A self-immolative and DT-diaphorase-activatable prodrug for drug-release tracking and therapy

Article abstract of DOI:10.1039/c7tb00266a

DT-diaphorase, which catalyzes the reduction of various biological substances like quinones, is overexpressed in some malignant tumors. However, exploiting this attractive property for the controlled release of an active drug from a prodrug is yet to be fully taken advantage of. Herein we report a DT-diaphorase-based prodrug for concomitant drug-release imaging and cancer chemotherapy. This prodrug system is composed of two camptothecin (CPT) moieties as the active anticancer drug, a DT-diaphorase-responsive quinone propionic acid moiety and a set of self-immolative linkers. The presence of DT-diaphorase leads to the release of two CPT molecules and restores the fluorescence of the latter, thereby realizing the fluorescence monitoring of the DT-diaphorase level as well as the tracking of CPT release. Upon internalization by DT-diaphorase overexpressing cells, the prodrug can release fluorescent CPT and exhibit high cytotoxicity (half-maximal inhibitory concentration 0.71 μM) towards the cancer cells. This prodrug features on-demand enzyme-biomarker-triggered drug release as well as self-monitoring of drug release, therapeutic effect and biomarker level. This new strategy may provide an effective approach for constructing prodrugs with enhanced drug loading as well as controllability for drug release and tracking.

Full text of DOI:10.1039/c7tb00266a

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ARTICLE
A self-immolative and DT-diaphorase-activatable prodrug for
drug-release tracking and therapy
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
a
a
a
Bowen Li† , Peilian Liu† , Donghang Yan , Fang Zeng* and Shuizhu Wu*
DOI: 10.1039/x0xx00000x
DT-diaphorase, which catalyzes reduction of various biological substances like quinones, is overexpressed in some
malignant tumors. However, exploiting this attractive property for the controlled release of an active drug from prodrug
has yet to be fully taken advantage of. Herein we report a DT-diaphorase-based prodrug for concomitant drug-release
imaging and cancer chemotherapy. This prodrug system is composed of two camptothecin (CPT) moieties as the active
anticancer drug, a DT-diaphorase-responsive quinone propionic acid moiety and a set of self-immonlative linkers. The
presence of DT-diaphorase leads to the release of two CPT molecules and restore the fluorescence of the latter, thereby
realizing the fluorescent monitoring of DT-diaphorase level as well as the tracking of CPT release. Upon internalization by
DT-diaphorase overexpressing cells, the prodrug can release fluorescent CPT and exhibit high cytotoxicity (half-maximal
inhibitory concentration 0.71 μM) towards the cancer cells. This prodrug features on-demand enzyme-biomarker-triggered
drug release as well as self-monitoring of drug release, therapeutic effect and biomarker level. This new strategy may
provide an effective approach for constructing prodrugs with enhanced drug loading as well as controllability for drug
www.rsc.org/
release
and
tracking.
has recently attracted considerable attention due to its remarkable
high expression in some malignant tumors, such as non-small cell
lung carcinoma, liver cancers and breast carcinomas; and in some
cancer cells highly elevated DT-diaphorase levels (up to 50-folds
1
. Introduction
Over the past several decades, chemotherapy remains a front-line
therapy for cancer. However, a major problem of chemotherapy is
its severe side-effects (high toxicity, non-specificity and etc.) to
1
2
compared to normal cells) have been observed. Despite the fact
that the DT-diaphorase level assay has been employed for cancer
1
normal cells. Development of prodrug strategies for overcoming
1
3
diagnosis, there are few reports on the exploitation of this level
side-effects have been deemed to be a promising approach to
1
3d-f
2
difference for developing prodrug.
indirectly enhance therapeutic efficiency; and the even more
preferable strategy is to develop delivery systems which can
On the other hand, self-immolative prodrugs, which have been
regarded as new platforms for drug delivery, show a valuable ability
to achieve controllable release of multiple drug molecules with a
3
achieve real-time tracking of the drug’s release and localization.
A prodrug, which can be triggered by the unique tumor
microenvironment and monitored by an imaging reagent, is an
elegant strategy in biomedicine and thus can significantly improve
1
4
single bond cleavage. Some of these prodrugs comprise self-
immolative linkers, drug molecules as the tail units, as well as an
enzyme substrate as the trigger; and following the enzymatic
activation, a cascade of self-eliminations take place, thereby
excluding the active drug as a result of a number of subsequent
1
,2
the therapeutic efficacy of anticancer drugs.
