Overcoming resistance to cisplatin by inhibition of glutathione S-transferases (GSTs) with ethacraplatin micelles in?vitro and in?vivo

Article abstract of DOI:10.1016/j.biomaterials.2017.08.021

Platinum-based DNA-adducting agents are used extensively in the clinic for cancer chemotherapy. However, the anti-tumor efficacy of these drugs is severely limited by cisplatin resistance, and this can lead to the failure of chemotherapy. One of cisplatin

Full text of DOI:10.1016/j.biomaterials.2017.08.021

Accepted Manuscript
Overcoming resistance to cisplatin by inhibition of glutathione S-transferases (GSTs)
with ethacraplatin micelles in vitro and in vivo
Shuyi Li, Chan Li, Shubin Jin, Juan Liu, Xiangdong Xue, Ahmed Shaker Eltahan,
Jiadong Sun, Jingjie Tan, Jinchen Dong, Xing-Jie Liang
PII:
S0142-9612(17)30531-8
10.1016/j.biomaterials.2017.08.021
JBMT 18222
DOI:
Reference:
To appear in: Biomaterials
Received Date: 6 April 2017
Revised Date: 9 July 2017
Accepted Date: 14 August 2017
Please cite this article as: Li S, Li C, Jin S, Liu J, Xue X, Eltahan AS, Sun J, Tan J, Dong J, Liang X-J,
Overcoming resistance to cisplatin by inhibition of glutathione S-transferases (GSTs) with ethacraplatin
micelles in vitro and in vivo, Biomaterials (2017), doi: 10.1016/j.biomaterials.2017.08.021.
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ACCEPTED MANUSCRIPT
Overcoming resistance to cisplatin by inhibition
of glutathione S-transferases (GSTs) with
ethacraplatin micelles in vitro and in vivo
Shuyi Lia,b,1, Chan Li*,a,b,1, Shubin Jinc, Juan Liud, Xiangdong Xuea, Ahmed Shaker
Eltahana,b, Jiadong Suna, Jingjie Tana, Jinchen Donga and Xing-Jie Liang*,a,b
a
Laboratory of Controllable Nanopharmaceuticals, Chinese Academy of Sciences
(CAS) Center for Excellence in Nanoscience; and CAS Key Laboratory for
Biomedical Effects of Nanomaterials and Nanosafety, National Center for
Nanoscience and Technology of China, No.11, First North Road, Zhongguancun,
Beijing, 100190, P. R. China.
b University of Chinese Academy of Sciences, Beijing 100049, P. R. China
c
Beijing Municipal Institute of Labour Protection, No.55 Taoranting Road, Xicheng
District, Beijing, 100054, P. R. China.
d Tissue Engineering Lab, Beijing Institute of Transfusion Medicine, Beijing 1000850,
P. R. China.
1S.L. and C.L. contributed equally to this work.
*Corresponding author:
Xing-Jie Liang, Tel: +86-010-82545569; Fax: +86-010-62656765; Email:
liangxj@nanoctr.cn;
Chan Li, Tel: +86-010-82545615; Fax: +86-010-62656765; Email: lic@nanoctr.cn.

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Abstract
Platinum-based DNA-adducting agents are used extensively in the clinic for cancer
chemotherapy. However, the anti-tumor efficacy of these drugs is severely limited by
cisplatin resistance, and this can lead to the failure of chemotherapy. One of cisplatin
resistance mechanisms is associated with overexpression of glutathione S-transferases
(GSTs), which would accelerate the deactivation of cisplatin and decrease its
antitumor efficiency. Nanoscale micelles encapsulating ethacraplatin, a conjugate of
cisplatin and ethacrynic acid (an effective GSTs inhibitor), can enhance the
accumulation of active cisplatin in cancer cells by inhibiting the activity of GSTs and
circumventing deactivation of cisplatin. In vitro and in vivo results provide strong
evidence that GSTs inhibitor-modified cisplatin prodrug combined with nanoparticle
encapsulation favor high effective platinum accumulation, significantly enhanced
antitumor efficacy against cisplatin-resistant cancer and decreased system toxicity. It
is believed that these ethacraplatin-loaded micelles have the ability of overcoming
resistance of cancers toward cisplatin and will improve the prospects for
chemotherapy of cisplatin-resistant cancers in the near future.
cisplatin resistance│ glutathione S-transferases (GSTs)│ ethacraplatin│ drug delivery
1. Introduction
Cancer endangers the health of humans around the world [1]. Chemotherapy is one
of the leading choices for cancer treatment. Three platinum-based DNA-adducting
agents, cisplatin, carboplatin, and oxaliplatin, are most widely used in standard-of-
care chemotherapy for many types of cancer. However, in the clinic, platinum-based

