Intracellular enzyme-activatable prodrug for real-time monitoring of chlorambucil delivery and imaging

Article abstract of DOI:10.1016/j.cclet.2017.04.024

Carboxylesterase, a necessary enzyme in various mammalian cells, has been employed in various biological applications. Herein, we designed and synthesized a novel carboxylesterase-based prodrug, which can realize simultaneous drug-release imaging and cancer chemotherapy. This prodrug comprises three parts: coumarin as the fluorophore and the cleavable architecture, chlorambucil as the anticancer drug, and acetyl group as the enzyme-responsive unit. The presence of carboxylesterase leads to the activation of coumarin fluorescence, and this fluorescence serves as the reporting signal for assessing the enzyme level and drug release. Moreover, the prodrug was incorporated in liposome for monitoring drug release and chemotherapeutic effect in living cells. Upon internalization by HeLa cells, the prodrug can release chlorambucil and exhibit high cytotoxicity. This approach may provide some helpful insights for enhancing therapeutic effect and tracking the release of prodrug.

Full text of DOI:10.1016/j.cclet.2017.04.024

Accepted Manuscript
Title: Intracellular enzyme-activatable prodrug for real-time
monitoring of chlorambucil delivery and imaging
Author: Meng Ni Wen-Jun Zeng Xin Xie Ze-Lin Chen Hao
Wu Chang-Min Yu Bo-Wen Li
PII:
S1001-8417(17)30161-4
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.cclet.2017.04.024
CCLET 4060
To appear in:
Chinese Chemical Letters
Received date:
Revised date:
Accepted date:
19-2-2017
4-4-2017
20-4-2017
Please cite this article as: M. Ni, W.-J. Zeng, X. Xie, Z.-L. Chen, H.
Wu, C.-M. Yu, B.-W. Li, Intracellular enzyme-activatable prodrug for real-time
monitoring of chlorambucil delivery and imaging, Chinese Chemical Letters (2017),
http://dx.doi.org/10.1016/j.cclet.2017.04.024
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Graphical Abstract
Intracellular enzyme-activatable prodrug for real-time monitoring of chlorambucil delivery and imaging
†
†
*
Meng Ni , Wen-Jun Zeng , Xin Xie, Ze-Lin Chen, Hao Wu, Chang-Min Yu, Bo-Wen Li
College of Materials Science & Engineering, South China University of Technology, Guangzhou 510640, China
A new carboxylesterase-activatable prodrug has been developed for enzyme-biomarker-triggered drug release and in situ
monitoring of drug release, therapeutic effect and biomarker level.
Page 1 of 10

Original article
Intracellular enzyme-activatable prodrug for real-time monitoring of chlorambucil
delivery and imaging
†
†
∗
Meng Ni , Wen-Jun Zeng , Xin Xie, Ze-Lin Chen, Hao Wu, Chang-Min Yu, Bo-Wen Li
College of Materials Science & Engineering, South China University of Technology, Guangzhou 510640, China
ARTICLE INFO
ABSTRACT
Article history:
Carboxylesterase, a necessary enzyme in various mammalian cells, has been employed in
various biological applications. Herein, we designed and synthesized a novel carboxylesterase-
based prodrug, which can realize simultaneous drug-release imaging and cancer chemotherapy.
This prodrug comprises three parts: coumarin as the fluorophore and the cleavable architecture,
chlorambucil as the anticancer drug, and acetyl group as the enzyme-responsive unit. The
presence of carboxylesterase leads to the activation of coumarin fluorescence, and this
fluorescence serves as the reporting signal for assessing the enzyme level and drug release.
Moreover, the prodrug was incorporated in liposome for monitoring drug release and
chemotherapeutic effect in living cells. Upon internalization by HeLa cells, the prodrug can
release chlorambucil and exhibit high cytotoxicity. This approach may provide some helpful
insights for enhancing therapeutic effect and tracking the release of prodrug.
Received 22 February 2017
Received in revised form 4 April 2017
Accepted 14 April 2017
Available online
Keywords:
Prodrug
Carboxylesterase
Chlorambucil
Fluorescent imaging
Liposomes
1
. Introduction
Chemotherapy is at the forefront of treatment against cancer because of its high efficiency and convenience [1,2]. Unfortunately,
conventional chemotherapy has shown several side effects including poor bioavailability, high toxicity and non-specificity [3-5]. To
address these problems, the developments of prodrug strategies for sidestepping side effects have been used to directly enhance
therapeutic efficiency; these strategies usually involve drug delivery systems, which can be activated by endogenous or exogenous
substances and release the anticancer drug in tumor tissues [6-9]. However, in situ monitoring of the drug’s release and localization in
tumor is yet to be fully exploited.