The special
4
environments of tumor cells include lower pH, intracellular
5
6-8
9
reduction, over-expressed enzymes, active oxygen species, or
15,16
1
0
steps.
The prodrugs characterized by biomarker-triggered on-
hypoxia. Among the over-expressed enzymes, DT-diaphorase is a
cytosolic flavoprotein which catalyzes an obligatory two-electron
reduction of some biological substances like quinones, by utilizing
nicotinamide adenine dinucleotide (NADH)/nicotinamide adenine
demand drug release through self-immolative reactions, which can
simultaneously monitor drug release/therapeutic effect and the
biomarker level, are highly desirable.
1
1
dinucleotide phosphate (NADPH) as the co-factors. DT-diaphorase
Camptothecin, a natural product originally extracted from
camptotheca acuminata, which stabilizes the covalent
attachment of topoisomerase I to single-strand DNA breaks,
can disrupt the DNA replication process in cancer cells, thus
1
7a
demonstrating strong anticancer activity.
This anticancer
drug has demonstrated to be effective against many types of
tumors, but its poor water solubility and its rapid conversion to
a less active carboxylate form under physiological conditions
1
7b
severely limit its clinical application. Hence, development of
a
new prodrug based camptothecin derivative, which
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integrates biomarker-triggered self-immolative drug release
and concomitant in-situ monitoring of drug release/biomarker 2.1 Materials
level, may circumvent these limitations and greatly improve 2,6-Dihydroxy
methyl-4-methyl
phenol,
t-
1
7c
therapeutic efficacy.
butyldimethylchlorosilane (TBSCl), imidazole, 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide hydrochloricde (EDC), 4-
dimethylaminopyridine (DMAP), p-toluenesulfonic acid
2
monohydrate (PTSA•H O) and triphosgene were purchased
from Sigma-Aldrich Reagents without further purification. DT-
diaphorase (lyophilized powder, recombinant, expressed in E.
coli) and β-nicotinamide adenine dinucleotide reduced
disodium salt (NADH) were purchased from Sigma-Aldrich.
Dichloromethane (DCM), N, N-dimethyl-formamide (DMF) was
2
dried with CaH and distilled under nitrogen atmosphere. The
water used throughout the experiments was the triple-distilled
water which was further treated by ion exchange columns and
then by a Milli-Q water purification system. Other solvents
used in this study were analytical grade reagents and used
without further purification.
Scheme 1. Schematic overview of the prodrug for detecting DT-
diaphorase, real-time drug tracing and anticancer action.
2
.2 Prodrug synthesis
Previously, we successfully designed a DT-diaphorase-based
molecular prodrug for concomitant drug-release imaging and Synthesis of compound 1-3: Compound 1-3 were synthesized
1
8
cancer chemotherapy in the prodrug system, one molecule of active according to the procedures reported elsewhere.
1
3f
drug was coupled with quinone propionic acid moiety. In an effort
to further improve the loading capacity of the molecular prodrug,
we postulate that, by employing a self-immolative unit with
multiple reaction sites like 1,3-dihydromethyl-2-phenol, we could
simultaneously incorporate more than one active drug molecules
and the triggering group, thus establishing a DT-diaphorase-based
prodrug system with enhanced drug loading and eventually better
therapeutic efficacy. To substantiate this idea, herein, we
demonstrate a molecular prodrug with high drug load and
enhanced efficacy by adopting a self-immolative architecture, as
illustrated in Scheme 1. This prodrug system, which is an enzyme-
biomarker triggered release system, is composed of two anticancer
drug camptothecin (CPT) moieties as the active anticancer drug, a
DT-diaphorase-responsive quinone propionic acid and a set of self-
immonlative linkers (Scheme 1 and Scheme 2). For this prodrug,
due to the photoinduced electron transfer (PET) process the CPT’s
Synthesis of 2,6-bis(((tert-butyldimethylsilyl)oxy)methyl)-4-
methylphenol (4).
