A sequential enzyme-activated and light-triggered pro-prodrug nanosystem for cancer detection and therapy

Article abstract of DOI:10.1039/c7tb01989k

DT-diaphorase is a cytosolic flavoenzyme whose level is strongly elevated in a number of tumor types. Incorporating a DT-diaphorase's substrate in the structure of anticancer drugs may facilitate cancer detection and therapy. Herein, we developed a novel pro-prodrug nanosystem for cancer detection and therapy, which features enzyme-activated fluorescence emission and subsequent light-triggered drug release. The pro-prodrug molecule comprises an anticancer drug methotrexate (MTX), an enzyme (DT-diaphorase) responsive quinone propionic acid moiety and a light-activatable coumarinyl. In the absence of DT-diaphorase, the quinone propionic acid moiety quenches the fluorescence of coumarin via photoinduced electron transfer (PET) and blocks the photocleavage pathway. DT-diaphorase can annihilate the effect of PET and restore the fluorescence of coumarin. This fluorescence serves as the reporting signal for assessing the enzyme biomarker level and discriminates tumor cells from normal cells, and subsequently photocontrollable release of the active drug, MTX, can be activated via one- or two-photon irradiation. This pro-prodrug nanosystem shows strong cytotoxicity toward cancer cells and a negligible effect on normal cells. This strategy provides a new platform for constructing nanosystems for cancer detection and subsequent on-demand selective killing of cancer cells via both internal- and external-stimuli activation.

Full text of DOI:10.1039/c7tb01989k

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Guoping Chen et al.
Regulating the stemness of mesenchymal stem cells by tuning
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A sequential enzyme-activated and light-triggered pro-prodrug
nanosystem for cancer detection and therapy
Received 00th January 20xx,
Accepted 00th January 20xx
Zelin Chen†, Bowen Li†, Xin Xie, Fang Zeng*, Shuizhu Wu*
DT-diaphorase is a cytosolic flavoenzyme whose level is strongly elevated in a number of tumor types. Incorporating a DT-
diaphorase’s substrate in the structure of anticancer drugs may facilitate cancer detection and therapy. Herein, we
developed a novel pro-prodrug nanosystem for cancer detection and therapy, which features enzyme-activated
fluorescent emission and subseqent light-triggered drug release. The pro-prodrug molecule comprises an anticancer drug
methotrexate (MTX), an enzyme (DT-diaphorase) responsive quinone propionic acid moiety and a light-activatable
coumarinyl. In the absence of DT-diaphorase, quinone propionic acid moiety quenchs the fluorescence of coumarin via
photoinduced electron transfer (PET) and blocks the photocleavage pathway. DT-diaphorase can annihilate the effect of
PET and restore the fluorescence of coumarin. This fluorescence serves as the reporting signal for assessing the enzyme
biomarker level and discriminates tumor cells from normal cells, and subsequently photocontrollable release of active
drug MTX can be activated via one- or two-photon irradiation. This pro-prodrug nanosystem show strong cytotoxicity
toward cancer cells and ignorable effect on normal cells. This strategy provides a new platform for constructing
nanosystems for cancer detection and subsequent on-demand selective killing of cancer cells via both internal- and
external-stimuli activation.
DOI: 10.1039/x0xx00000x
www.rsc.org/
DT-diaphorase, an important detoxifying enzyme, is widely
distributed in a variety of tissues and can catalyze an obligatory two
electron reduction of a broad range of substrates (quinones) using
1. Introduction
Traditional chemotherapy have shown accredited efficacy for
cancer treatments but is often hindered by several side-effects (high
toxicity, non-specificity and etc.) to normal cells.[1-4] On-demand
treatment upon effective cancer detection or discrimination of
cancer cells from normal cells would greatly reduce high toxicity of
traditional chemotherapy resulted from non-specificity of drugs. In
addition, prodrugs, which can be activated by unique
microenvironment in the tumor, strive to develop a new strategy for
bating the side-effects.[5-10] The traditional pursuit of prodrug has
largely focused on special internal stimuli in the tumor, such as high
levels of biothiols,[11-17] lower pH,[18-24] reactive oxygen species[25-32]
and various overexpressed enzymes.[33-41] Whereas, recent
researches revealed that the activation of prodrug in cancer cells
may be successively controlled by internal and external stimuli,[42-45]
and this kind of prodrug is also described as a “pro-prodrug”. A new
strategy for the pro-prodrug is that it is first actviated by the
differential expression of internal stimuli thus forming the prodrug,
then this prodrug can discriminate tumor cells from normal cells
and then release the anticancer drugs via external stimuli.[46-52]
either nicotinamide adenine dinucleotide (NADH) or nicotinamide
adenine dinucleotide phosphate (NADPH) as electron donors.[53-55]
Many literature reports indicated that DT-diaphorase is
overexpressed in many tumor cells/tissues, such as breast cancer,
non-small cell lung cancer, colorectal cancer and cervical cancer.[56]
All these cancer cells have been found to exhibit strongly elevated
DT-diaphorase concentrations (up to 50-fold compared to normal
cells).[57-58] On account of the huge difference of the DT-diaphorase
level between tumor and normal tissues, the enzyme has attracted