Prodrug might become a promising approach to integrate chemotherapy and diagnostics, and this approach has become attractive for
activating and monitoring drug release at the unique tumor microenvironment, thereby the therapeutic effect of anticancer drugs could
be significantly improved [10-14]. The special environments of tumor cells include intracellular reduction [15-17], lower pH [18,19]
hypoxia [20,21], active oxygen species [22,23] or over-expressed enzymes [24,25]. Among them, over-expressed enzymes in cancer
cells draw particular interest as an anticancer drug-releasing trigger (mechanism) because of the differences of the enzymes’ levels
between tumor and normal cells.
Cleavable prodrugs have shown excellent capability to mediate the release of drug molecules and fluorophore simultaneously via the
cleavage of a single linker [26]. A series of cleavable linkers for prodrugs have been developed in recent years, which indicated that
cleavable prodrugs are conductive to monitoring drug release and therapeutic effect as well as the trigger (usually biomarker) level
[
27,28]. Recently, coumarin has been adopted as a suitable fluorophore in biochemistry, which show low cytotoxicity, high quantum
yield, easy modification and some optical beneficial characteristics (ICT effect, photo-responsive feature, two-photon excitation and so
on). Many coumarin derivatives have been widely reported as fluorescent probes for detection of different biomarkers or assessment of
drug release [29-31].
The liposomes, as a FDA-approved drug delivery carrier, have been widely utilized for drug delivery [32-34]. Liposomes are defined
as self-assembled vesicles, which are composed of one or multiple concentric lipid bilayers and enclose an aqueous core. The
advantages of utilizing liposomes as drug carrier include improving solubility for the encapsulated drug, reducing the drug’s side-
effects and toxicity, protecting the drug against chemical and biological degradation during the drug delivery, and enhancing
biocompatibility [35].
Carboxylesterase (CaE) mediates the hydrolysis of carboxylic acid ester into alcohols and acids [36-38]. This enzyme acts not only
as a catalyst that has been widely used in organic synthesis or industrial production, but also as a key participant that works in drug
metabolism and detoxification. CaE has been found in most of human tissues, especially in liver, kidney, heart, small intestine and
blood. It has been reported that CaE is overexpressed in some types of cancer cells, and the CaE level is significantly different between
—
——
∗
Corresponding author.
E-mail address: msli860316@mail.scut.edu.cn.
†
These authors contributed equally to this work.
Page 2 of 10

these cancer cells and normal tissues [39,40]. CaE also has been employed as a biomarker; and to date, a number of CaE level assays
have been employed for cancer diagnosis [41,42], but there are still few reports on the exploitation of this enzyme as a trigger for
prodrug.
Considering the physiological and pathological significance of CaE, herein we fabricated a novel prodrug for which the drug release
is achieved via employing the overexpressed CaE in tumor cells as the trigger (activating means), as shown in Scheme 1. This prodrug
system contains an acetyl unit as the responsive unit toward CaE and the coumarin unit as the scaffold for building the cleavable linker
and the reporter fluorophore for monitoring the release of anticancer drug (chlorambucil). This CaE-activatable prodrug system has
several striking features: 1) The coumarin scaffold not only acts as the reporting fluorophore for assessing CaE level but also as a
cleavable architecture for releasing the drug; 2) The acetyl group serves as the enzyme-responsive unit and quencher for coumarin’s
fluorescence via intramolecular charge transfer (ICT) [43]; 3) To evaluate the prodrug’s performance, the prodrug is encapsulated into
liposomes, which can enhance aqueous solubility/dispersibility and in the meantime reduce the nonspecific side effects. As for the
prodrug, upon activation by the enzyme CaE, the ICT effect is diminished by the cleavage of the bond between the fluorophore
(coumarin) and the quencher (acetyl group); due to the cleavable reaction, the active drug and the fluorophore are released
simultaneously; consequently, the coumarin’s fluorescence is restored.