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agents only show excellent efficacy in the early stage of chemotherapy, and their
effectiveness subsequently becomes weakened by the development of cisplatin
resistance. Thus, cisplatin resistance is a key factor leading to chemotherapy failure.
In some types of cancer, the mechanism of cisplatin resistance is mainly associated
with the intracellular overexpression of glutathione S-transferases (GSTs) [2, 3].
GSTs are crucial detoxification enzymes that are capable of catalyzing the
conjugation of glutathione (GSH) with a multitude of xenobiotics, one of which is
cisplatin. Cisplatin undergoes detoxification by GSH more readily in resistant cells
due to the overexpression of GSTs. Hence, GSTs have been regarded as promising
drug targets to reverse cisplatin resistance in cancers. Especially, it has been reported
that the expression of GST-π is positively correlated with cisplatin resistance of
tumors.[2, 3] Subsequently, some strategies have been successfully developed to
inhibit their activity [4-6].
One smart approach is to suppress intratumoral GST activity by designing
bifunctional Pt(IV) prodrugs that have greater stability and are activated locally. This
allows for enhanced accumulation of active platinum(II) at the target sites. In
addition, Pt(IV) prodrugs generally exhibit improved properties such as lipophilicity
and active targeting as a result of modification of the axial ligands. More importantly,
Pt(IV) prodrugs with dual-threat capabilities, such as mitaplatin and Pt(IV)(α-
TOS)(OEt), provide a more effective therapeutic option because they can target
mitochondria and genomic DNA simultaneously [7-9].
Ethacraplatin (EA-Pt) is a bifunctional Pt(IV) prodrug that releases its ethacrynic
acid (EA) moiety, an effective GSTs inhibitor, and active platinum(II) metal center by

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reduction in cancer cells. It can potently inhibit GSTs, thereby enhancing
accumulation of active cytotoxic Pt(II) in the cancer cell, which facilitates generation
of platinum-DNA adducts, and consequently reverses drug resistance. This suggests
that ethacraplatin possesses a great potential for therapy of cisplatin-resistant cancer.
However, ethacraplatin is a hydrophobic molecule with poor water solubility. A
previous study showed that ethacraplatin had no better long-term (>24 h) anticancer
efficacy in vitro than cisplatin, which may impede its further application in vivo [10].
It is imperative to find an approach to overcome these limitations. Nanotechnology
has developed rapidly in the past two decades, and has displayed tremendous promise
for developing cancer therapies. Various drug delivery systems have been explored
which have opened up new prospects for altering the properties of drugs, alleviating
their systemic cytotoxicity and enhancing their anti-cancer efficacy in vitro and in
vivo [11-20]. Lippard and colleagues encapsulated mitaplatin into nanoparticles for
cancer therapy [21]. Their results demonstrated that this nanoparticle formation
strategy was beneficial in reducing the side effects of mitaplatin by decreasing
unwanted accumulation of platinum in the kidneys. In addition, encapsulation of
mitaplatin in nanoparticles did not compromise the long-term chemotherapeutic
effect. Lin and coworkers developed a self-assembly delivery system with pegylated
Zn pyrophosphate nanoscale coordination polymers (NCPs), which are capable of
carrying cisplatin or oxaliplatin prodrugs with high levels of drug loading [22]. Data
from a multitude of in vivo experiments indicated that these two novel drug delivery
systems are advantageous over free drugs in biodistribution and therapeutic efficacy.
Some nanoparticles physically loading cisplatin were also developed [23]. Duan et al
fabricated cisplatin-loaded EGF modified mPEG-PLGA-PLL nanoparticles [24],
which can improve antitumor efficiency for SKOV3 cancer in vitro and in vivo, and
reduce nephrotoxicity and systemic toxicity. Alexiou et al utilized superparamagnetic
iron oxide nanoparticles (SPIONs) with a dextran and hyaluronic acid (HA) coating to
deliver cisplatin [25]. The developed carrier reserved superparamagnetic property and
possessed high biocompatibility. The cisplatin-loaded nanoparticle showed
comparable antitumor activity to cisplatin. This work represents a promising

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application of magnetic drug targeting for cancer therapy. They have potential for
further translation in the clinic in the future.
Here we employed an FDA-approved adjuvant, 1,2-distearoyl-sn-glycero-3-
phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000), to
form micelles and deliver ethacraplatin (EA-Pt). The ethacraplatin-containing
micelles (M-EA-Pt) were demonstrated to improve the poor water-solubility of free
ethacraplatin, increase the drug content at the tumor site by the enhanced permeability
and retention (EPR) effect, and improve the antitumor activity without increasing
system toxicity in cisplatin-resistant cancer models in vitro and in vivo.
2. Materials and Methods
2.1. Materials
Cisplatin was purchased from Sigma-Aldrich Company (St. Louis, MO). Ethacrynic
acid was purchased from Enzo Life Sciences Inc (NY, USA). 1,2-distearoyl-sn-
glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-
PEG2000) was purchased from Lipoid GmbH (Ludwigshafen, Germany). Coumarin 6
was purchased from J&K Scientific Ltd (Beijing, China). Methanol was purchased
from Avantor Performance Materials Inc (PA, USA).
2.2. Synthesis and characterization of ethacraplatin
Ethcraplatin was synthesized according to the literature. Briefly, cis, cis, trans-
Pt(NH3)2Cl2(OH)2 was first synthesized. Cisplatin (500 mg) was added to water (10
mL) to form a suspension, then 30% hydrogen peroxide (4 mL) was added and stirred
at 50 °C. After 1 h, the suspension was stored at 4 °C overnight. The precipitated solid