2,6-Dihydroxy methyl-4-methyl phenol (1.5 g, 8.9 mmol) and
imidazole (1.81 g, 26.3 mmol) was dissolved in 10 mL THF and
then added to TBSCl (3.3 g, 22 mmol) in batches. After stirring
for 3 h at room temperature, the solvent was removed and
diluted with ethyl acetate. The organic phase was washed with
2 4
water and brine, dried over anhydrous Na SO overnight and
concentrated in vacuo. The crude product was purified by
column chromatography on silica gel (DCM:hexanes 1:10) to
1
give 4 (3.24 g, 92% yield) as a colorless oil. H NMR (600 MHz,
3
CDCl , ppm): δ 7.90 (s, 1H), 6.78 (s, 2H), 4.70 (s, 4H), 2.13 (s,
3
H), 0.82 (s, 18H).
fluorescence is efficiently quenched by the quinone propionic acid. Synthesis of 2,6-bis(((tert-butyldimethylsilyl)oxy)methyl)-4-
The presence of DT-diaphorase and the cofactor leads to the methylphenyl (4-nitrophenyl) carbonate (5) .
release of the two CPT molecules and restore the fluorescence of TEA (210 μL, 2.1 mmol) and trace DMAP were added to a
the laꢀer (as demonstrated in Scheme 1 and Scheme S1, ESI†), solution of compound
4
(200 mg, 0.49 mmol) in
o
thereby affording the two CPT moieties as the reporter of DT- dichloromethane and then stirring at 0 C for 0.5 h. 4-
diaphorase level and their own release. Moreover, the quinone
propionic acid moiety acts as the trigger for the drug release and
ensures eventual fluorescent restoration, it can be selectively
Nitrophenyl chloroformate was dissolved in dichloromethane
and slowly added in the solution. After 0.5 h of reaction, the
mixture was diluted with ethyl acetate, washed with water,
1
4
reduced by DT-diaphorase into hydroquinone,
which will
2 4
dried over anhydrous Na SO and concentrated in vacuo. The
subsequently be cleaved and turn into lactone, thereby causing the
release of the active and fluorescent CPT via a 1,4-elimination
reacꢁon (Scheme S1, ESI†). Therefore, this prodrug can realize on-
demand enzyme-biomarker-triggered drug release as well as self-
monitoring of drug release, therapeutic effect and biomarker level.
crude product was purified by column chromatography on
silica gel (EtOAc:hexanes 1:100) to give 5 (170 mg, 60% yield)
1
as a yellow oil. H NMR (600 MHz, CDCl
3
, ppm) δ 8.32 (d, J =
9
2
C
.2 Hz, 2H), 7.49 (d, J = 9.2 Hz, 2H), 7.22 (s, 2H), 4.72 (s, 4H),
.38 (s, 3H), 0.92 (s, 18H), 0.09 (s, 12H). HR-MS (ESI): calcd for
+
28
H
43NO
7
Si
2
([M+H] ) 562.2651, found: 562.2656.
2. Experimental Section
2
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Synthesis of N-(2-((1-(2,6-bis(((tert-butyldimethylsilyl)oxy)-
methyl)-4-methylphenoxy)ethyl)amino)ethyl)-N,3-dimethyl-
according to the supplier’s (ATCC) protocols. The two cell lines
were incubated with 10% FBS (fetal bovine serum, GIBCO) -
containing Dulbecco’s modified eagle medium (DMEM) with
1% penicillin and streptomycin (GIBCO) at 37 °C in a humidified
3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-
yl)butanamide (6).
2
Compound 5 (2.6 g, 4.4 mmol) and compound 3 (770 mg, 2.4 atmosphere containing 5% of CO . When the cell density
mmol) were dissolved in 10 mL DMF and TEA (1 ml, 10 mmol) reached 70-80% of confluence, a subculture was considered
was then slowly added into the solution. After 3 h, the solution complete. The medium was changed approximately every 1 to
was diluted with dichloromethane, washed with 1 N HCl and 2 days.
saline. The organic layer was collected and dried over MgSO
4
and concentrated in vacuo. The crude product was purified by 2.4 Cell imaging
column chromatography on silica gel (EtOAc:hexanes 1:4) to
1
give 6 (820 mg, 52% yield) as a yellow oil. H NMR (600 MHz, A549 and L929 cells were incubated in DMEM supplemented
CDCl
3
, ppm) δ 7.18 (s, 2H), 4.61 (s, 4H), 3.45 (d, J = 29.5 Hz, 4H), with 10% FBS for 24 h incubation. Then cells were seeded in a
3
.11-2.90 (m, 8H), 2.34 (dd, J = 8.8, 6.3 Hz, 3H), 2.13 (dd, J = 14.0, 6-well plate and each well containing polylysine-coated cell
5
.3 Hz, 3H), 1.94 (t, J = 12.4 Hz, 6H), 1.44 (s, 6H), 0.92 (s, 18H), 0.08 culture glass slides. Cells on glass slides was allowed to 50–
+
(
s, 12H). HR-MS (ESI): calcd for C40H66N O Si ([M+H] ) 743.4481, 70% confluence and washed with DMEM, and the cells were
2
7
2
found: 743.4479.