2
. Results and discussion
2
.1. Synthesis
The synthesis route for the prodrug 4 (AC-Cou-CBL) is depicted in Scheme 2. Compound 1 was synthesized from 2,4-
dihydroxybenzaldehyde, sodium propionate and propionic anhydride in the presence of piperidine. Then compound 1 was reacted with
acetic anhydride to yield compound 2. Bromination of compound 2 was accomplished with N-bromosuccinimide (NBS) and benzoyl
peroxide (BPO), affording compound 3. Finally, compound 4 was prepared by nucleophilic reaction of chlorambucil (CBL, anticancer
1
drug) with compound 3. The structures of all the synthesized compounds were confirmed by H NMR and HR-Mass spectrometry (Fig.
S1-S9 in Supporting information). In addition, for comparison, a control compound 8 (Pro-Cou-CBL, namely the prodrug with the
1
acetyl group being replaced by an ether group), and the characterizations for the control compound ( H NMR and HR-Mass) are shown
in Fig. S10-16 (Supporting information).
2
.2. Drug release studies
To verify the release of anticancer drug CBL from the prodrug AC-Cou-CBL was triggered by CaE, UV and fluorescence spectra
were recorded under physiological conditions (37 °C, pH 7.4, 10 mmol/L phosphate buffer). As shown in Fig. S17 (Supporting
information), the strong absorption of AC-Cou-CBL and 2-OH-coumarin are at 320 nm and 370 nm. The fluorescence of AC-Cou-
CBL shows weak emission at 462 nm due to the ICT effect resulted from the presence of acetyl moiety in the coumarin scaffold;
however, a 400-fold enhancement in emission intensity at 462 nm occurs in the presence of CaE, and this spectrum is in accord with
that of 2-OH-coumarin (Fig. 1A, fluorescence at 462 nm). In addition, the time and concentration dependence of the AC-Cou-CBL
fluorescence were also investigated, as shown in Fig. 1B and 1C. The fluorescence at 462 nm increases 400-fold due to the
biochemical reaction activated by CaE, reaching a plateau in 100 min. On stepwise addition of CaE (0 U/L to 200 U/L) to AC-Cou-
CBL, the fluorescence intensity at 462 nm increases until it reaches a saturation point at 200 U/L. To check whether the fluorescence
signal of the released fluorophore (2-OH-coumarin) can be employed to monitor drug release, we conducted the experiments to
investigate the correlation between the fluorescence signal and drug release. Fig. S18 shows the correlation analysis of fluorescence
intensity for varied concentrations of 2-OH-Coumarin (0-5 µmol/L) as well as that of AC-Cou-CBL (0-5 µmol/L) in the presence of
1
00 U/L CaE; the linear relationship clearly indicates that, the fluorescence of the released and thus activated 2-OH-coumarin can
serves as the reporting signal for detecting the enzyme level and monitoring drug release. All these results indicate that CBL can be
released from AC-Cou-CBL successfully in the presence of CaE.
In order to confirm the release of CBL from AC-Cou-CBL upon the activation by CaE, HPLC and high resolution MS spectrum
were employed for verification (Fig. 1D, Fig. 2, and Fig. S19 in Supporting information). As shown in Fig. 1D, CBL can be released
from the prodrug in the presence of CaE (100 U/L) based on HPLC measurement, whereas no drug release can be detected in the
absence of CaE. In Fig. 2, the retention time of AC-Cou-CBL, 2-OH-coumarin and CBL are identified at 2.12 min, 1.53 min and 1.75
min respectively in HPLC chromatogram. Futhermore, AC-Cou-CBL was reacted with CaE and then a small aliquot was subjected to
+
+
HR-MS analysis. Ionic peaks corresponding to CBL ([M+H] = 304.0875), 2-OH-coumarin ([M+H] = 193.0497) and AC-Cou-CBL
(
+
[M+H] = 520.1309) can be observed in the HR-MS spectra, clearly indicating that the active drug CBL could be released from the
AC-Cou-CBL (Fig. S19). These results provide evidence that the anticancer drug CBL is indeed released from the prodrug through
CaE mediated cleavable reaction.
2
.3. Selectivity
Fig. S20 (Supporting information) shows the effect of pH on CaE-activated reaction of AC-Cou-CBL. In the presence of CaE,
obvious increment of emission intensity can be detected and remains relatively stable in the pH range of 4-10. In the absence of CaE,
the emission intensity of AC-Cou-CBL is weak and keeps stable over a pH range of 4-8 and there is slight increase of fluorescence
Page 3 of 10

intensity at pH range of 9-10. These results show that, under physiological conditions, AC-Cou-CBL may be useful as a prodrug for
releasing CBL in tumor tissues where CaE is overexpressed.
