, 105.1, 11.7, 116.6, 122.8, 129.0, 135.2, 141.0,
153.8, 162.6, 176.3. HR MS [MꢀI]+:m/z calcd 951.3092, found
951.3092.
RESULTS AND DISCUSSION
Scheme 1. Synthesis of prodrug 4
Synthesis of compound 2
To a solution of compound 1 (0.37 g, 0.5 mmol) in MeOH
were added SnCl2ꢂH2O (2.25 g, 10 mmol) and hydrochloric
acid (3 mL). The mixture was stirred at 70 °C under Ar over
night. Methanol was evaporated, and the residue was separated
in CH2Cl2 and water. The aqueous layer was extracted with
CH2Cl2. The combined organic layers were dried over anhyꢀ
drous sodium sulfate and evaporated. The crude product was
purified via chromatography (silica gel) eluting with CH2Cl2:
1
MeOH (20:1) to afford 0.11 g (42 %) of the compound 2. H
NMR (400 MHz, CDCl3 ): δ 1.48 (s, 3H), 1.77 (s, 6H), 1.94 (s,
2H), 2.65 (s, 2H), 2.75 (s, 2H), 3.48 (s, 2H), 4.08 (s, 1H), 5.90
(d, J = 16Hz, 1H), 6.68 (s, 2H), 6.92 (s, 1H), 7.06 (d, J = 8Hz,
1H), 7.14 (s, 2H), 7.39ꢀ7.41 (m, 3H), 8.53 (d, J = 16Hz, 1H).
13C NMR (100 MHz, CDCl3 ): δ 12.5, 24.2, 28.3, 49.5, 79.7,
97.6, 100.1, 111.7, 113.3, 114.5, 115.2, 122.7, 129.1, 130.3,
+
139.1, 141.4, 142.0, 155.7, 162.9, 173.1. HR MS [MꢀI] : m/z
Conditions: (a) mꢀNitrophenol, Et3N, DMF, RT. (b) SnCl2,
HCl, MeOH, 70 °C. (c) 4ꢀNitrophenyl chloroformate, bisꢀ(2ꢀ
hydroxyethyl)disulfide, pyridine, THF (d) CPT, DMAP, triꢀ
phosgene, CH2Cl2.
calcd. 397.2274, found 397.2282.
Synthesis of compound 3
To a solution of the 4ꢀnitrophenyl chloroformate (0.1g, 0.5
mmol) in THF (1 mL) was added dropwise the mixture of
compound 2 (26.2 mg, 0.05 mmol) and pyridine (20 ꢃL) in dry
CH2Cl2 (4 mL). The reaction mixture was stirred at 0 °C for 2
h. Then, the above mixture was added dropwise into a solution
of bis (2ꢀhydroxyethyl) disulfide (100ꢃL) and pyridine (1 mL)
in THF (3 mL), the mixture was stirred room temperature unꢀ
der Ar for 12 h. The solvents were evaporated, and the residue
was separated in CH2Cl2 and water. The aqueous layer was
extracted with CH2Cl2. The combined organic layers were
dried and evaporated. The crude product was purified via
chromatography (neutral alumina) eluting with CH2Cl2:
The synthesis of prodrug 4 is depicted in Scheme 1. The merꢀ
ocyanine dye was synthesized from Cy7.Cl following the preꢀ
viously reported procedures.28ꢀ31 Compound 2 was reacted
with triphosgene, followed by treatment with 2, 2’ꢀ
dithiodiethanol to afford compound 3. Finally, prodrug 4 was
obtained by coupling of CPT with compound 3 in the presence
of triphosgene. The overall chemical structures of the prodrug
and the intermediate compounds were confirmed by 1H NMR,
13C NMR and HRMS (Supporting Information).
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Analytical Chemistry
Scheme 2. Proposed CPT Release Mechanism of the Acti-
vatable Prodrug 4
Figure 2. (a) Fluorescence response of prodrug 4 (17.5 ꢃM)
toward GSH, DTT, Cys, Hcy, other amino acids (Trp, Lys,
Met, Glu, Thr, Pro) and metal ions, 1 mM for each. (b) Fluoꢀ
rescence intensity at 702 nm of 4 with and without GSH as a
function of pH. Each spectrum was acquired 1 h after expoꢀ
sure to GSH (40 equiv.) at room temperature in HEPES buffer
(pH = 7.4) with λex = 660 nm.