considerable attention as
a potential candidate for cancer
diagnosis.[59-62] Our group has demonstrated that DT-diaphorase
responsive produrg is benifical for cancer chemotherapy; however,
there are no reports on the exploitation of the DT-diaphorase for
developing pro-prodrug for imaging cancer cells and possible
therapy.[63-64]
Methotrexate (MTX), as a folate reductase inhibitor, mainly
inhibits dihydrofolate reductase thus leaving dihydrofolic acid not
being able to be reduced to tetrahydrofolic acid (a cofactor in
anabolism of amino acids and nucleic acids), which accordingly
leads to DNA biosynthesis being inhibited. It is one of the most
widely used drugs in medical oncology for treatment of several
types of cancer.[65-66] However, many obstacles, including toxic side
effects toward normal cells, drug resistance, and acute and chronic
toxicity, still remain that impede its clinical application. Besides, its
poor water solubility also constitutes a hurdle in terms of wide-
ranging clinical application of MTX.[67-69] The methotrexate-based
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2. Experimental Section
2.1 Materials
prodrugs are an effective approach to overcome these
shortcomings, as well as improving therapeutic efficacy, but its poor
water solubility is still a major trouble for cancer treatment.[70-71]
m-Dihydroxybenzene, ethyl 4-chloroacetoacetate, methanesulfonic
acid, trimethylhydroquinone (TMHQ), methyl 3,3-dimethylacrylate,
Toward the aim of overcoming the above limitations, herein,
N-bromosuccinimide(NBS),
ethylcarbodiimide hydrochloride
1-(3-dimethylaminopropyl)-3-
(EDC) and 4-
we developed
a novel theranostic pro-prodrug for cancer
detection/imaging and therapy in DT-diaphorase over-expressed
tumor cells; the MTX-based pro-prodrug nanosystem features the
enzyme-activated fluorescence and subsequent light-triggered on-
demand drug release. This pro-prodrug contains three parts:
anticancer drug methotrexate (MTX), coumarin and quinone
propionic acid (Scheme 1). In this pro-prodrug, the prodrug
(coumarin derivatives) serves as the fluorophore to assess DT-
diaphorase level upon enzymatic reaction (enzyme activated) which
would be conducive to cancer detection/imaging, and also as a
phototrigger (upon unlocking) to release the active drug. Quinone
propionic acid unit, an electron accepter, is a key part of the pro-
prodrug that can lock the coumarin phototrigger and swich off its
fluorescence. In this design, as for the locked state, the coumarin
phototrigger and fluorescence are completely banned via
photoinduced electron transfer (PET) to quinone propionic acid unit.
On the contrary, the unlocked state which is activated only by DT-
diaphorase and monitored by coumarin fluorescence can release
the MTX by either one-photon visible light or two-photon
nearinfrared (NIR) light. The pro-prodrug was also loaded into the
liposomes, which could improve the biocompatibility and reduce
the side-effects, especially for enhancing the aqueous solubility of
MTX drug.[72-74] This new strategy integrating both internal and
external triggers offers a powerful tool for possible effective cancer
treatment. To the best of our knowledge, this is the first report on
the imaging and therapeutic effect of enzyme-activated light-
triggered pro-prodrug nanosystem.
dimethylaminopyridine(DMAP) were purchased from Aladdin
Reagent without further purification. 1,2-Distearoyl-sn-glycero-3-
phosphoethanolamine-N- [amino(polyethylene glycol)-2000] (DSPE-
PEG 2000) was provided by Thermo-scientific. DT-diaphorase
(lyophilized powder, recombinant, expressed in E. coli) and β-
nicotinamide adenine dinucleotide reduced disodium salt (NADH)
were purchased from Sigma-Aldrich. Water used in the experiments
is deionized water. Dichloromethane (DCM) and N, N-dimethyl-
formamide (DMF) was dried with CaH2 and distilled under nitrogen
atmosphere. Other solvents used in this study were analytical grade
reagents and used without further purification.
2.2 Pro-prodrug synthesis
Synthesis of 4-(chloromethyl)-7-hydroxy-2H-chromen-2-one
(1).
m-Dihydroxybenzene (4.40 g, 40 mmol) and ethyl 4-
chloroacetoacetate (7.90 g, 48 mmol) in concentrated H2SO4
solution (70%, 40 mL) were stirred for 12 h at 0 oC. The resulting
solution was poured slowly into ice water (300 mL) and a large
amount of white solid precipitated. The resulting precipitate was
filtered and washed with deionized water, followed by drying to
give compound 1 (7.27 g, 86% yield) as a white powder. 1H NMR
(600 MHz, DMSO-d6, ppm) δ 10.67 (s, 1H), 7.68 (d, J = 8.8 Hz, 1H),
6.85 (dd, J = 8.7, 2.4 Hz, 1H), 6.76 (d, J = 2.3 Hz, 1H), 6.43 (s, 1H),
4.96 (s, 2H). HR-MS (ESI): calcd for C10H7ClO3 ([M-H]-) 209.0006,
found: 209.0002.
Synthesis of 7-hydroxy-4-(hydroxymethyl)-2H-chromen-2-one
(2).
Compound 1 (4.20 g, 20 mmol) was added into deionized water (30
mL) and refluxed for two days. After the completion of reaction, the
solvent was removed and diluted with EtOAc and the organic phase
was washed with water and brine, dried over anhydrous Na2SO4 and
concentrated in vacuo. The crude product was purified by column
chromatography on silica gel (Methanol:DCM 1:10) to give
compound 2 (2.56 g, 67%) as a white powder. 1H NMR (600 MHz,
DMSO-d6, ppm) δ 10.53 (s, 1H), 7.52 (d, J = 8.7 Hz, 1H), 6.77 (dd, J =
8.7, 2.4 Hz, 1H), 6.73 (d, J = 2.3 Hz, 1H), 6.24 (t, J = 1.4 Hz, 1H), 5.59
(s, 1H), 4.70 (d, J = 1.3 Hz, 2H). HR-MS (ESI): calcd for C10H8O4 ([M-
H]-) 191.0345, found: 191.0345.
Synthesis of 6-hydroxy-4,4,5,7,8-pentamethylchroman-2-one
(3).