incubated with the prodrug in a humidified incubator (37 °C,
% CO ) for 12 h or 24 h. Afterwards, the 6-well plate were
N-(2-((1-(2,6-bis(hydroxymethyl)-4- washed with PBS for three times and the glass slides were
ethyl)-N,3-dimethyl-3-(2,4,5- taken out. Fluorescence images were obtained using an
5
2
Synthesis
of
methylphenoxy)ethyl)amino)
trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)butanamide (7). Olympus IX71 inverted fluorescence microscope equipped
Compounds 6 (500 mg, 1 mmol) and PTSA•H O (45 mg, 0.25 with a DP72 color CCD.
2
mmol) were mixed and stirred in methanol (5 mL) at room
temperature under nitrogen for 3 h. After completion of 2.5 Cell viability assay
reaction, the solvent was removed at room temperature. The
crude product was purified by column chromatography on Cells (5000 cells/well) were seeded in 96-well plates and
silica gel (EtOAc:hexanes 1:3) to give 7 (560 mg, 90% yield) as a incubated with the prodrug of varied concentrations from 0.1
1
yellow solid. H NMR (600 MHz, CDCl ,ppm) δ 7.23-7.18 (m, 2H), to 80 μM at 37 °C. The cells without the treatment were used
3
4
3
1
.52 (dd, J = 28.8, 14.7 Hz, 4H), 3.65-3.42 (m, 4H), 3.08 (ddd, J = 47.8, as control. After incubation for 48 h, the wells were washed
3.7, 19.2 Hz, 8H), 2.35 (t, J= 3.6 Hz, 3H), 2.16-2.07 (m, 3H), 1.98- three times with PBS buffer and treated with DMEM medium
-
.81 (m, 6H), 1.48-1.36 (m, 6H). MS(ESI): m/z 513.27 [M-H] .
containing 0.5 mg/mL MTT for another 4 h. The resulting
formazan crystal was dissolved in 150 μL of DMSO after
carefully removing the medium and the absorbance was
Synthesis of the prodrug 8 (the prodrug).
CPT (102 mg, 0.285 mmol) and DMAP (107 mg, 0.85 mmol) recorded at 570 nm. The cell viability assays were performed
were suspended in 10 mL anhydrous dichloromethane at ice using a Thermo MK3 ELISA plate reader. The independent
water under nitrogen. Triphosgene (34 mg, 0.114 mmol) was experiments performed in in six replicates were used to obtain
rapidly added and stirred for 30 min at room temperature. the statistical mean and standard deviation. As for the
Then compound 7 (60 mg, 0.12mmol) was dissolved in 5 mL dicoumarol-involved MTT assays, the cells were co-treated
anhydrous dichloromethane and stirred overnight. The with 15 μM dicoumarol and the prodrug of varied
mixture was diluted with ethyl acetate and washed with water, concentrations for 24 h before being subject to MTT assay.
saturated NaCl solution, dried over anhydrous Na SO and
2
4
evaporated to dryness. The crude product was purified by 2.6 Apoptosis Analysis by Annexin V-FITC and Propidium
column chromatography on silica gel (EtOAc) to give 8 (61 mg, Iodide (PI) Double Staining
1
3% yield) as a yellow solid. H NMR (600 MHz, CDCl ) δ 8.42 (s,
3
3
2
7
H), 8.23 (s, 2H), 7.96 (s, 2H), 7.86 (s, 2H), 7.70 (s, 2H), 7.36 (s, 2H), A549 and L929 cells were seeded in 6-well plate at the density
6
.16 (s, 2H), 5.66 (s, 2H), 5.40 (s, 2H), 5.30 (s, 4H), 5.05 (s, 2H), 4.96 of 1.0 ×10 cells/well for 12 h at 37 °C. When the cell density
(
s, 2H), 3.44 (t, J = 55.5 Hz, 4H), 3.08-2.75 (m, 8H), 2.30- 2.12 (m, reached 80-90% of confluence, the medium was replaced with
7
H), 2.02 (s, 4H), 1.81 (s, 6H), 1.32 (s, 6H), 0.98 (s, 6H). HR-MS (ESI): different concentrations of prodrug or CPT for 24 h. The cells
+
calcd for C70
H
66
N
6
O17 ([M+H] ) 1263.4557, found: 1263.4565.