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To evaluate the possible application of 4 in real biological
systems, the fluorescence response of 4 toward other biologiꢀ
cally relevant analytes, such as amino acids and metal ions,
was investigated. As shown in Figure 2a, no significant
changes in fluorescence intensity were observed upon addition
of the nonthiol amino acids and metal ions to prodrug 4. Altꢀ
hough, cysteine (Cys), dithiothreitol (DTT), and homocysteine
(Hcy) were found to cause a fluorescence response of 4 similar
to that of GSH, the potential interference of these compounds
can be neglected because of their relatively low concentrations
in the biologically systems. The pH effect on the GSHꢀinduced
fluorescence changes of 4 was also investigated. As shown in
Figure 2b, prodrug 4 remained stable and emits a weak fluoꢀ
rescence within 5.5ꢀ8.0 pH range. After treatment by 40 equiv
of GSH, the fluorescence at 702 nm was activated and reached
saturation in the 6.7ꢀ8.0 pH range. The above results indicated
that prodrug 4 can be applied as a GSHꢀinduced theranostics
system under physiological pH conditions with high selectiviꢀ
ty over potential interference by various biological compounds.
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The proposed CPT release mechanism of the activatable proꢀ
drug 4 is shown in Scheme 2. In the presence of GSH, the
disulfide bond of prodrug 4 is cleaved. Then, the intermediates
participate in the intramolecular cyclization44ꢀ45 and release the
NIR dye and the active drug CPT. In the high resolution mass
spectrometry (MS) spectra, the intrinsic peak (951.3097) of
prodrug 4 disappeared, and new peaks at 397.2273 (correꢀ
sponding to dye 2) and 349.1177 (corresponding to antiꢀcancer
drug CPT) were observed upon interaction of 40 equiv of GSH
with prodrug 4 (Figure. S1). These results are fully consistent
with our proposed drug release mechanism.
Figure1. (a) Fluorescence changes of prodrug 4 after treatment
with increasing concentrations of GSH (0ꢀ100 equiv). (b)
Changes in fluorescence intensity at 702 nm as a function of
GSH concentration. Each spectrum was acquired 1 h after
exposure to GSH at room temperature in HEPES buffer (pH =
7.4) with λex = 660 nm.
To verify that GSH was able to cleave the disulfide bond of
the prodrug 4 and consequently, activate the NIR fluorophore,
the fluorescence spectra of 4, upon addition of 0ꢀ100 equiv of
GSH, were investigated under optimum conditions (Figure.S2ꢀ
S4). As seen in Figure 1a, the fluorescence at 702 nm gradualꢀ
ly increased upon a stepwise addition of GSH and reached
saturation upon an addition of 40 equiv of GSH. The fluoresꢀ
cence intensity increased by approximately five times and
shows good linearity with the concentration of GSH over a
wide range (0ꢀ0.7 mM). This observation indicated that the
disulfide linker of the prodrug 4 can be effectively cleaved by
GSH. Therefore, the high concentration of GSH in cancer cells
may lead to the release of CPT and the NIR fluorescence turnꢀ
on.
Figure 3 Cellular fluorescence images of prodrug 4ꢀtreated
HepG2 and HLꢀ7702 cells. (a) HepG2 cells pretreated by
NEM (A), HLꢀ7702 cells (B), HepG2 cells were incubated
with prodrug 4 (C), HepG2 cells were pretreated with GSH
followed incubated with prodrug 4 (D). (b) Quantitative analyꢀ
sis of fluorescence intensity in cells incubated with prodrug 4.
(c) Confocal microscopic images of colocalized experiment in
HepG2 cells. Fluorescence images of HepG2 cells contained
with mitochondria tracker (E), fluorescence images of HepG2
cells contained with 4 (17.5 ꢃM) for 1 h (F). Overlay of
merged images of E and F (G). Fluorescence intensity profile
of regions of interest (green line in G) across the lines (H).
The photostability of NIR fluorophores in the theranostic proꢀ
drug is crucial for practical application in bioimaging. The
photoꢀstability of prodrug 4 treated by GSH was investigated
by timeꢀsequential scanning of living cells. After 500 s of conꢀ
tinuous irradiation with a 633 nm laser, no obvious changes
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