Scheme 1. Schematic overview of a pro-prodrug nanosystem for
imaging and therapy. Before enzyme-activation, in the locked state
Trimethylhydroquinone (3.0 g, 20 mmol) and methyl 3,3-
dimethylacrylate (2.74 g, 24 mmol) were added to methanesulfonic
acid (20 mL) and heated to 70 oC. The resulting solution was reacted
with vigorous stirring for 2 hours. After that, the reaction solution
was poured into water (100 mL), and then extracted with DCM and
the organic phase was washed with water and brine, dried over
anhydrous Na2SO4, followed by drying to give compound 3 (3.76 g,
80%) as a brown powder. 1H NMR (600 MHz, CDCl3, ppm) δ 4.57 (s,
pro-prodrug, the coumarin is
a dormant phototrigger with
quenched fluorescence; while enzymatic reaction leads to the
cleavage of quinone propionic acid group, which not only restores
the fluorescence of coumarin but also make it an active
phototrigger, leading to the release of active drug.
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1H), 2.55 (s, 2H), 2.36 (s, 3H), 2.22 (s, 3H), 2.18 (s, 3H), 1.46 (s, 6H). Synthesis
HR-MS (ESI): calcd for C14H18O3 ([M-H]-) 233.1178, found: 233.1174.
of
2-(4-(((2,4-diaminopteridin-6-
yl)methyl)(methyl)amino)benzamido)pentanedioic acid (7,
HO-COU-MTX, the prodrug molecule).
Synthesis of 3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-
1,4-dienyl)butanoic acid (4)
Compund 6 (16 mg, 0.018 mmol) was dissolved in 10 mL phosphate
buffer (pH 7.4, 10 mM) containing 1% (v/v) DMSO. In the presence
Compound 3 (2.34 g, 10 mmol) and N-bromosuccinimide (1.78 g,10 of DT-diaphorase and its cofactor NADH (100 μM), the resulting
mmol) were dissolved in 10% aqueous acetonitrile. The resulting solution was allowed to react at 37 oC for 24 h. The reaction solution
solution was stirred at room temperature for 4 h. The solvent was was extracted with DCM and purified by column chromatography on
removed and diluted with DCM and the organic phase was washed silica gel (Methanol:DCM, 1:20) to give compund 7 (7 mg, 59% yield)
with water and brine, dried over anhydrous Na2SO4 and as a yellow powder. The entire operation is completed in dark
concentrated in vacuo. The crude product was purified by column conditions. 1H NMR (600 MHz, DMSO-d6, ppm) δ 12.27 (s, 1H), 10.67
chromatography on silica gel (DCM) to give compound 4 (1.93 g, (s, 1H), 8.58 (s, 1H), 8.19 (d, J = 7.7 Hz, 1H), 7.73 (d, J = 9.0 Hz, 2H),
1
77%) as a yellow powder. H NMR (600 MHz, CDCl3, ppm) δ 3.02 (s, 7.68 (d, J = 8.8 Hz, 1H), 7.58 (d, J = 111.0 Hz, 2H), 6.86 – 6.84 (m,
2H), 2.14 (s, 3H), 1.94 (dd, J = 18.2, 1.0 Hz, 6H), 1.43 (s, 6H). HR-MS 1H), 6.83 (d, J = 9.1 Hz, 2H), 6.76 (d, J = 2.3 Hz, 1H), 6.67 (s, 2H), 6.42
(ESI): calcd for C14H18O4 ([M-H]-) 249.1127, found: 249.1125.
(s, 1H), 4.96 (s, 2H), 4.79 (s, 2H), 4.35 (ddd, J = 9.8, 7.7, 5.0 Hz, 1H),
3.21 (s, 3H), 2.32 (t, J = 7.5 Hz, 2H), 2.06-1.90 (m, 2H). HR-MS (ESI):
Synthesis of 4-(hydroxymethyl)-2-oxo-2H-chromen-7-yl 3- calcd for C30H38N8O8 ([M-H]-) 627.1952, found: 627.1954.
(2H-chromen-7-yl-3-)methyl-3-(2,4,5-trimethyl-3,6-
dioxocyclohexa-1,4-dienyl)butanoate (5).
Compound 2 (1.0 g, 5.2 mmol) and compound 4 (1.56 g, 6.2 mmol) 2.3 Fabrication of liposomal nanoparticles (LIP-DT-COU-MTX, the
were dissolved in 20 mL anhydrous DMF and DCM mixed solvent pro-prodrug nanosystem)
(VDMF/VDCM, 2:8), and then EDC (1.19 g, 6.2 mmol) and catalytic The liposomal nanoparticles were prepared by
a solvent
amount of DMAP were added to the solution. After stirring for 4 h evaporation method. DT-COU-MTX (8 mg) was dissolved in 200 μL
at room temperature, the solvent was removed and diluted with of DMSO and DSPE-PEG 2000 (8 mg) was dissolved in 1.8 mL of THF.
DCM. The organic phase was washed with water and brine, dried The solutions were added to ice water under sonication for 30 min
over anhydrous Na2SO4 and concentrated in vacuo. The crude for emulsification. After the solution was stirred at room
product was purified by column chromatography on silica gel temperature for 24 hours, the solvent was removed under vacuum.
(EtOAc:Hexanes 1:3) to give compound 5 (1.57 g, 71% yield) as a Next, the residual solution was subjected to one stage of dialysis
yellow powder. 1H NMR (600 MHz, CDCl3, ppm) δ 7.49 (d, J = 8.7 Hz, against water (2 L) using cellulose membrane with a 1000 molecular
1H), 7.04 (d, J = 2.2 Hz, 1H), 6.94 (dd, J = 8.7, 2.3 Hz, 1H), 6.59 (t, J = weight cutoff. Liposomes are obtained by ultracentrifugation at
1.4 Hz, 1H), 4.87 (d, J = 1.5 Hz, 2H), 3.29 (s, 2H), 2.18 (s, 3H), 1.94 (d, 12,000 rpm for 10 min at 4 oC. Finally, the collected nanoparticles
J = 3.4 Hz, 6H), 1.53 (s, 6H). HR-MS (ESI): calcd for C24H24O7 ([M-H]-) were stored at 4 oC for use.