were then washed with PBS and harvested after trypsinization
and centrifugation at 2000 rpm for 10 min. Subsequently, the
cells were collected and resuspended in 1 ml PBS and then
mixed with Annexin V Binding buffer (400 μL) containing 5 μL
2.3 Cell culture
A549 cells (human nonsmall cell lung cancer) and L929 cells Annexin V-FITC and 10 μL PI. Finally, the flow cytometry
murine aneuploid fibrosarcoma cells) were maintained
(
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analyses were performed using BD Accuri C6 flow cytometer soluble in common organic solvents and slightly soluble in water;
and the data were analyzed using the BD Biosciences software. while CPT exhibited poor solubility in these solvents (Table S2, ESI†).
2
.7 Measurements
1
3.2 Drug release studies in vitro
H NMR spectra were recorded on a Bruker Avance600 MHz
NMR spectrometer. All chemical shifts are reported in ppm
value using the peak of residual proton signals of TMS as an
internal reference. Mass spectra were obtained on a Bruker
Esquire HCT Plus mass spectrometer. High resolution mass
spectra were obtained on AB Sciex Triple TOF 5600+ mass
spectrometer. UV-vis spectra were recorded on a Hitachi U-
To demonstrate the release of active drug CPT from the prodrug in
the presence of DT-diaphorase, absorption and fluorescence
spectra for the prodrug solutions were determined under
physiological condition. The absorption spectra were shown in Fig.
S10, which indicate that both the prodrug and CPT show a strong
absorption band at 365 nm. Fig. 1A shows the fluorescence spectra
of CPT, the prodrug and the prodrug in the presence of DT-
diaphorase. This prodrug molecule is designed as an electron
donor-acceptor system in which the initial photoexcitation is
localized on the acceptor. In the absence of DT-Diaphorase, the
HOMO of the donor (CPT moiety) lies higher in energy than that of
the acceptor (quinone propionic acid moiety) and can transfer an
electron to the acceptor’s HOMO. This photoinduced electron
transfer (PET) process competes favorably with radiative decay to
the ground state, substantially diminishing the fluorescence
quantum yield and thus substantially quenching the CPT’s
fluorescence. In the presence of DT-diaphorase, a 6-fold increase in
fluorescence intensity at 446 nm can be observed in the presence
of DT-diaphorase and its cofactor NADH (100 μM) at 37 °C in
phosphate buffer (pH 7.4, 10 mM), and this spectrum is similar to
that of CPT. This is because, the released CPT restores its
fluorescence in the absence of PET process due to drug release, and
hence the released drug (restored fluorescence) can act as a turn-
on fluorescent reporter for drug release. In addition, a red-shift in
fluorescence band can be observed upon treatment with the
enzyme; and this red-shift is due to the cleavage of the carbonate
bond which leads to the change in the “push-pull” electronic
configuration of CPT and eventually causes the red-shift in
fluorescence band.
3010 UV-vis spectrophotometer. Fluorescence spectra were
recorded on a Hitachi F-4600 fluorescence spectrophotometer.
Fluorescence images were obtained using an Olympus IX 71
with a DP72 color CCD.
3
. Results and discussion
3.1 Synthesis of the prodrug
The synthesis route for the prodrug (compound 8) is shown in
Scheme 2. Compound 1-3 were synthesized according to previously
1
8
reported methods. 2,6-Dihydroxy-4-methyl phenol was allowed to
react with tert-butyldimethylsilyl chloride to yield compound 4.
Compound 5 was then obtained through a nucleophilic reaction
between 4-nitrophenyl chloroformate and compound 4; afterwards
compound 5 was allowed to react with 3 via a transesterification
reaction to give a key intermediate 6. Compound 7 was obtained by
deprotection of 6. Finally, the CPT was incorporated into compound
7
in the presence of triphosgene to yield the prodrug (compound 8).