To assess whether other biologically relevant substances will affect the cleavage reaction of AC-Cou-CBL upon activation by CaE,
AC-Cou-CBL was reacted with these substances respectively. As depicted in Fig. 3A, for the prodrug, significant fluorescence
intensity at 462 nm can only be observed in the presence of CaE, while there is no obvious enhancement for other potential
interferents. In addition, we also assessed the performance of this carboxylesterase-based prodrug in human serum (which contains
various proteins and could show possible background to hydrolysis of the prodrug). As shown in Fig. 3B, the prodrug in human serum
with 100 U/L CaE displays remarkable changes in fluorescence intensity at 462 nm and this result is similar to that of 2-OH-Coumarin
(5 µmol/L) in human serum. In order to enhance the biocompatibility and reduce side-effects, we constructed a liposome nanoparticle
containing AC-Cou-CBL (referred to as LIP-AC-Cou-CBL, Fig. S21 in Supporting information) and evaluated its performance in
human serum. Fig. 3B shows that the fluorescence intensity of LIP-AC-Cou-CBL without the addition of CaE is lower than that of AC-
Cou-CBL in human serum, and this result indicates a higher stability of the liposomal nanoparticle. Meanwhile, we also investigated
the LIP-AC-Cou-CBL with 100U/L CaE in serum, and the result shows that the fluorescence intensity is much weaker than that of AC-
Cou-CBL upon addition of CaE; and this indicates that the liposome can offer protection for the prodrug. All these results demonstrate
that, the fluorescence enhancement resulted from the cleavage reaction is indeed caused by CaE, and also provides further evidence
that CBL and 2-OH-coumarin can be released from the prodrug AC-Cou-CBL.
2
.4. Drug release studies in HeLa cells
HeLa cells were employed for drug release studies in cells. Upon incubation with AC-Cou-CBL for 0.5 h, we can observe blue
intracellular fluorescence in HeLa cells, and much brighter fluorescence can be detected for 1 h of incubation, as shown in Fig. 4A. In
order to improve aqueous solubility/dispersibility and enhance cellular uptake of the AC-Cou-CBL, we also evaluated LIP-AC-Cou-
CBL performance in cells. As for the liposomal nanoparticles, much stronger fluorescence can be detected in HeLa cells (Fig. 4B).
Therefore, the prodrug can be used to monitor the drug release process in HeLa cells, given the fact that the fluorophore 2-OH-
coumarin can be effectively released from the prodrug.
To investigate the therapeutic effect of LIP-AC-Cou-CBL against cancer cells, the cytotoxicity of HeLa cells was assessed using
typical MTT assays (Fig. 4C). The prodrug shows high cytotoxicity to HeLa cells and the half-maximal inhibitory concentration (IC )
5
0
of the prodrug is 15.2 µg/mL. In Fig S25 (Supporting information), we also performed the cytotoxicity for 2-OH-coumarin and
employed CBL as a positive control, the results indicate that the IC of CBL is 9.6 µmol/L and 2-OH-Coumarin (IC > 80 µmol/L)
50
50
exhibits low cytotoxicity. Furthermore, flow cytometry studies were used to further verify the cell apoptosis of LIP-AC-Cou-CBL with
varied concentrations of the prodrug (0 µg/mL to 80 µg/mL). After incubation for 2 h, the apoptotic percentage (including the early and
late apoptosis, Q1 and Q4 in Fig. 4D) induced by LIP-AC-Cou-CBL gradually increases (from 0.7% to 73.4%), as the prodrug
concentration is increased. All these results demonstrate that the prodrug shows quite good therapeutic effect in HeLa cells.
2
.5. Verification of the release mechanism by control compound
To further verify the release mechanism, the ester bond (between CBL and coumarin of AC-Cou-CBL) can be hydrolyzed by CaE
and CBL can be released. Therefore, we synthesized a control compound 8 (Pro-Cou-CBL) to investigate whether or not the release of
CBL is caused by the cleavable reactions starting from the hydrolysis of the ester bond. As shown in Scheme S1, the control compound
Pro-Cou-CBL which has no ester-responsive group (acetyl group) was treated with CaE (100U/L) for 1 h, and then monitored by
fluorescence and HPLC. As is expected, we can’t observe the enhancement of fluorescence (Fig. S22 in Supporting information) and
the CBL peak in HPLC chromatogram (Fig. S23 in Supporting information). Meanwhile, we also evaluate the performance of the LIP-
Pro-Cou-CBL (Fig. S21) in HeLa cells. The cell cytotoxicity and cell imaging experimental results demonstrate that Pro-Cou-CBL
can’t release coumarin fluorophore and the anticancer drug CBL (Fig. 4C and Fig. S24 in Supporting information). These results prove
that the release of drug is cause by the cleavable reactions triggered by CaE.