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Pt(NH3)2Cl2(OH)2 was filtered off and washed with cold water and ethanol. Light
yellow crystals of Pt(NH3)2Cl2(OH)2 were obtained. Next, excess oxalyl chloride (2.5
mL) was mixed with ethacrynic acid (500 mg) and refluxed at 70 °C for 60 min.
Unreacted oxalyl chloride was removed under vacuum. In order to remove the oxalyl
chloride completely, dry THF (2 × 5 mL) was added and removed by vacuum
extraction. When the extraction procedure was complete, ethacrynic acid chloride
remained as a pale yellow oil-like compound. The obtained ethacrynic acid chloride
was mixed with Pt(NH3)2Cl2(OH)2 (116 mg) in dry THF (5 mL) and stirred at 70 °C.
After approximately 40 min, THF was removed under vacuum. Acetone (5 mL) was
then added, followed by water (25 mL), and the mixture was stored at 4 °C overnight.
After removal of the acetone under vacuum, the water was discarded. The residue was
washed twice with water, then dried at 60 °C. The solid left was resuspended in
excess diethyl ether and filtered. The final product EA-Pt is an off-white solid.
Molar masses and molar mass distributions of EA-Pt were determined on a Bruker
Amazon SL electrospray ionization mass spectrometer (ESI-MS). Nuclear magnetic
resonance (NMR) spectra were registered on a Bruker AVANCE III NMR
spectrometer. Ultraviolet spectra (UV-Vis) were recorded on a LabTech Bluestar A
ultraviolet spectrometer (LabTech, China). The purity analysis of EA-Pt was carried
out by high-performance liquid chromatography (HPLC, LC-20A, Shimadzu). EA-Pt
was analyzed on an Eclipse XDB-C18 analytical column (250 mm × 4.6 mm, 5 ꢀm,
Agilent). The gradient was varied from 85% to 95% methanol (the water fraction
varied from 15% to 5% accordingly). The flow rate was 1.5 mL/min at 40 °C, and the

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gradient procedure lasted for 20 min. The UV-Vis detector was set at the wavelength
of 275 nm.
2.3. Preparation of ethacraplatin-loaded nanomicelles
Ethacraplatin-loaded micelles (M-EA-Pt) were synthesized from DSPE-PEG2000
and ethacraplatin by the film dispersion method. Ethacraplatin and DSPE-PEG2000
(1:7, w/w) were dispersed in 5 mL methanol. After removing the solvent by vacuum
rotary evaporation, a dry film was formed, which was then hydrated with deionized
water at room temperature for 10 min. Coumarin 6-loaded nanomicelles were
prepared according to the method described above. The morphology of the micelles
was measured by electron microscopy. The hydrated diameter of the micelles was
detected by dynamic light scattering (DLS) using a Zetasizer 5000 (Malvern
Instruments, Malvern, Worcestershire, UK). After formation of M-EA-Pt, the free
drug that was not encapsulated into the micelles was removed by filtration using 0.22
ꢀm membrane. The quantity of ethacraplatin in the M-EA-Pt was determined using
NexION 300X inductively coupled plasma mass spectroscopy (ICP-MS, PerkinElmer,
USA). The encapsulation efficiency (EE) and drug loading efficiency (DLE) were
determined using the formulas below:
weight of loaded EA-Pt
Encapsulation efficiency (%) =
× 100%
weight of EA-Pt in feed
weight of loaded EA-Pt
Drug loading efficiency (%) = weight of EA-Pt-loaded micelles × 100%
2.4. Ethacraplatin release from micelles

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The release profile of M-EA-Pt was detected by dialysis using minidialysis tubes
with a molecular mass cut off of 6 kDa - 8 kDa (Millipore, USA). A suspension of M-
EA-Pt in water was divided equally (100 ꢀL) into multiple minidialysis tubes. All
these minidialysis tubes were soaked in a same beaker containing 2 L PBS (pH 7.4)
and stirred in the dark at 37 °C. After 0, 1, 4, 6, 16, 25, 48, 72, 96, 120 and 144 h, one
minidialysis tube was withdrawn. The micellular suspension remained in each
minidialysis tube was transferred and the platinum content was quantified by ICP-MS
after digestion using aqua regia. The percentage of EA-Pt released at different time
(Px) is calculated as follows: Px = (Pt0-Ptx) / Pt0 × 100%, Pt0 is the initial platinum
content added in the tube, and Ptx is the platinum content remained in the tube at
different time. All experiments were carried out in triplicate.
2.5. Cell culture
The parent cell line BEL7404 and its cisplatin-resistant derivative BEL 7404-CP20
were presented as gifts by Michael M. Gottesman’s laboratory at NCI, NIH.
BEL7404-CP20 cells were cultured in medium including 5 µg/mL cisplatin. Both cell
lines were both cultured in an atmosphere containing 5% CO2 at 37 °C using DMEM-
high glucose (Wisent, Canada) with 10% fetal bovine serum (Wisent, Canada), 100
units/mL penicillin, and 100 µg/mL streptomycin (Gibico, Invitrogen). For in vitro
experiments, ethacraplatin was pre-dissolved in ethanol and further diluted with
DMEM-high glucose to the required concentration. Cisplatin and M-EA-Pt were pre-
dissolved in PBS and further diluted with fresh medium to the same concentration.
For in vivo experiments, ethacraplatin was pre-dissolved in ethanol and further diluted

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with saline to the required concentration. Cisplatin and M-EA-Pt were dissolved in
saline.
2.6. GST-π Expression
Expression of GST-π was detected by immunofluorescence. BEL7404 or
BEL7404-CP20 cells were fixed with 4% cold paraformaldehyde for 15 min. The
cells were blocked for 1 h in 1% BSA PBS and then incubated with monoclonal
antibody against GST-π (1:100, Abcam, Cambridge, UK) overnight. The membrane
was washed with PBS and then incubated for 1 h with goat anti-mouse secondary
antibody conjugated with FITC (1:1000, zsBio, Beijing, China). The nuclei were
stained with DAPI (10 µg/mL, Invitrogen, USA) for 1 min after washing three times
with PBS. Cells were visualized with a confocal laser-scanning microscope (LSM710,
Carl Zeiss) with excitation at 405 nm for DAPI and 488 nm for FITC. Cells that were
incubated with secondary antibody only were regarded as the control.
Expression of GST-π in BEL7404 or BEL7404-CP20 cells was also determined by
western blotting. Cells were dissolved in RIPA buffer containing protease inhibitor
cocktail (cOmplete, Roche, Switzerland) to obtain proteins. After centrifugation, Cell
lysates were collected after centrifugation. Then, protein concentration was
determined by BCA Protein Assay kit (Thermo Fisher, USA). Proteins were separated
by electrophoresis using 10% SDS-PAGE gel and then transferred onto PVDF
membrane. Membranes were exposed to monoclonal antibody against GST-π (1:200,
Abcam, Cambridge, UK) overnight at 4 °C. After washing, membranes were treated
with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000, zsBio,