The structures of all the intermediates and the final product were
1
1
confirmed by H nuclear magnetic resonance ( H NMR) and mass
spectrometry (Fig. S1–S9, ESI†). The zeta potential for the prodrug
and CPT were determined and the result is given in Fig. S10, which
indicates that in aqueous medium with 1% DMSO, the two
compounds were negatively charged. The solubility of the prodrug
and CPE was investigated, and it was found that the prodrug was
Furthermore, the concentration and time dependence of the
prodrug’s fluorescence spectra were also recorded, as shown in Fig.
1
B and 1C. Upon addition of 60 μg/mL DT-diaphorase into the
solution with 5 μM of prodrug, the fluorescence intensity at 446 nm
gradually increases with time and reaches a plateau in 24 h (Fig.1
B).
4
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O
O
O
Boc
N
O
O
O
O
O
H
N
Boc
N
H
N
N
H
N
c
N
OH
a
b
N
H
1
2
O
O
3
O
R
1
2
O N
OH
OH
O
O
O
f
HO
OH
d
TBSO
OTBS
e
O
O
O
N
O
2
N
O
Cl
N
O
TBSO
O
OTBS
R
2
TBSO
OTBS
4
5
6
O
O
O
O
O
O
g
N
h
O
O
O
N
N
O
O
O
N
O
O
O
O
O
HO
OH
O
O
O
O
N
N
7
N
8
N
Scheme 2. Synthetic route for the prodrug (compound 8). Reagents and Conditions: a) (Boc)
2
1
O, DCM, r.t., overnight; b) R , EDC, pyridine,
r.t., overnight; c) 5% TFA, DCM, r.t., 3 h; d) TBSCl, DCM, imidazole, THF, r.t., 3 h; e) R
r.t., 3h; g) PTSA, r.t., 3 h; h) CPT, Triphosgene, DMAP, DCM, 0 C to r.t., overnight.
2
, TEA, DMAP, DCM, 0 oC to r.t., 1 h; f) 3, TEA, DMF,
o
On the other hand, on gradual addition of DT-diaphorase (0 DT-diaphorase (0-80 μg/mL). Inset: Change in fluorescence
intensity at 446 nm as
a
function of DT-diaphorase
μg/mL to 80 μg/mL) to the solution of prodrug, the fluorescence
intensity at 446 nm increases until it reaches a saturation point
at 60 μg/mL. These results suggest the successful release of CPT
from the prodrug.
concentration. (D) Percentage of CPT (as determined by HPLC)
released from the prodrug (5 μM) as a function of time in the
presence or absence of DT-diaphorase (60 μg/mL). The cofactor
NADH was used for all measurements. All fluorescence spectra
data were obtained at 37 °C in phosphate buffer (pH 7.4, 10
mM) containing 1% (v/v) DMSO. Excitation wavelength: 365 nm.
To further confirm the release of CPT from the prodrug in the
presence
of
DT-diaphorase,
high-performance
liquid
chromatography (HPLC) was employed and high resolution MS
spectrum was recorded as well (Fig. 1D, Fig. 2, and Fig. 3). One
can find in Fig. 1D that, CPT is gradually released from the
prodrug in the presence of DT-diaphorase (60 μg/mL), whereas
there is no CPT detected in the absence of DT-diaphorase. As
shown in Fig. 2, the retention time of prodrug and CPT are
monitored and identified at 4.74 min and 1.63 min in HPLC
chromatogram. For the prodrug, upon treatment with DT-
diaphorase (60 μg/mL), the peak intensity at 4.74 min
(
corresponding to the prodrug) decreases and a new strong peak
emerges at 1.63 min which well matches that for CPT. Moreover,
the anticipated release of CPT as active cancer drug was also
proved by HR-MS (ESI) analyses, as shown in Fig. 3. There is an
Figure 1. (A) Fluorescence spectra of the prodrug (5 μM), CPT (5
μM) and prodrug (5 μM) with DT-diaphorase (60 μg/mL). (B)
Time course of fluorescence of the prodrug (5 μM) in the
presence of DT-diaphorase (60 μg/mL). Inset: Change in
fluorescence intensity at 446 nm as a function of time after DT-
diaphorase (60 μg/mL) treatment. (C) Fluorescent spectra for
+
ionic peak corresponding to the prodrug ([M+H] =1263.4567) in
+
the HR-MS spectrum as well as that for CPT ([M+H] =349.1192)
after the prodrug being treated with DT-diaphorase and NADH,
which provides additional evidence for the release of CPT from
prodrug (5 μM) after treatment with varied concentrations of the prodrug.