3
. Conclusion
In this work, we developed a CaE-activatable prodrug, which can release anticancer drug CBL and restore the fluorescence of the
coumarin fluorophore (2-OH-Coumarin) through cleavable reaction in the presence of CaE. The prodrug was also encapsulated in
liposomes for improving stable and enhancing biocompatibility; upon incubation with cancer cells, these liposomal nanoparticles can
release the coumarin fluorophore and exhibit quite good therapeutic effect. This strategy may provide some helpful insights for
designing prodrugs using enzyme as the trigger.
4
. Experimental
4
.1. Materials
2
,4-Dihydroxybenzaldehyde, sodium propionate, N-bromosuccinimide (NBS), benzoyl peroxide (BPO), chlorambucil (CBL) and
esterase were purchased from Sigma-Aldrich Reagents without further purification. Human serum was supplied by The Sixth Affiliated
Hospital, Sun Yat-sen University. N,N-Dimethyl-formamide (DMF) was dried with CaH and distilled under nitrogen atmosphere. The
2
Page 4 of 10

water used was the triple-distilled water which was further treated by ion exchange columns. Other solvents used in this study were
analytical grade reagents and used without further purification.
4
.2. Measurements
1
13
H NMR and C NMR spectra were recorded on a Bruker AV 500MHz or 600 MHz NMR spectrometer. High resolution mass
spectra were obtained on AB Sciex Triple TOF 5600+ mass spectrometer. UV-vis spectra were recorded at 37 °C on a Hitachi U-3010
UV-vis spectrophotometer. Fluorescence spectra were recorded at 37 °C on a Hitachi F-4600 fluorescence spectrophotometer.
Fluorescence images were obtained using an Olympus IX 71 with a DP72 color CCD. The particle size and distribution were
determined through dynamic light scattering (DLS) on a Malvern Nano-ZS90 particle size analyzer at a fixed angle of 90° at 25 °C.
Transmission electronic microscopy (TEM) experiments were carried out by mounting a drop (~15 µL) of the solution onto a carbon-
coated copper grid and observed on a JEM-2010HR transmission electron microscopy (Japan).
4
.3. Synthesis of compound 1-4
Synthesis of 7-hydroxy-3-methyl-2H-chromen-2-one (1): A mixture of 2,4-dihydroxybenzaldehyde (1.5 g, 10.8 mmol), sodium
propionate (2.25 g, 23.4 mmol), piperidine (150 µL, 1.5 mmol) and propionic anhydride (4 mL, 31.04 mmol) was refluxed at 140 °C
for 6 hours and then poured into ice. Then a 0.1 mol/L solution of HCl was added in the aqueous mixture, which yielded a milky
flocculent precipitate. The precipitate was filtered and treated under stirring with concentrated H SO (3 mL) for 30 minutes. The
2
4
obtained mixture was poured into ice again to afford brown residue, which was purified by column chromatography over silica gel
1
eluting with petroleum ether/ethyl acetate (2:1) to afford 1 (1.5 g, 82 % yield) as pale solid. H NMR (500 MHz, -DMSO-d ): δ 10.37
6
(
s, 1H), 7.74 (d, 1H, J = 8.8 Hz), 7.45-7.39 (m, 1H), 6.76 (dd, 1H, J = 8.4, 2.2 Hz), 6.70 (d, 1H, J = 2.0 Hz,), 2.03 (s, 3H). HR-MS
+
(ESI): calcd. for C H O ([M+H] ) 175.0401, found: 175.0403.
10
8
3
Synthesis of 3-methyl-2-oxo-2H-chromen-7-yl acetate (2): To the solution of 1 (1.76 g, 10 mmol) in 50 mL CH Cl were added
2
2
Ac O (19 mL, 20 mmol) and 3 mL pyridine. The mixture was stirred for 12 hours at room temperature and concentrated subsequently.