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Beijing, China) for 1 hour at room temperature and detected by chemiluminescence
imaging system (ChemiDoc Touch , BioradꢀUSA). β-actin was used as a protein-
loading control.
2.7. GST activity
BEL7404 and BEL7404-CP20 cancer cells (1.5 × 105) were seeded in 35 mm2
dishes and further cultured for 24 h. Then, the cells were harvested using a rubber
policeman and centrifuged at 1000 rpm. The cell pellets were resuspended in 30 µL
cold buffer (100 mM potassium phosphate, pH 7.0, including 2 mM EDTA)
containing protease inhibitor. The whole cells were lysed by freezing and thawing,
and centrifuged at 10000 g for 15 min at 4 °C. The supernatant was removed for assay.
The GST activity was detected using a Glutathinone-S-Transferase (GST) Assay Kit
(Sigma-Aldrich, USA) and the protein content was measured using a BCA Protein
Assay Kit (Thermo Fisher, USA). All experiments were carried out in triplicate.
2.8. Inhibition of GST activity in vitro
BEL7404-CP20 cells (1.5 × 105) were seeded in 35 mm2 dishes and further
cultured for 24 h. After that, the cells were treated with fresh medium containing
cisplatin, ethacraplatin or M-EA-Pt at platinum concentrations of 50 µM. After 4 h,
the cells were harvested and treated as described in the previous paragraph. Finally,
the supernatant was used to determine the residual GST activity. Untreated cells in
culture medium were used as the control. All experiments were carried out in
triplicate.

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2.9. Internalization and distribution of micelles in vitro
Firstly, a fluorescent molecule coumarin 6 (C6) was encapsulated into DSPE-
PEG2000 micelles to monitor the cellular uptake of the micelles. Next, BEL7404 cells
or BEL7404-CP20 cells were seeded in 35 mm2 dishes (105 cells per dish) and
cultured in DMEM-high glucose medium. After 48 h, the cells were exposed to 0.5
ꢀg/mL free C6 or C6 Micelles (M-C6) in culture medium. After incubation for 30 min,
the cells were rinsed repeatedly with PBS. The acidic organelles were then stained
with LysoTracker® Deep Red (1:1000, Eugene, Oregon, USA) for 15 min. After
three washes with PBS, the cell nuclei were stained with Hoechst 33342 (10 µg/mL,
Sigma, USA) for 15 min. Thereafter, the live cells were imaged by confocal
microscopy (LSM710, Carl Zeiss) with excitation at 405 nm for Hoechest 33342, 488
nm for coumarin 6 and 633 nm for LysoTracker® Deep Red. Statistical analysis data
of the mean fluorescence intensity of C6 was obtained by Image J software.
2.10. Analysis of platinum-DNA adduct formation
BEL 7404-CP 20 cells were grown at a concentration of 107 cells in 100 mm2
dishes for 24 h. Then cells were incubated with fresh medium containing PBS,
cisplatin, free EA-Pt, or M-EA-Pt at platinum concentrations of 50 µM. After 4 h, the
cells were rinsed 3 times with PBS, trypsinized, and centrifuged for 3 min at 1000 g.
The genomic DNA was extracted using a GenElute Mammalian Genomic DNA
Miniprep Kit. The DNA concentration was determined by UV−visible spectroscopy.

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After digestion, the platinum content was quantified by ICP-MS. All experiments
were carried out in triplicate.
2.11. In vitro antitumor activity
BEL7404 and BEL7404-CP20 were grown in 96-well plates overnight in DMEM-
high glucose medium. Cells were then incubated with various concentrations of free
ethacraplatin, M-EA-Pt or cisplatin at Pt concentrations of 0.5, 5, 20, 40, 80, 120, 160
and 200 µM for 48 h. Next, the cells were incubated for 3 h with 100 µL of 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (final concentration
0.5 mg/mL). Finally, the medium was substituted with 100 ꢀL of DMSO. The
absorbance at 570 nm was measured with a microplate reader (Tecan, Durham, USA).
All experiments were performed in triplicate.
2.12. In vivo antitumor activity
Female NOD SCID mice (18-20 g) were bought from Beijing Vital River
Laboratories. All care and handling of animals was performed with the approval of the
Animal Ethics Committee of the Medical School, Peking University (Beijing, China).
Cisplatin resistant BEL7404-CP20 liver cancer cells were suspended in 100 ꢀL of
PBS and Matrigel (Corning, USA) at a 1:1 ratio (v/v), then 107 cells were
subcutaneously injected into the right flanks of NOD SCID mice When the tumor
volume reached 50-100 mm3, five independent in vivo antitumor activity experiments
with n = 6 mice per group were carried out. Cisplatin, free EA-Pt or M-EA-Pt was
administered at a Pt dosage of 1.3 mg/kg by tail vein injection. The groups treated