423.1444, found: 423.1447.
The determination of the drug loading rate is based on the
measurement of the absorption spectra. The lyophilized DT-COU-
Synthesis of
2-(4-(((2,4-diaminopteridin-6- MTX-loaded nanoparticles were dissolved in 1mL of DMSO and
yl)methyl)(methyl)amino)benzamido)-5-((7-(3-methyl-3-
(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanoyloxy)-
2-oxo-2H-chromen-4-yl)methoxy)-5-oxopentanoic acid (6, DT-
COU-MTX, the pro-prodrug molecule).
subsequently tested for its absorbance at 380 nm. We define the
drug loading rate (DLR) as follows:
DLR = (mass of loaded DT-COU-MTX)/(mass of nanoparticles) x
100%
The determined DLR value was 49.6%.
MTX (300 mg, 0.66 mmol) and compound 5 (616 mg, 1.45 mmol)
were dissolved in 10 mL anhydrous DMF and DCM mixed solvent
(VDMF/VDCM, 2:8), and then EDC (279 mg, 1.45 mmol) and catalytic
amount of DMAP were added to the solution. After stirring for 24 h
at room temperature under nitrogen, the solvent was removed and
diluted with DCM. The organic phase was washed with water and
brine, dried over anhydrous Na2SO4 and concentrated in vacuo. The
crude product was purified by column chromatography on silica gel
(Methanol:DCM 1:20) to give compund 6 (85 mg, 15% yield) as a
light yellow powder. 1H NMR (600 MHz, DMSO-d6, ppm) δ 12.33 (s,
1H), 8.57 (s, 1H), 8.19 (d, J = 7.7 Hz, 1H), 7.88 (d, J = 8.7 Hz, 1H), 7.73
(d, J = 9.0 Hz, 2H), 7.56 (d, J = 132.5 Hz, 2H), 7.21 (d, J = 2.3 Hz, 1H),
7.11 (dd, J = 8.7, 2.3 Hz, 1H), 6.83 (d, J = 9.1 Hz, 2H), 6.68 (s, 2H),
6.68 (s, 1H), 5.02 (s, 2H), 4.79 (s, 2H), 4.35 (ddd, J = 9.7, 7.7, 5.0 Hz,
1H), 3.22 (s, 2H), 3.21 (s, 3H), 2.32 (t, J = 7.5 Hz, 2H), 2.13 (s, 3H),
2.09-1.91 (m, 2H), 1.88 (dd, J = 6.4, 1.0 Hz, 6H), 1.49 (s, 6H).HR-MS
(ESI): calcd for C24H24O7 ([M-H]-) 859.3052, found: 859.3050.
2.4 Cell culture
A549 cells (human non-small cell lung cancer cells), HeLa cells
(Human cervical cancer cells) and L929 cells (murine aneuploid
fibrosarcoma cells) were used for all in vitro cell based studies.
A549 and L929 cells were cultured using RPMI 1640 medium,
while HeLa cells were cultured using Dulbecco’s modified eagle
medium (DMEM). Both media contained 10 % FBS (Fetal
Bovine Serum), and 1% penicillin and streptomycin (GIBCO).
The medium is replaced at regular intervals (1 to 2 days). All
three cell lines were incubated in an environment with a
temperature of 37 °C and a carbon dioxide concentration of
5%.
2.5 Cell imaging
The three cell lines (A549, L929 and HeLa) were cultured 24
hours later and seeded into a six-well plate containing
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polylysine-coated cell culture glass slides. When the cell level, the A549 and HeLa cells were incubated with different
density was 50%-70%, the medium was replaced and washed concentrations of LIP-DT-COU-MTX (10 μg/mL to 100 μg/mL) for 2
with PBS, followed by treatment with LIP-DT-COU-MTX for 1 h h, then the cells were under light irradiation for 30 min (LED lamp,
or 2 h. After that the glass slides were taken out and washed with the wavelength range of 400–450 nm at the power of 10 mW
with PBS, followed by fluorescence imaging experiments. cm−2, blue light), and afterwards the cells were further incubated for
Fluorescence images were obtained using an Olympus IX71 9.5 h. The experiments involving dicoumarol (DIC) inhibition were
inverted fluorescence microscope with a DP72 color CCD.
conducted as follows: the cells were first pretreated with DIC (20
μM) for 4 h and then incubated with LIP-DT-COU-MTX (70 μg/mL)
2.6 Fluorescence generation of LIP-DT-COU-MTX measured by under the same conditions (with the same light irradiation
flow cytometry conditions). In the end, the cells were washed with PBS and
A549 and HeLa cells were seeded in 6-well plate at a density of harvested after trypsinization and centrifugation at 2000 rpm for 10
o
1.0 x 106 cells / well for 12 h at 37 C. Cells were exposed to min. Subsequently, the cells were collected and resuspended in 1
LIP-DT-COU-MTX (20 μg/mL) and incubated for 1 h and 2 h mL PBS and then mixed with Annexin V Binding buffer (400 μL)
respectively. The cells were then washed with PBS and containing 5 μL Annexin V-FITC and 10 μL PI. Finally, the flow
harvested after trypsinization and centrifugation. Approximate cytometry analyses were performed using BD Accuri C6 flow
10,000 cells were analysed on a BD CS6 flow cytometer cytometer and the data were analyzed using the BD Biosciences
equipped with a 370 nm argon laser.
software.