2
The residue was extracted with CH Cl (30 mL, 3 times) and the organic layer was washed with water and saturated aqueous NaCl
2
2
respectively and then dried over anhydrous Na SO . After the evaporation of the solvent, the residue was then purified by column
2
4
1
chromatography over silica gel eluting with petroleum ether/ethyl acetate (4:1) to afford 2 (2.10 g, 96 % yield) as white solid. H NMR
(
600 MHz, CDCl ): δ 7.51 (s, 1H), 7.42 (d, 1H, J = 8.4 Hz), 7.09 (d, 1H, J = 2.1 Hz), 7.02 (dd, 1H, J = 8.4, 2.2 Hz), 2.34 (s, 3H), 2.21
3
+
(d, 3H, J = 0.9 Hz). HR-MS (ESI): calcd. for C H O ([M+H] ) 219.0652, found: 219.0654.
12
10
4
Synthesis of acetic acid 3-bromomethyl-2-oxo-2H-chromen-7-yl ester (3): To the solution of 2 (1.09 g, 5 mmol) in 30 mL carbon
tetrachloride were added N-bromosuccinimide (NBS) (0.89 g, 5 mmol) and 40 mg benzoyl peroxide (BPO). The mixture was stirred
under reflux for 4 hours. After cooling down to room temperature, the insoluble residue was filtered off and the filtrate was
concentrated and then the residue was then purified by column chromatography over silica gel eluting with petroleum ether/ethyl
1
acetate (4:1) to afford 3 (1.15 g, 77 % yield) as white solid. H NMR (600 MHz, CDCl ): δ 7.85 (s, 1H), 7.51 (d, 1H, J = 8.5 Hz), 7.14
3
+
(
d, 1H, J = 2.1 Hz), 7.08 (dd, 1H, J = 8.5, 2.1 Hz), 4.42 (s, 2H), 2.35 (s, 3H). HR-MS (ESI): calcd. for C H BrO ([M+H] ) 296.9757,
12
9
4
found: 296.9756.
Synthesis of 4-{4-[bis-(2-chloro-ethyl)-amino]-phenyl}-butyric acid 7-acetoxy-2-oxo-2H-chromen-3-ylmethyl ester (4): To the
solution of 3 (99 mg, 0.33 mmol) in 3 mL dry N,N-dimethylformamide (DMF) were added chlorambucil (CBL) (101 mg, 0.33 mmol)
and potassium carbonate (23 mg, 0.165 mmol). The mixture was stirred at room temperature for 3 hours and extracted with 20 mL
CH Cl and the organic layer was washed with water, saturated aqueous 50 mL NaCl respectively and dried over anhydrous Na SO .
2
2
2
4
After the evaporation of the solvent, the residue was then purified by column chromatography over silica gel eluting with petroleum
1
ether/ethyl acetate (4:1) to afford 4 (150 mg, 86.5 % yield) as pale yellow solid. H NMR (600 MHz, CDCl ): δ 7.73 (s, 1H), 7.50 (d,
3
1
H, J = 8.4 Hz), 7.13 (d, 1H, J = 2.1 Hz), 7.10-7.04 (m, 3H), 6.63 (d, 2H, J = 8.5 Hz), 5.05 (s, 2H), 3.70 (t, 4H, J = 7.1 Hz), 3.62 (t, 4H,
13
J = 7.0 Hz), 2.58 (t, 2H, J = 7.5 Hz), 2.42 (t, 2H, J = 7.5 Hz), 2.34 (s, 3H), 2.01-1.89 (m, 2H). C NMR (125 MHz, CDCl ): δ 173.12,
3
1
3
68.68, 159.93, 154.14, 153.16, 144.41, 140.35, 130.36, 129.72, 128.74, 123.18, 118.64, 116.57, 112.21, 110.26, 61.02, 53.61, 40.53,
+
3.93, 33.46, 26.63, 21.13. HR-MS (ESI): calcd. for C H Cl NO ([M+H] ) 520.1298, found: 520.1304.