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with saline and empty micelles were regarded as controls. M-EA-Pt was synthesized
freshly, and the platinum content was determined before every injection. Tumors were
measured in two dimensions twice every week and the total volume was calculated
2
using the following formula: tumor volume = length×width . The body weights of the
2
mice were determined every other day to monitor potential side effects.
2.13. Hemolysis experiment
Fresh blood was collected from healthy female NOD SCID mice by retro-orbital
bleeds. Red blood cells (RBCs) were isolated from 0.8 mL fresh blood by
centrifuging at 10,000g for 5 min. The RBCs were repeatedly rinsed with saline until
no color was observed in the supernatant. RBCs were then suspended in 15 mL saline.
RBC suspension (0.5 mL) was mixed with 0.5 mL of saline containing different
concentrations of M-EA-Pt at 25, 50, 75, 100 and 200 ꢀg/mL to give final
concentrations of 12.5, 25, 37.5, 50 and 100 ꢀg/mL, respectively. The negative
control was 0.5 mL RBC suspension mixed with 0.50 mL saline and the positive
control was 0.5 mL RBC suspension mixed with 0.50 mL distilled water. The samples
were mixed gently, kept at room temperature for 2 h, and then centrifuged at 10,000g
for 5 min. The absorbance of the supernatant at 540 nm was recorded in the 96-well
plate. The percentage of hemolysis was evaluated as follows:
Abssample - Absnegativeꢁ control
Hemolysis % = Abs
× 100%
positiveꢁ control - Absnegativeꢁ control
2.14. Histological study and blood biochemical analysis

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After completion of the in vivo antitumor activity experiment, blood was collected
by retro-orbital bleeds and centrifuged at 5000 g for 10 min. The blood sera were then
transferred into several new tubes. The blood serum samples were analyzed for renal
function markers (blood urea nitrogen (BUN) and creatinine (Cr)) in the Beijing
Lawke Health Laboratory Center for Clinical Laboratory Development.
After the blood was collected, all the mice were euthanized, and the primary organs
were excised and fixed in 10% formalin solution. The organ samples were then
analyzed in the Beijing Lawke Health Laboratory Center for Clinical Laboratory
Development by hematoxylin and eosin (H&E) staining. The stained slides were
observed by light microscopy (EVOS XL Core, Thermo Fisher, USA).
2.15. Intratumoral microdistribution of platinum
1 × 107 cisplatin-resistant BEL7404-CP20 liver cancer cells were subcutaneously
inoculated into the right flanks of female NOD SCID mice. After the tumor volume
reached around 150 mm3, the mice were randomly divided into four group (n = 3).
Cisplatin, free ethacraplatin or M-EA-Pt was injected intravenously into mice at a Pt
dosage of 10 µmol/kg. The mice, which were used as controls, were intravenously
injected with saline. After 24 h, all the mice were euthanized and the tumors were
excised and digested in aqua regia at 300 °C. Finally, the platinum content in the
tumors was determined by ICP-MS.
3. Results and discussion
3.1. Synthesis and characterization of micelles encapsulating ethacraplatin

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Ethacraplatin was synthesized according to the procedures reported previously [10].
1
The chemical structure and purity of ethacraplatin was verified by its ESI-MS, H
NMR, UV-vis and HPLC spectra (Fig. S1-S4). Ethacraplatin was encapsulated within
DSPE-PEG2000 micelles by a film dispersion method (Fig. 1A). DSPE forms the
hydrophobic core inside the micelle that creates a suitable environment for
encapsulating ethacraplatin, while the role of PEG is to provide the hydrophilic ends
on the surface that improve the water solubility of the whole drug-loaded micelle. In
addition, the circulation time of micelles can also be increased because of the
hydrophilic PEG ends, which provide steric stabilization and stealth properties.
Fig. 1. Characterization of micelles encapsulating ethacraplatin (M-EA-Pt). (A) Schematic
illustration of M-EA-Pt. (B) TEM image of M-EA-Pt after staining with 1% uranyl acetate. (C)
Size distribution of M-EA-Pt. (D) Release profiles of ethacraplatin from micelles at pH 7.4 and
37 °C. Released platinum was quantified using ICP-MS. Scale bar = 200 nm.

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Micelles were prepared using different weight ratios of ethacraplatin (EA-Pt) to
DSPE-PEG2000 (mEA-Pt : mDSPE-PEG2000) (Table S1). The resulting micelles were
evaluated for their average hydrodynamic diameters, polydispersity indices (PDI),
encapsulation efficiencies and drug loading efficiencies, which were calculated
according to the Pt content as quantified by ICP-MS. When the mEA-Pt : mDSPE-PEG2000
ratio decreased, the diameter of the micelles became smaller, while the encapsulation
efficiency increased. We found that it was difficult for our system to form micelles
when mEA-Pt : mDSPE-PEG2000 was smaller than 1:7. Micelles with mEA-Pt : mDSPE-PEG2000
= 1:7 were used in the following study because they had the highest encapsulation
efficiency together with an ideal drug loading capacity and PDI (Table S1). According
to previous reports [26-28], the resulting particles are of an appropriate size to target
passively tumor tissue through the EPR effect.
The morphologies of ethacraplatin-loaded micelles (M-EA-Pt) were examined by
TEM. M-EA-Pt micelles were uniform spheres, with a diameter of approximately 40
nm (Fig. 1B). The particle diameters determined by TEM are smaller than the
hydrodynamic diameters determined by DLS, due to stretching of the hydrophilic
PEG ends and swelling of the micelle cores in aqueous medium (Fig. 1C).
3.2. Controlled release of ethacraplatin in vitro
Next, the release profile of ethacraplatin from micelles was evaluated. Aliquots of
M-EA-Pt micelles were dialyzed against phosphate buffered saline (PBS) at pH 7.4
and 37 °C. The released platinum content was quantified using ICP-MS. The
percentage of encapsulated platinum released over time is presented in Fig. 1D. There