2.7 Cell viability assay
2.9 Measurements
Cells (5000 cells/well) were seeded in 96-well plates. After 24 h of 1H NMR spectra were recorded on a Bruker Avance 600 MHz NMR
incubation at 37 °C, cells were washed with PBS buffer, and then the spectrometer. All chemical shifts are reported in ppm value using
PBS was replaced with fresh medium containing a series of LIP-DT- the peak of residual proton signals of TMS as an internal reference.
COU-MTX samples from 0.2 to 70 μg/mL or MTX from 0.1 to 30 Mass spectra were obtained on a Bruker Esquire HCT Plus mass
μg/mL for 2 h at 37 °C, then the cells were placed under light spectrometer. High resolution mass spectra were obtained on Sciex
irradiation for 30 min (LED lamp, with the wavelength range of 400– X500R mass spectrometer. UV-vis spectra were recorded on a
450 nm at the power of 10 mW cm−2, blue light), and afterwards the Hitachi U-3010 UV-vis spectrophotometer. Fluorescence spectra
cells were further incubated for 48 h. The experiments involving were recorded on a Hitachi F-4600 fluorescence spectrophotometer.
dicoumarol (DIC) inhibition were conducted as follows: first the cells Fluorescence images were obtained using an Olympus IX 71 with a
were pretreated with DIC (20 μM) for 4 h and then incubated with DP72 color CCD. HPLC analyses were performed on Agilent
different concentrations of LIP-DT-COU-MTX (0.2 to 70 μg/mL) 1260. The two-photon experiments were performed by
under the same conditions. In addition, to evaluate the toxicity of irradiating with a fs mode-locked Ti:sapphire laser system
DIC, the cells were only incubated with DIC (20 μM) for 48 h. For the (output beam
photocytotoxicity experiment, the cells were irradiated by visible Flow cytometry were recorded on a BD CS6 flow cytometer.
light for 30 min (LED lamp, with the wavelength range of 400–450 he particle size and distribution were determined based on
≈120 fs duration and 1 kHz repetition rate).
T
nm at the power of 10 mW cm−2, blue light). After irradiation, the dynamic light scattering (DLS) mechanism on a Malvern Nano-
cells were again incubated for 48 h. For the dark cytotoxicity assay, ZS90 particle size analyzer at a fixed angle of 90° at 25°C.
the cells were incubated with LIP-DT-COU-MTX without irradiation. Transmission electronic microscopy (TEM) experiments were
After incubation for 48 h, the cells were washed three times with carried out by mounting a drop (~15 μL) of the solution onto a
PBS buffer and treated with DMEM medium or RPMI 1640 medium carbon-coated copper grid and observed on a JEM-2010HR
containing 0.5 mg/mL MTT for another 4 h. The resulting formazan transmission electron microscopy.
crystal was dissolved in 150 μL of DMSO after carefully removing
the medium and the absorbance was recorded at 570 nm. The cell
3. Results and discussion
viability assays were performed using a Thermo MK3 ELISA plate
reader. The independent experiments performed in in six replicates 3.1 Synthesis of the Pro-prodrug
were used to obtain the statistical mean and standard deviation.
The synthesis route for the pro-prodrug (DT-COU-MTX, 6) is shown
in Scheme 2. Compounds 1-4 were synthesized according to
previously reported methods.[75-77] 7-Hydroxy-4-(hydroxymethyl)-
2H-chromen-2-one was allowed to react with quinone propionic
2.8 Apoptosis analysis by Annexin V-FITC and Propidium
Iodide (PI) double staining
A549, HeLa and L929 cells were seeded in 6-well plate at the density acid to yield compound 5. The pro-prodrug (DT-COU-MTX, 6) was
of 1.0 ×106 cells/well for 24 h at 37 °C. When the A549 and L929 obtained by esterification between MTX and compound 5.
cells density reached 80-90% of confluence, the medium was Subsequently, under dark conditions, the DT-COU-MTX react with
replaced with 70 μg/mL of LIP-DT-COU-MTX or 20 μg/mL of MTX for DT-diaphorase to yield the prodrug (HO-COU-MTX, 7), as shown in
2 h, then the cells were under light irradiation for 30 min (LED Figure S1. The structures of all the intermediates and the final
lamp, with the wavelength range of 400–450 nm at the power of 10 product were confirmed by 1H nuclear magnetic resonance (1H
mW cm−2, blue light), and afterwards the cells were further NMR ) and high-resolution mass spectrometry (HR-MS) (Figures S2-
incubated for 9.5 h. For evaluating the effects of the nanosystem 15, ESI†).
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Scheme 2. Synthetic route for the pro-prodrug (DT-COU-MTX). Reagents and Conditions: a) Ethyl 4-chloroacetoacetate, 70% H2SO4, 0 oC, 12
h; b) H2O, reflux, 48 h; c) Methyl 3,3-dimethylacrylate, methanesulfonic acid, 70 oC, 1.5 h; d) 10% aqueous acetonitrile solution, NBS, r. t., 1
h; e) DMF/DCM, EDC, DMAP,
3.2 Fluorescent response of the DT-COU-MTX toward DT- Figure 1B, which is about 50-fold of its original intensity. On the
diaphorase other hand, with a gradual addition of DT-diaphorase (0 μg/mL to
r
.t.,
4
h; f) Methotrexate, DMF/DCM, EDC, DMAP, N2, r. t., 24
h
To determine whether this DT-COU-MTX is a highly specific senor 40 μg/mL) to the solution of DT-COU-MTX, the fluorescence
for DT-diaphorase, we investigated its spectral in simulated intensity at 475 nm increases until it reaches a saturation point at
physiological conditions. First, absorption and fluorescence spectra 20 μg/mL (Figure 1C). These results indicate that the fluorescence
for the DT-COU-MTX solutions were determined and shown in of DT-COU-MTX is restored in the prensence of DT-diaphorase and
Figure S16 and Figure 1A, which indicate that both the DT-COU-MTX the HO-COU-MTX undergoes further photolysis to produce
and MTX show a strong absorption band at 320 nm and 380 nm. coumarin and MTX.