26
27
2
6
4
.4. In vitro drug release study
The experiments for in vitro drug release were conducted in PBS solutions (pH 7.4, 10 mmol/L) containing 2% (v/v) DMSO or in
human serum. The final concentration of the serum in the test solution is 10-fold diluted. Fluorescent spectra of AC-Cou-CBL (5
µmol/L) reacted with or without CaE (100 U/L) for 1 h in both PBS solutions and human serum were recorded. Fluorescence spectra of
AC-Cou-CBL (5 µmol/L) reacted with CaE (100 U/L) for varied time (0-120 min) or varied CaE concentrations (0-200 U/L) in 1 h in
PBS and varied AC-Cou-CBL concentrations (0-5 µmol/L) with CaE (100 U/L) for 1 h in human serum were recorded. Fluorescent
spectra of LIP- AC-Cou-CBL (40 µg/mL) with or without CaE (100 U/L) in human serum were recorded. Fluorescent spectra of varied
2
4
-OH-Coumarin (0-5 µmol/L) were recorded as well. The excitation wavelength was set at 405 nm and emission was recorded from
20-600 nm with excitation and emission slits of 5 nm, volt 500 V.
4
.5. Fabrication of liposomal nanoparticles (LIP-AC-Cou-CBL or LIP-Pro-Cou-CBL)
Page 5 of 10

A DMSO (100 µL) solution of AC-Cou-CBL or Pro-Cou-CBL (10 mg) was diluted with THF (1.9 mL) and the DSPE-PEG 2000
(10 mg) was also dissolved in 1 mL THF, which were added dropwise into 20 mL water under sonication for 20 min at 4 °C. The
mixture was then stirred at room temperature for 24 hours. After stirring, THF was removed at 37 °C under reduced pressure and the
residual solution underwent dialysis with water (cut-off MW 1000). The suspension was centrifuged at 12,000 rpm for 15 min at 4 °C
to collect the nanoparticles. After washing three times, the nanoparticles were resuspended in the water (10 mL).
4
.6. Cell cultures
Cervical cancer (HeLa) cell lines were incubated in DMEM medium (10% FBS and 1% penicillin/streptomycin) in a humidified
incubator containing 5% CO at 37 °C.
2
4
.7. Cellular uptake of AC-Cou-CBL and LIP-AC-Cou-CBL in HeLa cells
In vitro uptake of AC-Cou-CBL and LIP-AC-Cou-CBL was studied with the usage of fluorescence microscopy in HeLa cells. HeLa
cells were cultured in a 30 mm 6-well plate with the polylysine-coated cell culture glass slides inside in complete DMEM. The cells
were cultured at 37 °C in a humidified 5 % CO incubator. After 24 h incubation, the culture medium was removed, and then the cells
2
were washed with PBS buffer three times and subsequently AC-Cou-CBL (10 µmol/L) and the diluted LIP-AC-Cou-CBL solutions (40
µg/mL) with DMEM were added. The cells were continuously cultured for 0.5 and 1 h respectively at 37 °C. Then, after culture
medium being removed, the cells were washed with PBS buffer for three times and the glass slides were taken out to image on an
Olympus IX71 inverted fluorescence microscope equipped with a DP72 color CCD.
4
.8. Cell viability assay (MTT assay)
The cell viability experiment to evaluate the LIP-AC-Cou-CBL，LIP-Pro-Cou-CBL, CBL and 2-OH-coumarin cytotoxicity towards
HeLa cell line was carried out by MTT viability assay. In the MTT assay, cells well were seeded in a 96-well plate in 200 µL of
DMEM medium. The cells were continued to incubate for 24 hours. 200 µL PBS was used to wash each well after the removal of
DMEM medium. 200 µL of a medium containing LIP-AC-Cou-CBL (from 0 to 150 µg/mL) was added respectively. The cells were
incubated for 48 hours. After the removal of DMEM medium, 20 µL of 5 mg/mL MTT assay stock solution in PBS was added into
each well and the cells were incubated for another 4 hours, then the medium containing unreacted MTT was removed. 200 µL of
DMSO was added into each well to dissolve the obtained blue formazan crystals, and the absorbance at 490 nm was recorded on a
Thermo MK3 ELISA plate reader to calculate the cell viability.
4
.9. Apoptosis with flow cytometry assays
HeLa cells were seeded in a 6-well plate with 2 mL of complete DMEM in each well and incubated for 24 h. After the removal of
DMEM medium and washed with PBS, various concentrations of LIP-AC-Cou-CBL (0 to 80 µg/mL) were added to each well
respectively and incubated at 37 °C for 2 hours. HeLa cells without the treatment were used as a control. Floating and attached cells
were both collected and washed with PBS for a quantified determination. The cells were stained with Alexa Fluor 488 FITC-
conjugated annexin V and PI according to the manufacturer’s instructions to evaluate cell apoptosis. Finally, the flow cytometry
analyses were performed using BD Accuri C6 flow cytometer and the data were analyzed using the BD Biosciences software.