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is no obvious burst release, a drawback of micelle-based delivery system, as only 14%
of the total platinum was released in the first 1 hour. After 72 hours of incubation,
approximately 85% of the total platinum was released (Fig. 1D). The data
demonstrated that these M-EA-Pt micelles are an optimal platform for sustained
release of platinum.
3.3. Inhibition of the activity of GSTs in vitro
To begin with, immunofluorescence was used to confirm the expression of GST-π
(a member of the GST family), which was significantly related with the cisplatin
resistance of tumor, in the hepatocellular carcinoma cell line BEL7404 and its
cisplatin-resistant derivative BEL7404-CP20 (Fig. 2A). GST-π was expressed at
much higher levels in the BEL7404-CP20 cells than in the parental cells. This was
also confirmed by Western Blot (Fig. 2B). Next, the activity of GSTs in BEL7404
and BEL7404-CP20 cells was verified. The data demonstrated that the activity of
GSTs in BEL7404-CP20 was at a significantly higher level (Fig. 2C). These results
are consistent with previous reports that GSTs, especially GST-π, contribute to the
cisplatin resistance [3-6].
Thereafter, the residual GST activity was determined in BEL7404-CP20 cells after
incubation with 50 ꢀM cisplatin, free EA-Pt or M-EA-Pt for 4 h (Fig. 2D). After
incubation, marked inhibition of GST activity was observed in the cells incubated
with free EA-Pt and M-EA-Pt. The GST activity in the cells incubated with cisplatin
was only slightly lower than that in control group (Fig. 2D). This result indicated that
free ethacraplatin and ethacraplatin-encapsulated micelles were able to enter live

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mammalian cells and significantly inhibit the GST activity. The activity of GSTs in
the BEL7404-CP20 cancer cells incubated with M-EA-Pt was significantly lower than
in the cells incubated with free EA-Pt. This might benefit from the higher uptake of
M-EA-Pt by cells, which was due to endocytosis of nanomicelles, and the resultant
higher GSTs inhibition than free EA-Pt. These results indicated that M-EA-Pt had
stronger ability than free EA-Pt in GSTs inhibition.
Fig. 2. GST-π expression and GST activity in cells. (A) Detection of GST-π expression (green) in
BEL7404 and BEL7404-CP20 cells by confocal fluorescence microscopy. Cells were pretreated
with (+) or without (−) a primary monoclonal GST antibody, then incubated with goat anti-mouse
secondary antibody conjugate with FITC (green). Nuclei are stained with DAPI (blue). (B)
Western blot analysis of GST-π in BEL7404 and BEL7404-CP20 cells. β-actin represented a
protein-loading control. (C) Activity of GSTs in BEL7404 and BEL7404-CP20 cells. (D) Residual

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GST activity of BEL7404-CP20 cells after exposure to cisplatin, free EA-Pt or M-EA-Pt at Pt
concentrations of 50 ꢀM. The control group was untreated cells. Data are presented as means ±
SD (n = 3), ***P < 0.001.
3.4. Anti-cancer efficacy in vitro
The anti-cancer efficacy of M-EA-Pt was evaluated in BEL7404 and BEL7404-
CP20 cells. The two cancer cell lines were incubated with cisplatin, free EA-Pt and
M-EA-Pt at different platinum concentrations for 48 h and the cell viability was
assessed by MTT assay. For the cisplatin-sensitive cancer cells, the anticancer
efficacy of cisplatin is superior to free ethacraplatin and M-EA-Pt (Fig. S5). For the
cisplatin-resistant cancer cells, free ethacraplatin and M-EA-Pt outperformed cisplatin
(Fig. 3A). For both EA-Pt and M-EA-Pt, the resistance factors (RF) are approximately
1, which means that the cisplatin resistance is almost overcome (Table 1).
Furthermore, IC50 of M-EA-Pt (33.0 µM) was lower than that of free ethacraplatin
(58.9 µM) in resistant cells and this phenomenon was also observed in the cisplatin-
sensitive cancer cells. The empty micelles formed by DSPE-PEG2000 showed
ignorable cytotoxicity toward BEL7404 and BEL7404-CP20 cells (Fig. S6). These
results highlighted that micelle formation enhanced the anticancer efficacy of
ethacraplatin in cancer cells, especially in cisplatin-resistant cells.

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Fig. 3. Evaluation of in vitro antitumor activity. (A) In vitro antitumor activity of cisplatin, free
EA-Pt and M-EA-Pt toward BEL7404-CP20 cells. (B) BEL7404-CP20 cells were incubated with
F-C6 and M-C6, then the cellular internalization and distribution of C6 were imaged by confocal
fluorescence microscopy. The control group was untreated cells. DNA is labeled by Hoechst
(blue). Endosomes/lysosomes are labeled by Lystotracker (red). (C) Statistical analysis of the