Fluorescence spectra were recorded at 37 °C in phosphate buffer
(pH 7.4, 10 mM) in the presence of cofactor NADH (100 μM), as
shown in Figure 1A-C. In Figure 1A , the HO-COU-MTX exhibits the
strong fluorescence at 475 nm, and the DT-COU-MTX shows the
insignificant fluorescence as a result of the PET effect due to the
existenece of quinone propionic acid moiety. In the presence of 20
μg/mL DT-diaphorase, strong fluorescence (a 50-fold increase in
fluorescence intensity) at 475 nm can be observed and this
spectrum is similar to that of HO-COU-MTX. While the HO-COU-
MTX was irradiated with blue light for up to 40 min, a blue-shift
in fluorescence band can be observed and the new emission peak
display at about 465 nm, corresponding to the emission of the
released coumarin (compound 2, Figures 1A and S17). As for
evaluating enzymatic properties of the DT-COU-MTX in vitro, the
concentration and time dependence of the HO-COU-MTX
fluorescence spectra were also recorded, as shown in Figures 1B
and 1C. With the increasing incubating time, the fluorescence at
475 nm increases steadily and reaches a plateau in 120 min in
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Figure 1. (A) Fluorescence spectra of the DT-COU-MTX (5 μM), the HPLC analysis as shown in Figure S19. These results indicate that the
DT-COU-MTX (5 μM) in the presence of DT-diaphorase (20 μg/mL), HO-COU-MTX (the product from the reaction between DT-COU-MTX
the HO-COU-MTX (5 μM) with or without irradiation and coumarin and the enzyme DT-diaphorase) can serve as a fluorescence probe
(5 μM). (B) Time course of fluorescence of the DT-COU-MTX (5 μM) for DT-diaphorase level and release the anticaner drug MTX by one-
in the presence of DT-diaphorase (20 μg/mL). Inset: Change in photon or two-photon irradiation.
fluorescence intensity at 475 nm as a function of time after DT-
diaphorase (20 μg/mL) treatment. (C) Fluorescent spectra for DT-
COU-MTX (5 μM) after treatment with varied concentrations of DT-
diaphorase (0-40 μg/mL). Inset: Change in fluorescence intensity at
475 nm as
a function of DT-diaphorase concentration. (D)
Percentage of MTX (as determined by HPLC) released from the DT-
COU-MTX or HO-COU-MTX (5 μM) as a function of time under
irradiation. The cofactor NADH (100 μM) was used for all
measurements. All fluorescence spectra data were obtained at 37 oC
in phosphate buffer (pH 7.4, 10 mM) containing 1% (v/v) DMSO.
Excitation wavelength: 405 nm.
3.3 Drug release studies in vitro
In order to further explore the release of MTX from DT-COU-MTX in
the presence of DT-diaphorase under irradiation, high-performance
liquid chromatography (HPLC, one-photon irradiation) was
employed and high resolution MS spectrum (HR-MS, two-photon
irradiation) was recorded in Figure 1D, Figures 2A-C . As shown in
Figure 1D, upon irradiation conditions (one-photon), MTX is
gradually released from the HO-COU-MTX, and the release rate in
the irradiation time of 40 min reaches the maximum, up to about
78%; while the DT-COU-MTX is quite stable under irradiation with
no MTX released. In addition, the DT-COU-MTX with irradiation time
was extended to 48 h, and the released rate is still negligible as
shown in Figure S18. This confirms that the PET effect can
effectively lock the photochemical reactions and the DT-COU-MTX is
stable with irradiation in the absence of DT-diaphorase. As shown in
Figure 2A and 2B, in the presence of DT-diaphorase, the DT-COU-
MTX, compound 3 (quinone propionic acid will be reduced by DT-
diaphorase into lactone, with the same structure as compound 3)
and HO-COU-MTX give rise to peaks at 1.80, 2.27 and 1.16 min
respectively in HPLC chromatogram. Under the visible light
irradiation conditions (one-photon), the HO-COU-MTX, coumarin
and MTX give rise to peaks at 1.26, 1.58 and 1.06 min respectively in
HPLC chromatogram. As it is known that, the coumarinyl can be
activated by two-photon near-infrared (NIR) irradiation at 800 nm.
Compared to one-photon irradiation, two-photon irradiation is
more beneficial for biological applications, as it can ensure deeper
tissue penetration with less photo-induced damages.[78-80]
Moreover, we further evaluated the drug release of two-photon
process by HR-MS. DT-COU-MTX was reacted with DT-diaphorase
and then underwent irradiation using a Coherent Legend Elite Ti:
sapphire regenerative amplifier system as the femtosecond laser
source at 800 nm, afterwards a small aliquot was subjected to HR-
MS analysis. Ionic peaks corresponding to MTX ([HR-MS-H]-=
453.1630), coumarin ([HR-MS-H]- = 191.0344) and HO-COU-MTX
([HR-MS-H]- = 627.1954) can be observed in the HR-MS spectra. The
above experimental results show that the DT-COU-MTX can detect
the level of DT-diaphorase via the fluorescent emission intensity,
and further release the MTX under irradiation. On the other hand,
the light-triggered controllability for the drug release from the DT-
COU-MTX upon enzyme activation have been demonstrated using
Figure 2. Typical HPLC chromatogram of (A) the DT-COU-MTX, HO-
COU-MTX, compound 3 and the DT-COU-MTX (10 μM) incubated
with DT-diaphorase (20 μg/mL) and its cofactor NADH (100 μM) for
2 h; (B) the HO-COU-MTX, MTX, coumarin and the HO-COU-MTX
treated with irradiatioon for 30 min. Peaks in the chromatograms
were detected by monitoring the absorption at 380 nm. The mobile
phase for A was 30/70 acetonitrile/methanol and the mobile phase
for B was 10/20/70 water/acetonitrile/methanol and the flow rate
was 1.0 mL/min. (C) MTX release (as determined by HR-MS) as a
function of two-photon irradiation.