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3
.3 Selectivity and anti-interference performance of prodrug
To assess the selectivity of the prodrug towards DT-
diaphorase over some potential interferents, the prodrug was
allowed to respectively incubate with DT-diaphorase, some
metal ions, amino acids, biological reductants or other enzymes
for 24 h, and the fluorescent intensity at 446 nm for the samples
were then recorded. As shown in Fig. 4I, none of the potential
interferents can cause remarkable fluorescence enhancement.
This result confirms that this prodrug shows quite good
selectivity toward DT-diaphorase over other competitive
substances and also demonstrates that the fluorescence
enhancement as a result of CPT release was induced by DT-
diaphorase.
Figure 2. Typical HPLC chromatogram of the prodrug (A), CPT (B)
and the prodrug (10 μM) incubated with DT-diaphorase (60
μg/mL) and cofactor NADH for 24 h (C). Peaks in the
chromatograms were detected by monitoring the absorption at
365 nm. The mobile phase was 80/20 acetonitrile/water and the
flow rate was 1.0 mL/min.
Figure 3. HR-MS (ESI) analyses for the prodrug (10 μM) upon incubation with DT-diaphorase (60 μg/mL) and cofactor NADH for 24 h.
6
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the pH range of 4-10. This result shows that the DT-diaphorase-
linked camptothecin conjugate (prodrug ) may be useful as an
8
indicating agent for delivering camptothecin to cancer cells at
biological pH.
3
.4 Drug release studies in DT-diaphorase over-expressing cell
We investigated the release of CPT in
a DT-diaphorase
overexpressing cell line (A549 cell) or a normal cell line without
overexpression of DT-diaphorase (L929 cell) by using fluorescent
microscope, and the results are presented in Fig. 5. For A549 cells,
upon 12 h of incubation with the prodrug (10 μM), blue intracellular
fluorescence can be observed; and after 24 h of incubation, the
fluorescence become very bright, as shown in Fig. 5A. On the other
hand, pretreatment of A549 cells with dicoumarol (a DT-diaphorase
inhibitor) causes the disappearance of the intracellular fluorescence
(
Fig. 5B). While for the L929 cells in which DT-diaphorase is not
overexpressed, the incubation with the prodrug for 24 h cannot
induce detectable blue fluorescence under a similar experimental
condition (Fig. 5B). We can thus conclude that the prodrug can be
used to monitor the CPT release in DT-diaphorase over-expressing
cells.
Figure 4. I) Fluorescence intensity for the prodrug (5 μM) at 446 nm
in phosphate buffer (pH 7.4, 10 mM, containing 1% (v/v) DMSO) in
the presence of various relevant species for 24 h. Excitation
wavelength: 365 nm. (60 μg/mL for DT-diaphorase (DT),
Nitroreductase (NTR), Azoreductase (AZD), Alkaline phosphatase
(
ALP), Carboxylesterase (CAE), 5 mM for AA, Hcy, GSH, Gly, Arg, Phe
and Cys, 1 mM for others). II) Fluorescence intensity at 446 nm of
the prodrug (5μM) as a function of pH in the absence or presence of
DT-diaphorase (60 μg/mL) in phosphate buffer (10 mM, containing
1
% (v/v) DMSO). The fluorescent intensities were recorded after
exposure to DT-diaphorase and NADH for 24 h at 37 °C. Excitation
wavelength: 365 nm.
The response of the prodrug to DT-diaphorase under varied pH
values was investigated and the result is given in Fig. 4II. In the
absence of DT-diaphorase, the fluorescence of prodrug is found
stable over the pH range from 3 to 10; whereas in the presence of
DT-diaphorase and cofactor, an enhancement of fluorescence
intensities are observed, and the values remain fairly stable within
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expressing cancer cells (A549) and normal cells (L929), the
cytotoxicity of prodrug is assessed using typical MTT assays and the
flow cytometry experiments. The viabilities for A549 and L929 cells
upon treatment with the prodrug assessed using MTT assays are
shown in Fig. 6I. The prodrug shows high cytotoxicity towards DT-
diaphorase over-expressing A549 cells with a molar half-maximal
inhibitory concentration (IC50) of 0.71 μM, while that for our
prodrug system with only one drug moiety is around 1.2 μM, and
the IC50 for CPT is around 0.70 μM, suggesting that incorporating
two CPT onto one prodrug molecule can enhance anticancer
efficacy. On the other hand, in terms of equivalent value of active
drug, the current prodrug only has a comparable IC50 (1.4 μM)
compared to that of our previous one-CPT prodrug. Moreover, as
expected, the prodrug exhibits a much higher IC50 value (> 80 μM)
towards L929 cells, indicating the prodrug is of low cytotoxicity
towards normal cells in which DT-diaphorase is not over-expressed.