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mean fluorescence intensity of C6 from the confocal fluorescence microscopy data. (D) Platinum
content in the genomic DNA of BEL7404-CP20 cells after incubation with cisplatin, free EA-Pt or
M-EA-Pt at a Pt concentration of 50 µM for 4 h. Data are presented as means ± SD (n = 3), *P <
0.05, **P < 0.01, ***P < 0.001. Scale bar = 20 µm.
Table 1. Comparison of IC50 and drug-resistance factor of cisplatin, free EA-Pt, M-EA-Pt and
empty micelles in BEL7404 and BEL7404-CP20 cells. Data are presented as means ± SD (n = 3).
IC50 (ꢀM)
BEL7404-CP20
Resistance Factor
Test Compound
(RF)
6.26
1.08
1.14
-
BEL7404
12.2 ± 0.9
55.0 ± 0.8
28.9 ± 0.5
> 200
Cisplatin
EA-Pt
76.4 ± 2.4
58.9 ± 0.5
33.0 ± 0.5
114
M-EA-Pt
Empty Micelles
In sensitive cancer cell lines, cisplatin is more active than EA-Pt and M-EA-Pt. This
is due to the following two reasons. 1. Since GSTs are not highly expressed in
sensitive cell lines, cisplatin can exert its activity normally, while the GSTs inhibition
ability of EA-Pt and M-EA-Pt don’t contribute to a higher level of active platinum in
nuclei. 2. On the other hand, the cytotoxicity of EA-Pt and M-EA-Pt are slightly
limited by the rate and efficiacy of their transformation in cells to cisplatin. However,
in resistant cancer cell lines, EA-Pt and M-EA-Pt outperformed cisplatin. The highly
expressed GSTs in resistant cell lines would accelerate the deactivation of cisplatin,
but they have much less effect on EA-Pt and M-EA-Pt that have ability of GSTs
inhibition. Although the process of their transformation to active species in cells

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slightly decreased the efficiency of EA-Pt and M-EA-Pt, the circumvention of EA-Pt
and M-EA-Pt from inactivation by GSTs/GSH pathway endowed them with higher
cytotoxicity than cisplatin in resistant cell lines.
3.5. Internalization and distribution of micelles in vitro
The internalization of micelles was investigated at the subcellular level using
confocal laser scanning microscopy (CLSM). As ethacraplatin does not possess
fluorescent properties, a fluorescent molecule coumarin 6 (C6) was encapsulated
within the DSPE-PEG2000 micelles for the observation of micelle behavior in cells.
Thereafter, BEL7404 and BEL7404-CP20 cells were incubated with free C6 (F-C6)
and C6-loaded micelles (M-C6). After 30 min, stronger green fluorescence from C6
was observed in the cells incubated with M-C6 than in the cells treated with F-C6
(Fig. 3B-C, Fig. S7). This result clearly indicated that M-C6 was more rapidly and
effectively internalized than F-C6 into the cancer cells. Micelle formation
significantly increased the intracellular C6 accumulation in both cisplatin-resistant
and parental cancer cells. Furthermore, most of the fluorescent signals from M-C6
were co-localized with endosomes/lysosomes labeled by the LysoTracker Deep Red
marker, suggesting that internalization of the micelles was related to endocytic
pathways [29]. Consequently, we conclude that micelle formation is capable of
enhancing the GST inhibitory activity and the anticancer efficacy of ethacraplatin by
increasing drug uptake by cancer cells, especially by cisplatin-resistant cancer cells
(Scheme 1).

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Scheme 1. Scheme of release of EA-Pt in cisplatin-resistant cells after endocytosis of M-EA-Pt.
The diagram shows the mechanism by which ethacrynic acid (EA) inhibits glutathione S-
transferases (GSTs) to circumvent the cisplatin resistance of cancer cells.
3.6. Quantification of platinum-DNA adduct formation
It is generally known that cell death caused by cisplatin mainly derives from
formation of platinum-DNA adducts. To quantify and compare the content of
platinum-DNA adducts, we exposed BEL7404-CP20 cancer cells to 50 µM cisplatin,
free EA-Pt or M-EA-Pt for 4 h. The genomic DNA was isolated using a DNA
miniprep kit. The formation of platinum-DNA adducts was determined by quantifying

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the Pt content in the genomic DNA by ICP-MS. Compared with cells treated with
cisplatin, the DNA from cells treated with both free EA-Pt and M-EA-Pt exhibited
significantly higher levels of platinum-DNA adducts (Fig. 3D). These data suggested
that, comparing with cisplatin, free EA-Pt and M-EA-Pt have more active platinum
species to enter into nucleis, which owed to their GSTs inhibition ability. Moreover,
cells treated with M-EA-Pt contained 2.76 times more platinum-DNA adducts than
that treated with free EA-Pt, which corroborated our hypothesis that the M-EA-Pt
formulation might be capable of enhancing the antitumor activity of EA-Pt in the
cisplatin-resistant cancer cells due to a higher level of DNA damage. This is
consistent with the higher internalization of M-C6 in cells.
3.7. Anti-cancer efficacy in vivo
As M-EA-Pt showed promising results in cisplatin-resistant cancer cells in the in
vitro experiment, we next tested the anticancer efficacy in vivo. First of all, the
excellent blood compatibility of M-EA-Pt was verified (Fig. S8). After that, we
assessed the anticancer efficacy of M-EA-Pt in the cisplatin-resistant BEL7404-CP20
human liver cancer subcutaneous mouse model. BEL7404-CP20 tumor-bearing mice
were treated by intravenous injections of saline, empty micelles, cisplatin, free EA-Pt
and M-EA-Pt (platinum dose of 1.3 mg/kg) every 3 days. When the tumor growth was
monitored for 30 days, no obvious inhibition of tumor growth was observed in the
mice treated with cisplatin or free EA-Pt compared with the control groups (mice
treated with saline and empty micelles) (Fig. 4A). However, M-EA-Pt significantly
repressed tumor growth compared to the controls. The antitumor efficiency was