Acknowledgment
We gratefully acknowledge the financial support by the Science and Technology Planning Project of Guangzhou (No.
2
01607020015) and the Science and Technology Planning Project of Guangdong Province (No. 2014A010105009).
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Page 7 of 10

Scheme 1. Structure of prodrug and mechanism of this prodrug for drug release and imaging in HeLa cells.
Cl
N
HO
OH
O
a
b
O
O
Br
c
d
O
O
Cl
CHO
HO
O
O
O
O
O
O
O
O
O
O
O
1
2
3
4
(AC-Cou-CBL)
Scheme 2. Synthetic route for the prodrug AC-Cou-CBL (compound 4). Reagents and Conditions: a) sodium propionate, piperidine, propionic anhydride, 140
°
C, 6 h; b) CH
2
Cl
2
, Ac
2
O and pyridine, r.t., 12 h; c) N-Bromosuccinimide (NBS), benzoyl peroxide (BPO), CCl
4
, reflux, 4 h; d) chlorambucil (CBL), DMF,
potassium carbonate, r.t., 3 h.
Page 8 of 10

Fig. 1. (A) Fluorescence spectra of the AC-Cou-CBL (5 µmol/L), 2-OH-coumarin (5 µmol/L) and AC-Cou-CBL (5 µmol/L) with CaE (100 U/L) for 1 h. (B)
Time-dependent fluorescence spectra for the AC-Cou-CBL (5 µmol/L) with CaE (100 U/L). Inset: Change in fluorescence intensity at 462 nm as a function of
time after CaE (100 U/L) treatment. (C) Fluorescent spectra for AC-Cou-CBL (5 µmol/L) with CaE (0-200 U/L) for 1 h. Inset: Change in fluorescence intensity
at 462 nm as a function of CaE concentration. (D) Percentage of CBL (as determined by HPLC) released from the AC-Cou-CBL (5 µmol/L) as a function of
time with or without CaE (100 U/L).
Fig. 2. Typical HPLC chromatogram of the AC-Cou-CBL (10 µmol/L) incubated with CaE (100 U/L) (A), 2-OH-Coumarin (B), AC-Cou-CBL (C) and CBL
(D). Peaks in the chromatograms were detected by monitoring the absorption at 320 nm (CBL and AC-Cou-CBL), 370 nm (2-OH-Coumarin). The mobile phase
was 80/20 MeOH/water and the flow rate was 1.0 mL/min.
Fig. 3. (A) Fluorescence intensity for the prodrug (5 µmol/L) at 462 nm in phosphate buffer (pH 7.4, 10 mmol/L, containing 2% (v/v) DMSO) in the presence of
various relevant species for 1 h. Excitation wavelength: 405 nm. (100 U/L for Carboxylesterase (CaE), nitroreductase (NTR), azoreductase (AZD), alkaline
Page 9 of 10

phosphatase (ALP) and DT-diaphorase (DTD) respectively; 50 mmol/L for Hcy, GSH, Cys, Lys, Phe, Arg, DTT and Gly respectively; 100 mmol/L for others
respectively). (B) Fluorescence intensity at 462 nm. (a): AC-Cou-CBL (5 µmol/L) in serum without the addition of CaE for 1 h; (b): 2-OH-Coumarin in serum
without the addition of CaE for 1 h; (c): AC-Cou-CBL (5 µmol/L) in serum with the addition of CaE (100U/L) for 1 h; (d): LIP-AC-Cou-CBL (40 µg/mL) in
serum without the addition of CaE for 1 h；(e) LIP-AC-Cou-CBL (40 µg/mL) in serum with the addition of CaE (100U/L) for 1 h.
Fig. 4. Fluorescent microscopic images for HeLa cells in the absence (the control) or presence of the AC-Cou-CBL (10 µmol/L) (A), LIP-AC-Cou-CBL (40
µg/mL) (B) with the incubation time of 0.5 h or 1 h respectively. (C) Cell viability profiles for HeLa cell line treated with the LIP-AC-Cou-CBL and LIP-Pro-
Cou-CBL of varied concentrations for 48 hours. (D) Flow cytometry analysis for apoptosis of HeLa cells induced by LIP-Pro-Cou-CBL (0-80 µg/mL)
nanoparticles.
Page 10 of 10