3.4 Anti-interference performance of DT-COU-MTX
In order to investigate possible application of DT-COU-MTX in
biological systems, the selectivity experiments were performed by
DT-diaphorase and some potential interferents (such as some metal
ions, amino acids, biological reductants or enzymes), and the results
are depicted in Figure 3A. Upon incubating with DT-diaphorase or
possible interferents for 2 h, the remarkable enhancement of
fluorescence at 475 nm can be observed in the presence of DT-
diaphorase, while no obvious fluorescence change can be detected
with the potential interferents. The results demonstrate that the
DT-COU-MTX exhibit a good selectivity for DT-diaphorase under
physiological conditions. In addition, the pH effect on the reaction
of DT-COU-MTX and the DT-diaphorase were investigated, as shown
in Figure 3B. In the absence of DT-diaphorase,the fluorescence
intensities at 475 nm is weak and keeps stable over the pH range
from 3 to 9; whereas in the presence of DT-diaphorase and its
cofactor, an obvious increment of fluorescence intensity can be
detected, and the values remain fairly stable within the pH range of
3-9. The results of this experiment demonstrate that DT-COU-MTX
may be a potentially potent tool for the effective detection of DT-
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diaphorase and release the delivery of anticancer drugs in a lines (L929) to evaluate the efficacy of the LIP-DT-COU-MTX on cell
physiological environment.
viability (Figure 5A). Upon light irradiation, compared to the high
viability of L929, the LIP-DT-COU-MTX shows strong cytotoxicity
towards HeLa and A549 cells, the IC50 (concentration inhibiting cell
growth to 50% of control) value was determined as 32.66 μg/mL for
HeLa cells and 20.07 μg/mL for A549 cells. While the LIP-DT-COU-
MTX had a half inhibitory concentration of much greater than 70
μg/mL for L929 cells. MTT assays were also performed for A549,
HeLa and L929 cells co-treated with dicoumarol (DIC, an inhibitor of
DT-diaphorase) and then treatments with the LIP-DT-COU-MTX
under the same condition, and the results are presented in Figure
5A. Upon pretreatment with DIC, the cells with DT-diaphorase
overexpression (A549 cells and HeLa cells) or normal cells (L929
cells) without the enzyme overexpression all exhibit high viabilities
(over 80% at 70 μg/mL LIP-DT-COU-MTX). In the control
experiment, the IC50 value of MTX for HeLa cells and A549 cells were
9.31 and 5.22 μg/mL, respectively (Figure S23). The results show
that the half-inhibitory concentration of LIP-DT-COU-MTX for cancer
cells has the same order of magnitude as that of MTX. Moreover, as
shown in Figure S24, the cell viability for the three cell lines upon
exposure to blue light irradiation but not treated with the LIP-DT-
COU-MTX was also measured, and the blue light alone leads to
insignificant cytotoxicity. These results reveal that the cytotoxicity of
LIP-DT-COU-MTX derives from the light-triggered drug release after
enzymatic reaction. In order to further verify the effect of
irradiation on drug release, three groups of cells were treated with
LIP-DT-COU-MTX without irradiation as a control in Figure S25. In
the absence of light irradiation, the cells viabilities of the three
groups of cells were relatively high. In addition, the DIC cytotoxicity
experiments show that the DIC (20 μM) leads to insignificant
cytotoxicity (Figure S26). These results prove that the LIP-DT-COU-
MTX only produces therapeutic effect when the DT-diaphorase and
phototrigger conditions are available at the same time.
Figure 3. (A) The fluorescence histogram of the DT-COU-MTX after
treatment with different substances in phosphate buffer (pH 7.4, 10
mM, containing 1% (v/v) DMSO) for 2 h. Excitation wavelength: 405
nm. (20 μg/mL for DT-diaphorase (DT), Nitroreductase (NTR),
Azoreductase (AZD) and Alkaline phosphatase (ALP); 5 mM for AA,
Hcy, GSH, Gly, Arg, Phe and Cys; 1 mM for others). (B) The
fluorescence profile of the DT-COU-MTX at different pH values after
DT-diaphorase (20 μg/mL) or without DT-diaphorase treatment in
phosphate buffer (10 mM, containing 1% (v/v) DMSO). The
fluorescent intensities were recorded after exposure to DT-
diaphorase and NADH for 2 h at 37 °C. Excitation wavelength: 405
nm.
3.5 Imaging and pro-apoptotic effects in DT-diaphorase
overexpressing cells
Subsequently, we explored the imaging effect of DT-COU-MTX in DT-
diaphorase overexpressing cell lines (A549 cells and HeLa cells) or
normal cell line without overexpression of DT-diaphorase (L929
cells) by using fluorescent microscope. In order to improve aqueous
solubility or dispersibility and enhance cellular uptake of the MTX-
based pro-prodrug,[81-85] we loaded DT-COU-MTX into the liposome
carrier, forming a pro-prodrug nanosystem (termed as LIP-DT-COU-
MTX). The particle size distribution (DLS) and TEM images of LIP-DT-
COU-MTX are shown in Figure S20. After that, we evaluated LIP-DT-
COU-MTX performance in cells and the results are presented in
Figures 4A-B. For A549 cells, upon 1 h of incubation with the LIP-DT-
COU-MTX (20 μg/mL), intracellular blue fluorescence can be
observed; and after 2 h of incubation, the fluorescence become
very bright, as shown in Figure 4A. The intracellular imaging of HeLa
cells is similar to that of A549 cells as shown in Figure S21. For the
function of DT-diaphorase on LIP-DT-COU-MTX, A549 cells were
pretreated with dicoumarol (a DT-diaphorase inhibitor) and the
fluorescence was disappeared in the cells (Figure 4B). Meanwhile
for the L929 cells in which DT-diaphorase is not overexpressed, the
incubation with the pro-prodrug nanoparticles for 2 h cannot induce
detectable blue fluorescence under similar experimental conditions
as shown in Figure 4B. The capability of LIP-DT-COU-MTX in DT-
diaphorase detection in cells was further confirmed by flow
cytometry analysis as shown in Figure S22. The above results
demonstrate that this nanosystem can effectively discriminate
cancer cells from normal cells and serve as a theranostic agent for
cancer cells.