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further confirmed by histological analysis of tumor tissues using the hematoxylin and
eosin (H&E) staining method. A number of vacuoles in cytoplasm and obvious cell
shrinkage and nuclear condensation were only observed in the M-EA-Pt-treated
group, while the other groups did not exhibited these changes in the tumor tissue,
which indicated M-EA-Pt can induce high level of apoptosis in vivo (Fig. 4E). This
result strongly supported our hypothesis that M-EA-Pt overcame cisplatin resistance,
thereby achieving enhanced anticancer efficacy. Moreover, no significant loss of body
weight was observed in the M-EA-Pt treatment group, compared with the 10% body
weight loss in the cisplatin group (Fig. 4B). In addition, only two mice survived in the
cisplatin-treated group at the end of the treatment, once again indicating that cisplatin
exhibits fatal side effects (Fig. 4C). These results suggested that M-EA-Pt overcomes
cisplatin-resistance of tumor, circumvents possible side effects associated with
cisplatin and exhibit significantly enhanced inhibition efficiency against cisplatin-
resistant tumor over cisplatin and free EA-Pt.
Cisplatin is obviously powerless to treat cisplatin-resistant cancers, since the highly
expressed GSTs in cisplatin-resistant cancers would partly deactivate cisplatin and
decrease the effective platinum content in nucleis. Free EA-Pt shows antitumor
activity and overcomes drug resistance in the in vitro assay, but is not effective
enough to fight resistant cancer cells in vivo as water solubility is critical for drug
availability after injection. These phenomena emphasize the fact that GSTs inhibition
and micelle formation are crucially important to achieve the antitumor activity of M-
EA-Pt micelles in the treatment of cisplatin-resistant cancers.

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Fig. 4. In vivo antitumor efficacy study. (A) In vivo tumor growth inhibition curves of BEL7404-
CP20 tumor-bearing mice treated with saline, empty micelles, cisplatin, free EA-Pt or M-EA-Pt
twice a week for 4 weeks. Data are presented as means ± SD (n = 6). (B) Relative body weight of
BEL7404-CP20 tumor-bearing mice treated with saline, empty micelles, cisplatin, free EA-Pt or

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M-EA-Pt over the course of the treatment. Data are presented as means ± SD (n = 6). (C) Survival
curve of BEL7404-CP20 tumor-bearing mice over the course of the treatment. Data are presented
as means ± SD (n = 6). (D) Intratumoral microdistribution of platinum in BEL7404-CP20 tumor
tissues, as measured by ICP-MS, 24 h post-administration of cisplatin, free EA-Pt or M-EA-Pt.
Data are presented as means ± SD (n = 3). (E) Histological study of BEL7404-CP20 tumor tissues.
*P < 0.05, **P < 0.01. Scale bar = 50 µm.
Next, the toxicity of the different treatments was further ascertained by histological
study and blood biochemistry analysis. After completion of the study, histological
analysis of sections of heart, liver, spleen, lung and kidney was performed by the
H&E staining method. The blood of mice in all the treatment groups was collected to
assess the levels of creatinine (Cr) and blood urea nitrogen (BUN). Marked changes
were only observed in the cisplatin-treated group (Fig. 5A). Histological staining
revealed that cisplatin administration caused hyaline cast filling in the renal tubules,
which indicated renal failure (Fig. 5B-C). Meanwhile, These data are consistent with
a previous report that nephrotoxicity is the dose-limiting toxicity of cisplatin [30]. No
apparent nephrotoxicity was detected in the other treated groups. Overall, the above-
mentioned results demonstrated that our system is an outstanding candidate with high
efficiency and low toxicity for chemotherapy of cisplatin-resistant cancer.

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Fig. 5. In vivo toxicity of cisplatin, free EA-Pt and M-EA-Pt. (A) Blood analysis of urea nitrogen
(BUN) and creatinine (Cr) at completion of the treatment with cisplatin, free EA-Pt or M-EA-Pt.
Saline was administered as a control. (B) Histological study of kidney sections at completion of
the treatment with cisplatin, free EA-Pt or M-EA-Pt. Saline was administered as a control. Black
arrows indicate hyaline cast filling in the renal tubules. (C) Histological study of heart, liver,
spleen and lung of BEL7404-CP20 tumor-bearing mice at completion of the treatment with
cisplatin, free EA-Pt or M-EA-Pt. Saline was administered as a control. Data are presented as
means ± SD (n = 3). Scale bar = 100 µm.
Finally, the intratumoral microdistribution of platinum in BEL7404-CP20 tumor
tissues was investigated (Fig. 4D). The data demonstrated that the level of Pt in the
tumor tissue was much higher in the M-EA-Pt-treated mice than in the mice treated
with free EA-Pt. This may be because the EPR effect promotes the accumulation of
nanosized micelles. The higher accumulation of platinum in the tumor contributed to
the improved antitumor activities of M-EA-Pt.
4. Conclusion
In summary, we encapsulated a GST inhibitor axial-conjugated Pt(IV) prodrug,
named ethacraplatin, into the DSPE-PEG2000 micelles for treatment of cisplatin-
resistant cancer. The obtained ethacraplatin-loaded micelles have uniform size, high
monodispersity and controlled release properties. The micelles were able to inhibit
GST activity in cancer cells more strongly than the free drug. Our data proved that
unlike the free drug, this developed M-EA-Pt system has the potency to tackle
cisplatin resistance and inhibit cisplatin-resistant tumor growth in vitro and in vivo.