To further verify the role of DT-diaphorase in the prodrug’s action,
MTT assays were performed for A549 and HeLa cells co-treated
with dicoumarol (an inhibitor of DT-diaphorase) and the prodrug for
2
4 hours, and the result is presented in Fig. S12. Upon pretreatment
with dicoumarol, the cells all exhibit high viabilities (over 80% at 10
μM of prodrug). This result suggests that the prodrug is activated by
DT-diaphorase. Furthermore, the flow cytometry studies were
performed to further verify the pro-apoptotic effect of the prodrug
towards DT-diaphorase-overexpressing cells. For L929 cells, after
incubation with the prodrug for 24 h, the apoptotic percentage
(
including the early and late apoptosis, Q1 and Q4 in Fig. 6II and 6III)
is 9.4%; whereas incubation with unmodified CPT leads to a much
higher apoptotic percentage of 44.7%. While for A549 cells, the
apoptotic percentage reaches to 67.4% upon treatment with the
prodrug, compared to 53% upon treatment with CPT. The effect of
the prodrug’s concentration (1 μM to 20 μM) on pro-apoptotic
effect towards A549 cells was also assessed by the flow cytometry
(
Fig. 6III). With the increasing concentration of the prodrug, the
apoptotic percentage of A549 cells gradually increases from 26.0 %
to 87.1%. The results from flow cytometry are in conformity with
the MTT results in that the prodrug exhibits higher cytotoxicity
towards DT-diaphorase over-expressing cells.
Figure 5. A) Fluorescent microscopic images for A549 cells in the
absence (the control) or presence of the prodrug (10 μM, with the
incubation time of 12 h or 24 h respectively) B). Fluorescence
images of A549 cells and L929 cells upon incubation with the
prodrug (10 μM) for 24 h, and of A549 cells pretreated with 20 μM
of dicoumarol (the inhibitor of DT-diaphorase) for 30 min followed
by the incubation of the prodrug (10 μM) for 24 h.
It is known that, the low drug loading of prodrug and
correspondingly the low drug level locally at the disease site is an
obstacle to optimal therapeutic effect. For this prodrug, the drug
load is 55.0%, while that for our previous system is 46.0%. This
prodrug
8 which can release two equivalent CPT moieties from a
single prodrug molecule could enhance the drug concentration
locally at disease site through efficient enzyme-biomarker triggered
self-immolative release. To investigate the therapeutic effect of this
high drug-loading prodrug
8 against the DT-diaphorase over-
8
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the Fundamental Research Funds for the Central Universities
2015ZY013).
(
Notes and references
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4
2
Figure. 6 (I) Cell viabilities for A549 and L929 cell lines upon
3
4
treatment
with
the
prodrug
of
varied
concentrations(incubation for 48 h). (II) Flow cytometry
analysis: representative percent distribution of A549 and L929
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4
. Conclusions
In summary, we have successfully developed a self-immolative
DT-diaphorase activatable prodrug that can realize on-demand
biomarker-triggered drug release and simultaneously monitor
drug release/therapeutic effect and the biomarker level. This
prodrug has several advantageous features: firstly one prodrug
molecule contains two CPT moieties, which greatly improves
drug loading and correspondingly the therapeutic effect;
secondly CPT moieties in the prodrug not only serve as the
active drug but also the turn-on fluorescent reporter for
monitoring and imaging of the released drug and enzyme-
biomarker level; thirdly the quinone propionic acid moiety of
the prodrug not only acts as the trigger for biomarker-
responsive on-demand drug release but also as a strong
quencher of CPT fluorescence via photoinduced electron
transfer (PET), which ensures the eventual fluorescence turn-
on imaging and tracking. This new strategy may offer useful
insights for developing prodrugs with enhanced drug loading
as well as controllability for drug release and tracking.
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We acknowledge the financial support by NSFC (21574044,
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A DT-diaphorase-activatable theranostic prodrug has been developed for visualizing
the release of active drug and enhancing the therapeutic effect.