Figure 4. Fluorescent microscopic images for A549 cells that has
been treated with
a LIP-DT-COU-MTX (20 μg/mL, with the
incubation time of 1 h or 2 h respectively) or without a LIP-DT-COU-
MTX (the control). (B) Fluorescent microscopic images for A549 cells
and L929 cells upon incubation with the LIP-DT-COU-MTX (20
μg/mL) for 2 h, and images of A549 cells pretreated with 20 μM of
dicoumarol (the inhibitor of DT-diaphorase) for 30 min and then
with the incubation of the LIP-DT-COU-MTX (20 μg/mL) for 2 h.
Furthermore, the pro-apoptotic effect of the LIP-DT-COU-MTX
towards DT-diaphorase-overexpressing cells with light irradiation
was evaluated by the flow cytometry studies. As shown in Figure 5B,
the apoptotic percentage (including the early and late apoptosis, Q4
and Q1 in Figure 5B) is 9.0% after incubation with the LIP-DT-COU-
We next performed MTT assays for DT-diaphorase
overexpressing cancer cell lines (HeLa and A549) and normal cell
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analysis of A549 and L929 treated LIP-DT-COU-MTX (70 μg/mL) or
MTX (20 μg/mL). (C) Flow cytometry diagram of A549 cells
respectively treated with the LIP-DT-COU-MTX of varied
concentrations (10 μg/mL to 100 μg/mL). In the dual staining
diagrams, vital cells are negative for both PI and annexin V (in region
Q3); early apoptotic cells are PI negative and annexin V positive (in
Q4); late apoptotic/dead cells are positive for both PI and annexin V
(in Q1), while the damaged cells locate in region Q2.
MTX in normal cells (L929 cells); while for the unmodified MTX, the
apoptotic percentage is up to 36.4%, threefold higher than that of
the nanosystem. As for A549 cells, the apoptotic percentage
reaches to 55.5% and 68.7% upon treatment with LIP-DT-COU-MTX
and MTX. In Figure 5C, we also investigated the concentration
dependence of LIP-DT-COU-MTX for pro-apoptotic efficiency
towards A549 cells by the flow cytometry. The subsequent addition
of LIP-DT-COU-MTX (10 μg/mL to 100 μg/mL) can induce
remarkable increase in apoptotic percentage of A549 cells (17.5 %
to 73.8%). Similar results were obtained for the same experiment
with HeLa cells in Figure S27. As shown in Figure S28, the results
from the experiment involving DIC inhibition show that the
apoptotic percentage of the cells pretreated with DIC (20 μM) is
lower (approximately 5% at 70 μg/mL LIP-DT-COU-MTX ), but those
treated with LIP-DT-COU-MTX alone is higher, this is consistent with
the results of MTT experiments. All these results of flow cytometry
have proven that LIP-DT-COU-MTX have potent therapeutic effects
on cancer cells with overexpression of DT-diaphorase with the
visible light irradiation.
4. Conclusions
In summary, we successfully synthesized an enzyme-activated
and
light-triggered
pro-prodrug
nanosystem
for
detection/imaging and therapy. This pro-prodrug has several
advantageous features: Firstly, the PET effect by quinone
propionate can quench the fluorescence and block photolysis
of coumarin, which improves the stability and controllability of
prodrugs and avoid premature release of the anticancer drugs;
secondly, the fluorescence of prodrug (HO-COU-MTX) can
discriminate the DT-diaphorase overexpressing tumor cells
from aerobic healthy cells, and then controllable release of the
anticancer drug (MTX) by one or two photon activation can be
achieved; thirdly, the pro-prodrug was also encapsulated in
liposomes for enhancing biocompatibility and improving
aqueous solubility. All the experiments show that the pro-prodrug
only releases the MTX via the sequential controls of DT-diaphorase
and light irradiation. This strategy may offer an approach for
developing light-triggered pro-prodrug using enzyme as the
activator.
The above results demonstrate that this nanosystem can serve
as a DT-diaphorase probe to discriminate tumor cells from normal
cells, and it only exhibits strong cytotoxicity for DT-diaphorase
overexpressing cells. Moreover, this nanosystem has the
advantageous properties of releasing anti-cancer drugs under the
two-photon irradiation. Hence, this nanosystem could be developed
as a helpful tool for imaging and therapy.
Acknowledgements
We acknowledge the financial support by NSFC (21574044,
21474031 and 51673066), the Science and Technology
Planning Project of Guangzhou (Project No. 201607020015),
the Science and Technology Planning Project of Guangdong
(Project No. 2014A010105009) and the Natural Science
Foundation of Guangdong Province (2016A030312002).
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Table of Contents
A sequential enzyme-activated and light-triggered pro-prodrug
nanosystem for cancer detection and therapy
Zelin Chen†, Bowen Li†, Xin Xie, Fang Zeng*, Shuizhu Wu*
A sequential enzyme-activated and light-triggered pro-prodrug has been developed
for cancer biomarker detection and on-demand therapy
.